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McDonnell, Donald Patrick

Overview:

The research in our group is focused on the development and application of mechanism based approaches to identify novel therapeutics for use in the treatment and prevention of hormonally responsive cancers. Specifically we are interested in the pharmaceutical exploitation of the estrogen and androgen receptors as therapeutic targets in breast and prostate cancers and in defining how these receptors influence the pathogenesis of these diseases. These efforts have led to the discovery of several drugs that are currently being evaluated in the clinic as cancer therapeutics, and to the identification of potential biomarkers and predictors of response that can help to target the use of these new drugs. Most recently we have explored approaches to treat triple negative breast cancer and have identified an important pathway that links obesity/dyslipidemia and cancer risk.

Positions:

Chair, Department of Pharmacology & Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Glaxo-Wellcome Professor of Molecular Cancer Biology, in the School of Medicine

Pharmacology & Cancer Biology
School of Medicine

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Professor in Medicine

Medicine, Endocrinology, Metabolism, and Nutrition
School of Medicine

Core Faculty in Innovation & Entrepreneurship

Duke Innovation & Entrepreneurship
Institutes and Provost's Academic Units

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1988

Ph.D. — Baylor College of Medicine

News:

Grants:

Targeting convergent oncogenic signaling during AR inhibition to overcome metastasis and immune evasion in prostate cancer

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
August 01, 2019
End Date
July 31, 2024

Duke University Program in Environmental Health

Administered By
Environmental Sciences and Policy
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2013
End Date
June 30, 2024

Identification of Novel Targets in Treatment Resistant Prostate Cancer

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
September 18, 2017
End Date
September 17, 2022

Targeting tumor-specific apoptosis regulation in advanced ER+ breast cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Collaborator
Start Date
August 15, 2019
End Date
August 14, 2022

Building Interdisciplinary Research Careers in Women's Health

Administered By
Obstetrics and Gynecology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 26, 2002
End Date
July 31, 2022

Building Interdisciplinary Research Careers in Women's Health

Administered By
Obstetrics and Gynecology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 26, 2002
End Date
July 31, 2022

Cancer cell intrinsic and extrinsic actions of steroid hormones in breast tumors

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Principal Investigator
Start Date
June 15, 2018
End Date
June 14, 2022

Training Program in Developmental and Stem Cell Biology

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2001
End Date
April 30, 2022

Defining the Rules of Breast Cancer Cell Traffic Through Bone

Administered By
Medicine, Hematologic Malignancies and Cellular Therapy
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
January 09, 2017
End Date
December 31, 2021

Identification of actionable networks promoting breast cancer progression and brain metastasis

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Collaborator
Start Date
September 01, 2018
End Date
August 31, 2021

Translational Research in Surgical Oncology

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
January 01, 2002
End Date
August 31, 2021

Regulatory principles of the Warburg Effect

Administered By
Pharmacology & Cancer Biology
Role
Collaborator
Start Date
July 01, 2017
End Date
June 30, 2021

The Role of Epigenetic Plasticity in Breast Cancer Recurrence

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
March 01, 2018
End Date
April 30, 2021

RIP140 as a mediator of Estrogen Receptor action in tumor associated macrophages

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2017
End Date
August 30, 2020

RIP140 as a mediator of Estrogen Receptor action in tumor associated macrophages

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2017
End Date
August 30, 2020

Immune modulation of CaMKK2 in the ovarian tumor microenvironment

Administered By
Obstetrics and Gynecology, Gynecologic Oncology
Role
Mentor
Start Date
July 01, 2019
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Targeting the GRHL2/AGR2/LYPD3 axis in breast cancer

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
April 01, 2018
End Date
March 31, 2020

Targeting precursor neural (N)-cadherin to eliminate chemotherapy-resistant triple-negative breast tumor cells

Administered By
Surgery, Plastic, Maxillofacial, and Oral Surgery
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
March 01, 2017
End Date
February 29, 2020

Role of Cholesterol Efflux Blockade in Mycobacterial Infection In Vivo

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
February 01, 2017
End Date
January 31, 2020

ER and LXR as mediators of the pathobiology of hypercholesterolemia

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 01, 1997
End Date
January 31, 2020

Pharmacology Industry Internships for Ph.D. Students

Administered By
Pharmacology & Cancer Biology
AwardedBy
American Society for Pharmacology and Experimental Therapeutics
Role
Participating Faculty Member
Start Date
January 01, 2017
End Date
December 31, 2019

Hypercholesterolemia as a modifier of the tumor microenvironment in breast cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Principal Investigator
Start Date
September 30, 2016
End Date
September 30, 2019

Targeting differential apoptosis regulation in triple negative breast cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Collaborator
Start Date
September 30, 2016
End Date
September 29, 2019

Development of mechanistically distinct androgen receptor antagonists and degraders for the treatment of advanced castra

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Principal Investigator
Start Date
September 30, 2015
End Date
September 29, 2019

Developing in vivo active probes for CAMKK2 to treat cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
August 01, 2017
End Date
July 31, 2019

Endocrinology and Metabolism Training Program

Administered By
Medicine, Endocrinology, Metabolism, and Nutrition
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2014
End Date
June 30, 2019

Duke University Program in Environmental Health

Administered By
Environmental Sciences and Policy
AwardedBy
National Institute of Environmental Health Sciences
Role
Mentor
Start Date
July 01, 2013
End Date
June 30, 2019

Macrophages and Treatment-Resistant NVAMD

Administered By
Ophthalmology, Vitreoretinal Diseases & Surgery
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
May 01, 2015
End Date
April 30, 2019

Effects of dietary methionine restriction on tumor growth and epigenetics

Administered By
Pharmacology & Cancer Biology
Role
Co-Mentor
Start Date
January 01, 2018
End Date
December 31, 2018

Fibulin-3 as a Novel Biomarker and Trget in the Breast Tumor Microenvironment

Administered By
Medicine, Medical Oncology
Role
Key Faculty
Start Date
December 01, 2015
End Date
November 30, 2018

A Novel Function for ALK4 in Suppressing Breast Cancer Progression

Administered By
Medicine, Medical Oncology
Role
Co-Mentor
Start Date
August 20, 2015
End Date
August 19, 2018

Developing small-molecule probes for protein arginine methyltransferases

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
July 01, 2015
End Date
June 30, 2018

Seahorse XFe96 Extracellular Flux Analyzer

Administered By
Pediatrics, Endocrinology
Role
Major User
Start Date
June 07, 2017
End Date
June 06, 2018

Targeting the synthetic essential kinases of breast cancers

Administered By
Molecular Genetics and Microbiology
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
April 15, 2015
End Date
April 14, 2018

Optimization of ERalpa-LBD-based CAR-T switch

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
August 04, 2017
End Date
February 03, 2018

The role of ERRalpha/PGC-1 in disease pathogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2006
End Date
January 31, 2018

CaMKK2: a druggable target that regulates macrophage function in breast cancer

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
October 05, 2013
End Date
October 04, 2017

Control of the apoptosome in epithelial ovarian cancer to enhance chemotherapy

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 01, 2014
End Date
August 31, 2017

Evaluation of the therapeutic potential of combining VT464 and G1T38 in treatment-resistant prostate and breast cancer models

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
January 19, 2016
End Date
July 31, 2017

Kinase Target of Diverse Cell Surface Receptors in Cancer Invasion and Metastasis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
July 21, 2011
End Date
April 30, 2017

Lipid Activated Nuclear Receptors in Age-Related Macular Degeneration

Administered By
Ophthalmology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
May 01, 2011
End Date
April 30, 2017

Metabolism of Effector T cells as a Target in SLE

Administered By
Pharmacology & Cancer Biology
AwardedBy
Vanderbilt University
Role
Principal Investigator
Start Date
September 01, 2015
End Date
January 31, 2017

BC123577: Targeting Epigenetics Therapy for Estrogen Receptor Negative Breast Cancers

Administered By
Chemistry
Role
Co Investigator
Start Date
September 30, 2013
End Date
September 29, 2016

Targeting the CDK 4/6 axis in Castration Resistant Prostate Cancer

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
February 16, 2015
End Date
August 31, 2016

Targeting aerobic glycolysis in T-ALL

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2014
End Date
July 15, 2016

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Identification of a novel target for treating breast cancer metastasis

Administered By
Pharmacology & Cancer Biology
Role
Co-Mentor
Start Date
August 01, 2014
End Date
February 29, 2016

Evaluation of VT-464 in AR positive and ER positive breast cancer models

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
February 01, 2015
End Date
January 31, 2016

Androgen receptor degradation as a therapeutic modality in castrate resistant prostate cancer

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
July 01, 2015
End Date
December 31, 2015

T cell metabolism as a determinant of differentiation in allergic asthma

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
April 08, 2011
End Date
August 31, 2015

Clinical Oncology Research Career Development Program

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 29, 2009
End Date
July 31, 2015

Determination of the antiandrogenic properties of VT-464, a novel CYP17 lyase inhibitor

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
May 20, 2014
End Date
May 31, 2015

B cell metabolism in activation and autoantibody production

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
PD/PI
Start Date
June 01, 2013
End Date
May 31, 2015

Molecular pharmacology of bazedoxifene and pipendoxifene in breast cancer

Administered By
Pharmacology & Cancer Biology
Role
Principal Investigator
Start Date
November 28, 2013
End Date
November 27, 2014

27-hydroxycholesterol as a link between obesity and breast cancer pathogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
January 01, 2013
End Date
August 31, 2014

Live-Animal Micro-CT System

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2012
End Date
May 14, 2014

Molecular Determinants of Androgen Receptor Pharmacology

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 15, 2003
End Date
November 30, 2013

Nonclassical signaling of the androgen receptor polyproline domain

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
July 01, 2009
End Date
June 30, 2010

G Protein Involvement in Oncogenesis and Metastasis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
February 01, 2004
End Date
July 31, 2009

Integrated instrument systems for maintenance and delivery of RNAi libraries

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
February 15, 2008
End Date
February 14, 2009

The Androgen Receptor: Cofactor Interface

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2002
End Date
June 30, 2005

Development and Use of Systems to Study Estrogen Action

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2002
End Date
December 31, 2002

RAR-Beta Mediated Apoptosis in Breast Cancer Cells

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 22, 1995
End Date
August 31, 2000
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Awards:

Robert R. Ruffolo Career Achievement Award in Pharmacology. ASPET.

Type
Other
Awarded By
ASPET
Date
January 01, 2017

Member. National Academy of Medicine.

Type
National
Awarded By
National Academy of Medicine
Date
January 01, 2016

Distinguished Alumni Award. Baylor College of Medicine.

Type
Other
Awarded By
Baylor College of Medicine
Date
January 01, 2013

Roy O. Greep Award. The Endocrine Society.

Type
Other
Awarded By
The Endocrine Society
Date
January 01, 2013

AAAS Fellows. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2011

MERIT Award. National Institutes of Health.

Type
National
Awarded By
National Institutes of Health
Date
January 01, 2005

Publications:

Constitutively active ESR1 mutations in gynecologic malignancies and clinical response to estrogen-receptor directed therapies.

OBJECTIVE: Endocrine therapy is often considered as a treatment for hormone-responsive gynecologic malignancies. In breast cancer, activating mutations in the estrogen receptor (mutESR1) contribute to therapeutic resistance to endocrine therapy, especially aromatase inhibitors (AIs). The purpose of this study was to evaluate the frequency and clinical relevance of ESR1 genomic alterations in gynecologic malignancies. METHODS: DNA from FFPE tumor tissue obtained during routine clinical care for 9645 gynecologic malignancies (ovary, fallopian tube, uterus, cervix, vagina, vulvar, and placenta) was analyzed for all classes of genomic alterations (base substitutions (muts), insertions, deletions, rearrangements, and amplifications) in ESR1 by hybrid capture next generation sequencing. A subset of alterations was characterized in laboratory-based transcription assays for response to endocrine therapies. RESULTS: A total of 295 ESR1 genomic alterations were identified in 285 (3.0%) cases. mutESR1 were present in 86 (0.9%) cases and were more common in uterine compared to other cancers (2.0% vs <1%, respectively p < 0.001). mutESR1 were enriched in carcinomas with endometrioid versus serous histology (4.4% vs 0.2% respectively, p < 0.0001 in uterine and 3.5% vs 0.3% respectively, p = 0.0004 in ovarian carcinomas). In three of four patients with serial sampling, mutESR1 emerged under the selective pressure of AI therapy. Despite decreased potency of estrogen receptor (ER) antagonists in transcriptional assays, clinical benefit was observed following treatment with selective ER-targeted therapy, in one case lasting >48 months. CONCLUSIONS: While the prevalence of ESR1 mutations in gynecologic malignancies is low, there are significant clinical implications useful in guiding therapeutic approaches for these cancers.

Authors
Gaillard, SL; Andreano, KJ; Gay, LM; Steiner, M; Jorgensen, MS; Davidson, BA; Havrilesky, LJ; Alvarez Secord, A; Valea, FA; Colon-Otero, G; Zajchowski, DA; Chang, C-Y; McDonnell, DP; Berchuck, A; Elvin, JA
MLA Citation
Gaillard, Stéphanie L., et al. “Constitutively active ESR1 mutations in gynecologic malignancies and clinical response to estrogen-receptor directed therapies..” Gynecol Oncol, vol. 154, no. 1, July 2019, pp. 199–206. Pubmed, doi:10.1016/j.ygyno.2019.04.010.
PMID
30987772
Source
pubmed
Published In
Gynecol Oncol
Volume
154
Issue
1
Publish Date
2019
Start Page
199
End Page
206
DOI
10.1016/j.ygyno.2019.04.010

Inhibition of ERRα Prevents Mitochondrial Pyruvate Uptake Exposing NADPH-Generating Pathways as Targetable Vulnerabilities in Breast Cancer.

Most cancer cells exhibit metabolic flexibility, enabling them to withstand fluctuations in intratumoral concentrations of glucose (and other nutrients) and changes in oxygen availability. While these adaptive responses make it difficult to achieve clinically useful anti-tumor responses when targeting a single metabolic pathway, they can also serve as targetable metabolic vulnerabilities that can be therapeutically exploited. Previously, we demonstrated that inhibition of estrogen-related receptor alpha (ERRα) significantly disrupts mitochondrial metabolism and that this results in substantial antitumor activity in animal models of breast cancer. Here we show that ERRα inhibition interferes with pyruvate entry into mitochondria by inhibiting the expression of mitochondrial pyruvate carrier 1 (MPC1). This results in a dramatic increase in the reliance of cells on glutamine oxidation and the pentose phosphate pathway to maintain nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis. In this manner, ERRα inhibition increases the efficacy of glutaminase and glucose-6-phosphate dehydrogenase inhibitors, a finding that has clinical significance.

Authors
Park, S; Safi, R; Liu, X; Baldi, R; Liu, W; Liu, J; Locasale, JW; Chang, C-Y; McDonnell, DP
MLA Citation
Park, Sunghee, et al. “Inhibition of ERRα Prevents Mitochondrial Pyruvate Uptake Exposing NADPH-Generating Pathways as Targetable Vulnerabilities in Breast Cancer..” Cell Rep, vol. 27, no. 12, June 2019, pp. 3587-3601.e4. Pubmed, doi:10.1016/j.celrep.2019.05.066.
PMID
31216477
Source
pubmed
Published In
Cell Reports
Volume
27
Issue
12
Publish Date
2019
Start Page
3587
End Page
3601.e4
DOI
10.1016/j.celrep.2019.05.066

CaMKK2 in myeloid cells is a key regulator of the immune-suppressive microenvironment in breast cancer.

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8+ T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2-/- mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2-/- macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8+ T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.

Authors
Racioppi, L; Nelson, ER; Huang, W; Mukherjee, D; Lawrence, SA; Lento, W; Masci, AM; Jiao, Y; Park, S; York, B; Liu, Y; Baek, AE; Drewry, DH; Zuercher, WJ; Bertani, FR; Businaro, L; Geradts, J; Hall, A; Means, AR; Chao, N; Chang, C-Y; McDonnell, DP
MLA Citation
Racioppi, Luigi, et al. “CaMKK2 in myeloid cells is a key regulator of the immune-suppressive microenvironment in breast cancer..” Nat Commun, vol. 10, no. 1, June 2019. Pubmed, doi:10.1038/s41467-019-10424-5.
PMID
31164648
Source
pubmed
Published In
Nature Communications
Volume
10
Issue
1
Publish Date
2019
Start Page
2450
DOI
10.1038/s41467-019-10424-5

Decoding the Inversion Symmetry Underlying Transcription Factor DNA-Binding Specificity and Functionality in the Genome.

Understanding why a transcription factor (TF) binds to a specific DNA element in the genome and whether that binding event affects transcriptional output remains a great challenge. In this study, we demonstrate that TF binding in the genome follows inversion symmetry (IS). In addition, the specific DNA elements where TFs bind in the genome are determined by internal IS within the DNA element. These DNA-binding rules quantitatively define how TFs select the appropriate regulatory targets from a large number of similar DNA elements in the genome to elicit specific transcriptional and cellular responses. Importantly, we also demonstrate that these DNA-binding rules extend to DNA elements that do not support transcriptional activity. That is, the DNA-binding rules are obeyed, but the retention time of the TF at these non-functional DNA elements is not long enough to initiate and/or maintain transcription. We further demonstrate that IS is universal within the genome. Thus, IS is the DNA code that TFs use to interact with the genome and dictates (in conjunction with known DNA sequence constraints) which of those interactions are functionally active.

Authors
Coons, LA; Burkholder, AB; Hewitt, SC; McDonnell, DP; Korach, KS
MLA Citation
Coons, Laurel A., et al. “Decoding the Inversion Symmetry Underlying Transcription Factor DNA-Binding Specificity and Functionality in the Genome..” Iscience, vol. 15, May 2019, pp. 552–91. Pubmed, doi:10.1016/j.isci.2019.04.006.
PMID
31152742
Source
pubmed
Published In
Iscience
Volume
15
Publish Date
2019
Start Page
552
End Page
591
DOI
10.1016/j.isci.2019.04.006

HOXB13 interaction with MEIS1 modifies proliferation and gene expression in prostate cancer.

BACKGROUND: The recurrent p.Gly84Glu germline mutation (G84E) in HOXB13 is consistently associated with prostate cancer (PCa), although the mechanisms underlying such linkage remain elusive. The majority of the PCa-associated HOXB13 mutations identified are localized to two conserved domains in HOXB13 that have been shown to mediate the interaction with MEIS cofactors belonging to the TALE family of homeodomain transcription factors. In this study, we sought to interrogate the biochemical and functional interactions between HOXB13 and MEIS in prostatic cells with a goal of defining how the HOXB13-MEIS complex impacts PCa pathobiology and define the extent to which the oncogenic activity of G84E is related to its effect on HOXB13-MEIS interaction/function. METHODS: HOXB13 and MEIS paralog expression in prostate epithelial cells and PCa cell lines was characterized by qPCR and immunoblot analyses. HOXB13 and MEIS1 co-expression in human prostate tissue was confirmed by IHC, followed by co-IP mapping of HOXB13-MEIS1 interactions. Proliferation of the PCa cell line LAPC4 following shRNA-mediated knockdown of each gene or both genes was assessed using DNA- and metabolic-based assays. Transcriptional targets of HOXB13 and MEIS1 were identified by gene expression profiling and qPCR. Finally, protein stability of HOXB13 in the context of MEIS1 was determined using pulse-chase assays. RESULTS: HOXB13 and MEIS1 are co-expressed and interact in prostate cells. Both of the putative MEIS interacting domains (MID) within HOXB13 were shown to be capable of mediating the interaction between HOXB13 and MEIS1 independently and such interactions were not influenced by the G84E mutation. The inhibitory effect of either HOXB13 or MEIS1 knockdown on cellular proliferation was augmented by knockdown of both genes, and MEIS1 knockdown abolished HOXB13-driven regulation of BCHE and TNFSF10 mRNA expression. Notably, we demonstrated that MEIS1 stabilized the HOXB13 protein in LAPC4 cells. CONCLUSIONS: Our study provides evidence for functional HOXB13-MEIS1 interactions in PCa. MEIS1 may contribute to the cancer-promoting actions of HOXB13 in cellular proliferation and gene regulation by prolonging HOXB13 half-life. Our data demonstrates that G84E is not a loss-of-function mutation that interferes with HOXB13 stability or ability to interact with MEIS1.

Authors
Johng, D; Torga, G; Ewing, CM; Jin, K; Norris, JD; McDonnell, DP; Isaacs, WB
MLA Citation
Johng, Dorhyun, et al. “HOXB13 interaction with MEIS1 modifies proliferation and gene expression in prostate cancer..” Prostate, vol. 79, no. 4, Mar. 2019, pp. 414–24. Pubmed, doi:10.1002/pros.23747.
PMID
30560549
Source
pubmed
Published In
Prostate
Volume
79
Issue
4
Publish Date
2019
Start Page
414
End Page
424
DOI
10.1002/pros.23747

Targeting mutant estrogen receptors.

A drug used in hormone replacement therapy can target estrogen receptors that have become resistant to breast cancer treatments.

Authors
Wardell, SE; Norris, JD; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “Targeting mutant estrogen receptors..” Elife, vol. 8, Jan. 2019. Pubmed, doi:10.7554/eLife.44181.
PMID
30648967
Source
pubmed
Published In
Elife
Volume
8
Publish Date
2019
DOI
10.7554/eLife.44181

27-Hydroxycholesterol, an Endogenous SERM, and Risk of Fracture in Postmenopausal Women: A Nested Case-Cohort Study in the Women's Health Initiative.

27-Hydroxycholesterol (27HC) is a purported, novel endogenous SERM. In animal models, 27HC has an anti-estrogen effect in bone, and 17β-estradiol mitigates this effect. 27HC in relation to fracture risk has not been investigated in humans. Depending on the level of bioavailable 17β-estradiol (bioE2 ), 27HC may increase fracture risk in postmenopausal women and modify the fracture risk reduction from menopausal hormone therapy (MHT). To test these a priori hypotheses, we conducted a nested case-cohort study of 868 postmenopausal women within the Women's Health Initiative Hormone Therapy (WHI-HT) trials. The WHI-HT tested conjugated equine estrogens versus placebo and separately conjugated equine estrogens plus progestin versus placebo. Fracture cases were 442 women who had an adjudicated incident hip or clinical vertebral fracture during the WHI-HT follow-up. The subcohort included 430 women randomly selected at WHI-HT baseline, four of whom had a subsequent fracture. Of the 868 women, 266 cases and 219 non-cases were assigned to the placebo arms. Cox models estimated hazard ratios for incident fracture in relation to pre-randomization circulating levels of 27HC and 27HC/bioE2 molar ratio. Models adjusted for age, race/ethnicity, total cholesterol, bioE2 , sex hormone-binding globulin, 25-hydroxyvitamin D, diabetes, osteoporosis, prior MHT use, BMI, falls history, and prior fracture. In women assigned to placebo arms, those in the middle and the highest tertiles of 27HC/bioE2 had an up to 1.9-fold (95% confidence intervals, 1.25 to 2.99) greater risk of fracture than women in the lowest tertile. In women assigned to MHT arms, fracture risk increased with continuous 27HC/bioE2 levels but not with categorical levels. 27HC levels alone were not associated with fracture risk. 27HC and 27HC/bioE2 did not modify the fracture risk reduction from MHT. In postmenopausal women, circulating levels of 27HC relative to bioE2 may identify those at increased risk of fracture. © 2018 American Society for Bone and Mineral Research.

Authors
Chang, P-Y; Feldman, D; Stefanick, ML; McDonnell, DP; Thompson, BM; McDonald, JG; Lee, JS
MLA Citation
Chang, Po-Yin, et al. “27-Hydroxycholesterol, an Endogenous SERM, and Risk of Fracture in Postmenopausal Women: A Nested Case-Cohort Study in the Women's Health Initiative..” J Bone Miner Res, vol. 34, no. 1, Jan. 2019, pp. 59–66. Pubmed, doi:10.1002/jbmr.3576.
PMID
30138538
Source
pubmed
Published In
J Bone Miner Res
Volume
34
Issue
1
Publish Date
2019
Start Page
59
End Page
66
DOI
10.1002/jbmr.3576

Thyroid hormone receptor and ERRα coordinately regulate mitochondrial fission, mitophagy, biogenesis, and function.

Thyroid hormone receptor β1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and β-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.

Authors
Singh, BK; Sinha, RA; Tripathi, M; Mendoza, A; Ohba, K; Sy, JAC; Xie, SY; Zhou, J; Ho, JP; Chang, C-Y; Wu, Y; Giguère, V; Bay, B-H; Vanacker, J-M; Ghosh, S; Gauthier, K; Hollenberg, AN; McDonnell, DP; Yen, PM
MLA Citation
Singh, Brijesh K., et al. “Thyroid hormone receptor and ERRα coordinately regulate mitochondrial fission, mitophagy, biogenesis, and function..” Sci Signal, vol. 11, no. 536, June 2018. Pubmed, doi:10.1126/scisignal.aam5855.
PMID
29945885
Source
pubmed
Published In
Sci Signal
Volume
11
Issue
536
Publish Date
2018
DOI
10.1126/scisignal.aam5855

Dysregulation of mitochondrial dynamics proteins are a targetable feature of human tumors.

Altered mitochondrial dynamics can broadly impact tumor cell physiology. Using genetic and pharmacological profiling of cancer cell lines and human tumors, we here establish that perturbations to the mitochondrial dynamics network also result in specific therapeutic vulnerabilities. In particular, through distinct mechanisms, tumors with increased mitochondrial fragmentation or connectivity are hypersensitive to SMAC mimetics, a class of compounds that induce apoptosis through inhibition of IAPs and for which robust sensitivity biomarkers remain to be identified. Further, because driver oncogenes exert dominant control over mitochondrial dynamics, oncogene-targeted therapies can be used to sensitize tumors to SMAC mimetics via their effects on fission/fusion dynamics. Collectively, these data demonstrate that perturbations to the mitochondrial dynamics network induce targetable vulnerabilities across diverse human tumors and, more broadly, suggest that the altered structures, activities, and trafficking of cellular organelles may facilitate additional cancer therapeutic opportunities.

Authors
Anderson, GR; Wardell, SE; Cakir, M; Yip, C; Ahn, Y-R; Ali, M; Yllanes, AP; Chao, CA; McDonnell, DP; Wood, KC
MLA Citation
Anderson, Grace R., et al. “Dysregulation of mitochondrial dynamics proteins are a targetable feature of human tumors..” Nat Commun, vol. 9, no. 1, Apr. 2018. Pubmed, doi:10.1038/s41467-018-04033-x.
PMID
29700304
Source
pubmed
Published In
Nature Communications
Volume
9
Issue
1
Publish Date
2018
Start Page
1677
DOI
10.1038/s41467-018-04033-x

Discovery of LSZ102, a Potent, Orally Bioavailable Selective Estrogen Receptor Degrader (SERD) for the Treatment of Estrogen Receptor Positive Breast Cancer.

In breast cancer, estrogen receptor alpha (ERα) positive cancer accounts for approximately 74% of all diagnoses, and in these settings, it is a primary driver of cell proliferation. Treatment of ERα positive breast cancer has long relied on endocrine therapies such as selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs). The steroid-based anti-estrogen fulvestrant (5), the only approved SERD, is effective in patients who have not previously been treated with endocrine therapy as well as in patients who have progressed after receiving other endocrine therapies. Its efficacy, however, may be limited due to its poor physicochemical properties. We describe the design and synthesis of a series of potent benzothiophene-containing compounds that exhibit oral bioavailability and preclinical activity as SERDs. This article culminates in the identification of LSZ102 (10), a compound in clinical development for the treatment of ERα positive breast cancer.

Authors
Tria, GS; Abrams, T; Baird, J; Burks, HE; Firestone, B; Gaither, LA; Hamann, LG; He, G; Kirby, CA; Kim, S; Lombardo, F; Macchi, KJ; McDonnell, DP; Mishina, Y; Norris, JD; Nunez, J; Springer, C; Sun, Y; Thomsen, NM; Wang, C; Wang, J; Yu, B; Tiong-Yip, C-L; Peukert, S
MLA Citation
Tria, George S., et al. “Discovery of LSZ102, a Potent, Orally Bioavailable Selective Estrogen Receptor Degrader (SERD) for the Treatment of Estrogen Receptor Positive Breast Cancer..” J Med Chem, vol. 61, no. 7, Apr. 2018, pp. 2837–64. Pubmed, doi:10.1021/acs.jmedchem.7b01682.
PMID
29562737
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
61
Issue
7
Publish Date
2018
Start Page
2837
End Page
2864
DOI
10.1021/acs.jmedchem.7b01682

Validation of histone deacetylase 3 as a therapeutic target in castration-resistant prostate cancer.

BACKGROUND: Whereas the androgen receptor (AR) signaling axis remains a therapeutic target in castration-resistant prostate cancer (CRPC), the emergence of AR mutations and splice variants as mechanisms underlying resistance to contemporary inhibitors of this pathway highlights the need for new therapeutic approaches to target this disease. Of significance in this regard is the considerable preclinical data, indicating that histone deacetylase (HDAC) inhibitors may have utility in the treatment of CRPC. However, the results of clinical studies using HDAC inhibitors (directed against HDAC1, 2, 3, and 8) in CRPC are equivocal, a result that some have attributed to their ability to induce an epithelial to mesenchymal transition (EMT) and neuroendocrine differentiation. We posited that it might be possible to uncouple the beneficial effects of HDAC inhibitors on AR signaling from their undesired activities by targeting specific HDACs as opposed to using the pan-inhibitor strategy that has been employed to date. METHODS: The relative abilities of pan- and selective-Class I HDAC inhibitors to attenuate AR-mediated target gene expression and proliferation were assessed in several prostate cancer cell lines. Small interfering RNA (siRNA)-mediated knockdown approaches were used to confirm the importance of of HDAC 1, 2, and 3 expression in these processes. Further, the ability of each HDAC inhibitor to induce the expression of EMT markers (RNA and protein) and EMT-like phenotype(s) (migration) were also assessed. The anti-tumor efficacy of a HDAC3-selective inhibitor, RGFP966, was compared to the pan-HDAC inhibitor Suberoylanilide Hydroxamic Acid (SAHA) in the 22Rv1 xenograft model. RESULTS: Using genetic and pharmacological approaches we demonstrated that a useful inhibition of AR transcriptional activity, absent the induction of EMT, could be achieved by specifically inhibiting HDAC3. Significantly, we also determined that HDAC3 inhibitors blocked the activity of the constitutively active AR V7-splice variant and inhibited the growth of xenograft tumors expressing this protein. CONCLUSIONS: Our studies provide strong rationale for the near-term development of specific HDAC3 inhibitors for the treatment of CRPC.

Authors
McLeod, AB; Stice, JP; Wardell, SE; Alley, HM; Chang, C-Y; McDonnell, DP
MLA Citation
McLeod, Abigail B., et al. “Validation of histone deacetylase 3 as a therapeutic target in castration-resistant prostate cancer..” Prostate, vol. 78, no. 4, Mar. 2018, pp. 266–77. Pubmed, doi:10.1002/pros.23467.
PMID
29243324
Source
pubmed
Published In
Prostate
Volume
78
Issue
4
Publish Date
2018
Start Page
266
End Page
277
DOI
10.1002/pros.23467

Neomorphic ERα Mutations Drive Progression in Breast Cancer and Present a Challenge for New Drug Discovery.

In this issue of Cancer Cell, Jeselsohn et al. dissect the function of several of the most clinically important estrogen receptor alpha mutants associated with endocrine therapy resistance in breast cancer and demonstrate that they manifest disease-relevant neomorphic activities that likely contribute to tumor pathogenesis.

Authors
McDonnell, DP; Norris, JD; Chang, C-Y
MLA Citation
McDonnell, Donald P., et al. “Neomorphic ERα Mutations Drive Progression in Breast Cancer and Present a Challenge for New Drug Discovery..” Cancer Cell, vol. 33, no. 2, Feb. 2018, pp. 153–55. Pubmed, doi:10.1016/j.ccell.2018.01.014.
Website
http://hdl.handle.net/10161/16118
PMID
29438688
Source
pubmed
Published In
Cancer Cell
Volume
33
Issue
2
Publish Date
2018
Start Page
153
End Page
155
DOI
10.1016/j.ccell.2018.01.014

Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance.

The androgen receptor is a major driver of prostate cancer and inhibition of its transcriptional activity using competitive antagonists, such as enzalutamide remains a frontline therapy for prostate cancer management. However, the majority of patients eventually develop drug resistance. We propose that targeting the androgen receptor for degradation via Proteolysis Targeting Chimeras (PROTACs) will be a better therapeutic strategy for targeting androgen receptor signaling in prostate cancer cells. Here we perform a head-to-head comparison between a currently approved androgen receptor antagonist enzalutamide, and its PROTAC derivative, ARCC-4, across different cellular models of prostate cancer drug resistance. ARCC-4 is a low-nanomolar androgen receptor degrader able to degrade about 95% of cellular androgen receptors. ARCC-4 inhibits prostate tumor cell proliferation, degrades clinically relevant androgen receptor point mutants and unlike enzalutamide, retains antiproliferative effect in a high androgen environment. Thus, ARCC-4 exemplifies how protein degradation can address the drug resistance hurdles of enzalutamide.

Authors
Salami, J; Alabi, S; Willard, RR; Vitale, NJ; Wang, J; Dong, H; Jin, M; McDonnell, DP; Crew, AP; Neklesa, TK; Crews, CM
MLA Citation
Salami, Jemilat, et al. “Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance..” Commun Biol, vol. 1, 2018. Pubmed, doi:10.1038/s42003-018-0105-8.
PMID
30271980
Source
pubmed
Published In
Communications Biology
Volume
1
Publish Date
2018
Start Page
100
DOI
10.1038/s42003-018-0105-8

The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells.

Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8+T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia.High cholesterol is a risk factor for breast cancer recurrence. Here the authors show that cholesterol promotes breast cancer metastasis via its metabolite 27-hydroxycholesterol (27HC) that acts on immune myeloid cells residing at the distal metastatic sites, thus promoting an immune suppressive environment.

Authors
Baek, AE; Yu, Y-RA; He, S; Wardell, SE; Chang, C-Y; Kwon, S; Pillai, RV; McDowell, HB; Thompson, JW; Dubois, LG; Sullivan, PM; Kemper, JK; Gunn, MD; McDonnell, DP; Nelson, ER
MLA Citation
Baek, Amy E., et al. “The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells..” Nat Commun, vol. 8, no. 1, Oct. 2017. Pubmed, doi:10.1038/s41467-017-00910-z.
PMID
29021522
Source
pubmed
Published In
Nature Communications
Volume
8
Issue
1
Publish Date
2017
Start Page
864
DOI
10.1038/s41467-017-00910-z

A Predictive Model for Selective Targeting of the Warburg Effect through GAPDH Inhibition with a Natural Product.

Targeted cancer therapies that use genetics are successful, but principles for selectively targeting tumor metabolism that is also dependent on the environment remain unknown. We now show that differences in rate-controlling enzymes during the Warburg effect (WE), the most prominent hallmark of cancer cell metabolism, can be used to predict a response to targeting glucose metabolism. We establish a natural product, koningic acid (KA), to be a selective inhibitor of GAPDH, an enzyme we characterize to have differential control properties over metabolism during the WE. With machine learning and integrated pharmacogenomics and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond the status of individual genes.

Authors
Liberti, MV; Dai, Z; Wardell, SE; Baccile, JA; Liu, X; Gao, X; Baldi, R; Mehrmohamadi, M; Johnson, MO; Madhukar, NS; Shestov, AA; Chio, IIC; Elemento, O; Rathmell, JC; Schroeder, FC; McDonnell, DP; Locasale, JW
MLA Citation
Liberti, Maria V., et al. “A Predictive Model for Selective Targeting of the Warburg Effect through GAPDH Inhibition with a Natural Product..” Cell Metab, vol. 26, no. 4, Oct. 2017, pp. 648-659.e8. Pubmed, doi:10.1016/j.cmet.2017.08.017.
PMID
28918937
Source
pubmed
Published In
Cell Metab
Volume
26
Issue
4
Publish Date
2017
Start Page
648
End Page
659.e8
DOI
10.1016/j.cmet.2017.08.017

DNA Sequence Constraints Define Functionally Active Steroid Nuclear Receptor Binding Sites in Chromatin.

Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin-interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications.

Authors
Coons, LA; Hewitt, SC; Burkholder, AB; McDonnell, DP; Korach, KS
MLA Citation
Coons, Laurel A., et al. “DNA Sequence Constraints Define Functionally Active Steroid Nuclear Receptor Binding Sites in Chromatin..” Endocrinology, vol. 158, no. 10, Oct. 2017, pp. 3212–34. Pubmed, doi:10.1210/en.2017-00468.
PMID
28977594
Source
pubmed
Published In
Endocrinology
Volume
158
Issue
10
Publish Date
2017
Start Page
3212
End Page
3234
DOI
10.1210/en.2017-00468

Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling.

Despite the advances in the diagnosis and treatment of breast cancer, breast cancers still cause significant mortality. For some patients, especially those with triple-negative breast cancer, current treatments continue to be limited and ineffective. Therefore, there remains an unmet need for a novel therapeutic approach. One potential strategy is to target the altered metabolic state that is rewired by oncogenic transformation. Specifically, this rewiring may render certain outside nutrients indispensable. To identify such a nutrient, we performed a nutrigenetic screen by removing individual amino acids to identify possible addictions across a panel of breast cancer cells. This screen revealed that cystine deprivation triggered rapid programmed necrosis, but not apoptosis, in the basal-type breast cancer cells mostly seen in TNBC tumors. In contrast, luminal-type breast cancer cells are cystine-independent and exhibit little death during cystine deprivation. The cystine addiction phenotype is associated with a higher level of cystine-deprivation signatures noted in the basal type breast cancer cells and tumors. We found that the cystine-addicted breast cancer cells and tumors have strong activation of TNFα and MEKK4-p38-Noxa pathways that render them susceptible to cystine deprivation-induced necrosis. Consistent with this model, silencing of TNFα and MEKK4 dramatically reduces cystine-deprived death. In addition, the cystine addiction phenotype can be abrogated in the cystine-addictive cells by miR-200c, which converts the mesenchymal-like cells to adopt epithelial features. Conversely, the introduction of inducers of epithelial-mesenchymal transition (EMT) in cystine-independent breast cancer cells conferred the cystine-addiction phenotype by modulating the signaling components of cystine addiction. Together, our data reveal that cystine-addiction is associated with EMT in breast cancer during tumor progression. These findings provide the genetic and mechanistic basis to explain how cystine deprivation triggers necrosis by activating pre-existing oncogenic pathways in cystine-addicted TNBC with prominent mesenchymal features.

Authors
Tang, X; Ding, C-K; Wu, J; Sjol, J; Wardell, S; Spasojevic, I; George, D; McDonnell, DP; Hsu, DS; Chang, JT; Chi, J-T
MLA Citation
Tang, X., et al. “Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling..” Oncogene, vol. 36, no. 30, July 2017, pp. 4235–42. Pubmed, doi:10.1038/onc.2016.394.
PMID
27869167
Source
pubmed
Published In
Oncogene
Volume
36
Issue
30
Publish Date
2017
Start Page
4235
End Page
4242
DOI
10.1038/onc.2016.394

Impact of 27-hydroxylase (CYP27A1) and 27-hydroxycholesterol in breast cancer.

The impact of systemic 27-hydroxycholesterol (27HC) and intratumoral CYP27A1 expression on pathobiology and clinical response to statins in breast cancer needs clarification. 27HC is an oxysterol produced from cholesterol by the monooxygenase CYP27A1, which regulates intracellular cholesterol homeostasis. 27HC also acts as an endogenous selective estrogen receptor (ER) modulator capable of increasing breast cancer growth and metastasis. 27HC levels can be modulated by statins or direct inhibition of CYP27A1, thereby attenuating its pro-tumorigenic activities. Herein, the effect of statins on serum 27HC and tumor-specific CYP27A1 expression was evaluated in 42 breast cancer patients treated with atorvastatin within a phase II clinical trial. Further, the associations between CYP27A1 expression with other primary tumor pathological features and clinical outcomes were studied in two additional independent cohorts. Statin treatment effectively decreased serum 27HC and deregulated CYP27A1 expression in tumors. However, these changes were not associated with anti-proliferative responses to statin treatment. CYP27A1 was heterogeneously expressed among primary tumors, with high expression significantly associated with high tumor grade, ER negativity and basal-like subtype. High CYP27A1 expression was independently prognostic for longer recurrence-free and overall survival. Importantly, the beneficial effect of high CYP27A1 in ER-positive breast cancer seemed limited to women aged ≤50 years. These results establish a link between CYP27A1 and breast cancer pathobiology and prognosis and propose that the efficacy of statins in reducing serum lipids does not directly translate to anti-proliferative effects in tumors. Changes in other undetermined serum or tumor factors suggestively mediate the anti-proliferative effects of statins in breast cancer.

Authors
Kimbung, S; Chang, C-Y; Bendahl, P-O; Dubois, L; Thompson, JW; McDonnell, DP; Borgquist, S
MLA Citation
Kimbung, Siker, et al. “Impact of 27-hydroxylase (CYP27A1) and 27-hydroxycholesterol in breast cancer..” Endocr Relat Cancer, vol. 24, no. 7, July 2017, pp. 339–49. Pubmed, doi:10.1530/ERC-16-0533.
PMID
28442559
Source
pubmed
Published In
Endocr Relat Cancer
Volume
24
Issue
7
Publish Date
2017
Start Page
339
End Page
349
DOI
10.1530/ERC-16-0533

Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer.

The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase-selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel's direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor-bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC.

Authors
Norris, JD; Ellison, SJ; Baker, JG; Stagg, DB; Wardell, SE; Park, S; Alley, HM; Baldi, RM; Yllanes, A; Andreano, KJ; Stice, JP; Lawrence, SA; Eisner, JR; Price, DK; Moore, WR; Figg, WD; McDonnell, DP
MLA Citation
Norris, John D., et al. “Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer..” J Clin Invest, vol. 127, no. 6, June 2017, pp. 2326–38. Pubmed, doi:10.1172/JCI87328.
PMID
28463227
Source
pubmed
Published In
J Clin Invest
Volume
127
Issue
6
Publish Date
2017
Start Page
2326
End Page
2338
DOI
10.1172/JCI87328

CDK4/6 Therapeutic Intervention and Viable Alternative to Taxanes in CRPC.

Resistance to second-generation androgen receptor (AR) antagonists and CYP17 inhibitors in patients with castration-resistant prostate cancer (CRPC) develops rapidly through reactivation of the androgen signaling axis and has been attributed to AR overexpression, production of constitutively active AR splice variants, or the selection for AR mutants with altered ligand-binding specificity. It has been established that androgens induce cell-cycle progression, in part, through upregulation of cyclin D1 (CCND1) expression and subsequent activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Thus, the efficacy of the newly described CDK4/6 inhibitors (G1T28 and G1T38), docetaxel and enzalutamide, was evaluated as single agents in clinically relevant in vitro and in vivo models of hormone-sensitive and treatment-resistant prostate cancer. CDK4/6 inhibition (CDK4/6i) was as effective as docetaxel in animal models of treatment-resistant CRPC but exhibited significantly less toxicity. The in vivo effects were durable and importantly were observed in prostate cancer cells expressing wild-type AR, AR mutants, and those that have lost AR expression. CDK4/6i was also effective in prostate tumor models expressing the AR-V7 variant or the AR F876L mutation, both of which are associated with treatment resistance. Furthermore, CDK4/6i was effective in prostate cancer models where AR expression was lost. It is concluded that CDK4/6 inhibitors are a viable alternative to taxanes as therapeutic interventions in endocrine therapy-refractory CRPC.Implications: The preclinical efficacy of CDK4/6 monotherapy observed here suggests the need for near-term clinical studies of these agents in advanced prostate cancer. Mol Cancer Res; 15(6); 660-9. ©2017 AACR.

Authors
Stice, JP; Wardell, SE; Norris, JD; Yllanes, AP; Alley, HM; Haney, VO; White, HS; Safi, R; Winter, PS; Cocce, KJ; Kishton, RJ; Lawrence, SA; Strum, JC; McDonnell, DP
MLA Citation
Stice, James P., et al. “CDK4/6 Therapeutic Intervention and Viable Alternative to Taxanes in CRPC..” Mol Cancer Res, vol. 15, no. 6, June 2017, pp. 660–69. Pubmed, doi:10.1158/1541-7786.MCR-17-0028.
PMID
28209757
Source
pubmed
Published In
Mol Cancer Res
Volume
15
Issue
6
Publish Date
2017
Start Page
660
End Page
669
DOI
10.1158/1541-7786.MCR-17-0028

Impact of the sterol 27-hydroxylase (CYP27A1) and 27-hydroxycholesterol on tumor-pathological features, prognosis and response to statin treatment in breast cancer

Authors
Kimbung, S; Chang, C-Y; Bendahl, P-O; Dubois, L; Thompson, WJ; McDonnell, DP; Borgquist, S
MLA Citation
Kimbung, S., et al. “Impact of the sterol 27-hydroxylase (CYP27A1) and 27-hydroxycholesterol on tumor-pathological features, prognosis and response to statin treatment in breast cancer.” Annals of Oncology, vol. 28, OXFORD UNIV PRESS, 2017.
Source
wos
Published In
Annals of Oncology
Volume
28
Publish Date
2017

Discovery of an Acrylic Acid Based Tetrahydroisoquinoline as an Orally Bioavailable Selective Estrogen Receptor Degrader for ERα+ Breast Cancer.

Tetrahydroisoquinoline 40 has been identified as a potent ERα antagonist and selective estrogen receptor degrader (SERD), exhibiting good oral bioavailability, antitumor efficacy, and SERD activity in vivo. We outline the discovery and chemical optimization of the THIQ scaffold leading to THIQ 40 and showcase the racemization of the scaffold, pharmacokinetic studies in preclinical species, and the in vivo efficacy of THIQ 40 in a MCF-7 human breast cancer xenograft model.

Authors
Burks, HE; Abrams, T; Kirby, CA; Baird, J; Fekete, A; Hamann, LG; Kim, S; Lombardo, F; Loo, A; Lubicka, D; Macchi, K; McDonnell, DP; Mishina, Y; Norris, JD; Nunez, J; Saran, C; Sun, Y; Thomsen, NM; Wang, C; Wang, J; Peukert, S
MLA Citation
Burks, Heather E., et al. “Discovery of an Acrylic Acid Based Tetrahydroisoquinoline as an Orally Bioavailable Selective Estrogen Receptor Degrader for ERα+ Breast Cancer..” J Med Chem, vol. 60, no. 7, Apr. 2017, pp. 2790–818. Pubmed, doi:10.1021/acs.jmedchem.6b01468.
PMID
28296398
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
60
Issue
7
Publish Date
2017
Start Page
2790
End Page
2818
DOI
10.1021/acs.jmedchem.6b01468

CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer.

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662-73. ©2017 AACR.

Authors
Alfaqih, MA; Nelson, ER; Liu, W; Safi, R; Jasper, JS; Macias, E; Geradts, J; Thompson, JW; Dubois, LG; Freeman, MR; Chang, C-Y; Chi, J-T; McDonnell, DP; Freedland, SJ
MLA Citation
Alfaqih, Mahmoud A., et al. “CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer..” Cancer Res, vol. 77, no. 7, Apr. 2017, pp. 1662–73. Pubmed, doi:10.1158/0008-5472.CAN-16-2738.
PMID
28130224
Source
pubmed
Published In
Cancer Res
Volume
77
Issue
7
Publish Date
2017
Start Page
1662
End Page
1673
DOI
10.1158/0008-5472.CAN-16-2738

MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

Authors
Christenson, JL; Butterfield, KT; Spoelstra, NS; Norris, JD; Josan, JS; Pollock, JA; McDonnell, DP; Katzenellenbogen, BS; Katzenellenbogen, JA; Richer, JK
MLA Citation
Christenson, Jessica L., et al. “MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression..” Horm Cancer, vol. 8, no. 2, Apr. 2017, pp. 69–77. Pubmed, doi:10.1007/s12672-017-0285-6.
PMID
28194662
Source
pubmed
Published In
Horm Cancer
Volume
8
Issue
2
Publish Date
2017
Start Page
69
End Page
77
DOI
10.1007/s12672-017-0285-6

Abstract P3-07-14: Targeting ERR-α regulated lactate metabolism eliminates drug-resistant breast cancer cells

Authors
Quayle, L; Park, S; McDonnell, DP; Ottewell, PD; Holen, I
MLA Citation
Quayle, L., et al. “Abstract P3-07-14: Targeting ERR-α regulated lactate metabolism eliminates drug-resistant breast cancer cells.” Poster Session Abstracts, American Association for Cancer Research, 2017. Crossref, doi:10.1158/1538-7445.sabcs16-p3-07-14.
Source
crossref
Published In
Poster Session Abstracts
Publish Date
2017
DOI
10.1158/1538-7445.sabcs16-p3-07-14

Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer.

Targeted inhibitors of the human epidermal growth factor receptor 2 (HER2), such as trastuzumab and lapatinib, are among the first examples of molecularly targeted cancer therapy and have proven largely effective for the treatment of HER2-positive breast cancers. However, approximately half of those patients either do not respond to these therapies or develop secondary resistance. Although a few signaling pathways have been implicated, a comprehensive understanding of mechanisms underlying HER2 inhibitor drug resistance is still lacking. To address this critical question, we undertook a concerted approach using patient expression data sets, HER2-positive cell lines, and tumor samples biopsied both before and after trastuzumab treatment. Together, these methods revealed that high expression and activation of a specific subset of receptor tyrosine kinases (RTKs) was strongly associated with poor clinical prognosis and the development of resistance. Mechanistically, these RTKs are capable of maintaining downstream signal transduction to promote tumor growth via the suppression of cellular senescence. Consequently, these findings provide the rationale for the design of therapeutic strategies for overcoming drug resistance in breast cancer via combinational inhibition of the limited number of targets from this specific subset of RTKs.

Authors
Alexander, PB; Chen, R; Gong, C; Yuan, L; Jasper, JS; Ding, Y; Markowitz, GJ; Yang, P; Xu, X; McDonnell, DP; Song, E; Wang, X-F
MLA Citation
Alexander, Peter B., et al. “Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer..” J Biol Chem, vol. 292, no. 2, Jan. 2017, pp. 748–59. Pubmed, doi:10.1074/jbc.M116.754960.
Website
http://hdl.handle.net/10161/15359
PMID
27903634
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
292
Issue
2
Publish Date
2017
Start Page
748
End Page
759
DOI
10.1074/jbc.M116.754960

Chemotherapy enriches for an invasive triple-negative breast tumor cell subpopulation expressing a precursor form of N-cadherin on the cell surface.

BACKGROUND: Although most triple-negative breast cancer (TNBC) patients initially respond to chemotherapy, residual tumor cells frequently persist and drive recurrent tumor growth. Previous studies from our laboratory and others' indicate that TNBC is heterogeneous, being composed of chemo-sensitive and chemo-resistant tumor cell subpopulations. In the current work, we studied the invasive behaviors of chemo-resistant TNBC, and sought to identify markers of invasion in chemo-residual TNBC. METHODS: The invasive behavior of TNBC tumor cells surviving short-term chemotherapy treatment in vitro was studied using transwell invasion assays and an experimental metastasis model. mRNA expression levels of neural cadherin (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment. RESULTS: TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy treatment. CONCLUSIONS: TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease.

Authors
Nelson, ER; Li, S; Kennedy, M; Payne, S; Kilibarda, K; Groth, J; Bowie, M; Parilla-Castellar, E; de Ridder, G; Marcom, PK; Lyes, M; Peterson, BL; Cook, M; Pizzo, SV; McDonnell, DP; Bachelder, RE
MLA Citation
Nelson, Erik R., et al. “Chemotherapy enriches for an invasive triple-negative breast tumor cell subpopulation expressing a precursor form of N-cadherin on the cell surface..” Oncotarget, vol. 7, no. 51, Dec. 2016, pp. 84030–42. Pubmed, doi:10.18632/oncotarget.12767.
PMID
27768598
Source
pubmed
Published In
Oncotarget
Volume
7
Issue
51
Publish Date
2016
Start Page
84030
End Page
84042
DOI
10.18632/oncotarget.12767

PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation.

Therapies that efficiently induce apoptosis are likely to be required for durable clinical responses in patients with solid tumors. Using a pharmacological screening approach, we discovered that combined inhibition of B cell lymphoma-extra large (BCL-XL) and the mammalian target of rapamycin (mTOR)/4E-BP axis results in selective and synergistic induction of apoptosis in cellular and animal models of PIK3CA mutant breast cancers, including triple-negative tumors. Mechanistically, inhibition of mTOR/4E-BP suppresses myeloid cell leukemia-1 (MCL-1) protein translation only in PIK3CA mutant tumors, creating a synthetic dependence on BCL-XL This dual dependence on BCL-XL and MCL-1, but not on BCL-2, appears to be a fundamental property of diverse breast cancer cell lines, xenografts, and patient-derived tumors that is independent of the molecular subtype or PIK3CA mutational status. Furthermore, this dependence distinguishes breast cancers from normal breast epithelial cells, which are neither primed for apoptosis nor dependent on BCL-XL/MCL-1, suggesting a potential therapeutic window. By tilting the balance of pro- to antiapoptotic signals in the mitochondria, dual inhibition of MCL-1 and BCL-XL also sensitizes breast cancer cells to standard-of-care cytotoxic and targeted chemotherapies. Together, these results suggest that patients with PIK3CA mutant breast cancers may benefit from combined treatment with inhibitors of BCL-XL and the mTOR/4E-BP axis, whereas alternative methods of inhibiting MCL-1 and BCL-XL may be effective in tumors lacking PIK3CA mutations.

Authors
Anderson, GR; Wardell, SE; Cakir, M; Crawford, L; Leeds, JC; Nussbaum, DP; Shankar, PS; Soderquist, RS; Stein, EM; Tingley, JP; Winter, PS; Zieser-Misenheimer, EK; Alley, HM; Yllanes, A; Haney, V; Blackwell, KL; McCall, SJ; McDonnell, DP; Wood, KC
MLA Citation
Anderson, Grace R., et al. “PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation..” Sci Transl Med, vol. 8, no. 369, Dec. 2016. Pubmed, doi:10.1126/scitranslmed.aae0348.
Website
http://hdl.handle.net/10161/13335
PMID
27974663
Source
pubmed
Published In
Sci Transl Med
Volume
8
Issue
369
Publish Date
2016
Start Page
369ra175
DOI
10.1126/scitranslmed.aae0348

CYP27A1/27HC/SREBP2 axis is a novel therapeutic target in prostate cancer

Authors
Alfaqih, M; Nelson, ER; Liu, W; Safi, R; Jasper, JS; Macias, E; Geradts, J; Chang, CY; McDonnell, DP; Freedland, S
MLA Citation
Alfaqih, M., et al. “CYP27A1/27HC/SREBP2 axis is a novel therapeutic target in prostate cancer.” European Journal of Cancer, vol. 69, Elsevier BV, 2016, pp. S42–43. Crossref, doi:10.1016/s0959-8049(16)32708-3.
Source
crossref
Published In
European Journal of Cancer
Volume
69
Publish Date
2016
Start Page
S42
End Page
S43
DOI
10.1016/s0959-8049(16)32708-3

Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer.

Clinical resistance to the second-generation antiandrogen enzalutamide in castration-resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights an unmet medical need for next-generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide) and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR-CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm, bound to heat shock proteins. Thus, we have identified third-generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC.

Authors
Pollock, JA; Wardell, SE; Parent, AA; Stagg, DB; Ellison, SJ; Alley, HM; Chao, CA; Lawrence, SA; Stice, JP; Spasojevic, I; Baker, JG; Kim, SH; McDonnell, DP; Katzenellenbogen, JA; Norris, JD
MLA Citation
Pollock, Julie A., et al. “Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer..” Nat Chem Biol, vol. 12, no. 10, Oct. 2016, pp. 795–801. Pubmed, doi:10.1038/nchembio.2131.
PMID
27501397
Source
pubmed
Published In
Nat Chem Biol
Volume
12
Issue
10
Publish Date
2016
Start Page
795
End Page
801
DOI
10.1038/nchembio.2131

Identification and Therapeutic Inhibition of Pathways Driving Resistance to Targeted Breast Cancer Therapies

Authors
Wood, KC; Anderson, GR; Wardell, SE; Jasper, JS; Stein, EM; Martz, CA; Alley, HM; Haney, V; Yllanes, A; Blackwell, KL; McCall, SJ; McDonnell, DP
MLA Citation
Wood, Kris C., et al. “Identification and Therapeutic Inhibition of Pathways Driving Resistance to Targeted Breast Cancer Therapies.” Journal of Womens Health, vol. 25, no. 9, MARY ANN LIEBERT, INC, Sept. 2016, pp. 976–976.
Source
wos
Published In
Journal of Women'S Health (2002)
Volume
25
Issue
9
Publish Date
2016
Start Page
976
End Page
976

MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presents a major challenge in the clinic due to its lack of reliable prognostic markers and targeted therapies. Accumulating evidence strongly supports the notion that microRNAs (miRNAs) are involved in tumorigenesis and could serve as biomarkers for diagnostic purposes. To identify miRNAs that functionally suppress metastasis of TNBC, we employed a concerted approach with selecting miRNAs that display differential expression profiles from bioinformatic analyses of breast cancer patient databases and validating top candidates with functional assays using breast cancer cell lines and mouse models. We have found that miR-148a exhibits properties as a tumor suppressor as its expression is inversely correlated with the ability of both human and mouse breast cancer cells to colonize the lung in mouse xenograft tumor models. Mechanistically, miR-148a appears to suppress the extravasation process of cancer cells, likely by targeting two genes WNT1 and NRP1 in a cell non-autonomous manner. Importantly, lower expression of miR-148a is detected in higher-grade tumor samples and correlated with increased likelihood to develop metastases and poor prognosis in subsets of breast cancer patients, particularly those with TNBC. Thus, miR-148a is functionally defined as a suppressor of breast cancer metastasis and may serve as a prognostic biomarker for this disease.

Authors
Xu, X; Zhang, Y; Jasper, J; Lykken, E; Alexander, PB; Markowitz, GJ; McDonnell, DP; Li, Q-J; Wang, X-F
MLA Citation
Xu, Xin, et al. “MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer..” Oncotarget, vol. 7, no. 15, Apr. 2016, pp. 20381–94. Pubmed, doi:10.18632/oncotarget.7953.
PMID
26967387
Source
pubmed
Published In
Oncotarget
Volume
7
Issue
15
Publish Date
2016
Start Page
20381
End Page
20394
DOI
10.18632/oncotarget.7953

ERRα-Regulated Lactate Metabolism Contributes to Resistance to Targeted Therapies in Breast Cancer.

Imaging studies in animals and in humans have indicated that the oxygenation and nutritional status of solid tumors is dynamic. Furthermore, the extremely low level of glucose within tumors, while reflecting its rapid uptake and metabolism, also suggests that cancer cells must rely on other energy sources in some circumstances. Here, we find that some breast cancer cells can switch to utilizing lactate as a primary source of energy, allowing them to survive glucose deprivation for extended periods, and that this activity confers resistance to PI3K/mTOR inhibitors. The nuclear receptor, estrogen-related receptor alpha (ERRα), was shown to regulate the expression of genes required for lactate utilization, and isotopomer analysis revealed that genetic or pharmacological inhibition of ERRα activity compromised lactate oxidation. Importantly, ERRα antagonists increased the in vitro and in vivo efficacy of PI3K/mTOR inhibitors, highlighting the potential clinical utility of this drug combination.

Authors
Park, S; Chang, C-Y; Safi, R; Liu, X; Baldi, R; Jasper, JS; Anderson, GR; Liu, T; Rathmell, JC; Dewhirst, MW; Wood, KC; Locasale, JW; McDonnell, DP
MLA Citation
Park, Sunghee, et al. “ERRα-Regulated Lactate Metabolism Contributes to Resistance to Targeted Therapies in Breast Cancer..” Cell Rep, vol. 15, no. 2, Apr. 2016, pp. 323–35. Pubmed, doi:10.1016/j.celrep.2016.03.026.
PMID
27050525
Source
pubmed
Published In
Cell Reports
Volume
15
Issue
2
Publish Date
2016
Start Page
323
End Page
335
DOI
10.1016/j.celrep.2016.03.026

Abstract P3-14-04: Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo

Authors
Ellison, SJ; Norris, JD; Wardell, S; Eisner, JR; Hoekstra, WJ; Stagg, DB; Alley, HM; Moore, WR; McDonnell, DP
MLA Citation
Ellison, S. J., et al. “Abstract P3-14-04: Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo.” Poster Session Abstracts, American Association for Cancer Research, 2016. Crossref, doi:10.1158/1538-7445.sabcs15-p3-14-04.
Source
crossref
Published In
Poster Session Abstracts
Publish Date
2016
DOI
10.1158/1538-7445.sabcs15-p3-14-04

Efficacy of SERD/SERM Hybrid-CDK4/6 Inhibitor Combinations in Models of Endocrine Therapy-Resistant Breast Cancer.

PURPOSE: Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)-positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy-resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD- or SSH-CDK4/6 inhibitor combinations in models of endocrine therapy-resistant ESR1(+) breast cancer. EXPERIMENTAL DESIGN: SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy-resistant ESR1(+) breast cancer. RESULTS: The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone. CONCLUSIONS: A SERD- or SSH-palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. See related commentary by DeMichele and Chodosh, p. 4999.

Authors
Wardell, SE; Ellis, MJ; Alley, HM; Eisele, K; VanArsdale, T; Dann, SG; Arndt, KT; Primeau, T; Griffin, E; Shao, J; Crowder, R; Lai, J-P; Norris, JD; McDonnell, DP; Li, S
MLA Citation
Wardell, Suzanne E., et al. “Efficacy of SERD/SERM Hybrid-CDK4/6 Inhibitor Combinations in Models of Endocrine Therapy-Resistant Breast Cancer..” Clin Cancer Res, vol. 21, no. 22, Nov. 2015, pp. 5121–30. Pubmed, doi:10.1158/1078-0432.CCR-15-0360.
PMID
25991817
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
21
Issue
22
Publish Date
2015
Start Page
5121
End Page
5130
DOI
10.1158/1078-0432.CCR-15-0360

Evaluation of the pharmacological activities of RAD1901, a selective estrogen receptor degrader.

Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains a first-line treatment for estrogen receptor 1 (ESR1) positive breast cancer. However, tumor resistance limits the duration of response. The clinical efficacy of fulvestrant, a selective ER degrader (SERD) that triggers receptor degradation, has confirmed that ESR1 often remains engaged in endocrine therapy resistant cancers. Recently developed, selective ER modulators (SERMs)/SERD hybrids (SSHs) that facilitate ESR1 degradation in breast cancer cells and reproductive tissues have been advanced as an alternative treatment for advanced breast cancer, particularly in the metastatic setting. RAD1901 is one SSH currently being evaluated clinically that is unique among ESR1 modulators in that it readily enters the brain, a common site of breast cancer metastasis. In this study, RAD1901 inhibited estrogen activation of ESR1 in vitro and in vivo, inhibited estrogen-dependent breast cancer cell proliferation and xenograft tumor growth, and mediated dose-dependent downregulation of ESR1 protein. However, doses of RAD1901 insufficient to induce ESR1 degradation were shown to result in the activation of ESR1 target genes and in the stimulation of xenograft tumor growth. RAD1901 is an SSH that exhibits complex pharmacology in breast cancer models, having dose-dependent agonist/antagonist activity displayed in a tissue-selective manner. It remains unclear how this unique pharmacology will impact the utility of RAD1901 for breast cancer treatment. However, being the only SERD currently known to access the brain, RAD1901 merits evaluation as a targeted therapy for the treatment of breast cancer brain metastases.

Authors
Wardell, SE; Nelson, ER; Chao, CA; Alley, HM; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “Evaluation of the pharmacological activities of RAD1901, a selective estrogen receptor degrader..” Endocr Relat Cancer, vol. 22, no. 5, Oct. 2015, pp. 713–24. Pubmed, doi:10.1530/ERC-15-0287.
PMID
26162914
Source
pubmed
Published In
Endocr Relat Cancer
Volume
22
Issue
5
Publish Date
2015
Start Page
713
End Page
724
DOI
10.1530/ERC-15-0287

Identification of a Novel Coregulator, SH3YL1, That Interacts With the Androgen Receptor N-Terminus.

Nuclear receptor (NR)-mediated transcriptional activity is a dynamic process that is regulated by the binding of ligands that induce distinct conformational changes in the NR. These structural alterations lead to the differential recruitment of coregulators (coactivators or corepressors) that control the expression of NR-regulated genes. Here, we show that a stretch of proline residues located within the N-terminus of androgen receptor (AR) is a bona fide coregulator binding surface, the disruption of which reduces the androgen-dependent proliferation and migration of prostate cancer (PCa) cells. Using T7 phage display, we identified a novel AR-interacting protein, Src homology 3 (SH3)-domain containing, Ysc84-like 1 (SH3YL1), whose interaction with the receptor is dependent upon this polyproline domain. As with mutations within the AR polyproline domain, knockdown of SH3YL1 attenuated androgen-mediated cell growth and migration. RNA expression analysis revealed that SH3YL1 was required for the induction of a subset of AR-modulated genes. Notable was the observation that ubinuclein 1 (UBN1), a key member of a histone H3.3 chaperone complex, was a transcriptional target of the AR/SH3YL1 complex, correlated with aggressive PCa in patients, and was necessary for the maximal androgen-mediated proliferation and migration of PCa cells. Collectively, these data highlight the importance of an amino-terminal activation domain, its associated coregulator, and downstream transcriptional targets in regulating cellular processes of pathological importance in PCa.

Authors
Blessing, AM; Ganesan, S; Rajapakshe, K; Ying Sung, Y; Reddy Bollu, L; Shi, Y; Cheung, E; Coarfa, C; Chang, JT; McDonnell, DP; Frigo, DE
MLA Citation
Blessing, Alicia M., et al. “Identification of a Novel Coregulator, SH3YL1, That Interacts With the Androgen Receptor N-Terminus..” Mol Endocrinol, vol. 29, no. 10, Oct. 2015, pp. 1426–39. Pubmed, doi:10.1210/me.2015-1079.
PMID
26305679
Source
pubmed
Published In
Mol Endocrinol
Volume
29
Issue
10
Publish Date
2015
Start Page
1426
End Page
1439
DOI
10.1210/me.2015-1079

Small-Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging.

Androgen receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of selective androgen receptor degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small-molecule AR ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to second-generation AR antagonists.

Authors
Gustafson, JL; Neklesa, TK; Cox, CS; Roth, AG; Buckley, DL; Tae, HS; Sundberg, TB; Stagg, DB; Hines, J; McDonnell, DP; Norris, JD; Crews, CM
MLA Citation
Gustafson, Jeffrey L., et al. “Small-Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging..” Angew Chem Int Ed Engl, vol. 54, no. 33, Aug. 2015, pp. 9659–62. Pubmed, doi:10.1002/anie.201503720.
PMID
26083457
Source
pubmed
Published In
Angew Chem Int Ed Engl
Volume
54
Issue
33
Publish Date
2015
Start Page
9659
End Page
9662
DOI
10.1002/anie.201503720

Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106

Authors
Alwarawrah, Y; Hughes, P; Safi, R; McDonnell, DP; Spector, NL; Haystead, TA
MLA Citation
Alwarawrah, Yazan, et al. “Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106.” Experimental and Molecular Therapeutics, American Association for Cancer Research, 2015. Crossref, doi:10.1158/1538-7445.am2015-2703.
Source
crossref
Published In
Experimental and Molecular Therapeutics
Publish Date
2015
DOI
10.1158/1538-7445.am2015-2703

Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice.

Pregnancy promotes physiological adaptations throughout the body, mediated by the female sex hormones progesterone and estrogen. Changes in the metabolic properties of skeletal muscle enable the female body to cope with the physiological challenges of pregnancy and may also be linked to the development of insulin resistance. We conducted global microarray, proteomic, and metabolic analyses to study the role of the progesterone receptor and its transcriptional regulator, smoothelin-like protein 1 (SMTNL1) in the adaptation of skeletal muscle to pregnancy. We demonstrate that pregnancy promotes fiber-type changes from an oxidative to glycolytic isoform in skeletal muscle. This phenomenon is regulated through an interaction between SMTNL1 and progesterone receptor, which alters the expression of contractile and metabolic proteins. smtnl1(-/-) mice are metabolically less efficient and show impaired glucose tolerance. Pregnancy antagonizes these effects by inducing metabolic activity and increasing glucose tolerance. Our results suggest that SMTNL1 has a role in mediating the actions of steroid hormones to promote fiber switching in skeletal muscle during pregnancy. Our findings also bear on the management of gestational diabetes that develops as a complication of pregnancy in ~4% of women.

Authors
Lontay, B; Bodoor, K; Sipos, A; Weitzel, DH; Loiselle, D; Safi, R; Zheng, D; Devente, J; Hickner, RC; McDonnell, DP; Ribar, T; Haystead, TA
MLA Citation
Lontay, Beata, et al. “Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice..” J Biol Chem, vol. 290, no. 29, July 2015, pp. 17985–98. Pubmed, doi:10.1074/jbc.M115.658120.
PMID
26048986
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
290
Issue
29
Publish Date
2015
Start Page
17985
End Page
17998
DOI
10.1074/jbc.M115.658120

Oral Selective Estrogen Receptor Downregulators (SERDs), a Breakthrough Endocrine Therapy for Breast Cancer.

Drugs that inhibit estrogen receptor alpha (ERα) or that block the production of estrogens remain frontline interventions in the treatment and management of breast cancer at all stages. However, resistance to endocrine therapies, especially in the setting of advanced disease, remains an impediment to durable clinical responses. Although the mechanisms underlying resistance to existing agents are complex, preclinical studies suggest that selective estrogen receptor downregulators (SERDs), molecules which eliminate ERα expression, may have particular utility in the treatment of breast cancers that have progressed on tamoxifen and/or aromatase inhibitors. The discovery and development of orally bioavailable SERDs provide the opportunity to evaluate the utility of eliminating ERα expression in advanced metastatic breast cancers.

Authors
McDonnell, DP; Wardell, SE; Norris, JD
MLA Citation
McDonnell, Donald P., et al. “Oral Selective Estrogen Receptor Downregulators (SERDs), a Breakthrough Endocrine Therapy for Breast Cancer..” J Med Chem, vol. 58, no. 12, June 2015, pp. 4883–87. Pubmed, doi:10.1021/acs.jmedchem.5b00760.
PMID
26039356
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
58
Issue
12
Publish Date
2015
Start Page
4883
End Page
4887
DOI
10.1021/acs.jmedchem.5b00760

Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer.

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. In vivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Authors
Allensworth, JL; Evans, MK; Bertucci, F; Aldrich, AJ; Festa, RA; Finetti, P; Ueno, NT; Safi, R; McDonnell, DP; Thiele, DJ; Van Laere, S; Devi, GR
MLA Citation
Allensworth, Jennifer L., et al. “Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer..” Mol Oncol, vol. 9, no. 6, June 2015, pp. 1155–68. Pubmed, doi:10.1016/j.molonc.2015.02.007.
PMID
25769405
Source
pubmed
Published In
Mol Oncol
Volume
9
Issue
6
Publish Date
2015
Start Page
1155
End Page
1168
DOI
10.1016/j.molonc.2015.02.007

Direct effects of the selective CYP17 lyase (L) inhibitor, VT-464, on the androgen receptor (AR) and its oral activity in an F876L tumor mouse xenograft model.

Authors
Moore, WR; Norris, JD; Wardell, S; Eisner, JR; Hoekstra, WJ; Schotzinger, RJ; McDonnell, DP
MLA Citation
Moore, William R., et al. “Direct effects of the selective CYP17 lyase (L) inhibitor, VT-464, on the androgen receptor (AR) and its oral activity in an F876L tumor mouse xenograft model..” Journal of Clinical Oncology, vol. 33, no. 7_suppl, American Society of Clinical Oncology (ASCO), 2015, pp. 263–263. Crossref, doi:10.1200/jco.2015.33.7_suppl.263.
Source
crossref
Published In
Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
Volume
33
Issue
7_suppl
Publish Date
2015
Start Page
263
End Page
263
DOI
10.1200/jco.2015.33.7_suppl.263

Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways.

The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR(-/-) and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR(-/-) mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1(+) ) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C-C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that are consistent with a possible role of AhR in wet AMD. The data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus; GEO Submission No. GSE56983, NCBI Tracking System No. 17021116.

Authors
Choudhary, M; Kazmin, D; Hu, P; Thomas, RS; McDonnell, DP; Malek, G
MLA Citation
Choudhary, Mayur, et al. “Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways..” J Pathol, vol. 235, no. 1, Jan. 2015, pp. 101–12. Pubmed, doi:10.1002/path.4433.
PMID
25186463
Source
pubmed
Published In
J Pathol
Volume
235
Issue
1
Publish Date
2015
Start Page
101
End Page
112
DOI
10.1002/path.4433

Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

Authors
Martz, CA; Ottina, KA; Singleton, KR; Jasper, JS; Wardell, SE; Peraza-Penton, A; Anderson, GR; Winter, PS; Wang, T; Alley, HM; Kwong, LN; Cooper, ZA; Tetzlaff, M; Chen, P-L; Rathmell, JC; Flaherty, KT; Wargo, JA; McDonnell, DP; Sabatini, DM; Wood, KC
MLA Citation
Martz, Colin A., et al. “Systematic identification of signaling pathways with potential to confer anticancer drug resistance..” Sci Signal, vol. 7, no. 357, Dec. 2014. Pubmed, doi:10.1126/scisignal.aaa1877.
PMID
25538079
Source
pubmed
Published In
Sci Signal
Volume
7
Issue
357
Publish Date
2014
Start Page
ra121
DOI
10.1126/scisignal.aaa1877

Delineation of a FOXA1/ERα/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer.

UNLABELLED: Tamoxifen, a selective estrogen receptor (ER) modulator (SERM), remains a frontline clinical therapy for patients with ERα-positive breast cancer. However, the relatively rapid development of resistance to this drug in the metastatic setting remains an impediment to a durable response. Although drug resistance likely arises by many different mechanisms, the consensus is that most of the implicated pathways facilitate the outgrowth of a subpopulation of cancer cells that can either recognize tamoxifen as an agonist or bypass the regulatory control of ERα. Notable in this regard is the observation here and in other studies that expression of anterior gradient homology 2 (AGR2), a known proto-oncogene and disulfide isomerase, was induced by both estrogen (17β-estradiol, E2) and 4-hydroxytamoxifen (4OHT) in breast cancer cells. The importance of AGR2 expression is highlighted here by the observation that (i) its knockdown inhibited the growth of both tamoxifen-sensitive and -resistant breast cancer cells and (ii) its increased expression enhanced the growth of ERα-positive tumors in vivo and increased the migratory capacity of breast cancer cells in vitro. Interestingly, as with most ERα target genes, the expression of AGR2 in all breast cancer cells examined requires the transcription factor FOXA1. However, in tamoxifen-resistant cells, the expression of AGR2 occurs in a constitutive manner, requiring FOXA1, but loses its dependence on ER. Taken together, these data define the importance of AGR2 in breast cancer cell growth and highlight a mechanism where changes in FOXA1 activity obviate the need for ER in the regulation of this gene. IMPLICATIONS: These findings reveal the transcriptional interplay between FOXA1 and ERα in controlling AGR2 during the transition from therapy-sensitive to -resistant breast cancer and implicate AGR2 as a relevant therapeutic target.

Authors
Wright, TM; Wardell, SE; Jasper, JS; Stice, JP; Safi, R; Nelson, ER; McDonnell, DP
MLA Citation
Wright, Tricia M., et al. “Delineation of a FOXA1/ERα/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer..” Mol Cancer Res, vol. 12, no. 12, Dec. 2014, pp. 1829–39. Pubmed, doi:10.1158/1541-7786.MCR-14-0195.
PMID
25100862
Source
pubmed
Published In
Mol Cancer Res
Volume
12
Issue
12
Publish Date
2014
Start Page
1829
End Page
1839
DOI
10.1158/1541-7786.MCR-14-0195

The estrogen receptor as a mediator of the pathological actions of cholesterol in breast cancer.

Despite increased survivorship among patients, breast cancer remains the most common cancer among women and is the second leading cause of cancer death in women. The magnitude of this problem provides a strong impetus for new chemopreventative strategies and/or lifestyle changes that reduce cancer incidence. It is of significance, therefore, that several studies positively correlate obesity to the development of breast cancer. Importantly, obesity is also highly associated with elevated cholesterol, and cholesterol itself is a risk factor for breast cancer. Furthermore, patients taking statins demonstrate a lower breast cancer incidence and decreased recurrence. The recent observation that 27-hydroxycholesterol (27HC) is produced in a stoichiometric manner from cholesterol, together with our recent demonstration that it exerts partial agonist activity on both the estrogen and liver X receptors, suggested a potential mechanistic link between hyper-cholesterolemia and breast cancer incidence. Using genetic and pharmacological approaches, we have recently shown that elevation of circulating 27HC significantly increases tumor growth and metastasis in murine models of breast cancer. Further, we have demonstrated in appropriate animal models that the impact of high-fat diet on tumor pathogenesis can be mitigated by statins or by small molecule inhibitors of CYP27A1. These findings suggest that pharmacological or dietary modifications that lower total cholesterol, and by inference 27HC, are likely to reduce the impact of obesity/metabolic syndrome on breast cancer incidence.

Authors
McDonnell, DP; Chang, C-Y; Nelson, ER
MLA Citation
McDonnell, D. P., et al. “The estrogen receptor as a mediator of the pathological actions of cholesterol in breast cancer..” Climacteric, vol. 17 Suppl 2, Dec. 2014, pp. 60–65. Pubmed, doi:10.3109/13697137.2014.966949.
PMID
25320023
Source
pubmed
Published In
Climacteric
Volume
17 Suppl 2
Publish Date
2014
Start Page
60
End Page
65
DOI
10.3109/13697137.2014.966949

Cholesterol and breast cancer pathophysiology.

Cholesterol is a risk factor for breast cancer although the mechanisms by which this occurs are not well understood. One hypothesis is that dyslipidemia results in increased cholesterol content in cell membranes, thus impacting upon membrane fluidity and subsequent signaling. In addition, studies demonstrate that the metabolite, 27-hydroxycholesterol (27HC), can function as an estrogen, increasing the proliferation of estrogen receptor (ER)-positive breast cancer cells. This was unexpected because 27HC and other oxysterols activate the liver X receptors (LXR), resulting in a reduction of intracellular cholesterol. Resolution of this paradox will require dissection of the molecular mechanisms by which ER and LXR converge in breast cancer cells. Regardless, the observation that 27HC influences breast cancer provides a rationale for strategies that target cholesterol metabolism.

Authors
Nelson, ER; Chang, C-Y; McDonnell, DP
MLA Citation
Nelson, Erik R., et al. “Cholesterol and breast cancer pathophysiology..” Trends Endocrinol Metab, vol. 25, no. 12, Dec. 2014, pp. 649–55. Pubmed, doi:10.1016/j.tem.2014.10.001.
PMID
25458418
Source
pubmed
Published In
Trends Endocrinol Metab
Volume
25
Issue
12
Publish Date
2014
Start Page
649
End Page
655
DOI
10.1016/j.tem.2014.10.001

From empirical to mechanism-based discovery of clinically useful Selective Estrogen Receptor Modulators (SERMs).

Our understanding of the molecular mechanisms underlying the pharmacological actions of estrogen receptor (ER) ligands has evolved considerably in recent years. Much of this knowledge has come from a detailed dissection of the mechanism(s) of action of the Selective Estrogen Receptor Modulators (SERMs) tamoxifen and raloxifene, drugs whose estrogen receptor (ER) agonist/antagonist properties are influenced by the cell context in which they operate. These studies have revealed that notwithstanding differences in drug pharmacokinetics, the activity of an ER ligand is determined primarily by (a) the impact that a given ligand has on the receptor conformation and (b) the ability of structurally distinct ER-ligand complexes to interact with functionally distinct coregulators. Exploitation of the established relationships between ER structure and activity has led to the development of improved SERMs with more favorable therapeutic properties and of tissue-selective estrogen complexes, drugs in which a SERM and an ER agonist are combined to yield a blended activity that results in distinct clinical profiles. Remarkably, endogenous ligands that exhibit SERM activity have also been identified. One of these ligands, 27-hydroxycholesterol (27HC), has been shown to manifest ER-dependent pathological activities in the cardiovascular system, bone and mammary gland. Whereas the physiological activity of 27HC remains to be determined, its discovery highlights how cells have adopted mechanisms to allow the same receptor ligand complex to manifest different activities in different cells, and also how these processes can be exploited for new drug development.

Authors
Wardell, SE; Nelson, ER; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “From empirical to mechanism-based discovery of clinically useful Selective Estrogen Receptor Modulators (SERMs)..” Steroids, vol. 90, Nov. 2014, pp. 30–38. Pubmed, doi:10.1016/j.steroids.2014.07.013.
PMID
25084324
Source
pubmed
Published In
Steroids
Volume
90
Publish Date
2014
Start Page
30
End Page
38
DOI
10.1016/j.steroids.2014.07.013

Copper signaling axis as a target for prostate cancer therapeutics.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.

Authors
Safi, R; Nelson, ER; Chitneni, SK; Franz, KJ; George, DJ; Zalutsky, MR; McDonnell, DP
MLA Citation
Safi, Rachid, et al. “Copper signaling axis as a target for prostate cancer therapeutics..” Cancer Res, vol. 74, no. 20, Oct. 2014, pp. 5819–31. Pubmed, doi:10.1158/0008-5472.CAN-13-3527.
Website
http://hdl.handle.net/10161/9192
PMID
25320179
Source
pubmed
Published In
Cancer Res
Volume
74
Issue
20
Publish Date
2014
Start Page
5819
End Page
5831
DOI
10.1158/0008-5472.CAN-13-3527

Abstract 3311: The cholesterol/ 27-hydroxycholesterol axis is a novel therapeutic target in castrate resistant prostate cancer

Authors
Alfaqih, MA; Nelson, ER; Safi, R; Jasper, J; Chang, C-Y; Freedland, SJ; McDonnell, DP
MLA Citation
Alfaqih, Mahmoud A., et al. “Abstract 3311: The cholesterol/ 27-hydroxycholesterol axis is a novel therapeutic target in castrate resistant prostate cancer.” Molecular and Cellular Biology, American Association for Cancer Research, 2014. Crossref, doi:10.1158/1538-7445.am2014-3311.
Source
crossref
Published In
Molecular and Cellular Biology
Publish Date
2014
DOI
10.1158/1538-7445.am2014-3311

Abstract 4570: Role of ERRalpha in ovarian cancer

Authors
Stevens, EV; Whitaker, R; Guinet, A; Chang, C-Y; Grenier, C; Marks, J; McDonnell, DP; Murphy, SK; Berchuck, A; Gaillard, S
MLA Citation
Stevens, Ellen V., et al. “Abstract 4570: Role of ERRalpha in ovarian cancer.” Experimental and Molecular Therapeutics, American Association for Cancer Research, 2014. Crossref, doi:10.1158/1538-7445.am2014-4570.
Source
crossref
Published In
Experimental and Molecular Therapeutics
Publish Date
2014
DOI
10.1158/1538-7445.am2014-4570

Endocrine Society 2014 Laureate Awards.

Authors
Gustafsson, J-A; Katzenellenbogen, B; Jameson, JL; Roth, J; Melmed, S; McDonnell, D; Wartofsky, L; Crowley, WF; Griz, LH; Becker, C; Moore, DD; Drucker, D; Singh, N; O'Malley, B
MLA Citation
Gustafsson, Jan-Ake, et al. “Endocrine Society 2014 Laureate Awards..” Horm Cancer, vol. 5, no. 5, Oct. 2014, pp. 339–55. Pubmed, doi:10.1007/s12672-014-0193-y.
PMID
25091407
Source
pubmed
Published In
Horm Cancer
Volume
5
Issue
5
Publish Date
2014
Start Page
339
End Page
355
DOI
10.1007/s12672-014-0193-y

Obesity, cholesterol metabolism, and breast cancer pathogenesis.

Obesity and altered lipid metabolism are risk factors for breast cancer in pre- and post-menopausal women. These pathologic relationships have been attributed in part to the impact of cholesterol on the biophysical properties of cell membranes and to the influence of these changes on signaling events initiated at the membrane. However, more recent studies have indicated that the oxysterol 27-hydroxycholesterol (27HC), and not cholesterol per se, may be the primary biochemical link between lipid metabolism and cancer. The enzyme responsible for production of 27HC from cholesterol, CYP27A1, is expressed primarily in the liver and in macrophages. In addition, significantly elevated expression of this enzyme within breast tumors has also been observed. It is believed that 27HC, acting through the liver X receptor in macrophages and possibly other cells, is involved in maintaining organismal cholesterol homeostasis. It has also been shown recently that 27HC is an estrogen receptor agonist in breast cancer cells and that it stimulates the growth and metastasis of tumors in several models of breast cancer. These findings provide the rationale for the clinical evaluation of pharmaceutical approaches that interfere with cholesterol/27HC synthesis as a means to mitigate the impact of cholesterol on breast cancer pathogenesis. Cancer Res; 74(18); 4976-82. ©2014 AACR.

Authors
McDonnell, DP; Park, S; Goulet, MT; Jasper, J; Wardell, SE; Chang, C-Y; Norris, JD; Guyton, JR; Nelson, ER
MLA Citation
McDonnell, Donald P., et al. “Obesity, cholesterol metabolism, and breast cancer pathogenesis..” Cancer Res, vol. 74, no. 18, Sept. 2014, pp. 4976–82. Pubmed, doi:10.1158/0008-5472.CAN-14-1756.
PMID
25060521
Source
pubmed
Published In
Cancer Res
Volume
74
Issue
18
Publish Date
2014
Start Page
4976
End Page
4982
DOI
10.1158/0008-5472.CAN-14-1756

Use of phage display to identify novel mineralocorticoid receptor-interacting proteins.

The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter.

Authors
Yang, J; Fuller, PJ; Morgan, J; Shibata, H; McDonnell, DP; Clyne, CD; Young, MJ
MLA Citation
Yang, Jun, et al. “Use of phage display to identify novel mineralocorticoid receptor-interacting proteins..” Mol Endocrinol, vol. 28, no. 9, Sept. 2014, pp. 1571–84. Pubmed, doi:10.1210/me.2014-1101.
PMID
25000480
Source
pubmed
Published In
Mol Endocrinol
Volume
28
Issue
9
Publish Date
2014
Start Page
1571
End Page
1584
DOI
10.1210/me.2014-1101

Bisphenol AP Is a Novel AR Down-Regulator in Castrate Resistant Prostate Cancer Cells Via Modulation of Nuclear Receptors' Networks

Authors
Stossi, F; Bolt, MJ; Dandekar, RD; Putluri, V; Kaushik, AK; Chang, C-Y; Mancini, MM; Sreekumar, A; McDonnell, DP; Mancini, MA
MLA Citation
Stossi, Fabio, et al. “Bisphenol AP Is a Novel AR Down-Regulator in Castrate Resistant Prostate Cancer Cells Via Modulation of Nuclear Receptors' Networks.” Endocrine Reviews, vol. 35, no. 3, ENDOCRINE SOC, June 2014.
Source
wos
Published In
Endocrine Reviews
Volume
35
Issue
3
Publish Date
2014

Structurally Novel Antagonists of Androgen Receptors with Elevated Potency for Antiandrogen-Resistant Receptor Mutants in Prostate Cancer: Tetra-Aryl-Substituted Cyclobutanes

Authors
Katzenellenbogen, JA; Pollock, JA; Parent, AA; Wardell, SE; McDonnell, DP; Norris, JD
MLA Citation
Katzenellenbogen, John A., et al. “Structurally Novel Antagonists of Androgen Receptors with Elevated Potency for Antiandrogen-Resistant Receptor Mutants in Prostate Cancer: Tetra-Aryl-Substituted Cyclobutanes.” Endocrine Reviews, vol. 35, no. 3, ENDOCRINE SOC, 2014.
Source
wos
Published In
Endocrine Reviews
Volume
35
Issue
3
Publish Date
2014

Structurally Novel Antagonists of Androgen Receptors with Elevated Potency for Antiandrogen-Resistant Receptor Mutants in Prostate Cancer: Tetra-Aryl-Substituted Cyclobutanes

Authors
Katzenellenbogen, JA; Pollock, JA; Parent, AA; Wardell, SE; McDonnell, DP; Norris, JD
MLA Citation
Katzenellenbogen, John A., et al. “Structurally Novel Antagonists of Androgen Receptors with Elevated Potency for Antiandrogen-Resistant Receptor Mutants in Prostate Cancer: Tetra-Aryl-Substituted Cyclobutanes.” Endocrine Reviews, vol. 35, no. 3, ENDOCRINE SOC, 2014.
Source
wos
Published In
Endocrine Reviews
Volume
35
Issue
3
Publish Date
2014

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Authors
Scholtz, EL; Krishnan, S; Ball, BA; Corbin, CJ; Moeller, BC; Stanley, SD; McDowell, KJ; Hughes, AL; McDonnell, DP; Conley, AJ
MLA Citation
Scholtz, Elizabeth L., et al. “Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone..” Proc Natl Acad Sci U S A, vol. 111, no. 9, Mar. 2014, pp. 3365–70. Pubmed, doi:10.1073/pnas.1318163111.
PMID
24550466
Source
pubmed
Published In
Proc Natl Acad Sci U S A
Volume
111
Issue
9
Publish Date
2014
Start Page
3365
End Page
3370
DOI
10.1073/pnas.1318163111

ELF3 is a repressor of androgen receptor action in prostate cancer cells.

The androgen receptor (AR) has a critical role in the development and progression of prostate cancer (PC) and is a major therapeutic target in this disease. The transcriptional activity of AR is modulated by the coregulators with which it interacts, and consequently deregulation of cofactor expression and/or activity impacts the expression of genes whose products can have a role in PC pathogenesis. Here we report that E74-like factor 3 (ELF3), a member of the ETS family of transcription factors, is a repressor of AR transcriptional activity. Exogenous expression of ELF3 represses AR transcriptional activity when assessed using reporter-based transfection assays or when evaluated on endogenous AR target genes. Conversely, ELF3 knock down increases the AR transcriptional activity. Biochemical dissection of this activity indicates that it results from the physical interaction between ELF3 and AR and that this interaction inhibits the recruitment of AR to specific androgen response elements within target gene promoters. Significantly, we observed that depletion of ELF3 expression in LNCaP cells promotes cell migration, whereas increased ELF3 expression severely inhibits tumor growth in vitro and in a mouse xenograft model. Taken together, these results suggest that modulation of ELF3 expression and/or AR/ELF3 interaction may have utility in the treatment of PC.

Authors
Shatnawi, A; Norris, JD; Chaveroux, C; Jasper, JS; Sherk, AB; McDonnell, DP; Giguère, V
MLA Citation
Shatnawi, A., et al. “ELF3 is a repressor of androgen receptor action in prostate cancer cells..” Oncogene, vol. 33, no. 7, Feb. 2014, pp. 862–71. Pubmed, doi:10.1038/onc.2013.15.
PMID
23435425
Source
pubmed
Published In
Oncogene
Volume
33
Issue
7
Publish Date
2014
Start Page
862
End Page
871
DOI
10.1038/onc.2013.15

4,4'-Unsymmetrically substituted 3,3'-biphenyl alpha helical proteomimetics as potential coactivator binding inhibitors.

A series of unsymmetrically substituted biphenyl compounds was designed as alpha helical proteomimetics with the aim of inhibiting the binding of coactivator proteins to the nuclear hormone receptor coactivator binding domain. These compounds were synthesized in good overall yields in seven steps starting from 2-bromoanisole. The final products were evaluated using cotransfection reporter gene assays and mammalian two-hybrid competitive inhibition assays to demonstrate their effectiveness as competitive binding inhibitors. The results from this study indicate that these proteomimetics possess the ability to inhibit coactivator-receptor interactions, but via a mixed mode of inhibition.

Authors
Weiser, PT; Chang, C-Y; McDonnell, DP; Hanson, RN
MLA Citation
Weiser, Patrick T., et al. “4,4'-Unsymmetrically substituted 3,3'-biphenyl alpha helical proteomimetics as potential coactivator binding inhibitors..” Bioorg Med Chem, vol. 22, no. 2, Jan. 2014, pp. 917–26. Pubmed, doi:10.1016/j.bmc.2013.10.051.
PMID
24360824
Source
pubmed
Published In
Bioorg Med Chem
Volume
22
Issue
2
Publish Date
2014
Start Page
917
End Page
926
DOI
10.1016/j.bmc.2013.10.051

27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology.

Hypercholesterolemia is a risk factor for estrogen receptor (ER)-positive breast cancers and is associated with a decreased response of tumors to endocrine therapies. Here, we show that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol and an ER and liver X receptor (LXR) ligand, increases ER-dependent growth and LXR-dependent metastasis in mouse models of breast cancer. The effects of cholesterol on tumor pathology required its conversion to 27HC by the cytochrome P450 oxidase CYP27A1 and were attenuated by treatment with CYP27A1 inhibitors. In human breast cancer specimens, CYP27A1 expression levels correlated with tumor grade. In high-grade tumors, both tumor cells and tumor-associated macrophages exhibited high expression levels of the enzyme. Thus, lowering circulating cholesterol levels or interfering with its conversion to 27HC may be a useful strategy to prevent and/or treat breast cancer.

Authors
Nelson, ER; Wardell, SE; Jasper, JS; Park, S; Suchindran, S; Howe, MK; Carver, NJ; Pillai, RV; Sullivan, PM; Sondhi, V; Umetani, M; Geradts, J; McDonnell, DP
MLA Citation
Nelson, Erik R., et al. “27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology..” Science, vol. 342, no. 6162, Nov. 2013, pp. 1094–98. Pubmed, doi:10.1126/science.1241908.
PMID
24288332
Source
pubmed
Published In
Science
Volume
342
Issue
6162
Publish Date
2013
Start Page
1094
End Page
1098
DOI
10.1126/science.1241908

Aryl hydrocarbon receptor deficiency causes dysregulated cellular matrix metabolism and age-related macular degeneration-like pathology.

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that regulates xenobiotic metabolism and detoxification. Herein, we report a previously undescribed role for the AhR signaling pathway as an essential defense mechanism in the pathogenesis of early dry age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We found that AhR activity and protein levels in human retinal pigment epithelial (RPE) cells, cells vulnerable in AMD, decrease with age. This finding is significant given that age is the most established risk factor for development of AMD. Moreover, AhR(-/-) mice exhibit decreased visual function and develop dry AMD-like pathology, including disrupted RPE cell tight junctions, accumulation of RPE cell lipofuscin, basal laminar and linear-like deposit material, Bruch's membrane thickening, and progressive RPE and choroidal atrophy. High-serum low-density lipoprotein levels were also observed in AhR(-/-) mice. In its oxidized form, this lipoprotein can stimulate increased secretion of extracellular matrix molecules commonly found in deposits from RPE cells, in an AhR-dependent manner. This study demonstrates the importance of cellular clearance via the AhR signaling pathway in dry AMD pathogenesis, implicating AhR as a potential target, and the mouse model as a useful platform for validating future therapies.

Authors
Hu, P; Herrmann, R; Bednar, A; Saloupis, P; Dwyer, MA; Yang, P; Qi, X; Thomas, RS; Jaffe, GJ; Boulton, ME; McDonnell, DP; Malek, G
MLA Citation
Hu, Peng, et al. “Aryl hydrocarbon receptor deficiency causes dysregulated cellular matrix metabolism and age-related macular degeneration-like pathology..” Proc Natl Acad Sci U S A, vol. 110, no. 43, Oct. 2013, pp. E4069–78. Pubmed, doi:10.1073/pnas.1307574110.
PMID
24106308
Source
pubmed
Published In
Proc Natl Acad Sci U S A
Volume
110
Issue
43
Publish Date
2013
Start Page
E4069
End Page
E4078
DOI
10.1073/pnas.1307574110

Bazedoxifene exhibits antiestrogenic activity in animal models of tamoxifen-resistant breast cancer: implications for treatment of advanced disease.

PURPOSE: There is compelling evidence to suggest that drugs that function as pure estrogen receptor (ER-α) antagonists, or that downregulate the expression of ER-α, would have clinical use in the treatment of advanced tamoxifen- and aromatase-resistant breast cancer. Although such compounds are currently in development, we reasoned, based on our understanding of ER-α pharmacology, that there may already exist among the most recently developed selective estrogen receptor modulators (SERM) compounds that would have usage as breast cancer therapeutics. Thus, our objective was to identify among available SERMs those with unique pharmacologic activities and to evaluate their potential clinical use with predictive models of advanced breast cancer. EXPERIMENTAL DESIGN: A validated molecular profiling technology was used to classify clinically relevant SERMs based on their impact on ER-α conformation. The functional consequences of these observed mechanistic differences on (i) gene expression, (ii) receptor stability, and (iii) activity in cellular and animal models of advanced endocrine-resistant breast cancer were assessed. RESULTS: The high-affinity SERM bazedoxifene was shown to function as a pure ER-α antagonist in cellular models of breast cancer and effectively inhibited the growth of both tamoxifen-sensitive and -resistant breast tumor xenografts. Interestingly, bazedoxifene induced a unique conformational change in ER-α that resulted in its proteasomal degradation, although the latter activity was dispensable for its antagonist efficacy. CONCLUSION: Bazedoxifene was recently approved for use in the European Union for the treatment of osteoporosis and thus may represent a near-term therapeutic option for patients with advanced breast cancer.

Authors
Wardell, SE; Nelson, ER; Chao, CA; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “Bazedoxifene exhibits antiestrogenic activity in animal models of tamoxifen-resistant breast cancer: implications for treatment of advanced disease..” Clin Cancer Res, vol. 19, no. 9, May 2013, pp. 2420–31. Pubmed, doi:10.1158/1078-0432.CCR-12-3771.
PMID
23536434
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
19
Issue
9
Publish Date
2013
Start Page
2420
End Page
2431
DOI
10.1158/1078-0432.CCR-12-3771

The molecular mechanisms underlying the pharmacological actions of estrogens, SERMs and oxysterols: implications for the treatment and prevention of osteoporosis.

Estrogen therapy and hormone therapy are effective options for the prevention and treatment of osteoporosis, although because of their significant side effect profile, long term use for these applications is not recommended. Whereas SERMs (Selective Estrogen Receptor Modulators) exhibit a more favorable side effect profile, the currently available medicines in this class are substantially less effective in bone than classical estrogens. However, the results of substantial efforts that have gone into defining the mechanisms that underlie the pharmacology of estrogens, antiestrogens and SERMs have informed the development of the next generation of SERMs and have led to the development of TSECs (Tissue Selective Estrogen Complexes), a new class of ER-modulator. Further, the recent determination that the oxysterol 27-hydroxycholesterol functions as an endogenous SERM has highlighted an unexpected link between hypercholesterolemia and bone biology and must be considered in any discussions of ER-pharmacology. This review considers the most recent progress in our understanding of ER pharmacology and how this has and will be translated into new medicines for the treatment and prevention of osteoporosis.

Authors
Nelson, ER; Wardell, SE; McDonnell, DP
MLA Citation
Nelson, Erik R., et al. “The molecular mechanisms underlying the pharmacological actions of estrogens, SERMs and oxysterols: implications for the treatment and prevention of osteoporosis..” Bone, vol. 53, no. 1, Mar. 2013, pp. 42–50. Pubmed, doi:10.1016/j.bone.2012.11.011.
PMID
23168292
Source
pubmed
Published In
Bone
Volume
53
Issue
1
Publish Date
2013
Start Page
42
End Page
50
DOI
10.1016/j.bone.2012.11.011

Abstract PR3: The cholesterol metabolite 27-hydroxycholesterol increases breast cancer tumor growth and metastasis

Authors
Nelson, ER; Wardell, SE; Howe, MK; Carver, NJ; Umetani, M; McDonnell, DP
MLA Citation
Nelson, Erik Russell, et al. “Abstract PR3: The cholesterol metabolite 27-hydroxycholesterol increases breast cancer tumor growth and metastasis.” Determinants of Tumor Metastasis, American Association for Cancer Research, Feb. 2013. Crossref, doi:10.1158/1538-7445.tim2013-pr3.
Source
crossref
Published In
Determinants of Tumor Metastasis
Publish Date
2013
DOI
10.1158/1538-7445.tim2013-pr3

Androgens promote prostate cancer cell growth through induction of autophagy.

Androgens regulate both the physiological development of the prostate and the pathology of prostatic diseases. However, the mechanisms by which androgens exert their regulatory activities on these processes are poorly understood. In this study, we have determined that androgens regulate overall cell metabolism and cell growth, in part, by increasing autophagy in prostate cancer cells. Importantly, inhibition of autophagy using either pharmacological or molecular inhibitors significantly abrogated androgen-induced prostate cancer cell growth. Mechanistically, androgen-mediated autophagy appears to promote cell growth by augmenting intracellular lipid accumulation, an effect previously demonstrated to be necessary for prostate cancer cell growth. Further, autophagy and subsequent cell growth is potentiated, in part, by androgen-mediated increases in reactive oxygen species. These findings demonstrate a role for increased fat metabolism and autophagy in prostatic neoplasias and highlight the potential of targeting underexplored metabolic pathways for the development of novel therapeutics.

Authors
Shi, Y; Han, JJ; Tennakoon, JB; Mehta, FF; Merchant, FA; Burns, AR; Howe, MK; McDonnell, DP; Frigo, DE
MLA Citation
Shi, Yan, et al. “Androgens promote prostate cancer cell growth through induction of autophagy..” Mol Endocrinol, vol. 27, no. 2, Feb. 2013, pp. 280–95. Pubmed, doi:10.1210/me.2012-1260.
PMID
23250485
Source
pubmed
Published In
Mol Endocrinol
Volume
27
Issue
2
Publish Date
2013
Start Page
280
End Page
295
DOI
10.1210/me.2012-1260

Regulation of Bone Cell Function by Estrogens

The biological actions of estrogens are manifest in cells expressing either of two genetically and functionally distinct estrogen receptors (ERs). Although generally considered reproductive hormones, estrogens are also key regulators of processes involved in bone homeostasis and in the cardiovascular and central nervous systems. Drugs that regulate the activity of the ERs are widely used as contraceptives, components of hormone therapy regimens in menopausal women, and breast cancer treatments/chemopreventives. Importantly, the mechanisms by which estrogens work in different tissues are not the same, and these differences can be exploited in the development of selective estrogen receptor modulators (SERMs), compounds whose relative agonist/antagonist activity can differ in different tissues. More recently developed are the tissue selective estrogen complexes (TSECs), comprised of a SERM and an estrogen. These drugs effectively treat hot flashes, exhibit useful estrogenic activity in the bone, and are inactive in the uterus and breast. Therefore, these drugs are likely to be advantageous over current hormone therapy regimens in which a progestin is required. In this chapter, a discussion of our current understanding of ER action is presented, as well as how this information has enabled a shift from empirical- to mechanism-based discovery of ER modulators. © 2013 Elsevier Inc. All rights reserved.

Authors
Wardell, SE; McDonnell, DP; Nelson, ER
MLA Citation
Wardell, S. E., et al. Regulation of Bone Cell Function by Estrogens. Jan. 2013, pp. 329–44. Scopus, doi:10.1016/B978-0-12-415853-5.00014-5.
Source
scopus
Publish Date
2013
Start Page
329
End Page
344
DOI
10.1016/B978-0-12-415853-5.00014-5

Convergence of oncogenic and hormone receptor pathways promotes metastatic phenotypes.

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

Authors
Augello, MA; Burd, CJ; Birbe, R; McNair, C; Ertel, A; Magee, MS; Frigo, DE; Wilder-Romans, K; Shilkrut, M; Han, S; Jernigan, DL; Dean, JL; Fatatis, A; McDonnell, DP; Visakorpi, T; Feng, FY; Knudsen, KE
MLA Citation
Augello, Michael A., et al. “Convergence of oncogenic and hormone receptor pathways promotes metastatic phenotypes..” J Clin Invest, vol. 123, no. 1, Jan. 2013, pp. 493–508. Pubmed, doi:10.1172/JCI64750.
PMID
23257359
Source
pubmed
Published In
J Clin Invest
Volume
123
Issue
1
Publish Date
2013
Start Page
493
End Page
508
DOI
10.1172/JCI64750

Molecular pathways: the metabolic regulator estrogen-related receptor α as a therapeutic target in cancer.

The estrogen-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily of transcription factors whose activity is regulated by the expression level and/or activity of its obligate coregulators, peroxisome proliferator-activated receptor γ coactivator-1 α and β (PGC-1α or PGC-1β). Under normal physiologic conditions, and in responding to different environmental stimuli, the ERRα/PGC-1 complex is involved in regulating metabolic homeostasis under conditions of high energy demand in brown adipocytes, proliferating T cells, and muscle. Interestingly, increased expression and activity of the ERRα/PGC-1 axis has also been shown to correlate with unfavorable clinical outcomes in both breast and ovarian tumors. The observation that ERRα activity is manifest in all breast tumor subtypes with particularly high activity being evident in ERα-negative, HER2-positive, and triple-negative breast cancers has raised significant interest in targeting this receptor for the treatment of those breast cancers for which therapeutic options are limited.

Authors
Chang, C-Y; McDonnell, DP
MLA Citation
Chang, Ching-yi, and Donald P. McDonnell. “Molecular pathways: the metabolic regulator estrogen-related receptor α as a therapeutic target in cancer..” Clin Cancer Res, vol. 18, no. 22, Nov. 2012, pp. 6089–95. Pubmed, doi:10.1158/1078-0432.CCR-11-3221.
PMID
23019305
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
18
Issue
22
Publish Date
2012
Start Page
6089
End Page
6095
DOI
10.1158/1078-0432.CCR-11-3221

3,3'-Disubstituted bipolar biphenyls as inhibitors of nuclear receptor coactivator binding.

A series of bipolar biphenyl compounds was synthesized as proteomimetic analogs of the LXXLL penta-peptide motif responsible for the binding of coactivator proteins to the nuclear hormone receptor coactivator binding domain. These compounds were subjected to multiple in vitro assays to evaluate their effectiveness as competitive binding inhibitors. The results from this initial study indicate that these proteomimetics possess the ability to inhibit this protein-protein interaction.

Authors
Weiser, PT; Williams, AB; Chang, C-Y; McDonnell, DP; Hanson, RN
MLA Citation
Weiser, Patrick T., et al. “3,3'-Disubstituted bipolar biphenyls as inhibitors of nuclear receptor coactivator binding..” Bioorg Med Chem Lett, vol. 22, no. 21, Nov. 2012, pp. 6587–90. Pubmed, doi:10.1016/j.bmcl.2012.09.007.
PMID
23017882
Source
pubmed
Published In
Bioorg Med Chem Lett
Volume
22
Issue
21
Publish Date
2012
Start Page
6587
End Page
6590
DOI
10.1016/j.bmcl.2012.09.007

Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERα-dependent and -independent manners.

Lysine specific demethylase 1 (LSD1, also known as KDM1) is a histone modifying enzyme that regulates the expression of many genes important in cancer progression and proliferation. It is present in various transcriptional complexes including those containing the estrogen receptor (ER). Indeed, inhibition of LSD1 activity and or expression has been shown to attenuate estrogen signaling in breast cancer cells in vitro, implicating this protein in the pathogenesis of cancer. Herein we describe experiments that utilize small molecule inhibitors, phenylcyclopropylamines, along with small interfering RNA to probe the role of LSD1 in breast cancer proliferation and in estrogen-dependent gene transcription. Surprisingly, whereas we have confirmed that inhibition of LSD1 strongly inhibits proliferation of breast cancer cells, we have determined that the cytostatic actions of LSD1 inhibition are not impacted by ER status. These data suggest that LSD1 may be a useful therapeutic target in several types of breast cancer; most notably, inhibitors of LSD1 may have utility in the treatment of ER-negative cancers for which there are minimal therapeutic options.

Authors
Pollock, JA; Larrea, MD; Jasper, JS; McDonnell, DP; McCafferty, DG
MLA Citation
Pollock, Julie A., et al. “Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERα-dependent and -independent manners..” Acs Chem Biol, vol. 7, no. 7, July 2012, pp. 1221–31. Pubmed, doi:10.1021/cb300108c.
PMID
22533360
Source
pubmed
Published In
Acs Chem Biol
Volume
7
Issue
7
Publish Date
2012
Start Page
1221
End Page
1231
DOI
10.1021/cb300108c

Research resource: Transcriptional profiling in a cellular model of breast cancer reveals functional and mechanistic differences between clinically relevant SERM and between SERM/estrogen complexes.

Exploitation of the relationship between estrogen receptor (ER) structure and activity has led to the development of 1) selective ER modulators (SERM), compounds whose relative agonist/antagonist activities differ between target tissues; 2) selective ER degraders (SERD), compounds that induce a conformational change in the receptor that targets it for proteasomal degradation; and 3) tissue-selective estrogen complexes (TSEC), drugs in which a SERM and an ER agonist are combined to yield a blended activity that results in distinct clinical profiles. In this study, we have performed a comprehensive head-to-head analysis of the transcriptional activity of these different classes of ERM in a cellular model of breast cancer. Not surprisingly, these studies highlighted important functional differences and similarities among the existing SERM, selective ER degraders, and TSEC. Of particular importance was the identification of genes that were regulated by various TSEC combinations but not by an estrogen or SERM alone. Cumulatively, the findings of this analysis are informative with respect to the mechanisms by which ER is engaged by different enhancers/promoters and highlights how promoter context influences the pharmacological activity of ER ligands.

Authors
Wardell, SE; Kazmin, D; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “Research resource: Transcriptional profiling in a cellular model of breast cancer reveals functional and mechanistic differences between clinically relevant SERM and between SERM/estrogen complexes..” Mol Endocrinol, vol. 26, no. 7, July 2012, pp. 1235–48. Pubmed, doi:10.1210/me.2012-1031.
PMID
22570330
Source
pubmed
Published In
Mol Endocrinol
Volume
26
Issue
7
Publish Date
2012
Start Page
1235
End Page
1248
DOI
10.1210/me.2012-1031

Exploring the potential role of the oxidant-activated transcription factor aryl hydrocarbon receptor in the pathogenesis of AMD.

Authors
Malek, G; Dwyer, M; McDonnell, D
MLA Citation
Malek, Goldis, et al. “Exploring the potential role of the oxidant-activated transcription factor aryl hydrocarbon receptor in the pathogenesis of AMD..” Adv Exp Med Biol, vol. 723, 2012, pp. 51–59. Pubmed, doi:10.1007/978-1-4614-0631-0_8.
PMID
22183315
Source
pubmed
Published In
Advances in Experimental Medicine and Biology
Volume
723
Publish Date
2012
Start Page
51
End Page
59
DOI
10.1007/978-1-4614-0631-0_8

The oxysterol, 27-hydroxycholesterol, links cholesterol metabolism to bone homeostasis through its actions on the estrogen and liver X receptors.

Osteoporosis and age-related bone loss are important public health concerns. Therefore, there is a high level of interest in the development of medical interventions and lifestyle changes that reduce the incidence of osteoporosis and age-related bone loss. Decreased bone mineral density is associated with high cholesterol, and patients on statins have increased bone mineral densities, strongly implicating cholesterol as a negative regulator of bone homeostasis. In this study, using both molecular and pharmacological approaches, we have been able to demonstrate that the primary cholesterol metabolite, 27-hydroxycholesterol, through its actions on both estrogen receptors and liver X receptors, decreases osteoblast differentiation and enhances osteoclastogenesis, resulting in increased bone resorbtion in mice. Induction of the short heterodimer partner protein by estrogens in osteoblasts can attenuate the liver X receptor-mediated actions of 27-hydroxycholesterol in bone. These data establish a mechanistic link between cholesterol and bone quality, highlight an unexpected target of estrogens in osteoblasts, and define a signaling axis, the therapeutic exploitation of which is likely to yield novel antiosteoporotic drugs.

Authors
Nelson, ER; DuSell, CD; Wang, X; Howe, MK; Evans, G; Michalek, RD; Umetani, M; Rathmell, JC; Khosla, S; Gesty-Palmer, D; McDonnell, DP
MLA Citation
Nelson, Erik R., et al. “The oxysterol, 27-hydroxycholesterol, links cholesterol metabolism to bone homeostasis through its actions on the estrogen and liver X receptors..” Endocrinology, vol. 152, no. 12, Dec. 2011, pp. 4691–705. Pubmed, doi:10.1210/en.2011-1298.
PMID
21933863
Source
pubmed
Published In
Endocrinology
Volume
152
Issue
12
Publish Date
2011
Start Page
4691
End Page
4705
DOI
10.1210/en.2011-1298

Estrogen-related receptor-α is a metabolic regulator of effector T-cell activation and differentiation.

Stimulation of resting CD4(+) T lymphocytes leads to rapid proliferation and differentiation into effector (Teff) or inducible regulatory (Treg) subsets with specific functions to promote or suppress immunity. Importantly, Teff and Treg use distinct metabolic programs to support subset specification, survival, and function. Here, we describe that the orphan nuclear receptor estrogen-related receptor-α (ERRα) regulates metabolic pathways critical for Teff. Resting CD4(+) T cells expressed low levels of ERRα protein that increased on activation. ERRα deficiency reduced activated T-cell numbers in vivo and cytokine production in vitro but did not seem to modulate immunity through inhibition of activating signals or viability. Rather, ERRα broadly affected metabolic gene expression and glucose metabolism essential for Teff. In particular, up-regulation of Glut1 protein, glucose uptake, and mitochondrial processes were suppressed in activated ERRα(-/-) T cells and T cells treated with two chemically independent ERRα inhibitors or by shRNAi. Acute ERRα inhibition also blocked T-cell growth and proliferation. This defect appeared as a result of inadequate glucose metabolism, because provision of lipids, but not increased glucose uptake or pyruvate, rescued ATP levels and cell division. Additionally, we have shown that Treg requires lipid oxidation, whereas Teff uses glucose metabolism, and lipid addition selectively restored Treg--but not Teff--generation after acute ERRα inhibition. Furthermore, in vivo inhibition of ERRα reduced T-cell proliferation and Teff generation in both immunization and experimental autoimmune encephalomyelitis models. Thus, ERRα is a selective transcriptional regulator of Teff metabolism that may provide a metabolic means to modulate immunity.

Authors
Michalek, RD; Gerriets, VA; Nichols, AG; Inoue, M; Kazmin, D; Chang, C-Y; Dwyer, MA; Nelson, ER; Pollizzi, KN; Ilkayeva, O; Giguere, V; Zuercher, WJ; Powell, JD; Shinohara, ML; McDonnell, DP; Rathmell, JC
MLA Citation
Michalek, Ryan D., et al. “Estrogen-related receptor-α is a metabolic regulator of effector T-cell activation and differentiation..” Proc Natl Acad Sci U S A, vol. 108, no. 45, Nov. 2011, pp. 18348–53. Pubmed, doi:10.1073/pnas.1108856108.
PMID
22042850
Source
pubmed
Published In
Proc Natl Acad Sci U S A
Volume
108
Issue
45
Publish Date
2011
Start Page
18348
End Page
18353
DOI
10.1073/pnas.1108856108

The metabolic regulator ERRα, a downstream target of HER2/IGF-1R, as a therapeutic target in breast cancer.

A genomic signature designed to assess the activity of the estrogen-related receptor alpha (ERRα) was used to profile more than 800 breast tumors, revealing a shorter disease-free survival in patients with tumors exhibiting elevated receptor activity. Importantly, this signature also predicted the ability of an ERRα antagonist, XCT790, to inhibit proliferation in cellular models of breast cancer. Using a chemical genomic approach, it was determined that activation of the Her2/IGF-1R signaling pathways and subsequent C-MYC stabilization upregulate the expression of peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1β), an obligate cofactor for ERRα activity. PGC-1β knockdown in breast cancer cells impaired ERRα signaling and reduced cell proliferation, implicating a functional role for PGC-1β/ERRα in the pathogenesis of breast cancers.

Authors
Chang, C-Y; Kazmin, D; Jasper, JS; Kunder, R; Zuercher, WJ; McDonnell, DP
MLA Citation
Chang, Ching-yi, et al. “The metabolic regulator ERRα, a downstream target of HER2/IGF-1R, as a therapeutic target in breast cancer..” Cancer Cell, vol. 20, no. 4, Oct. 2011, pp. 500–10. Pubmed, doi:10.1016/j.ccr.2011.08.023.
PMID
22014575
Source
pubmed
Published In
Cancer Cell
Volume
20
Issue
4
Publish Date
2011
Start Page
500
End Page
510
DOI
10.1016/j.ccr.2011.08.023

Smoothelin-like 1 protein is a bifunctional regulator of the progesterone receptor during pregnancy.

During pregnancy, uterine smooth muscle (USM) coordinately adapts its contractile phenotype in order to accommodate the developing fetus and then prepare for delivery. Herein we show that SMTNL1 plays a major role in pregnancy to promote adaptive responses in USM and that this process is specifically mediated through interactions of SMTNL1 with the steroid hormone receptor PR-B. In vitro and in vivo SMTNL1 selectively binds PR and not other steroid hormone receptors. The physiological relationship between the two proteins was also established in global gene expression and transcriptional reporter studies in pregnant smtnl1(-/-) mice and by RNA interference in progesterone-sensitive cell lines. We show that the contraction-associated and progestin-sensitive genes (oxytocin receptor, connexin 43, and cyclooxygenase-2) and prolactins are down-regulated in pregnant smtnl1(-/-) mice. We suggest that SMTNL1 is a bifunctional co-regulator of PR-B signaling and thus provides a molecular mechanism whereby PR-B is targeted to alter gene expression patterns within USM cells to coordinately promote alterations in USM function during pregnancy.

Authors
Bodoor, K; Lontay, B; Safi, R; Weitzel, DH; Loiselle, D; Wei, Z; Lengyel, S; McDonnell, DP; Haystead, TA
MLA Citation
Bodoor, Khaldon, et al. “Smoothelin-like 1 protein is a bifunctional regulator of the progesterone receptor during pregnancy..” J Biol Chem, vol. 286, no. 36, Sept. 2011, pp. 31839–51. Pubmed, doi:10.1074/jbc.M111.270397.
PMID
21771785
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
286
Issue
36
Publish Date
2011
Start Page
31839
End Page
31851
DOI
10.1074/jbc.M111.270397

The turnover of estrogen receptor α by the selective estrogen receptor degrader (SERD) fulvestrant is a saturable process that is not required for antagonist efficacy.

It has become apparent of late that even in tamoxifen and/or aromatase resistant breast cancers, ERα remains a bona fide therapeutic target. Not surprisingly, therefore, there has been considerable interest in developing Selective ER Degraders (SERDs), compounds that target the receptor for degradation. Currently, ICI 182,780 (ICI, fulvestrant) is the only SERD approved for the treatment of breast cancer. However, the poor pharmaceutical properties of this injectable drug and its lack of superiority over second line aromatase inhibitors in late stage breast cancer have negatively impacted its clinical use. These findings have provided the impetus to develop second generation, orally bioavailable SERDs with which quantitative turnover of ERα in tumors can be achieved. Interestingly however, the contribution of SERD activity to fulvestrant efficacy is unclear, making it difficult to define the characteristics desired of the next generation of ER antagonists. It is of significance therefore, that we have determined that the antagonist activity of ICI and its ability to induce ERα degradation are not coupled processes. Specifically, our results indicate that it is the ability of ICI to interact with ERα and to (a) competitively displace estradiol and (b) induce a conformational change in ER incompatible with transcriptional activation that are likely to be the most important pharmacological characteristics of this drug. Collectively, these data argue for a renewed emphasis on the development of high affinity, orally bioavailable pure antagonists and suggest that SERD activity though proven effective may not be required for ERα antagonism in breast cancer.

Authors
Wardell, SE; Marks, JR; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “The turnover of estrogen receptor α by the selective estrogen receptor degrader (SERD) fulvestrant is a saturable process that is not required for antagonist efficacy..” Biochem Pharmacol, vol. 82, no. 2, July 2011, pp. 122–30. Pubmed, doi:10.1016/j.bcp.2011.03.031.
PMID
21501600
Source
pubmed
Published In
Biochem Pharmacol
Volume
82
Issue
2
Publish Date
2011
Start Page
122
End Page
130
DOI
10.1016/j.bcp.2011.03.031

Examination of the effects of small molecule inhibitors of human histone lysine-specific demethylase I in breast cancer cells

Authors
Pollock, JA; Larrea, MD; McDonnell, DP; McCafferty, DG
MLA Citation
Pollock, Julie A., et al. “Examination of the effects of small molecule inhibitors of human histone lysine-specific demethylase I in breast cancer cells.” Abstracts of Papers of the American Chemical Society, vol. 241, AMER CHEMICAL SOC, 2011.
Source
wos
Published In
Abstracts of Papers of the American Chemical Society
Volume
241
Publish Date
2011

Research resource: nuclear receptor atlas of human retinal pigment epithelial cells: potential relevance to age-related macular degeneration.

Retinal pigment epithelial (RPE) cells play a vital role in retinal physiology by forming the outer blood-retina barrier and supporting photoreceptor function. Retinopathies including age-related macular degeneration (AMD) involve physiological and pathological changes in the epithelium, severely impairing the retina and effecting vision. Nuclear receptors (NRs), including peroxisome proliferator-activated receptor and liver X receptor, have been identified as key regulators of physiological pathways such as lipid metabolic dysregulation and inflammation, pathways that may also be involved in development of AMD. However, the expression levels of NRs in RPE cells have yet to be systematically surveyed. Furthermore, cell culture lines are widely used to study the biology of RPE cells, without knowledge of the differences or similarities in NR expression and activity between these in vitro models and in vivo RPE. Using quantitative real-time PCR, we assessed the expression patterns of all 48 members of the NR family plus aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in human RPE cells. We profiled freshly isolated cells from donor eyes (in vivo), a spontaneously arising human cell line (in vitro), and primary cell culture lines (in vitro) to determine the extent to which NR expression in the cultured cell lines reflects that of in vivo. To evaluate the validity of using cell culture models for investigating NR receptor biology, we determined transcriptional activity and target gene expression of several moderately and highly expressed NRs in vitro. Finally, we identified a subset of NRs that may play an important role in pathobiology of AMD.

Authors
Dwyer, MA; Kazmin, D; Hu, P; McDonnell, DP; Malek, G
MLA Citation
Dwyer, Mary A., et al. “Research resource: nuclear receptor atlas of human retinal pigment epithelial cells: potential relevance to age-related macular degeneration..” Mol Endocrinol, vol. 25, no. 2, Feb. 2011, pp. 360–72. Pubmed, doi:10.1210/me.2010-0392.
PMID
21239617
Source
pubmed
Published In
Mol Endocrinol
Volume
25
Issue
2
Publish Date
2011
Start Page
360
End Page
372
DOI
10.1210/me.2010-0392

CaM kinase kinase beta-mediated activation of the growth regulatory kinase AMPK is required for androgen-dependent migration of prostate cancer cells.

While patients with advanced prostate cancer initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years. Although hormone-refractory disease is unresponsive to androgen-deprivation, androgen receptor (AR)-regulated signaling pathways remain active and are necessary for cancer progression. Thus, both AR itself and the processes downstream of the receptor remain viable targets for therapeutic intervention. Microarray analysis of multiple clinical cohorts showed that the serine/threonine kinase Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is both highly expressed in the prostate and further elevated in prostate cancers. Using cellular models of prostate cancer, we have determined that androgens (a) directly increase the expression of a CaMKKβ splice variant and (b) increase functional CaMKKβ protein levels as determined by the phosphorylation of both CaMKI and AMP-activated protein kinase (AMPK), two of CaMKKβ's primary substrates. Importantly, inhibition of the CaMKKβ-AMPK, but not CaMKI, signaling axis in prostate cancer cells by pharmacological inhibitors or siRNA-mediated knockdown blocks androgen-mediated migration and invasion. Conversely, overexpression of CaMKKβ alone leads to both increased AMPK phosphorylation and cell migration. Given the key roles of CaMKKβ and AMPK in the biology of prostate cancer cells, we propose that these enzymes are potential therapeutic targets in prostate cancer.

Authors
Frigo, DE; Howe, MK; Wittmann, BM; Brunner, AM; Cushman, I; Wang, Q; Brown, M; Means, AR; McDonnell, DP
MLA Citation
Frigo, Daniel E., et al. “CaM kinase kinase beta-mediated activation of the growth regulatory kinase AMPK is required for androgen-dependent migration of prostate cancer cells..” Cancer Res, vol. 71, no. 2, Jan. 2011, pp. 528–37. Pubmed, doi:10.1158/0008-5472.CAN-10-2581.
PMID
21098087
Source
pubmed
Published In
Cancer Res
Volume
71
Issue
2
Publish Date
2011
Start Page
528
End Page
537
DOI
10.1158/0008-5472.CAN-10-2581

Identification of ligand-selective peptide antagonists of the mineralocorticoid receptor using phage display.

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. The glucocorticoid cortisol binds the MR with equivalent affinity to that of the mineralocorticoids aldosterone and deoxycorticosterone. In nonepithelial tissues, including the myocardium, which do not express the cortisol-inactivating enzyme 11β hydroxysteroid dehydrogenase 2, cortisol has been implicated in the activation of MR. The mechanisms for ligand- and tissue-specific actions of the MR are undefined. Over the past decade, it has become clear that coregulator proteins are critical for nuclear receptor-mediated gene expression. A subset of these coregulators may confer specificity to MR-mediated responses. To evaluate whether different physiological ligands can induce distinct MR conformations that underlie differential coregulator recruitment and ligand-specific gene regulation, we utilized phage display technology to screen 10(8) 19mer peptides for their interaction with the MR in the presence of agonist ligands. We identified ligand-selective MR-interacting peptides that acted as potent antagonists of MR-mediated transactivation. This represents a novel mechanism of MR antagonism that may be manipulated in the rational design of a ligand- or tissue-selective MR modulator to treat diseases like heart failure without side effects such as hyperkalemia.

Authors
Yang, J; Chang, C-Y; Safi, R; Morgan, J; McDonnell, DP; Fuller, PJ; Clyne, CD; Young, MJ
MLA Citation
Yang, Jun, et al. “Identification of ligand-selective peptide antagonists of the mineralocorticoid receptor using phage display..” Mol Endocrinol, vol. 25, no. 1, Jan. 2011, pp. 32–43. Pubmed, doi:10.1210/me.2010-0193.
PMID
21106883
Source
pubmed
Published In
Mol Endocrinol
Volume
25
Issue
1
Publish Date
2011
Start Page
32
End Page
43
DOI
10.1210/me.2010-0193

Mechanisms of progesterone receptor inhibition of inflammatory responses in cellular models of breast cancer.

Both pro- and antimitogenic activities have been ascribed to progesterone receptor (PR) agonists and antagonists in breast cancer cells; however, the transcriptional responses that underlie these paradoxical functions are not apparent. Using nontransformed, normal human mammary epithelial cells engineered to express PR and standard microarray technology, we defined 2370 genes that were significantly regulated by the PR agonist R5020. Gene ontology (GO) analysis revealed that GO terms involved in inflammation and nuclear factor-κB (NF-κB) signaling were among the most significantly regulated. Interestingly, on those NF-κB responsive genes that were inhibited by agonist-activated PR, antagonists either 1) mimicked the actions of agonists or 2) reversed the inhibitory actions of agonists. This difference in pharmacological response could be attributed to the fact that although agonist- and antagonist-activated PR is recruited to NF-κB-responsive promoters, the physical presence of PR tethered to the promoter of some genes is sufficient for transcriptional inhibition, whereas on others, an agonist-activated PR conformation is required for inhibition of NF-κB signaling. Importantly, the actions of PR on the latter class of genes were reversed by an activation function-2-inhibiting, LXXLL-containing peptide. Consideration of the relative activities of these distinct antiinflammatory pathways in breast cancer may be instructive with respect to the likely therapeutic activity of PR agonists or antagonists in the treatment of breast cancer.

Authors
Kobayashi, S; Stice, JP; Kazmin, D; Wittmann, BM; Kimbrel, EA; Edwards, DP; Chang, C-Y; McDonnell, DP
MLA Citation
Kobayashi, Sakiko, et al. “Mechanisms of progesterone receptor inhibition of inflammatory responses in cellular models of breast cancer..” Mol Endocrinol, vol. 24, no. 12, Dec. 2010, pp. 2292–302. Pubmed, doi:10.1210/me.2010-0289.
PMID
20980435
Source
pubmed
Published In
Mol Endocrinol
Volume
24
Issue
12
Publish Date
2010
Start Page
2292
End Page
2302
DOI
10.1210/me.2010-0289

The molecular mechanisms underlying the pharmacological actions of ER modulators: implications for new drug discovery in breast cancer.

Our understanding of the molecular mechanisms underlying the pharmacological actions of estrogen receptor (ER) ligands has evolved considerably in recent years. Much of this knowledge has come from a detailed dissection of the mechanism(s) of action of the Selective Estrogen Receptor Modulators (SERMs) tamoxifen and raloxifene, so called for their ability to function as ER agonists or antagonists depending on the tissue in which they operate. These mechanistic insights have had a significant impact on the discovery of second generation SERMs, some of which are in late stage clinical development for the treatment/prevention of breast cancer as well as other estrogenopathies. In addition to the SERMs, however, have emerged the Selective Estrogen Degraders (SERDs), which as their name suggests, interact with and facilitate ER turnover in cells. One drug of this class, fulvestrant, has been approved as a third line treatment for ER-positive metastatic breast cancer. Whereas the first generation SERMs/SERDs were discovered in a serendipitous manner, this review will highlight how our understanding of the molecular pharmacology of ER ligands has been utilized in the development of the next generation of SERMs/SERDs, some of which are likely to have a major impact on the pharmacotherapy of breast cancer.

Authors
McDonnell, DP; Wardell, SE
MLA Citation
McDonnell, Donald P., and Suzanne E. Wardell. “The molecular mechanisms underlying the pharmacological actions of ER modulators: implications for new drug discovery in breast cancer..” Curr Opin Pharmacol, vol. 10, no. 6, Dec. 2010, pp. 620–28. Pubmed, doi:10.1016/j.coph.2010.09.007.
PMID
20926342
Source
pubmed
Published In
Curr Opin Pharmacol
Volume
10
Issue
6
Publish Date
2010
Start Page
620
End Page
628
DOI
10.1016/j.coph.2010.09.007

WNT11 expression is induced by estrogen-related receptor alpha and beta-catenin and acts in an autocrine manner to increase cancer cell migration.

Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, β-catenin (β-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by β-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or β-cat. Furthermore, silencing of ERRα and β-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/β-cat-dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/β-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and β-cat that influences the migratory capacity of cancer cells.

Authors
Dwyer, MA; Joseph, JD; Wade, HE; Eaton, ML; Kunder, RS; Kazmin, D; Chang, C-Y; McDonnell, DP
MLA Citation
Dwyer, Mary A., et al. “WNT11 expression is induced by estrogen-related receptor alpha and beta-catenin and acts in an autocrine manner to increase cancer cell migration..” Cancer Res, vol. 70, no. 22, Nov. 2010, pp. 9298–308. Pubmed, doi:10.1158/0008-5472.CAN-10-0226.
PMID
20870744
Source
pubmed
Published In
Cancer Res
Volume
70
Issue
22
Publish Date
2010
Start Page
9298
End Page
9308
DOI
10.1158/0008-5472.CAN-10-0226

An interdomain interaction of the androgen receptor is required for its aggregation and toxicity in spinal and bulbar muscular atrophy.

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function.

Authors
Orr, CR; Montie, HL; Liu, Y; Bolzoni, E; Jenkins, SC; Wilson, EM; Joseph, JD; McDonnell, DP; Merry, DE
MLA Citation
Orr, Christopher R., et al. “An interdomain interaction of the androgen receptor is required for its aggregation and toxicity in spinal and bulbar muscular atrophy..” J Biol Chem, vol. 285, no. 46, Nov. 2010, pp. 35567–77. Pubmed, doi:10.1074/jbc.M110.146845.
PMID
20826791
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
285
Issue
46
Publish Date
2010
Start Page
35567
End Page
35577
DOI
10.1074/jbc.M110.146845

The endogenous selective estrogen receptor modulator 27-hydroxycholesterol is a negative regulator of bone homeostasis.

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

Authors
DuSell, CD; Nelson, ER; Wang, X; Abdo, J; Mödder, UI; Umetani, M; Gesty-Palmer, D; Javitt, NB; Khosla, S; McDonnell, DP
MLA Citation
DuSell, Carolyn D., et al. “The endogenous selective estrogen receptor modulator 27-hydroxycholesterol is a negative regulator of bone homeostasis..” Endocrinology, vol. 151, no. 8, Aug. 2010, pp. 3675–85. Pubmed, doi:10.1210/en.2010-0080.
PMID
20501668
Source
pubmed
Published In
Endocrinology
Volume
151
Issue
8
Publish Date
2010
Start Page
3675
End Page
3685
DOI
10.1210/en.2010-0080

The Mineralocorticoid Receptor: Exploring Ligand-Specificity Using Peptide Phage Display

Authors
Yang, J; Clyne, CD; Safi, R; Chang, CY; Fuller, PJ; McDonnell, DP; Young, MJ
MLA Citation
Yang, J., et al. “The Mineralocorticoid Receptor: Exploring Ligand-Specificity Using Peptide Phage Display.” Endocrine Reviews, vol. 31, no. 3, ENDOCRINE SOC, 2010.
Source
wos
Published In
Endocrine Reviews
Volume
31
Issue
3
Publish Date
2010

Expression, purification and primary crystallographic study of human androgen receptor in complex with DNA and coactivator motifs.

The androgen receptor (AR) is a DNA-binding and hormone-activated transcription factor that plays critical roles in the development and progression of prostate cancer. The transcriptional function of AR is modulated by intermolecular interactions with DNA elements and coactivator proteins, as well as intramolecular interactions between AR domains; thus, the structural information from the full-length AR or a multi-domain fragment is essential for understanding the molecular basis of AR functions. Here we report the expression and purification of full-length AR protein and of a fragment containing its DNA-binding and ligand-binding domains connected by the hinge region in the presence of its natural ligand, dihydrotestosterone. Crystals of ligand-bound full-length AR and of the AR fragment in complex with DNA elements and coactivator motifs have been obtained and diffracted to low resolutions. These results help establish a foundation for pursuing further crystallographic studies of an AR/DNA complex.

Authors
Zhou, XE; Suino-Powell, K; Ludidi, PL; McDonnell, DP; Xu, HE
MLA Citation
Zhou, X. Edward, et al. “Expression, purification and primary crystallographic study of human androgen receptor in complex with DNA and coactivator motifs..” Protein Expr Purif, vol. 71, no. 1, May 2010, pp. 21–27. Pubmed, doi:10.1016/j.pep.2009.12.002.
PMID
19995608
Source
pubmed
Published In
Protein Expr Purif
Volume
71
Issue
1
Publish Date
2010
Start Page
21
End Page
27
DOI
10.1016/j.pep.2009.12.002

Expression Profiling of Nuclear Receptors in Human Retinal Pigment Epithelial Cells: Relevance to Age-Related Macular Degeneration

Authors
Malek, G; Hu, P; Dwyer, M; Cousins, SW; McDonnell, DP
MLA Citation
Malek, G., et al. “Expression Profiling of Nuclear Receptors in Human Retinal Pigment Epithelial Cells: Relevance to Age-Related Macular Degeneration.” Investigative Ophthalmology & Visual Science, vol. 51, no. 13, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2010.
Source
wos
Published In
Investigative Ophthalmology & Visual Science
Volume
51
Issue
13
Publish Date
2010

Oxidant Activation of the Transcription Factor Aryl Hydrocarbon Receptor Effects Mitochondrial Function in Retinal Pigment Epithelial Cells

Authors
Dwyer, M; McDonnell, DP; Hu, P; Cousins, S; Malek, G
MLA Citation
Dwyer, M., et al. “Oxidant Activation of the Transcription Factor Aryl Hydrocarbon Receptor Effects Mitochondrial Function in Retinal Pigment Epithelial Cells.” Investigative Ophthalmology & Visual Science, vol. 51, no. 13, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2010.
Source
wos
Published In
Investigative Ophthalmology & Visual Science
Volume
51
Issue
13
Publish Date
2010

Multimodal regulation of E2F1 gene expression by progestins.

An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR.

Authors
Wade, HE; Kobayashi, S; Eaton, ML; Jansen, MS; Lobenhofer, EK; Lupien, M; Geistlinger, TR; Zhu, W; Nevins, JR; Brown, M; Otteson, DC; McDonnell, DP
MLA Citation
Wade, Hilary E., et al. “Multimodal regulation of E2F1 gene expression by progestins..” Mol Cell Biol, vol. 30, no. 8, Apr. 2010, pp. 1866–77. Pubmed, doi:10.1128/MCB.01060-09.
PMID
20123965
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
30
Issue
8
Publish Date
2010
Start Page
1866
End Page
1877
DOI
10.1128/MCB.01060-09

Inhibition of aldehyde dehydrogenase expands hematopoietic stem cells with radioprotective capacity.

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units, verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes.

Authors
Muramoto, GG; Russell, JL; Safi, R; Salter, AB; Himburg, HA; Daher, P; Meadows, SK; Doan, P; Storms, RW; Chao, NJ; McDonnell, DP; Chute, JP
MLA Citation
Muramoto, Garrett G., et al. “Inhibition of aldehyde dehydrogenase expands hematopoietic stem cells with radioprotective capacity..” Stem Cells, vol. 28, no. 3, Mar. 2010, pp. 523–34. Pubmed, doi:10.1002/stem.299.
PMID
20054864
Source
pubmed
Published In
Stem Cells
Volume
28
Issue
3
Publish Date
2010
Start Page
523
End Page
534
DOI
10.1002/stem.299

The homeodomain protein HOXB13 specifies the cellular response to androgens

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “The homeodomain protein HOXB13 specifies the cellular response to androgens.” Endocrine Journal, vol. 57, JAPAN ENDOCRINE SOC, Mar. 2010, pp. S258–59.
Source
wos
Published In
Endocrine Journal
Volume
57
Publish Date
2010
Start Page
S258
End Page
S259

ERRgamma regulates cardiac, gastric, and renal potassium homeostasis.

Energy production by oxidative metabolism in kidney, stomach, and heart, is primarily expended in establishing ion gradients to drive renal electrolyte homeostasis, gastric acid secretion, and cardiac muscle contraction, respectively. In addition to orchestrating transcriptional control of oxidative metabolism, the orphan nuclear receptor, estrogen-related receptor gamma (ERRgamma), coordinates expression of genes central to ion homeostasis in oxidative tissues. Renal, gastric, and cardiac tissues subjected to genomic analysis of expression in perinatal ERRgamma null mice revealed a characteristic dysregulation of genes involved in transport processes, exemplified by the voltage-gated potassium channel, Kcne2. Consistently, ERRgamma null animals die during the first 72 h of life with elevated serum potassium, reductions in key gastric acid production markers, and cardiac arrhythmia with prolonged QT intervals. In addition, we find altered expression of several genes associated with hypertension in ERRgamma null mice. These findings suggest a potential role for genetic polymorphisms at the ERRgamma locus and ERRgamma modulators in the etiology and treatment of renal, gastric, and cardiac dysfunction.

Authors
Alaynick, WA; Way, JM; Wilson, SA; Benson, WG; Pei, L; Downes, M; Yu, R; Jonker, JW; Holt, JA; Rajpal, DK; Li, H; Stuart, J; McPherson, R; Remlinger, KS; Chang, C-Y; McDonnell, DP; Evans, RM; Billin, AN
MLA Citation
Alaynick, William A., et al. “ERRgamma regulates cardiac, gastric, and renal potassium homeostasis..” Mol Endocrinol, vol. 24, no. 2, Feb. 2010, pp. 299–309. Pubmed, doi:10.1210/me.2009-0114.
PMID
19965931
Source
pubmed
Published In
Mol Endocrinol
Volume
24
Issue
2
Publish Date
2010
Start Page
299
End Page
309
DOI
10.1210/me.2009-0114

Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation.

The current statistics associated with breast cancer continue to show a relatively high recurrence rate together with a poor survival for aggressive metastatic disease. These findings reflect, in part, the pharmaceutical intractability of processes involved in the metastatic process and highlight the need to identify additional drug targets for the treatment of late-stage disease. In the current study, we report that ligand activation of the aryl-hydrocarbon receptor (AhR) inhibits multiple aspects of the metastatic process in a panel of breast cancer cell lines that represent the major breast cancer subtypes. Specifically, it was observed that treatment with exogenous AhR agonists significantly inhibited cell invasiveness and motility in the Boyden chamber assay and inhibited colony formation in soft agar regardless of estrogen receptor (ER), progesterone receptor, or human epidermal growth factor receptor 2 status. Knockdown of the AhR using small interfering RNA duplexes demonstrated that the inhibition of invasiveness was receptor dependent and that endogenous receptor activity was protective in each cell type examined. The inhibition of invasiveness and anchorage-independent growth correlated with the ability of exogenous AhR agonists to promote differentiation. Finally, exogenous AhR agonists were able to promote differentiation in a putative mammary cancer stem cell line. Cumulatively, these results suggest that the AhR plays an important role in mammary epithelial differentiation and, as such, represent a promising therapeutic target for a range of phenotypically distinct human breast cancers.

Authors
Hall, JM; Barhoover, MA; Kazmin, D; McDonnell, DP; Greenlee, WF; Thomas, RS
MLA Citation
Hall, Julie M., et al. “Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation..” Mol Endocrinol, vol. 24, no. 2, Feb. 2010, pp. 359–69. Pubmed, doi:10.1210/me.2009-0346.
PMID
20032195
Source
pubmed
Published In
Mol Endocrinol
Volume
24
Issue
2
Publish Date
2010
Start Page
359
End Page
369
DOI
10.1210/me.2009-0346

Minireview: Nuclear receptors, hematopoiesis, and stem cells.

Nuclear receptors (NRs) regulate a panoply of biological processes, including the function and development of cells within the hematopoietic and immune system, such as erythrocytes, monocytes, and lymphocytes. Significantly less is known regarding the function of NRs in regulating the fate of hematopoietic stem cells (HSCs), the self-renewing, pluripotent cells that give rise to the entirety of the blood and immune systems throughout the lifetime of an individual. Several recent studies suggest, either directly or indirectly, a role for members of the NR family in regulating the differentiation and self-renewal of HSCs, embryonic stem cells, and induced pluripotent stem cells. Herein, we review in detail the function of specific NRs in controlling HSC and other stem cell fate and propose a framework through which these observations can be translated into therapeutic amplification of HSCs for clinical purposes.

Authors
Chute, JP; Ross, JR; McDonnell, DP
MLA Citation
Chute, John P., et al. “Minireview: Nuclear receptors, hematopoiesis, and stem cells..” Mol Endocrinol, vol. 24, no. 1, Jan. 2010, pp. 1–10. Pubmed, doi:10.1210/me.2009-0332.
PMID
19934345
Source
pubmed
Published In
Mol Endocrinol
Volume
24
Issue
1
Publish Date
2010
Start Page
1
End Page
10
DOI
10.1210/me.2009-0332

Regulation of aryl hydrocarbon receptor function by selective estrogen receptor modulators.

Selective estrogen receptor modulators (SERMs), such as tamoxifen (TAM), have been used extensively for the treatment and prevention of breast cancer and other pathologies associated with aberrant estrogen receptor (ER) signaling. These compounds exhibit cell-selective agonist/antagonist activities as a consequence of their ability to induce different conformational changes in ER, thereby enabling it to recruit functionally distinct transcriptional coregulators. However, the observation that SERMs can also regulate aspects of calcium signaling and apoptosis in an ER-independent manner in some systems suggests that some of the activity of drugs within this class may also arise as a consequence of their ability to interact with targets other than ER. In this study, we demonstrate that 4-hydroxy-TAM (4OHT), an active metabolite of TAM, directly binds to and modulates the transcriptional activity of the aryl hydrocarbon receptor (AHR). Of specific interest was the observation, that in the absence of ER, 4OHT can induce the expression of AHR target genes involved in estradiol metabolism, cellular proliferation, and metastasis in cellular models of breast cancer. The potential role for AHR in SERM pharmacology was further underscored by the ability of 4OHT to suppress osteoclast differentiation in vitro in part through AHR. Cumulatively, these findings provide evidence that it is necessary to reevaluate the relative roles of ER and AHR in manifesting the pharmacological actions and therapeutic efficacy of TAM and other SERMs.

Authors
DuSell, CD; Nelson, ER; Wittmann, BM; Fretz, JA; Kazmin, D; Thomas, RS; Pike, JW; McDonnell, DP
MLA Citation
DuSell, Carolyn D., et al. “Regulation of aryl hydrocarbon receptor function by selective estrogen receptor modulators..” Mol Endocrinol, vol. 24, no. 1, Jan. 2010, pp. 33–46. Pubmed, doi:10.1210/me.2009-0339.
PMID
19901195
Source
pubmed
Published In
Mol Endocrinol
Volume
24
Issue
1
Publish Date
2010
Start Page
33
End Page
46
DOI
10.1210/me.2009-0339

The homeodomain protein HOXB13 regulates the cellular response to androgens.

HOXB13 is a member of the homeodomain family of sequence-specific transcription factors and, together with the androgen receptor (AR), plays a critical role in the normal development of the prostate gland. We demonstrate here that, in prostate cancer cells, HOXB13 is a key determinant of the response to androgens. Specifically, it was determined that HOXB13 interacts with the DNA-binding domain of AR and inhibits the transcription of genes that contain an androgen-response element (ARE). In contrast, the AR:HOXB13 complex confers androgen responsiveness to promoters that contain a specific HOXB13-response element. Further, HOXB13 and AR synergize to enhance the transcription of genes that contain a HOX element juxtaposed to an ARE. The profound effects of HOXB13 knockdown on androgen-regulated proliferation, migration, and lipogenesis in prostate cancer cells highlight the importance of the observed changes in gene expression.

Authors
Norris, JD; Chang, C-Y; Wittmann, BM; Kunder, RS; Cui, H; Fan, D; Joseph, JD; McDonnell, DP
MLA Citation
Norris, John D., et al. “The homeodomain protein HOXB13 regulates the cellular response to androgens..” Molecular Cell, vol. 36, no. 3, Nov. 2009, pp. 405–16. Epmc, doi:10.1016/j.molcel.2009.10.020.
PMID
19917249
Source
epmc
Published In
Molecular Cell
Volume
36
Issue
3
Publish Date
2009
Start Page
405
End Page
416
DOI
10.1016/j.molcel.2009.10.020

Induction of Kruppel-like factor 5 expression by androgens results in increased CXCR4-dependent migration of prostate cancer cells in vitro.

Advanced prostate cancers preferentially metastasize to bone, suggesting that this tissue produces factors that provide a suitable microenvironment for prostate cancer cells. Recently, it has become clear that even in antiandrogen-resistant cancers, the androgen receptor (AR)-signaling axis is required for prostate cancer progression. Therefore, we hypothesized that AR may be involved in the regulation of pathways that are responsible for the homing of prostate cancer cells to select microenvironments. In support of this hypothesis, we have determined that chemokine (C-X-C motif) receptor 4 (CXCR4), the receptor for the chemokine CXCL12, is up-regulated in prostate cancer cells in response to androgens. Given that the levels of CXCL12 are elevated at sites of known prostate cancer metastases such as bone, these results suggest that androgens may influence prostate cancer metastasis. Specifically, we demonstrate that androgens increase the levels of both CXCR4 mRNA and functional protein in LNCaP prostate cancer cells. Importantly, androgens enhanced the migration of LNCaP cells toward a CXCL12 gradient, an effect that could be blocked by the specific CXCR4 antagonist AMD3100. Interestingly, CXCR4 is not directly regulated by androgens but rather is positively up-regulated by Krüppel-like factor 5 (KLF5), a transcription factor that we have shown to be an early, direct target of AR. Further, KLF5 is both required and sufficient for androgen-mediated CXCR4 expression and migration toward CXCL12. Taken together, these findings demonstrate that AR can utilize the CXCL12/CXCR4 axis through induction of KLF5 expression to promote prostate cancer progression and highlight the potential utility of CXCR4 antagonists as prostate cancer therapeutics.

Authors
Frigo, DE; Sherk, AB; Wittmann, BM; Norris, JD; Wang, Q; Joseph, JD; Toner, AP; Brown, M; McDonnell, DP
MLA Citation
Frigo, Daniel E., et al. “Induction of Kruppel-like factor 5 expression by androgens results in increased CXCR4-dependent migration of prostate cancer cells in vitro..” Mol Endocrinol, vol. 23, no. 9, Sept. 2009, pp. 1385–96. Pubmed, doi:10.1210/me.2009-0010.
PMID
19460858
Source
pubmed
Published In
Mol Endocrinol
Volume
23
Issue
9
Publish Date
2009
Start Page
1385
End Page
1396
DOI
10.1210/me.2009-0010

Fasting-induced hepatic production of DHEA is regulated by PGC-1alpha, ERRalpha, and HNF4alpha.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is involved in the coordinate induction of changes in gene expression in the liver that enable a homeostatic response to alterations in metabolic state, environmental cues, and nutrient availability. In exploring the specific pathways under PGC-1alpha regulation in the liver, we have made the surprising observation that this coactivator can induce the expression of CYP11A1 and CYP17A1, key rate-limiting enzymes involved in the initial steps of steroidogenesis. Both of these enzymes function to produce C(19)-steroids, converting cholesterol into pregnenolone, and then to dehydroepiandrosterone (DHEA). Estrogen-related receptor (ERR)-alpha mediates PGC-1alpha's induction of CYP11A1 and binds within the first intron of the CYP11A1 gene. Both ERR-alpha and hepatocyte nuclear factor-4alpha are required for PGC-1alpha-mediated induction of CYP17A1, and specific binding sites for these receptors have been identified in the regulatory regions of this gene. The potential physiological significance of these observations was highlighted in rats where fasting induced hepatic expression of PGC-1alpha and CYP17A1 and was associated with an increase in hepatic levels of DHEA. These data suggest that DHEA could be playing a role as an intracellular signaling molecule involved in modulating hepatic activity in response to fasting conditions.

Authors
Grasfeder, LL; Gaillard, S; Hammes, SR; Ilkayeva, O; Newgard, CB; Hochberg, RB; Dwyer, MA; Chang, C-Y; McDonnell, DP
MLA Citation
Grasfeder, Linda L., et al. “Fasting-induced hepatic production of DHEA is regulated by PGC-1alpha, ERRalpha, and HNF4alpha..” Mol Endocrinol, vol. 23, no. 8, Aug. 2009, pp. 1171–82. Pubmed, doi:10.1210/me.2009-0024.
PMID
19389810
Source
pubmed
Published In
Mol Endocrinol
Volume
23
Issue
8
Publish Date
2009
Start Page
1171
End Page
1182
DOI
10.1210/me.2009-0024

Inhibition of prostate cancer cell growth by second-site androgen receptor antagonists.

The impact of ligand binding on nuclear receptor (NR) structure and the ability of target cells to distinguish between different receptor-ligand complexes are key determinants of the pharmacological activity of NR ligands. However, until relatively recently, these mechanistic insights have not been used in a prospective manner to develop screens for NR modulators with specific therapeutic activities. Driven by the need for unique androgen receptor (AR) antagonists that retain activity in hormone-refractory prostate cancer, we developed and applied a conformation-based screen to identify AR antagonists that were mechanistically distinct from existing drugs of this class. Two molecules were identified by using this approach, D36 and D80, which interact with AR in a unique manner and allosterically inhibit AR agonist activity. Unlike the clinically important antiandrogens, casodex and hydroxyflutamide, both D36 and D80 block androgen action in cellular models of hormone-refractory prostate cancer. Mechanistically, these compounds further distinguish themselves from classical AR antagonists in that they do not promote AR nuclear translocation and quantitatively inhibit the association of AR with DNA even under conditions of overexpression. Although the therapeutic potential of these antiandrogens is apparent, it is the demonstration that it is possible, to modulate the interaction of cofactors with agonist-activated AR, using second-site modulators, that has the greatest potential with respect to the therapeutic exploitation of AR and other NRs.

Authors
Joseph, JD; Wittmann, BM; Dwyer, MA; Cui, H; Dye, DA; McDonnell, DP; Norris, JD
MLA Citation
Joseph, James D., et al. “Inhibition of prostate cancer cell growth by second-site androgen receptor antagonists..” Proc Natl Acad Sci U S A, vol. 106, no. 29, July 2009, pp. 12178–83. Pubmed, doi:10.1073/pnas.0900185106.
PMID
19574450
Source
pubmed
Published In
Proc Natl Acad Sci U S A
Volume
106
Issue
29
Publish Date
2009
Start Page
12178
End Page
12183
DOI
10.1073/pnas.0900185106

Characterization of antiestrogenic activity of the Chinese herb, Prunella vulgaris, using in vitro and in vivo (mouse xenograft) models (Biology of Reproduction 2009, 80, (375-383) DOI: 10.1095/biolreprod.107.065375)

Authors
Collins, NH; Lessey, EC; DuSell, CD; McDonnell, DP; Fowler, L; Palomino, WA; Illera, MJ; Yu, X; Mo, B; Houwing, AM; Lessey, BA
MLA Citation
Collins, N. H., et al. “Characterization of antiestrogenic activity of the Chinese herb, Prunella vulgaris, using in vitro and in vivo (mouse xenograft) models (Biology of Reproduction 2009, 80, (375-383) DOI: 10.1095/biolreprod.107.065375).” Biology of Reproduction, vol. 80, no. 6, June 2009. Scopus, doi:10.1095/biolreprod.109.078303.
Source
scopus
Published In
Biology of Reproduction
Volume
80
Issue
6
Publish Date
2009
Start Page
1306
DOI
10.1095/biolreprod.109.078303

Purification and characterization of recombinant human mineralocorticoid receptor.

The mineralocorticoid receptor (MR) plays a critical role in the maintenance of electrolyte homeostasis and blood pressure via direct effects on the distal nephron and the cardiovascular system. The MR also has an important role in the pathology of cardiovascular disease, particularly heart failure, and is therefore an attractive therapeutic target. However, renal side effects limit its use in the clinic. Previous studies of MR molecular pharmacology have been performed on its isolated ligand-binding domain (LBD); however, current evidence suggests that nuclear receptor LBDs behave differently in isolation, than in the context of the full-length receptor. To date, technical issues have precluded production of full-length MR, thereby preventing molecular and structural studies of the MR LBD in its natural context. Here, we describe expression and purification of full-length human MR (hMR). hMR was expressed in Sf9 insect cells with an N-terminal biotinylated (bt)-tag, and stabilised by addition of ligand. bt-hMR exhibited ligand-binding and transactivation properties similar to that of the native protein. Affinity purification using an avidin matrix yielded approximately 120mug MR protein from 0.5lt Sf9 culture, and the receptor was purified bound to either aldosterone or cortisol. Recombinant hMR had a molecular weight of 110-130kDa, bound an MR DNA response element in vitro and interacted with a known co-regulator, PGC-1alpha, in GST pull-down assays, indicating its functional activity. Availability of this reagent will now enable analysis of MR structure and ligand interactions in the context of the full-length receptor, a prerequisite for future development of ligand-selective MR antagonists for the treatment of cardiovascular disease.

Authors
Clyne, CD; Chang, C-Y; Safi, R; Fuller, PJ; McDonnell, DP; Young, MJ
MLA Citation
Clyne, Colin D., et al. “Purification and characterization of recombinant human mineralocorticoid receptor..” Mol Cell Endocrinol, vol. 302, no. 1, Apr. 2009, pp. 81–85. Pubmed, doi:10.1016/j.mce.2008.11.030.
PMID
19114086
Source
pubmed
Published In
Molecular and Cellular Endocrinology
Volume
302
Issue
1
Publish Date
2009
Start Page
81
End Page
85
DOI
10.1016/j.mce.2008.11.030

Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands.

The pharmacological activity of different nuclear receptor ligands is reflected by their impact on receptor structure. Thus, we asked whether differential presentation of protein-protein interaction surfaces on the androgen receptor (AR), a surrogate assay of receptor conformation, could be used in a prospective manner to define the pharmacological activity of bound ligands. To this end, we identified over 150 proteins/polypeptides whose ability to interact with AR is influenced in a differential manner by ligand binding. The most discriminatory of these protein-AR interactions were used to develop a robust compound-profiling tool that enabled the separation of ligands into functionally distinguishable classes. Importantly, the ligands within each class exhibited similar pharmacological activities, a result that highlights the relationship between receptor structure and activity and provides direction for the discovery of novel AR modulators.

Authors
Norris, JD; Joseph, JD; Sherk, AB; Juzumiene, D; Turnbull, PS; Rafferty, SW; Cui, H; Anderson, E; Fan, D; Dye, DA; Deng, X; Kazmin, D; Chang, C-Y; Willson, TM; McDonnell, DP
MLA Citation
Norris, John David, et al. “Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands..” Chemistry & Biology, vol. 16, no. 4, Apr. 2009, pp. 452–60. Epmc, doi:10.1016/j.chembiol.2009.01.016.
PMID
19389631
Source
epmc
Published In
Chemistry & Biology
Volume
16
Issue
4
Publish Date
2009
Start Page
452
End Page
460
DOI
10.1016/j.chembiol.2009.01.016

Estrogen-related receptor alpha induces the expression of vascular endothelial growth factor in breast cancer cells.

Estrogen-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor family of transcription factors. In addition to its function as a metabolic regulator, ERRalpha has been implicated in the growth and progression of several malignancies. In the setting of breast cancer, not only is ERRalpha a putative negative prognostic factor, but we have recently found that knock-down of its expression retards tumor growth in a xenograft model of this disease. The specific aspects of ERRalpha function that are responsible for its actions in breast cancer, however, remain unclear. Using the coactivator PGC-1alpha as a protein ligand to regulate ERRalpha activity, we analyzed the effects of this receptor on gene expression in the ERalpha-positive MCF-7 cell line. This analysis led to the identification of a large number of potential ERRalpha target genes, many of which were subsequently validated in other breast cancer cell lines. Importantly, we demonstrate in this study that activation of ERRalpha in several different breast cancer cell lines leads to a significant increase in VEGF mRNA expression, an activity that translates into an increase in VEGF protein secretion. The induction of VEGF results from the interaction of ERRalpha with specific ERR-responsive elements within the VEGF promoter. These findings suggest that ERRalpha-dependent induction of VEGF may contribute to the overall negative phenotype observed in tumors in which ERRalpha is expressed and provide validation for its use as a therapeutic target in cancer.

Authors
Stein, RA; Gaillard, S; McDonnell, DP
MLA Citation
Stein, Rebecca A., et al. “Estrogen-related receptor alpha induces the expression of vascular endothelial growth factor in breast cancer cells..” J Steroid Biochem Mol Biol, vol. 114, no. 1–2, Mar. 2009, pp. 106–12. Pubmed, doi:10.1016/j.jsbmb.2009.02.010.
PMID
19429439
Source
pubmed
Published In
J Steroid Biochem Mol Biol
Volume
114
Issue
1-2
Publish Date
2009
Start Page
106
End Page
112
DOI
10.1016/j.jsbmb.2009.02.010

Glucose metabolism as a target of histone deacetylase inhibitors.

The therapeutic efficacy of histone deacetylase inhibitors (HDACI) is generally attributed to their ability to alter gene expression secondary to their effects on the acetylation status of transcription factors and histones. However, because HDACIs exhibit similar transcriptional effects in most cells, the molecular basis for their therapeutic selectivity toward malignant cells is largely unknown. In this study, we report that HDACI, of distinct chemotypes, quantitatively inhibit glucose transporter 1 (GLUT1)-mediated glucose transport into multiple myeloma cells through both down-regulation of GLUT1 and inhibition of hexokinase 1 (HXK1) enzymatic activity. Unexpectedly, however, this inhibition of glucose utilization is accompanied by an increase in amino acid catabolism with no increase in fatty acid oxidation. Our findings suggest that an HDACI-induced change in carbon source preference could contribute to the therapeutic efficacy of these drugs by creating a pattern of fuel utilization that is incompatible with rapid tumor growth and survival. Furthermore, these results, which implicate glucose metabolism as a target of HDACI, suggest that caution should be exercised in attributing effects of this class of drug to primary alterations in gene transcription.

Authors
Wardell, SE; Ilkayeva, OR; Wieman, HL; Frigo, DE; Rathmell, JC; Newgard, CB; McDonnell, DP
MLA Citation
Wardell, Suzanne E., et al. “Glucose metabolism as a target of histone deacetylase inhibitors..” Mol Endocrinol, vol. 23, no. 3, Mar. 2009, pp. 388–401. Pubmed, doi:10.1210/me.2008-0179.
PMID
19106193
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
23
Issue
3
Publish Date
2009
Start Page
388
End Page
401
DOI
10.1210/me.2008-0179

Pharmacological manipulation of the RAR/RXR signaling pathway maintains the repopulating capacity of hematopoietic stem cells in culture.

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However, RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study, we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506, a selective RXR modulator, inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters, suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly, using combinatorial peptide phage display, we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation, and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture.

Authors
Safi, R; Muramoto, GG; Salter, AB; Meadows, S; Himburg, H; Russell, L; Daher, P; Doan, P; Leibowitz, MD; Chao, NJ; McDonnell, DP; Chute, JP
MLA Citation
Safi, Rachid, et al. “Pharmacological manipulation of the RAR/RXR signaling pathway maintains the repopulating capacity of hematopoietic stem cells in culture..” Mol Endocrinol, vol. 23, no. 2, Feb. 2009, pp. 188–201. Pubmed, doi:10.1210/me.2008-0121.
PMID
19106195
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
23
Issue
2
Publish Date
2009
Start Page
188
End Page
201
DOI
10.1210/me.2008-0121

Characterization of antiestrogenic activity of the Chinese herb, prunella vulgaris, using in vitro and in vivo (Mouse Xenograft) models.

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182,780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner--responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

Authors
Collins, NH; Lessey, EC; DuSell, CD; McDonnell, DP; Fowler, L; Palomino, WA; Illera, MJ; Yu, X; Mo, B; Houwing, AM; Lessey, BA
MLA Citation
Collins, Nancy H., et al. “Characterization of antiestrogenic activity of the Chinese herb, prunella vulgaris, using in vitro and in vivo (Mouse Xenograft) models..” Biol Reprod, vol. 80, no. 2, Feb. 2009, pp. 375–83. Pubmed, doi:10.1095/biolreprod.107.065375.
PMID
18923163
Source
pubmed
Published In
Biology of Reproduction
Volume
80
Issue
2
Publish Date
2009
Start Page
375
End Page
383
DOI
10.1095/biolreprod.107.065375

Estrogen-related receptor alpha is critical for the growth of estrogen receptor-negative breast cancer.

Expression of estrogen-related receptor alpha (ERRalpha) has recently been shown to carry negative prognostic significance in breast and ovarian cancers. The specific role of this orphan nuclear receptor in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor alpha (ERalpha) and ERRalpha initially suggested that these receptors may have similar transcriptional targets. Using the well-characterized ERalpha-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ERalpha-ERRalpha cross-talk using an unbiased microarray approach. In addition to generating a host of novel ERRalpha target genes, this study yielded the surprising result that most ERRalpha-regulated genes are unrelated to estrogen signaling. The relatively small number of genes regulated by both ERalpha and ERRalpha led us to expand our study to the more aggressive and less clinically treatable ERalpha-negative class of breast cancers. In this setting, we found that ERRalpha expression is required for the basal level of expression of many known and novel ERRalpha target genes. Introduction of a small interfering RNA directed to ERRalpha into the highly aggressive breast carcinoma MDA-MB-231 cell line dramatically reduced the migratory potential of these cells. Although stable knockdown of ERRalpha expression in MDA-MB-231 cells had no effect on in vitro cell proliferation, a significant reduction of tumor growth rate was observed when these cells were implanted as xenografts. Our results confirm a role for ERRalpha in breast cancer growth and highlight it as a potential therapeutic target for estrogen receptor-negative breast cancer.

Authors
Stein, RA; Chang, C-Y; Kazmin, DA; Way, J; Schroeder, T; Wergin, M; Dewhirst, MW; McDonnell, DP
MLA Citation
Stein, Rebecca A., et al. “Estrogen-related receptor alpha is critical for the growth of estrogen receptor-negative breast cancer..” Cancer Res, vol. 68, no. 21, Nov. 2008, pp. 8805–12. Pubmed, doi:10.1158/0008-5472.CAN-08-1594.
PMID
18974123
Source
pubmed
Published In
Cancer Res
Volume
68
Issue
21
Publish Date
2008
Start Page
8805
End Page
8812
DOI
10.1158/0008-5472.CAN-08-1594

Definition of Functionally Important Mechanistic Differences among Selective Estrogen Receptor Modulators

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “Definition of Functionally Important Mechanistic Differences among Selective Estrogen Receptor Modulators.” Menopause the Journal of the North American Menopause Society, vol. 15, no. 6, LIPPINCOTT WILLIAMS & WILKINS, 2008, pp. 1199–1199.
Source
wos
Published In
Menopause (New York, N.Y.)
Volume
15
Issue
6
Publish Date
2008
Start Page
1199
End Page
1199

27-Hydroxycholesterol: a potential endogenous regulator of estrogen receptor signaling.

The selective estrogen receptor modulators (SERMs) are synthetic pharmaceuticals, the relative agonist and antagonist activities of which are not equivalent in all cells. Their discovery has raised the possibility that endogenous small molecules might exist that have similar properties and could have important physiological roles. In support of this hypothesis is the recent demonstration that the oxysterol 27-hydroxycholesterol (27HC) interacts with and modulates the transcriptional activity of both estrogen receptor (ER) subtypes and that the relative agonist and antagonist activity of 27HC is influenced by both cell and promoter context. Although there is limited information available on the role of 27HC in classical estrogen-responsive tissues, that which is available in animal models of cardiovascular disease and cellular models of breast cancer support a role for this ligand in ER signaling. These results provide an interesting potential link between cholesterol (and cholesterol metabolism) and ER function, the physiological and pathological importance of which remains to be determined.

Authors
DuSell, CD; McDonnell, DP
MLA Citation
DuSell, Carolyn D., and Donald P. McDonnell. “27-Hydroxycholesterol: a potential endogenous regulator of estrogen receptor signaling..” Trends Pharmacol Sci, vol. 29, no. 10, Oct. 2008, pp. 510–14. Pubmed, doi:10.1016/j.tips.2008.07.003.
PMID
18722677
Source
pubmed
Published In
Trends in Pharmacological Sciences
Volume
29
Issue
10
Publish Date
2008
Start Page
510
End Page
514
DOI
10.1016/j.tips.2008.07.003

Development of a small-molecule serum- and glucocorticoid-regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic.

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference-mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer.

Authors
Sherk, AB; Frigo, DE; Schnackenberg, CG; Bray, JD; Laping, NJ; Trizna, W; Hammond, M; Patterson, JR; Thompson, SK; Kazmin, D; Norris, JD; McDonnell, DP
MLA Citation
Sherk, Andrea B., et al. “Development of a small-molecule serum- and glucocorticoid-regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic..” Cancer Res, vol. 68, no. 18, Sept. 2008, pp. 7475–83. Pubmed, doi:10.1158/0008-5472.CAN-08-1047.
PMID
18794135
Source
pubmed
Published In
Cancer Res
Volume
68
Issue
18
Publish Date
2008
Start Page
7475
End Page
7483
DOI
10.1158/0008-5472.CAN-08-1047

Differential effects of prostate cancer therapeutics on neuroendocrine transdifferentiation.

Androgen ablation therapy is widely used for the treatment of advanced prostate cancer. However, the effectiveness of this intervention strategy is generally short-lived as the disease ultimately progresses to a hormone-refractory state. In recent years, it has become clear that even in antiandrogen-resistant cancers the androgen receptor (AR) signaling axis is intact and is required for prostate cancer growth. Thus, there is a heightened interest in developing small molecules that function in part by down-regulating AR expression in tumors. Paradoxically, AR expression has been shown to be important in preventing the transdifferentiation of epithelial prostate cancer cells toward a neuroendocrine phenotype associated with tumor progression. Consequently, we have evaluated the relative effect of prostate cancer therapeutics that function in part by depleting AR levels on neuroendocrine differentiation in established cellular models of prostate cancer. These studies reveal that although histone deacetylase inhibitors can down-regulate AR expression they increase the expression of neuroendocrine markers and alter cellular morphology. Inhibition of AR signaling using classic AR antagonists or small interfering RNA-mediated AR ablation induces incomplete neuroendocrine differentiation. Importantly, the Hsp90 inhibitor geldanamycin effectively down-regulates AR expression while having no effect on neuroendocrine differentiation. Taken together, these data show that the phenotypic responses to pharmacologic agents used in the clinic to prevent the progression of prostate cancer are not equivalent, a finding of significant therapeutic importance.

Authors
Frigo, DE; McDonnell, DP
MLA Citation
Frigo, Daniel E., and Donald P. McDonnell. “Differential effects of prostate cancer therapeutics on neuroendocrine transdifferentiation..” Mol Cancer Ther, vol. 7, no. 3, Mar. 2008, pp. 659–69. Pubmed, doi:10.1158/1535-7163.MCT-07-0480.
PMID
18347151
Source
pubmed
Published In
Molecular Cancer Therapeutics
Volume
7
Issue
3
Publish Date
2008
Start Page
659
End Page
669
DOI
10.1158/1535-7163.MCT-07-0480

Nuclear receptor cofactor interactions as targets for new drug discovery

© 2008 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. The classical models of nuclear receptor pharmacology held that agonists functioned by binding to their cognate receptors facilitating their conversion from an inactive form to one that was capable of activating transcription. By extrapolation, it was considered that antagonists functioned by competitively inhibiting agonist binding, freezing the receptor in an inactive state. However, as early as 1967 when the biological actions of the “antiestrogen” tamoxifen were first described, it was clear that this simple model did not adequately describe estrogen receptor pharmacology. Indeed, tamoxifen is now classified as a Selective Estrogen Receptor Modulator (SERM), one of a group of compounds whose relative agonist/antagonist activity differs between cells. Similarly, tissue selective progesterone, androgen and glucococorticoid receptor modulators have also been identified. Significant progress has been made in defining the molecular mechanism(s) by which cells distinguish between agonists and antagonists and how some receptor modulators can manifest their actions in a cell-selective manner. The most important of these are (1) differences in the relative expression level of receptor isoforms or subtypes, (2) the impact which the bound ligand has on the structure of its cognate receptor, and (3) the complement of coactivators and corepressors in a target cell which can interact with the activated receptor. Exploitation of this complexity will lead to the development of novel classes of nuclear receptor modulators with useful therapeutic activities.

Authors
Grasfeder, LL; McDonnell, DP
MLA Citation
Grasfeder, L. L., and D. P. McDonnell. “Nuclear receptor cofactor interactions as targets for new drug discovery.” NR Coregulators and Human Diseases, 2008, pp. 572–99. Scopus, doi:10.1142/9789812819178_0019.
Source
scopus
Publish Date
2008
Start Page
572
End Page
599
DOI
10.1142/9789812819178_0019

The commercialism of academic research

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “The commercialism of academic research.” Nature Medicine, vol. 14, no. 10, Jan. 2008, pp. 1014–1014. Scopus, doi:10.1038/nm1008-1014.
Source
scopus
Published In
Nature Medicine
Volume
14
Issue
10
Publish Date
2008
Start Page
1014
End Page
1014
DOI
10.1038/nm1008-1014

27-hydroxycholesterol is an endogenous selective estrogen receptor modulator.

Selective estrogen receptor (ER) modulators (SERMs) are ER ligands whose relative agonist/antagonist activities vary in a cell- and promoter-dependent manner. The molecular basis underlying this selectivity can be attributed to the ability of these ligands to induce distinct alterations in ER structure leading to differential recruitment of coactivators and corepressors. Whether SERM activity is restricted to synthetic ligands or whether molecules exist in vivo that function in an analogous manner remains unresolved. However, the recent observation that oxysterols bind ER and antagonize the actions of 17beta-estradiol (E2) on the vascular wall suggests that this class of ligands may possess SERM activity. We demonstrate here that 27-hydroxycholesterol (27HC), the most prevalent oxysterol in circulation, functions as a SERM, the efficacy of which varies when assessed on different endpoints. Importantly, 27HC positively regulates both gene transcription and cell proliferation in cellular models of breast cancer. Using combinatorial peptide phage display, we have determined that 27HC induces a unique conformational change in both ERalpha and ERbeta, distinguishing it from E2 and other SERMs. Thus, as with other ER ligands, it appears that the unique pharmacological activity of 27HC relates to its ability to impact ER structure and modulate cofactor recruitment. Cumulatively, these data indicate that 27HC is an endogenous SERM with partial agonist activity in breast cancer cells and suggest that it may influence the pathology of breast cancer. Moreover, given the product-precursor relationship between 27HC and cholesterol, our findings have implications with respect to breast cancer risk in obese/hypercholesteremic individuals.

Authors
DuSell, CD; Umetani, M; Shaul, PW; Mangelsdorf, DJ; McDonnell, DP
MLA Citation
DuSell, Carolyn D., et al. “27-hydroxycholesterol is an endogenous selective estrogen receptor modulator..” Mol Endocrinol, vol. 22, no. 1, Jan. 2008, pp. 65–77. Pubmed, doi:10.1210/me.2007-0383.
PMID
17872378
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
22
Issue
1
Publish Date
2008
Start Page
65
End Page
77
DOI
10.1210/me.2007-0383

On the intractability of estrogen-related receptor alpha as a target for activation by small molecules.

The estrogen-related receptor alpha (ERRalpha) is a potential target for activation in the treatment of metabolic disease. To date, no small-molecule agonists of ERRalpha have been identified despite several high-throughput screening campaigns. We describe the synthesis and profiling of a small array of compounds designed on the basis of a previously reported agonist-bound crystal structure of the closely related receptor ERRgamma. The results suggest that ERRalpha may be intractable as a direct target for pharmacologic activation.

Authors
Hyatt, SM; Lockamy, EL; Stein, RA; McDonnell, DP; Miller, AB; Orband-Miller, LA; Willson, TM; Zuercher, WJ
MLA Citation
Hyatt, Stephen M., et al. “On the intractability of estrogen-related receptor alpha as a target for activation by small molecules..” J Med Chem, vol. 50, no. 26, Dec. 2007, pp. 6722–24. Pubmed, doi:10.1021/jm7012387.
PMID
18052088
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
50
Issue
26
Publish Date
2007
Start Page
6722
End Page
6724
DOI
10.1021/jm7012387

Molecular pharmacology of estrogen and progesterone receptors

This chapter provides an update on the mechanisms of action of estrogen and progesterone. Particularly important concepts include nongenomic effects, receptor isoforms that affect agonist/antagonist actions, and the role of coactivators and corepressors in various tissues. The steroid hormones estrogen and progesterone are low-molecular weight, lipophilic hormones that through their action as modulators of distinct signal transduction pathways, are involved in the regulation of reproductive function. These hormones are also important regulators in bone, the cardiovascular system, and the central nervous system. Despite their different roles in these systems, it has become apparent that estrogens and progestins are mechanistically similar. Insights gleaned from the study of each hormone have advanced the understanding of this class of molecules as a whole. Some of the recent mechanistic discoveries that have occurred in the field are highlighted and the subsequent changes in our understanding of the pharmacology of this class of steroid hormones are explored. © 2007 Elsevier Inc. All rights reserved.

Authors
Sherk, AB; McDonnell, DP
MLA Citation
Sherk, A. B., and D. P. McDonnell. Molecular pharmacology of estrogen and progesterone receptors. Dec. 2007, pp. 17–28. Scopus, doi:10.1016/B978-012369443-0/50004-1.
Source
scopus
Publish Date
2007
Start Page
17
End Page
28
DOI
10.1016/B978-012369443-0/50004-1

Definition of functionally important mechanistic differences among selective estrogen receptor down-regulators.

One subclass of antiestrogens, the selective estrogen receptor down-regulators (SERDs), have received considerable attention of late as they competitively inhibit estrogen binding and induce a rapid, proteasome-dependent degradation of the receptor. Contained within this class of molecules is the steroidal antiestrogen ICI182,780 (faslodex), recently approved for the treatment of metastatic cancer, and GW5638/DPC974, a SERD that is currently being evaluated in the clinic. Given that mechanistic differences between different selective estrogen receptor modulators have been translated into important clinical profiles, it was of interest to determine if the SERD subclass of ligands were likewise functionally or mechanistically distinguishable. In this study, we show that although the steroidal and nonsteroidal SERDs target ERalpha for degradation, the underlying mechanism(s) are different. Of note was the identification of a specific protein-protein interaction surface presented on ERalpha in the presence of the ICI182,780-activated receptor which is required for degradation. Interestingly, this surface is also presented on ERalpha in the presence of RU58,668, a SERD that is chemically distinct from ICI182,780. This surface is not required for GW5638-mediated degradation, and thus, this SERD seems to affect ERalpha down-regulation by a different mechanism. These data suggest that sequencing of therapies using drugs of this class is likely to be possible. Finally, because of the unmet need for orally active SERDS that function similarly to ICI182,780, we have used the insights from these mechanistic studies to develop and validate a high-throughput screen for compounds of this class with improved pharmaceutical properties.

Authors
Wittmann, BM; Sherk, A; McDonnell, DP
MLA Citation
Wittmann, Bryan M., et al. “Definition of functionally important mechanistic differences among selective estrogen receptor down-regulators..” Cancer Res, vol. 67, no. 19, Oct. 2007, pp. 9549–60. Pubmed, doi:10.1158/0008-5472.CAN-07-1590.
PMID
17909066
Source
pubmed
Published In
Cancer Research
Volume
67
Issue
19
Publish Date
2007
Start Page
9549
End Page
9560
DOI
10.1158/0008-5472.CAN-07-1590

The nuclear receptor-coactivator interaction surface as a target for peptide antagonists of the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARalpha, PPARdelta, and PPARgamma) constitute a family of nuclear receptors that regulates metabolic processes involved in lipid and glucose homeostasis. Although generally considered to function as ligand-regulated receptors, all three PPARs exhibit a high level of constitutive activity that may result from their stimulation by intracellularly produced endogenous ligands. Consequently, complete inhibition of PPAR signaling requires the development of inverse agonists. However, the currently available small molecule antagonists for the PPARs function only as partial agonists, or their efficacy is not sufficient to inhibit the constitutive activity of these receptors. Due to the lack of efficacious antagonists that interact with the ligand-binding domain of the PPARs, we decided to target an interaction that is central to nuclear receptor-mediated gene transcription: the nuclear receptor-coactivator interaction. We utilized phage display technology to identify short LXXLL-containing peptides that bind to the PPARs. Analysis of these peptides revealed a consensus binding motif consisting of HPLLXXLL. Cross-screening of these peptides for binding to other nuclear receptors enabled the identification of a high-affinity PPAR-selective peptide that has the ability to repress PPARgamma1-dependent transcription of transfected reporter genes. Most importantly, when introduced into HepG2 cells, the peptide inhibited the expression of endogenous PPARgamma1 target genes, adipose differentiation-related protein and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase 2. This work lends support for the rational development of peptidomimetics that block receptor-mediated transcription by targeting the nuclear receptor-coactivator interaction surface.

Authors
Mettu, NB; Stanley, TB; Dwyer, MA; Jansen, MS; Allen, JE; Hall, JM; McDonnell, DP
MLA Citation
Mettu, Niharika B., et al. “The nuclear receptor-coactivator interaction surface as a target for peptide antagonists of the peroxisome proliferator-activated receptors..” Mol Endocrinol, vol. 21, no. 10, Oct. 2007, pp. 2361–77. Pubmed, doi:10.1210/me.2007-0201.
PMID
17595321
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
21
Issue
10
Publish Date
2007
Start Page
2361
End Page
2377
DOI
10.1210/me.2007-0201

The molecular mechanisms underlying the proinflammatory actions of thiazolidinediones in human macrophages.

It is hypothesized that the antiinflammatory actions of peroxisome proliferator-activated receptors (PPARs) may explain the protective effect of these receptors in diabetes, atherosclerosis, cancer, and other inflammatory diseases. However, emerging evidence for proinflammatory activities of activated PPARs is concerning in light of new studies that associate PPAR modulators with an increased incidence of both cardiovascular events in humans and the sporadic formation of tumors in rodents. In an attempt to define the role of each PPAR subtype in inflammation, we made the unexpected observation that human PPARdelta is a positive regulator of inflammatory responses in both monocytes and macrophages. Notably, TNFalpha-stimulated cells administered PPARdelta agonists express and secrete elevated levels of inflammatory cytokines. Most surprising, however, was the finding that thiazolidinediones (TZDs) and other known PPARgamma ligands display different degrees of proinflammatory activities in a PPARgamma- and PPARalpha-independent manner via their ability to augment PPARdelta signaling. A series of mechanistic studies revealed that TZDs, at clinically relevant concentrations, bind and activate the transcriptional activity of PPARdelta. Collectively, these studies suggest that the observed proinflammatory and potentially deleterious effects of PPARgamma ligands may be mediated through an off-target effect on PPARdelta. These studies highlight the need for PPAR modulators with increased receptor subtype specificity. Furthermore, they suggest that differences in systemic exposure and consequently in the activation of PPARgamma and PPARdelta may explain why TZDs can exhibit both inflammatory and antiinflammatory activities in humans.

Authors
Hall, JM; McDonnell, DP
MLA Citation
Hall, Julie M., and Donald P. McDonnell. “The molecular mechanisms underlying the proinflammatory actions of thiazolidinediones in human macrophages..” Mol Endocrinol, vol. 21, no. 8, Aug. 2007, pp. 1756–68. Pubmed, doi:10.1210/me.2007-0060.
PMID
17488971
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
21
Issue
8
Publish Date
2007
Start Page
1756
End Page
1768
DOI
10.1210/me.2007-0060

The vitamin D receptor interacts preferentially with DRIP205-like LxxLL motifs.

The vitamin D receptor (VDR) mediates the biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) through its capacity to recruit coregulatory proteins. This interaction is mediated via a coregulatory LxxLL motif. We screened a combinatorial (x)7LxxLL(x)7 phage library with purified VDR to identify peptides that displayed high affinity and selectivity for VDR. These peptides contained the consensus sequence Lx E/H x H/F P L/M/I LxxLL and exhibited significant sequence similarity to the active LxxLL box found in DRIP205. Nearly all LxxLL peptides interacted in a ligand-dependent manner directly with human VDR. However, a pattern of selectivity of the peptides for other members of the nuclear receptor family was also observed. Interestingly, the interaction between the VDR and many of the peptides was differentially sensitive to a broad assortment of VDR ligands. Finally, several of these peptides were shown to inhibit activation of a 1,25(OH)2D3-sensitive reporter gene. These studies suggest that the LxxLL motif can interact directly with the VDR and that this interaction is regulated by chemically diverse vitamin D ligands.

Authors
Zella, LA; Chang, C-Y; McDonnell, DP; Pike, JW
MLA Citation
Zella, Lee A., et al. “The vitamin D receptor interacts preferentially with DRIP205-like LxxLL motifs..” Arch Biochem Biophys, vol. 460, no. 2, Apr. 2007, pp. 206–12. Pubmed, doi:10.1016/j.abb.2006.12.016.
PMID
17254542
Source
pubmed
Published In
Archives of Biochemistry and Biophysics
Volume
460
Issue
2
Publish Date
2007
Start Page
206
End Page
212
DOI
10.1016/j.abb.2006.12.016

Structural insights into corepressor recognition by antagonist-bound estrogen receptors.

Direct recruitment of transcriptional corepressors to estrogen receptors (ER) is thought to contribute to the tissue-specific effects of clinically important ER antagonists. Here, we present the crystal structures of two affinity-selected peptides in complex with antagonist-bound ERalpha ligand-binding domain. Both peptides adopt helical conformations, bind along the activation function 2 coregulator interaction surface, and mimic corepressor (CoRNR) sequence motif binding. Peptide binding is weak in a wild-type context but significantly enhanced by removal of ER helix 12. This region contains a previously unrecognized CoRNR motif that is able to compete with corepressors for binding to activation function 2, thereby providing a structural explanation for the poor ability of ER to directly interact with classical corepressors. Furthermore, the ability of other sequence motifs to mimic corepressor binding raises the possibility that coregulators do not necessarily require CoRNR motifs for direct recruitment to antagonist-bound ER.

Authors
Heldring, N; Pawson, T; McDonnell, D; Treuter, E; Gustafsson, J-A; Pike, ACW
MLA Citation
Heldring, Nina, et al. “Structural insights into corepressor recognition by antagonist-bound estrogen receptors..” J Biol Chem, vol. 282, no. 14, Apr. 2007, pp. 10449–55. Pubmed, doi:10.1074/jbc.M611424200.
PMID
17283072
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
282
Issue
14
Publish Date
2007
Start Page
10449
End Page
10455
DOI
10.1074/jbc.M611424200

Definition of the molecular basis for estrogen receptor-related receptor-alpha-cofactor interactions.

Estrogen receptor-related receptor-alpha (ERRalpha) is an orphan nuclear receptor that does not appear to require a classical small molecule ligand to facilitate its interaction with coactivators and/or hormone response elements within target genes. Instead, the apo-receptor is capable of interacting in a constitutive manner with coactivators that stimulate transcription by acting as protein ligands. We have screened combinatorial phage libraries for peptides that selectively interact with ERRalpha to probe the architecture of the ERRalpha-coactivator pocket. In this manner, we have uncovered a fundamental difference in the mechanism by which this receptor interacts with peroxisome proliferator-activated receptor-gamma coactivator-1alpha, as compared with members of the steroid receptor coactivator subfamily of coactivators. Our findings suggest that it may be possible to develop ERRalpha ligands that exhibit different pharmacological activities as a consequence of their ability to differentially regulate coactivator recruitment. In addition, these findings have implications beyond ERRalpha because they suggest that subtle alterations in the structure of the activation function-2 pocket within any nuclear receptor may enable differential recruitment of coactivators, an observation of notable pharmaceutical importance.

Authors
Gaillard, S; Dwyer, MA; McDonnell, DP
MLA Citation
Gaillard, Stéphanie, et al. “Definition of the molecular basis for estrogen receptor-related receptor-alpha-cofactor interactions..” Mol Endocrinol, vol. 21, no. 1, Jan. 2007, pp. 62–76. Pubmed, doi:10.1210/me.2006-0179.
PMID
17053040
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
21
Issue
1
Publish Date
2007
Start Page
62
End Page
76
DOI
10.1210/me.2006-0179

Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression.

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression.

Authors
Zhang, Y; Ma, K; Sadana, P; Chowdhury, F; Gaillard, S; Wang, F; McDonnell, DP; Unterman, TG; Elam, MB; Park, EA
MLA Citation
Zhang, Yi, et al. “Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression..” J Biol Chem, vol. 281, no. 52, Dec. 2006, pp. 39897–906. Pubmed, doi:10.1074/jbc.M608657200.
PMID
17079227
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
281
Issue
52
Publish Date
2006
Start Page
39897
End Page
39906
DOI
10.1074/jbc.M608657200

Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs.

In the absence of specific high-affinity agonists and antagonists, it has been difficult to define the target genes and biological responses attributable to many of the orphan nuclear receptors (ONRs). Indeed, it appears that many members of this receptor superfamily are not regulated by classical small molecules but rather their activity is controlled by interacting cofactors. Motivated by this finding, we have developed an approach to genetically isolate specific receptor-cofactor pairs in cells, allowing us to define the biological responses attributable to each complex. This is accomplished by using combinatorial peptide phage display to engineer the receptor interacting domain of each cofactor such that it interacts selectively with one nuclear receptor. In this study, we describe the customization of PGC-1alpha and its use to study the biology of the estrogen-related receptor alpha (ERRalpha) in cultured liver cells.

Authors
Gaillard, S; Grasfeder, LL; Haeffele, CL; Lobenhofer, EK; Chu, T-M; Wolfinger, R; Kazmin, D; Koves, TR; Muoio, DM; Chang, C-Y; McDonnell, DP
MLA Citation
Gaillard, Stéphanie, et al. “Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs..” Mol Cell, vol. 24, no. 5, Dec. 2006, pp. 797–803. Pubmed, doi:10.1016/j.molcel.2006.10.012.
PMID
17157261
Source
pubmed
Published In
Molecular Cell
Volume
24
Issue
5
Publish Date
2006
Start Page
797
End Page
803
DOI
10.1016/j.molcel.2006.10.012

International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors.

Authors
Lu, NZ; Wardell, SE; Burnstein, KL; Defranco, D; Fuller, PJ; Giguere, V; Hochberg, RB; McKay, L; Renoir, J-M; Weigel, NL; Wilson, EM; McDonnell, DP; Cidlowski, JA
MLA Citation
Lu, Nick Z., et al. “International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors..” Pharmacol Rev, vol. 58, no. 4, Dec. 2006, pp. 782–97. Pubmed, doi:10.1124/pr.58.4.9.
PMID
17132855
Source
pubmed
Published In
Pharmacological Reviews
Volume
58
Issue
4
Publish Date
2006
Start Page
782
End Page
797
DOI
10.1124/pr.58.4.9

Estrogen-related receptor alpha as a therapeutic target in cancer.

The orphan receptor estrogen-related receptor alpha (ERR alpha) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. This protein is structurally most related to the canonical estrogen receptor and has been shown to modulate estrogen signaling in some contexts. These observations have heightened interest in ERR alpha as a therapeutic target in both breast and ovarian cancer and in other estrogenopathies. This review details our present understanding of ERR alpha action with a view to highlight specific aspects of its signal-transduction pathway in breast cancer that may be amenable to pharmaceutical manipulation.

Authors
Stein, RA; McDonnell, DP
MLA Citation
Stein, R. A., and D. P. McDonnell. “Estrogen-related receptor alpha as a therapeutic target in cancer..” Endocr Relat Cancer, vol. 13 Suppl 1, Dec. 2006, pp. S25–32. Pubmed, doi:10.1677/erc.1.01292.
PMID
17259555
Source
pubmed
Published In
Endocrine Related Cancer
Volume
13 Suppl 1
Publish Date
2006
Start Page
S25
End Page
S32
DOI
10.1677/erc.1.01292

Mechanism-based discovery as an approach to identify the next generation of estrogen receptor modulators.

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “Mechanism-based discovery as an approach to identify the next generation of estrogen receptor modulators..” Faseb J, vol. 20, no. 14, Dec. 2006, pp. 2432–34. Pubmed, doi:10.1096/fj.06-1202ufm.
PMID
17142791
Source
pubmed
Published In
Faseb Journal : Official Publication of the Federation of American Societies for Experimental Biology
Volume
20
Issue
14
Publish Date
2006
Start Page
2432
End Page
2434
DOI
10.1096/fj.06-1202ufm

The use of conformational peptide probes to aid in the identification of novel PPAR selective modulators

Authors
Mettu, NB; Jansen, MS; Dwyer, MA; Hall, JM; Stanley, TB; McDonnell, DP
MLA Citation
Mettu, N. B., et al. “The use of conformational peptide probes to aid in the identification of novel PPAR selective modulators.” Inflammation Research, vol. 55, BIRKHAUSER VERLAG AG, Oct. 2006, pp. S126–27.
Source
wos
Published In
Inflammation Research : Official Journal of the European Histamine Research Society ... [Et Al.]
Volume
55
Publish Date
2006
Start Page
S126
End Page
S127

Research resource articles - A new feature for Molecular Endocrinology

Authors
Cidlowski, J; Birnbaumer, M; DeFranco, D; Hunzicker-Dunn, M; McDonnell, D; Pilch, P; Tsai, MJ; Haworth, M
MLA Citation
Cidlowski, J., et al. “Research resource articles - A new feature for Molecular Endocrinology.” Molecular Endocrinology, vol. 20, no. 9, Aug. 2006. Scopus, doi:10.1210/me.2006-8931.
Source
scopus
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
20
Issue
9
Publish Date
2006
Start Page
1971
DOI
10.1210/me.2006-8931

Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells.

Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.

Authors
Chute, JP; Muramoto, GG; Whitesides, J; Colvin, M; Safi, R; Chao, NJ; McDonnell, DP
MLA Citation
Chute, John P., et al. “Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells..” Proc Natl Acad Sci U S A, vol. 103, no. 31, Aug. 2006, pp. 11707–12. Pubmed, doi:10.1073/pnas.0603806103.
PMID
16857736
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
103
Issue
31
Publish Date
2006
Start Page
11707
End Page
11712
DOI
10.1073/pnas.0603806103

Human PXR forms a tryptophan zipper-mediated homodimer.

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal beta-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface beta-strands, generating a 10-stranded intermolecular beta-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR's ability to interact with DNA, RXRalpha, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor's recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor.

Authors
Noble, SM; Carnahan, VE; Moore, LB; Luntz, T; Wang, H; Ittoop, OR; Stimmel, JB; Davis-Searles, PR; Watkins, RE; Wisely, GB; LeCluyse, E; Tripathy, A; McDonnell, DP; Redinbo, MR
MLA Citation
Noble, Schroeder M., et al. “Human PXR forms a tryptophan zipper-mediated homodimer..” Biochemistry, vol. 45, no. 28, July 2006, pp. 8579–89. Pubmed, doi:10.1021/bi0602821.
PMID
16834332
Source
pubmed
Published In
Biochemistry
Volume
45
Issue
28
Publish Date
2006
Start Page
8579
End Page
8589
DOI
10.1021/bi0602821

Linking ligand-induced alterations in androgen receptor structure to differential gene expression: a first step in the rational design of selective androgen receptor modulators.

We have previously identified a family of novel androgen receptor (AR) ligands that, upon binding, enable AR to adopt structures distinct from that observed in the presence of canonical agonists. In this report, we describe the use of these compounds to establish a relationship between AR structure and biological activity with a view to defining a rational approach with which to identify useful selective AR modulators. To this end, we used combinatorial peptide phage display coupled with molecular dynamic structure analysis to identify the surfaces on AR that are exposed specifically in the presence of selected AR ligands. Subsequently, we used a DNA microarray analysis to demonstrate that differently conformed receptors facilitate distinct patterns of gene expression in LNCaP cells. Interestingly, we observed a complete overlap in the identity of genes expressed after treatment with mechanistically distinct AR ligands. However, it was differences in the kinetics of gene regulation that distinguished these compounds. Follow-up studies, in cell-based assays of AR action, confirmed the importance of these alterations in gene expression. Together, these studies demonstrate an important link between AR structure, gene expression, and biological outcome. This relationship provides a firm underpinning for mechanism-based screens aimed at identifying SARMs with useful clinical profiles.

Authors
Kazmin, D; Prytkova, T; Cook, CE; Wolfinger, R; Chu, T-M; Beratan, D; Norris, JD; Chang, C-Y; McDonnell, DP
MLA Citation
Kazmin, Dmitri, et al. “Linking ligand-induced alterations in androgen receptor structure to differential gene expression: a first step in the rational design of selective androgen receptor modulators..” Mol Endocrinol, vol. 20, no. 6, June 2006, pp. 1201–17. Pubmed, doi:10.1210/me.2005-0309.
PMID
16574741
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
20
Issue
6
Publish Date
2006
Start Page
1201
End Page
1217
DOI
10.1210/me.2005-0309

Estrogen-related receptor α as a therapeutic target

The utility of nuclear receptors (NRs) as targets for drug discovery is well recognized. Growing evidence suggests that ERRα, an orphan NR whose physiological and pathological roles remain under investigation, may have utility in the treatment of various cancer types as well as metabolic and osteopenic disorders. In this article, we review the literature on ERRα as a potential therapeutic target. Within this context we identify several key challenges in progressing small molecules which target ERRα, including the separation of efficacy and liability. Copyright © 2006 Prous Science.

Authors
Stein, R; Way, JM; McDonnell, DP; Zuercher, WJ
MLA Citation
Stein, R., et al. “Estrogen-related receptor α as a therapeutic target.” Drugs of the Future, vol. 31, no. 5, May 2006, pp. 427–36. Scopus, doi:10.1358/dof.2006.031.05.994706.
Source
scopus
Published In
Drugs of the Future
Volume
31
Issue
5
Publish Date
2006
Start Page
427
End Page
436
DOI
10.1358/dof.2006.031.05.994706

SARMS, SERMS, SGRM

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “SARMS, SERMS, SGRM.” Osteoporosis International, vol. 17, SPRINGER LONDON LTD, 2006, pp. S128–S128.
Source
wos
Published In
Osteoporosis International : a Journal Established as Result of Cooperation Between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the Usa
Volume
17
Publish Date
2006
Start Page
S128
End Page
S128

Structure-guided synthesis of tamoxifen analogs with improved selectivity for the orphan ERRgamma.

The design and synthesis of 4-hydroxytamoxifen (4-OHT) derivatives are described. The binding affinities of these compounds toward the orphan estrogen-related receptor gamma and the classical estrogen receptor alpha demonstrate that analogs bearing hydroxyalkyl groups display improved binding selectivity profiles compared with that of 4-OHT. An X-ray crystal structure of one of the designed compounds bound to ERRgamma LBD confirms the molecular basis of the selectivity.

Authors
Chao, EYH; Collins, JL; Gaillard, S; Miller, AB; Wang, L; Orband-Miller, LA; Nolte, RT; McDonnell, DP; Willson, TM; Zuercher, WJ
MLA Citation
Chao, Esther Y. H., et al. “Structure-guided synthesis of tamoxifen analogs with improved selectivity for the orphan ERRgamma..” Bioorg Med Chem Lett, vol. 16, no. 4, Feb. 2006, pp. 821–24. Pubmed, doi:10.1016/j.bmcl.2005.11.030.
PMID
16307879
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
16
Issue
4
Publish Date
2006
Start Page
821
End Page
824
DOI
10.1016/j.bmcl.2005.11.030

Crystal structure of the human LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity.

The DNA-binding and ligand-binding functions of nuclear receptors are localized to independent domains separated by a flexible hinge. The DNA-binding domain (DBD) of the human liver receptor homologue-1 (hLRH-1), which controls genes central to development and metabolic homeostasis, interacts with monomeric DNA response elements and contains an Ftz-F1 motif that is unique to the NR5A nuclear receptor subfamily. Here, we present the 2.2A resolution crystal structure of the hLRH-1 DBD in complex with duplex DNA, and elucidate the sequence-specific DNA contacts essential for the ability of LRH-1 to bind to DNA as a monomer. We show that the unique Ftz-F1 domain folds into a novel helix that packs against the DBD but does not contact DNA. Mutations expected to disrupt the positioning of the Ftz-F1 helix do not eliminate DNA binding but reduce the transcriptional activity of full-length LRH-1 significantly. Moreover, we find that altering the Ftz-F1 helix positioning eliminates the enhancement of LRH-1-mediated transcription by the coactivator GRIP1, an action that is associated primarily with the distantly located ligand-binding domain (LBD). Taken together, these results indicate that subtle structural changes in a nuclear receptor DBD can exert long-range functional effects on the LBD of a receptor, and significantly impact transcriptional regulation.

Authors
Solomon, IH; Hager, JM; Safi, R; McDonnell, DP; Redinbo, MR; Ortlund, EA
MLA Citation
Solomon, Isaac H., et al. “Crystal structure of the human LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity..” J Mol Biol, vol. 354, no. 5, Dec. 2005, pp. 1091–102. Pubmed, doi:10.1016/j.jmb.2005.10.009.
PMID
16289203
Source
pubmed
Published In
Journal of Molecular Biology
Volume
354
Issue
5
Publish Date
2005
Start Page
1091
End Page
1102
DOI
10.1016/j.jmb.2005.10.009

Coactivation of liver receptor homologue-1 by peroxisome proliferator-activated receptor gamma coactivator-1alpha on aromatase promoter II and its inhibition by activated retinoid X receptor suggest a novel target for breast-specific antiestrogen therapy.

Aromatase inhibitors target the production of estrogen in breast adipose tissue, but in doing so, also decrease estrogen formation in bone and other sites, giving rise to deleterious side effects, such as bone loss and arthralgia. Thus, it would be clinically useful to selectively inhibit aromatase production in breast. In this regard, we have determined that the orphan nuclear receptor liver receptor homologue-1 (LRH-1) is a specific transcriptional activator of aromatase gene expression in human breast preadipocytes but not in other tissues of postmenopausal women. In this study, we show that the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is a physiologically relevant modulator of LRH-1, and that its transcriptional activity can be inhibited effectively using receptor-interacting peptide antagonists that prevent PGC-1alpha recruitment. Interestingly, we note that all of these peptides also interact in an agonist-dependent manner with retinoid X receptor alpha (RXRalpha), suggesting that these two receptors may compete for limiting cofactors within target cells. In support of this hypothesis, we show that 9-cis-retinoic acid, acting through RXR, inhibits both the basal and PGC-1alpha-induced transcriptional activity of LRH-1. The importance of this finding was confirmed by showing that LRH-1-dependent, PGC-1alpha-stimulated regulation of aromatase gene expression in primary human breast preadipocytes was effectively suppressed by RXR agonists. We infer from these data that LRH-1 is a bona fide target whose inhibition would selectively block aromatase expression in breast, while sparing other sites of expression.

Authors
Safi, R; Kovacic, A; Gaillard, S; Murata, Y; Simpson, ER; McDonnell, DP; Clyne, CD
MLA Citation
Safi, Rachid, et al. “Coactivation of liver receptor homologue-1 by peroxisome proliferator-activated receptor gamma coactivator-1alpha on aromatase promoter II and its inhibition by activated retinoid X receptor suggest a novel target for breast-specific antiestrogen therapy..” Cancer Res, vol. 65, no. 24, Dec. 2005, pp. 11762–70. Pubmed, doi:10.1158/0008-5472.CAN-05-2792.
PMID
16357189
Source
pubmed
Published In
Cancer Research
Volume
65
Issue
24
Publish Date
2005
Start Page
11762
End Page
11770
DOI
10.1158/0008-5472.CAN-05-2792

Coactivation of liver receptor homologue-1 by peroxisome proliferator-activated receptor gamma coactivator-1 alpha on aromatase promoter II and its inhibition by activated retinoid X receptor suggest a novel target for breast-specific antiestrogen therapy

Authors
Safi, R; Kovacic, A; Gaillard, S; Murata, Y; Simpson, ER; McDonnell, DP; Clyne, CD
MLA Citation
Safi, R., et al. “Coactivation of liver receptor homologue-1 by peroxisome proliferator-activated receptor gamma coactivator-1 alpha on aromatase promoter II and its inhibition by activated retinoid X receptor suggest a novel target for breast-specific antiestrogen therapy.” Cancer Research, vol. 65, no. 24, AMER ASSOC CANCER RESEARCH, Dec. 2005, pp. 11762–70. Wos, doi:10.1158/0008-5472.CAN-05-2792.
Source
wos
Published In
Cancer Research
Volume
65
Issue
24
Publish Date
2005
Start Page
11762
End Page
11770
DOI
10.1158/0008-5472.CAN-05-2792

Coregulators in nuclear estrogen receptor action: from concept to therapeutic targeting.

Estrogens are key regulators of growth, differentiation, and the physiological functions of a wide range of target tissues, including the male and female reproductive tracts, breast, and skeletal, nervous, cardiovascular, digestive and immune systems. The majority of these biological activities of estrogens are mediated through two genetically distinct receptors, ERalpha and ERbeta, which function as hormone-inducible transcription factors. Over the past decade, it has become increasingly clear that the recruitment of coregulatory proteins to ERs is required for ER-mediated transcriptional and biological activities. These "coactivator" complexes enable the ERs to respond appropriately: 1) to hormones or pharmacological ligands, 2) interpret extra- and intra-cellular signals, 3) catalyze the process of chromatin condensation and 4) to communicate with the general transcription apparatus at target gene promoters. In addition to activating proteins, the existence of corepressors, proteins that function as negative regulators of ER activity in either physiological or pharmacological contexts, provides an additional level of complexity in ER action. This review also describes current efforts aimed at developing pharmaceutical agents that target ER-cofactor interactions as therapeutics for estrogen-associated pathologies.

Authors
Hall, JM; McDonnell, DP
MLA Citation
Hall, Julie M., and Donald P. McDonnell. “Coregulators in nuclear estrogen receptor action: from concept to therapeutic targeting..” Mol Interv, vol. 5, no. 6, Dec. 2005, pp. 343–57. Pubmed, doi:10.1124/mi.5.6.7.
PMID
16394250
Source
pubmed
Published In
Molecular Interventions
Volume
5
Issue
6
Publish Date
2005
Start Page
343
End Page
357
DOI
10.1124/mi.5.6.7

Development of peptide antagonists for the androgen receptor using combinatorial peptide phage display.

Under the auspices of the Nuclear Receptor Signaling Atlas (NURSA), we have undertaken to evaluate the feasibility of targeting nuclear receptor-coactivator surfaces for new drug discovery. The underlying objective of this approach is to provide the research community with reagents that can be used to modulate the transcriptional activity of nuclear receptors. Using combinatorial peptide phage display, we have been able to develop peptide antagonists that target specific nuclear receptor (NR)-coactivator binding surfaces. It can be appreciated that reagents of this nature will be of use in the study of orphan nuclear receptors for whom classical ligands have not yet been identified. In addition, because the interaction of coactivators with the receptor is an obligate step for NR transcriptional activity, it is anticipated that peptides that block these interactions will enable the definition of the biological and pharmacological significance of individual NR-coactivator interactions. In this report, we describe the use of this approach to develop antagonists of the androgen receptor by targeting its coactivator-binding pocket and their use to study the coactivator-binding surface of this receptor. Based on our findings, we believe that molecules that function by disrupting the androgen receptor-cofactor interactions will have use in the treatment of prostate cancer.

Authors
Chang, C-Y; Abdo, J; Hartney, T; McDonnell, DP
MLA Citation
Chang, Ching-Yi, et al. “Development of peptide antagonists for the androgen receptor using combinatorial peptide phage display..” Mol Endocrinol, vol. 19, no. 10, Oct. 2005, pp. 2478–90. Pubmed, doi:10.1210/me.2005-0072.
PMID
16051662
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
19
Issue
10
Publish Date
2005
Start Page
2478
End Page
2490
DOI
10.1210/me.2005-0072

Structural basis for an unexpected mode of SERM-mediated ER antagonism.

Tamoxifen is effective for the prevention and treatment of estrogen-dependent breast cancers, but is associated with an increased incidence of endometrial tumors. We report the crystal structure of the estrogen receptor alpha (ERalpha) ligand binding domain (LBD) bound to the structurally similar compound GW5638, which has therapeutic potential and does not stimulate the uterus. Like tamoxifen, GW5638 relocates the carboxy-terminal helix (H12) to the known coactivator-docking site in the ERalpha LBD. However, GW5638 repositions residues in H12 through specific contacts with the N terminus of this helix. In contrast to tamoxifen, the resulting increase in exposed hydrophobic surface of ERalpha LBD correlates with a significant destabilization of ERalpha in MCF-7 cells. Thus, the GW5638-ERalpha LBD structure reveals an unexpected mode of SERM-mediated ER antagonism, in which the stability of ERalpha is decreased through an altered position of H12. This dual mechanism of antagonism may explain why GW5638 can inhibit tamoxifen-resistant breast tumors.

Authors
Wu, Y-L; Yang, X; Ren, Z; McDonnell, DP; Norris, JD; Willson, TM; Greene, GL
MLA Citation
Wu, Ya-Ling, et al. “Structural basis for an unexpected mode of SERM-mediated ER antagonism..” Mol Cell, vol. 18, no. 4, May 2005, pp. 413–24. Pubmed, doi:10.1016/j.molcel.2005.04.014.
PMID
15893725
Source
pubmed
Published In
Molecular Cell
Volume
18
Issue
4
Publish Date
2005
Start Page
413
End Page
424
DOI
10.1016/j.molcel.2005.04.014

Identification and structure-activity relationship of phenolic acyl hydrazones as selective agonists for the estrogen-related orphan nuclear receptors ERRbeta and ERRgamma.

The first small molecule agonists of the estrogen-related receptors have been identified. GSK4716 (3) and GSK9089 (4) show binding to ERRgamma with remarkable selectivity over the classical estrogen receptors. Notably, in cell-based reporter assays, 3 mimics the protein ligand PGC-1alpha in activation of human ERRbeta and ERRgamma.

Authors
Zuercher, WJ; Gaillard, S; Orband-Miller, LA; Chao, EYH; Shearer, BG; Jones, DG; Miller, AB; Collins, JL; McDonnell, DP; Willson, TM
MLA Citation
Zuercher, William J., et al. “Identification and structure-activity relationship of phenolic acyl hydrazones as selective agonists for the estrogen-related orphan nuclear receptors ERRbeta and ERRgamma..” J Med Chem, vol. 48, no. 9, May 2005, pp. 3107–09. Pubmed, doi:10.1021/jm050161j.
PMID
15857113
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
48
Issue
9
Publish Date
2005
Start Page
3107
End Page
3109
DOI
10.1021/jm050161j

Androgen receptor-cofactor interactions as targets for new drug discovery.

Cofactor recruitment is a crucial regulatory step in nuclear receptor signal transduction. Given the obligate nature of interactions between cofactors and these receptors for transcriptional activity, it is likely that drugs that target coactivator interaction surfaces will function as pure antagonists with particular utility in the treatment of estrogen- and androgen-dependent cancers. Recent crystallographic analysis of one of the major protein-protein interaction surfaces on the androgen receptor has raised expectations that it will be possible to develop small-molecule antagonists that block cofactor interactions.

Authors
Chang, C-Y; McDonnell, DP
MLA Citation
Chang, Ching-yi, and Donald P. McDonnell. “Androgen receptor-cofactor interactions as targets for new drug discovery..” Trends Pharmacol Sci, vol. 26, no. 5, May 2005, pp. 225–28. Pubmed, doi:10.1016/j.tips.2005.03.002.
PMID
15860367
Source
pubmed
Published In
Trends in Pharmacological Sciences
Volume
26
Issue
5
Publish Date
2005
Start Page
225
End Page
228
DOI
10.1016/j.tips.2005.03.002

Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP.

The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 A of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1's control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.

Authors
Ortlund, EA; Lee, Y; Solomon, IH; Hager, JM; Safi, R; Choi, Y; Guan, Z; Tripathy, A; Raetz, CRH; McDonnell, DP; Moore, DD; Redinbo, MR
MLA Citation
Ortlund, Eric A., et al. “Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP..” Nat Struct Mol Biol, vol. 12, no. 4, Apr. 2005, pp. 357–63. Pubmed, doi:10.1038/nsmb910.
PMID
15723037
Source
pubmed
Published In
Nature Structural & Molecular Biology
Volume
12
Issue
4
Publish Date
2005
Start Page
357
End Page
363
DOI
10.1038/nsmb910

Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor.

The vitamin D receptor (VDR) is a ligand-responsive transcription factor that forms active, heterodimeric complexes with the 9-cis retinoic acid receptor (RXR) on vitamin D response elements (VDREs). Both proteins consist of an N-terminal DNA-binding domain, a C-terminal ligand-binding domain, and an intervening hinge region. The length requirements of the hinge for both transcriptional regulation and DNA binding have not been studied to date for any member of the steroid hormone superfamily. We have generated a series of internal deletion mutants of the VDR hinge and found that deletion of as few as five amino acids from the C-terminus of the hinge significantly reduces transcriptional activation in vivo. Replacing deleted residues in the C-terminus of the hinge with alanines restored activity, indicating that this section of the hinge acts as a sequence-independent spacer. The hinge region of VDR forms a long helix, and the geometric consequences of this structure may explain the requirement of the hinge region for transcriptional activity. Interestingly, all of the deletion mutants, even those that do not activate transcription, bind VDREs with equal and high affinity, indicating that the defect in these mutants is not their ability to bind VDREs. In contrast to VDR, constructs of RXR containing deletions of up to 14 amino acids in the hinge region exhibit near wild-type transcriptional activity. The ability to delete more of the RXR hinge may be related to the additional plasticity required by its role as the common heterodimer partner for nuclear receptors on differing DNA elements.

Authors
Shaffer, PL; McDonnell, DP; Gewirth, DT
MLA Citation
Shaffer, Paul L., et al. “Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor..” Biochemistry, vol. 44, no. 7, Feb. 2005, pp. 2678–85. Pubmed, doi:10.1021/bi0477182.
PMID
15709781
Source
pubmed
Published In
Biochemistry
Volume
44
Issue
7
Publish Date
2005
Start Page
2678
End Page
2685
DOI
10.1021/bi0477182

Gene expression profiling of breast cancer in relation to estrogen receptor status and estrogen-metabolizing enzymes: clinical implications.

Interactions between luminal epithelial cells and their surrounding microenvironment govern the normal development and function of the mammary gland. Estradiol plays a key role in abnormal intracellular signaling, which contributes to the development and progression of breast tumors. The present article summarizes the results from a microarray whole genome gene expression analysis as well as a quantitative analysis of the mRNA expression of members of the estradiol metabolic and signaling pathways in the tumors of postmenopausal breast cancer patients. The analysis of the variation in whole genome gene expression resulted in a tumor classification comprising several distinct groups with distinct expression of the estrogen receptor (ER). The parallel study on the expression of only nine mRNA transcripts of members of the estradiol pathways resulted in two main clusters, representing ER- and ER tumors. The mRNA expression of the estradiol-metabolizing enzymes did not follow the expression of the ER in all cases, leading to the recognition of several further subclasses of tumors. When the tumor classes obtained by whole genome gene expression analysis were compared with those obtained by independent quantitation of the estradiol-metabolizing enzymes, a statistically significant association between both classification groups was observed. These findings point to a possible association between development of a tumor with a particular expression profile and its capacity to synthesize estradiol as measured by the expression of the transcripts for the necessary key enzymes. Further, whole genome expression patterns were studied in 12 patients treated with anastrozole. Using significance analysis of microarrays, we identified 298 genes significantly differently expressed between partial response and progressive disease groups.

Authors
Kristensen, VN; Sørlie, T; Geisler, J; Langerød, A; Yoshimura, N; Kåresen, R; Harada, N; Lønning, PE; Børresen-Dale, A-L
MLA Citation
Kristensen, Vessela N., et al. “Gene expression profiling of breast cancer in relation to estrogen receptor status and estrogen-metabolizing enzymes: clinical implications..” Clin Cancer Res, vol. 11, no. 2 Pt 2, Jan. 2005, pp. 878s-883s.
PMID
15701881
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 Pt 2
Publish Date
2005
Start Page
878s
End Page
883s

Tamoxifen versus aromatase inhibitors for breast cancer prevention.

Long-term exposure to estradiol is associated with an increased risk of breast cancer, but the mechanisms responsible are not firmly established. The prevailing theory postulates that estrogens increase the rate of cell proliferation by stimulating estrogen receptor (ER)-mediated transcription, thereby increasing the number of errors occurring during DNA replication. An alternative theory suggests that estradiol is metabolized to quinone derivatives, which directly remove base pairs from DNA through a process called depurination. Error-prone DNA repair then results in point mutations. We postulate that both processes act in an additive or synergistic fashion. If correct, aromatase inhibitors would block both processes, whereas antiestrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than antiestrogens. Our initial studies showed that catechol-estrogen metabolites are formed in MCF-7 human breast cancer cells in culture. We then used an animal model that allows dissociation of ER-mediated function from the effects of estradiol metabolites and showed formation of genotoxic estradiol metabolites. We also examined the incidence of tumors formed in these ERalpha knockout mice bearing the Wnt-1 transgene. The absence of estradiol markedly reduced the incidence of tumors and delayed their onset. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER-mediated mechanisms to induce breast cancer. These findings support the possibility that aromatase inhibitors might be more effective than antiestrogens in preventing breast cancer.

Authors
Yue, W; Wang, J-P; Li, Y; Bocchinfuso, WP; Korach, KS; Devanesan, PD; Rogan, E; Cavalieri, E; Santen, RJ
MLA Citation
Yue, Wei, et al. “Tamoxifen versus aromatase inhibitors for breast cancer prevention..” Clin Cancer Res, vol. 11, no. 2 Pt 2, Jan. 2005, pp. 925s-930s.
PMID
15701888
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 Pt 2
Publish Date
2005
Start Page
925s
End Page
930s

Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: Conference summary statement

The Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer, co-chaired by Steven Come, MD, and Aman Buzdar, MD, was held in Cambridge, MA, July 21-22, 2004. The conference was organized with the objective of analyzing recent advances in basic, translational, and clinical research relating to endocrine manipulation of breast cancer, and examining the implications of these findings both for patient management and for future research. Conference attendees were selected based on their contributions to the basic or clinical understanding of breast cancer, and included internationally recognized researchers in the fields of molecular biology, pharmacology, epidemiology, and medical oncology. The conference, which combined brief scientific reports with extended periods of open discussion, focused upon the following issues: The biology of the estrogen receptor (ER) and how ligand-dependent conformational changes in the receptor and receptor down-regulation present new therapeutic opportunities. The mechanisms of resistance to both tamoxifen and the aromatase inhibitors. The scientific rationale, preclinical evidence, and clinical experience to date supporting the use of combination therapies to improve efficacy and delay resistance to endocrine-based treatment. Current understanding of the biology of transforming growth factor (TGF)-β and type 1 insulin-like growth factor (IGF-I) and their potential as therapeutic targets. Emerging data on aromatase inhibitors in early breast cancer: implications for clinical practice today. New drug development: preclinical and early clinical findings for the steroidal antiestrogen TAS-108. Aromatase inhibitors in breast cancer prevention. The protective effect of early pregnancy on the breast and the possible application of biological and genomic observations in this model to an alternative prevention strategy. Priorities for translational and clinical research. At the conclusion of the conference, the faculty convened to formulate the following summary statement.

Authors
Come, SE; Buzdar, AU; Ingle, JN; Arteaga, CL; Brodie, AM; Colditz, GA; Johnston, SRD; Kristensen, VN; Lønning, PE; McDonnell, DP; Osborne, CK; Russo, J; Santen, RJ; Yee, D; Hart, CS
MLA Citation
Come, S. E., et al. “Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: Conference summary statement.” Clinical Cancer Research, vol. 11, no. 2 II, Jan. 2005.
Source
scopus
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 II
Publish Date
2005

Estrogen, estrogen plus progestin therapy, and risk of breast cancer.

Epidemiologic evidence relating use of postmenopausal hormones to risk of breast cancer by nature relies on trends in prescribing practices. Data on the adverse effect of combination estrogen plus progestin used for long durations has only become available over the past decade. Evidence is reviewed relating estrogen alone and estrogen plus progestin to increased risk of breast cancer. Whereas current evidence indicates that longer duration of use increases risk of invasive breast cancer regardless of formulation, the rate of increase in risk is greater for combination estrogen plus progestin therapy. Although data are limited, continuous combined therapy and sequential therapy seem to have comparable impact on breast cancer risk. Combination therapy is more strongly related to lobular breast cancer than is estrogen alone. Unresolved issues remain about dose of estrogen and progestin in relation to risk, and about identification of women for whom short-term use to relieve menopausal symptoms may be safe and effective.

Authors
Colditz, GA
MLA Citation
Colditz, Graham A. “Estrogen, estrogen plus progestin therapy, and risk of breast cancer..” Clin Cancer Res, vol. 11, no. 2 Pt 2, Jan. 2005, pp. 909s-917s.
PMID
15701886
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 Pt 2
Publish Date
2005
Start Page
909s
End Page
917s

The molecular pharmacology of estrogen receptor modulators: Implications for the treatment of breast cancer

In addition to physiologic activities in the reproductive, skeletal, and central nervous systems, estrogens have been shown to play important roles in the aberrant cell proliferation observed in breast and reproductive tract cancers. Not surprisingly, pharmaceuticals that target different steps in the estrogen signal transduction pathway have found widespread use in the treatment of a wide variety of estrogen-linked disorders. The goal of this review is to outline what is known about the molecular pharmacology of the estrogen receptor and discuss how this information can be used to guide selection of drugs for a particular therapeutic application, and identify new targets where pharmaceutical exploitation could yield novel therapeutics.

Authors
McDonnell, DP; Yee, D; Santen, R; Osborne, K; Brodie, A
MLA Citation
McDonnell, D. P., et al. “The molecular pharmacology of estrogen receptor modulators: Implications for the treatment of breast cancer.” Clinical Cancer Research, vol. 11, no. 2 II, Jan. 2005.
Source
scopus
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 II
Publish Date
2005

The molecular pharmacology of estrogen receptor modulators: implications for the treatment of breast cancer.

In addition to physiologic activities in the reproductive, skeletal, and central nervous systems, estrogens have been shown to play important roles in the aberrant cell proliferation observed in breast and reproductive tract cancers. Not surprisingly, pharmaceuticals that target different steps in the estrogen signal transduction pathway have found widespread use in the treatment of a wide variety of estrogen-linked disorders. The goal of this review is to outline what is known about the molecular pharmacology of the estrogen receptor and discuss how this information can be used to guide selection of drugs for a particular therapeutic application, and identify new targets where pharmaceutical exploitation could yield novel therapeutics.

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “The molecular pharmacology of estrogen receptor modulators: implications for the treatment of breast cancer..” Clin Cancer Res, vol. 11, no. 2 Pt 2, Jan. 2005, pp. 871s-877s.
PMID
15701880
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 Pt 2
Publish Date
2005
Start Page
871s
End Page
877s

The molecular pharmacology of estrogen receptor modulators: Implications for the treatment of breast cancer

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “The molecular pharmacology of estrogen receptor modulators: Implications for the treatment of breast cancer.” Clinical Cancer Research, vol. 11, no. 2, AMER ASSOC CANCER RESEARCH, 2005, pp. 871S-877S.
Source
wos
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2
Publish Date
2005
Start Page
871S
End Page
877S

Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: conference summary statement.

Authors
Come, SE; Buzdar, AU; Ingle, JN; Arteaga, CL; Brodie, AM; Colditz, GA; Johnston, SRD; Kristensen, VN; Lønning, PE; McDonnell, DP; Osborne, CK; Russo, J; Santen, RJ; Yee, D; Hart, CS
MLA Citation
Come, Steven E., et al. “Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: conference summary statement..” Clin Cancer Res, vol. 11, no. 2 Pt 2, Jan. 2005, pp. 861s-864s.
PMID
15701878
Source
pubmed
Published In
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
Volume
11
Issue
2 Pt 2
Publish Date
2005
Start Page
861s
End Page
864s

Regulation of aromatase expression in the breast by LRH-1: a new potential target for breast cancer therapy

Authors
Simpson, ER; McDonnell, DP; Kovacic, A; Clyne, CD; Safi, R
MLA Citation
Simpson, E. R., et al. “Regulation of aromatase expression in the breast by LRH-1: a new potential target for breast cancer therapy.” Breast Cancer Research and Treatment, vol. 94, SPRINGER, 2005, pp. S236–S236.
Source
wos
Published In
Breast Cancer Research and Treatment
Volume
94
Publish Date
2005
Start Page
S236
End Page
S236

Single-step purification of full-length human androgen receptor.

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 microM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

Authors
Juzumiene, D; Chang, C-Y; Fan, D; Hartney, T; Norris, JD; McDonnell, DP
MLA Citation
Juzumiene, Dalia, et al. “Single-step purification of full-length human androgen receptor..” Nucl Recept Signal, vol. 3, 2005. Pubmed, doi:10.1621/nrs.03001.
PMID
16604169
Source
pubmed
Published In
Nuclear Receptor Signaling
Volume
3
Publish Date
2005
Start Page
e001
DOI
10.1621/nrs.03001

The molecular determinants of estrogen receptor pharmacology.

The classical models of steroid receptor pharmacology held that agonists functioned by binding to their cognate receptors, facilitating their conversion from an inactive form to one that was capable of activating transcription. By extrapolation, it was believed that antagonists functioned by competitively inhibiting agonist binding, freezing the receptor in an inactive state. However, as early as 1967 when the biologic actions of the "antiestrogen" tamoxifen were first described, it was clear that this simple model did not adequately describe estrogen receptor (ER) pharmacology. Since these initial observations, significant progress has been made in defining the molecular mechanism(s) by which cells distinguish between different ER ligands. The most important of these are: (i) differences in the relative expression level of the two ER subtypes, alpha and beta, in target tissues; (ii) the impact, which the bound ligand has on ER structure; and (iii) the complement of coactivators and corepressors in a target cell, which can interact with the activated receptor. This review describes how these advances have impacted our understanding of the pharmacologic activities of currently available ER ligands.

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “The molecular determinants of estrogen receptor pharmacology..” Maturitas, vol. 48 Suppl 1, Aug. 2004, pp. S7-12. Pubmed, doi:10.1016/j.maturitas.2004.03.006.
PMID
15337242
Source
pubmed
Published In
Maturitas
Volume
48 Suppl 1
Publish Date
2004
Start Page
S7
End Page
12
DOI
10.1016/j.maturitas.2004.03.006

Coactivator AIB1 links estrogen receptor transcriptional activity and stability.

Agonist-mediated degradation of estrogen receptor alpha (ERalpha) has been associated with its transcriptional activity. However, the mechanism by which ERalpha is targeted for degradation and whether there is a direct functional link between ERalpha stability and ERalpha-mediated transactivation have not been elucidated. Here we provide evidence that the p160 coactivator, AIB1, uniquely mediates agonist-induced, but not antagonist-induced, ERalpha degradation. We show that AIB1 recruitment by ERalpha is not only necessary but also sufficient to promote degradation. Suppression of AIB1 levels leads to ERalpha stabilization in the presence of 17beta-estradiol and, despite increased ERalpha levels, reduced recruitment of ERalpha to endogenous target gene promoters. In addition, association of RNA polymerase II with ERalpha target promoters is lost when AIB1 is suppressed, leading to inhibition of target gene transcription. AIB1 thus plays a dual role in regulating ERalpha activity, one in recruiting transcription factors including other coactivators involved in gene activation and the other in regulating ERalpha protein degradation mediated by the ubiquitin-proteosome machinery.

Authors
Shao, W; Keeton, EK; McDonnell, DP; Brown, M
MLA Citation
Shao, Wenlin, et al. “Coactivator AIB1 links estrogen receptor transcriptional activity and stability..” Proc Natl Acad Sci U S A, vol. 101, no. 32, Aug. 2004, pp. 11599–604. Pubmed, doi:10.1073/pnas.0402997101.
PMID
15289619
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
101
Issue
32
Publish Date
2004
Start Page
11599
End Page
11604
DOI
10.1073/pnas.0402997101

Hormone therapy: physiological complexity belies therapeutic simplicity.

The results of the Women's Health Initiative, a study anticipated to provide definitive answers about health benefits and risks of postmenopausal hormone therapy, have generated debate and confusion among clinicians, researchers, and the lay public. The ovarian hormones estrogen and progesterone, which decline at menopause, normally elicit complex tissue-specific responses throughout the body. Major advances are providing a detailed molecular definition of how that differential action is achieved. Here we review estrogen and progestin actions, discuss how effectively knowledge of steroid hormone endocrinology has been incorporated into clinical studies, and consider the impact on modern hormone therapy protocols and pharmaceutical development.

Authors
Turgeon, JL; McDonnell, DP; Martin, KA; Wise, PM
MLA Citation
Turgeon, Judith L., et al. “Hormone therapy: physiological complexity belies therapeutic simplicity..” Science, vol. 304, no. 5675, May 2004, pp. 1269–73. Pubmed, doi:10.1126/science.1096725.
PMID
15166356
Source
pubmed
Published In
Science
Volume
304
Issue
5675
Publish Date
2004
Start Page
1269
End Page
1273
DOI
10.1126/science.1096725

Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition.

In this study, we demonstrate that the pervasive xenobiotic methoxyacetic acid and the commonly prescribed anticonvulsant valproic acid, both short-chain fatty acids (SCFAs), dramatically increase cellular sensitivity to estrogens, progestins, and other nuclear hormone receptor ligands. These compounds do not mimic endogenous hormones but rather act to enhance the transcriptional efficacy of ligand activated nuclear hormone receptors by up to 8-fold in vitro and in vivo. Detailed characterization of their mode of action revealed that these SCFAs function as both activators of p42/p44 mitogen-activated protein kinase and as inhibitors of histone deacetylases at doses that parallel known exposure levels. Our results define a class of compounds that possess a dual mechanism of action and function as hormone sensitizers. These findings prompt an evaluation of previously unrecognized drug-drug interactions in women who are administered exogenous hormones while exposed to certain xenobiotic SCFAs. Furthermore, our study highlights the need to structure future screening programs to identify additional hormone sensitizers.

Authors
Jansen, MS; Nagel, SC; Miranda, PJ; Lobenhofer, EK; Afshari, CA; McDonnell, DP
MLA Citation
Jansen, Michelle S., et al. “Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition..” Proc Natl Acad Sci U S A, vol. 101, no. 18, May 2004, pp. 7199–204. Pubmed, doi:10.1073/pnas.0402014101.
PMID
15103026
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
101
Issue
18
Publish Date
2004
Start Page
7199
End Page
7204
DOI
10.1073/pnas.0402014101

Progesterone, estrogen and androgen receptor signaling pathways are complex and provide a wealth of opportunities for new drug discovery.

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “Progesterone, estrogen and androgen receptor signaling pathways are complex and provide a wealth of opportunities for new drug discovery..” Abstracts of Papers of the American Chemical Society, vol. 227, AMER CHEMICAL SOC, 2004, pp. U45–U45.
Source
wos
Published In
Abstracts of Papers of the American Chemical Society
Volume
227
Publish Date
2004
Start Page
U45
End Page
U45

Selective LXXLL peptides antagonize transcriptional activation by the retinoid-related orphan receptor RORgamma.

The retinoid-related orphan receptor gamma (RORgamma) has been shown to function as a positive regulator of transcription in many cell lines. Transcriptional activation by nuclear receptors involves recruitment of co-activators that interact with receptors through their LXXLL motifs (NR box). In this study, we analyze the interaction of RORgamma with the co-activator SRC1 and use a series of LXXLL-containing peptides to probe for changes in the conformation of the co-activator interaction surface of the RORgamma LBD. We demonstrate that the H3-4/H12 co-activator interaction surface of RORgamma displays a selectivity for LXXLL peptides that is distinct from those of other nuclear receptors. LXXLL peptides that interacted with RORgamma efficiently antagonized RORgamma-mediated transcriptional activation. Mutations E502Q and Y500F in H12, and K334A, Q347A, and I348D in H3 and H4 of RORgamma, severely impact the recruitment of LXXLL peptides. The effects of these mutations are consistent with predictions made on the basis of the structure of the RORgamma(LBD) derived through homology modeling. These peptide antagonists provide a useful tool to analyze the conformation changes in the RORgamma(LBD) and to study RORgamma receptor signaling.

Authors
Kurebayashi, S; Nakajima, T; Kim, S-C; Chang, C-Y; McDonnell, DP; Renaud, J-P; Jetten, AM
MLA Citation
Kurebayashi, Shogo, et al. “Selective LXXLL peptides antagonize transcriptional activation by the retinoid-related orphan receptor RORgamma..” Biochem Biophys Res Commun, vol. 315, no. 4, Mar. 2004, pp. 919–27. Pubmed, doi:10.1016/j.bbrc.2004.01.131.
PMID
14985100
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
315
Issue
4
Publish Date
2004
Start Page
919
End Page
927
DOI
10.1016/j.bbrc.2004.01.131

The jensen symposium; a tribute to a pioneer in the field of nuclear receptor biology.

The Jensen Symposium was held at the University of Cincinnati in December 2003 to honor the pioneering contributions of Dr. Elwood Jensen to the field of nuclear hormone action. Those in attendance were treated to an outstanding scientific program that served as an update of recent progress and illustrated the breadth of activity in the nuclear receptor field. Here we highlight recent findings presented at the Symposium that provide new insights into the mechanisms of nuclear receptor action and the diverse roles of members of the nuclear receptor superfamily in development and homeostasis.

Authors
Glass, CK; McDonnell, DP
MLA Citation
Glass, Christopher K., and Donald P. McDonnell. “The jensen symposium; a tribute to a pioneer in the field of nuclear receptor biology..” Mol Cell, vol. 13, no. 4, Feb. 2004, pp. 459–67.
PMID
14992717
Source
pubmed
Published In
Molecular Cell
Volume
13
Issue
4
Publish Date
2004
Start Page
459
End Page
467

E10. Interpreting the results of the women's health initiative within the context of our current understanding of oestrogen and progesterone action

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “E10. Interpreting the results of the women's health initiative within the context of our current understanding of oestrogen and progesterone action.” European Journal of Cancer, Supplement, vol. 2, no. 9 SPEC. ISS., Jan. 2004, pp. 35–40. Scopus, doi:10.1016/j.ejcsup.2004.08.050.
Source
scopus
Published In
European Journal of Cancer Supplements
Volume
2
Issue
9 SPEC. ISS.
Publish Date
2004
Start Page
35
End Page
40
DOI
10.1016/j.ejcsup.2004.08.050

Mining the complexities of nuclear receptor signaling for new drug targets.

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “Mining the complexities of nuclear receptor signaling for new drug targets..” Biology of Reproduction, SOC STUDY REPRODUCTION, 2004, pp. 81–81.
Source
wos
Published In
Biology of Reproduction
Publish Date
2004
Start Page
81
End Page
81

Increased expression of estrogen receptor beta in pachytene spermatocytes after short-term methoxyacetic acid administration.

Degeneration of primary spermatocytes by apoptosis occurs during normal spermatogenesis, as well as in several pathological conditions, including exposure to specific testicular toxicants. The mechanisms that regulate the death and survival of primary spermatocytes, however, are still not well understood. The recent localization of estrogen receptor beta (ERbeta) and P450 aromatase in pachytene spermatocytes suggests a role for estrogens in this step of spermatogenesis. Using a well-known model of pachytene spermatocyte apoptosis in adult rats consisting of the administration of methoxyacetic acid (MAA), we investigated the participation of ERbeta during the initial phase of apoptosis, prior to germ cell loss. Adult rats were treated with a single intraperitoneal dose of MAA, and DNA laddering analysis confirmed apoptotic cell death in the testis. In enriched germ cell fractions and testis from MAA-treated animals, ERbeta mRNA increased significantly at 3 and 6 hours, respectively. Next, stage-specific induction of ERbeta mRNA was demonstrated by use of laser capture microdissection of seminiferous tubules in combination with semiquantitative reverse transcription-polymerase chain reaction. The ERbeta protein also increased significantly after 6 hours and was mainly immunolocalized in the cytoplasm of pachytene spermatocytes of afflicted tubules. The cytoplasmic localization was confirmed by Western blot analysis of isolated cytoplasmic and nuclear fractions of testicular extracts. Finally, the MAA activation of ERbeta was tested in vitro in HepG2 cells cotransfected with ERbeta and a reporter construct that contained a consensus estrogen responsive element. Addition of MAA at similar doses used in vivo elicited a similar estrogenic activation as did estradiol at 1 nmol/L concentration. The present results raise the possibility that cytoplasmic ERbeta participates in the apoptotic process of pachytene spermatocytes induced by MAA. Whether MAA interacts with ERbeta in the cytoplasm of primary spermatocytes, preventing the progression of the first meiotic division, however, remains to be determined.

Authors
Tirado, OM; Selva, DM; Toràn, N; Suárez-Quian, CA; Jansen, M; McDonnell, DP; Reventós, J; Munell, F
MLA Citation
Tirado, Oscar M., et al. “Increased expression of estrogen receptor beta in pachytene spermatocytes after short-term methoxyacetic acid administration..” J Androl, vol. 25, no. 1, Jan. 2004, pp. 84–94.
PMID
14662790
Source
pubmed
Published In
Journal of Andrology
Volume
25
Issue
1
Publish Date
2004
Start Page
84
End Page
94

The impact of the women's health initiative on the search for improved treatments for the conditions associated with long-term oestrogen deprivation

Publication of the preliminary findings from the combination (oestrogen and progestogen) hormone therapy (HT) arm of the Women's Health Initiative (WHI) on the heels of the Heart and Estrogen/progestin Replacement Study (HERS) sent the field of menopausal medicine into turmoil. It is now considered that oestrogen therapy (ET)/HT should be used only to treat the shortterm climacteric conditions associated with menopause and that its use to treat conditions associated with long-term oestrogen deprivation (ie, cardiovascular disease, osteoporosis and delay of the onset of dementia and Alzheimer's disease) is no longer appropriate. As a result, there has been a dramatic decrease in the number of patients using ET/HT. With few new therapeutic options on the horizon to treat either the short or long-term consequences of oestrogen deprivation, it seems that progress in the treatment of the menopausal patient has taken a large step backwards. This review considers how the direction of research in the field of menopausal medicine has been shaped by the WHI and outlines what the future might hold.

Authors
McDonnell, DP; Miranda, P
MLA Citation
McDonnell, D. P., and P. Miranda. “The impact of the women's health initiative on the search for improved treatments for the conditions associated with long-term oestrogen deprivation.” Journal of the British Menopause Society, vol. 9, no. SUPPL. 2, Dec. 2003, pp. 4–10.
Source
scopus
Published In
Journal of the British Menopause Society
Volume
9
Issue
SUPPL. 2
Publish Date
2003
Start Page
4
End Page
10

Pharmacological uncoupling of androgen receptor-mediated prostate cancer cell proliferation and prostate-specific antigen secretion.

The androgen receptor (AR), a member of the nuclear receptor family, is a ligand-inducible transcription factor. In the prostate gland, androgens regulate the transcription of several genes that ultimately result in cell growth and differentiation. With a goal of developing tissue-selective AR modulators that can be used to treat prostate cancer and other androgenopathies, we have taken an approach to identify androgens that function in a manner distinct from the physiological androgens testosterone and dihydrotestosterone. Classical AR agonists function by binding to and inducing a conformational change in the receptor. This facilitates the obligate interaction of the amino and carboxyl terminus of the receptor, recruitment of coactivators, and subsequent regulation of target genes. On the basis of this paradigm, we screened a library of potential AR agonists for compounds that induce an "activating" conformational change in the receptor structure but that do not facilitate a high-affinity intermolecular interaction between the amino and carboxyl terminus. Compounds identified in this manner behaved as partial agonists of AR-mediated transcription in a variety of assays. Additional compounds were identified in this screen that did not allow the activation function-2 coactivator pocket to form and were demonstrated to function as weak agonists of AR-mediated transcription. Surprisingly, when we examined the ability of these compounds to induce cell proliferation, we observed that despite having different degrees of partial agonist activities on classical transcriptional responses (i.e., induction of prostate-specific antigen), these compounds were as efficacious as dihydrotestosterone in stimulating proliferation. The unexpected finding that AR-mediated transcription and proliferation can be uncoupled suggests that AR is not used in the same manner in all androgen-regulated biological processes.

Authors
Sathya, G; Chang, C-Y; Kazmin, D; Cook, CE; McDonnell, DP
MLA Citation
Sathya, Ganesan, et al. “Pharmacological uncoupling of androgen receptor-mediated prostate cancer cell proliferation and prostate-specific antigen secretion..” Cancer Res, vol. 63, no. 22, Nov. 2003, pp. 8029–36.
PMID
14633736
Source
pubmed
Published In
Cancer Research
Volume
63
Issue
22
Publish Date
2003
Start Page
8029
End Page
8036

Function and mode of action of nuclear receptors: Estrogen, progesterone, and vitamin D

Estrogens and progestins play important roles in regulating growth and differentiation of a wide range of cell types, in both reproductive and nonreproductive tissues. Not surprisingly, therefore, endocrine active substances that mimic the actions of these steroid hormones have been demonstrated to have untoward effects on the reproductive function of a variety of animals. Although direct links between exposure to andocrine active substances of this class and reproductive abnormalities in humans have been difficult to establish, the potential for harm by this class of chemicals warrants further investigation. This chapter summarizes our current understanding of the molecular pharmacology of the estrogen and progesterone receptors, ER and PR, beginning with a historical perspective of ER pharmacology and ending with a comparison of these receptors to the vitamin D receptor (VDR), a nonsteroid hormone nuclear receptor involved in regulating growth and development in non-reproductive tissues. Major topics discussed include receptor subtypes (or isoforms), receptor functional domains, ligand-binding characteristics, receptor structure, cofactor binding, effects of phosphorylation, and nonclassical modes of action. This discussion will demonstrate the need for developing novel screens for potential endocrine disruptors that incorporate our current understanding of nuclear receptor pharmacology.

Authors
Kimbrel, EA; McDonnell, DP
MLA Citation
Kimbrel, E. A., and D. P. McDonnell. “Function and mode of action of nuclear receptors: Estrogen, progesterone, and vitamin D.” Pure and Applied Chemistry, vol. 75, no. 11–12, Nov. 2003, pp. 1671–83.
Source
scopus
Published In
Pure and Applied Chemistry
Volume
75
Issue
11-12
Publish Date
2003
Start Page
1671
End Page
1683

Mining the complexities of the estrogen signaling pathways for novel therapeutics.

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “Mining the complexities of the estrogen signaling pathways for novel therapeutics..” Endocrinology, vol. 144, no. 10, Oct. 2003, pp. 4237–40. Pubmed, doi:10.1210/en.2003-0900.
PMID
14500558
Source
pubmed
Published In
Endocrinology
Volume
144
Issue
10
Publish Date
2003
Start Page
4237
End Page
4240
DOI
10.1210/en.2003-0900

Randomized controlled trial evidence that estrogen replacement therapy reduces the progression of subclinical atherosclerosis in healthy postmenopausal women without preexisting cardiovascular disease - Response

Authors
Herrington, DM; Howard, TD; Brosnihan, B; Hawkins, GA; Reboussin, DM; Xu, JF; Zheng, SQL; Meyers, DA; Bleeker, ER; McDonnell, DP; Li, XL
MLA Citation
Herrington, D. M., et al. “Randomized controlled trial evidence that estrogen replacement therapy reduces the progression of subclinical atherosclerosis in healthy postmenopausal women without preexisting cardiovascular disease - Response.” Circulation, vol. 108, no. 1, LIPPINCOTT WILLIAMS & WILKINS, July 2003, pp. E5–E5.
Source
wos
Published In
Circulation
Volume
108
Issue
1
Publish Date
2003
Start Page
E5
End Page
E5

Expression of functional estrogen receptor beta in locus coeruleus-derived Cath.a cells.

Estrogen may have an important role in the brain beyond the development and regulation of reproductive function. Gender differences in the incidence of depression suggest that regulation of mood represents one such action. The locus coeruleus, a brain stem noradrenergic nucleus implicated in mood regulation, concentrates [(3)H]estradiol, but expression of the two estrogen receptor (ER) subtypes (ERalpha and ERbeta) varies across species. Further, the role of each subtype in estrogen action on noradrenergic neurons is unknown. We examined the expression of ERs in the Cath.a (central-adrenergic-tyrosine-hydroxylase-expressing) cell line derived from mouse brain stem and found that they express ERbeta protein but not ERalpha protein. Transient transfection assays using an estrogen-responsive reporter gene indicate that ERbeta is functional. The pure estrogen antagonist ICI 182,780 completely abolished estrogen's effects. Selective ER modulator results suggest that ER in Cath.a cells behaves in a manner consistent with ERbeta pharmacology. R,R-Tetrahydrochrysene, an ERalpha agonist, had no effect on luciferase-driven activity in Cath.a cells. This study provides the first report of a cell line that spontaneously expresses functional ERbeta protein. Cath.a cells may prove to be a useful tool in elucidating basic pharmacologic properties of ERbeta. It may also help reveal the molecular mechanisms involved in mood regulation by estrogen.

Authors
Rincavage, HL; McDonnell, DP; Kuhn, CM
MLA Citation
Rincavage, Heather L., et al. “Expression of functional estrogen receptor beta in locus coeruleus-derived Cath.a cells..” Endocrinology, vol. 144, no. 7, July 2003, pp. 2829–35. Pubmed, doi:10.1210/en.2002-221120.
PMID
12810537
Source
pubmed
Published In
Endocrinology
Volume
144
Issue
7
Publish Date
2003
Start Page
2829
End Page
2835
DOI
10.1210/en.2002-221120

Synthetic LXXLL peptide antagonize 1,25-dihydroxyvitamin D3-dependent transcription.

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) through its ability to regulate cellular programs of gene expression. We identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides using a mammalian two-hybrid system and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. Peptides were identified that were reactive to RXR alone as well as to both VDR and RXR. Peptide fusion proteins were then examined in MC3T3 E1 cells for their ability to block induction of the osteocalcin promoter by 1,25(OH)(2)D(3) or stimulation of an RARE-TK reporter by 9-cis RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)(2)D(3)-dependent transcription by up to 75%. Peptides that interacted with RXR blocked 9-cis RA induced transcription. Two RXR-interacting peptides, however, were also found to block 1,25(OH)(2)D(3) response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)(2)D(3)-induced osteocalcin gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)(2)D(3).

Authors
Pike, JW; Pathrose, P; Barmina, O; Chang, C-Y; McDonnell, DP; Yamamoto, H; Shevde, NK
MLA Citation
Pike, J. Wesley, et al. “Synthetic LXXLL peptide antagonize 1,25-dihydroxyvitamin D3-dependent transcription..” J Cell Biochem, vol. 88, no. 2, Feb. 2003, pp. 252–58. Pubmed, doi:10.1002/jcb.10336.
PMID
12520523
Source
pubmed
Published In
Journal of Cellular Biochemistry
Volume
88
Issue
2
Publish Date
2003
Start Page
252
End Page
258
DOI
10.1002/jcb.10336

MTA-1 (metastasis associated protein-1) is upregulated in tamoxifen-resistant breast cancer

Authors
Blackwell, KL; Dewhirst, MW; McDonnell, DP; Dressman, H; Snyder, SA; Marks, JR
MLA Citation
Blackwell, K. L., et al. “MTA-1 (metastasis associated protein-1) is upregulated in tamoxifen-resistant breast cancer.” Breast Cancer Research and Treatment, vol. 82, KLUWER ACADEMIC PUBL, 2003, pp. S88–S88.
Source
wos
Published In
Breast Cancer Research and Treatment
Volume
82
Publish Date
2003
Start Page
S88
End Page
S88

Aromasin and its 17-hydro metabolite manifest androgenic activity which may be responsible for its bone protective effect.

Authors
Chang, CY; Jansen, M; Sathya, G; McDonnell, DP
MLA Citation
Chang, C. Y., et al. “Aromasin and its 17-hydro metabolite manifest androgenic activity which may be responsible for its bone protective effect..” Breast Cancer Research and Treatment, vol. 82, KLUWER ACADEMIC PUBL, 2003, pp. S103–S103.
Source
wos
Published In
Breast Cancer Research and Treatment
Volume
82
Publish Date
2003
Start Page
S103
End Page
S103

Application of random peptide phage display to the study of nuclear hormone receptors.

Authors
Chang, C-Y; Norris, JD; Jansen, M; Huang, H-J; McDonnell, DP
MLA Citation
Chang, Ching-yi, et al. “Application of random peptide phage display to the study of nuclear hormone receptors..” Methods Enzymol, vol. 364, 2003, pp. 118–42. Pubmed, doi:10.1016/s0076-6879(03)64007-3.
PMID
14631842
Source
pubmed
Published In
Methods in Enzymology
Volume
364
Publish Date
2003
Start Page
118
End Page
142
DOI
10.1016/s0076-6879(03)64007-3

Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic LXXLL peptide antagonists that target the activation domains of the vitamin D and retinoid X receptors.

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through its ability to regulate cellular programs of gene expression. Although RXR appears to participate as a heterodimeric partner with the VDR, absolute evidence for its role remains equivocal in vivo. To test this role and to investigate the requirement for comodulator interaction, we identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. We used a mammalian cell two-hybrid system to screen a series of nuclear receptor (NR)-reactive LXXLL peptides previously identified through phage display screening for hormone-dependent reactivity with either VDR or RXR. Three categories of peptides were identified: those reactive with both VDR and RXR, those selective for RXR, and those unreactive to either receptor. Peptide fusion proteins were then examined in MC3T3-E1 cells for their ability to block induction of the osteocalcin (OC) promoter by 1,25(OH)2D3 or stimulation of a retinoic acid response element-thymidine kinase (RARE-TK) reporter by 9-cis-RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)2D3-dependent transcription by up to 75%. Control LXXLL sequences derived from Src-1 and Grip also suppressed 1,25(OH)2D3-induced transactivation; peptides that interacted with RXR blocked 9-cis-RA-induced transcription. Interestingly, two RXR-interacting peptides were also found to block 1,25(OH)2D3 response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)2D3-induced OC gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)2D3.

Authors
Pathrose, P; Barmina, O; Chang, C-Y; McDonnell, DP; Shevde, NK; Pike, JW
MLA Citation
Pathrose, Peterson, et al. “Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic LXXLL peptide antagonists that target the activation domains of the vitamin D and retinoid X receptors..” J Bone Miner Res, vol. 17, no. 12, Dec. 2002, pp. 2196–205. Pubmed, doi:10.1359/jbmr.2002.17.12.2196.
PMID
12469913
Source
pubmed
Published In
Journal of Bone and Mineral Research
Volume
17
Issue
12
Publish Date
2002
Start Page
2196
End Page
2205
DOI
10.1359/jbmr.2002.17.12.2196

Problems for risk assessment of endocrine-active estrogenic compounds.

Estrogenic industrial compounds such as bisphenol A (BPA) and nonylphenol typically bind estrogen receptor (ER) alpha and ERBeta and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually greater than or equal to 1,000-fold lower than observed for 17Beta-estradiol. Risk assessment of estrogenic compounds on the basis of their potencies in simple reporter gene or binding assays may be inappropriate. For example, selective ER modulators (SERMs) represent another class of synthetic estrogens being developed for treatment of hormone-dependent problems. SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with an estrogen-responsive complement C3 promoter-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4 -hydroxytamoxifen; BPA; 2 ,4 ,6 -trichloro-4-biphenylol; 2 ,3 ,4 ,5 -tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane. The results show that synthetic and phytoestrogens are weakly estrogenic but induce distinct patterns of ER agonist/antagonist activities that are cell context- and promoter-dependent, suggesting that these compounds will induce tissue-specific (in vivo(ER agonist or antagonist activities. These results suggest that other receptors, such as the aryl hydrocarbon receptor, that also bind structurally diverse ligands may exhibit unique responses in vivo that are not predicted by standard in vitro bioassays.

Authors
Safe, SH; Pallaroni, L; Yoon, K; Gaido, K; Ross, S; McDonnell, D
MLA Citation
Safe, Stephen H., et al. “Problems for risk assessment of endocrine-active estrogenic compounds..” Environ Health Perspect, vol. 110 Suppl 6, Dec. 2002, pp. 925–29. Pubmed, doi:10.1289/ehp.02110s6925.
PMID
12634121
Source
pubmed
Published In
Environmental Health Perspectives
Volume
110 Suppl 6
Publish Date
2002
Start Page
925
End Page
929
DOI
10.1289/ehp.02110s6925

Identification and Characterization of Novel Estrogen Receptor-β-Sparing Antiprogestins.

Authors
Sathya, G; Jansen, MS; Nagel, SC; Cook, CE; McDonnell, DP
MLA Citation
Sathya, Ganesan, et al. “Identification and Characterization of Novel Estrogen Receptor-β-Sparing Antiprogestins..” Endocrinology, vol. 143, no. 8, Aug. 2002, pp. 3071–82. Pubmed, doi:10.1210/endo.143.8.8942.
PMID
28201467
Source
pubmed
Published In
Endocrinology
Volume
143
Issue
8
Publish Date
2002
Start Page
3071
End Page
3082
DOI
10.1210/endo.143.8.8942

Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists.

Several lines of evidence have indicated that the estrogen receptor (ER) can recruit the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT), to target genes in the presence of tamoxifen, suggesting a possible role for NCoR/SMRT in regulating ER pharmacology. However, a tamoxifen-dependent, direct interaction between NCoR/SMRT and ER in vitro has not been demonstrated. To investigate the possible involvement of different corepressors in the actions of antiestrogen-bound ER, we have constructed a phage display library that expresses 23-amino acid peptides containing the canonical CoRNR box motif in an otherwise random background. Screening of the CoRNR box library with apo-ER or ER treated with tamoxifen or ICI 182,780 led to the isolation of peptides whose ability to interact with ER was influenced by the nature of the bound ligand. Using a series of ERalpha mutants, we found that helix 12 was not required for the binding of CoRNR box peptides, whereas disruption of helixes 3 and 5 had a marked effect on peptide binding. One mutant, ER-L372R, lost the ability to interact with CoRNR box-containing peptides without affecting its binding to LXXLL motif-containing peptides. The estradiol- and tamoxifen-mediated transcriptional activity of ER-L372R was dramatically increased by 11- and 3-fold, respectively, compared with that of wild-type ERalpha. The ICI 182,780-mediated repressional activity of this mutant was also reduced by 4-fold compared with that of wild-type ERalpha. These results suggest that leucine 372 may be an important part of the interaction surface on ER that is responsible for corepressor binding. In addition, our data suggest that corepressors, other than NCoR/SMRT, may be involved in ER signaling.

Authors
Huang, H-J; Norris, JD; McDonnell, DP
MLA Citation
Huang, Huey-Jing, et al. “Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists..” Mol Endocrinol, vol. 16, no. 8, Aug. 2002, pp. 1778–92. Pubmed, doi:10.1210/me.2002-0089.
PMID
12145334
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
16
Issue
8
Publish Date
2002
Start Page
1778
End Page
1792
DOI
10.1210/me.2002-0089

Identification and characterization of novel estrogen receptor-beta-sparing antiprogestins.

The steroid hormones estrogen and progesterone together regulate the development and maintenance of the female reproductive system. The actions of these two hormones are mediated by their respective nuclear receptors located within overlapping cell populations in target organs. The molecular mechanism of action of these two hormones has been defined to a large extent using estrogen receptor (ER) and progesterone receptor (PR) antagonists. In the case of ER, the available antagonists are highly receptor selective. With respect to PR, however, the available antiprogestins also interact with the receptors for glucocorticoids, mineralocorticoids, and androgens. Whereas these cross-reactivities can usually be managed in studies of female reproductive function, it is the recent demonstration that RU486 is an effective antagonist of the beta-isoform of ER that suggested the need for more selective antiprogestins. In this study, we used cell-based transcriptional assays combined with screens using coactivator peptide analogs to identify two novel classes of antiprogestins that distinguish themselves from the antiprogestin RU486 in the manner they interact with PR. One class exhibits the characteristics of a pure antiprogestin in that its members bind to the receptor and induce a conformational change that prevents the presentation of two potential coactivator binding surfaces on the protein. The second class of compounds distinguish themselves from RU486 in that they are ERbeta sparing. When tested in vivo the ER-sparing antiprogestins were as effective as RU486 in suppressing superovulation. It is anticipated that the availability of these new antiprogestins will advance the studies of PR pharmacology in a manner similar to how the availability of selective ER modulators has helped the study of ER action.

Authors
Sathya, G; Jansen, MS; Nagel, SC; Cook, CE; McDonnell, DP
MLA Citation
Sathya, Ganesan, et al. “Identification and characterization of novel estrogen receptor-beta-sparing antiprogestins..” Endocrinology, vol. 143, no. 8, Aug. 2002, pp. 3071–82. Pubmed, doi:10.1210/endo.143.8.8942.
PMID
12130573
Source
pubmed
Published In
Endocrinology
Volume
143
Issue
8
Publish Date
2002
Start Page
3071
End Page
3082
DOI
10.1210/endo.143.8.8942

Elucidation of the molecular mechanism of action of selective estrogen receptor modulators.

The term selective estrogen receptor modulator (SERM) describes a group of pharmaceuticals that manifest estrogen receptor (ER) agonist activity in some tissues but opposes estrogen action in others. Although the name describing this class of drugs is new, the concept is not, as compounds exhibiting tissue-selective ER agonist/antagonist properties have been available for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of SERMs and develop them as target organ-selective ER agonists for the treatment of osteoporosis and other estrogenopathies. This realization has provided the impetus for research in this area, the progress of which is described in this review.

Authors
McDonnell, DP; Wijayaratne, A; Chang, C-Y; Norris, JD
MLA Citation
McDonnell, Donald P., et al. “Elucidation of the molecular mechanism of action of selective estrogen receptor modulators..” Am J Cardiol, vol. 90, no. 1A, July 2002, pp. 35F-43F. Pubmed, doi:10.1016/s0002-9149(01)02221-4.
PMID
12106639
Source
pubmed
Published In
The American Journal of Cardiology
Volume
90
Issue
1A
Publish Date
2002
Start Page
35F
End Page
43F
DOI
10.1016/s0002-9149(01)02221-4

Direct interactions between corepressors and coactivators permit the integration of nuclear receptor-mediated repression and activation.

The unliganded thyroid hormone receptor beta (TRbeta) represses the basal transcriptional activity of target genes, in part through interactions with the nuclear receptor corepressor (N-CoR). In this study we have identified a rather unexpected interaction between N-CoR and the nuclear receptor coactivator ACTR. We have demonstrated in vitro and in intact cells that N-CoR directly associates with ACTR and that the interaction surfaces on N-CoR and ACTR are distinct from those required for TR binding. The significance of this finding was demonstrated by showing that N-CoR facilitates an interaction between unliganded-TRbeta and ACTR. One possible consequence of the formation of the trimeric complex of N-CoR/ACTR/unliganded-TR is that N-CoR may raise the local concentration of ACTR at target gene promoters. In support of this hypothesis it was demonstrated that the presence of N-CoR can enhance TRbeta-mediated transcriptional activation. It is proposed, therefore, that TRbeta- mediated activation and repression are integrally linked in a manner that is not predicted by the current models of nuclear receptor action.

Authors
Li, X; Kimbrel, EA; Kenan, DJ; McDonnell, DP
MLA Citation
Li, Xiaolin, et al. “Direct interactions between corepressors and coactivators permit the integration of nuclear receptor-mediated repression and activation..” Mol Endocrinol, vol. 16, no. 7, July 2002, pp. 1482–91. Pubmed, doi:10.1210/mend.16.7.0860.
PMID
12089344
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
16
Issue
7
Publish Date
2002
Start Page
1482
End Page
1491
DOI
10.1210/mend.16.7.0860

The transcription factor B-Myb is maintained in an inhibited state in target cells through its interaction with the nuclear corepressors N-CoR and SMRT.

The B-Myb transcription factor has been implicated in coordinating the expression of genes involved in cell cycle regulation. Although it is expressed in a ubiquitous manner, its transcriptional activity is repressed until the G(1)-S phase of the cell cycle by an unknown mechanism. In this study we used biochemical and cell-based assays to demonstrate that the nuclear receptor corepressors N-CoR and SMRT interact with B-Myb. The significance of these B-Myb-corepressor interactions was confirmed by the finding that B-Myb mutants, which were unable to bind N-CoR, exhibited constitutive transcriptional activity. It has been shown previously that phosphorylation of B-Myb by cdk2/cyclin A enhances its transcriptional activity. We have now determined that phosphorylation by cdk2/cyclin A blocks the interaction between B-Myb and N-CoR and that mutation of the corepressor binding site within B-Myb bypasses the requirement for this phosphorylation event. Cumulatively, these findings suggest that the nuclear corepressors N-CoR and SMRT serve a previously unappreciated role as regulators of B-Myb transcriptional activity.

Authors
Li, X; McDonnell, DP
MLA Citation
Li, Xiaolin, and Donald P. McDonnell. “The transcription factor B-Myb is maintained in an inhibited state in target cells through its interaction with the nuclear corepressors N-CoR and SMRT..” Mol Cell Biol, vol. 22, no. 11, June 2002, pp. 3663–73. Pubmed, doi:10.1128/mcb.22.11.3663-3673.2002.
PMID
11997503
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
11
Publish Date
2002
Start Page
3663
End Page
3673
DOI
10.1128/mcb.22.11.3663-3673.2002

Connections and regulation of the human estrogen receptor.

Estrogen regulates a plethora of functionally dissimilar processes in a broad range of tissues. Recent progress in the study of the molecular mechanism of action of estrogen(s) has revealed why different cells can respond to the same hormone in a different manner. Three of these findings are of particular importance: (i) There are two genetically and functionally distinct estrogen receptors that have distinct expression patterns in vivo; (ii) the positive and negative transcriptional activities of these receptors require them to engage transcription cofactors (coactivators or corepressors) in target cells; and (iii) not all cofactors are functionally equivalent, nor are they expressed in the same manner in all cells. Thus, although the estrogen receptor is required for a cell to respond to an estrogenic stimulus, the nature and extent of that response are determined by the proteins, pathways, and processes with which the receptor interacts.

Authors
McDonnell, DP; Norris, JD
MLA Citation
McDonnell, Donald P., and John D. Norris. “Connections and regulation of the human estrogen receptor..” Science, vol. 296, no. 5573, May 2002, pp. 1642–44. Pubmed, doi:10.1126/science.1071884.
PMID
12040178
Source
pubmed
Published In
Science
Volume
296
Issue
5573
Publish Date
2002
Start Page
1642
End Page
1644
DOI
10.1126/science.1071884

Common estrogen receptor polymorphism augments effects of hormone replacement therapy on E-selectin but not C-reactive protein.

BACKGROUND: The estrogen receptor-alpha (ER-alpha) IVS1-401 polymorphism identifies a group of women (approximately 20%) who have augmented effects of hormone replacement therapy (HRT) on levels of HDL cholesterol. This study sought to determine if this augmentation extends to HRT regulation of E-selectin and C-reactive protein (CRP) and to explore possible mechanisms by which this polymorphism might influence estrogen action. METHODS AND RESULTS: Serum levels of soluble E-selectin and CRP were measured at baseline and 1 year in 264 postmenopausal women randomized to treatment with oral conjugated equine estrogen (0.625 mg/d), estrogen plus progestin (medroxyprogesterone acetate 2.5 mg/d), or placebo. Women with the ER-alpha IVS1-401 C/C genotype receiving HRT had nearly a 2-fold greater reduction in E-selectin compared with C/T or T/T women (P for interaction=0.02). In contrast, there was no augmentation of the HRT-associated increase in CRP among the C/C women compared with C/T or T/T women (P for interaction=0.54). Of luciferase reporter constructs containing sequences spanning the IVS1-401 T/C polymorphism, expression of the construct containing the C allele was enhanced >10-fold, with cotransfection of a constitutively expressed B-myb vector. In contrast, B-myb resulted in only a 2.5-fold increase in expression of the T allele construct. CONCLUSIONS: Women with the ER-alpha IVS1-401 C/C genotype have greater reductions in E-selectin but no further increases in CRP with HRT. The C allele produces a functional binding site for the transcription factor B-myb. The impact of this polymorphism on ER-alpha transcription and other estrogen-sensitive intermediate and clinical end points has not yet been established.

Authors
Herrington, DM; Howard, TD; Brosnihan, KB; McDonnell, DP; Li, X; Hawkins, GA; Reboussin, DM; Xu, J; Zheng, SL; Meyers, DA; Bleecker, ER
MLA Citation
Herrington, David M., et al. “Common estrogen receptor polymorphism augments effects of hormone replacement therapy on E-selectin but not C-reactive protein..” Circulation, vol. 105, no. 16, Apr. 2002, pp. 1879–82. Pubmed, doi:10.1161/01.cir.0000016173.98826.88.
PMID
11997270
Source
pubmed
Published In
Circulation
Volume
105
Issue
16
Publish Date
2002
Start Page
1879
End Page
1882
DOI
10.1161/01.cir.0000016173.98826.88

Evaluation of ligand-dependent changes in AR structure using peptide probes.

Mutations in the AR are frequently found in relapsed prostate cancers, some of which permit antiandrogens as well as nonandrogenic compounds to function as androgens. However, the molecular mechanism(s) by which these mutations enable this aberrant AR pharmacology is still unknown. To explore this issue, we used a series of LxxLL-containing peptides (L, leucine; x, any amino acid) to probe the conformation of the AF-2/coactivator binding pocket of AR and AR mutants when complexed with different ligands. We have identified in a previous study two peptides that bind to the wild-type AR in an agonist-dependent manner. Interestingly, we found these same peptides also interacted with several AR variants that are frequently found in antihormone refractory prostate cancers, in the presence of either androgens or antiandrogens. This suggests that the agonist activity of antiandrogens and other physiologically relevant ligands occurs because they, in the background of these mutations, allow AR-AF2 to adopt an active conformation. Initially, this result appeared to contradict the findings of others that suggest that coactivator binding to AR-AF2 is not required for AR activity. In probing this paradox further, however, we determined that the role of AR-AF2 appears to be to stabilize the overall structure of the receptor, allowing the amino terminus to interact with appropriate coactivators. This conclusion is supported by our finding that overexpression of the AF2-binding peptides blocks the interaction between the amino and carboxyl termini of AR but does not attenuate AR transcriptional activity. This can be explained by the fact that overexpression of the LxxLL-containing peptide or the amino terminus of AR appears to have a similar effect on the AR-ligand binding domain, as both have the ability to stabilize agonist binding by decreasing ligand off-rate. Thus, we believe that resistance in certain prostate cancers occurs as a consequence of receptor mutations that enable antagonist-and/or nonclassical ligand-bound AR to present a wild-type-like AF-2 conformation.

Authors
Chang, C-Y; McDonnell, DP
MLA Citation
Chang, Ching-Yi, and Donald P. McDonnell. “Evaluation of ligand-dependent changes in AR structure using peptide probes..” Mol Endocrinol, vol. 16, no. 4, Apr. 2002, pp. 647–60. Pubmed, doi:10.1210/mend.16.4.0818.
PMID
11923463
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
16
Issue
4
Publish Date
2002
Start Page
647
End Page
660
DOI
10.1210/mend.16.4.0818

A negative coregulator for the human ER.

ERalpha is a ligand-activated transcription factor and a key regulator of the processes involved in cellular proliferation and differentiation. In addition, aberrant ERalpha activity is linked to several pathological conditions including breast cancer. A complex network of coregulatory proteins is largely believed to determine the transcriptional activity of ERalpha. We report here the isolation of a protein, denoted RTA for repressor of tamoxifen transcriptional activity, which contains an RNA recognition motif and interacts with the receptor N-terminal activation domain. RTA interacts with RNA in vitro, and its overexpression inhibits the partial agonist activity manifest by the antiestrogen tamoxifen while minimally affecting E2-activated transcription. Mutation of the RNA recognition motif alters RNA binding specificity and results in a dominant negative form of RTA that leads to derepression of ERalpha transcriptional activity, allowing all classes of antiestrogens to manifest partial agonist activity and enhancing agonist efficacy. These findings suggest a role for RNA binding proteins as coregulatory factors of the nuclear receptor family and reveal a novel mechanism by which antiestrogens can manifest agonist activities in some tissues.

Authors
Norris, JD; Fan, D; Sherk, A; McDonnell, DP
MLA Citation
Norris, John D., et al. “A negative coregulator for the human ER..” Mol Endocrinol, vol. 16, no. 3, Mar. 2002, pp. 459–68. Pubmed, doi:10.1210/mend.16.3.0787.
PMID
11875103
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
16
Issue
3
Publish Date
2002
Start Page
459
End Page
468
DOI
10.1210/mend.16.3.0787

Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer.

Some aspects of ligand-regulated transcription activation by the estrogen receptor (ER) are associated with the estrogen-dependent formation of a hydrophobic cleft on the receptor surface. At least in vitro, this cleft is required for direct interaction of ER with an alpha helix, containing variants of the sequence LXXLL, found in many coactivators. In cells, it is unknown whether ER interactions with the different LXXLL-containing helices are uniformly similar or whether they vary with LXXLL sequence or activating ligand. Using fluorescence resonance energy transfer (FRET), we confirm in the physiological environment a direct interaction between the estradiol (E2)-bound ER and LXXLL peptides expressed in living cells as fusions with spectral variants of the green fluorescent protein. This interaction was blocked by a single amino acid mutation in the hydrophobic cleft. No FRET was detected when cells were incubated with the antiestrogenic ligands tamoxifen and ICI 182,780. E2, diethylstilbestrol, ethyl indenestrol A, and 6,4'-dihydroxyflavone all promoted FRET and activated ER-dependent transcription. Measurement of the level of FRET of ER with different LXXLL-containing peptides suggested that the orientations or affinities of the LXXLL interactions with the hydrophobic cleft were globally similar but slightly different for some activating ligands.

Authors
Weatherman, RV; Chang, C-Y; Clegg, NJ; Carroll, DC; Day, RN; Baxter, JD; McDonnell, DP; Scanlan, TS; Schaufele, F
MLA Citation
Weatherman, R. V., et al. “Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer..” Mol Endocrinol, vol. 16, no. 3, Mar. 2002, pp. 487–96. Pubmed, doi:10.1210/mend.16.3.0813.
PMID
11875107
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
16
Issue
3
Publish Date
2002
Start Page
487
End Page
496
DOI
10.1210/mend.16.3.0813

Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements.

Hormone-activated ERs (ERalpha and ERbeta) bind with high affinity to specific DNA sequences, estrogen response elements (EREs), located within the regulatory regions of target genes. Once considered to function solely as receptor tethers, there is an increasing amount of recent evidence to suggest that the sequence of the ERE can influence receptor activity. In this study, we have performed a systematic analysis of the role of different EREs in ER pharmacology. Specifically, by measuring ER activity on the vitellogenin A2, complement 3 gene, pS2, and lactoferrin EREs, we demonstrate that the activities of E2 and xenoestrogen ligands through ERalpha and ERbeta are significantly influenced by the nature of the response element. Using a series of ERalpha and ERbeta interacting peptides that contain the coactivator-binding motif LXXLL, we show that the type of ERE with which the receptor associates regulates the structure of the coactivator pocket on ER. Furthermore, using a novel ELISA developed to measure ER-coactivator interactions revealed that these different conformational states of ERalpha and ERbeta are functionally relevant, as they dictate receptor coactivator binding preference. Together, these results indicate that the DNA response element is a key regulator of receptor structure and biological activity and suggest the ERE sequence influences the recruitment of coactivators to the ER at target gene promoters. We propose that DNA-induced alteration of protein structure and coregulator recruitment may serve as a universal regulatory component for differential gene expression by other nuclear hormone receptors and unrelated transcription factors.

Authors
Hall, JM; McDonnell, DP; Korach, KS
MLA Citation
Hall, Julie M., et al. “Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements..” Mol Endocrinol, vol. 16, no. 3, Mar. 2002, pp. 469–86. Pubmed, doi:10.1210/mend.16.3.0814.
PMID
11875105
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
16
Issue
3
Publish Date
2002
Start Page
469
End Page
486
DOI
10.1210/mend.16.3.0814

Definition of the molecular and cellular mechanisms underlying the tissue-selective agonist/antagonist activities of selective estrogen receptor modulators.

The term selective estrogen receptor modulators describes a group of pharmaceuticals that function as estrogen receptor (ER) agonists in some tissues but that oppose estrogen action in others. Although the name for this class of drugs has been adopted only recently, the concept is not new, as compounds exhibiting tissue-selective ER agonist/antagonist properties have been around for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of these drugs and develop them as target organ-selective ER agonists for the treatment of osteoporosis and other estrogenopathies. This realization has provided the impetus for research in this area, the progress of which is discussed in this review.

Authors
McDonnell, DP; Connor, CE; Wijayaratne, A; Chang, C-Y; Norris, JD
MLA Citation
McDonnell, Donald P., et al. “Definition of the molecular and cellular mechanisms underlying the tissue-selective agonist/antagonist activities of selective estrogen receptor modulators..” Recent Prog Horm Res, vol. 57, 2002, pp. 295–316.
PMID
12017549
Source
pubmed
Published In
Recent Progress in Hormone Research
Volume
57
Publish Date
2002
Start Page
295
End Page
316

Glucocorticoids manifest androgenic activity in a cell line derived from a metastatic prostate cancer.

The pathophysiological mechanism(s) by which androgen independence develops in prostate cancer remains to be determined. The identification in many prostate cancer specimens of a mutant androgen receptor, T877A, with altered ligand specificity has provided an explanation for some treatment failures. The T877A mutant androgen receptor recognizes a number of nonandrogenic compounds, including certain estrogens, progestins, and even antiandrogens as androgens. However, a comprehensive screen for hormonal agents which display agonist activity on this mutant has not been performed. In this study, we characterized this clinically important receptor mutant further and found that it can be activated by a wide range of compounds, including a number of endogenous glucocorticoids. Among the most clinically relevant compounds identified are DOC and corticosterone, both of which can effectively activate the mutant receptor at concentrations normally found in blood. Dexamethasone, a synthetic glucocorticoid frequently used in various contexts for prostate cancer therapy, is also recognized as an androgen by the mutant receptor. These unexpected findings suggest the need to: (a) reassess the role of adrenally derived glucocorticoids in prostate cancer disease progression; and (b) recognize the potential for iatrogenic stimulation of disease progression with certain glucocorticoid interventions.

Authors
Chang, CY; Walther, PJ; McDonnell, DP
MLA Citation
Chang, C. Y., et al. “Glucocorticoids manifest androgenic activity in a cell line derived from a metastatic prostate cancer..” Cancer Res, vol. 61, no. 24, Dec. 2001, pp. 8712–17.
PMID
11751389
Source
pubmed
Published In
Cancer Research
Volume
61
Issue
24
Publish Date
2001
Start Page
8712
End Page
8717

Capitalizing on the complexities of estrogen receptor pharmacology in the quest for the perfect SERM.

The term Selective Estrogen Receptor Modulators (SERMs) has been used of late to describe a group of pharmaceuticals that manifest estrogen receptor (ER) agonist activity in some tissues, but that oppose estrogen action in others. Whereas the name describing this class of drugs is new, the concept is not. Indeed, compounds exhibiting tissue-selective ER agonist/antagonist properties have been around for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of these drugs and develop them as treatments for estrogenopathies where it is desirable to direct therapy to a specific estrogen-responsive target organ. This realization has provided the impetus for research in this area and has pushed the development and clinical use of this class of drugs. The objective of this review is to describe how the medical need for SERMs arose and how recent studies of the mechanism of action of the currently available drugs are paving the way for the development of novel drugs with improved selectivity.

Authors
McDonnell, DP; Chang, CY; Norris, JD
MLA Citation
McDonnell, D. P., et al. “Capitalizing on the complexities of estrogen receptor pharmacology in the quest for the perfect SERM..” Ann N Y Acad Sci, vol. 949, Dec. 2001, pp. 16–35. Pubmed, doi:10.1111/j.1749-6632.2001.tb03999.x.
PMID
11795348
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
949
Publish Date
2001
Start Page
16
End Page
35
DOI
10.1111/j.1749-6632.2001.tb03999.x

Development of an ER action indicator mouse for the study of estrogens, selective ER modulators (SERMs), and Xenobiotics.

We have developed a transgenic mouse that functions as a reporter of ER activity, termed ER action indicator (ERIN), by incorporating a transgene with an estrogen-responsive promoter (three copies of the vitellogenin estrogen response element with a minimal thymidine kinase promoter) linked to the reporter gene beta-galactosidase. Evaluation of ER activity in female ERIN mice demonstrated estrogen-inducible expression of the reporter gene in the uterus, pituitary, and hypothalamus; established targets of estrogen action. Importantly, we also identified ER activity in a number of nonclassical estrogen target tissues, including kidney, liver, adrenal, and thyroid gland. ERIN provides a system to measure the same end point (transgene regulation) in different target tissues, permitting separation of the contributions of cell- and promoter-specific factors in determining ER pharmacology. In this regard we observed that on this specific promoter the pituitary gland was 25-fold more sensitive than the uterus to the estrogen diethylstilbestrol, implying the existence of cell-specific factors that influence ligand sensitivity. Our studies also identified considerable difference in the efficacy and potency of ER ligands in the uterus when ER transcriptional activity was assayed vs. uterine weight gain. Specifically, we observed that the environmental estrogen bisphenol A was a potent agonist in stimulating ER transcriptional activity, whereas it exhibited little uterotropic activity. In contrast to bisphenol A, tamoxifen significantly increased uterine weight, but minimally induced ER reporter activity in this tissue. Given the results of these studies, we believe that ERIN will be a useful model to evaluate ER ligand pharmacology and will assist in defining the cellular and molecular mechanisms that determine agonist and antagonist activity.

Authors
Nagel, SC; Hagelbarger, JL; McDonnell, DP
MLA Citation
Nagel, S. C., et al. “Development of an ER action indicator mouse for the study of estrogens, selective ER modulators (SERMs), and Xenobiotics..” Endocrinology, vol. 142, no. 11, Nov. 2001, pp. 4721–28. Pubmed, doi:10.1210/endo.142.11.8471.
PMID
11606437
Source
pubmed
Published In
Endocrinology
Volume
142
Issue
11
Publish Date
2001
Start Page
4721
End Page
4728
DOI
10.1210/endo.142.11.8471

The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators.

The human estrogen receptor alpha-isoform (ERalpha) is a nuclear transcription factor that displays a complex pharmacology. In addition to classical agonists and antagonists, the transcriptional activity of ERalpha can be regulated by selective estrogen receptor modulators, a new class of drugs whose relative agonist/antagonist activity is determined by cell context. It has been demonstrated that the binding of different ligands to ERalpha results in the formation of unique ERalpha-ligand conformations. These conformations have been shown to influence ERalpha-cofactor binding and, therefore, have a profound impact on ERalpha pharmacology. In this study, we demonstrate that the nature of the bound ligand also influences the stability of ERalpha, revealing an additional mechanism by which the pharmacological activity of a compound is determined. Of note we found that although all ERalpha-ligand complexes can be ubiquitinated and degraded by the 26 S proteasome in vivo, the mechanisms by which they are targeted for proteolysis appear to be different. Specifically, for agonist-activated ERalpha, an inverse relationship between transcriptional activity and receptor stability was observed. This relationship does not extend to selective estrogen receptor modulators and pure antagonists. Instead, it appears that with these compounds, the determinant of receptor stability is the ligand-induced conformation of ERalpha. We conclude that the different conformational states adopted by ERalpha in the presence of different ligands influence transcriptional activity directly by regulating cofactor binding and indirectly by modulating receptor stability.

Authors
Wijayaratne, AL; McDonnell, DP
MLA Citation
Wijayaratne, A. L., and D. P. McDonnell. “The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators..” J Biol Chem, vol. 276, no. 38, Sept. 2001, pp. 35684–92. Pubmed, doi:10.1074/jbc.M101097200.
PMID
11473106
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
276
Issue
38
Publish Date
2001
Start Page
35684
End Page
35692
DOI
10.1074/jbc.M101097200

Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic peptide antagonists that target the activation domain of retinoid X receptor.

Authors
Pathrose, P; Chang, CY; McDonnell, DP; Pike, JW
MLA Citation
Pathrose, P., et al. “Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic peptide antagonists that target the activation domain of retinoid X receptor..” Journal of Bone and Mineral Research, vol. 16, AMER SOC BONE & MINERAL RES, Sept. 2001, pp. S184–S184.
Source
wos
Published In
Journal of Bone and Mineral Research
Volume
16
Publish Date
2001
Start Page
S184
End Page
S184

Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates.

The ubiquitin-protein ligase (E3), hRPF1/Nedd4, is a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity. Although previously characterized as a regulator of the stability of cytoplasmic proteins, hRPF1/Nedd4 has also been suggested to have a role in the nucleus. However, in light of the cytoplasmic localization of hRPF1/Nedd4, it is unclear whether bona fide nuclear substrates of hRPF1/Nedd4 exist, and if so, what mechanism may allow a cytoplasmic ubiquitin ligase to manifest nuclear activity. Our search for nuclear substrates led to the identification of the human proline-rich transcript, brain-expressed (hPRTB) protein, the ubiquitination and degradation of which is regulated by hRPF1/Nedd4. Interestingly, hPRTB colocalizes with the splicing factor SC35 in nuclear speckles. Finally, we demonstrate that hRPF1/Nedd4 is indeed capable of entering the nucleus; however, the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. Cumulatively, these findings highlight a nuclear role for the ubiquitin ligase hRPF1/Nedd4 and underscore cytoplasmic/nuclear localization as an important regulatory component of hRPF1/Nedd4-substrate recognition.

Authors
Hamilton, MH; Tcherepanova, I; Huibregtse, JM; McDonnell, DP
MLA Citation
Hamilton, M. H., et al. “Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates..” J Biol Chem, vol. 276, no. 28, July 2001, pp. 26324–31. Pubmed, doi:10.1074/jbc.M101205200.
PMID
11342538
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
276
Issue
28
Publish Date
2001
Start Page
26324
End Page
26331
DOI
10.1074/jbc.M101205200

The molecular pharmacology of estrogen receptor agonists, antagonists and SERMs

Authors
McDonnell, DP; Connor, CE; Hall, J; Chang, CY; Jansen, MS; Norris, JD
MLA Citation
McDonnell, D. P., et al. “The molecular pharmacology of estrogen receptor agonists, antagonists and SERMs.” Bone, vol. 28, no. 5, ELSEVIER SCIENCE INC, May 2001, pp. S68–S68.
Source
wos
Published In
Bone
Volume
28
Issue
5
Publish Date
2001
Start Page
S68
End Page
S68

Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor.

Tamoxifen inhibits estrogen receptor (ER) transcriptional activity by competitively inhibiting estradiol binding and inducing conformational changes in the receptor that may prevent its interaction with coactivators. In bone, the cardiovascular system, and some breast tumors, however, tamoxifen exhibits agonist activity, suggesting that the tamoxifen-ER complex is not recognized identically in all cells. We used phage display to demonstrate that the antiestrogen GW5638 induces a unique structural change in the ER. The biological significance of this conformational change was revealed in studies that demonstrated that tamoxifen-resistant breast tumor explants are not cross-resistant to GW5638. Because of these properties, this drug is currently being developed as a potential therapeutic for tamoxifen-resistant breast cancers.

Authors
Connor, CE; Norris, JD; Broadwater, G; Willson, TM; Gottardis, MM; Dewhirst, MW; McDonnell, DP
MLA Citation
Connor, C. E., et al. “Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor..” Cancer Res, vol. 61, no. 7, Apr. 2001, pp. 2917–22.
PMID
11306468
Source
pubmed
Published In
Cancer Research
Volume
61
Issue
7
Publish Date
2001
Start Page
2917
End Page
2922

Estrogen receptor-cofactor interactions as targets for novel drug discovery.

Authors
Norris, JD; Chang, C; McDonnell, DP
MLA Citation
Norris, J. D., et al. “Estrogen receptor-cofactor interactions as targets for novel drug discovery..” Ernst Schering Research Foundation Workshop, no. 34, Jan. 2001, pp. 181–201.
PMID
11394045
Source
epmc
Published In
Organ Selective Actions of Steroid Hormones
Issue
34
Publish Date
2001
Start Page
181
End Page
201

Toxicology of environmental estrogens.

It has been hypothesized that environmental contaminants that modulate endocrine signaling pathways may be causally linked to adverse health effects in humans. There has been particular concern regarding synthetic estrogens and their role in disrupting normal development of the male reproductive tract. Most estrogenic industrial compounds, such as bisphenol A (BPA) and nonylphenol, typically bind estrogen receptors alpha (ERalpha) and beta (ERbeta) and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually > or = 1,000-fold lower than observed for 17beta-estradiol (E2). Selective estrogen receptor modulators (SERMs) represent another class of synthetic estrogens that are being developed for treatment of hormone-dependent problems. The SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with a pC3-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4'-hydroxytamoxifen; BPA; 2',4',6'-trichloro-4-biphenylol; 2',3',4',5'-tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). The results show that synthetic and phytoestrogens induce distinct patterns of gene activation in HepG2 and U2 osteogenic sarcoma cells, suggesting that these compounds will induce tissue-specific in vivo ER agonist or antagonist activities. The predicted differences between these compounds, based on results of the in vitro bioassay, have been confirmed. For example, BPA inhibits E2-induced responses in the rodent uterus, and HPTE and structurally related compounds are ERalpha agonists and ERbeta antagonists in assays carried out in HepG2 and other cancer cell lines.

Authors
Safe, SH; Pallaroni, L; Yoon, K; Gaido, K; Ross, S; Saville, B; McDonnellc, D
MLA Citation
Safe, S. H., et al. “Toxicology of environmental estrogens..” Reprod Fertil Dev, vol. 13, no. 4, 2001, pp. 307–15.
PMID
11800169
Source
pubmed
Published In
Reproduction, Fertility, and Development
Volume
13
Issue
4
Publish Date
2001
Start Page
307
End Page
315

Development of peptide antagonists that target estrogen receptor beta-coactivator interactions.

The biological actions of estrogen are manifest through two genetically distinct estrogen receptors (ER alpha and ER beta) that display nonidentical expression patterns in target tissues. The phenotypic alterations in response to estrogens in mice disrupted for either or both of these receptors are not identical, suggesting that each subtype plays a unique role in ER-action. However, the lack of subtype-specific agonists and antagonists has made it difficult to define the processes that are regulated by ER alpha and/or ER beta. Previously, we have reported the identification and characterization of a series of LXXLL-containing peptide antagonists that block estrogen signaling by preventing the association of ER alpha with required coactivators. As expected, given the similarity of the coactivator binding pockets among nuclear receptors, most of the peptide antagonists identified inhibited the activity of multiple receptors. However, by altering sequences flanking the core LXXLL motif, some receptor selectivity was afforded. Building on this observation, we have screened combinatorial phage libraries, expressing peptides in the format X7LXXLLX7, for peptides that interact in a specific manner with ER beta. Using this approach, a series of highly specific, potent peptide antagonists have been identified that efficiently inhibit ER beta-mediated estrogen signaling when introduced into target cells. Interestingly, in cells where both ER subtypes were expressed, these ER beta antagonists were capable of attenuating ER action, suggesting that ER alpha and ER beta do indeed form functional heterodimeric complexes. We believe that suitably formulated versions of these peptides can be used to study ER beta action in vitro and in vivo. In addition, the unanticipated specificity of the peptides identified should serve as an impetus to investigate the use of this approach to develop peptide antagonists of other nuclear receptors and unrelated transcription factors.

Authors
Hall, JM; Chang, CY; McDonnell, DP
MLA Citation
Hall, J. M., et al. “Development of peptide antagonists that target estrogen receptor beta-coactivator interactions..” Mol Endocrinol, vol. 14, no. 12, Dec. 2000, pp. 2010–23. Pubmed, doi:10.1210/mend.14.12.0561.
PMID
11117531
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
14
Issue
12
Publish Date
2000
Start Page
2010
End Page
2023
DOI
10.1210/mend.14.12.0561

Temporally distinct and ligand-specific recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor.

Ligand binding to estrogen receptor (ER) is presumed to regulate the type and timing of ER interactions with different cofactors. Using fluorescence microscopy in living cells, we characterized the recruitment of five different green fluorescent protein (GFP)-labeled ER-interacting peptides to the distinct subnuclear compartment occupied by blue fluorescent protein (BFP)-labeled ER alpha. Different ligands promoted the recruitment of different peptides. One peptide was recruited in response to estradiol (E2), tamoxifen, raloxifene, or ICI 182,780 incubation whereas other peptides were recruited specifically by E2 or tamoxifen. Peptides containing different sequences surrounding the ER-interacting motif LXXLL were recruited with different time courses after E2 addition. Complex temporal kinetics also were observed for recruitment of the full-length, ER cofactor glucocorticoid receptor-interacting protein 1 (GRIP1); rapid, E2-dependent recruitment of GRIP1 was blocked by mutation of the GRIP1 LXXLL motifs to LXXAA whereas slower E2 recruitment persisted for the GRIP1 LXXAA mutant. This suggested the presence of multiple, temporally distinct GRIP 1 recruitment mechanisms. E2 recruitment of GRIP1 and LXXLL peptides was blocked by coincubation with excess ICI 182,780. In contrast, preformed E2/ER/GRIP1 and E2/ER/LXXLL complexes were resistant to subsequent ICI 182,780 addition whereas ICI 182,780 dispersed preformed complexes containing the GRIP1 LXXAA mutant. This suggested that E2-induced LXXLL binding altered subsequent ligand/ER interactions. Thus, alternative, ligand-selective recruitment and dissociation mechanisms with distinct temporal sequences are available for ER alpha action in vivo.

Authors
Schaufele, F; Chang, CY; Liu, W; Baxter, JD; Nordeen, SK; Wan, Y; Day, RN; McDonnell, DP
MLA Citation
Schaufele, F., et al. “Temporally distinct and ligand-specific recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor..” Mol Endocrinol, vol. 14, no. 12, Dec. 2000, pp. 2024–39. Pubmed, doi:10.1210/mend.14.12.0572.
PMID
11117532
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
14
Issue
12
Publish Date
2000
Start Page
2024
End Page
2039
DOI
10.1210/mend.14.12.0572

Development of peptide antagonists that target estrogen receptor-cofactor interactions.

We have developed a series of high-affinity peptide antagonists that inhibit the transcriptional activity of both subtypes of the human estrogen receptor (ERalpha and ERbeta). We believe that it will be possible to develop these peptides, or corresponding peptidomimetic derivatives, into pharmaceuticals for use in the treatment of breast cancer and other estrogenopathies. It is anticipated that drugs of this type could be used in combination with classical antiestrogens, such as tamoxifen, to achieve a complete blockage of ER-transcriptional activity. Although ER has been the primary target of our studies to date, it is likely that the insights gained from this work will apply to other nuclear receptors and transcription factors.

Authors
McDonnell, DP; Chang, CY; Norris, JD
MLA Citation
McDonnell, D. P., et al. “Development of peptide antagonists that target estrogen receptor-cofactor interactions..” J Steroid Biochem Mol Biol, vol. 74, no. 5, Nov. 2000, pp. 327–35.
PMID
11162941
Source
pubmed
Published In
The Journal of Steroid Biochemistry and Molecular Biology
Volume
74
Issue
5
Publish Date
2000
Start Page
327
End Page
335

Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: structure-activity studies.

We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE) with estrogen receptor alpha (ERalpha), ERbeta, and the androgen receptor (AR). In this study, we characterize the ERalpha, ERbeta, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERalpha, ERbeta, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17beta-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2, 2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERalpha agonist activity and ERbeta and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERalpha agonist activity and did not alter ERbeta or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERalpha and ERbeta agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERalpha and ERbeta activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.

Authors
Gaido, KW; Maness, SC; McDonnell, DP; Dehal, SS; Kupfer, D; Safe, S
MLA Citation
Gaido, K. W., et al. “Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: structure-activity studies..” Mol Pharmacol, vol. 58, no. 4, Oct. 2000, pp. 852–58.
PMID
10999957
Source
pubmed
Published In
Molecular Pharmacology
Volume
58
Issue
4
Publish Date
2000
Start Page
852
End Page
858

Modulation of estrogen receptor-alpha transcriptional activity by the coactivator PGC-1.

A transcriptional coactivator of the peroxisome proliferator-activated receptor-gamma (PPARgamma), PPARgamma-coactivator-1(PGC-1) interacts in a constitutive manner with the hinge domain of PPARgamma and enhances its transcriptional activity. In this study we demonstrate that PGC-1 is a coactivator of estrogen receptor-alpha (ERalpha)-dependent transcriptional activity. However the mechanism by which PGC-1 interacts with ERalpha is different from that of PPARgamma. Specifically, it was determined that the carboxyl terminus of PGC-1 interacts in a ligand-independent manner with the ERalpha hinge domain. In addition, an LXXLL motif within the amino terminus of PGC-1 was shown to interact in an agonist-dependent manner with the AF2 domain within the carboxyl terminus of ERalpha. The ability of PGC-1 to associate with and potentiate the transcriptional activity of an ERalpha-AF2 mutant that is unable to interact with the p160 class of coactivators suggests that this coactivator may have a unique role in estrogen signaling. It is concluded from these studies that PGC-1 is a bona fide ERalpha coactivator, which may serve as a convergence point between PPARgamma and ERalpha signaling.

Authors
Tcherepanova, I; Puigserver, P; Norris, JD; Spiegelman, BM; McDonnell, DP
MLA Citation
Tcherepanova, I., et al. “Modulation of estrogen receptor-alpha transcriptional activity by the coactivator PGC-1..” J Biol Chem, vol. 275, no. 21, May 2000, pp. 16302–08. Pubmed, doi:10.1074/jbc.M001364200.
PMID
10748020
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
275
Issue
21
Publish Date
2000
Start Page
16302
End Page
16308
DOI
10.1074/jbc.M001364200

The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding.

The human progesterone receptor (PR) exists as two functionally distinct isoforms, hPRA and hPRB. hPRB functions as a transcriptional activator in most cell and promoter contexts, while hPRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of steroid hormone receptor transcriptional activity. Although the precise mechanism of hPRA-mediated transrepression is not fully understood, an inhibitory domain (ID) within human PR, which is necessary for transrepression by hPRA, has been identified. Interestingly, although ID is present within both hPR isoforms, it is functionally active only in the context of hPRA, suggesting that the two receptors adopt distinct conformations within the cell which allow hPRA to interact with a set of cofactors that are different from those recognized by hPRB. In support of this hypothesis, we identified, using phage display technology, hPRA-selective peptides which differentially modulate hPRA and hPRB transcriptional activity. Furthermore, using a combination of in vitro and in vivo methodologies, we demonstrate that the two receptors exhibit different cofactor interactions. Specifically, it was determined that hPRA has a higher affinity for the corepressor SMRT than hPRB and that this interaction is facilitated by ID. Interestingly, inhibition of SMRT activity, by either a dominant negative mutant (C'SMRT) or histone deacetylase inhibitors, reverses hPRA-mediated transrepression but does not convert hPRA to a transcriptional activator. Together, these data indicate that the ability of hPRA to transrepress steroid hormone receptor transcriptional activity and its inability to activate progesterone-responsive promoters occur by distinct mechanisms. To this effect, we observed that hPRA, unlike hPRB, was unable to efficiently recruit the transcriptional coactivators GRIP1 and SRC-1 upon agonist binding. Thus, although both receptors contain sequences within their ligand-binding domains known to be required for coactivator binding, the ability of PR to interact with cofactors in a productive manner is regulated by sequences contained within the amino terminus of the receptors. We propose, therefore, that hPRA is transcriptionally inactive due to its inability to efficiently recruit coactivators. Furthermore, our experiments indicate that hPRA interacts efficiently with the corepressor SMRT and that this activity permits it to function as a transdominant repressor.

Authors
Giangrande, PH; Kimbrel, EA; Edwards, DP; McDonnell, DP
MLA Citation
Giangrande, P. H., et al. “The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding..” Mol Cell Biol, vol. 20, no. 9, May 2000, pp. 3102–15. Pubmed, doi:10.1128/mcb.20.9.3102-3115.2000.
PMID
10757795
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
9
Publish Date
2000
Start Page
3102
End Page
3115
DOI
10.1128/mcb.20.9.3102-3115.2000

Selective estrogen receptor modulators (SERMs): A first step in the development of perfect hormone replacement therapy regimen.

Until 1986, our understanding of estrogen receptor (ER) action was based on information derived from in vitro biochemical analyses and in vivo correlations. With the cloning of the human ER cDNA, the reconstitution of ER responsive transcription units in heterologous cells has permitted the genetic dissection of the ER signal transduction pathway. The recent discovery of ER beta and a multitude of adaptor proteins (coactivators and corepressors) has expanded the potential explanation for tissue-selective activities. The current concept of ER action includes a rheostat-like action of the receptor due to conformational changes in the ligand receptor complex that depend on the nature of the bound ligand. This conformational change also determines subsequent adaptor protein interactions. Recognition of the tissue-specific activities of tamoxifen, the first selective ER modulator (SERM), led to the development of new SERMs (raloxifene and toremifene) with greater tissue selectivities. A knowledge of the key adaptor proteins expressed within each ER target cell will allow mechanism-based screening of selective ER modulators. These future "designer estrogens" of the next millennium will be used for specific applications in the central nervous, cardiovascular, bone and reproductive systems.

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “Selective estrogen receptor modulators (SERMs): A first step in the development of perfect hormone replacement therapy regimen..” J Soc Gynecol Investig, vol. 7, no. 1 Suppl, Jan. 2000, pp. S10–15.
PMID
10732322
Source
pubmed
Published In
Journal of the Society for Gynecologic Investigation
Volume
7
Issue
1 Suppl
Publish Date
2000
Start Page
S10
End Page
S15

Selective Estrogen Receptor Modulators (SERMs): A First Step in the Development of Perfect Hormone Replacement Therapy Regimen

Authors
McDonnell, DP
MLA Citation
McDonnell, Donald P. “Selective Estrogen Receptor Modulators (SERMs): A First Step in the Development of Perfect Hormone Replacement Therapy Regimen.” Journal of the Society for Gynecologic Investigation, vol. 7, no. 1_suppl, SAGE Publications, 2000, pp. S10–15. Crossref, doi:10.1177/1071557600007001s05.
Source
crossref
Published In
Journal of the Society for Gynecologic Investigation
Volume
7
Issue
1_suppl
Publish Date
2000
Start Page
S10
End Page
S15
DOI
10.1177/1071557600007001s05

Comparative Analyses of Mechanistic Differences Among Antiestrogens1.

Authors
Wijayaratne, AL; Nagel, SC; Paige, LA; Christensen, DJ; Norris, JD; Fowlkes, DM; McDonnell, DP
MLA Citation
Wijayaratne, Ashini L., et al. “Comparative Analyses of Mechanistic Differences Among Antiestrogens1..” Endocrinology, vol. 140, no. 12, Dec. 1999, pp. 5828–40. Pubmed, doi:10.1210/endo.140.12.7164.
PMID
28200824
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
12
Publish Date
1999
Start Page
5828
End Page
5840
DOI
10.1210/endo.140.12.7164

Differential Interaction of the Methoxychlor Metabolite 2,2-Bis-(p-Hydroxyphenyl)-1,1,1-Trichloroethane with Estrogen Receptors α and β1.

Authors
Gaido, KW; Leonard, LS; Maness, SC; Hall, JM; McDonnell, DP; Saville, B; Safe, S
MLA Citation
Gaido, Kevin W., et al. “Differential Interaction of the Methoxychlor Metabolite 2,2-Bis-(p-Hydroxyphenyl)-1,1,1-Trichloroethane with Estrogen Receptors α and β1..” Endocrinology, vol. 140, no. 12, Dec. 1999, pp. 5746–53. Pubmed, doi:10.1210/endo.140.12.7191.
PMID
28200584
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
12
Publish Date
1999
Start Page
5746
End Page
5753
DOI
10.1210/endo.140.12.7191

The Estrogen Receptor β-Isoform (ERβ) of the Human Estrogen Receptor Modulates ERα Transcriptional Activity and Is a Key Regulator of the Cellular Response to Estrogens and Antiestrogens1.

Authors
Hall, JM; McDonnell, DP
MLA Citation
Hall, Julie M., and Donald P. McDonnell. “The Estrogen Receptor β-Isoform (ERβ) of the Human Estrogen Receptor Modulates ERα Transcriptional Activity and Is a Key Regulator of the Cellular Response to Estrogens and Antiestrogens1..” Endocrinology, vol. 140, no. 12, Dec. 1999, pp. 5566–78. Pubmed, doi:10.1210/endo.140.12.7179.
PMID
28200448
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
12
Publish Date
1999
Start Page
5566
End Page
5578
DOI
10.1210/endo.140.12.7179

Comparative analyses of mechanistic differences among antiestrogens.

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERalpha) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug. If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results. Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility. Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (40HT), idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of peptides that recognize different surfaces on ERalpha, we have found that following binding to ERalpha, each ligand induces a distinct ERalpha-ligand conformation. Furthermore, transcriptional assays indicate that each ERalpha-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 40HT-mediated activities. Relative binding assays have shown that the affinity of these ligands for ERalpha is not always representative of their inhibitory activity. Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins. Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor. Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied. However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene. These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERalpha function.

Authors
Wijayaratne, AL; Nagel, SC; Paige, LA; Christensen, DJ; Norris, JD; Fowlkes, DM; McDonnell, DP
MLA Citation
Wijayaratne, A. L., et al. “Comparative analyses of mechanistic differences among antiestrogens..” Endocrinology, vol. 140, no. 12, Dec. 1999, pp. 5828–40. Pubmed, doi:10.1210/endo.140.12.7164.
PMID
10579349
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
12
Publish Date
1999
Start Page
5828
End Page
5840
DOI
10.1210/endo.140.12.7164

Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Authors
Chang, CY; Norris, JD; Grøn, H; Paige, LA; Hamilton, PT; Kenan, DJ; Fowlkes, D; McDonnell, DP
MLA Citation
Chang, C. Y., et al. “Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta..” Mol Cell Biol, vol. 19, no. 12, Dec. 1999, pp. 8226–39. Pubmed, doi:10.1128/mcb.19.12.8226.
PMID
10567548
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
12
Publish Date
1999
Start Page
8226
End Page
8239
DOI
10.1128/mcb.19.12.8226

Differential interaction of the methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane with estrogen receptors alpha and beta.

Concern that some chemicals in our environment may affect human health by disrupting normal endocrine function has prompted research on interactions of environmental contaminants with steroid hormone receptors. We compared the activity of 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor, at estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). Human hepatoma cells (HepG2) were transiently transfected with either human or rat ERalpha or ERbeta plus an estrogen-responsive, complement 3-luciferase construct containing a complement 3 gene promoter sequence linked to a luciferase reporter gene. After transfection, cells were treated with various concentrations of HPTE in the presence (for detecting antagonism) or absence (for detecting agonism) of 17beta-estradiol. HPTE was a potent ERalpha agonist in HepG2 cells, with EC50 values of approximately 5 x 10(-8) and 10(-8) M for human and rat ERalpha, respectively. In contrast, HPTE had minimal agonist activity with either human or rat ERbeta and almost completely abolished 17beta-estradiol-induced ERbeta-mediated activity. Moreover, HPTE behaved as an ERalpha agonist and an ERbeta antagonist with other estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa cells. This study demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.

Authors
Gaido, KW; Leonard, LS; Maness, SC; Hall, JM; McDonnell, DP; Saville, B; Safe, S
MLA Citation
Gaido, K. W., et al. “Differential interaction of the methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane with estrogen receptors alpha and beta..” Endocrinology, vol. 140, no. 12, Dec. 1999, pp. 5746–53. Pubmed, doi:10.1210/endo.140.12.7191.
PMID
10579340
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
12
Publish Date
1999
Start Page
5746
End Page
5753
DOI
10.1210/endo.140.12.7191

The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens.

The human estrogen receptor alpha (ERalpha) and the recently identified ERbeta share a high degree of amino acid homology; however, there are significant differences in regions of these receptors that would be expected to influence transcriptional activity. Consequently, we compared the mechanism(s) by which these receptors regulate target gene transcription, and evaluated the cellular consequences of coexpression of both ER subtypes. Previously, it has been determined that ERalpha contains two distinct activation domains, ERalpha-AF-1 and ERalpha-AF-2, whose transcriptional activity is influenced by cell and promoter context. We determined that ERbeta, like ERalpha, contains a functional AF-2, however, the ERbeta-AF-2 domain functions independently within the receptor. Of additional significance was the finding that ERbeta does not contain a strong AF-1 within its amino-terminus but, rather, contains a repressor domain that when removed, increases the overall transcriptional activity of the receptor. The importance of these findings was revealed when it was determined that ERbeta functions as a transdominant inhibitor of ERalpha transcriptional activity at subsaturating hormone levels and that ERbeta decreases overall cellular sensitivity to estradiol. Additionally, the partial agonist activity of tamoxifen manifest through ERalpha in some contexts was completely abolished upon coexpression of ERbeta. In probing the mechanisms underlying ERbeta-mediated repression of ERalpha transcriptional activity we have determined that 1) ERalpha and ERbeta can form heterodimers within target cells; and 2) ERbeta interacts with target gene promoters in a ligand-independent manner. Cumulatively, these data indicate that one role of ERbeta is to modulate ERalpha transcriptional activity, and thus the relative expression level of the two isoforms will be a key determinant of cellular responses to agonists and antagonists.

Authors
Hall, JM; McDonnell, DP
MLA Citation
Hall, J. M., and D. P. McDonnell. “The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens..” Endocrinology, vol. 140, no. 12, Dec. 1999, pp. 5566–78. Pubmed, doi:10.1210/endo.140.12.7179.
PMID
10579320
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
12
Publish Date
1999
Start Page
5566
End Page
5578
DOI
10.1210/endo.140.12.7179

The Molecular Pharmacology of SERMs.

Estrogen-containing medicines have been used successfully for the past 50 years for the treatment of conditions associated with menopause. Although initially considered a reproductive hormone, millions of years of clinical exposure to estrogen(s) have indicated that its influence extends to a variety of target tissues not generally considered to be involved in reproduction. Specifically, estrogen has positive actions in the skeleton, the cardiovascular system and possibly the central nervous system, activities that combine to have a positive impact on mortality and morbidity. However, despite the medical benefits afforded by estrogen replacement therapy, the number of women who initiate or remain on therapy for longer than one year is relatively small. This is due in part to the fear that estrogens increase the risk for breast cancer. Consequently, it was realized several years ago that novel estrogen receptor modulators were needed, which would retain the beneficial effects of estrogens in most target organs but be inactive in the breast. Although the perfect tissue-selective estrogen remains to be identified, progress in this direction has been made. In the past year, for example, we have seen selective estrogen receptor modulators (SERMs) enter into the clinic for the prevention of osteoporosis. Compounds of this class, which function as estrogens in the skeletal system but oppose estrogen action in the breast, are the first step in developing the perfect hormone replacement therapy. This review summarizes the complex pharmacology of the SERMs and illustrates how they differ mechanistically from estradiol, the physiological ligand of the estrogen receptor.

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “The Molecular Pharmacology of SERMs..” Trends Endocrinol Metab, vol. 10, no. 8, Oct. 1999, pp. 301–11.
PMID
10481160
Source
pubmed
Published In
Trends Endocrinol Metab
Volume
10
Issue
8
Publish Date
1999
Start Page
301
End Page
311

Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae.

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

Authors
Beaudenon, SL; Huacani, MR; Wang, G; McDonnell, DP; Huibregtse, JM
MLA Citation
Beaudenon, S. L., et al. “Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae..” Mol Cell Biol, vol. 19, no. 10, Oct. 1999, pp. 6972–79. Pubmed, doi:10.1128/mcb.19.10.6972.
PMID
10490634
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
10
Publish Date
1999
Start Page
6972
End Page
6979
DOI
10.1128/mcb.19.10.6972

Peptide antagonists of the human estrogen receptor.

Estrogen receptor alpha transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor alpha. When these peptides were coexpressed with estrogen receptor alpha in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen-partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor alpha transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor alpha antagonists for the treatment of tamoxifen-refractory breast cancers.

Authors
Norris, JD; Paige, LA; Christensen, DJ; Chang, CY; Huacani, MR; Fan, D; Hamilton, PT; Fowlkes, DM; McDonnell, DP
MLA Citation
Norris, J. D., et al. “Peptide antagonists of the human estrogen receptor..” Science, vol. 285, no. 5428, July 1999, pp. 744–46. Pubmed, doi:10.1126/science.285.5428.744.
PMID
10426998
Source
pubmed
Published In
Science (New York, N.Y.)
Volume
285
Issue
5428
Publish Date
1999
Start Page
744
End Page
746
DOI
10.1126/science.285.5428.744

Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

Authors
Tetel, MJ; Giangrande, PH; Leonhardt, SA; McDonnell, DP; Edwards, DP
MLA Citation
Tetel, M. J., et al. “Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo..” Mol Endocrinol, vol. 13, no. 6, June 1999, pp. 910–24. Pubmed, doi:10.1210/mend.13.6.0300.
PMID
10379890
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
13
Issue
6
Publish Date
1999
Start Page
910
End Page
924
DOI
10.1210/mend.13.6.0300

Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium.

Estrogens and growth factors such as epidermal growth factor (EGF) act as mitogens promoting cellular proliferation in the breast and in the reproductive tract. Although it was considered originally that these agents manifested their mitogenic actions through separate pathways, there is a growing body of evidence suggesting that the EGF and estrogen-mediated signaling pathways are intertwined. Indeed, it has been demonstrated recently that 17beta-estradiol (E2) can induce a rapid activation of mitogen-activated protein kinase (MAPK) in mammalian cells, an event that is independent of both transcription and protein synthesis. In this study, we have used a pharmacological approach to dissect this novel pathway in MCF-7 breast cancer cells and have determined that in the presence of endogenous estrogen receptor, activation of MAPK by E2 is preceded by a rapid increase in cytosolic calcium. The involvement of intracellular calcium in this process was supported by the finding that the presence of EGTA and Ca2+-free medium did not affect the activation of MAPK by E2 and, additionally, that this response was blocked by the addition of the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. Cumulatively, these data indicate that the estrogen receptor, in addition to functioning as a transcription factor, is also involved, through a nongenomic mechanism, in the regulation of both intracellular calcium homeostasis and MAPK-signaling pathways. Although nongenomic actions of estrogens have been suggested by numerous studies in the past, the ability to link estradiol and the estrogen receptor to a well defined signaling pathway strongly supports a physiological role for this activity.

Authors
Improta-Brears, T; Whorton, AR; Codazzi, F; York, JD; Meyer, T; McDonnell, DP
MLA Citation
Improta-Brears, T., et al. “Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium..” Proc Natl Acad Sci U S A, vol. 96, no. 8, Apr. 1999, pp. 4686–91. Pubmed, doi:10.1073/pnas.96.8.4686.
PMID
10200323
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
96
Issue
8
Publish Date
1999
Start Page
4686
End Page
4691
DOI
10.1073/pnas.96.8.4686

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta.

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs alpha and beta and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER alpha and ER beta ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

Authors
Paige, LA; Christensen, DJ; Grøn, H; Norris, JD; Gottlin, EB; Padilla, KM; Chang, CY; Ballas, LM; Hamilton, PT; McDonnell, DP; Fowlkes, DM
MLA Citation
Paige, L. A., et al. “Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta..” Proc Natl Acad Sci U S A, vol. 96, no. 7, Mar. 1999, pp. 3999–4004. Pubmed, doi:10.1073/pnas.96.7.3999.
PMID
10097152
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
96
Issue
7
Publish Date
1999
Start Page
3999
End Page
4004
DOI
10.1073/pnas.96.7.3999

The Novel Progesterone Receptor Antagonists RTI 3021-012 and RTI3021-022 Exhibit Complex Glucocorticoid Receptor Antagonist Activities: Implications for the Development of Dissociated Antiprogestins.

Authors
Wagner, BL; Pollio, G; Giangrande, P; Webster, JC; Breslin, M; Mais, DE; Cook, CE; Vedeckis, WV; Cidlowski, JA; McDonnell, DP
MLA Citation
Wagner, B. L., et al. “The Novel Progesterone Receptor Antagonists RTI 3021-012 and RTI3021-022 Exhibit Complex Glucocorticoid Receptor Antagonist Activities: Implications for the Development of Dissociated Antiprogestins..” Endocrinology, vol. 140, no. 3, Mar. 1999, pp. 1449–58. Pubmed, doi:10.1210/endo.140.3.6581.
PMID
28204331
Source
pubmed
Published In
Endocrinology
Volume
140
Issue
3
Publish Date
1999
Start Page
1449
End Page
1458
DOI
10.1210/endo.140.3.6581

The A and B isoforms of the human progesterone receptor: two functionally different transcription factors encoded by a single gene.

In humans, the biological response to progesterone is mediated by two forms of the progesterone receptor (hPR-A; 94kDa and hPR-B; 114kDa). These two isoforms are transcribed from distinct, estrogen-inducible promoters within a single-copy progesterone receptor (PR) gene; the only difference between them is that the first 164 amino acids of hPR-B are absent in hPR-A. In most cell lines, hPR-A functions as a transcriptional repressor of progesterone-responsive promoters, whereas hPR-B functions as a transcriptional activator of the same genes. The observation, made in the early 1990s, that shorter isoforms of some transcriptional activators can act as transrepressors of the transcriptional activity of the larger isoforms, initiated a line of investigation that led to the discovery that hPR-A is a strong transrepressor of hPR-B activity. Interestingly, hPR-A also functions as a transdominant repressor of the transcriptional activity of the estrogen, glucocorticoid, androgen, and mineralocorticoid receptors. A specific inhibitory domain (ID) within hPR-A responsible for this activity has been mapped to the extreme amino terminus of the receptor. Interestingly, although this inhibitory domain is contained within both PR isoforms, its activity is manifest only in the context of hPR-A. The identification of a discrete inhibitory region within hPR-A, whose activity was masked in the context of hPR-B, suggests that these two receptor isoforms may interact with different proteins (transcription factors, co-activators, co-repressors) within the cell. In support of this hypothesis, we have recently observed that the co-repressor SMRT (silencing mediator of retinoid and thyroid receptors) interacts much more tightly with hPR-A than with hPR-B. This important finding led to the initial conclusion that the ability of hPR-A to repress hPR-B transcriptional activity could occur as a consequence of hPR-B/A heterodimerization, where the presence of SMRT in the complex could prevent transcriptional activation. The observation, however, that hPR-A also inhibits human estrogen receptor (hER) transcriptional activity, a receptor with which hPR-A is not able to heterodimerize, suggests that there must be additional complexity. This chapter outlines what is known about the mechanism of action of hPR-A and hPR-B and how this knowledge has enhanced our understanding of PR pharmacology.

Authors
Giangrande, PH; McDonnell, DP
MLA Citation
Giangrande, P. H., and D. P. McDonnell. “The A and B isoforms of the human progesterone receptor: two functionally different transcription factors encoded by a single gene..” Recent Prog Horm Res, vol. 54, 1999, pp. 291–313.
PMID
10548881
Source
pubmed
Published In
Recent Progress in Hormone Research
Volume
54
Publish Date
1999
Start Page
291
End Page
313

Wyeth lederle recent advances in conjugated estrogens estrogen action 1998: new insights. new drugs, new issues

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “Wyeth lederle recent advances in conjugated estrogens estrogen action 1998: new insights. new drugs, new issues.” International Journal of Fertility and Menopausal Studies, vol. 43, no. 4, Dec. 1998.
Source
scopus
Published In
International Journal of Fertility and Menopausal Studies
Volume
43
Issue
4
Publish Date
1998
Start Page
222

Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol.

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.

Authors
Gould, JC; Leonard, LS; Maness, SC; Wagner, BL; Conner, K; Zacharewski, T; Safe, S; McDonnell, DP; Gaido, KW
MLA Citation
Gould, J. C., et al. “Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol..” Mol Cell Endocrinol, vol. 142, no. 1–2, July 1998, pp. 203–14.
PMID
9783916
Source
pubmed
Published In
Molecular and Cellular Endocrinology
Volume
142
Issue
1-2
Publish Date
1998
Start Page
203
End Page
214

Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells.

Recent reports have raised new concerns that chemicals in our environment may disrupt normal reproduction and development through inhibition of androgen receptor function. This heightened concern has also increased our need for methods that allow us to characterize chemical interaction with the androgen receptor. In this report we describe an androgen receptor assay that utilizes the HepG2 human hepatoma cell line transiently transfected with the human androgen receptor and an androgen-responsive reporter. We used this assay to characterize the interaction with the androgen receptor of several steroidal and nonsteroidal chemicals, including isomers of DDT and methoxychlor. Chemicals were tested either in the absence (for determining agonist activity) or presence of 10(-7) M dihydrotestosterone (for determining antagonist activity). Testosterone and dihydrotestosterone were equally potent agonists in this assay. Estradiol and progesterone displayed partial agonist/antagonist activity. Flutamide was a complete agonist, whereas its hydroxylated metabolite, hydroxyflutamide, only partially antagonized and displayed some agonist activity at 10(-6) M and above. o,p'-DDT, o,p'-DDE, o,p'-DDD, p,p'-DDT, p,p'-DDE, and p, p'-DDD all behaved as antagonists at concentrations above 10(-6) M. p,p'-DDE also showed some agonist activity at 10(-5) M. Methoxychlor was only weakly antagonistic while its hydroxylated metabolite, HPTE, was approximately 10-fold more potent. Our results demonstrate that the HepG2 assay is a sensitive and specific method for detecting chemical interaction with the androgen receptor. This reporter gene assay, which we have used to characterize interaction with both the estrogen and androgen receptors, coupled with more extensive in vivo studies, should be useful for determining the role of multiple steroid receptors in the mechanism of action of endocrine active chemicals.

Authors
Maness, SC; McDonnell, DP; Gaido, KW
MLA Citation
Maness, S. C., et al. “Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells..” Toxicol Appl Pharmacol, vol. 151, no. 1, July 1998, pp. 135–42. Pubmed, doi:10.1006/taap.1998.8431.
PMID
9705896
Source
pubmed
Published In
Toxicology and Applied Pharmacology
Volume
151
Issue
1
Publish Date
1998
Start Page
135
End Page
142
DOI
10.1006/taap.1998.8431

Potency of combined estrogenic pesticides: Synergy between synthetic oestrogens?

These studies each show that certain binary mixtures of weak, environmental estrogens act additively. The results were obtained by testing mixtures of these compounds in a variety of estrogen assays. These studies were prompted by a now retracted report that such binary mixtures act synergistically, not simply additively. The structures of the compounds used in these studies are shown below.

Authors
Ramamoorthy, K; Wang, F; Chen, IC; Safe, S; Norris, JD; McDonnell, DP; Gaido, KW; Bocchinfuso, WP; Korach, KS; Ashby, J; Lefevre, PA; Odum, J; Harris, CA; Routledge, EJ; Sumpter, JP
MLA Citation
Ramamoorthy, K., et al. “Potency of combined estrogenic pesticides: Synergy between synthetic oestrogens?.” Chemtracts, vol. 11, no. 4, Apr. 1998, pp. 306–08.
Source
scopus
Published In
Chemtracts
Volume
11
Issue
4
Publish Date
1998
Start Page
306
End Page
308

Enhancement of estrogen receptor transcriptional activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology.

The human estrogen receptor (ER) contains two major activation functions (AFs) responsible for its transcriptional activity. One of these, activation function 2 (AF-2), located within the hormone-binding domain (HBD), has been shown to mediate the ligand-dependent transcriptional activity of ER as well as other members of the nuclear receptor superfamily. Recently, proteins interacting with the HBD of several nuclear receptors have been cloned. One of these proteins, glucocorticoid receptor interacting protein (GRIP-1), has been shown to interact with ER and was originally hypothesized to mediate its transcriptional activity through AF-2. However, we find in this study that the transcriptional activity of ER, containing mutations in the AF-2 core sequence, can be enhanced by coexpression of the coactivator GRIP-1, suggesting that this protein may not rely solely on the AF-2 domain for interaction. We propose, therefore, that the HBD of ER either contains multiple binding sites that are necessary for association with GRIP-1 or, alternatively, that this coactivator contacts the receptor in an undetermined region within the HBD. Importantly, these studies demonstrate also that mutations or deletion of AF-2 alter the ligand pharmacology of the receptor such that ER loses the ability to discriminate between agonists and antagonists. Interestingly, on these mutant receptors GRIP-1 still functions as a coactivator independent of the nature of the bound ligand. It is likely, therefore, that the C-terminal AF-2 domain may function as a molecular switch allowing the wild-type receptor to discriminate between agonists and antagonists as well as providing a surface with which associated proteins can interact.

Authors
Norris, JD; Fan, D; Stallcup, MR; McDonnell, DP
MLA Citation
Norris, J. D., et al. “Enhancement of estrogen receptor transcriptional activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology..” J Biol Chem, vol. 273, no. 12, Mar. 1998, pp. 6679–88. Pubmed, doi:10.1074/jbc.273.12.6679.
PMID
9506965
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
273
Issue
12
Publish Date
1998
Start Page
6679
End Page
6688
DOI
10.1074/jbc.273.12.6679

The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor.

Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.

Authors
Wagner, BL; Norris, JD; Knotts, TA; Weigel, NL; McDonnell, DP
MLA Citation
Wagner, B. L., et al. “The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor..” Mol Cell Biol, vol. 18, no. 3, Mar. 1998, pp. 1369–78. Pubmed, doi:10.1128/mcb.18.3.1369.
PMID
9488452
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
18
Issue
3
Publish Date
1998
Start Page
1369
End Page
1378
DOI
10.1128/mcb.18.3.1369

Definition of the molecular mechanism of action of tissue-selective oestrogen-receptor modulators.

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “Definition of the molecular mechanism of action of tissue-selective oestrogen-receptor modulators..” Biochem Soc Trans, vol. 26, no. 1, Feb. 1998, pp. 54–60.
PMID
10909757
Source
pubmed
Published In
Biochemical Society Transactions
Volume
26
Issue
1
Publish Date
1998
Start Page
54
End Page
60

Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor.

In humans, the biological response to progesterone is mediated by two distinct forms of the progesterone receptor (human (h) PR-A, 94 kDa and hPR-B, 114 kDa). These two isoforms are transcribed from distinct estrogen-inducible promoters within a single copy PR gene; the only difference between them is that the first 164 amino acids of hPR-B (B-upstream sequence) are absent in hPR-A. In most cell lines such as MCF-7 (human breast cancer cells), CV-1 (monkey kidney fibroblasts), and HeLa (human cervical carcinoma cells), hPR-A functions as a transcriptional repressor, whereas hPR-B functions as a transcriptional activator of progesterone-responsive genes. Interestingly, in these cell contexts, hPR-A also acts as a trans-dominant repressor of the transcriptional activity of other steroid hormone receptors. In contrast to hPR-A, which functions predominantly as a ligand-dependent transcriptional repressor, we show in this study that the A isoform of the chicken PR (cPR-A) lacks this trans-dominant repressor function and is a transcriptional activator in all contexts examined. By constructing chimeras between the N-terminal domains of the chicken and human PR, we mapped the trans-dominant repressor function of hPR-A to the first 140 amino acids of the protein. Notably, when this 140-amino acid "repressor" domain is placed onto chicken PR-A, the activity of the latter changes from a transcriptional activator to a repressor. Interestingly, however, this "repressor domain" is necessary, but not sufficient, for trans-repression as it is inactive when it is tethered to a heterologous protein. This suggests that the trans-repression function is comprised not only of the repressor domain of hPR-A but also requires the context of the receptor to function. The identification of a discrete inhibitory region within hPR-A which is transferable to another receptor implies that this region interacts with a set of transcription factors or adaptors that are distinct from those recognized by hPR-B, the identification of which will be required to define the mechanism by which hPR-A modulates steroid hormone receptor transcriptional activity. Thus, although chickens and humans both produce two very similar forms of the progesterone receptor, it is clear from these studies that the mechanism of action of progesterone in these two systems is quite different.

Authors
Giangrande, PH; Pollio, G; McDonnell, DP
MLA Citation
Giangrande, P. H., et al. “Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor..” J Biol Chem, vol. 272, no. 52, Dec. 1997, pp. 32889–900. Pubmed, doi:10.1074/jbc.272.52.32889.
PMID
9407067
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
272
Issue
52
Publish Date
1997
Start Page
32889
End Page
32900
DOI
10.1074/jbc.272.52.32889

Additive estrogenic activities of a binary mixture of 2',4',6'-trichloro- and 2',3',4',5'-tetrachloro-4-biphenylol.

The estrogenic activity of 2',4',6'-trichloro-4-biphenylol (HO-PCB3), 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB4), and an equimolar mixture of both compounds (HO-PCB3/HO-PCB4) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 cells, and in a yeast-based reporter gene assay. Treatment of the animals with 17beta-estradiol (E2) (0.02 microg/kg/day x3) resulted in increased uterine wet weight, peroxidase activity and progesterone receptor binding. Treatment with 18, 73, 183 or 366 micromol/kg (x3) doses of HO-PCB3, HO-PCB4, or HO-PCB3/HO-PCB4 (equimolar) caused a dose-dependent increase in estrogenic activity; a maximal-induced response was not observed at any dose and the activity of the mixture was additive. Binding of E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 to the mouse uterine estrogen receptor (ER) was determined in a competitive binding assay using [3H]E2 as the radioligand. The IC50 values were 1.1 x 10(-8), 3.4 x 10(-6), 9.9 x 10(-7), and 4.25 x 10(-6) m, respectively. HO-PCB3 and HO-PCB4 maximally induced MCF-7 cell proliferation, rat creatine kinase, and human complement C3 (C3-LUC) reporter gene activity at concentrations of 10(-5) to 10(-6) m, and these compounds were 10(3) to 10(4) less potent than E2. The HO-PCB3/HO-PCB4 mixture was active at the high concentration (10(-5) m) and was additive for these responses. HO-PCB3 and HO-PCB4 also exhibited estrogenic activity in human HepG2 cells cotransfected with C3-LUC and an ER expression plasmid, and the estrogenic activity of the HO-PCB mixture was additive. Similar results were obtained in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. The effects of variable ER expression on the potential synergistic interactions of HO-PCB3/HO-PCB4 were investigated in HepG2 cells cotransfected with C3-LUC (405 ng/well) and variable amounts of ER expression plasmid (270, 27, 2.7, or 0.27 ng/well). The results show that as ER levels decreased, the magnitude of the induction response by E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 also decreased. However, the activities of the HO-PCB mixture were additive at high and low levels of ER. Similar results were obtained in MDA-MB-231 cells cotransfected with C3-LUC and variable amounts of ER expression plasmid. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus; MCF-7, HepG2, and MDA-MBA-231 human cancer cells; and a yeast based-reporter gene assay, both HO-PCB3 and HO-PCB4 exhibited estrogenic activity. The estrogenic activity of an equimolar mixture of these compounds was additive at high and low levels of ER expression.

Authors
Ramamoorthy, K; Vyhlidal, C; Wang, F; Chen, I; Safe, S; McDonnell, DP; Leonard, LS; Gaido, KW
MLA Citation
Ramamoorthy, K., et al. “Additive estrogenic activities of a binary mixture of 2',4',6'-trichloro- and 2',3',4',5'-tetrachloro-4-biphenylol..” Toxicol Appl Pharmacol, vol. 147, no. 1, Nov. 1997, pp. 93–100. Pubmed, doi:10.1006/taap.1997.8281.
PMID
9356311
Source
pubmed
Published In
Toxicology and Applied Pharmacology
Volume
147
Issue
1
Publish Date
1997
Start Page
93
End Page
100
DOI
10.1006/taap.1997.8281

Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone.

The estrogen receptor (ER) mixed agonists tamoxifen and raloxifene have been shown to protect against bone loss in ovariectomized rats. However, the mechanism by which these compounds manifest their activity in bone is unknown. We have used a series of in vitro screens to select for compounds that are mechanistically distinct from tamoxifen and raloxifene in an effort to define the properties of an ER modulator required for bone protection. Using this approach, we identified a novel high affinity ER antagonist, GW5638, which when assayed in vitro functions as an ER antagonist, inhibiting the agonist activity of estrogen, tamoxifen, and raloxifene and reversing the "inverse agonist" activity of the pure antiestrogen ICI182,780. Thus, GW5638 appears to function as an antagonist in these in vitro systems, although in a manner distinct from other known ER modulators. Predictably, therefore, GW5638 alone displays minimal uterotropic activity in ovariectomized rats, but will inhibit the agonist activity of estradiol in this environment. Unexpectedly, however, this compound functions as a full ER agonist in bone and the cardiovascular system. These data suggest that the mechanism by which ER operates in different cells is not identical, and that classical agonist activity is not required for the bone protective activity of ER modulators.

Authors
Willson, TM; Norris, JD; Wagner, BL; Asplin, I; Baer, P; Brown, HR; Jones, SA; Henke, B; Sauls, H; Wolfe, S; Morris, DC; McDonnell, DP
MLA Citation
Willson, T. M., et al. “Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone..” Endocrinology, vol. 138, no. 9, Sept. 1997, pp. 3901–11. Pubmed, doi:10.1210/endo.138.9.5358.
PMID
9275080
Source
pubmed
Published In
Endocrinology
Volume
138
Issue
9
Publish Date
1997
Start Page
3901
End Page
3911
DOI
10.1210/endo.138.9.5358

Evaluation of a recombinant yeast cell estrogen screening assay.

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment.

Authors
Coldham, NG; Dave, M; Sivapathasundaram, S; McDonnell, DP; Connor, C; Sauer, MJ
MLA Citation
Coldham, N. G., et al. “Evaluation of a recombinant yeast cell estrogen screening assay..” Environ Health Perspect, vol. 105, no. 7, July 1997, pp. 734–42. Pubmed, doi:10.1289/ehp.97105734.
PMID
9294720
Source
pubmed
Published In
Environmental Health Perspectives
Volume
105
Issue
7
Publish Date
1997
Start Page
734
End Page
742
DOI
10.1289/ehp.97105734

Identification of a third autonomous activation domain within the human estrogen receptor.

Using a genetic selection system established in the yeast Saccharomyces cerevisiae, we have isolated, by random mutagenesis of the human estrogen receptor (ER), six mutants that display constitutive transcriptional activity. All of the mutants identified contained single base insertions or deletions leading to frameshift mutations, resulting in receptor truncations within the hormone-binding domain between amino acids (aa) 324-351. Interestingly, an ER mutant (aa 1-282) was transcriptionally inactive in yeast, suggesting that a domain important for transcriptional activity lies between aa 282 and 351 within human ER. Deletions representative of the mutants isolated in the yeast system were created in mammalian expression vectors and examined for transcriptional activity in animal cells to determine the physiological relevance of this domain. Receptors truncated at aa 282 were either weakly active or inactive; however, an ER deletion at aa 351 was approximately 50% as active as wild type ER (induced with estrogen). Furthermore, a chimeric receptor consisting of the DNA binding domain of GAL4 fused to aa 282-351 of the human ER was transcriptionally active on a GAL4 reporter. We conclude, therefore, that an autonomous activation domain (referred to as AF2a), functional in both yeast and mammalian cells, lies between aa 282-351 of the human ER.

Authors
Norris, JD; Fan, D; Kerner, SA; McDonnell, DP
MLA Citation
Norris, J. D., et al. “Identification of a third autonomous activation domain within the human estrogen receptor..” Mol Endocrinol, vol. 11, no. 6, June 1997, pp. 747–54. Pubmed, doi:10.1210/mend.11.6.0008.
PMID
9171238
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
11
Issue
6
Publish Date
1997
Start Page
747
End Page
754
DOI
10.1210/mend.11.6.0008

Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism.

The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.

Authors
Ramamoorthy, K; Wang, F; Chen, IC; Norris, JD; McDonnell, DP; Leonard, LS; Gaido, KW; Bocchinfuso, WP; Korach, KS; Safe, S
MLA Citation
Ramamoorthy, K., et al. “Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism..” Endocrinology, vol. 138, no. 4, Apr. 1997, pp. 1520–27. Pubmed, doi:10.1210/endo.138.4.5056.
PMID
9075711
Source
pubmed
Published In
Endocrinology
Volume
138
Issue
4
Publish Date
1997
Start Page
1520
End Page
1527
DOI
10.1210/endo.138.4.5056

Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay.

There is a concern that chemicals in our environment are affecting human health by disrupting normal endocrine function. Much of the concern has focused on chemicals that can interact directly with steroid hormone receptors. We have used a yeast-based assay to assess chemical interactions with the estrogen, androgen, and progesterone receptors. The yeast transformants used in this study contained the human estrogen, androgen, or progesterone receptor along with the appropriate steroid responsive elements upstream of the beta-galactosidase reporter gene. Chemicals were added to yeast cultures in doses ranging from 10(-12) to 10(-4) M and following incubation, the yeasts were then lysed and assayed for beta-galactosidase activity. Diethylstilbesterol and 17-beta estradiol were most active in the estrogen receptor assay, followed by the phytoestrogen, coumestrol. p-Nonylphenol and bisphenol A were approximately 5000- and 15,000-fold less active, respectively, than estradiol. Methoxychlor, DDT and its metabolites, o,p'-DDD, and o,p'-DDE ranged in potency from 5 to 24 X 10(6) less potent than estradiol. Testosterone and dihydrotestosterone were most potent in the androgen receptor assay, followed by estradiol and progesterone. p,p'-DDE was approximately 10(6)-fold less potent than testosterone. None of the industrial chemicals tested interacted with the progesterone receptor. These data demonstrate the utility of using yeast-based receptor assays for detecting chemical interaction with steroid receptors and these assays should serve as a useful component of an in vitro-in vivo strategy to assess the effects of chemicals on endocrine function.

Authors
Gaido, KW; Leonard, LS; Lovell, S; Gould, JC; Babaï, D; Portier, CJ; McDonnell, DP
MLA Citation
Gaido, K. W., et al. “Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay..” Toxicol Appl Pharmacol, vol. 143, no. 1, Mar. 1997, pp. 205–12. Pubmed, doi:10.1006/taap.1996.8069.
PMID
9073609
Source
pubmed
Published In
Toxicology and Applied Pharmacology
Volume
143
Issue
1
Publish Date
1997
Start Page
205
End Page
212
DOI
10.1006/taap.1996.8069

Potency of combined estrogenic pesticides.

Authors
Ramamoorthy, K; Wang, F; Chen, IC; Safe, S; Norris, JD; McDonnell, DP; Gaido, KW; Bocchinfuso, WP; Korach, KS
MLA Citation
Ramamoorthy, K., et al. “Potency of combined estrogenic pesticides..” Science, vol. 275, no. 5298, Jan. 1997, pp. 405–06. Pubmed, doi:10.1126/science.275.5298.405.
PMID
9005556
Source
pubmed
Published In
Science (New York, N.Y.)
Volume
275
Issue
5298
Publish Date
1997
Start Page
405
End Page
406
DOI
10.1126/science.275.5298.405

BRCA1 expression is not directly responsive to estrogen.

Expression of the breast cancer susceptibility gene, BRCA1, is induced by 17-beta estradiol (E2) in estrogen receptor containing breast cancer cell lines. Our previous studies have shown that BRCA1 transcription is also regulated with the cell cycle, reaching maximal levels just before the onset of DNA synthesis. In this study, we have examined whether the estrogen induction of BRCA1 is direct or is a result of the mitogenic activity of the hormone. Four lines of evidence lead us to conclude that E2 induces BRCA1 primarily through an increase in DNA synthesis: (1) The kinetics and magnitude of induction are different from the directly E2 inducible gene, pS2; (2) Induction of BRCA1, but not pS2, is blocked by cycloheximide indicating that de novo protein synthesis is required; (3) Other hormonal and growth factor treatments that induce DNA synthesis have a similar effect, including IGF-1, EGF and DNA synthetic flares induced by tamoxifen and retinoic acid; (4) BRCA1 genomic fragments near the 5' end of the gene containing putative estrogen response elements fail to respond to E2 when transfected into breast cancer cell lines. The most consistent explanation for these findings and other published studies is that BRCA1 transcription is induced as a result of the mitogenic activity of E2 in estrogen receptor positive cells.

Authors
Marks, JR; Huper, G; Vaughn, JP; Davis, PL; Norris, J; McDonnell, DP; Wiseman, RW; Futreal, PA; Iglehart, JD
MLA Citation
Marks, J. R., et al. “BRCA1 expression is not directly responsive to estrogen..” Oncogene, vol. 14, no. 1, Jan. 1997, pp. 115–21. Pubmed, doi:10.1038/sj.onc.1200808.
PMID
9010238
Source
pubmed
Published In
Oncogene
Volume
14
Issue
1
Publish Date
1997
Start Page
115
End Page
121
DOI
10.1038/sj.onc.1200808

Erratum: Identification of the sequences within the human coplement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity (Molecular Endocrinology (1996) 10 (1605-1616))

Authors
Norris, J; Fan, D; McDonnell, DP
MLA Citation
Norris, J., et al. “Erratum: Identification of the sequences within the human coplement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity (Molecular Endocrinology (1996) 10 (1605-1616)).” Molecular Endocrinology, vol. 11, no. 3, Jan. 1997.
Source
scopus
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
11
Issue
3
Publish Date
1997
Start Page
341

Analysis of the molecular pharmacology of estrogen receptor agonists and antagonists provides insights into the mechanism of action of estrogen in bone.

Authors
McDonnell, DP; Norris, JD
MLA Citation
McDonnell, D. P., and J. D. Norris. “Analysis of the molecular pharmacology of estrogen receptor agonists and antagonists provides insights into the mechanism of action of estrogen in bone..” Osteoporos Int, vol. 7 Suppl 1, 1997, pp. S29–34.
PMID
9205643
Source
pubmed
Published In
Osteoporosis International : a Journal Established as Result of Cooperation Between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the Usa
Volume
7 Suppl 1
Publish Date
1997
Start Page
S29
End Page
S34

Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity.

The promoter of the human C3 gene has been shown to be responsive to stimulation by both estrogen and tamoxifen-activated estrogen receptor (ER) in transcriptional assays reconstituted in mammalian cells. Using a series of deletions and point mutations, we have determined that the agonist activity of these two compounds was dependent upon the direct interaction of ER with each of three estrogen response elements (EREs) contained within this promoter. One of these sequences, ERE1 resembles the canonical vitellogenin A2-ERE whereas the other two, ERE2 and ERE3, do not display significant homology to known EREs. Using gene transfer studies it was shown that these sequences are necessary and sufficient for ER-mediated transcription. Interestingly, using in vitro receptor/DNA-binding assays we demonstrated that neither ERE1, ERE2, or ERE3 alone formed high-affinity complexes with purified ER; however when a promoter fragment containing all three sequences was used, specific, high-affinity ER-DNA interactions were observed. It was not surprising, therefore, that, when assayed individually on a heterologous promoter, these sequences function as weak EREs but together they act in a synergistic manner to create a strong ER-dependent enhancer. It has been suggested that tamoxifen mediates its partial agonist activity through AP-1 at target promoters. However, the fact that purified ER can bind directly to the estrogen-responsive sequences within the C3 promoter, and that tamoxifen activity on this promoter is unaffected by AP-1 coexpression, indicates that at least on some promoters tamoxifen can manifest partial agonist activity through a classical ER/ ERE- mediated mechanism.

Authors
Fan, JD; Wagner, BL; McDonnell, DP
MLA Citation
Fan, J. D., et al. “Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity..” Mol Endocrinol, vol. 10, no. 12, Dec. 1996, pp. 1605–16. Pubmed, doi:10.1210/mend.10.12.8961270.
PMID
8961270
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
10
Issue
12
Publish Date
1996
Start Page
1605
End Page
1616
DOI
10.1210/mend.10.12.8961270

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Authors
Wagner, BL; Pollio, G; Leonhardt, S; Wani, MC; Lee, DY; Imhof, MO; Edwards, DP; Cook, CE; McDonnell, DP
MLA Citation
Wagner, B. L., et al. “16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity..” Proc Natl Acad Sci U S A, vol. 93, no. 16, Aug. 1996, pp. 8739–44. Pubmed, doi:10.1073/pnas.93.16.8739.
PMID
8710941
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
93
Issue
16
Publish Date
1996
Start Page
8739
End Page
8744
DOI
10.1073/pnas.93.16.8739

Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors.

We have developed a system in Saccharomyces cerevisiae in which agonist-dependent transcriptional activity of the human progesterone receptor (hPR) is elevated to the point that it compromises cell growth. Screens for suppressors of this phenotype led to the demonstration that RSP5 is involved in hPR transactivation. Expression of RSP5 in yeast cells potentiated hPR and human glucocorticoid receptor (hGR) transcriptional activity and increased the efficacy of weak agonists of these receptors. Remarkably, expression of this yeast protein in mammalian cells had a similar effect on PR and GR transcriptional activity. Importantly, a human homolog of RSP5, hRPF1, functioned identically in mammalian cells. Previously, it has been demonstrated that RSP5 overexpression in yeast cells suppressed mutations within SPT3, a protein which interacts with the TATA-box-binding protein (TBP), suggesting that RSP5 and SPT3 operate in the same regulatory pathway. In support of this observation, we have shown that SPT3 enhances the activity of RSP5 on GR and PR when tested in yeast or mammalian cells. We conclude from these experiments that the regulatory pathways in which RSP5 and SPT3 operate in yeast cells are conserved in higher eukaryotes. Additionally, since SPT3 has been shown to contact yeast TBP directly and is the likely homolog of human TBP-associated factor TAFII18, we propose that RSP5/hRPF1 and SPT3 establish a functional link between activated PR and GR and the general transcription apparatus.

Authors
Imhof, MO; McDonnell, DP
MLA Citation
Imhof, M. O., and D. P. McDonnell. “Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors..” Mol Cell Biol, vol. 16, no. 6, June 1996, pp. 2594–605. Pubmed, doi:10.1128/mcb.16.6.2594.
PMID
8649367
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
16
Issue
6
Publish Date
1996
Start Page
2594
End Page
2605
DOI
10.1128/mcb.16.6.2594

The molecular pharmacology of ovarian steroid receptors.

Authors
Vegeto, E; Wagner, BL; Imhof, MO; McDonnell, DP
MLA Citation
Vegeto, E., et al. “The molecular pharmacology of ovarian steroid receptors..” Vitam Horm, vol. 52, 1996, pp. 99–128.
PMID
8909158
Source
pubmed
Published In
Vitamins and Hormones
Volume
52
Publish Date
1996
Start Page
99
End Page
128

Impact of process changes on the physical properties and formability of silver metal oxide contact materials

The physical characteristics of electrical contact materials are influenced by the method and process parameters used in their manufacture. This study examines internally oxidized silver metal oxide contact materials manufactured using two casting methods (vertical, semi-continuous casting and horizontal, continuous casting), grain refining additives, and variations to the internal oxidation temperature. The impact of these changes on the physical characteristics (oxide particle size and distribution, grain structure, and mechanical properties) and the effect on formability was evaluated in order to understand the consequences of these changes on the ability to fabricate finished electrical contacts. The results indicate that additives and oxidation temperature influence formability by altering the oxide particle size, geometry, and distribution. However, the method of casting had little or no influence on the formability of the material.

Authors
Gardener, J; McDonnell, D
MLA Citation
Gardener, J., and D. McDonnell. “Impact of process changes on the physical properties and formability of silver metal oxide contact materials.” Electrical Contacts, Proceedings of the Annual Holm Conference on Electrical Contacts, Dec. 1995, pp. 373–80.
Source
scopus
Published In
Electrical Contacts, Proceedings of the Annual Holm Conference on Electrical Contacts
Publish Date
1995
Start Page
373
End Page
380

Identification of a new subclass of Alu DNA repeats which can function as estrogen receptor-dependent transcriptional enhancers.

We have utilized a genetic selection system in yeast to identify novel estrogen-responsive genes within the human genome and to define the sequences in the BRCA-1 gene responsible for its estrogen responsiveness. This approach led to the identification of a new subclass within the Alu family of DNA repeats which have diverged from known Alu sequences and have acquired the ability to function as estrogen receptor-dependent enhancers. Importantly, these new elements confer receptor-dependent estrogen responsiveness to a heterologous promoter when assayed in mammalian cells. This transcriptional activity can be attenuated by the addition of either of three different classes of estrogen receptor antagonists, indicating that these elements function as classical estrogen receptor-dependent enhancers. Furthermore, this enhancer activity is restricted to a specific subset of DNA repeats because consensus Alu elements of four major subfamilies do not respond to the estrogen receptor. Previously, most Alu sequences have been considered to be functionally inert. However, this work provides strong evidence that a significant subset can confer estrogen responsiveness upon a promoter within which they are located. Clearly, Alu sequences must now be considered as important contributors to the regulation of gene transcription in estrogen receptor-containing cells.

Authors
Norris, J; Fan, D; Aleman, C; Marks, JR; Futreal, PA; Wiseman, RW; Iglehart, JD; Deininger, PL; McDonnell, DP
MLA Citation
Norris, J., et al. “Identification of a new subclass of Alu DNA repeats which can function as estrogen receptor-dependent transcriptional enhancers..” J Biol Chem, vol. 270, no. 39, Sept. 1995, pp. 22777–82. Pubmed, doi:10.1074/jbc.270.39.22777.
PMID
7559405
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
270
Issue
39
Publish Date
1995
Start Page
22777
End Page
22782
DOI
10.1074/jbc.270.39.22777

Cellular mechanisms which distinguish between hormone- and antihormone-activated estrogen receptor.

The use of reverse genetics has permitted a definition of the structural features within estrogen receptor required for its productive association with the transcription apparatus. These sequences, transactivation function 1 (TAF1) in the amino terminus and TAF2 at the carboxyl terminus, display distinct transcriptional functions. Using specific receptor mutations it has been shown that on some promoters both TAF1 and TAF2 are required for maximal transcriptional activity, whereas on others, additional factors bound to the target promoter can functionally substitute for TAF1 or TAF2. Estrogen functions as an ER agonist by promoting functional synergism between TAF1 and TAF2. Conversely, 4-OH-tamoxifen inhibits TAF2 activity and functions as an antagonist in cell contexts where TAF2 is required. Alternatively, if a 'TAF2 function' is supplied by another factor, 4-OH tamoxifen can manifest ER agonist activity. These data indicate that alterations in the cellular expression of proteins which mimic TAF1 or TAF2 activity can have a profound effect on the pharmacology of ER modulators. Thus the identification of the cellular proteins which interact with ER and its TAF regions will allow a definition of the mechanism used by the cell to distinguish between hormone- and antihormone-activated estrogen receptor.

Authors
McDonnell, DP; Dana, SL; Hoener, PA; Lieberman, BA; Imhof, MO; Stein, RB
MLA Citation
McDonnell, D. P., et al. “Cellular mechanisms which distinguish between hormone- and antihormone-activated estrogen receptor..” Ann N Y Acad Sci, vol. 761, June 1995, pp. 121–37. Pubmed, doi:10.1111/j.1749-6632.1995.tb31374.x.
PMID
7625716
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
761
Publish Date
1995
Start Page
121
End Page
137
DOI
10.1111/j.1749-6632.1995.tb31374.x

Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens.

We have developed a series of in vitro models with which to evaluate the biological activity of estrogen receptor (ER) agonists and antagonists. Using a protease digestion assay we show that the conformational changes induced within ER are distinct for agonists and antagonists. However, this assay is unable to discriminate between pure antagonists like ICI164,384 and partial agonists such as 4-OH tamoxifen or keoxifene. Using a chimeric ER-VP16 construct, we demonstrate that both pure antagonists and partial agonists deliver ER to its DNA target within cells. However, the ability of the DNA-bound receptor to activate transcription in the presence of a given antagonist is dependent on cell and promoter context. These data, suggesting functional differences among ER antagonists, were confirmed by additional experiments demonstrating that their ability to modulate the transcriptional activity of a series of ER mutants is dramatically different. Depending on the cell and promoter context and the particular ER form expressed, 4-OH tamoxifen and the related compound, keoxifene, functioned as partial agonists. Importantly, the transcriptional profiles of these two compounds were dissimilar, suggesting that they are functionally different from each other and from ICI164,384, which does not display agonist activity under any context examined. Our results reveal functional differences between these clinically important antiestrogens and suggest that the distinct biologies manifest by these compounds in vivo relate to their ability to differentially regulate ER function.

Authors
McDonnell, DP; Clemm, DL; Hermann, T; Goldman, ME; Pike, JW
MLA Citation
McDonnell, D. P., et al. “Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens..” Mol Endocrinol, vol. 9, no. 6, June 1995, pp. 659–69. Pubmed, doi:10.1210/mend.9.6.8592512.
PMID
8592512
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
9
Issue
6
Publish Date
1995
Start Page
659
End Page
669
DOI
10.1210/mend.9.6.8592512

Definition of the critical cellular components which distinguish between hormone and antihormone activated progesterone receptor.

The steroid hormone progesterone is a key modulator of the cellular processes associated with the maintenance and development of female reproductive function. The biological activity of this hormone is mediated by specific nuclear receptors located in target cell nuclei which upon activation are capable of modulating the transcriptional activity of promoters containing progesterone response elements. Abnormalities in the progesterone receptor (PR) signal transduction pathway are implicated in pathological states such as breast cancer, endometriosis, and uterine fibroids. As a result of the medical need to modulate PR transcriptional activity, antiprogestins, compounds which oppose the actions of progesterone and novel progesterone receptor agonists, have been developed. This review outlines our current understanding of the critical cellular components which define the pharmacology of progesterone receptor agonists and antagonists, and how this information will impact the discovery and development of additional therapeutics.

Authors
Clemm, DL; Macy, BL; Santiso-Mere, D; McDonnell, DP
MLA Citation
Clemm, D. L., et al. “Definition of the critical cellular components which distinguish between hormone and antihormone activated progesterone receptor..” J Steroid Biochem Mol Biol, vol. 53, no. 1–6, June 1995, pp. 487–95.
PMID
7626499
Source
pubmed
Published In
The Journal of Steroid Biochemistry and Molecular Biology
Volume
53
Issue
1-6
Publish Date
1995
Start Page
487
End Page
495

Unraveling the human progesterone receptor signal transduction pathway Insights into antiprogestin action.

The steroid hormone progesterone is a key modulator of the cellular processes that are required for the development and maintenance of reproductive function. Produced primarily by ovarian granulosa cells, it mediates its biological activity throughout the body by interacting with specific high-affinity nuclear receptors located in target cell nuclei. These receptors are latent transcription factors, which, upon binding progesterone, are capable of interacting with specific recognition sequences within target gene promoters. The consequence of these interactions are determined by the cell and promoter context of the DNA-bound receptor. Abnormalities in the progesterone receptor (PR) signal transduction pathway are implicated in pathological states such as breast cancer, endometriosis, and uterine fibroids. Consequently, as a result of the medical need to modulate PR transcriptional activity, antiprogestins, compounds that oppose the actions of progesterone, have been developed. Recent advances in our understanding of the molecular mechanism of action of progesterone have revealed the likely mechanisms by which antiprogestins manifest their biological activity. It is anticipated that this new information will facilitate the discovery and development of additional antiprogestins that may demonstrate superior therapeutic profiles.

Authors
McDonnell, DP
MLA Citation
McDonnell, D. P. “Unraveling the human progesterone receptor signal transduction pathway Insights into antiprogestin action..” Trends Endocrinol Metab, vol. 6, no. 4, May 1995, pp. 133–38.
PMID
18406695
Source
pubmed
Published In
Trends in Endocrinology and Metabolism: Tem
Volume
6
Issue
4
Publish Date
1995
Start Page
133
End Page
138

Advances in our understanding of ligand-activated nuclear receptors

During the past year, our understanding of signal transduction pathways mediated by nuclear receptors has advanced significantly. Progress was due, in part, to the identification of ligands for orphan receptors, to the discovery of proteins that interact with nuclear receptors and to genetic analyses in animal models using gene-knockout techniques. © 1995.

Authors
Wen, DX; McDonnell, DP
MLA Citation
Wen, D. X., and D. P. McDonnell. “Advances in our understanding of ligand-activated nuclear receptors.” Current Opinion in Biotechnology, vol. 6, no. 5, Jan. 1995, pp. 582–89. Scopus, doi:10.1016/0958-1669(95)80096-4.
Source
scopus
Published In
Current Opinion in Biotechnology
Volume
6
Issue
5
Publish Date
1995
Start Page
582
End Page
589
DOI
10.1016/0958-1669(95)80096-4

Development of tissue-selective estrogen receptor modulators

Authors
McDonnell, DP; Lieberman, BA; Norris, J
MLA Citation
McDonnell, D. P., et al. “Development of tissue-selective estrogen receptor modulators.” Organ Selective Actions of Steroid Hormones, edited by D. T. Baird et al., vol. 16, SPRINGER-VERLAG BERLIN, 1995, pp. 1–28.
Source
wos
Published In
Organ Selective Actions of Steroid Hormones
Volume
16
Publish Date
1995
Start Page
1
End Page
28

Comparison of the switching behavior of internally oxidized and powder metallurgical silver metal oxide contact materials

The switching behavior of powder metallurgical and internally oxidized silver tin oxide based and silver cadmium oxide based contact materials was compared. Computer supported analysis of contact performance (contact weld force, changes in electrical resistance and temperature) coupled with an analysis of the impact of electrical switching on physical characteristics (resistance to contact erosion, degradation of contact surface) was examined. Internally oxidized materials exhibited superior erosion resistance while powder metallurgical materials were slightly better regarding weld resistance. Both manufacturing methods produced materials with comparable electrical resistance and temperature performance. Silver cadmium oxide compositions tended to perform better with respect to electrical resistance and temperature while silver tin oxide based materials had slightly improved erosion and weld resistance. However, the relative oxide level and manufacturing method had the dominant influence on contact performance. The results indicate that consideration to both the manufacturing method as well as the composition should be given when choosing an electrical contact material.

Authors
McDonnell, D
MLA Citation
McDonnell, D. “Comparison of the switching behavior of internally oxidized and powder metallurgical silver metal oxide contact materials.” Electrical Contacts, Proceedings of the Annual Holm Conference on Electrical Contacts, Dec. 1994, pp. 253–60.
Source
scopus
Published In
Electrical Contacts, Proceedings of the Annual Holm Conference on Electrical Contacts
Publish Date
1994
Start Page
253
End Page
260

Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay.

We hypothesized that estradiol levels are higher in prepubertal girls than in prepubertal boys and that this greater secretion of estradiol might drive the more rapid epiphyseal development and earlier puberty in girls. Since previous estradiol assays have lacked adequate sensitivity to test the hypothesis of higher estradiol levels in girls, we developed a new ultrasensitive assay to measure estrogen levels. The assay uses a strain of Saccharomyces cerevisiae genetically engineered for extreme sensitivity to estrogen. Yeast were transformed with plasmids encoding the human estrogen receptor and an estrogen-responsive promoter fused to the structural gene for beta-galactosidase. Ether extracts of 0.8 ml of serum were incubated with yeast for 8 h and the beta-galactosidase response was used to determine estrogen bioactivity relative to estradiol standards prepared in charcoal-stripped plasma. The assay was highly specific for estradiol with < 3% cross-reactivity with estrone, estriol, or estradiol metabolites. The detection limit was < 0.02 pg/ml estradiol equivalents (100-fold lower than existing assays). Using this assay, we measured estrogen levels in 23 prepubertal boys (9.4 +/- 2.0 yr) and 21 prepubertal girls (7.7 +/- 1.9 [SD] yr). The estrogen level in girls, 0.6 +/- 0.6 pg/ml estradiol equivalents, was significantly greater than the level in boys, 0.08 +/- 0.2 pg/ml estradiol equivalents (P < 0.05). We conclude that the ultrasensitive recombinant cell bioassay for estrogen is approximately 100-fold more sensitive than previous estradiol assays, that estrogen levels are much lower prepubertally, in both sexes, than reported previously, and that prepubertal girls have 8-fold higher estrogen levels than prepubertal boys.

Authors
Klein, KO; Baron, J; Colli, MJ; McDonnell, DP; Cutler, GB
MLA Citation
Klein, K. O., et al. “Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay..” J Clin Invest, vol. 94, no. 6, Dec. 1994, pp. 2475–80. Pubmed, doi:10.1172/JCI117616.
PMID
7989605
Source
pubmed
Published In
The Journal of Clinical Investigation
Volume
94
Issue
6
Publish Date
1994
Start Page
2475
End Page
2480
DOI
10.1172/JCI117616

The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells.

The biological response to progesterone is mediated by two distinct forms of the human progesterone receptor (hPR-A and hPR-B). In most cell contexts, hPR-B functions as a transcriptional activator of progesterone-responsive genes, whereas hPR-A functions as a transcriptional inhibitor of all steroid hormone receptors. We have created mutations within the carboxyl terminus of hPR which differentially effect the transcriptional activity of hPR-B in a cell- and promoter-specific manner. Analogous mutations, when introduced into hPR-A, have no effect on its ability to inhibit the transcriptional activity of other steroid hormone receptors. The observed differences in the structural requirements for hPR-B and hPR-A function suggest that transcriptional activation and repression by PR are mediated by two separate pathways within the cell. In support of this hypothesis, we have shown that hPR-A mediated repression of human estrogen receptor (hER) transcriptional activity is not dependent on hER expression level but depends largely on the absolute expression level of hPR-A. Thus, it appears that hPR-A inhibits hER transcriptional activity as a consequence of a noncompetitive interaction of hPR-A with either distinct cellular targets or different contact sites on the same target. We propose that hPR-A expression facilitates a ligand-dependent cross-talk among sex steroid receptor signaling pathways within the cell. It is likely, therefore, that alterations in the expression level of hPR-A or its cellular target can have profound effects on the physiological or pharmacological responses to sex steroid hormone receptor ligands.

Authors
Wen, DX; Xu, YF; Mais, DE; Goldman, ME; McDonnell, DP
MLA Citation
Wen, D. X., et al. “The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells..” Mol Cell Biol, vol. 14, no. 12, Dec. 1994, pp. 8356–64. Pubmed, doi:10.1128/mcb.14.12.8356.
PMID
7969170
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
14
Issue
12
Publish Date
1994
Start Page
8356
End Page
8364
DOI
10.1128/mcb.14.12.8356

Human and rat peroxisome proliferator activated receptors (PPARs) demonstrate similar tissue distribution but different responsiveness to PPAR activators.

We have isolated a human peroxisomal proliferator activated receptor (hPPAR) from a human liver cDNA library. Based on sequence analysis, we have determined that this cDNA encodes the human PPAR alpha. When assayed in a reconstituted hPPAR responsive transcription system in mammalian CV-1 cells, this receptor was shown to be transcriptionally activated by hypolipidemic agents like clofibric acid, and ETYA (5,8,11,14-eicosatetraynoic acid; a synthetic arachidonic acid homolog). When analyzed in CV-1 cells, the rat PPAR alpha was similarly transcriptionally regulated. However, when assayed in a human liver cell line (HepG2) we noticed that ETYA was a more efficient activator of hPPAR alpha than rPPAR alpha. Thus, factors other than the receptor are important in determining the cellular responsiveness to this class of compounds. Interestingly, WY-14,643, another peroxisome proliferator, was a much more potent activator of rPPAR alpha than human PPAR alpha when assayed in both cell lines. This may explain in part why certain fibrates are potent hepatocarcinogens in rodents. Northern analysis indicates that hPPAR alpha and rPPAR alpha are well expressed in heart, kidney and liver. We further demonstrate that hPPAR alpha and human retinoid X receptor alpha synergistically interact to bind and transactivate through a peroxisomal proliferator response element. Thus in a similar cell and promoter context the rat and human PPARs show a differential response to certain activators. Cumulatively these data suggest that differential ligand responsiveness does not provide a complete explanation for the different biological effects exhibited by hypolipidemic drugs when administered to humans and rats.

Authors
Mukherjee, R; Jow, L; Noonan, D; McDonnell, DP
MLA Citation
Mukherjee, R., et al. “Human and rat peroxisome proliferator activated receptors (PPARs) demonstrate similar tissue distribution but different responsiveness to PPAR activators..” J Steroid Biochem Mol Biol, vol. 51, no. 3–4, Nov. 1994, pp. 157–66.
PMID
7981125
Source
pubmed
Published In
The Journal of Steroid Biochemistry and Molecular Biology
Volume
51
Issue
3-4
Publish Date
1994
Start Page
157
End Page
166

Definition of the cellular mechanisms which distinguish between hormone and antihormone activated steroid receptors.

Steroid hormones are key regulatory molecules required for the coordinated regulation of the events associated with growth, differentiation and maintenance of cellular homeostasis. A large number of clinical abnormalities have been shown to be associated with defects in sex steroid hormone production or in the way the cell responds to these hormonal stimuli. As a consequence of the need to modulate the action of the sex steroids, several antihormones, compounds which oppose the action of the natural hormones, have been developed. These antihormones have found widespread application in the treatment of breast and prostate cancers, endometriosis and uterine fibroids. Of late, considerable progress has been made in defining the precise molecular mechanism of action of steroid hormones and their corresponding antihormones. It is anticipated that this information will impact the discovery and development of novel antihormones with improved therapeutic profiles.

Authors
McDonnell, DP; Clemm, DL; Imhof, MO
MLA Citation
McDonnell, D. P., et al. “Definition of the cellular mechanisms which distinguish between hormone and antihormone activated steroid receptors..” Semin Cancer Biol, vol. 5, no. 5, Oct. 1994, pp. 327–36.
PMID
7849261
Source
pubmed
Published In
Seminars in Cancer Biology
Volume
5
Issue
5
Publish Date
1994
Start Page
327
End Page
336

Potentiation of progesterone receptor-mediated transcription by the immunosuppressant FK506.

The nontransformed steroid receptors contain several non-steroid binding proteins, such as hsp90, hsp70, and p59. Recently, we and others have shown that p59 (FKBP59) is an immunophilin which binds two potent immunosuppressants, FK506 and rapamycin. This raises the possibility that FK506 or rapamycin may modify the function of steroid receptors. To develop this line of inquiry, we chose a yeast model system in which the human progesterone receptor form B (hPR-B) was cotransformed with a reporter gene. The reporter contains two copies of a progesterone response element/glucocorticoid response element (PRE/GRE) upstream of the CYC1 promoter which are linked to the lacZ gene of Escherichia coli. We found that FK506 potentiated the ability of progesterone in activating transcription. To gain insight into the mechanism of FK506's regulation of PR action, we questioned whether calcineurin is involved, because it has been shown that FK506 is a specific inhibitor of calcineurin, a Ca(2+)- and calmodulin-regulated phosphatase, through the formation of an FKBP12-FK506-calcineurin-calmodulin complex. We found that 15-O-desmethyl-FK520, an FK506 analogue which is an excellent ligand of FKBP12, but a poor inhibitor of calcineurin, failed to induce the same effect as FK506. We also found that calmidazolium, a calmodulin antagonist, mimicked FK506's action. Furthermore, immunoblot analysis showed that both FK506 and calmidazolium potentiated the effect of progesterone in decreasing the mobility of hPR-B upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This suggests that FK506 and calmidazolium may cooperate with progesterone in increasing the level of hPR-B phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Tai, PK; Albers, MW; McDonnell, DP; Chang, H; Schreiber, SL; Faber, LE
MLA Citation
Tai, P. K., et al. “Potentiation of progesterone receptor-mediated transcription by the immunosuppressant FK506..” Biochemistry, vol. 33, no. 35, Sept. 1994, pp. 10666–71. Pubmed, doi:10.1021/bi00201a014.
PMID
7521210
Source
pubmed
Published In
Biochemistry
Volume
33
Issue
35
Publish Date
1994
Start Page
10666
End Page
10671
DOI
10.1021/bi00201a014

Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells.

A powerful and versatile system for the identification of novel response elements for members of the intracellular receptor family is presented as applied to the human estrogen receptor. In the past, a limited number of estrogen response elements (EREs) have been functionally identified in the promoter regions of estrogen-regulated genes. From these a consensus ERE has been defined that is identical to the ERE of the Xenopus laevis vitellogenin gene, i.e., 5'-GGTCA NNN TGACC-3'. In order to investigate without bias the range of sequences that could function as EREs in vivo, we have developed a genetic selection in yeast expressing the human estrogen receptor (hER) and transformed with a random oligonucleotide library in a vector where expression of a selectable marker requires insertion of an upstream activating sequence. More than 1,000,000 transformants were screened and of 726 clones that contained activating sequences, 65 were found to be hormone-dependent. Sequencing revealed that the majority contained at least one 4/5 match to a canonical ERE half-site, but only one contained a full consensus ERE as previously defined. Some contained half-sites arranged as direct repeats. Twelve elements were further characterized to compare estrogen activation in yeast and mammalian cells and in vitro binding to hER. The results of these studies reveal that sequences that bind weakly to hER in vitro are fully functional as EREs in yeast and are conditionally responsive to estrogen in mammalian cells. In addition, an element was identified that is more sensitive to the partial agonist activities of tamoxifen and nafoxidine than is the consensus ERE, indicating that not only promoter context but the sequence of the binding site itself can allow distinction between receptor activated by agonist and that activated by antagonist.

Authors
Dana, SL; Hoener, PA; Wheeler, DA; Lawrence, CB; McDonnell, DP
MLA Citation
Dana, S. L., et al. “Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells..” Mol Endocrinol, vol. 8, no. 9, Sept. 1994, pp. 1193–207. Pubmed, doi:10.1210/mend.8.9.7838152.
PMID
7838152
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
8
Issue
9
Publish Date
1994
Start Page
1193
End Page
1207
DOI
10.1210/mend.8.9.7838152

Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment.

The brain isozyme of creatine kinase (CKB) is a major component of the estrogen-induced proteins in the rat uterus. Hormonal specificity of this response was studied in cotransfection assays using the rat CKB promoter linked to the bacterial chloramphenicol acetyltransferase gene. Response was specific for estrogen as 17 beta-estradiol in the presence of estrogen receptor dramatically stimulated the CKB promoter. This induction was completely blocked by the estrogen antagonist ICI 164,384. Nuclear receptors for progesterone, androgen, glucocorticoid and vitamin D did not significantly activate the CKB promoter in the presence of their respective ligands. Creatine kinase (CK) activity was analyzed in decidualized mouse uterus to assess estrogenic activity in vivo. Upon oil stimulation, uterine horns of day 4 pseudopregnant mice underwent a dramatic outgrowth in response to endogenous progesterone. This response was accompanied by a significant decrease in CK activity from a control value of 1.44 +/- 0.25 to 0.38 +/- 0.08 IU/mg protein (P < 0.001), indicating that the action of estrogen was suppressed. Treatment of females one day prior to oil-stimulation with progesterone receptor antagonists, RU486 (Mifepristone) or ZK299 (Onapristone), or with a monoclonal antibody to progesterone (DB3), abolished decidualization, and also restored the CK activity to the control value. These results suggest that CK can be used as a specific cellular marker to detect unopposed estrogen action in the mouse uterus associated with progesterone withdrawal or receptor blockade.

Authors
Crombie, DL; Mukherjee, R; McDonnell, DP; Hayes, JS; Wang, MW
MLA Citation
Crombie, D. L., et al. “Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment..” J Steroid Biochem Mol Biol, vol. 49, no. 2–3, June 1994, pp. 123–29.
PMID
8031708
Source
pubmed
Published In
The Journal of Steroid Biochemistry and Molecular Biology
Volume
49
Issue
2-3
Publish Date
1994
Start Page
123
End Page
129

RU486 exerts antiestrogenic activities through a novel progesterone receptor A form-mediated mechanism.

The human progesterone receptor (hPR) exists in two distinct forms in most cells, hPR-A and hPR-B. Both receptor isoforms exhibit distinct biological functions and demonstrate a cell- and promoter-specific ability to regulate gene transcription. Interestingly, in cell contexts where PR-A is transcriptionally inactive, it acts as a progesterone-dependent inhibitor of estrogen receptor function. Coexpression of the human estrogen receptor with the A form (but not the B form) of the human progesterone receptor resulted in a ligand-dependent inhibition of estrogen receptor-mediated gene transcription. The antiprogestins RU486 (MIfepristone) and ZK98299 (Onapristone) and related antiprogestins were all effective "noncompetitive" inhibitors of the estrogen receptor in this assay as none of these compounds interacted directly with the estrogen receptor. This observation may explain in part the observed tissue-specific antiestrogenic effects of RU486 and further indicates that the antiestrogenic activities of antiprogestins may be intrinsic to their biological function. This important new information defines novel activities of progesterone receptor ligands and may alter the way in which we define progesterone receptor modulators for future clinical applications. In addition, these data reveal that the A form of the progesterone receptor plays a key role in modulating estrogen receptor function in cells where both receptors are expressed.

Authors
McDonnell, DP; Goldman, ME
MLA Citation
McDonnell, D. P., and M. E. Goldman. “RU486 exerts antiestrogenic activities through a novel progesterone receptor A form-mediated mechanism..” J Biol Chem, vol. 269, no. 16, Apr. 1994, pp. 11945–49.
PMID
8163495
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
269
Issue
16
Publish Date
1994
Start Page
11945
End Page
11949

The human progesterone receptor A-form functions as a transcriptional modulator of mineralocorticoid receptor transcriptional activity.

The human progesterone receptor (hPR) exists as two distinct molecular forms in most cells, hPR-A and -B. These receptor isoforms display distinct biological functions and demonstrate a cell and promoter specific ability to regulate gene transcription. In cellular contexts where hPR-A is transcriptionally inactive it can function as a ligand dependent inhibitor of mineralocorticoid receptor (MR) transcriptional activity. Inhibition occurs by a non-competitive mechanism as direct binding to MR is not required. Interestingly, PR agonists differ in their ability to facilitate the inhibitory function of hPR-A, suggesting that a specific receptor conformation may be preferred for this activity. Those compounds derived from 19-nor-testosterone are the most effective. The antiprogestins RU486, ZK98299 and ZK112993 are effective MR antagonists in the presence of coexpressed hPR-A. The mechanism of hPR-A mediated inhibition of MR transcriptional activity is unknown. We propose that inhibition results from a competition of hPR-A with MR for a common transcription factor and that the association of hPR-A with this factor is not transcriptionally productive.

Authors
McDonnell, DP; Shahbaz, MM; Vegeto, E; Goldman, ME
MLA Citation
McDonnell, D. P., et al. “The human progesterone receptor A-form functions as a transcriptional modulator of mineralocorticoid receptor transcriptional activity..” J Steroid Biochem Mol Biol, vol. 48, no. 5–6, Apr. 1994, pp. 425–32.
PMID
8180103
Source
pubmed
Published In
The Journal of Steroid Biochemistry and Molecular Biology
Volume
48
Issue
5-6
Publish Date
1994
Start Page
425
End Page
432

Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions

We have used a series of human estrogen receptor (ER) mutants to evaluate the cell- and promoter-specific transcriptional activities of the TAF1 and TAF2 transactivation regions within the human ER. We show that the manifestation of TAF1 or TAF2 function depends strongly upon promoter context; on certain promoters, both the TAF1 and TAF2 activators are required for wild-type transcriptional activity, whereas on other promoters, the TAF1 and TAF2 activators function independently. Using these constructs, we show that the antagonist activity of the triphenylethylene-derived antiestrogens, e.g. tamoxifen, arises from their intrinsic inability to activate ER TAF2 function. However, on certain promoters, these antiestrogens efficiently activate gene transcription through ER. Consistent with this observation, the TAF2 function of the ER is not required on all promoters. In these TAF2- independent promoter contexts, TAF2 function may be provided by a separate transcription factor bound to the promoter. These data suggest that 1) TAF1 may be the major transcriptional activator of the ER; and 2) TAF2 functions as a transcriptional facilitator. On promoters where TAF2 function is provided independently of the ER, the TAF1 function of the ER can function independently of TAF2 activity, allowing triphenylethylene-derived antiestrogens to demonstrate partial agonist activity. These observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.

Authors
Tzukerman, MT; Esty, A; Santiso-Mere, D; Danielian, P; Parker, MG; Stein, RB; Pike, JW; McDonnell, DP
MLA Citation
Tzukerman, M. T., et al. “Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions.” Molecular Endocrinology, vol. 8, no. 1, Jan. 1994, pp. 21–30. Scopus, doi:10.1210/me.8.1.21.
Source
scopus
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
8
Issue
1
Publish Date
1994
Start Page
21
End Page
30
DOI
10.1210/me.8.1.21

Estrogen receptor mediated repression of rat gonadotropin releasing hormone (GnRH) promoter activity in hypothalamic cells

Authors
Kepa, JK; Neeley, CI; Jacobsen, BM; Bruder, JM; McDonnell, DP; Leslie, KK; Wierman, ME
MLA Citation
Kepa, J. K., et al. “Estrogen receptor mediated repression of rat gonadotropin releasing hormone (GnRH) promoter activity in hypothalamic cells.” Endocrine, vol. 2, no. 10, Jan. 1994, pp. 947–56.
Source
scopus
Published In
Endocrine
Volume
2
Issue
10
Publish Date
1994
Start Page
947
End Page
956

Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions.

We have used a series of human estrogen receptor (ER) mutants to evaluate the cell- and promoter-specific transcriptional activities of the TAF1 and TAF2 transactivation regions within the human ER. We show that the manifestation of TAF1 or TAF2 function depends strongly upon promoter context; on certain promoters, both the TAF1 and TAF2 activators are required for wild-type transcriptional activity, whereas on other promoters, the TAF1 and TAF2 activators function independently. Using these constructs, we show that the antagonist activity of the triphenylethylene-derived antiestrogens, e.g. tamoxifen, arises from their intrinsic inability to activate ER TAF2 function. However, on certain promoters, these antiestrogens efficiently activate gene transcription through ER. Consistent with this observation, the TAF2 function of the ER is not required on all promoters. In these TAF2-independent promoter contexts, TAF2 function may be provided by a separate transcription factor bound to the promoter. These data suggest that 1) TAF1 may be the major transcriptional activator of the ER; and 2) TAF2 functions as a transcriptional facilitator. On promoters where TAF2 function is provided independently of the ER, the TAF1 function of the ER can function independently of TAF2 activity, allowing triphenylethylene-derived antiestrogens to demonstrate partial agonist activity. These observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.

Authors
Tzukerman, MT; Esty, A; Santiso-Mere, D; Danielian, P; Parker, MG; Stein, RB; Pike, JW; McDonnell, DP
MLA Citation
Tzukerman, M. T., et al. “Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions..” Mol Endocrinol, vol. 8, no. 1, Jan. 1994, pp. 21–30. Pubmed, doi:10.1210/mend.8.1.8152428.
PMID
8152428
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
8
Issue
1
Publish Date
1994
Start Page
21
End Page
30
DOI
10.1210/mend.8.1.8152428

Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells

A powerful and versatile system for the identification of novel response elements for members of the intracellular receptor family is presented as applied to the human estrogen receptor. In the past, a limited number of estrogen response elements (EREs) have been functionally identified in the promoter regions of estrogen-regulated genes. From these a consensus ERE has been defined that is identical to the ERE of the Xenopus laevis vitellogenin gene, i.e., 5'-GGTCA NNN TGACC-3'. In order to investigate without bias the range of sequences that could function as EREs in vivo, we have developed a genetic selection in yeast expressing the human estrogen receptor (hER) and transformed with a random oligonucleotide library in a vector where expression of a selectable marker requires insertion of an upstream activating sequence. More than 1,000,000 transformants were screened and of 726 clones that contained activating sequences, 65 were found to be hormone- dependent. Sequencing revealed that the majority contained at least one 4/5 match to a canonical ERE half-site, but only one contained a full consensus ERE as previously defined. Some contained half-sites arranged as direct repeats. Twelve elements were further characterized to compare estrogen activation in yeast and mammalian cells and in vitro binding to hER. The results of these studies reveal that sequences that bind weakly to hER in vitro are fully functional as EREs in yeast and are conditionally responsive to estrogen in mammalian cells. In addition, an element was identified that is more sensitive to the partial agonist activities of tamoxifen and nafoxidine than is the consensus ERE, indicating that not only promoter context but the sequence of the binding site itself can allow distinction between receptor activated by agonist and that activated by antagonist.

Authors
Dana, SL; Hoener, PA; Wheeler, DA; Lawrence, CB; McDonnell, DP
MLA Citation
Dana, S. L., et al. “Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells.” Molecular Endocrinology, vol. 8, no. 9, 1994, pp. 1193–207. Scival, doi:10.1210/me.8.9.1193.
Source
scival
Published In
Molecular Endocrinology
Volume
8
Issue
9
Publish Date
1994
Start Page
1193
End Page
1207
DOI
10.1210/me.8.9.1193

Comparison of the switching behavior of silver metal oxide contact materials

An investigation of the switching behavior of internally oxidized silver cadmium oxide and silver tin, indium oxide contact materials was undertaken in which detailed, computer supported analysis of changes in the performance (contact weld force, electrical resistance and temperature change) was evaluated. In addition, the impact of electrical switching on the physical characteristics of the material (contact erosion and surface morphology) was examined. The silver tin, indium oxide materials exhibited significantly higher resistance to erosion than the silver cadmium oxide materials. The resistance to welding as well as changes to electrical resistance and temperature were comparable and were more dependent on oxide level than on materials. The results suggest that internally oxidized silver tin, indium oxide could serve as a non-toxic replacement for internally oxidized silver cadmium oxide.

Authors
McDonnell, D; Gardener, J; Gondusky, J
MLA Citation
McDonnell, D., et al. “Comparison of the switching behavior of silver metal oxide contact materials.” Electrical Contacts, Proceedings of the Annual Holm Conference on Electrical Contacts, Dec. 1993, pp. 37–43.
Source
scopus
Published In
Electrical Contacts, Proceedings of the Annual Holm Conference on Electrical Contacts
Publish Date
1993
Start Page
37
End Page
43

The mechanism of action of steroid hormones: a new twist to an old tale.

Steroid hormones, vitamins, and thyroid hormone are potent chemical messengers that exert dramatic effects on cell differentiation, homeostasis, and morphogenesis. These molecules, though diverse in structure, share a mechanistically similar mode of action. The effector molecules diffuse across cellular membranes and bind to specific high affinity receptors in the target cell nuclei. This interaction results in the conversion of an inactive receptor to one that can interact with the regulatory regions of target genes and modulate the rate of transcription of specific gene sets. The recent cloning and characterization of the functional receptors for these hormones has been enlightening as to the individual steps involved in steroid signal transduction. In addition, emerging evidence suggests that receptor function can be influenced by cell and promoter context indicating that it may be possible to develop tissue specific or tissue-restricted drugs. The concept that a single receptor can modulate gene transcription in a cell-specific manner is of great medical and pharmaceutical importance. The focus of this review is to highlight the recent developments in the steroid receptor field and to illustrate the novel approaches been undertaken to identify novel pharmaceuticals.

Authors
McDonnell, DP; Clevenger, B; Dana, S; Santiso-Mere, D; Tzukerman, MT; Gleeson, MA
MLA Citation
McDonnell, D. P., et al. “The mechanism of action of steroid hormones: a new twist to an old tale..” J Clin Pharmacol, vol. 33, no. 12, Dec. 1993, pp. 1165–72.
PMID
8126251
Source
pubmed
Published In
Journal of Clinical Pharmacology
Volume
33
Issue
12
Publish Date
1993
Start Page
1165
End Page
1172

Nuclear hormone receptors as targets for new drug discovery.

There are two basic types of receptor transducing systems: those which utilize membrane bound receptors and are activated at the cell surface by the appropriate hormone and transmit their signal to the internae of the cell via a second messenger (i.e. cAMP), and those that utilize internal, cytoplasmic or nuclear receptors (intracellular receptors) which upon activation by hormones interact directly with DNA and alter the genetic program of a cell. This review focuses on the mechanism of action of these intracellular receptors and discusses how such an understanding is expected to facilitate the discovery of new therapeutic agents.

Authors
McDonnell, DP; Vegeto, E; Gleeson, MA
MLA Citation
McDonnell, D. P., et al. “Nuclear hormone receptors as targets for new drug discovery..” Biotechnology (N Y), vol. 11, no. 11, Nov. 1993, pp. 1256–61.
PMID
7764187
Source
pubmed
Published In
Bio/Technology
Volume
11
Issue
11
Publish Date
1993
Start Page
1256
End Page
1261

Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function.

Two distinct isoforms of the human progesterone receptor (hPR-A and hPR-B) have been identified previously. They differ only in that hPR-B contains an additional 164 amino acids at the amino terminus. Among various species these two forms arise as a result of either alternate initiation of translation from the same mRNA or by transcription from alternate promoters within the same gene. In order to understand the reason for their existence, we studied the transcriptional capacity of these individual receptors and observed that their activity was influenced strongly by cell and promoter context. More surprising was the observation that in promoter and cell contexts where hPR-A was inactive, it acted as a potent trans-dominant repressor of hPR-B-mediated transcription. This event occurred at substoichiometric concentrations of hPR-A and was hormone dependent. Human PR-A was not a general repressor of ligand-mediated transcription, as it had no effect on vitamin D receptor function. Interestingly, hPR-A but not hPR-B was capable of a similar inhibition of glucocorticoid, androgen, and mineralocorticoid receptor-mediated gene transcription. This suggests a specific role for the hPR-A isoform in this regulatory process. The trans-dominant effects of hPR-A were induced also by the antiprogestins ZK112993 and ZK98299 and a DNA binding defective hPR-A mutant, suggesting that the inhibitory function of hPR-A does not require DNA binding. The dual role of hPR-A as an activator or repressor of transcription defines a potential mechanism by which cells can generate dissimilar responses to a single hormone and provides a molecular explanation for the existence of two distinct forms of the hPR.

Authors
Vegeto, E; Shahbaz, MM; Wen, DX; Goldman, ME; O'Malley, BW; McDonnell, DP
MLA Citation
Vegeto, E., et al. “Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function..” Mol Endocrinol, vol. 7, no. 10, Oct. 1993, pp. 1244–55. Pubmed, doi:10.1210/mend.7.10.8264658.
PMID
8264658
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
7
Issue
10
Publish Date
1993
Start Page
1244
End Page
1255
DOI
10.1210/mend.7.10.8264658

Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae.

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of methylmalonyl-CoA to succinyl-CoA. Previous reports have described cloning and sequencing of a cDNA for human methylmalonyl-CoA mutase. This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones. We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae. This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues.

Authors
Andrews, E; Jansen, R; Crane, AM; Cholin, S; McDonnell, D; Ledley, FD
MLA Citation
Andrews, E., et al. “Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae..” Biochem Med Metab Biol, vol. 50, no. 2, Oct. 1993, pp. 135–44.
PMID
7903149
Source
pubmed
Published In
Biochemical Medicine and Metabolic Biology
Volume
50
Issue
2
Publish Date
1993
Start Page
135
End Page
144

Chicken progesterone receptor expressed in Saccharomyces cerevisiae is correctly phosphorylated at all four Ser-Pro phosphorylation sites.

This study describes the phosphorylation of chicken progesterone receptor (cPR) produced in yeast, Saccharomyces cerevisiae, and examines the dependence of specific phosphorylations on hormone and DNA binding. The chicken progesterone receptor is expressed in vivo as two forms, cPRB and a smaller form, cPRA. Characterization of the phosphorylation sites in the cPRB form expressed in yeast shows that progesterone receptor is phosphorylated on the three serines (Ser211, Ser260, and Ser530) reported previously in chicken oviduct. An additional site which was phosphorylated in response to hormone was also detected and was subsequently identified as Ser367. Although cPRB and cPRA are phosphorylated identically in chicken oviduct, cPRA expressed in yeast is phosphorylated on Ser211, Ser260, and Ser367, but phosphorylation of Ser530 is almost undetectable. In contrast, cPRB expressed in yeast is phosphorylated on all four sites. No phosphorylations were found in or near the region required for hormone binding, indicating that phosphorylation is not required for hormone binding. In order to determine whether any of the phosphorylations were DNA-dependent, phosphorylation was also studied using cPRA containing a partial deletion of the DNA binding domain. Two of the sites, Ser211 and Ser367, showed reduced phosphorylation in this mutant, suggesting a possible requirement for DNA binding activity for the phosphorylation of these sites. To our knowledge, this is one of the first demonstrations that a eucaryotic protein expressed in yeast is correctly phosphorylated.

Authors
Poletti, A; Conneely, OM; McDonnell, DP; Schrader, WT; O'Malley, BW; Weigel, NL
MLA Citation
Poletti, A., et al. “Chicken progesterone receptor expressed in Saccharomyces cerevisiae is correctly phosphorylated at all four Ser-Pro phosphorylation sites..” Biochemistry, vol. 32, no. 37, Sept. 1993, pp. 9563–69. Pubmed, doi:10.1021/bi00088a007.
PMID
8373763
Source
pubmed
Published In
Biochemistry
Volume
32
Issue
37
Publish Date
1993
Start Page
9563
End Page
9569
DOI
10.1021/bi00088a007

Positive regulation of the vitamin D receptor by its cognate ligand in heterologous expression systems.

Hormonal vitamin D3 is a major regulator of calcium metabolism and is involved in basic cellular processes, such as those of proliferation and differentiation. These actions are mediated via an intracellular vitamin D3 receptor (VDR), which is a member of the evergrowing steroid hormone receptor superfamily. The interaction between the vitamin D3 ligand and its receptor is thought to be through a classic steroid hormone mechanism. It is notable, however, that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has also been documented as an agent that directly up-regulates endogenous VDR in both intact animals and cultured cells. In this report, we confirm that the levels of recombinantly expressed VDR produced in transiently transfected COS-1 cells also increase several-fold when the cells are treated with 1,25-(OH)2D3. Additionally, we show that a similar pattern is exhibited in a Saccharomyces cerevisiae expression system. This indicates that the mechanism for VDR up-regulation is conserved in both yeast and mammalian cells. Our results show that up-regulation by 1,25-(OH)2D3 is specific to the VDR, and that 1,25-(OH)2D3 does not affect other expressed receptor proteins, such as those for estrogen and progesterone. Finally, we demonstrate that the mechanism of up-regulation apparently occurs at the level of the protein and is most likely due to altered stability of the occupied receptor. Our observations lead us to propose that in addition to the classically viewed role of hormone in receptor activation, 1,25-(OH)2D3 may serve to amplify signal response via homologous up-regulation.

Authors
Santiso-Mere, D; Sone, T; Hilliard, GM; Pike, JW; McDonnell, DP
MLA Citation
Santiso-Mere, D., et al. “Positive regulation of the vitamin D receptor by its cognate ligand in heterologous expression systems..” Mol Endocrinol, vol. 7, no. 7, July 1993, pp. 833–39. Pubmed, doi:10.1210/mend.7.7.8413308.
PMID
8413308
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
7
Issue
7
Publish Date
1993
Start Page
833
End Page
839
DOI
10.1210/mend.7.7.8413308

Creation of an active estrogen-responsive element by a single base change in the flanking sequence of a cellular oncogene: a possible mechanism for hormonal carcinogenesis?

Estrogens are considered to act as promoters in a multistep process of hormonal carcinogenesis, although the molecular mechanisms by which these hormones act in tumorigenesis are unclear at present. Estradiol is known to induce expression of certain proto-oncogenes, and this led us to examine potential regulatory regions of the cellular c-fos oncogene. The 5'-flanking region of the murine c-fos contains a 13-bp palindromic sequence (GGTCTnnnAGACC) with striking homology to the consensus estrogen-responsive element (ERE) GGTCAnnnTGACC. However, the c-fos sequence did not bind the human estrogen receptor or confer hormonal responsiveness in a yeast-based transcriptional test system. Importantly, a single base change in the fifth position of the c-fos sequence (GGTCTnnnAGACC to GGTCA/GnnnAGACC) produced an element that bound the estrogen receptor and conferred estrogen-dependent transcriptional activation of a reporter gene. This suggests a specific hypothesis by which estrogens could act as tumor promoters. In this paradigm, the regulatory region of the cellular oncogenes, tumor suppressor genes, and growth-factor genes contain inactive sequences with close homologies to hormone-responsive elements. Initiation occurs when some agent (e.g., a chemical carcinogen) causes a mutation in such a sequence to create a functional hormone-responsive element. Estrogens, acting through their receptors and the mutated element, can then activate the target gene to stimulate cell proliferation and increase the population of initiated cells.

Authors
Nawaz, Z; Stancel, GM; McDonnell, DP; Hyder, SM
MLA Citation
Nawaz, Z., et al. “Creation of an active estrogen-responsive element by a single base change in the flanking sequence of a cellular oncogene: a possible mechanism for hormonal carcinogenesis?.” Mol Carcinog, vol. 7, no. 2, 1993, pp. 76–82.
PMID
8457291
Source
pubmed
Published In
Molecular Carcinogenesis
Volume
7
Issue
2
Publish Date
1993
Start Page
76
End Page
82

Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function

Two distinct isoforms of the human progesterone receptor (hPR-A and hPR-B) have been identified previously. They differ only in that hPR-B contains an additional 164 amino acids at the amino terminus. Among various species these two forms arise as a result of either alternate initiation of translation from the same mRNA or by transcription from alternate promoters within the same gene. In order to understand the reason for their existence, we studied the transcriptional capacity of these individual receptors and observed that their activity was influenced strongly by cell and promoter context. More surprising was the observation that in promoter and cell contexts where hPR-A was inactive, it acted as a potent trans-dominant represser of hPR-B-mediated transcription. This event occurred at substoichiometric concentrations of hPR-A and was hormone dependent. Human PR-A was not a general represser of ligand-mediated transcription, as it had no effect on vitamin D receptor function. Interestingly, hPR-A but not hPR-B was capable of a similar inhibition of glucocorticoid, androgen, and mineralocorticoid receptor-mediated gene transcription. This suggests a specific role for the hPR-A isoform in this regulatory process. The trans-dominant effects of hPR-A were induced also by the antiprogestins ZK112993 and ZK98299 and a DNA binding defective hPR-A mutant, suggesting that the inhibitory function of hPR-A does not require DNA binding. The dual role of hPR-A as an activator or represser of transcription defines a potential mechanism by which cells can generate dissimilar responses to a single hormone and provides a molecular explanation for the existence of two distinct forms of the hPR. Copyright © 1993 by The Endocrine Society.

Authors
Vegeto, E; Shahbaz, MM; Wen, DX; Goldman, ME; O'Malley, BW; McDonnell, DP
MLA Citation
Vegeto, E., et al. “Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function.” Molecular Endocrinology, vol. 7, no. 10, 1993, pp. 1244–55. Scival, doi:10.1210/me.7.10.1244.
Source
scival
Published In
Molecular Endocrinology
Volume
7
Issue
10
Publish Date
1993
Start Page
1244
End Page
1255
DOI
10.1210/me.7.10.1244

Identification of a negative regulatory function for steroid receptors.

This report describes the identification of a negative regulator of estrogen and progesterone receptor function. Using a reconstituted estrogen-responsive transcription system in Saccharomyces cerevisiae, we have identified a "repressor function," which when mutated, increases the transcriptional activity of the estrogen and progesterone receptors. In the case of the estrogen receptor this mutation increases the sensitivity of estrogen-mediated activation by at least four orders of magnitude. Analysis of derivatives of the estrogen receptor indicated that this repressor specifically affects the transcription activity of the TAF1 activation domain of the estrogen receptor. The repressor was cloned by complementation and identified as SSN6, a previously described mediator of glucose repression in yeast. Our results indicate that SSN6 is likely to be involved also in the repression of other cellular activators. Interestingly, deletion of the SSN6 protein allows the antiestrogens ICI 164384 and nafoxidine to behave as more potent agonists of estrogen receptor function, while RU486 also becomes a more potent activator of progesterone receptor function. These data suggest that in wild-type cells the role of hormone is twofold: it promotes DNA binding of the receptor and it also induces a conformational change in the receptor which overcomes the effects of this repressor function.

Authors
McDonnell, DP; Vegeto, E; O'Malley, BW
MLA Citation
McDonnell, D. P., et al. “Identification of a negative regulatory function for steroid receptors..” Proc Natl Acad Sci U S A, vol. 89, no. 22, Nov. 1992, pp. 10563–67. Pubmed, doi:10.1073/pnas.89.22.10563.
PMID
1438251
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
89
Issue
22
Publish Date
1992
Start Page
10563
End Page
10567
DOI
10.1073/pnas.89.22.10563

Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene.

We have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyltransferase, linked to regions of mouse c-fos, to identify a specific estrogen response element (ERE) in this protooncogene. This element is located in the untranslated 3'-flanking region of the c-fos gene, 5 kilobases (kb) downstream from the c-fos promoter and 1.5 kb downstream of the poly(A) signal. This element confers estrogen responsiveness to chloramphenicol acetyltransferase reporters linked to both the herpes simplex virus thymidine kinase promoter and the homologous c-fos promoter. Deletion analysis localized the response element to a 200-base pair fragment which contains the element GGTCACCACAGCC that resembles the consensus ERE sequence GGTCACAGTGACC originally identified in Xenopus vitellogenin A2 gene. A synthetic 36-base pair oligodeoxynucleotide containing this c-fos sequence conferred estrogen inducibility to the thymidine kinase promoter. The corresponding sequence also induced reporter activity when present in the c-fos gene fragment 3 kb from the thymidine kinase promoter. Gel-shift experiments demonstrated that synthetic oligonucleotides containing either the consensus ERE or the c-fos element bind human estrogen receptor obtained from a yeast expression system. However, the mobility of the shifted band is faster for the fos-ERE-complex than the consensus ERE complex suggesting that the three-dimensional structure of the protein-DNA complexes is different or that other factors are differentially involved in the two reactions. When the 5'-GGTCA sequence present in the c-fos ERE is mutated to 5'-TTTCA, transcriptional activation and receptor binding activities are both lost. Mutation of the CAGCC-3' element corresponding to the second half-site of the c-fos sequence also led to the loss of receptor binding activity, suggesting that both half-sites of this element are involved in this function. The estrogen induction mediated by either the c-fos or the consensus ERE was blunted by the antiestrogen tamoxifen. Based on these studies, we believe the 3'-fos ERE sequence we have identified may be a major cis-acting element involved in the physiological regulation of the gene by estrogens in vivo.

Authors
Hyder, SM; Stancel, GM; Nawaz, Z; McDonnell, DP; Loose-Mitchell, DS
MLA Citation
Hyder, S. M., et al. “Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene..” J Biol Chem, vol. 267, no. 25, Sept. 1992, pp. 18047–54.
PMID
1517237
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
267
Issue
25
Publish Date
1992
Start Page
18047
End Page
18054

Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast.

The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.

Authors
Pham, TA; Hwung, YP; Santiso-Mere, D; McDonnell, DP; O'Malley, BW
MLA Citation
Pham, T. A., et al. “Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast..” Mol Endocrinol, vol. 6, no. 7, July 1992, pp. 1043–50. Pubmed, doi:10.1210/mend.6.7.1508220.
PMID
1508220
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
6
Issue
7
Publish Date
1992
Start Page
1043
End Page
1050
DOI
10.1210/mend.6.7.1508220

Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Wooge, CH; Nilsson, GM; Heierson, A; McDonnell, DP; Katzenellenbogen, BS
MLA Citation
Wooge, C. H., et al. “Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells..” Mol Endocrinol, vol. 6, no. 6, June 1992, pp. 861–69. Pubmed, doi:10.1210/mend.6.6.1495491.
PMID
1495491
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
6
Issue
6
Publish Date
1992
Start Page
861
End Page
869
DOI
10.1210/mend.6.6.1495491

The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor.

The human progesterone receptor form B (hPR-B) was expressed in Saccharomyces cerevisiae together with a specific reporter plasmid. To understand the mechanism underlying antagonist ligand activity, libraries of hormone binding domain (HBD)-mutated hPR-B molecules were prepared. A mutant receptor was identified that had lost the ability to bind either progesterone or R5020; it could still bind RU486 and, surprisingly, fully activated transcription in the presence of this "antagonist" and other antiprogestins. When this receptor mutant was assayed in mammalian cells, RU486 again demonstrated agonistic activity. Sequence analysis indicated that the mutant phenotype was due to truncation of the carboxy (C)-terminal 42 aa. We conclude that amino acids in the extreme C-terminal region are required for the receptor to bind progesterone, while antagonists bind to a site located more N-terminal of the HBD. Our results suggest that the extreme C-terminal region of the receptor contains an inhibitory function that silences receptor transactivation in the absence of agonist and in the presence of antagonist.

Authors
Vegeto, E; Allan, GF; Schrader, WT; Tsai, MJ; McDonnell, DP; O'Malley, BW
MLA Citation
Vegeto, E., et al. “The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor..” Cell, vol. 69, no. 4, May 1992, pp. 703–13. Pubmed, doi:10.1016/0092-8674(92)90234-4.
PMID
1586949
Source
pubmed
Published In
Cell
Volume
69
Issue
4
Publish Date
1992
Start Page
703
End Page
713
DOI
10.1016/0092-8674(92)90234-4

A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae.

A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Poletti, A; Weigel, NL; McDonnell, DP; Schrader, WT; O'Malley, BW; Conneely, OM
MLA Citation
Poletti, A., et al. “A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae..” Gene, vol. 114, no. 1, May 1992, pp. 51–58. Pubmed, doi:10.1016/0378-1119(92)90706-u.
PMID
1316867
Source
pubmed
Published In
Gene
Volume
114
Issue
1
Publish Date
1992
Start Page
51
End Page
58
DOI
10.1016/0378-1119(92)90706-u

Modulation of progesterone receptor binding to progesterone response elements by positioned nucleosomes.

In cells, steroid hormone receptors interact with target enhancer elements on nucleosomes to regulate transcription of genes. To elucidate how nucleosomes can potentially regulate the interactions of steroid receptors with steroid response elements, we have examined the effects of nucleosome positioning and histone source on the binding of the progesterone receptor to DNA elements on nucleosomes reconstituted in vitro. We find that the affinity of the receptor for its response element is dependent on the position of the element within the nucleosome, but not on the histone source, active or inactive chromatin. Our results suggest that the strength of DNA-histone interactions within the nucleosome modulates the binding of progesterone receptor to response elements. Thus, nucleosome positioning is likely to influence the function of steroid receptors in vivo.

Authors
Pham, TA; McDonnell, DP; Tsai, MJ; O'Malley, BW
MLA Citation
Pham, T. A., et al. “Modulation of progesterone receptor binding to progesterone response elements by positioned nucleosomes..” Biochemistry, vol. 31, no. 5, Feb. 1992, pp. 1570–78. Pubmed, doi:10.1021/bi00120a039.
PMID
1737013
Source
pubmed
Published In
Biochemistry
Volume
31
Issue
5
Publish Date
1992
Start Page
1570
End Page
1578
DOI
10.1021/bi00120a039

Structural requirements for high affinity ligand binding by estrogen receptors: A comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 ± 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 ± 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 ± 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast. Full length and truncated receptors expressed in yeast, as well as the bacterially expressed ER, were as stable as the full length MCF-7 ER, with minimal loss of the initial binding capacity of the unoccupied receptor even after 10 h at 25 C. In contrast, there was no binding of either reversibly (estradiol) or covalently attaching (ketononestrol aziridine, tamoxifen aziridine) ligands to a 42-amino acid synthetic peptide (human ER amino acids 510-551) comprising a portion of the hormone binding domain considered essential for ligand binding and which encompasses Cys-530, shown previously to be the amino acid covalently labeled with ER affinity labeling ligands. These studies demonstrate that the hormone binding domain of the ER alone (amino acids 302-595) is sufficient to bind ligand with good affinity (ca. 30% that of full length ER) and with appropriate ligand structural specificity. Such expressed truncated proteins should be valuable in further studies to characterize the three-dimensional structure of the ligand binding pocket of the receptor.

Authors
Wooge, CH; Nilsson, GM; Heierson, A; McDonnell, DP; Katzenellenbogen, BS
MLA Citation
Wooge, C. H., et al. “Structural requirements for high affinity ligand binding by estrogen receptors: A comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.” Molecular Endocrinology, vol. 6, no. 6, 1992, pp. 861–69. Scival, doi:10.1210/me.6.6.861.
Source
scival
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
6
Issue
6
Publish Date
1992
Start Page
861
End Page
869
DOI
10.1210/me.6.6.861

Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast

The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.

Authors
Pham, TA; Hwung, YP; Santiso-Mere, D; McDonnell, DP; O'Malley, BW
MLA Citation
Pham, T. A., et al. “Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast.” Molecular Endocrinology, vol. 6, no. 7, 1992, pp. 1043–50. Scival, doi:10.1210/me.6.7.1043.
Source
scival
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
6
Issue
7
Publish Date
1992
Start Page
1043
End Page
1050
DOI
10.1210/me.6.7.1043

Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors

It is fairly well accepted that the presence of estrogen receptor (ER) and progesterone receptor (PgR) identifies breast cancer patients with a lower risk of relapse and better overall survival. But patients with discordant receptors, the ER+/PgR- phenotype, are often intermediate in clinical response. We focused upon this group of patients and have identified a truncated ER which is abundant in some ER+/PgR- breast tumors and which inhibits the binding of wild-type ER to its cognate response element. This variant interferes in a dominant negative manner with wild-type ER function and may represent a mechanism for modulation of estrogen responsiveness.

Authors
Fuqua, SAW; Fitzgerald, SD; Allred, DC; Elledge, RM; Nawaz, Z; McDonnell, DP; O'Malley, BW; Greene, GL; McGuire, WL
MLA Citation
Fuqua, S. A. W., et al. “Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors.” Cancer Research, vol. 52, no. 2, 1992, pp. 483–86.
Source
scival
Published In
Cancer Research
Volume
52
Issue
2
Publish Date
1992
Start Page
483
End Page
486

Identification of novel steroid-response elements.

A rapid method for defining novel steroid-responsive elements has been developed. Large libraries of degenerate oligonucleotides were analyzed using a yeast-based screen to identify estrogen-responsive DNA sequences. From a library of 40,000 recombinants, seven estrogen-responsive clones were identified. When sequenced, these elements showed remarkable diversity and were different from the consensus vitellogenin A2 ERE. One surprising result was the presence of the two half sites as direct repeats in some of the clones. This implies that in vivo estrogen receptor can bind and transactivate yeast genes through response elements in which the two half sites align as direct repeats. This protocol requires no purified protein and specifically selects for functional response elements. It has a wide application in the study of any transcription factor/DNA interaction.

Authors
Nawaz, Z; Tsai, MJ; McDonnell, DP; O'Malley, BW
MLA Citation
Nawaz, Z., et al. “Identification of novel steroid-response elements..” Gene Expr, vol. 2, no. 1, 1992, pp. 39–47.
PMID
1617301
Source
pubmed
Published In
Gene Expression
Volume
2
Issue
1
Publish Date
1992
Start Page
39
End Page
47

Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo.

The activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes. To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae. We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions. These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter. Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes. Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D. (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo.

Authors
Pham, TA; Hwung, YP; McDonnell, DP; O'Malley, BW
MLA Citation
Pham, T. A., et al. “Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo..” J Biol Chem, vol. 266, no. 27, Sept. 1991, pp. 18179–87.
PMID
1917950
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
266
Issue
27
Publish Date
1991
Start Page
18179
End Page
18187

In situ distinction between steroid receptor binding and transactivation at a target gene.

We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen. Inhibition occurs thought the disruption of complex cooperative interactions among the GAL4 molecules in this reporter. The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding. Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand. In contrast, this latter receptor form will not activate transcription. Our results are consistent with a two-step process for receptor activation. Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form. Second, it converts receptor to a transcriptionally competent form. With use of this yeast model system, these two steps can be distinguished in situ.

Authors
McDonnell, DP; Nawaz, Z; O'Malley, BW
MLA Citation
McDonnell, D. P., et al. “In situ distinction between steroid receptor binding and transactivation at a target gene..” Mol Cell Biol, vol. 11, no. 9, Sept. 1991, pp. 4350–55. Pubmed, doi:10.1128/mcb.11.9.4350.
PMID
1875926
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
11
Issue
9
Publish Date
1991
Start Page
4350
End Page
4355
DOI
10.1128/mcb.11.9.4350

High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae.

Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undergraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.

Authors
McDonnell, DP; Nawaz, Z; Densmore, C; Weigel, NL; Pham, TA; Clark, JH; O'Malley, BW
MLA Citation
McDonnell, D. P., et al. “High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae..” J Steroid Biochem Mol Biol, vol. 39, no. 3, Sept. 1991, pp. 291–97.
PMID
1911419
Source
pubmed
Published In
The Journal of Steroid Biochemistry and Molecular Biology
Volume
39
Issue
3
Publish Date
1991
Start Page
291
End Page
297

Antiestrogen can establish nonproductive receptor complexes and alter chromatin structure at target enhancers.

We describe in this report experiments in vivo that demonstrate that antiestrogens promote DNA binding of the estrogen receptor without efficiently inducing transcription. When the receptor is modified to carry a foreign unregulated transactivation domain, transcription can be induced efficiently by both estrogen and antiestrogens. Under apparent saturation conditions, antihormone-receptor complexes binding to responsive enhancer elements elicit only a low level of transcription. In addition, we show that both estrogen and an antiestrogen, nafoxidine, effect very similar alterations in chromatin structure at a responsive promoter. These results indicate that in vivo steroid receptor action can be regulated subsequent to the DNA binding step, by regulating interactions with the target transcriptional machinery. In this regard, antihormones can function by establishing receptor-DNA complexes that are transcriptionally nonproductive.

Authors
Pham, TA; Elliston, JF; Nawaz, Z; McDonnell, DP; Tsai, MJ; O'Malley, BW
MLA Citation
Pham, T. A., et al. “Antiestrogen can establish nonproductive receptor complexes and alter chromatin structure at target enhancers..” Proc Natl Acad Sci U S A, vol. 88, no. 8, Apr. 1991, pp. 3125–29. Pubmed, doi:10.1073/pnas.88.8.3125.
PMID
2014231
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
88
Issue
8
Publish Date
1991
Start Page
3125
End Page
3129
DOI
10.1073/pnas.88.8.3125

Variant human breast tumor estrogen receptor with constitutive transcriptional activity

Since progesterone receptor (PgR) is normally induced by estrogen, breast cancers lacking estrogen receptor (ER) would also be expected to lack PgR. However, a small percentage of breast cancers are ER- yet PgR+. These tumors might possess an ER which is defective in estrogen binding but is still functional in stimulating estrogen-responsive genes such as PgR. We have now detected such a variant, lacking exon 5 of the hormone-binding domain, using complementary DNA amplified by the polymerase chain reaction. This variant was the predominate ER RNA expressed in three ER-/PgR+ tumors. Furthermore, the variant ER constitutively activates transcription of a normally estrogen-dependent gene construct in yeast cells. The variant ER could explain the expression of PgR in certain tumors and have therapeutic implications.

Authors
Fuqua, SAW; Fitzgerald, SD; Chamness, GC; Tandon, AK; McDonnell, DP; Nawaz, Z; O'Malley, BW; McGuire, WL
MLA Citation
Fuqua, S. A. W., et al. “Variant human breast tumor estrogen receptor with constitutive transcriptional activity.” Cancer Research, vol. 51, no. 1, 1991, pp. 105–09.
Source
scival
Published In
Cancer Research
Volume
51
Issue
1
Publish Date
1991
Start Page
105
End Page
109

Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies.

We have cloned a cDNA encoding the human vitamin D receptor (VDR) into a high copy yeast plasmid controlled transcriptionally by the copper-inducible metallothionein (CUP-1) promoter to produce YEpV1. Introduction of this plasmid in the protease deficient Saccharomyces cerevisiae strain BJ 3505 and subsequent growth in the presence of copper and 1,25-dihydroxyvitamin D3 leads to the synthesis of intact VDR comprising over 0.5% of total soluble protein. The VDR was purified to near homogeneity from similarly induced yeast cultures by ammonium sulfate precipitation, and sequential DNA-cellulose and DEAE-Sephadex chromatography, and then characterized for physical and functional properties. The purified VDR associated with a specific synthetic DNA sequence comprising the vitamin D response element as assayed through bandshift analysis. Binding, however, required the presence of a mammalian cell protein factor that also enhances vitamin D response element interaction by mammalian cell-derived VDR. Polyclonal antibodies raised in rabbits against the purified VDR further retarded the receptor/nuclear factor/DNA complex in these analyses. These studies, together with our previous experiments that demonstrate reconstitution of a vitamin D-dependent transcription system in yeast, show that the VDR can be produced and purified from yeast in a functional form.

Authors
Sone, T; McDonnell, DP; O'Malley, BW; Pike, JW
MLA Citation
Sone, T., et al. “Expression of human vitamin D receptor in Saccharomyces cerevisiae. Purification, properties, and generation of polyclonal antibodies..” J Biol Chem, vol. 265, no. 35, Dec. 1990, pp. 21997–2003.
PMID
2174892
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
265
Issue
35
Publish Date
1990
Start Page
21997
End Page
22003

Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3.

The regulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3 is mediated by the vitamin D receptor and a cis-acting DNA response element that has been identified within the 5' region of the osteocalcin promoter. In this report, we show that vitamin D receptors derived from nuclear extracts of mammalian cells bind directly to this cis-acting element in vitro and do so in a manner requiring hormone. Vitamin D receptors derived from reticulocyte lysate translations in vitro or from extracts of a Saccharomyces cerevisiae strain that expresses the recombinant protein also bind the osteocalcin responsive element, but only when nuclear extracts of mammalian cells are provided. The vitamin-D-receptor-DNA-binding accessory factor is isolated by salt extraction, labile to temperature, and sensitive to tryptic digestion. These studies suggest that the high-affinity interaction of the vitamin D receptor with the osteocalcin vitamin D response element in vitro requires both 1,25-dihydroxyvitamin D3 and an accessory protein derived from the mammalian cell nucleus.

Authors
Liao, J; Ozono, K; Sone, T; McDonnell, DP; Pike, JW
MLA Citation
Liao, J., et al. “Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3..” Proc Natl Acad Sci U S A, vol. 87, no. 24, Dec. 1990, pp. 9751–55. Pubmed, doi:10.1073/pnas.87.24.9751.
PMID
2175914
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
87
Issue
24
Publish Date
1990
Start Page
9751
End Page
9755
DOI
10.1073/pnas.87.24.9751

High level expression of a truncated chicken progesterone receptor in Escherichia coli.

Using a novel Escherichia coli system we have successfully overexpressed a region of the chicken progesterone receptor which encodes both the DNA- and hormone-binding domains. The expression system produces the truncated receptor fragment as an in-frame fusion with ubiquitin. This strategy greatly enhances both the solubility and stability of fusion proteins expressed in E. coli. Synthesis has been further improved by induction of the lambda PL promoter with nalidixic acid at low growth temperatures (less than or equal to 30 degrees C) rather than use of conventional heat induction protocols. We can produce 10 mg of receptor fragment/liter of cells using this system, and we estimate that at least 0.3 mg of this receptor material is biologically active, as assessed by DNA-binding and hormone-binding assays. Receptor produced in this manner is almost indistinguishable from authentic oviduct progesterone receptor using the criteria of hormone-binding specificity and affinity and binding to a progesterone response element. This expression system offers a cheap convenient method for the production of mg amounts of biologically active derivatives of progesterone receptor for biochemical studies.

Authors
Power, RF; Conneely, OM; McDonnell, DP; Clark, JH; Butt, TR; Schrader, WT; O'Malley, BW
MLA Citation
Power, R. F., et al. “High level expression of a truncated chicken progesterone receptor in Escherichia coli..” J Biol Chem, vol. 265, no. 3, Jan. 1990, pp. 1419–24.
PMID
2153132
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
265
Issue
3
Publish Date
1990
Start Page
1419
End Page
1424

Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae).

The cDNAs encoding full-length chicken oviduct progesterone receptor B (PRB) and a truncated receptor (C1C2) lacking the amino-terminal domain were expressed in yeast (Saccharomyces cerevisiae) using a ubiquitin fusion system. The expression of the fusion protein is under the control of a copper-responsive yeast metallothionein promoter, and the fusion protein is subsequently cleaved by the yeast host enzyme to produce receptor protein. Western immunoblot analyses of yeast extracts containing full-length PRB revealed a polypeptide co-migrating with authentic chicken oviduct PRB. Using a polyclonal antibody (907) directed against the "hinge" region of the authentic chicken progesterone receptor, a 42-kDa polypeptide was detected by Western analysis in yeast extracts containing C1C2 receptors. Standard hormone binding assays indicated that these receptors produced in yeast cells exhibited steroid binding affinity and specificity characteristic of the authentic chicken progesterone receptor. To test for progesterone receptor-mediated activation of transcription in yeast, reporter plasmids were constructed to transform yeast cells expressing PRB or C1C2 receptors. The reporter gene contained two copies of a progesterone response element upstream of the yeast proximal CYC1 promoter fused to the beta-galactosidase gene of Escherichia coli. The induction of beta-galactosidase activity by PRB and C1C2 was strictly dependent on specific ligand and the presence of a progesterone response element. However, overproduced C1C2 receptors had an adverse effect on the transcription of the lacZ gene. It was found that when overproduced C1C2 was activated by progesterone, an inhibitory effect on normal yeast cell growth was evident. These observations suggest that C1C2 is a potent trans-acting factor in yeast and that the amino-terminal domain of the chicken progesterone receptor may play a role in selective modulation of target gene activation.

Authors
Mak, P; McDonnell, DP; Weigel, NL; Schrader, WT; O'Malley, BW
MLA Citation
Mak, P., et al. “Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae)..” J Biol Chem, vol. 264, no. 36, Dec. 1989, pp. 21613–18.
PMID
2689442
Source
pubmed
Published In
The Journal of Biological Chemistry
Volume
264
Issue
36
Publish Date
1989
Start Page
21613
End Page
21618

Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae.

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

Authors
McDonnell, DP; Pike, JW; Drutz, DJ; Butt, TR; O'Malley, BW
MLA Citation
McDonnell, D. P., et al. “Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae..” Mol Cell Biol, vol. 9, no. 8, Aug. 1989, pp. 3517–23. Pubmed, doi:10.1128/mcb.9.8.3517.
PMID
2552296
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
9
Issue
8
Publish Date
1989
Start Page
3517
End Page
3523
DOI
10.1128/mcb.9.8.3517

Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression.

The human vitamin D receptor (VDR) has been cloned recently. Two cDNAs comprising the full-length VDR were spliced, cloned into a mammalian expression vector, and transiently expressed in COS-1 cells. The protein product exhibited properties consistent with that observed for receptor in human cells. A series of 5'- and 3'-deletions of the full-length VDR cDNA was prepared and evaluated. Native DNA binding was localized to a peptide fragment (residues 1-114) whose most prominent feature is the cysteine rich region proven to represent the DNA binding domain in other steroid receptors. Steroid binding-competence required synthesis of a peptide that initiated C-terminal to the DNA-binding domain at residue 114 and which contained the remaining 313 residues. To determine the location of elements within the receptor necessary for transcription, an osteocalcin gene promoter-chloramphenicol acetyltransferase reporter gene was cotransfected together with wild type or mutant VDR cDNAs and the latter's effect on chloramphenicol acetyltransferase activity was assessed. Cotransfection of wild type receptor alone resulted in efficient transcription of the reporter plasmid. However, synthesis of a peptide containing the DNA binding domain as well as 76 residues carboxy terminal to this region exhibited some degree of activity, albeit constitutive. These results suggest that the functional domains of the VDR are similar to that of other steroid receptors and that these domains participate in the transcriptional regulation of the human osteocalcin gene.

Authors
McDonnell, DP; Scott, RA; Kerner, SA; O'Malley, BW; Pike, JW
MLA Citation
McDonnell, D. P., et al. “Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression..” Mol Endocrinol, vol. 3, no. 4, Apr. 1989, pp. 635–44. Pubmed, doi:10.1210/mend-3-4-635.
PMID
2542779
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
3
Issue
4
Publish Date
1989
Start Page
635
End Page
644
DOI
10.1210/mend-3-4-635

Human vitamin D receptor mutations: identification of molecular defects in hypocalcemic vitamin D resistant rickets.

Authors
Hughes, M; Malloy, P; Kieback, D; McDonnell, D; Feldman, D; Pike, JW; O'Malley, B
MLA Citation
Hughes, M., et al. “Human vitamin D receptor mutations: identification of molecular defects in hypocalcemic vitamin D resistant rickets..” Adv Exp Med Biol, vol. 255, 1989, pp. 491–503. Pubmed, doi:10.1007/978-1-4684-5679-0_52.
PMID
2618873
Source
pubmed
Published In
Advances in Experimental Medicine and Biology
Volume
255
Publish Date
1989
Start Page
491
End Page
503
DOI
10.1007/978-1-4684-5679-0_52

Cloning and expression of full-length cDNA encoding human vitamin D receptor.

Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3' noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of approximately equal to 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

Authors
Baker, AR; McDonnell, DP; Hughes, M; Crisp, TM; Mangelsdorf, DJ; Haussler, MR; Pike, JW; Shine, J; O'Malley, BW
MLA Citation
Baker, A. R., et al. “Cloning and expression of full-length cDNA encoding human vitamin D receptor..” Proc Natl Acad Sci U S A, vol. 85, no. 10, May 1988, pp. 3294–98. Pubmed, doi:10.1073/pnas.85.10.3294.
PMID
2835767
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
85
Issue
10
Publish Date
1988
Start Page
3294
End Page
3298
DOI
10.1073/pnas.85.10.3294

Molecular biology of the vitamin D hormone.

Authors
Haussler, MR; Mangelsdorf, DJ; Komm, BS; Terpening, CM; Yamaoka, K; Allegretto, EA; Baker, AR; Shine, J; McDonnell, DP; Hughes, M
MLA Citation
Haussler, M. R., et al. “Molecular biology of the vitamin D hormone..” Recent Prog Horm Res, vol. 44, 1988, pp. 263–305.
PMID
2851156
Source
pubmed
Published In
Recent Progress in Hormone Research
Volume
44
Publish Date
1988
Start Page
263
End Page
305

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor.

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.

Authors
McDonnell, DP; Pike, JW; O'Malley, BW
MLA Citation
McDonnell, D. P., et al. “The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor..” J Steroid Biochem, vol. 30, no. 1–6, 1988, pp. 41–46.
PMID
2838696
Source
pubmed
Published In
Journal of Steroid Biochemistry
Volume
30
Issue
1-6
Publish Date
1988
Start Page
41
End Page
46

Immunoselection of cDNAs to avian intestinal calcium binding protein 28K and a novel calmodulin-like protein: assessment of mRNA regulation by the vitamin D hormone.

Calcium's role in a variety of cellular processes has been well documented. The storage, distribution, and delivery of calcium are regulated by a family of binding proteins including troponin C, calmodulin, parvalbumin, and vitamin D dependent calcium binding protein (CaBP-28), all of which have evolved from a common ancestral gene. To evaluate vitamin D regulation of gene transcription, a CaBP-28 cDNA (767 base pairs) was isolated from a chicken intestine lambda gt11 library utilizing a polyvalent CaBP-28 antibody as a probe. Coincident with the identification of the CaBP-28 cDNA, a group of cDNAs also was isolated (with the anti-CaBP-28 antibody) that demonstrated 84% nucleotide homology and 99% deduced amino acid homology with chicken brain calmodulin (CaM). This new CaM-like cDNA was named neoCaM. There is little nucleotide homology between the CaBP-28 cDNA and neoCaM. The CaBP-28 cDNA hybridizes with three transcripts of 2000, 2900, and 3300 bases which are dramatically induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], while the neoCaM cDNA recognizes three distinct (from CaBP-28) transcripts. Two of these mRNAs are 1400 and 1800 bases as described for brain CaM, but another larger 4000-base transcript is detected with neoCaM. Neither the CaM nor the neoCaM transcript reveals any modulation by 1,25(OH)2D3. Herein, we discuss the possible significance of not only the isolation of both cDNAs with a single antibody but also the relation of neoCaM to other well-characterized CaM cDNAs.

Authors
Mangelsdorf, DJ; Komm, BS; McDonnell, DP; Pike, JW; Haussler, MR
MLA Citation
Mangelsdorf, D. J., et al. “Immunoselection of cDNAs to avian intestinal calcium binding protein 28K and a novel calmodulin-like protein: assessment of mRNA regulation by the vitamin D hormone..” Biochemistry, vol. 26, no. 25, Dec. 1987, pp. 8332–38. Pubmed, doi:10.1021/bi00399a046.
PMID
3442659
Source
pubmed
Published In
Biochemistry
Volume
26
Issue
25
Publish Date
1987
Start Page
8332
End Page
8338
DOI
10.1021/bi00399a046

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D.

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.

Authors
McDonnell, DP; Mangelsdorf, DJ; Pike, JW; Haussler, MR; O'Malley, BW
MLA Citation
McDonnell, D. P., et al. “Molecular cloning of complementary DNA encoding the avian receptor for vitamin D..” Science, vol. 235, no. 4793, Mar. 1987, pp. 1214–17. Pubmed, doi:10.1126/science.3029866.
PMID
3029866
Source
pubmed
Published In
Science (New York, N.Y.)
Volume
235
Issue
4793
Publish Date
1987
Start Page
1214
End Page
1217
DOI
10.1126/science.3029866

Structural organization and regulation of the chicken estrogen receptor.

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.

Authors
Maxwell, BL; McDonnell, DP; Conneely, OM; Schulz, TZ; Greene, GL; O'Malley, BW
MLA Citation
Maxwell, B. L., et al. “Structural organization and regulation of the chicken estrogen receptor..” Mol Endocrinol, vol. 1, no. 1, Jan. 1987, pp. 25–35. Pubmed, doi:10.1210/mend-1-1-25.
PMID
2901032
Source
pubmed
Published In
Molecular Endocrinology (Baltimore, Md.)
Volume
1
Issue
1
Publish Date
1987
Start Page
25
End Page
35
DOI
10.1210/mend-1-1-25

Analysis of repetitive sequence elements containing tRNA-like sequences.

Several repetitive sequence elements from diverse species share extensive sequence homology with tRNA molecules. Analysis of the tRNA-like sequences within these elements suggest that they have originated from authentic tRNA sequences. Elements containing tRNA-like sequences can be divided into three distinct groups whose members share extensive sequence homology, have similar sequence organization and have unique species distribution. We suggest that these three groups represent independent examples of retroposon families that have originated from tRNAs.

Authors
Lawrence, CB; McDonnell, DP; Ramsey, WJ
MLA Citation
Lawrence, C. B., et al. “Analysis of repetitive sequence elements containing tRNA-like sequences..” Nucleic Acids Res, vol. 13, no. 12, June 1985, pp. 4239–52. Pubmed, doi:10.1093/nar/13.12.4239.
PMID
3839306
Source
pubmed
Published In
Nucleic Acids Research
Volume
13
Issue
12
Publish Date
1985
Start Page
4239
End Page
4252
DOI
10.1093/nar/13.12.4239
Show More

Research Areas:

  • 3T3 Cells
  • 8-Bromo Cyclic Adenosine Monophosphate
  • ADP-ribosyl Cyclase 1
  • Acetates
  • Acetyl Coenzyme A
  • Acetyltransferases
  • Adolescent
  • Adult
  • Age Factors
  • Aging
  • Aldehyde Dehydrogenase
  • Allosteric Regulation
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Androgen Receptor Antagonists
  • Androgens
  • Antigens, CD
  • Antigens, CD38
  • Antigens, Nuclear
  • Antineoplastic Agents
  • Antineoplastic Agents, Hormonal
  • Apoptosis
  • Aromatase
  • Aryl Hydrocarbon Receptor Nuclear Translocator
  • Atlases as Topic
  • Autocrine Communication
  • Benzhydryl Compounds
  • Benzoates
  • Binding Sites
  • Biological Transport
  • Biomimetic Materials
  • Blotting, Western
  • Bone and Bones
  • Breast
  • Breast Neoplasms
  • Bruch Membrane
  • COS Cells
  • Cadherins
  • Calcium
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Carcinoma, Hepatocellular
  • Cardiovascular System
  • Cell Line, Tumor
  • Cell Nucleus
  • Cell Proliferation
  • Cercopithecus aethiops
  • Chemokine CXCL12
  • Child
  • Cholestanetriol 26-Monooxygenase
  • Cholesterol
  • Cholesterol Side-Chain Cleavage Enzyme
  • Chromans
  • Chromatin
  • Chromatin Immunoprecipitation
  • Chrysenes
  • Cinnamates
  • Cluster Analysis
  • Combinatorial Chemistry Techniques
  • Consensus Sequence
  • Cytoprotection
  • DNA Primers
  • DNA-Binding Proteins
  • Dehydroepiandrosterone
  • Dieldrin
  • Diethylstilbestrol
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Drug Design
  • Drug Discovery
  • Drug Evaluation, Preclinical
  • Drug Interactions
  • Drug Partial Agonism
  • Drug Resistance
  • Drug Resistance, Neoplasm
  • Drug Screening Assays, Antitumor
  • Drug Synergism
  • E2F1 Transcription Factor
  • Enhancer Elements, Genetic
  • Enzyme Activation
  • Enzyme Inhibitors
  • Enzyme-Linked Immunosorbent Assay
  • Estradiol
  • Estradiol Antagonists
  • Estradiol Congeners
  • Estrenes
  • Estriol
  • Estrogen Antagonists
  • Estrogen Receptor Modulators
  • Estrogen Receptor alpha
  • Estrogen Receptor beta
  • Estrogen Replacement Therapy
  • Estrogens
  • Estrogens, Non-Steroidal
  • Estrone
  • Extracellular Matrix
  • Female
  • Flow Cytometry
  • Furylfuramide
  • Gene Expression
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Gene Expression Regulation, Neoplastic
  • Gene Library
  • Genes, Reporter
  • Glucose
  • Glucose Transporter Type 1
  • Gonadal Steroid Hormones
  • Green Fluorescent Proteins
  • HCT116 Cells
  • HEK293 Cells
  • Haplorhini
  • HeLa Cells
  • Heat-Shock Proteins
  • Hematopoiesis
  • Hepatocyte Nuclear Factor 4
  • Hexokinase
  • Histone Acetyltransferases
  • Histone Deacetylase Inhibitors
  • Homeodomain Proteins
  • Hormone Antagonists
  • Humans
  • Hydroxycholesterols
  • Hydroxymethylglutaryl-CoA Synthase
  • Immunoblotting
  • Immunohistochemistry
  • Immunosuppressive Agents
  • Indoles
  • Induced Pluripotent Stem Cells
  • Inflammatory Breast Neoplasms
  • Insecticides
  • Insulin-Like Growth Factor I
  • Interleukin-1beta
  • Intracellular Signaling Peptides and Proteins
  • Kruppel-Like Transcription Factors
  • Leupeptins
  • Ligands
  • Lipofuscin
  • Lipoproteins, LDL
  • Locus Coeruleus
  • MCF-7 Cells
  • MSX1 Transcription Factor
  • Macrophages
  • Macular Degeneration
  • Male
  • Mammary Glands, Human
  • Metribolone
  • Mice
  • Mice, Congenic
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Inbred NOD
  • Mice, Knockout
  • Mice, Nude
  • Mice, SCID
  • Mice, Transgenic
  • Microscopy, Electron
  • Middle Aged
  • Mifepristone
  • Mitochondria
  • Mitogen-Activated Protein Kinases
  • Molecular Conformation
  • Molecular Sequence Data
  • Molecular Targeted Therapy
  • Multiple Myeloma
  • Mutagenesis, Site-Directed
  • NF-kappa B
  • Norepinephrine
  • Nuclear Receptor Co-Repressor 1
  • Nuclear Receptor Co-Repressor 2
  • Nuclear Receptor Coactivator 1
  • Nuclear Receptor Coactivator 2
  • Nuclear Receptor Coactivators
  • Organ Size
  • Orphan Nuclear Receptors
  • Osteoblasts
  • Osteoclasts
  • Osteogenesis
  • Osteoporosis
  • Ovariectomy
  • Oxidation-Reduction
  • PPAR gamma
  • Peptide Library
  • Peroxidase
  • Peroxisome Proliferator-Activated Receptors
  • Phenols
  • Pigment Epithelium of Eye
  • Plicamycin
  • Polymerase Chain Reaction
  • Progesterone
  • Progestins
  • Promegestone
  • Promoter Regions, Genetic
  • Prostate-Specific Antigen
  • Prostatic Neoplasms
  • Protein Conformation
  • Protein Kinase Inhibitors
  • Protein Kinases
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases
  • Proteins
  • Proteolysis
  • RNA Interference
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Raloxifene
  • Raloxifene Hydrochloride
  • Rats
  • Rats, Sprague-Dawley
  • Rats, Wistar
  • Reactive Oxygen Species
  • Receptor Cross-Talk
  • Receptor, IGF Type 1
  • Receptor, erbB-2
  • Receptors, Androgen
  • Receptors, Aryl Hydrocarbon
  • Receptors, CXCR4
  • Receptors, Calcitriol
  • Receptors, Cytoplasmic and Nuclear
  • Receptors, Estrogen
  • Receptors, Glucocorticoid
  • Receptors, Progesterone
  • Receptors, Retinoic Acid
  • Receptors, Steroid
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Repetitive Sequences, Nucleic Acid
  • Repressor Proteins
  • Response Elements
  • Retinal Dehydrogenase
  • Retinal Pigment Epithelium
  • Retinoblastoma Protein
  • Retinoid X Receptor alpha
  • Retinoid X Receptors
  • Retinoids
  • Reverse Transcriptase Polymerase Chain Reaction
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Selective Estrogen Receptor Modulators
  • Sequence Deletion
  • Species Specificity
  • Stem Cell Transplantation
  • Steroid 17-alpha-Hydroxylase
  • Steroid Hydroxylases
  • Steroids
  • Stilbenes
  • Structure-Activity Relationship
  • T-Lymphocytes
  • Tacrolimus Binding Proteins
  • Tamoxifen
  • Thiazolidinediones
  • Tight Junctions
  • Toxaphene
  • Trans-Activators
  • Transcription Factor AP-1
  • Transcription Factors
  • Transcription, Genetic
  • Transcriptional Activation
  • Transfection
  • Translocation, Genetic
  • Tretinoin
  • Tumor Burden
  • Two-Hybrid System Techniques
  • Ubiquitin-Protein Ligase Complexes
  • Ubiquitin-Protein Ligases
  • Up-Regulation
  • Uterus
  • Vascular Endothelial Growth Factor A
  • Wnt Proteins
  • Young Adult
  • beta Catenin