Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-2039 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. FGF19 protects hepatocellular carcinoma cells against endoplasmic reticulum stress via activation of FGFR4-GSK3β-Nrf2 signaling Yong Teng1,2#*, Huakan Zhao3,4#, Lixia Gao1, Wenfa Zhang3, Austin Y Shull5, Chloe Shay6 1. Department of Oral Biology, Augusta University, Augusta, GA, USA 2. Georgia Cancer Center, Augusta University, Augusta, GA, USA 3. School of Life Sciences, Chongqing University, Chongqing, China 4. Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing, China 5. Department of Biology, Presbyterian College, Clinton, SC, USA 6. Emory Children’s Center, Emory University, Atlanta, GA, USA # These authors contributed equally to this work Running Title: Role of FGF19 in ER stress Key Words: FGF19, FGFR4, Nrf2, HCC, ER stress, anticancer Abbreviations list: AARE: amino-acid-response element; ANOVA: analysis of variance; ARE: antioxidant response elements; ATF4: activating transcription factor 4; ChIP-qPCR: chromatin immunoprecipitation quantitative-PCR; CV: cyclic voltammetry; DMSO: dimethyl sulfoxide; EMT: epithelial-mesenchymal transition; ER: endoplasmic reticulum; FGF19: fibroblast growth factor 19; FGFR4: FGF receptor 4; GSK3β: glycogen synthase kinase •− 3β; HCC: hepatocellular carcinoma; Nrf2: nuclear factor E2-related factor 2; O2 : superoxide; PBS: phosphate-buffered saline; ROS: reactive free radicals; RT-PCR: reverse transcription polymerase chain reaction; SOD: superoxide dismutase; TG: thapsigargin; TM: tunicamycin; UPR: unfolded protein response Financial support: This work was supported in part by Dental College of Georgia Special Funding Initiative (to Y.T.). *Author for correspondence: Yong Teng, PhD, Department of Oral Biology Augusta University, 1120 15th Street, Augusta, GA 30912 Tel: +17064465611, Fax: +17067219415, E-mail: [email protected] Disclosure of Potential Conflict of Interest: The authors declare no competing financial interests 1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-2039 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT: The tumor microenvironment induces endoplasmic reticulum (ER) stress in tumor cells, an event that can promote progression, but it is unknown how tumor cells adapt to this stress. In this study, we show that the fibroblast growth factor FGF19, a gene frequently amplified in hepatocellular carcinoma (HCC), facilitates a survival response to ER stress. Levels of FGF19 expression were increased in stressed HCC cells in culture and in a mouse xenograft model. Induction of ER stress required the transcription factor ATF4, which directly bound the FGF19 promoter. In cells where ER stress was induced, FGF19 overexpression promoted HCC cell survival and increased resistance to apoptosis, whereas FGF19 silencing counteracted these effects. Mechanistic investigations implicated glycogen synthase kinase-3β in regulating nuclear accumulation of the stress-regulated transcription factor Nrf2 activated by FGF19. Our findings show how FGF19 provides a cytoprotective role against ER stress by activating a FGFR4-GSK3β-Nrf2 signaling cascade, with implications for targeting this signaling node as a candidate therapeutic regimen for HCC management. 2 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-2039 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Fibroblast growth factor 19 (FGF19), a member of the hormone-like FGF family, has activity as an ileum-derived postprandial enterokine regulating liver metabolism (1). FGF19 predominantly signals through FGF receptor 4 (FGFR4) in the presence of coreceptor β-Klotho to efficiently activate downstream signaling pathways (2). In humans, increased expression of FGF19 is critical for the development and progression of several cancer types including hepatocellular carcinoma (HCC), breast cancer, prostate cancer, thyroid cancer and cholangiocarcinoma (3-6). FGF19 was recently identified as a driver oncogene in HCC, a cancer known for its high incidence in cancer-related deaths (3,7). FGF19 regulates a variety of hepatocyte cellular functions, such as bile acid metabolism, proliferation, differentiation, and epithelial-mesenchymal transition (EMT) in a FGFR4-dependent manner (8-10). In mice, aberrant signaling through Fgf15, the mouse orthologue to human FGF19, has been implicated in HCC development in a context of chronic liver injury and fibrosis (11). Elevated endoplasmic reticulum (ER) stress is observed in solid tumors as a consequence of microenvironment changes (12). When ER stress occurs, ER functions are altered, and a number of molecular actions collectively identified as the “unfolded protein response” (UPR) are activated to counteract ER-associated damage (13). Several indications suggest that cancer cell adaptation to adverse conditions largely relies on the ability of a cell to perturb ER stress-associated regulatory networks and prevent ER stress-induced cell death (14,15). It has been reported that either mRNA or secreted protein levels of FGF19 was increased in intestinal epithelial Caco-2 cells following treatment of ER inducers Thapsigargin (TG) or Tunicamycin (TM) (16). However, the molecular mechanisms underlying upregulation of FGF19 under ER stress have not been explored in detail. 3 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-2039 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Nuclear factor E2-related factor 2 (Nrf2), a key mediator involved in anti-oxidative, anti-inflammatory, and mitochondrial protection, can activate an array of genes encoding antioxidant and detoxification enzymes through selective transcriptional binding within antioxidant response elements (ARE) (17). Nrf2 regulatory signals serve protective roles in hepatic inflammation, fibrosis, and regeneration (18). Abundant expression of Nrf2 has been identified in different cancer types including HCC, which may associate with cancer cell proliferation, invasion, and chemoresistance (19,20). The self-protective, antioxidant response of Nrf2 is a complex and highly orchestrated pathophysiological process (18,21), and glycogen synthase kinase 3β (GSK3β) serves as an integration point of a myriad of signaling pathways during the control of Nrf2 activity (22,23). Recent evidence in HCC cells indicates that GSK3β can physically interact with Nrf2 and inhibit Nrf2 activity by nuclear export and subsequent proteasomal degradation in response to late-phase oxidative stress (24). Our previous study has demonstrated that the FGF19/FGFR4 axis inactivates GSK3β by increasing phosphorylation levels of GSK3β in HCC cells (10). Thus, such coordinating events lead us to determine whether a functional relationship between FGF19 and Nrf2 takes place in the tumor microenvironment, specifically within the context of elevated ER stress. Here we show for the first time that FGF19 is translationally activated by ER stress both in cultured HCC cells and in a mouse-xenograft model. The ER stress-associated activation of FGF19 transcription requires activating transcription factor 4 (ATF4), which increasingly binds to the amino-acid-response element (AARE) within the FGF19 promoter during ER stress. As well, we demonstrate that depletion of FGF19 decreases HCC cell survival and enhances ROS-associated apoptosis upon ER stress. Additionally, FGF19 facilitates the nuclear accumulation of Nrf2 through the FGFR4-GSK3β signaling, which plays a central role in providing FGF19-dependent cytoprotection to HCC cells against ER stress. Thus, by taking into account its cytoprotective role in the tumor microenvironment, FGF19 may represent a promising molecular target in which its inhibition could be used therapeutically against HCC development. 4 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-2039 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MATERIALS AND METHODS Cell culture and standard assays HL7702, HepG2, and Hep3B cell lines were obtained from the American Type Culture Collection (ATCC). MHCC97L and MHCC97H cell lines were purchased from the Cell Bank of Chinese Academy of Sciences (CBCAS). All cell lines were maintained in the growth medium according to the manufacturer’s instructions and passage <5 were used in this study. Real-time reverse transcription polymerase chain reaction (RT-PCR), Western blot, transient transfections, lentiviral transduction, and luciferase reporter assays were carried out as described previously (10,25-29). The gene-specific primers used in real-time RT-PCR analysis are listed in Table S1. In vivo ER stress model All experimental procedures were approved