FGF19 Protects Hepatocellular Carcinoma Cells Against Endoplasmic Reticulum Stress Via Activation of FGFR4-Gsk3β-Nrf2 Signaling

Home , FGF19

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

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.

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.

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.

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 by the Institutional Animal Care and Use Committee (IACUC) at Augusta University. Six-week-old BALB/c athymic nude mice were inoculated subcutaneously in the

double hind flanks with 5×106 HCC cells per 100 µl suspended in dilute Matrigel 1:1 in ice cold

phosphate-buffered saline (PBS). Dimethyl sulfoxide (DMSO) or 10 μM TG was intratumorally injected

(left for DMSO and right for TG) when the xenografts reached ~150 mm3. The xenografts were harvested at

8 hours post-injection (hpi) and homogenized for real-time RT-PCR analysis.

Constructs, antibodies, inhibitors and other reagents

The full-length cDNA of human FGF19, FGFR4, and ATF4 were individually cloned into a pcDNA3.1 (+)

expression vector (Life Technologies, Carlsbad, CA). The ARE-luciferase reporter containing three copies of

ARE sequences (in head-to-tail orientation) were generated using the pGL3 vector (Promega, Madison, WI,

USA). Twenty-one base pair siRNA duplexes targeting human Nrf2 gene

(5’-GUAAGAAGCCAGAUGUUAATT-3’), ATF4 gene (5’-GCCUAGGUCUCUUAGAUGATT-3’) and a standard control (Dharmacon siCONTROL nontargeting siRNA) were synthesized by Dharmacon RNA

Technologies (Lafayette, CO). FGF19, ATF4 and β-Actin antibodies were purchased from Abcam

(Cambridge, MA). Antibodies against Nrf2, p-GSK3β (Ser9), GRP78，c-PARP, Lami B, HO-1 and Bcl-2 were procured from Cell Signaling Technologies (Beverly, MA). GSK3β inhibitors TWS119 and Tideglusib,

PI3K inhibitor LY294002, JNK inhibitor SP600125, and MEK inhibitor U0126 were obtained from

Selleckchem (Houston, TX). TG and TM, acetaminophen, silymarin, superoxide dismutase (SOD), ethanol,

® H2O2, DMSO and DAPI were purchased from Sigma-Aldrich (St. Louis, MO). NE-PER Nuclear and

Cytoplasmic Extraction Reagent Kit was purchased from Thermo Scientific (Waltham, MA) and used according to the manufacturer's instructions.

Cell proliferation, viability and apoptosis

Cell proliferation and viability at the indicated incubation times were determined by CellTiter 96® AQueous

One Solution Cell Proliferation Kit and CellTiter-Glo® Luminescent Cell Viability Kit (Promega, Madison,

WI), respectively. Apoptosis was determined by the FITC Annexin V/Dead Cell Apoptosis Kit with FITC

annexin V and PI (Life Technologies, Carlsbad, CA) and analyzed by flow cytometry using the BD

FACSCalibur system (BD Biosciences, Franklin Lakes, NJ). For DAPI staining, cells were stained with 0.5

g/ml DAPI for 1 hour after fixation with 4 % paraformaldehyde. The stained cells were washed with PBS

and examined by fluorescence microscopy (Olympus Corporation, Tokyo, Japan). Apoptotic cells were

identified by condensation and fragments of nuclei. The apoptotic cells in seven randomly selected fields

from triplicate chambers were counted in each experiment under a horizon.

•− Electrochemical detection of O2

Electrochemical detection of free radical levels released from cells was established in our previous work

(10,25,26,30). Briefly, 5×105 HCC cells were seeded in 6-well plates in the presence or absence of various

•− ER stress inducers. Cyclic voltammetry (CV) was used to monitor cellular superoxide (O2 ) generation on

CHI760D electrochemical station (ChenHua Instruments, Wuhan, China). SOD was applied to verify current

•− changes caused by O2 . Percentages of peak (potential=0.7V; current enhancement) were compared and

calculated against the control curve.

Chromatin immunoprecipitation quantitative-PCR (ChIP-qPCR)

ChIP assays were performed using a ChIP Kit (Abcam, Cambridge, MA) as we previously described. Briefly,

chromatin from cells was crosslinked with 1% formaldehyde for 10 min at room temperature, sheared to an

average size of 500bp, and immunoprecipitated with an ATF4 antibody (Abcam). The ChIP-qPCR primers

(Forward: 5’-GGAGGCTTGATGCAATCCCGATAA-3’; Reverse:

5’-GGCAGCCTTATATAGTAGCGCCTT-3’) were designed to amplify a proximal promoter region

containing the putative ATF4 binding element AARE (5’-CTTGATGCA-3’) at the FGF19 promoter. Each

immunoprecipitated DNA sample was quantified using qPCR and all ChIP-qPCR signals were normalized

to the input.

Statistical analysis

Statistical analyses were performed with unpaired Student’s t test for two group comparisons and one-way

analysis of variance (ANOVA) for multi-group comparisons. Data were presented as mean ± S.E.M of at

least three independent experiments. Gene expression data were obtained from GEO database and the

Pearson’s correlation coefficient was used to analyze the correlation between the expression levels of two

genes. A p-value of 0.05 or less was considered to be significant.

RESULTS

ER stress induces FGF19 expression in HCC cells in vitro and in vivo

FGF19 is highly expressed in HCC, and the UPR is always activated in HCC, raising the possibility that ER

stress-dependent alteration in FGF19 expression is the one of the main mechanisms that underlie liver

tumorigenesis. We first tested whether ER stress influenced FGF19 expression in HCC cell lines. HepG2,

Hep3B, MHCC97L, and MHCC97H cells were treated with ER stress inducers TG and TM, respectively.

Both TG and TM treatment led to a significant increase in FGF19 expression levels in all cell lines

examined (Figure 1A and 1B), concomitant with increasing levels of the ER stress marker GRP78 (Figure

1B). The expression of FGF19 in normal human HL7702 hepatocytes was also increased following TG

treatment (Figure 1A and 1B). No significant changes of FGFR4 levels were observed in the presence or

absence of ER stress (Figure 1B). We next examined the expression status of FGF19 using different triggers

of ER stress. Consistent with TG treatment, H2O2, ethanol, and acetaminophen were also able to induce

FGF19 upregulation in HepG2 and MHCC97L cells in a dose-dependent manner (Figure 1C).

To determine whether elevated FGF19 expression was associated with ER stress in vivo, we developed

HepG2 and MHCC97L xenografts in nude mice and then induced ER stress through intratumoral injection

of TG. TG treatment led to a significant increase (p=0.007) in FGF19 expression levels in the isolated tumor

cells compared with vehicle treatment (Figure 1D). Silymarin, a mix of natural flavonoids, exhibits potent

antioxidant function in repressing ER stress (31). We treated MHCC97L cells with silymarin in the presence of TG or TM for 24 hours. Decreased expression levels of FGF19 were found in our co-treatments, as

compared to those with TG or TM single drug treatment (Figure 1E and 1F). These results suggest that

blocking cell oxidation abrogates FGF19 upregulation in ER stress. The mouse Fgf15 gene is homologous

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. with the human FGF19 gene and is not expressed in normal mouse liver (11). No Fgf15 expression was detected out in the mouse liver tissues treated with TG, CCl4 or acetaminophen, although Grp78 was markedly upregulated after treatment (Supplementary Figure S1).

ATF4 is required for ER stress-induced FGF19 upregulation

ATF4 is a critical ER stress-inducible transcription factor (27) implicated as a regulator of ER stress-induced

FGF19 upregulation in intestinal epithelial cells (32). Increased ATF4 expression levels were observed in

HepG2 and MHCC97L cells following TG exposure (Figure 2A and 2B), suggesting that ATF4 may contribute to an ER stress-induced increase in FGF19 mRNA in HCC cells. To further address this observation, we then knocked down ATF4 using siRNA in HepG2 cells and determined FGF19 expression levels with or without TG treatment. Loss of ATF4 expression attenuated FGF19 upregulation induced by

TG (Figure 2C and 2D). In contrast, overexpression of ATF4 upregulated FGF19 expression in MHCC97L cells (Figure 2E). To further determine whether ATF4 transcriptionally regulates FGF19, we performed ChIP assays using a ChIP-grade ATF4 antibody, which demonstrated a specific, direct interaction of the ATF4 protein with the AARE within the FGF19 promoter in MHCC97L cells (Figure 2F). As expected, increased levels of ATF4 at the FGF19 promoter binding site were seen following TG treatment (Figure 2F).

Consistently, analysis of microarray datasets from HCC patients revealed a clear positive correlation between ATF4 and FGF19 levels (Supplementary Figure S2). Taken together, these results indicate that

ATF4 is upregulated during ER stress, which in turn, transcriptionally activates FGF19 in HCC cells.

Upregulation of FGF19 defends HCC cells against ER stress

We next determined the potential role of FGF19 in ER stress. Forced expression of FGF19 enhanced cell survival and inhibited apoptosis in TG-treated MHCC97L cells (Figure 3A and 3B). Given that excessive

•− reactive free radicals (ROS), such as O2 , are released to activate stress-response pathways and induce

•− defense mechanisms (33), we developed sensitive electrochemical biosensors to monitor O2 generation in

•− cancer cells in situ (Supplementary Figure S3). Intracellular O2 was increased in TG-treated MHCC97L

cells (Figure 3C), and this increase was abrogated when FGF19 was overexpressed (Figure 3C), suggesting

•− that FGF19-mediated induction of O2 levels may contribute to ER stress-induced consequences in HCC

•− cells. SOD alternately catalyzes the dismutation (or partitioning) of O2 into either O2 or H2O2 (34). In these

assays, SOD treatment abrogated current changes trigged by TG (Figure 3C), demonstrating the specificity

of the electrochemical biosensors.

In contrast, knockdown of FGF19 led to a dramatic decrease in survival rate and an increase in

apoptosis in TG-treated MHCC97H cells compared with siRNA no-target control (siNC) treated cells

•− (Figure 3D and 3E). Electrochemical analysis showed that FGF19 depletion enhanced O2 release following

TG treatment (Figure 3F). These findings support a notion that high expression levels of FGF19 help to

defend HCC cells against ER stress. A similar tendency was observed in H2O2-treated HCC cells

(Supplementary Figure S4), indicating that FGF19 plays a pivotal role in ER stress response.

The FGF19-FGFR4 axis is critical for the resistance of HCC cells to ER stress

The FGF19-FGFR4 signaling has been implicated in the development and progression of HCC (2,35). Our

previous studies have shown that overexpression of FGF19 in HCC cells enriched secreted FGF19 levels in

the supernatant to promote cell growth and migration through activation of FGFR4 (10,25). We have

generated FGFR4 knockout MHCC97L cells using a CRISPR-Cas9 system (10). Using these cells, we

determined the possible involvement of FGFR4 in ER stress. In the TG treatment, the phenotypes shown in

•− FGF19 knockdown cells (Figure 3), such as decreased cell survival, increased capacity of O2 release, and

enhanced cell apoptosis, were also observed in FGFR4 knockout cells (Figure 4A-4C). To evaluate whether

FGF19-FGFR4 axis contributes to the resistance of HCC cells to ER stress, we overexpressed the FGF19 gene in FGFR4 knockout MHCC97L cells before TG treatment. As expected, increased FGF19 expression

could not protect HCC cells against ER stress when FGFR4 was depleted (Figure 4D-4F), demonstrating that FGFR4 is a required component during this process. Together, these observations reveal that ER stress can be buffered by mitigating the activation of the FGF19-FGFR4 axis in HCC cells.

FGF19 promotes nuclear accumulation of Nrf2 through FGFR4/GSK3β signaling

Nrf2 is a transcription factor known to regulate expression of a large array of antioxidant and cytoprotective

genes to mediate cellular stress responses (36). Since FGF19 impacts the protection of HCC cells against ER

stress, we examined the involvement of Nrf2 in the FGF19 regulatory network. FGF19 overexpression resulted in a significant increase in Nrf2 protein levels in HepG2 and MHCC97L cells, while FGF19

knockdown reduced Nrf2 levels in MHCC97H cells (Figure 5A). The anti-apoptosis gene Bcl-2 and anti-oxidant gene HO-1 are downstream targets of Nrf2 (37). In the presence of TG, both genes showed higher expression levels in FGF19 overexpressing cells and lower expression levels in FGF19 knockdown

cells as compared to the respective controls (Figure 5A). FGFR4 knockout attenuated Nrf2-dependent

expression of Bcl-2 and HO-1 in FGF19 overexpressing MHCC97L cells (Figure 5B), indicating that Nrf2

pathway is heavily dependent on FGF19/FGFR4 activation in HCC cells. Surprisingly, mRNA levels of Nrf2

did not change in HCC cells regardless of FGF19 expression levels (Figure 5A), which prompted us to investigate whether upregulation of FGF19 facilitated nuclear accumulation of Nrf2. Compared with control

cells, enriched Nrf2 protein levels were found in the purified nuclear fraction in FGF19 overexpressing

MHCC97L cells with concomitant reduction of Nrf2 in cytosol (Figure 5C).

To further explore that increased FGF19 activates Nrf2-mediated activation of ARE-dependent genes, we constructed a luciferase reporter containing a triplicate of ARE sequence motifs (Figure 5D).

Co-transfection of full-length FGF19 cDNA with the ARE reporter into MHCC97L cells led to increased

luciferase activities (Figure 5D). Similar results were also observed when the full-length FGFR4 cDNA

and the ARE luciferase reporter were co-transfected (Figure 5D). To determine the potential clinical

relevance of these genes in HCC patients, we assessed GEO datasets and investigated the association

between the expression levels of FGF19 and HO-1 or Bcl-2 in HCC clinical samples. This analysis showed a

positive linear association either between FGF19 and HO-1 transcript levels as well as between FGF19 and

Bcl-2 (Supplementary Figure S2).

To elucidate the molecular mechanisms responsible for FGF19-induced Nrf2 accumulation, the status of

Keap1 and GSK3β were determined, two critical regulators of Nrf2. As shown in Figure 5A, enhanced

phospho-GSK3β levels, but not Keap1 levels, were found in FGF19-overexpressing HCC cells,

demonstrating that FGF19 facilitates Nrf2 nuclear accumulation via inactivation of GSK3β by

phosphorylation at specific residues. We thus inactivated GSK3β activity using GSK3β inhibitors TW119 and Tideglusib and observed attenuated FGF19-dependent Nrf2 accumulation (Figure 5E). Unlike the

GSK3β inhibitors, however, treatment with PI3K, ERK and JNK inhibitors did not affect luciferase activities

of ARE reporter in MHCC97L cells where FGF19 was overexpressed (Figure 5F). Taken together, these

results demonstrate that GSK3β contributes to Nrf2 nuclear accumulation triggered by ER stress-induced

FGF19-FGFR4 activation in HCC cells.

FGF19 is essential for Nrf2-dependent cytoprotection in HCC cells under ER stress

Persistent accumulation of Nrf2 in the nucleus has been implicated in enhancing oncogenesis (38). Since

high expression of FGF19 promotes nuclear translocation of Nrf2, we depleted Nrf2 in FGF19 overexpressing MHCC97L cells to examine the functional role of Nrf2 in FGF19-dependent cell survival.

Knockdown of Nrf2 using siRNA remarkably attenuated the proliferation rate induced by FGF19

overexpression (Supplementary Figure S5). Most importantly, in the treatment of TG, Nrf2 knockdown

•− blunted the increase in FGF19-dependent cell survival (Figure 6A) and disrupted the ability to remove O2

(Figure 6B). The same phenotypes were also observed in the presence of H2O2 (Supplementary Figure S6).

Furthermore, overexpression of FGF19 enhanced resistance to apoptotic cell death triggered by TG, but this

was blunted when Nrf2 was depleted in these cells (Figure 6C and 6D). Loss of Nrf2 expression also

attenuated the FGF19-dependent induction of HO-1 and Bcl-2 in TG-treated MHCC97L cells (Figure 6E).

Nrf2 is positioned at the interface of oxidative and ER stress pathways (38). To determine whether

FGF19 promotes Nrf2 to translocate into nucleus during ER stress, HepG2 or MHCC97H cells were treated

with TG in the presence or absence of FGF19 expression to determine the accumulation status of Nrf2

within the nucleus. Based on our results, TG-induced FGF19 upregulation was associated with increased nuclear accumulation of Nrf2, whereas Nrf2 levels in the nucleus were reduced to almost beyond detection when FGF19 was depleted (Figure 6F). These observations indicate that FGF19 plays a cytoprotection role

through activation of Nrf2 in HCC cells when under ER stress.

DISCUSSION

ER stress has been implicated in the pathogenesis of viral hepatitis, insulin resistance, hepatosteatosis,

nonalcoholic steatohepatitis, and other liver diseases that increase risk of HCC (39,40). ER stress is also present in HCC when the tumor microenvironment experiences nutrient deprivation, hypoxia, and imbalance between production and removal of ROS (41,42). However, the mechanisms underlying cellular adaptation

to ER stress during development and progression of HCC are still unclear. Our study showed that ER stress

promoted ATF4-mediated transcriptional activation of FGF19 in HCC cells and consequently enhanced the

prosurvival and anti-apoptotic response of HCC cells during ER stress (Figure 7). These findings

demonstrate that FGF19 is involved in tumorigenesis and adaptation to extreme environments, which may

represent a legitimate target for cancer therapeutics.

In this work, we revealed a novel functional role of FGF19 in promoting HCC cell survival under acute

ER stress. FGF19 secreted from HCC cells functions as endocrine or paracrine factors to activate the FGFR4

receptor residing on the surface of HCC cells to increase GSK3β phosphorylation and, in turn, promote Nrf2

translocation to the nucleus (Figure 7). Nuclear accumulation of Nrf2 transcriptionally activates target genes

such as HO-1 and Bcl-2 in HCC cells, leading to a significant decrease in ROS-associated apoptosis induced by ER stress (Figure 7). Thus, it appears that HCC cells create a dependence on the FGF19 stress response

pathway to maintain a permissive growth environment. Therefore, inhibition of the FGF19-FGFR4 axis may

prevent tumor development.

Induction of apoptosis is a prominent mode of cytotoxicity for many chemotherapeutic drugs. Apoptosis

occurs by distinct pathways that involve the cell surface, mitochondria, or ER. Certain drugs, such as

sorafenib, the first-line systemic therapy for advanced HCC, can induce ER stress-mediated apoptosis

independent of the MEK-ERK pathway (43,44). Our group has recently demonstrated that hyperactivation

of FGF19-FGFR4 axis is one of the main mechanisms of sorafenib resistance in HCC cells, and blocking it

can overcome the resistance to sorafenib (25). The present study implies that FGF19-FGFR4-GSK3β-Nrf2 signaling cascades may be a main pathway involved in sorafenib-induced ER stress.

Either FGF19 or FGFR4 represents a promising target in developing novel cancer therapies against

FGF19-FGFR4-dependent cancers. FGF19 is an endocrine hormone of the FGF family that regulates bile

acid, carbohydrate, lipid, and energy metabolism (10,45). Although a neutralizing antibody specific against

FGF19 (1A6) has been developed to block the binding of FGF19 to FGFR4 and neutralize the tumorigenic

oncogenic activity of FGF19 in HCC cells, it has an unexpected effect in preventing the beneficial functions

of FGF19 in bile acid homeostasis (4,45). To achieve successful therapy, M70, an engineered and

nontumorigenic FGF19 variant carrying 3 amino acid substitutions (A30S, G31S, and H33L) and a 5 amino

acid deletion, has been developed (45,46). Unlike 1A6, M70 can effectively block the tumorigenic effects

associated with wild-type FGF19, without compromising its role in bile acid homeostasis (45,46).

Nevertheless, the other encompassing for disrupting FGF19-FGFR4 interaction is by specifically targeting

FGFR4 activation. BLU9931, the first highly selective anti-FGFR4 inhibitor, exhibits strong ability in suppressing FGFR4 activity and decreasing tumor size in HCC xenograft models that maintain an intact

FGFR4 signaling pathway (47).

In summary, we present evidence that ER stress-induced FGF19 upregulation and FGFR4 activation

facilitates HCC cell survival and anti-apoptosis via the FGFR4-GSK3β-Nrf2 signaling cascade. Thereby, our findings make FGF19 an interesting, emerging molecular target for potential therapeutic intervention for patients with HCC.

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Figure legends

Figure 1. ER stress induces FGF19 upregulation in HCC cells. (A, B) Normal hepatocytes cell line

HL7702 and HCC cell lines HepG2, Hep3B, MHCC97L and MHCC97H were treated with 1 μM TG or 1

μg/ml TM for 24 hours. RNA and protein from treated cells were collected for real-time RT-PCR (A) and

Western blot analysis (B). (C) HepG2 and MHCC97L cells were treated with the indicated doses of H2O2, ethanol, or acetaminophen for 24 hours, respectively. RNA from treated cells was collected for real-time

RT-PCR analysis. (D) Six-week-old nude mice were inoculated subcutaneously in the double hind flanks

with 5×106 HepG2 or MHCC97L. When the xenografts reached ~150 mm3, DMSO and 10 μM TG were

intratumorally injected, respectively. At 8 hpi, the xenografts were harvested and homogenized for real-time

RT-PCR analysis (n=6). A permutation test was used to determine a significant difference in FGF19

expression levels between the two groups. (E, F) MHCC97L cells were treated with TG or TM for 24 hours,

in the presence or absence of 10 μM silymarin. RNA and protein from treated cells were collected for real-time RT-PCR (E) and Western blot analysis (F). *p<0.05, **p<0.01, ***p<0.001. N.S. indicates no

significant difference.

Figure 2. ATF4 transcriptionally activates FGF19 gene in HCC cells under ER stress. (A, B) HepG2 and MHCC97L cells were treated with 1 μM TG for 24 hours, and ATF4 expression levels were determined

by real-time RT-PCR (A) and Western blot analysis (B). (C, D) HepG2 cells were transfected with siRNA targeted against luciferase (siNC) or ATF4 (siATF4) for 60 hours. RNA and protein from these cells were harvested for real-time RT-PCR (C) and Western blot analysis (D). (E) MHCC97L cells were transfected

with empty vector (EV) or vector containing full-length of ATF4 (ATF4 O/E) for 72 hours. FGF19

expression levels were determined by Western blot. (F) To determine whether FGF19 is the target of ATF4,

ChIP assays were performed using IgG and ATF4 antibodies to recover DNA binding sequences from

HepG2 cell lysates, followed by qPCR using the primers that were designed to the regions containing the corresponding AARE (left panel). ChIP assays were also used to measure the FGF19 levels in ATF4

immunocomplex in the presence or absence of 1 μM TG (right panel). **p<0.01, ***p<0.001.

Figure 3. FGF19 promotes HCC cell survival and reduces ROS-associated apoptosis under ER stress.

(A) MHCC97L cells transfected with empty vector (EV) or vector containing full-length of FGF19 (FGF19

O/E) were treated with the indicated doses of TG for 24 hours, and cell viability was determined by MTS

assays. (B, C) MHCC97L cells transfected with empty vector (EV) or vector containing full length of

FGF19 (FGF19 O/E) were treated with 1 μM TG for 24 hours. Cell apoptosis was determined by flow

cytometry with PI and Annexin V FITC staining (B, left panel) and Western blot with c-PARP antibody (B,

•− right panel), and cellular release of O2 was determined by electrochemical analysis (C). (D) MHCC97H cells transfected with siRNA targeted against luciferase (siNC) or FGF19 (shFGF19-1 and shFGF19-2) were

treated with indicated doses of TG for 24 hours. Cell viability was determined by MTS assays. (E, F)

MHCC97H cells transfected with shRNA targeted against GFP (shNC) or FGF19 (shFGF19-1 and

shFGF19-2) were treated with 1 μM TG for 24 hours. Cell apoptosis was determined by flow cytometry

with PI and Annexin V FITC staining (E, left panel) and Western blot with c-PARP antibody (E, right panel),

•− and cellular release of O2 was determined by electrochemical analysis (F). *p<0.05, **p<0.01,

***p<0.001.

Figure 4. FGFR4 is essential for the role of FGF19 in HCC cells under ER stress. (A-C) FGFR4 knockout (FGFR4 KO) and wild-type (WT) MHCC97L cells were treated with 1 μM TG for 24 hours. Cell viability was determined by MTS assays (A), cell apoptosis was determined by flow cytometry with PI and

Annexin V FITC staining (B, left panel) and Western blot with c-PARP antibody (B, right panel), and

•− cellular release of O2 was determined by electrochemical analysis (C). (D-F) FGFR4 knockout MHCC97L cells were transfected with empty vector (EV) or vector containing full-length of FGF19 (FGF19 O/E), followed by treatment with 1 μM TG for 24 hours. Cell viability was determined by MTS assays (D), cell apoptosis was determined by flow cytometry with PI and Annexin V FITC staining (E, left panel) and

•− Western blot with c-PARP antibody (E, right panel), and cellular release of O2 was determined by

electrochemical analysis (F). **p<0.01, N.S. indicates no significant difference.

Figure 5. FGF19 facilitates nuclear accumulation of Nrf2 through FGFR4-GSK3β signaling. (A)

HepG2 and MHCC97L cells were transfected with empty vector (EV) or vector containing full-length of

FGF19 (FGF19 O/E), whereas MHCC97H cells were transfected with shRNA targeted against GFP (shNC)

or FGF19 (shFGF19-1 and shFGF19-2). RNA and protein from these cells were collected for real-time

RT-PCR (left panel) and Western blot analysis (right panel). (B) FGFR4 knockout (FGFR4 KO) and

wild-type (WT) MHCC97L cells were transfected with empty vector (EV) or vector containing full-length

of FGF19 (FGF19 O/E). The levels of Nrf2, HO-1 and Bcl-2 were determined by Western blot. (C) Cytosol

and nucleus were isolated from MHCC97L cells transfected with empty vector (EV) or vector containing

full-length of FGF19 (FGF19 O/E). The levels of Nrf2 and FGF19 were determined by Western blot. Lamin

B serves as a control of nuclear protein. (D) FGF19 and FGFR4 overexpressing MHCC97L cells (FGF19

O/E and FGFR4 O/E) were transfected with a luciferase reporter containing triple ARE for 48 hours.

Luciferase activities of ARE were determined using the Promega Dual Luciferase Assay Kits. (E)

MHCC97H cells transfected with shRNA targeted against GFP (shNC) or FGF19 (shFGF19-1 and

shFGF19-2) were treated with 10 μM of TWS119 or 1 μM Tideglusib, respectively. Protein from these cells

was collected for Western blot analysis. (F) FGF19 overexpressing MHCC97L cells (FGF19 O/E) and

control cells (EV) were transfected with a luciferase reporter containing triple ARE for 24 hours, before the

treatment of 10 μM LY294002, 10 μM SP600125, or 1 μM U0126, respectively. Luciferase activities of

ARE were determined using the Promega Dual Luciferase Assay Kits. *p<0.05, **p<0.01, ***p<0.001, N.S.

indicates no significant difference.

Figure 6. Nrf2 is required for FGF19-mediated cytoprotection in HCC cells under ER stress. (A-D)

FGF19 overexpressing MHCC97L cells (FGF19 O/E) and control cells (EV) were transfected with siRNA targeted against luciferase (siNC) or Nrf2 (siNrf2), followed by treatment with 1 μM TG. Cell viability was

•− determined by MTS assays (A), cellular release of O2 was determined by electrochemical analysis (B), and cell apoptosis was determined by flow cytometry with PI and Annexin V FITC staining (C) and Western blot with c-PARP antibody (D). (E) FGF19 overexpressing MHCC97L cells (FGF19 O/E) and control cells (EV) were transfected with siRNA targeted against luciferase (siNC) or Nrf2 (siNrf2), followed by treatment with

1 μM TG for 24 hours. The levels of Nrf2, HO-1 and Bcl-2 were determined by Western blot. (F) HepG2 and MHCC97H cells were transfected with shRNA targeted against GFP (shNC) or FGF19 (shFGF19-1 and shFGF19-2). The levels of Nrf2, HO-1 and Bcl-2, in the presence or absence of 1 μM TG, were determined by Western blot. *p<0.05, **p<0.01.

Figure 7. Schematic representation of FGF19 signaling pathway in HCC cells under ER stress. Tumor

microenvironment factors promotes ER stress induction, which upregulates FGF19 at transcriptional levels

in HCC cells through increased ATF4 protein levels bound to its promoter. Self-regulated secretion of

FGF19 (autocrine), together with endocrine and paracrine FGF19, constitutively binds and activates FGFR4 on the surface of HCC cells, which in turn, activates GSK3β phosphorylation and promotes nuclear translocation of Nrf2. As a result, nuclear accumulation of Nrf2 induces oncogenesis through transcriptional activation of its targets HO-1, Bcl-2 and other pro-survival genes.

FGF19 protects hepatocellular carcinoma cells against endoplasmic reticulum stress via activation of FGFR4-GSK3 β -Nrf2 signaling

Yong Teng, Huakan zhao, Lixia Gao, et al.

Cancer Res Published OnlineFirst September 26, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-2039

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