Jen-Tsan Chi

Overview:

We are using functional genomic approaches to investigate the nutrient signaling and stress adaptations of cancer cells when exposed to various nutrient deprivations and microenvironmental stress conditions. Recently, we focus on two areas. First, we are elucidating the genetic determinants and disease relevance of ferroptosis, a newly recognized form of cell death. Second, we have identified the mammalian stringent response pathway which is highly similar to bacterial stringent response, but with some very interesting twists and novel mechanisms.

A. The genetic determinants and disease relevance of ferroptosis

Ferroptosis is a newly recognized form of cell death that is characterized by iron dependency and lipid peroxidation. The importance of ferroptosis is being recognized in many human diseases, including cancers, ischemia injuries, and neurodegeneration. Previously, we have identified the profound cystine addiction of renal cell carcinoma (1), breast cancer cells (2, 3), and ovarian cancer cells (4). Based on the concept that cystine deprivation triggers the ferroptosis due to the unopposed oxidative stresses, we have performed functional genomic screens to identify many novel genetic determinants of ferroptosis. For example, we have found that DNA damage response and ATM kinase regulate ferroptosis via affecting iron metabolism (5). This finding supports the potential of ionizing radiation to trigger DNA damage response and synergize with ferroptosis to treat human cancers. In addition, we found that ferroptosis is highly regulated by cell density. When cells are grown at low density, they are highly susceptible to ferroptosis. In contrast, the same cells become resistant to ferroptosis when grown at high density and confluency. we have found the Hippo pathway effectors TAZ and YAP are responsible for the cell density-dependent ferroptosis (4, 6, 7). Right now, we are pursuing several other novel determinants of ferroptosis that will reveal surprising insights into this new form of cell death.

B. A new stress pathway – mammalian stress response

All living organisms encounter a wide variety of nutrient deprivations and environmental stresses. Therefore, all organisms have developed various mechanisms to respond and promote survival under stress. In bacteria, the main strategy is “stringent response” triggered by the accumulation of the alarmone (p)ppGpp (shortened to ppGpp below) via regulation of its synthetase RelA and its hydrolase SpoT (8). The ppGpp binds to the transcription factor DksA and RNA polymerase to orchestrate extensive transcriptional changes that repress proliferation and promote stress survival (8, 9). While highly conserved among bacteria, the stringent response had not been reported in metazoans. However, a recent study identified Drosophila and human MESH1 (Metazoan SpoT Homolog 1) as the homologs of the ppGpp hydrolase domain of the bacterial SpoT (10). Both MESH1 proteins exhibit ppGpp hydrolase activity, and the deletion of Mesh1 in Drosophila led to a transcriptional response reminiscent of the bacterial stringent response (10). Recently, we have found that the genetic removal of MESH1 in tumor cells triggers extensive transcriptional changes and confers protection against oxidative stress-induced ferroptosis (11). Importantly, MESH1 removal also triggers proliferative arrest and other robust anti-tumor effects. Therefore, MESH1 knockdown leads to both stress survival and proliferation arrest, two cardinal features highly reminiscent of the bacterial stringent response. Therefore, we termed this pathway as “mammalian stringent response” (12). We have found that NADPH is the relevant MESH1 in the contexts of ferroptosis (13). Now, we are investigating how MESH1 removal leads to proliferation of arrests and anti-tumor phenotypes. Furthermore, we have found several other substrates of MESH1. We are investigating their function using culture cells, MESH1 KO mice, and other model organisms.

 

C. Genomic and single cell RNA analysis of Red Blood Cells

Red blood cells (RBC) are responsible for oxygen delivery to muscles during vigorous exercise. Therefore, many doping efforts focus on increasing RBC number and function to boost athletic performance during competition. For many decades, RBC were thought to be merely identical “sacs of hemoglobin” with no discernable differences due to factors such as age or pre-transfusion storage time. Additionally, because RBC lose their nuclei during terminal differentiation, they were not believed to retain any genetic materials.  These long-held beliefs have now been disproven and the results have significant implications for detecting autologous blood transfusion (ABT) doping in athletes.  We were among the first to discover that RBCs contain abundant and diverse species of RNAs. Using this knowledge, we subsequently optimized protocols and performed genomic analysis of the RBC transcriptome in sickle cell disease; these results revealed that heterogeneous RBCs could be divided into several subpopulations, which had implications for the mechanisms of malaria resistance. As an extension of these studies, we used high resolution Illumina RNA-Seq approaches to identify hundreds of additional known and novel microRNAs, mRNAs, and other RNA species in RBCs. This dynamic RBC transcriptome represents a significant opportunity to assess the impact that environmental factors (such as pre-transfusion refrigerate storage) on the RBC transcriptome. We have now identified a >10-fold change in miR-720 as well as several other RNA transcripts whose levels are significantly altered by RBC storage (14) which gained significant press coverage. We are pursuing the genomic and single cell analysis of RNA transcriptome in the context of blood doping, sickle cell diseases and other red cell diseases.

 

 

 

 

1.         Tang X, Wu J, Ding CK, Lu M, Keenan MM, Lin CC, et al. Cystine Deprivation Triggers Programmed Necrosis in VHL-Deficient Renal Cell Carcinomas. Cancer Res. 2016;76(7):1892-903.

2.         Tang X, Ding CK, Wu J, Sjol J, Wardell S, Spasojevic I, et al. Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling. Oncogene. 2017;36(30):4379.

3.         Lin CC, Mabe NW, Lin YT, Yang WH, Tang X, Hong L, et al. RIPK3 upregulation confers robust proliferation and collateral cystine-dependence on breast cancer recurrence. Cell Death Differ. 2020.

4.         Yang WH, Huang Z, Wu J, Ding C-KC, Murphy SK, Chi J-T. A TAZ-ANGPTL4-NOX2 axis regulates ferroptotic cell death and chemoresistance in epithelial ovarian cancer. Molecular Cancer Research. 2019: molcanres.0691.2019.

5.         Chen PH, Wu J, Ding CC, Lin CC, Pan S, Bossa N, et al. Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism. Cell Death Differ. 2019.

6.         Yang W-H, Chi J-T. Hippo pathway effectors YAP/TAZ as novel determinants of ferroptosis. Molecular & Cellular Oncology. 2019:1699375.

7.         Yang WH, Ding CKC, Sun T, Hsu DS, Chi JT. The Hippo Pathway Effector TAZ Regulates Ferroptosis in Renal Cell Carcinoma Cell Reports. 2019;28(10):2501-8.e4.

8.         Potrykus K, Cashel M. (p)ppGpp: still magical? Annu Rev Microbiol. 2008;62:35-51.

9.         Kriel A, Bittner AN, Kim SH, Liu K, Tehranchi AK, Zou WY, et al. Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance. Mol Cell. 2012;48(2):231-41.

10.       Sun D, Lee G, Lee JH, Kim HY, Rhee HW, Park SY, et al. A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nat Struct Mol Biol. 2010;17(10):1188-94.

11.       Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-72.

12.       Ding C-KC, Rose J, Wu J, Sun T, Chen K-Y, Chen P-H, et al. Mammalian stringent-like response mediated by the cytosolic NADPH phosphatase MESH1. bioRxiv. 2018.

13.       Ding C-KC, Rose J, Sun T, Wu J, Chen P-H, Lin C-C, et al. MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis. Nature Metabolism. 2020.

14.       Yang WH, Doss JF, Walzer KA, McNulty SM, Wu J, Roback JD, et al. Angiogenin-mediated tRNA cleavage as a novel feature of stored red blood cells. Br J Haematol. 2018.

 

 

Positions:

Associate Professor in Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Assistant Professor of Medicine

Medicine, Rheumatology and Immunology
School of Medicine

Assistant Professor in Radiation Oncology

Radiation Oncology
School of Medicine

Associate Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1991

National Taiwan University (Taiwan)

Ph.D. 2000

Stanford University

Postdoctoral Research, Biochemistry

Stanford University

Grants:

Metabolic regulation of KLHL proteins through O-glycosylation

Administered By
Molecular Genetics and Microbiology
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Storage-specific erythrocyte gene signatures to detect autologous transfusion

Administered By
Molecular Genetics and Microbiology
Awarded By
Partnership for Clean Competition
Role
Principal Investigator
Start Date
End Date

Detect autologous transfusion by novel separation and characterization of RBC storage exosomes

Administered By
Molecular Genetics and Microbiology
Awarded By
Partnership for Clean Competition
Role
Principal Investigator
Start Date
End Date

Small RNA transcriptome as novel approaches to detect autologous blood transfusion

Administered By
Molecular Genetics and Microbiology
Awarded By
World Anti-Doping Agency
Role
Principal Investigator
Start Date
End Date

Comparison of oxidant damage, Nrf2 characteristics, and gene modification of cord blood versus plerixafor-mobilized adult CD34+ cells from sickle cell disease patients

Administered By
Molecular Genetics and Microbiology
Awarded By
New York Blood Center
Role
Principal Investigator
Start Date
End Date

Publications:

A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and notch signaling.

The development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS) - an aggressive soft tissue sarcoma of childhood - has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.
Authors
Slemmons, KK; Deel, MD; Lin, Y-T; Oristian, KM; Kuprasertkul, N; Genadry, KC; Chen, P-H; Chi, J-TA; Linardic, CM
MLA Citation
URI
https://scholars.duke.edu/individual/pub1482836
PMID
34004824
Source
pubmed
Published In
Biology Open
Published Date
DOI
10.1242/bio.050211

A genomic analysis of cellular responses and adaptions to extracellular acidosis

Even though lactic acidosis is a prominent feature of solid tumors, we have limited understanding of the mechanisms by which lactic acidosis influences the genetic, epigenetic, proteomic, and metabolic phenotypes of cancer cells. This chapter aims to (1) briefly outline the tumor microenvironment and how acidity relates to its biology; (2) briefly discuss traditional hypothesis-driven or single-gene studies that have explored cancer cells’ responses to acidosis or lactic acidosis; (3) explain what we have learned from “-omics” approaches that have been applied to studying cellular response to acid and lactic acid; and (4) reflect on the projections of these studies in (2) and (3) to in vivo human tumor biology and how we can use this information to better inform disease treatments.
Authors
Keenan, MM; Lin, CC; Ashley Chi, JT
MLA Citation
Keenan, M. M., et al. “A genomic analysis of cellular responses and adaptions to extracellular acidosis.” Molecular Genetics of Dysregulated PH Homeostasis, 2014, pp. 135–57. Scopus, doi:10.1007/978-1-4939-1683-2_8.
URI
https://scholars.duke.edu/individual/pub1498140
Source
scopus
Published Date
Start Page
135
End Page
157
DOI
10.1007/978-1-4939-1683-2_8

Application of bioluminescence resonance energy transfer-based cell tracking approach in bone tissue engineering.

Bioluminescent imaging (BLI) has emerged as a popular in vivo tracking modality in bone regeneration studies stemming from its clear advantages: non-invasive, real-time, and inexpensive. We recently adopted bioluminescence resonance energy transfer (BRET) principle to improve BLI cell tracking and generated the brightest bioluminescent signal known to date, which thus enables more sensitive real-time cell tracking at deep tissue level. In the present study, we brought BRET-based cell tracking strategy into the field of bone tissue engineering for the first time. We labeled rat mesenchymal stem cells (rMSCs) with our in-house BRET-based GpNLuc reporter and evaluated the cell tracking efficacy both in vitro and in vivo. In scaffold-free spheroid 3D culture system, using BRET-based GpNLuc labeling resulted in significantly better correlation to cell numbers than a fluorescence based approach. In scaffold-based 3D culture system, GpNLuc-rMSCs displayed robust bioluminescence signals with minimal background noise. Furthermore, a tight correlation between BLI signal and cell number highlighted the robust reliability of using BRET-based BLI. In calvarial critical sized defect model, robust signal and the consistency in cell survival evaluation collectively supported BRET-based GpNLuc labeling as a reliable approach for non-invasively tracking MSC. In summary, BRET-based GpNLuc labeling is a robust, reliable, and inexpensive real-time cell tracking method, which offers a promising direction for the technological innovation of BLI and even non-invasive tracking systems, in the field of bone tissue engineering.
Authors
Wang, L; Lee, DJ; Han, H; Zhao, L; Tsukamoto, H; Kim, Y-I; Musicant, AM; Parag-Sharma, K; Hu, X; Tseng, HC; Chi, J-T; Wang, Z; Amelio, AL; Ko, C-C
MLA Citation
Wang, Lufei, et al. “Application of bioluminescence resonance energy transfer-based cell tracking approach in bone tissue engineering.J Tissue Eng, vol. 12, Jan. 2021, p. 2041731421995465. Pubmed, doi:10.1177/2041731421995465.
URI
https://scholars.duke.edu/individual/pub1475405
PMID
33643604
Source
pubmed
Published In
J Tissue Eng
Volume
12
Published Date
Start Page
2041731421995465
DOI
10.1177/2041731421995465

Molecular genetics of dysregulated pH homeostasis

Most biological reactions and functions occur within a narrow range of pH. Any changes in the pH have great impacts on the biological function at every level, including protein folding, enzymatic activities and proliferation, and cell death. Therefore, maintaining the pH homeostasis at the local or systemic level is one of the highest priorities for all multicellular organisms. Many redundant mechanisms are in place to maintain the pH homeostasis, a topic that is well covered in scientific literature and in medical textbooks. However, when the pH homeostasis is disrupted in various physiological adaptations and pathological situations, resulting acidity may trigger significant pathophysiological events, and modulate disease outcomes. Therefore, understanding how various cells sense and react to acidity have broad impact in a wide variety of human diseases including cancer, stroke, myocardial infarction, diabetes, and renal and infectious diseases. In this book, many investigators have summarized the molecular genetics on the detailed mechanisms by which different mammalian cells sense and respond to acidity. These chapters cover the acidity with broad impact in biological understanding and human diseases and review various sensing mechanisms and cellular responses to pH alterations in both physiological (taste, pain) and pathological (ischemia and cancers) settings. Furthermore, the authors present a broad spectrum of investigative approaches to cellular response to acidosis in a wide variety of human diseases.
Authors
MLA Citation
Ashley Chi, J. T. Molecular genetics of dysregulated pH homeostasis. 2014, pp. 1–160. Scopus, doi:10.1007/978-1-4939-1683-2.
URI
https://scholars.duke.edu/individual/pub1498141
Source
scopus
Published Date
Start Page
1
End Page
160
DOI
10.1007/978-1-4939-1683-2

Introduction: Molecular genetics of acid sensing and response

Since most biological reactions in human body occur within narrow ranges around neutral environments, any changes in the pH environment have great impacts on a wide variety of functions, including gene expression, protein folding, enzymatic activities, cell proliferation, and cell death. When the pH homeostasis is disrupted and results in tissue acidity, how various cell types sense and respond in their physiology, metabolism, and gene expression play a dramatic role in modulating disease outcomes. Therefore, understanding how various cells sense and react to pH imbalance have broad impact in a wide variety of human diseases, including cancer, stroke, myocardial infarction, diabetes, and renal and infectious diseases. In this book, several experts in the field have highlighted various aspects of the molecular genetics on how mammalian cells sense and respond to acidosis and their implications in the normal physiological adaptations and pathogenesis. These reviews highlight at least three levels of complexity in the acid sensing and response among different cell types and disease settings.
Authors
Lin, CC; Keenan, MM; Ashley Chi, JT
MLA Citation
Lin, C. C., et al. “Introduction: Molecular genetics of acid sensing and response.” Molecular Genetics of Dysregulated PH Homeostasis, 2014, pp. 1–7. Scopus, doi:10.1007/978-1-4939-1683-2_1.
URI
https://scholars.duke.edu/individual/pub1498142
Source
scopus
Published Date
Start Page
1
End Page
7
DOI
10.1007/978-1-4939-1683-2_1