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:

Single Cell Analysis of Stored Red Blood Cells Using Ultra-High Throughput Holographic Cytometry.

Holographic cytometry is introduced as an ultra-high throughput implementation of quantitative phase imaging of single cells flowing through parallel microfluidic channels. Here, the approach was applied for characterizing the morphology of individual red blood cells during storage under regular blood bank conditions. Samples from five blood donors were examined, over 100,000 cells examined for each, at three time points. The approach allows high-throughput phase imaging of a large number of cells, greatly extending our ability to study cellular phenotypes using individual cell images. Holographic cytology images can provide measurements of multiple physical traits of the cells, including optical volume and area, which are observed to consistently change over the storage time. In addition, the large volume of cell imaging data can serve as training data for machine-learning algorithms. For the study here, logistic regression was used to classify the cells according to the storage time points. The analysis showed that at least 5000 cells are needed to ensure accuracy of the classifiers. Overall, results showed the potential of holographic cytometry as a diagnostic tool.
Authors
Park, H-S; Price, H; Ceballos, S; Chi, J-T; Wax, A
MLA Citation
Park, Han-Sang, et al. “Single Cell Analysis of Stored Red Blood Cells Using Ultra-High Throughput Holographic Cytometry.Cells, vol. 10, no. 9, Sept. 2021. Pubmed, doi:10.3390/cells10092455.
URI
https://scholars.duke.edu/individual/pub1497079
PMID
34572104
Source
pubmed
Published In
Cells
Volume
10
Published Date
DOI
10.3390/cells10092455

Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.
Authors
Donlin, LT; Rao, DA; Wei, K; Slowikowski, K; McGeachy, MJ; Turner, JD; Meednu, N; Mizoguchi, F; Gutierrez-Arcelus, M; Lieb, DJ; Keegan, J; Muskat, K; Hillman, J; Rozo, C; Ricker, E; Eisenhaure, TM; Li, S; Browne, EP; Chicoine, A; Sutherby, D; Noma, A; Accelerating Medicines Partnership RA/SLE Network,; Nusbaum, C; Kelly, S; Pernis, AB; Ivashkiv, LB; Goodman, SM; Robinson, WH; Utz, PJ; Lederer, JA; Gravallese, EM; Boyce, BF; Hacohen, N; Pitzalis, C; Gregersen, PK; Firestein, GS; Raychaudhuri, S; Moreland, LW; Holers, VM; Bykerk, VP; Filer, A; Boyle, DL; Brenner, MB; Anolik, JH
MLA Citation
Donlin, Laura T., et al. “Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue.” Arthritis Res Ther, vol. 20, no. 1, July 2018, p. 139. Pubmed, doi:10.1186/s13075-018-1631-y.
URI
https://scholars.duke.edu/individual/pub1489554
PMID
29996944
Source
pubmed
Published In
Arthritis Research & Therapy
Volume
20
Published Date
Start Page
139
DOI
10.1186/s13075-018-1631-y

Inhibiting xCT/SLC7A11 induces ferroptosis of myofibroblastic hepatic stellate cells but exacerbates chronic liver injury.

BACKGROUND & AIMS: The outcome of liver injury is dictated by factors that control the accumulation of myofibroblastic (activated) hepatic stellate cells (MF-HSCs) but therapies that specifically block this process have not been discovered. We evaluated the hypothesis that MF-HSCs and liver fibrosis could be safely reduced by inhibiting the cysteine/glutamate antiporter xCT. METHODS: xCT activity was disrupted in both HSC lines and primary mouse HSCs to determine its effect on HSC biology. For comparison, xCT expression and function were also determined in primary mouse hepatocytes. Finally, the roles of xCT were assessed in mouse models of liver fibrosis. RESULTS: We found that xCT mRNA levels were almost a log-fold higher in primary mouse HSCs than in primary mouse hepatocytes. Further, primary mouse HSCs dramatically induced xCT as they became MF, and inhibiting xCT blocked GSH synthesis, reduced growth and fibrogenic gene expression and triggered HSC ferroptosis. Doses of xCT inhibitors that induced massive ferroptosis in HSCs had no effect on hepatocyte viability in vitro, and xCT inhibitors reduced liver fibrosis without worsening liver injury in mice with acute liver injury. However, TGFβ treatment up-regulated xCT and triggered ferroptosis in cultured primary mouse hepatocytes. During chronic liver injury, xCT inhibitors exacerbated injury, impaired regeneration and failed to improve fibrosis, confirming that HSCs and hepatocytes deploy similar mechanisms to survive chronic oxidative stress. CONCLUSIONS: Inhibiting xCT can suppress myofibroblastic activity and induce ferroptosis of MF-HSCs. However, targeting xCT inhibition to MF-HSCs will be necessary to exploit ferroptosis as an anti-fibrotic strategy.
Authors
Du, K; Oh, SH; Dutta, RK; Sun, T; Yang, W-H; Chi, J-T; Diehl, AM
MLA Citation
Du, Kuo, et al. “Inhibiting xCT/SLC7A11 induces ferroptosis of myofibroblastic hepatic stellate cells but exacerbates chronic liver injury.Liver Int, vol. 41, no. 9, Sept. 2021, pp. 2214–27. Pubmed, doi:10.1111/liv.14945.
URI
https://scholars.duke.edu/individual/pub1482835
PMID
33991158
Source
pubmed
Published In
Liver Int
Volume
41
Published Date
Start Page
2214
End Page
2227
DOI
10.1111/liv.14945

Non-canonical role of Hippo tumor suppressor serine/threonine kinase 3 STK3 in prostate cancer.

Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo tumor suppressor pathway that regulates Yes-associated protein 1 (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulates YAP1/TAZ inhibition and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyperactivation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. Additionally, high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate-resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth, and Matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo, and biochemical analysis suggests a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative to XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemoprotective effects. Our results indicate that STK3 has a non-canonical role in PC progression and that inhibition of STK3 may have a therapeutic potential for PC that merits further investigation.
Authors
Schirmer, AU; Driver, LM; Zhao, MT; Wells, CI; Pickett, JE; O'Bryne, SN; Eduful, BJ; Yang, X; Howard, L; You, S; Devi, GR; DiGiovanni, J; Freedland, SJ; Chi, J-T; Drewry, DH; Macias, E
MLA Citation
Schirmer, Amelia U., et al. “Non-canonical role of Hippo tumor suppressor serine/threonine kinase 3 STK3 in prostate cancer.Mol Ther, Aug. 2021. Pubmed, doi:10.1016/j.ymthe.2021.08.029.
URI
https://scholars.duke.edu/individual/pub1494995
PMID
34450249
Source
pubmed
Published In
Molecular Therapy : the Journal of the American Society of Gene Therapy
Published Date
DOI
10.1016/j.ymthe.2021.08.029

The regulation of ferroptosis by MESH1 through the activation of the integrative stress response.

All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation "alarmone" (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response. However, the function of MESH1 remains unknown until our recent discovery of MESH1 as the first cytosolic NADPH phosphatase that regulates ferroptosis. To further understand whether MESH1 knockdown triggers a similar transcriptional response in mammalian cells, here, we employed RNA-Seq to analyze the transcriptome response to MESH1 knockdown in human cancer cells. We find that MESH1 knockdown induced different genes involving endoplasmic reticulum (ER) stress, especially ATF3, one of the ATF4-regulated genes in the integrative stress responses (ISR). Furthermore, MESH1 knockdown increased ATF4 protein, eIF2a phosphorylation, and induction of ATF3, XBPs, and CHOP mRNA. ATF4 induction contributes to ~30% of the transcriptome induced by MESH1 knockdown. Concurrent ATF4 knockdown re-sensitizes MESH1-depleted RCC4 cells to ferroptosis, suggesting its role in the ferroptosis protection mediated by MESH1 knockdown. ATF3 induction is abolished by the concurrent knockdown of NADK, implicating a role of NADPH accumulation in the integrative stress response. Collectively, these results suggest that MESH1 depletion triggers ER stress and ISR as a part of its overall transcriptome changes to enable stress survival of cancer cells. Therefore, the phenotypic similarity of stress tolerance caused by MESH1 removal and NADPH accumulation is in part achieved by ISR to regulate ferroptosis.
Authors
Lin, C-C; Ding, C-KC; Sun, T; Wu, J; Chen, K-Y; Zhou, P; Chi, J-T
MLA Citation
Lin, Chao-Chieh, et al. “The regulation of ferroptosis by MESH1 through the activation of the integrative stress response.Cell Death Dis, vol. 12, no. 8, July 2021, p. 727. Pubmed, doi:10.1038/s41419-021-04018-7.
URI
https://scholars.duke.edu/individual/pub1488796
PMID
34294679
Source
pubmed
Published In
Cell Death & Disease
Volume
12
Published Date
Start Page
727
DOI
10.1038/s41419-021-04018-7