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Therapeutics, Targets, and Chemical Biology

Separating Tumorigenicity from Bile Acid Regulatory Activityfor Endocrine Hormone FGF19

Mei Zhou, Xueyan Wang, Van Phung, Darrin A. Lindhout, Kalyani Mondal, Jer-Yuan Hsu, Hong Yang,Mark Humphrey, Xunshan Ding, Taruna Arora, R. Marc Learned, Alex M. DePaoli, Hui Tian, and Lei Ling

AbstractHepatocellular carcinoma (HCC), one of the leading causes of cancer-related death, develops from premalignant

lesions in chronically damaged livers. Although it is well established that FGF19 acts through the receptor complexFGFR4-b-Klotho (KLB) to regulate bile acid metabolism, FGF19 is also implicated in the development of HCC. Inhumans, FGF19 is amplified in HCC and its expression is induced in the liver under cholestatic and cirrhoticconditions. Inmice, ectopic overexpression of FGF19 drives HCC development in a process that requires FGFR4. Inthis study, we describe an engineered FGF19 (M70) that fully retains bile acid regulatory activity but does notpromote HCC formation, demonstrating that regulating bile acidmetabolism is distinct and separable from tumor-promoting activity. Mechanistically, we show that FGF19 stimulates tumor progression by activating the STAT3pathway, an activity eliminated by M70. Furthermore, M70 inhibits FGF19-dependent tumor growth in a rodentmodel. Our results suggest that selectively targeting the FGF19–FGFR4 pathway may offer a tractable approach toimprove the treatment of chronic liver disease and cancer. Cancer Res; 74(12); 3306–16. �2014 AACR.

IntroductionFGF19 (also called FGF15 in rodents) is an endocrine

hormone of the FGF family that regulates bile acid, carbo-hydrate, lipid, and energy metabolism (1). FGF19 selectivelybinds to FGFR4, which can be further enhanced by core-ceptor KLB (2–5). The interaction between FGF19 andFGFR4 is crucial in repressing hepatic expression of cho-lesterol 7a-hydroxylase (CYP7A1), the first and rate-limitingenzyme in the conversion of cholesterol into bile acids (6–9).Interestingly, FGF19–FGFR4 signaling is also implicated inhepatocellular tumorigenesis. In humans, FGF19 is coam-plified with cyclin D1 (CCND1) at approximately 15% fre-quency in hepatocellular carcinoma (HCC) on 11q13.3 (10).Clonal growth and tumorigenicity of HCC cells harboring the11q13.3 amplicon can be inhibited by RNAi-mediated knock-down of FGF19, as well as an anti-FGF19 antibody (10).Transgenic mice with ectopic expression of FGF19 in skel-etal muscle develop HCC at the age of 10- to 12-month-old(11). This tumorigenic activity is thought to be mediated bythe liver-enriched FGFR4, because inactivation of FGFR4 viagene knockout or by a neutralizing antibody reduces tumorburden in FGF19 transgenic mice (12, 13). Therefore, target-

ing the FGF19–FGFR4 pathway has the potential for treatingHCC. However, early efforts have encountered setbacks.Severe toxicity was observed in non-human primates by ananti–FGF19-neutralizing antibody due to on-target inhibi-tion of endogenous FGF19, leading to dysregulation of bileacid metabolism (14). Hence, alternative approaches areneeded to overcome these barriers to developing successfultherapy.

In an effort to eliminate the tumorigenic activity of FGF19without compromising its beneficial role in bile acid homeo-stasis, we established an in vivo liver tumorigenicity model inmice to evaluate FGF19-induced hepatocarcinogenicity. Usingan adeno-associated virus (AAV)–mediated gene deliveryapproach (15), we introduced FGF19 transgene in mice andevaluated a panel of FGF19 variants in vivo to identify tumor-free variants. In this article, we show that one such variant,M70, fully retains the biologic activity of FGF19 in regulatingbile acid homeostasis while devoid of tumorigenicity. Notably,we found that M70 binds to and activates FGFR4, which isassumed to mediate FGF19-associated tumorigenicity (13, 16).Mechanistically, we show that FGF19 stimulates tumor pro-gression by activating the STAT3 pathway, an activitycompletely eliminated by M70. Furthermore, M70 inhibitsFGF19-dependent tumor growth in a rodent model.

Materials and MethodsDNA constructs

Human FGF19 (NM_005117), human FGFR4 (NM_022963),mouse FGFR4 (NM_008011), human KLB (NM_175737), andmouse KLB (NM_031180) cDNAs were purchased from Gene-copoeia. Mutations were introduced in the FGF19 cons-tructs using the QuickChange Site-Directed Mutagenesis Kit(Stratagene).

Authors' Affiliation: NGMBiopharmaceuticals, Inc., South San Francisco,California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Lei Ling, NGM Biopharmaceuticals, Inc., 630Gateway Boulevard, South San Francisco, CA 94080. Phone: 650-243-5546; Fax: 650-583-1646; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0208

�2014 American Association for Cancer Research.

CancerResearch

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on April 30, 2021. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 11, 2014; DOI: 10.1158/0008-5472.CAN-14-0208

http://cancerres.aacrjournals.org/

AAV ProductionAAV293 cells (Agilent Technologies) were cultured in

DMEM (Mediatech) supplemented with 10% FBS and 1�antibiotic–antimycotic solution (Mediatech). The cells weretransfected with three plasmids [AAV transgene, pHelper(Agilent Technologies), and AAV2/9] for viral production. Viralparticles were purified using a discontinued iodixanal (Sigma-Aldrich) gradient and resuspended in PBS with 10% glyceroland stored at �80�C. Viral titer or genome copy number wasdetermined by qPCR.

Animal experimentsAll animal studieswere approved by the Institutional Animal

Care and Use Committee at NGM. Mice were housed in apathogen-free animal facility at 22�C under controlled 12-hourlight/12-hour dark cycle. All mice were kept on standard chowdiet (Harlan Laboratories; Teklad 2918) and autoclaved waterad libitum. Male mice were used unless otherwise specified.C57BL/6J, FVB/NJ, BDF, ob/ob, and db/db mice were pur-chased from The Jackson Laboratory. Heterozygous rasH2transgenic mice were obtained from Taconic. All animalsreceived a single 200 mL intravenous injection of 3 � 1011

genome copies of AAV via tail vein on day 1. Animals wereeuthanized and livers were collected 24 or 52 weeks afterdosingwith AAV. Liver weight and liver tumor nodule numberswere recorded upon necropsy.

Histologic and immunohistochemical analysisFormalin-fixed paraffin-embedded tissue sections were

stained with hematoxylin and eosin (H&E) for histologicassessment at Nova Pathology. For immunohistology, anti-PCNA (Dako), anti-Ki67 (Dako), anti-glutamine synthetase(Thermo Fisher Scientific), or anti–b-catenin antibodies (CellSignaling Techology) were used. Biotinylated secondary anti-body, ABC-HRP reagent and DAB colorimetric peroxidasesubstrate (Vector Laboratories) were used for detection.

Measurement of serum FGF19 proteinWhole blood was collected from mouse tail snips into

capillary tubes (Becton Dickenson). Levels of human FGF19and variants were measured in serum using an ELISA assay(Biovendor). The assay recognizes both FGF19 and M70 in anindistinguishable manner.

Clinical chemistrySerum levels of liver enzymes (ALT, AST, and ALKP), trigly-

cerides, total cholesterol, HDL-C and LDL-C were measuredusing enzymatic reactions on COBAS Integra 400 clinicalanalyzer (Roche Diagnostics). Concentrations of total bileacids in serum were determined using a 3a-hydroxysteroiddehydrogenase method (Diazyme).

Gene expression analysisTotal RNA was extracted from tissues or cells using the

RNeasy Kit (Qiagen). qRT-PCR analysis was performed usingQuantiTect multiplex qRT-PCR master mix (Qiagen) andpremade primers and probes (Life Technologies). Reactionswere performed in triplicates on an Applied Biosystems7900HT Sequence Detection System. Relative mRNA levels

were calculated by the comparative threshold cycle methodusing GAPDH as the internal standard.

Expression of recombinant proteinsFGF19 and M70 were produced in E. coli and purified as

described withmodification (17). Methionine was added to themature peptides to facilitate expression in bacteria. Proteinswere then purified to homogeneity via successive rounds ofion-exchange and hydrophobic interaction chromatography.Protein sequence was confirmed via LC/MS and monodisper-sity via SEC-HPLC (TOSOH TSKgelG3000).

Surface plasmon resonance assaysSurface plasmon resonance (SPR) experiments were per-

formed on a Biacore T200 instrument at 25�C. For directbinding between FGFR4 and ligands, anti-Fc antibody wasimmobilized on a CM5 sensor chip using standard amine-coupling procedure and mouse FGFR4(ECD)-Fc (R&D Sys-tems) was captured subsequently. KD values were determinedusing the Biacore T200 evaluation software V.1 using a 1:1binding model.

Solid-phase binding assayNinety-six–well ELISA plates (Thermo Fisher Scientific)

were coated with goat-anti-Fc, blocked with 3% BSA, and thenincubated with 1 mg/mL mouse FGFR4(ECD)-Fc (R&D Sys-tems) in PBS containing 3% BSA. The plates were incubatedwith various concentrations of FGF19 or M70 [in the presenceof 20 mg/mL heparin (Sigma) and 1 mg/mL mouse KLB (R&DSystems)] in PBS/3% BSA. The bound proteins were detectedusing biotinylated FGF19-specific polyclonal antibody (R&DSystems) followed by streptavidin–horseradish peroxidase andthe TMB peroxidase colorigenic substrate (KPL).

Luciferase assaysRat L6 myoblasts (ATCC) were cultured in DMEM supple-

mented with 10% FBS at 37�C under 5% CO2. Cells weretransiently transfected with expression vectors encodingmouse KLB, mouse FGFR4, GAL4-Elk1 transcriptional activa-tor (pFA2-Elk1; Stratagene), and firefly luciferase reporter–driven GAL4-binding sites (pFR-luc; Stratagene) using Fugene6 transfection reagent (Roche Applied Science).

CYP7A1 expression in primary hepatocytes and in micePrimary hepatocytes from mouse or rat livers (Life Tech-

nologies) were plated on collagen I-coated 96-well plates(Becton Dickinson) and incubated overnight in Williams' Emedia supplemented with 100 nmol/L dexamethasone and0.25 mg/mL Matrigel. Cells were treated with recombinantFGF19 or M70 proteins for 24 hours. CYP7A1 expression in celllysates was determined by qRT-PCR analysis. For assessingCYP7A1 regulation in vivo, 12-week-old db/db mice wereinjected intraperitoneally (i.p.) with FGF19 or M70 proteins.Mice were euthanized 4 hours after dosing and livers wereharvested for qRT-PCR analysis.

In vivo signaling analysisThe db/db mice (9–11-week-old; The Jackson Laboratory)

were given intraperitoneal injections (1mg/kg) of FGF19 orM70

Separating Tumorigenicity from Bile Acid Activity for FGF19

www.aacrjournals.org Cancer Res; 74(12) June 15, 2014 3307




recombinant proteins. Livers were collected after injection andsnap-frozen in liquid nitrogen. Signaling proteins were detectedin liver lysates with antibodies to pSTAT3 (Cell Signaling Tech-nology #9145), STAT3 (Cell Signaling Technology #8768), orantibody cocktail I (Cell Signaling Technology #5301).

Statistical analysisAll results are expressed as the mean � SEM. One-way

ANOVA followed by the Dunnett post-test was used to com-pare data from multiple groups (GraphPad Prism). Whenindicated, unpaired the Student t test was used to comparetwo treatment groups. A P value of 0.05 or smaller wasconsidered statistically significant.

ResultsAn AAV-mediated transgene system for evaluation ofhepatocellular tumorigenesis in vivo

AAV-mediated gene delivery provides a means to achi-eve continuous transgene expression without inflammatory

responses that are commonly associated with other viralvectors (18). Sustained expression of up to 1 year has beenobserved with the AAV gene delivery method when introducedinto adult mice (19). The first AAV vector was recentlyapproved as a treatment for a genetic disorder in humans (20).

In the previously reported FGF19 transgenic models, FGF19was ectopically expressed in the skeletal muscle, a nonphysio-logic site of FGF19 expression (8, 11). Under pathologic con-ditions, such as cirrhosis or cholestasis, FGF19 expression isinduced in the liver (21–24). As an alternative to the conven-tional approach of generating transgenic mice, we introducedFGF19 via AAV in 6 to 12-week-old mice (Fig. 1A). The primarytissue of transgene expression using this method is liver (25).

Multiple mouse strains were evaluated for latency androbustness of FGF19-mediated liver tumor formation (Table1). A control AAV virus encoding GFP (AAV–GFP), did notpromote liver tumor formation in the mouse strains tested(Table 1). In general, mice injected with AAV–GFP exhibitedsimilar phenotype as saline-injected animals (data not shown).

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Figure 1. An AAV-mediated transgene system for studying hepatocellular tumorigenesis. A, a diagram of the experimental protocol. Mice were given a singleinjection of 3 � 1011 genome copies of AAV–FGF19 via tail vein when 6- to 12-week-old. Mice were sacrificed 24 or 52 weeks later for liver tumor analysis.ITR, inverted terminal repeat; EF1a, elongation factor 1a promoter. B, representative livers of db/db mice 24 weeks after administration of AAV–FGF19.A control virus encoding GFP (control) is included. C, serum levels of FGF19 were measured by ELISA at 1, 4, 12, and 24 weeks after AAV administrationin db/db mice (n ¼ 5). D, liver tumor multiplicity and size in db/db mice (n ¼ 15) expressing FGF19 transgene. Tumors per liver were counted and maximaltumor sizes were measured. E, histologic and immunohistochemical characterization of FGF19-induced liver tumors in db/db mice. FGF19-inducedneoplastic cells are strongly glutamine synthetase–positive. Tumors (T) are outlinedbydotted lines. All values representmean�SEM. ��,P<0.001; �,P<0.05denote significant differences versus the control group by the two-tailed t test.

Zhou et al.

Cancer Res; 74(12) June 15, 2014 Cancer Research3308




For simplicity, only results from AAV–GFP-injected animalswere shown as controls in following studies. Interestingly, thetumor latency varied depending upon the mouse geneticbackground. In particular, among severalmouse strains tested,db/db mice exhibited the shortest latency and high tumorpenetrance, with the appearance of multiple, large, raisedtumor nodules protruding from the liver surface 24 weeksfollowing AAV–FGF19 delivery (Fig. 1B). This finding is con-sistent with the observation that mutations in the leptinreceptor are frequently found in cirrhotic livers and are linkedto HCC in human (26, 27). The db/db mice, which carry agenetic defect in the leptin receptor (28), provide a clinicallyrelevant genetic context for evaluating candidate HCC-pro-moting genes.AAV-mediated delivery of wild-type FGF19 transgene into

db/dbmice resulted in high circulating levels of FGF19, reach-ing approximately 1 mg/mL 1 week after a single tail veininjection and persisting throughout the 24-week study period(Fig. 1C). Twenty-four weeks after gene delivery, the mice wereeuthanized and subjected to necropsy. Visible tumor noduleson the entire surface of the liver were counted (Fig. 1D). Themaximum diameter of the liver tumor nodules was recorded(Fig. 1D). Occasionally, a few liver tumor nodules wereobserved in db/db mice injected with control virus or saline,probably reflecting an increased background level of hepatictumorigenesis in this genetic model (Fig. 1D; data not shown).Microscopic examination classified the AAV–FGF19-

induced in situ liver tumors as solid HCC, which resembledthose reported in FGF19 transgenic animals (Fig. 1E).Cellular proliferative status, examined by immunohisto-chemical staining for Ki-67 and PCNA, indicated that thetumors were highly proliferative. Similar to the tumorsobserved in FGF19 transgenic mice, liver tumors in AAV–FGF19 mice were glutamine synthetase–positive, suggestiveof a pericentral origin (Fig. 1E; ref. 11). Liver tumors fromAAV–FGF19 mice also showed increased nuclear stainingfor b-catenin (Fig. 1E). Taken together, these data suggestthat AAV-mediated transgene expression in mice provides arobust system to evaluate FGF19-induced hepatocarcino-genesis in vivo.

M70 is an engineered, nontumorigenic FGF19Using the AAV-mediated gene delivery method, we evalu-

ated a panel of FGF19 variants in db/db mice for their tumor-igenicity. A FGF19 variant carrying 3 amino acid substitutions(A30S, G31S, andH33L) and a 5-amino acid deletion, referred asM70, was selected for further studies (Fig. 2A).

Whereas ectopic expression of FGF19 promoted significantliver tumor formation indb/dbmice (15.6� 2.8 tumor nodules/liver), livers frommice with high systemic exposure to M70 for24 weeks were completely free of hepatic tumor nodules (Fig.2B). FGF19-expressing mice exhibited a significant increase inliver weight (Fig. 2C), which closely correlates with liver tumorburden. In contrast, mice expressing M70 did not show anyincrease in liver weight (Fig. 2C). Similar results were obtainedwhen the liver-to-body weight ratio was calculated (Fig. 2Dand Fig. 2E). Average serum concentration of M70 was 2 to 3mg/mL in these mice, about 10,000-fold higher than circulatingFGF19 levels in human (Fig. 2F). Histologic analysis of liversfrom M70-expressing mice showed no evidence of neoplasticlesions associated with FGF19 overexpression in mice, includ-ing hepatocellular dysplasia, hepatocellular adenomas, or HCC(Fig. 2G). Moreover, as indicated by Ki-67 staining, overexpres-sion of M70 did not promote hepatocellular proliferationobserved in mice expressing FGF19 (Fig. 2G). Furthermore,although liver tumor lesions in FGF19-expressingmice becameprominently stained for glutamine synthetase (a marker forpericentral hepatocytes), no increased expression of glutaminesynthease was observed in the liver of M70-expressing mice(Fig. 2G). Finally, no liver toxicity, as reflected by serum levels ofliver enzymes, was observed following 24 weeks of exposure toM70 (Fig. 2H). Additional serum parameters (e.g., glucose,triglycerides, and cholesterol) are included in SupplementaryTable S1. Taken together, these results demonstrate that M70lacks the ability to promote hepatocellular tumorigenesis indb/db mice.

We also evaluated the liver tumorigenic potential ofM70 in arasH2 transgenic mouse model. CB6F1-RasH2 mice hemizy-gous for a human H-RAS transgene have been extensively usedas an accelerated alternative to the conventional 2-year car-cinogenicity assessment in rodents (29). Sensitive to genotoxic

Table 1. FGF19 promotes hepatocarcinogenesis in multiple mouse models

FGF19 Control

Mouse strain 24 Weeks 52 Weeks 24 Weeks 52 Weeks

C57BL6/J 0/5 4/5 (80%) 0/5 0/5BDF 0/5 5/5 (100%) 0/5 0/5FVB/N 0/5 1/5 (20%) 0/5 0/5ob/ob 4/5 (80%) n.d. 0/5 n.d.db/db 5/5 (100%) n.d. 0/5 n.d.

NOTE: Various strains ofmice (6–12-week-old) were injectedwith 3� 1011 genomecopies of AAV vectors encodingFGF19or a controlgene (GFP). Tumor incidencewasdetermined at 24or 52weeks after AAVadministration andexpressedas number of animalswith livertumor over the total number of animals in the group.Abbreviation: n.d., not determined.






and nongenotoxic carcinogens, rasH2 mice develop bothspontaneous and induced neoplasms earlier than wild-typemice. Because activation of RAS signaling pathway is frequent-ly observed in human HCC (30), this strain provides a relevantgenetic background for studying hepatocarcinogenicity.

During the course of a 52-week study, rasH2mice expressingFGF19 or M70 had a significant reduction of body weight gaincompared with control mice (Fig. 3A). After 52 weeks ofcontinuous exposure, clear differences in liver morphologywere observed in mice expressing FGF19 compared with thoseexpressing M70. Gross morphologic changes, including theappearance of multiple tumor nodules, were observed in thelivers of mice expressing FGF19 (Fig. 3B). In contrast, the liversfrom mice expressing M70 showed normal gross morphologyandwere completely free of tumor nodules (Fig. 3B). A low levelof spontaneous liver tumor formation was observed in controlrasH2 mice (Fig. 3B). Moreover, M70-expressing animalsshowed a dramatic decrease in liver weight compared withmice expressing FGF19 (Fig. 3C).M70 also normalized the ratioof liver to bodyweight in rasH2mice (Fig. 3D). The serum levelsof FGF19 and M70 in these mice are comparable, 155� 28 and209 � 22 ng/mL, respectively (Fig. 3E).

H&E-stained liver sections from these mice were evaluatedfor the presence of neoplastic lesions (Fig. 3F). In addition, anti-

glutamine synthetase staining was carried out as a marker ofFGF19-induced liver tumor (Fig. 3F). rasH2 mice expressingFGF19 displayed a variety of cellular abnormalities, includinghepatocellular adenoma and HCCs. Remarkably, none of thelivers from mice expressing M70 exhibited histologic evidenceof neoplastic lesions (Fig. 3F). Consistent with results ofhistologic analysis, increased hepatic expression of Ki-67 andAFP, an embryonic hepatic protein often induced in HCC (31),was observed in FGF19-expressing rasH2mice, but not in miceexpressing M70 (Fig. 3G).

In summary, unlike FGF19, prolonged exposure to highlevels of M70 did not promote liver tumor formation, in eitherdb/db or rasH2 mice.

M70 binds and activates FGFR4 in vitro and in vivoTo elucidate the molecular mechanism that underlies the

inability of M70 to induce liver tumors, we assessed theinteraction of M70 to the known receptor complex for FGF19.SPR analysis was used to measure direct binding of either M70or FGF19 to FGFR4. As shown in Fig. 4A and B, the affinity ofM70 for FGFR4 was comparable with that of FGF19 (dissoci-ation constant KD ¼ 134 � 47 and 167 � 5 nmol/L, respec-tively). Using a solid-phase assay, M70 interacted with theFGFR4–KLB receptor complex (Fig. 4C), with the presence of

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Figure 2. M70 is a tumor-free FGF19 variant after continuous exposure in db/db mice for 24 weeks. Alignment of protein sequences of M70 and FGF19 inthe N-terminal region. Mutations introduced intoM70 are underlined. Number of tumors per liver (B), liver weight (C), and the ratio of liver to bodyweight (D) ofdb/db mice (n ¼ 5) expressing FGF19 or M70 for 24 weeks. Growth curve (E) and serum levels of transgene expression (F) were also determined. G,representative liver sections from db/db mice after 24 weeks of transgene expression. H&E staining and immunohistochemical detection of Ki-67 andglutamine synthetase of liver tissue sections were included. Tumors (T) are outlined by dotted lines. H, serum levels of liver enzymes (ALT, alanineaminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; n ¼ 5) were measured before termination of the study. All valuesrepresent mean � SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001 denotes significant differences versus the control group by one-way ANOVA followed by theDunnett post-test.

Zhou et al.





KLB dramatically increased ligand-receptor affinity. The dis-sociation constant of M70 binding to the FGFR4–KLB receptorcomplex, indicated a high-affinity interaction thatwas virtuallyidentical to that of FGF19 (KD ¼ 2.14 and 2.49 nmol/L;respectively).We also evaluated the ability of M70 to activate its receptors

in a cell-based assay using rat L6 cells transfected with an FGF-responsive GAL-Elk1 luciferase reporter (9, 32). In this assay,effective binding of a ligand to FGFR results in the activation ofthe endogenous ERK kinase pathway, leading to subsequentactivation of a chimeric transcriptional activator comprising ofan Elk1 activation domain and a GAL4 DNA–binding domain.L6 cells lack functional FGFR or KLB and are only responsive toFGF19 when cotransfected with cognate receptors (data notshown). M70 activated intracellular signaling pathways in L6cells coexpressing FGFR4 and KLB as effectively as FGF19(EC50¼ 38 and 52 pmol/L forM70 and FGF19, respectively; Fig.4D). In contrast, signaling in cells transfectedwith FGFR4 alonewasmuch less responsive to either ligand, showing a >500-foldreduction in potency upon addition of either FGF19 or M70(Fig. 4D). These results suggest that the formation of a ternarycomplex between FGFR4–KLB coreceptors and the cognateligands is important for potent activation of intracellularsignaling.In addition, we analyzed FGFR4 pathway activation in

Hep3B, a human HCC cell line that expresses KLB and,among the FGFR isoforms, predominantly FGFR4. Recom-

binant M70 protein induced phosphorylation and activationof ERK with a similar potency and efficacy as wild-typeFGF19 (EC50 ¼ 0.38 and 0.37 nmol/L for M70 and FGF19,respectively; Fig. 4E).

Finally, we assessed a physiologically relevant activity ofM70. FGF19 have been implicated in the regulation of hepaticbile acid metabolism in humans and in rodents (7, 8). FGF19potently represses hepatic expression of CYP7A1, in a processthat requires FGFR4 (8, 9). We evaluated the ability of M70 toregulate CYP7A1 in primary hepatocytes. Upon addition tothe culture media, M70 effectively repressed CYP7A1 expres-sion in primary hepatocytes derived from mouse or rat liver,showing an activity comparable with that of wild-type FGF19(Fig. 4F).

To evaluate the acute effects of M70 administration onhepatic expression of CYP7A1 in vivo, mice were injected i.p.with recombinantM70 or FGF19 protein at doses ranging from0.001 to 10 mg/kg (Fig. 4G). A single i.p. injection of M70potently suppressed the expression of CYP7A1 mRNA with anED50 value of 1.29 mg/kg (Fig. 4G). Consistent with the repres-sion of bile acid synthesis, serum levels of total bile acids werelower in mice with long-term exposure to M70 (Fig. 4H). Thesedata demonstrate that systemic administration of M70 canpotently and rapidly trigger FGFR4-mediated response in vivo.

In summary,M70 andwild-type FGF19 exhibit a comparableprofile of biologic activity that leads to the activation of ERKand suppression of CYP7A1.

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Figure 3. No liver tumor formation in rasH2mice treated with M70 for 52 weeks. Growth curve (A), number of tumors per liver (B), liver weight (C), liver-to-bodyweight ratios (D), and serum levels of M70 or FGF19 (E) of rasH2 mice (n¼ 9) expressing FGF19 or M70 transgenes for 52 weeks. F, representative images oflivers stained with H&E or anti-glutamine synthetase. Portal (p) and central (c) veins are indicated. Tumors (T) are outlined by dotted lines. G, qRT-PCRanalysis of Ki-67 andAFPexpression in the liver. All values representmean�SEM. �,P<0.05; ��,P <0.01; ��,P <0.001denotes significant differences versusthe control group by one-way ANOVA followed by the Dunnett post-test.






M70 exhibits differential signaling pathway activationcompared with FGF19

M70 binds to the FGFR4 receptor complex and activates theintracellular signaling pathway leading to CYP7A1 repression,but does not promote liver tumor formation in either db/db orrasH2 mouse models. The identification and characterizationof M70 allows us to define two distinct and separable biologicprocesses regulated by the FGF19–FGFR4 pathway, bile acidhomeostasis, and tumorigenesis.

To elucidate the molecular basis for this lack of tumor-igenic potential, we analyzed the activation of key signalingproteins involved in tumorigenesis, including ERK, PI3K/AKT, STATs, and WNT/b-catenin pathways. M70 and FGF19proteins (1 mg/kg) were injected i.p. into db/db mice. Liverswere collected and phosphorylation of signaling proteins

was measured by immunoblotting. Consistent with theability of both molecules to signal in cultured primaryhepatocytes, FGF19 and M70 stimulated ERK phosphoryla-tion to a similar extent in liver tissues in vivo. In line withprevious reports on the role of FGF19 in modulating hepaticprotein synthesis (33), both wild-type FGF19 and M70induced phosphorylation of ribosomal S6 protein (Fig. 5).These data are consistent with results described in theprevious sections that M70 retains activity on the FGFR4–KLB receptor complex. Neither M70 or FGF19 had any effecton hepatic levels of phosphorylated AKT, nor did theysignificantly activate GSK3b and b-catenin at any of thetime points tested (Fig. 5).

Remarkably, FGF19 induced STAT3 phosphorylation 2hours after dosing (Fig. 5A). This effect lasted to 4 hours after

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Figure 4. M70 binds and activates FGFR4 in vitro and in vivo. A, Biacore SPR assay of the interaction between FGF19 and FGFR4. Left column shows bindingcurves obtained over a range of FGF19 concentrations, whereas right column shows the steady state fits of the data for obtaining KD values. B, bindingof M70 to FGFR4 by Biacore. C, solid-phase binding of M70 or FGF19 to the FGFR4–KLB receptor complex. D, receptor activation by M70 or FGF19.L6 cells were transiently transfected with FGFR4 and ELK-luciferase reporter in the presence or absence of KLB. E, M70 induces ERK phosphorylation inHep3B cells. F, M70 represses CYP7A1 expression in primary hepatocytes of mouse or rat origin. G, repression of hepatic CYP7A1 expression by M70in mice. Twelve-week-old db/db mice were injected i.p. with recombinant M70 or FGF19 protein. Hepatic CYP7A1 expression was evaluated byqRT-PCR. Dose–response curves of CYP7A1 repression in mice are shown. H, M70 reduces serum levels of total bile acids. Serum samples were taken fromdb/dbmice (n¼ 5) 24 weeks following administration of AAV vectors expressing FGF19, M70, or control virus. All values represent mean� SEM. �, P < 0.05denotes significant differences versus the control group by one-way ANOVA followed by the Dunnett post-test.

Zhou et al.





dosing (data not shown). In contrast, M70 completely lackedthe ability to induce STAT3 phosphorylation (Fig. 5A). ThepSTAT3 activation by FGF19 is likely due to noncell autono-mous mechanisms on the liver, because no induction ofpSTAT3 was observed 15 minutes after protein injection orin primary mouse hepatocyte culture (data not shown). Con-sistent with these observations of differential STAT3 phos-phorylation and activation, STAT3 target genes, includingSurvivin, Cyclin D1, and Bcl-XL, were dramatically upregulatedin db/db and rasH2 livers expressing FGF19, but not M70 (Fig.5B and C). Because STAT3 is an oncogene frequently activatedin HCC (34), its activation by FGF19 provides a plausiblemechanism for FGF19-induced hepatocarcinogenicity. Con-versely, the inability of M70 to activate the STAT3 pathwaycould contribute to its lack of tumorigenicity in vivo.Thus, M70 only activates a subset of signaling pathways

downstream of its receptors. This property, a hallmark ofselective modulator or "biased agonist" (35), suggests thatM70 may act as a selective modulator of FGFR4 to regulatemetabolism without causing tumorigenicity.

M70 inhibits FGF19-mediated tumor formationOur observations suggest that M70 behaves as a selective

modulator (or "biased ligand") to activate the metabolic sig-

naling of, but not the tumorigenic pathway from, FGFR4. Next,we examined whether the biased agonism ofM70 could inhibitFGF19-dependent tumor formation.

The db/db mice were injected with AAV–FGF19, with orwithout 10-fold molar excess of AAV-M70. Mice were necrop-sied 24 weeks after transgene expression and the livers wereexcised for analysis. Although ectopic expression of FGF19 indb/db mice promoted the formation of tumor nodules on thehepatic surface, livers from mice expressing both FGF19 andM70 were completely free of tumor nodules (Fig. 6A). Liverweights of mice coexpressing FGF19 and M70 were signifi-cantly lower relative to mice expressing FGF19 only (Fig. 6B).The ratios of liver to body weight in M70 and FGF19 cotreatedmicewere not significantly different from those of controlmice(Fig. 6C). The serum levels of FGF19 were 94� 12 ng/mL whendosed alone, and the combined serum level of FGF19 and M70was 453� 169 ng/mL (Fig. 6D). Histologic analysis of the liversconfirmed that, unlike FGF19-expressing mice, mice coexpres-sing M70 and FGF19 did not exhibit any histologic evidence ofliver tumors (Fig. 6E). These data clearly demonstrate thatM70can effectively block tumor formation induced by wild-typeFGF19.

These results are consistent with the notion thatM70 acts asa biased ligand that is capable of antagonizingwild-type FGF19

A

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Figure 5. Differential activation of cell signaling pathways by M70 and FGF19 in vivo. A, livers were harvested from db/db mice (n ¼ 6) injected i.p. withsaline, FGF19, or M70 proteins 2 hours after injection. Liver lysates were examined by Western blot analysis for expression and phosphorylation of theindicated proteins. Each lane represents an individual mouse. Rab11 served as a loading control. B, livers were harvested from db/dbmice (n¼ 5) 24 weeksfollowing administration of AAV vectors expressing FGF19 or M70 transgenes. STAT3 target genes (Survivin and Cyclin D1) mRNA levels in livers weremeasured by qRT-PCR. C, qPCR showing expression of mRNAs for STAT3 target genes (Survivin, Cyclin D, and Bcl-XL) in rasH2 mice 52 weeks followingadministration of AAV vectors expressing FGF19 or M70 transgenes. Results are represented as fold expression relative to control animals. All valuesrepresent mean � SEM. ��, P < 0.01; ��, P < 0.001 denotes significant differences versus the control group by one-way ANOVA followed by theDunnett post-test.






in tumorigenic signaling, and demonstrates the potential ofusing a selective modulator, such as M70, to suppress FGF19-dependent tumor growth.

DiscussionFGF19 is an endocrine hormone of the FGF family that

regulates bile acid, carbohydrate, lipid, and energy metabolism

(33, 36, 37). Among FGFRs, FGF19 binds selectively to FGFR4(2). A direct link between FGF19 and FGFR4 in hepatocellularoncogenesis is established in a mouse study in which FGF19-mediated liver tumorigenesis was abrogated in FGFR4 KOmice (13). Although predominantly expressed in the ileum,FGF19 is induced in liver under cholestatic conditions causedby either destruction or obstruction of bile duct, cirrhosis orbile acid accumulation. Because chronic liver cirrhosis andcholestasis often trigger compensatory regeneration and HCC,FGF19 might present a missing link between liver injury,regeneration, and cancer.

HCC represents an important unmet medical need (38).Given the potential role of FGF19 in HCC development, effortshave been devoted to developing therapies by targeting FGF19.Indeed, a neutralizing anti-FGF19 monoclonal antibody wasdeveloped and demonstrated antitumor activity in xenograftmodels (22). However, such strategy has suffered setback dueto serious adverse effects. Administration of this antibody tocynomolgus monkeys led to dose-related liver toxicity accom-panied by severe diarrhea (14). This adverse effect is apparentlydue to on-target inhibition of endogenous FGF19, resulting inincreased hepatic bile acid synthesis, elevated serum bile acid,perturbation of enterohepatic circulation, and the develop-ment of diarrhea and liver toxicity (14). Thus, alternativeapproaches must be developed.

Through a high-throughput in vivo screen, we identified anengineered FGF19 variant M70 that does not cause any livertumors even after prolonged exposure at supraphysiologiclevels in mice. Although there were previous reports of gen-erating tumor-free FGF19 variants (9, 32), those variants werespecifically designed to eliminate FGFR4 binding and, there-fore, impaired in regulating bile acid metabolism. In contrast,M70 retains the ability to maintain bile acid homeostasis.Importantly, we show that not only doesM70 lack tumorigenicpotential, but that it can effectively block the tumorigeniceffects associated with wild-type FGF19.

Themajor difference betweenM70 and FGF19 resides in theN-terminus of the protein. Each FGF family protein consists ofthe structurally conserved central globular domain, and theflanking N-terminal and C-terminal segments that are struc-turallyflexible and are divergent in primary sequences (1). In X-ray crystal structures of multiple FGF/FGFR complexes, the N-terminal segment of the FGF molecule makes specific contactwith the FGFR and is believed to play an important roledetermining the specificity of the FGF–FGFR interaction (1).Through systematic efforts using an in vivo screen, we foundthat changing three amino acids at the N-terminus coupledwith a 5-amino acid deletion eliminated tumorigenicity with-out impairing its ability to activate FGFR4-dependent processsuch as bile acid regulation.

To elucidate the molecular basis for the lack of tumorigenicpotential of M70, we carried out in vivo analyses to assess theactivation of key signaling proteins involved in tumorigenesis,including ERK, PI3K/AKT, STATs, and WNT/b-catenin path-ways. We found that FGF19, but not M70, activates STAT3 inmouse liver. STAT3 is a major player in hepatocellular onco-genesis (34). Phosphorylated (i.e., activated) STAT3 is found inapproximately 60% of HCC in humans (39), and correlates with

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Figure 6. M70 inhibits FGF19-induced tumor growth in db/dbmice. A–D,11-week-old db/dbmice (n¼ 5) were injected with AAV–FGF19 (3� 1010

genome copies) in the absence or presence of M70 (3 � 1011 genomecopies). Liver tumor score (A), liver weight (B), the ratio of liver to bodyweight (C), and serum levels of transgene expression (D)were determined24 weeks later. E, histology of livers of mice expressing FGF19 orcotreated with M70. Liver sections were stained with H&E or anti-glutamine synthetase, amarker for FGF19-induced liver tumors. Portal (p)and central (c) veins are indicated. Tumors (T) are outlined by dotted lines.All values represent mean � SEM. �, P < 0.05 denotes significantdifferences versus the control group by one-way ANOVA followed by theDunnett post-test; ##, P < 0.01 denotes significant differences by thetwo-tailed t test.

Zhou et al.





poor prognosis in patients with HCC (30). Constitutively activeSTAT3 acts as an oncogene in cellular transformation (40).Hepatocyte-specific ablation of STAT3 prevented HCC devel-opment in mice (39). Inhibitors of STAT3 activation block thegrowth of human cancer cells and are being tested in the clinicfor treating various cancers, including HCC (41–43). However,the events that lead to STAT3 activation in humanHCC are notknown. IL-6, among other inflammatory cytokines, is postu-lated to be themajor STAT3 activator in the liver (39). Here, weshow that FGF19 also activates STAT3 signaling in vivo, aneffect that could be directly mediated by the FGFR4 receptorcomplex, or indirectly through induction of cytokines orgrowth factors. The precise mechanism for FGF19-inducedSTAT3 activation remains to be investigated. Although previ-ous studies reported that FGF19 seems to have a strong effectonb-catenin activation in cultured cell lines (10, 12), we did notobserve an impact of FGF19 on levels of dephosphorylated (i.e.,activated) b-catenin or P-GSK3b in an acute in vivo setting inmice. At present, the basis for these disparate observations isunclear.Our data demonstrate that M70 exhibits the pharmacologic

characteristics of a "biased ligand" or a selective modulator.M70 exhibits bias toward certain FGFR4 signaling pathways(e.g., CYP7A1, pERK) to the relative exclusion of others (e.g.,tumor, pSTAT3). Studies of G protein-coupled receptors andion channels have demonstrated that selective receptor mod-ulators offer opportunities forfine-tuning biologic responses ina manner that is not attainable via classic orthosteric mechan-isms (44). As a selective FGFR4 modulator, M70 antagonizesthe oncogenic activity of FGF19 in HCC.FGF19 demonstrates an array of biologic effects. The ther-

apeutic potential for FGF19 includes the treatment of chronicliver diseases, as well as obesity and diabetes (45). However, the

carcinogenicity of FGF19 challenges the development of anFGF19 therapeutic for chronic use. With the identification ofM70, an engineered FGF19 devoid of tumorigenicity withpotent metabolic properties, therapeutic benefits could beachieved without unwanted side effects. Our study opens newavenues for modulating the FGF19 pathway to treat cancer,diseases with bile acid dysregulation, type II diabetes, andother metabolic disorders.

Disclosure of Potential Conflicts of InterestThe authors have ownership interest (including patents) in NGM

Biopharmaceuticals.

Authors' ContributionsConception and design: X. Wang, D.A. Lindhout, K. Mondal, X. Ding, A.M.DePaoli, H. Tian, L. LingDevelopment of methodology: M. Zhou, X. Wang, D.A. Lindhout, J.-Y. Hsu,X. Ding, T. AroraAcquisition of data (provided animals, acquired and managedpatients, provided facilities, etc.): M. Zhou, X. Wang, V. Phung, K. Mondal,M. Humphrey, T. AroraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Zhou, X. Wang, V. Phung, K. Mondal, A.M.DePaoli, L. LingWriting, review, and/or revision of themanuscript:M. Zhou, X. Wang, D.A.Lindhout, T. Arora, R.M. Learned, A.M. DePaoli, L. LingAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): X. Wang, H. Yang, R.M. LearnedStudy supervision: X. Wang, R.M. Learned, H. Tian, L. LingPerformed experiments and reviewed the manuscript: X. Ding

AcknowledgmentsThe authors thank Jin-Long Chen and Thomas Parsons for advice and

insightful discussion, Suzanne Crawley, Sara Zong, and Hadas Galon-Tillemanfor technical support, Krishna Allemneni for coordinating histology and pathol-ogy assessments of tissue sections, and the NGMvivarium staff for the care of theanimals used in the studies.

Received January 27, 2014; revised March 26, 2014; accepted April 4, 2014;published OnlineFirst April 11, 2014.

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2014;74:3306-3316. Published OnlineFirst April 11, 2014.Cancer Res Mei Zhou, Xueyan Wang, Van Phung, et al. Endocrine Hormone FGF19Separating Tumorigenicity from Bile Acid Regulatory Activity for

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