TargetingFGFRPathwayinHumanHepatocellular Carcinoma ... · FGFR3 expression may contribute to...

Cancer Biology and Signal Transduction

Targeting FGFRPathway inHumanHepatocellularCarcinoma: Expressing pFGFR and pMET forAntitumor ActivityJae-Cheol Jo1,2,3, EunKyoungChoi1,2, Jae-Sik Shin1,2, Jai-HeeMoon1,2, Seung-WooHong1,2,Ha-Reum Lee1,2,4, Seung-Mi Kim1,2, Soo-A Jung1,2, Dae-Hee Lee1,2, Seang Hwan Jung1,2,Sun-Hye Lee1,5, Jeong Eun Kim1,2, Kyu-pyo Kim1,2, Yong Sang Hong1,2, Young-Ah Suh1,5,Se Jin Jang1,5, Eun Kyung Choi1,6, Jung Shin Lee1,2, Dong-Hoon Jin1,2,7, and TaeWon Kim1,2

Abstract

The MET receptor tyrosine kinase, the receptor for hepatocytegrowth factor (HGF), has been implicated in cancer growth,invasion, migration, angiogenesis, and metastasis in a broadvariety of human cancers, including human hepatocellularcarcinoma (HCC). Recently, MET was suggested to be a poten-tial target for the personalized treatment of HCC with an activeHGF–MET signaling pathway. However, the mechanisms ofresistance to MET inhibitors need to be elucidated to provideeffective treatment. Here, we show that HCC cells exhibitdifferent sensitivities to the MET inhibitor PHA665752,depending on the phosphorylation status of FGFR. Treatmentof cells expressing both phospho-FGFR and phospho-METwith the inhibitor PHA665752 did not cause growth inhibitionand cell death, whereas treatment with AZD4547, a pan-FGFRinhibitor, resulted in decreased colony formation and cleavage

of caspase-3. Moreover, silencing of endogenous FGFR1 andFGFR2 by RNAi of HCC cells expressing phospho-FGFR,phospho-FGFR2, and phospho-MET overcame the resistanceto PHA665752 treatment. Treatment of primary cancer cellsfrom patients with HCC expressing both phospho-FGFR andphospho-MET with PHA665752 did not induce cell death,whereas AZD4547 treatment induced cell death through thecleavage of caspase-3. In addition, treatment of cells resistant toPHA665752 with AZD4547 abrogated the activation of down-stream effectors of cell growth, proliferation, and survival. Onthe basis of these results, we conclude that the FGFR pathway iscritical for HCC survival, and that targeting this pathway withAZD4547 may be beneficial for the treatment of patients withHCC-expressing phospho-FGFR and phospho-MET. Mol CancerTher; 14(11); 2613–22. �2015 AACR.

IntroductionHuman hepatocellular carcinoma (HCC) is the third leading

cause of cancer-related deaths worldwide (1). The prognosis of

patients with HCC remains poor, with a 1-year survival rate lessthan 50%, despite recent improvements in overall survival (OS).Treatmentwith amolecular-targeted agent, themultikinase inhib-itor sorafenib, has improved the OS in patients with HCC, andsorafenib is the only targeted agent shown to prolong the OS inrandomized controlled trials (2, 3). Nevertheless, sorafenib hasdemonstrated modest efficacy in patients with HCC in clinicalpractice. Therefore, a better understanding of the cellularmechan-isms in the development and proliferation of HCC is essential.

Hepatocytegrowth factor (HGF)–induced activationof the recep-tor tyrosine kinase (RTK), MET, triggers a variety of cellularresponses, including proliferation, survival, cytoskeleton rearrange-ments, cell–cell dissociation, and motility (4). Although manystudies have demonstrated that MET overexpression is associatedwith poor outcomes in HCC (5), definitive evidence that METinhibition is an effective strategy for the treatment of HCC has notbeen established. However, You and colleagues (6) suggested thatMET is a potential target for the personalized treatment ofHCCwithactiveHGF/METsignaling, bydemonstrating that inhibitionofMETcan significantly inhibit the growth of MET-positive HCC tumors.

Preclinical data have demonstrated that FGFR-mediated sig-naling may play a role in the cellular mechanisms underlying thepathogenesis of HCC (7). The main FGFRs expressed in livertissues are FGFR3 and FGFR4, and several studies have suggestedthat these receptorsmay be involved inHCC tumorigenesis (8, 9).Moreover, the levels and activities of several FGFRs are known tobe altered inHCC. For example, somaticmutations of FGFR1have

1Innovative Cancer Research, ASAN Institute for Life Science, AsanMedical Center, Seoul, Republic of Korea. 2Department of Oncology,University of Ulsan College of Medicine, Asan Medical Center, Seoul,Republic of Korea. 3Department of Hematology and Oncology, UlsanUniversity Hospital, University of Ulsan College of Medicine, Ulsan,RepublicofKorea. 4DepartmentofLifeSciences,SookmyungWomen'sUniversity, Seoul, Republic of Korea. 5Department of Pathology, Uni-versity of Ulsan College of Medicine, Asan Medical Center, Seoul,Republic of Korea. 6Department of Radiation Oncology, University ofUlsan College of Medicine, Asan Medical Center, Seoul, Republic ofKorea. 7Department of Convergence Medicine, University of UlsanCollege of Medicine, Asan Medical Center, Seoul, Republic of Korea.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

J.-C. Jo, E.K. Choi, and J.-S. Shin contributed equally to this article.

Corresponding Authors: Tae Won Kim, Department of Oncology, University ofUlsanCollegeofMedicine, AsanMedical Center, 88Olympicro-43gil, Songpa-gu,Seoul, 138-736, Republic of Korea. Phone: 82-2-3010-3910; Fax: 82-2-3010-6961;E-mail: [email protected]; and Dong-Hoon Jin, Department of Conver-genceMedicine, University ofUlsanCollege ofMedicine, AsanMedical Center, 88Olympicro-43gil, Songpa-gu, Seoul, 138-736, Republic of Korea. Phone: 82-2-3010-2643; Fax: 82-2-3010-4665; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0780

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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been found in several types of cancers, including gliomas and lungtumors (10, 11). In the case of FGFR2, its mutations have beenfound in endometrial carcinomas and gastric cancers (12, 13),whereas those of FGFR3 or FGFR4 have been found in bladdercarcinomas, multiple myelomas (14–16), and primary rhabdo-myosarcomas (17).

Overexpression of the FGFR2 protein in tumors has beenassociated with advanced clinical stages (18). Another studyreported that FGFR2 overexpression in HCC tumors was asso-ciated with a high Ki-67 proliferation index and high levels ofserum alpha-fetoprotein, and was linked to the development ofmultiple sites of metastasis (19). In addition, overexpression ofFGFR3 has been associated with a higher grade and poorerdifferentiation status in HCC tumors, suggesting that increasedFGFR3 expression may contribute to advanced HCC tumori-genesis (20). Thus, FGFRs may act as potent regulators in avariety of cancers. A study evaluating clinical HCC samplesstrongly suggested that FGFR4 was important for the prolifer-ation and survival of HCC tumor cells, as well as for thesecretion of alpha-fetoprotein (21).

Although the biologic roles of the inter-RTK signaling networksare still unclear, FGFR and MET have been recently reported tointeract with each other to promote cell growth or acquiredresistance to specific inhibitors in gastric cancer cell lines (22).In the present study, we therefore evaluated the response of HCCcell lines and primary HCC cells to a MET inhibitor, to determinewhether inhibition of the FGFR pathway can overcome theresistance to MET inhibitors.

Materials and MethodsCell culture and reagents

All human liver carcinoma cells were purchased from the ATCCwhoauthenticated the identity of the cell lines, except for SNU449

and SNU475, which were obtained from the KCLB (Korean CellLine Bank, Seoul, Republic of Korea). All human liver carcinomacell lines were maintained in RPMI-1640 medium or DMEMmedium (GIBCO BRL) containing 10% FBS (GIBCO BRL), 100U penicillin, and 100 mg/mL streptomycin in a 5%CO2 incubatorat 37�C. PHA665752, a specific MET inhibitor, was purchasedfrom Selleck Chemicals LLC, and AZD4547, a pan-FGFR inhib-itor, was kindly provided by AstraZeneca. Co., Ltd.

Human primary hepatocellular carcinoma cell cultureSpecimens of human primary hepatocellular carcinoma cancer

tissues were obtained from patients after written informed con-sent and approval by the Research Ethics Board at Asan MedicalCenter. The study protocol followed the tenets of the Declarationof Helsinki. Tumor specimens were minced with scissors, anddigested by incubation in MEM (GIBCO BRL) containing 1 mg/mL collagenase (Sigma-Aldrich, St. Louis) for 2 hours at 37�C.Cells were washed with medium containing 10% FBS, followedby washing with PBS (pH 7.4) to remove FBS. Next, the cells wereplated in Human Hepatocyte Basal Medium (HBM; Lonza) andcultured in a humidified incubator under 5% CO2 at 37�C.

Cell viabilityCells (3 � 105 cells/plate) were seeded in 60-mm plates and

treated with various amounts of PHA665752 or AZD4547 for 48hours. Cell viability was determined using the trypan blue dyeexclusion method.

Clonogenic assaySNU449 and SK-Hep-1 cells, seeded at 3� 102 cells per well in

6-well plates, were treated with AZD4547 or PHA665752 at aconcentration of 0.01�2 mmol/L for 24 hours. The cells werecultured for 14 days. Colonies were fixed with 10% formalin and

Figure 1.Effects of MET or pan-FGFR inhibitor on HCC cell lines. A, expression levels of phospho-MET and phospho-FGFR were evaluated by Western blotting of extractsof HCC cell lines with anti-pMET or pFGFR antibodies. b-Actin was used as a loading control. B, various HCC cell lines were seeded in 96-well plates andtreatedwith aMET inhibitor (PHA665752) or a pan-FGFR inhibitor (AZD4547). After incubation for 72 hours, MTS assayswere performed. C, differential effects of theMET inhibitor (PHA665752) or pan-FGFR inhibitor (AZD4547) on SNU449 and SK-HEP-1 cells. SNU449 (i) and SK-HEP-1 (ii) cells were treated with differentconcentrations of PHA665752 or AZD4547 for 48 hours. Cell death was determined using the trypan blue dye exclusion assay. Cells were harvested andanalyzed by Western blotting using antibody against cleaved caspase-3. b-Actin was used as a loading control. The graphs present the mean � SD of threeseparate experiments performed in triplicate; � , P < 0.05; �� , P < 0.01. D, colony formation assays were performed using SNU449 and SK-HEP-1 cell linestreated with the indicated concentrations of PHA665752 or AZD4547 (top). Data, mean � SD. Representative images are shown (bottom); � , P < 0.05.

Figure 2.Differential inhibition of downstreamsignaling molecules of HCC cell linestreated with MET or the pan-FGFRinhibitor. SNU449 (A) and SK-HEP-1 (B)cells were treated with MET or thepan-FGFR inhibitor at the indicatedconcentrations for 48 hours. Cell lysateswere analyzed by Western blottingusing antibodies against phospho-MET,MET, phospho-FRS2, phospho-AKT,AKT, phospho-ERK, and ERK. b-Actinwas used as a loading control.

AZD4547 Can Overcome Resistance to MET Inhibitor

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stained with 0.01% crystal violet dye solution. The numbers ofcolony-forming cells with a diameter �1 mm were counted.

DNA, siRNA, and transfectionMET, FGFR-1, and FGFR-2 cDNAs were purchased from Ori-

gene. The mutated MET T1191I DNA was generated using a PCR-based quick change site-directed mutagenesis kit (Intron Biotech-nology) and wild-type MET. All DNA transfections were per-formed using Lipofectamine 2000 reagent (Invitrogen) accordingto the manufacturer's instructions. For siRNA transfection, cellswere transfected using Lipofectamine RNAi MAX reagent (Invi-trogen) according to the manufacturer's protocol. The siRNAsequences for the transfection study were the following:FGFR1-siRNA, 50-GAA GUG CAU ACA CCG AGA C-30; FGFR2-siRNA, 50-AAG UGC UGG CUC UGU UCA AUG-30; FGFR3-siRNA, 50-AGA CGA TGCCAC TGA CAAG-30; and FGFR4-siRNA,50-AUA CGG ACA UCA UCC UGU A-30.

Immunoblot and immunoprecipitationTotal cellular proteins (20mg)were subjected to SDS-PAGE and

transferred to polyvinylidene difluoride membranes. The mem-branes were blocked with 5% non-fat dry milk in TBS-T buffer(20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 0.1%Tween 20) and incubated with the following antibodies: anti-Met, anti-FGFR2, and anti–b-actin antibodies (Santa Cruz Bio-technology); anti–phospho-Met (Y1234/1235), anti–phospho-FGFR (pan-pFGFR), anti–phospho-FRS2 (pFRS2), anti–phos-pho-AKT, anti–phospho-ERK, anti–phospho-tyrosine, anti-AKT,anti-ERK, anti-cleaved caspase 3 antibodies (Cell Signaling Tech-nology); anti-FGFR1 and anti-AFP antibodies (Abcam); and anti-FGFR3 and anti-FGFR4 (Abnova Corporation) antibodies. Pri-mary antibodies were detected with horseradish peroxidase–con-jugated goat anti-mouse or goat anti-rabbit secondary antibody(Cell Signaling Technology), as appropriate, and immunoreactiveproteins were visualized using an enhanced ECL detection reagent(GE Healthcare Bio-Sciences).

For immunoprecipitation analyses, cell lysates were immu-noprecipitated with anti-FGFR1, anti-FGFR2, anti-FGFR3, or

anti-FGFR4 antibodies for 12 hours at 4�C. Protein A/G Plus-Sepharose beads (20 mL; Santa Cruz Biotechnology) were thenadded, and the mixtures were incubated for an additional2 hours at 4�C. The immunoprecipitates were washed withNonidet P-40 lysis buffer, boiled in 2� SDS sample buffer, andanalyzed by Western blot analysis using anti–phospho-tyrosineantibody.

Immunofluorescence analysisHuman primary hepatocellular carcinoma cells were seeded on

glass coverslips. The cells werefixed in 4%paraformaldehyde, andpermeabilized-blocked, then incubated with primary antibodies.Following washing, the cells were incubated with Alexa488- orAlexa594-labeled secondary antibodies, and the cells were stainedbriefly with DAPI. After washing, the coverslips were mounted onglass slides using mounting solution. The slides were analyzed byconfocal microscopy (Carl Zeiss).

ImmunohistochemistryThe pMET and pFGFR in tumor tissues and tissue microarrays

(TMAs) were detected using immunohistochemistry (IHC). TheHCC TMAs were purchased from US Biomax, Inc. IHC wasperformed according to the manufacturer's protocol. Antigenretrieval was performed by heating the samples in citrate buffer(pH 6.0) in a microwave. The reaction products were developedwith diaminobenzidine (Dako), followed by counterstainingwith hematoxylin.

Statistical analysisAll datawere analyzed using two-tailed Student t tests. The level

of statistical significance was set at P < 0.05.

ResultsHuman hepatocellular carcinoma cancer cells expressresistance to a MET inhibitor dependent on thephosphorylation status of FGFR

Itwas recently reported that FGFR-expressing gastric cancer cellsare sensitive to a MET inhibitor (22), implying that some

Figure 3.Phosphorylation of FGFR1 and FGFR2mediates the resistance of the METinhibitor in human HCC cells. A,SNU449 and SK-HEP-1 cells wereharvested and immunoprecipitatedwith antibodies against FGFR1,FGFR2, FGFR3, and FGFR4. Theimmunoprecipitates were subjected toWestern blot analysis using a phospho-tyrosine antibody. The MKN45, KatoIII,HT29, and Huh7 cell lines were used aspositive controls for each antibody. B,SNU449 (i) and SK-HEP-1 (ii) cells weretreatedwith MET or pan-FGFR inhibitorand immunoprecipitated withantibodies directed against FGFR1 orFGFR2. The immunoprecipitates weresubjected to Western blot analysisusing a phospho-tyrosine antibody.

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association exists betweenMETand FGFRagainst the sensitivity tothe MET inhibitor in a variety of cancer cells. We thereforeinvestigated the dependence of the inhibitory effects of a METinhibitor on the status of FGFR in human hepatocellular carci-noma cells. First, we examined the phosphorylation status ofMETand FGFR, including the expression levels of both proteins inmultiple human hepatocellular carcinoma cells. Two of them, SK-Hep-1 and SNU449 cells, expressed both phospho-MET andphospho-FGFR (Fig. 1A). We examined the inhibitory effects oftreatment with a MET inhibitor (PHA665752) or a pan-FGFR

inhibitor (AZD4547) in various humanhepatocellular carcinomacells. The results demonstrated that SNU449 and SK-Hep-1 cellswere sensitive to AZD4547, exhibiting IC50 values of 82 and 84nmol/L, respectively. By contrast, these cell lines were resistant toPHA665752.Other cell lineswere resistant to the two compounds(Fig. 1B). On the basis of the IC50 data, we determined theconcentration of AZD4547, and in parallel, determined the con-centration of PHA665752. These human hepatocellular carcino-ma cells were selected to determine whether the differences insensitivities to MET inhibitor were dependent on the presence of

Figure 4.Silencing of FGFR1 or FGFR2 overcomes resistance to MET inhibitor. SNU449 (A) and SK-HEP-1 (B) cells were transfected with siRNA directed againstFGFR1 or scrambled. siRNA (sc) for 48 hours. The transfected cells were treated with the indicated concentrations of PHA665752. Cell death was evaluatedusing the trypan blue dye exclusion assay. Cells were harvested and analyzed byWestern blotting using antibodies directed against phospho-MET, phospho-FRS2,and cleaved caspase-3; b-actin was used as loading control. SNU449 (C) and SK-HEP-1 (D) cells were transfected with scrambled siRNA or FGFR2-specificsiRNA for 24 hours, and then treatedwith PHA665752 at the indicated concentrations for an additional 48 hours. Cell deathwas determined using the trypan blue dyeexclusion assay. Cells were harvested, and immunoblot analyses were performed to determine the expression of phospho-MET, phospho-FRS2, and cleavedcaspase-3; b-actin was used as loading control. Error bars indicate the SD of three individual experiments; � , P < 0.05; �� , P < 0.01. SNU449 (E) and SK-Hep1 (F) cellswere transfected with FGFR1 siRNA and/or FGFR2 siRNA for 24 hours and treated with the specific MET inhibitor PHA665752 (2 mmol/L) for 48 hours. Celldeathwas determined using the trypan blue dye exclusionmethod. Cell lysateswere prepared forWestern blot analysis using antibodies directed against FGFR1 andFGFR2, phospho-FRS2, and cleaved caspase-3; b-actin was used as loading control. Graph, mean � SD; �� , P < 0.01.






phospho-FGFR, and were therefore treated with various doses ofeach inhibitor that targets MET or FGFR. The results showed thatcell growth was inhibited by AZD4547 at lower concentrations,and cell death was induced at higher concentrations (>1 mmol/L; Fig. 1C and Supplementary Fig. S1). Notably, there was almostno change in the population of dead cells in response toPHA665752, a selective MET inhibitor, whereas the populationof dead cells after the treatment with AZD4547, a pan-FGFRinhibitor, dramatically increased (Fig. 1Ci). AZD4547 alsoinduced the cleavage of pro–caspase-3 to yield active caspase-3,but not in cells treated with PHA665752 (Fig. 1Cii). To analyzethe effects of AZD4547 or PHA665752 on caspase-3 cleavage, werepeated the Western blot analysis. AZD4547 treatment at 1 or 2mmol/L led to caspase-3 cleavage (Fig. 1C, bottom). To furthercharacterize the inhibitory effects of each inhibitor, AZD4547 orPHA665752, on cells that express both phospho-MET and phos-pho-FGFR, we performed colony-forming assays. Colony forma-tion in SNU449 and SK-Hep1 cells was significantly reduced byAZD4547, whereas a decrease in colony formation was notobserved with PHA665752 in both cell lines (Fig. 1D). Takentogether, the results indicate that differences in sensitivity to METinhibitor are dependent on the phosphorylation status of FGFR inhuman hepatocarcinoma cells.

MET inhibitor does not inhibit downstream signaling inhepatocarcinoma cells expressing pMET and pFGFR

As shown in Fig. 1, SNU449 and SK-Hep1 cells were insen-sitive to PHA665752, a selective MET inhibitor, whereas

both cell lines were sensitive to AZD4547, a pan-FGFR inhib-itor. Based upon these results, we investigated the effectsof each inhibitor on their downstream signaling pathways.Treatment with AZD4547 caused a decrease of FRS2 (FGFRsubstrate 2), AKT, and ERK phosphorylation in SNU449 cells.However, phosphorylation of these proteins did not decreaseafter exposure to PHA665752 (Fig. 2A). AZD4547 also blockedthe phosphorylation of FRS2, AKT, and ERK in SK-Hep1 cells,whereas PHA665752 did not (Fig. 2B). These results indicatedthat inhibition of FGFR inhibits the phosphorylation of thedownstream signaling molecules FRS2, AKT, and ERK in hep-atoma cells expressing pMET and pFGFR.

FGFR inhibitor, but not MET inhibitor, causes inhibition ofFGFR1 or FGFR2 phosphorylation in human hepatocarcinomacells

We examined the phosphorylation status of FGFR1, FGFR2,FGFR3, and FGFR4 in SNU449 and SK-Hep1 cells that expressboth pMET and pFGFR. Notably, phosphorylation of FGFR1 andFGFR2 was observed in both cell types, whereas FGFR3 andFGFR4 phosphorylation was not detected (Fig. 3A), implyingthat phospho-FGFR1 and FGFR2 may affect sensitivity to theMET inhibitor. We next examined the inhibition of FGFR1 andFGFR2 phosphorylation after treatment of these cells withAZD4547 or PHA665752 using an immunoprecipitation assay.AZD4547 treatment led to decreases in both p-FGFR1 and FGFR2in the two selected cell lines, but PHA665752 treatment did notinduce a decrease (Fig. 3B).

Figure 5.Effect of the expression of MET andFGFR1 or FGFR2on the sensitivity of theMET inhibitor in p-FGFR and p-METdouble-negative human HCC cells. A,PLC/PRF5 cells were transfected withFGFR1 and MET T1191I-expressingplasmids for 24 hours and treated withbFGF (basic fibroblast growth factor) at20 ng/mL and AZD4547, a pan-FGFRinhibitor, or PHA665752, a specific METinhibitor, at the indicatedconcentrationsfor another 48 hours. Cell death wasdetermined using the trypan blue dyeexclusion method. The column anderror bars represent the mean � SD;�� , P < 0.001. Cell lysates wereprepared for Western blot analysesusing antibodies directed againstFGFR1, phospho-FRS2, phospho-MET,and cleaved caspase-3; b-actin wasused as loading control. B, cells werecotransfected with FGFR2 and METT1191I constructs for 48 hours, treatedwith bFGF at 20 ng/mL, and AZD4547or PHA665752 at the indicatedconcentrations for an additional 48hours. The population of dead cells wasevaluated by the trypan blue exclusionassay. The column and error bars, mean� SD; �� , P < 0.01. Cell lysates wereprepared for Western blot analysesusing antibodies directed againstFGFR2, phospho-FRS2, phospho-MET,and cleaved caspase-3; b-actin wasused as loading control.

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Silencing of FGFR1 or FGFR2 overcomes resistance to the METinhibitor

We next investigated the effects of members of the FGFRfamily on the sensitivity to MET inhibitor. We analyzed thedependence of FGFR1 or FGFR2 on the sensitivity to METinhibitor using constructs expressing FGFR1 or FGFR2 siRNA.Initially, SNU449 cells were transfected with FGFR1-siRNA orscrambled siRNA, followed by PHA665752 treatment. Thedead cell population was significantly increased in FGFR1-siRNA–treated cells following PHA665752 treatment, but notin scrambled siRNA-treated cells (Fig. 4Ai). In addition, thecleavage of caspase-3 was significantly increased in FGFR1-siRNA–treated cells following PHA665752 treatment, and thephosphorylation level of FRS2 was decreased after exposure toPHA665752 (Fig. 4Aii). The results showed that silencing ofFGFR1 in SK-Hep1 cells increases cell death (Fig. 4B), indicat-ing that inhibition of FGFR1 can overcome the resistance toMET inhibitor.

As shown in Fig. 4A and B, SNU449 and SK-Hep1 cellsexpressed phospho-FGFR2 as well as phospho-FGFR1. Thus, weinvestigated the effect of depletion of FGFR2 by siRNA on thesensitivity to the MET inhibitor. Knockdown of FGFR2 led toincreased cell death in SNU449 (Fig. 4Ci) and SK-Hep1 cells (Fig.4Di) in response to PHA665752, but not in scrambled siRNA-treated cells, as seenwith FGFR1-siRNA. The cleavage of caspase-3also increased in FGFR2-siRNA–treated cells (Fig. 4Cii and Dii),indicating that inhibitionof FGFR2 canovercome the resistance toMET inhibitor. To further confirm these conclusions, we cotrans-fected cells that coexpressed pMET and pFGFR with FGFR1 and 2siRNAs. Cell death was significantly higher in cells cotransfectedwith FGFR1and2 siRNAs than in cells transfectedwith FGFR1or 2siRNAs (Fig. 4Ei and Fi). Double knockdown also significantlyincreased the cleaved form of caspase-3 compared with singleknockdown (Fig. 4Eii and Fii). We next examined the effects ofFGFR3 and FGFR4 knockdown on the sensitivity to a METinhibitor. Silencing FGFR3 and FGFR4 did not increase cell deathin the absence of PHA665752 (Supplementary Fig. S2), indicatingthat FGFR3 and FGFR4donot affect sensitivity to aMET inhibitor.In addition, MET inhibition induced cell death in the absence ofAZD4547 (Supplementary Fig. S3). These results indicate thatFGFR1 and 2 silencing enhances MET inhibitor sensitivity inhuman hepatocarcinoma cells.

FGFR1 or FGFR 2 confers resistance to the MET inhibitorTo further characterize the dependence of FGFR1 or FGFR2 on

the sensitivity to the MET inhibitor, we cotransfected PLC/PRF5cells that did not express pMET and pFGFR, with a constructexpressing FGFR1 cDNA and a MET CA (constitutively active)mutant or with FGFR2 cDNA and a MET CAmutant, followed bytreatment with PHA665752 or AZD4547. There was almost noinduction of cell death induced by PHA665752 in cells expressingFGFR1 cDNA and a MET CA mutant, whereas treatment ofAZD4547 led to increased cell death (Fig. 5Ai). Similarly, thedead cell population in cells expressing FGFR2 cDNA and a METCA mutant was significantly increased by AZD4547, but not byPHA665752 (Fig. 5Bii). In addition, caspase-3 cleavage wassignificantly increased in cells expressing FGFR1 or FGFR2 cDNAand a MET CAmutant following AZD4547, but not PHA665752,treatment (Fig. 5Ai and Bii). Moreover, c-MET altered the growthof these cells (Supplementary Fig. S4). Together, these resultsindicate that FGFR1 and FGFR2 activities are associated with

resistance to the MET inhibitor in human HCC cells expressingpMET and pFGFR.

AZD4547 induces cell death in human primaryhepatocarcinoma cells expressing pMET and pFGFR

On the basis of the above results, we examined the effect of eachinhibitor, PHA665752 or AZD4547, on primary cancer cells frompatients with hepatocarcinomas. We first analyzed the phosphor-ylation status of MET or FGFR. Two of 14 primary HCC cells,78019A and 3226B (patient numbers), coexpressed pMET andpFGFR (Fig. 6Ai). We then performed immunofluorescence anal-ysis on primary HCC cells. The 78019A and 3226B cells expressedpMET, pFGFR, and AFP (Fig. 6Aii). Human primary HCC cells,which include stromal cells and tumor cells, were obtained fromHCC patient tissues. We analyzed the drug sensitivity of cells inthe absence or presence of Hep-Par-1 as a HCC marker. Approx-imately 18% of human primary HCC cells were found to be Hep-Par-1 positive. AZD4547 treatment led to a dose-dependentcaspase-3 cleavage in Hep-Par-1–positive cells, but not in Hep-Par-1–negative cells. The Hep-Par-1–negative cells were almostalways stromal cells, with no induction of cell death by treatmentwith AZD4547. These results were confirmed by immunofluo-rescence analysis, showing that Hep-Par-1–positive cells dis-played sensitivity to AZD4547 at 1, 2, and 5 mmol/L (Fig. 6B andC). In addition, AZD4547 induced a decrease of pFRS2, but notpMET, in 3226B and 78019A cells (Fig. 6D and E), indicating thatAZD4547 can induce cell death in human primary hepatocarci-noma cells expressing pMET and pFGFR.

Next, we examined the inhibitory effect of each inhibitoron human primary hepatocarcinoma cells expressing pMETalone. PHA665752 induced a decrease of pMET, indicatingthat the MET inhibitor can act on human primary hepatocarci-noma cells expressing pMET alone (Fig. 6F and G). However,the concentration at which significant induction of caspase-3cleavage was observed may not be clinically achievable withAZD4547.

DiscussionThe RTK,MET, that controls cell growth, proliferation, survival,

and motility, is constitutively activated and amplified in severalcancer types, including non–small cell lung cancers, hepatocarci-nomas, and gastric cancers (23). Thus, MET is considered aclinically important target for therapies against these types ofcancers. Agents targeting MET have been evaluated and haverecently entered clinical trials. Major problems of therapies target-ing tyrosine kinases involve the lack of response to treatment ofmany tumors, and/or the drug resistance that eventually develops.In the present study, we show that AZD4547, a pan-FGFR inhib-itor, can overcome the resistance to MET inhibitor in humanhepatocarcinoma cells.

It was recently reported that activation of RTK in gastric cancercells mediates resistance to MET inhibition, implicating thatsome association exists between RTK and MET during the induc-tion of drug resistance. Specifically, Kataoka and colleagues (22)reported that TKI appears to be more effective against humangastric cancer, by blocking inter-RTK signaling networks thatdepend on MET or FGFR2. Foretinib (GSK1363089), a multi-kinase inhibitor of MET and VEGFRs, shows different sensitiv-ities that are dependent on FGFR expression (22). The FGFRinhibitor that has been most extensively studied in patients with







Jo et al.




HCC is brivanib, an ATP-competitive dual inhibitor of VEGFRand FGFR1–3 (24). Despite strong preclinical and phase II dataof HCC (25, 26), brivanib failed in large randomized phase IIItrials in both the first- and second-line settings for patients withadvanced HCC (27, 28). Nonetheless, FGFR inhibition remainsan attractive therapeutic target for HCC, and requires furtherinvestigation of its possible clinical applications. Efforts con-tinue to explore the use of multikinase inhibitors that alsotarget FGFR in patients with advanced HCC, and ongoing first-line trials include a randomized phase III study of lenvatinibcompared with sorafenib, a randomized phase II trial of dovi-tinib compared with sorafenib in the Asia-Pacific region, and aphase I/II trial of nintedanib in combination with sorafenib.Conversely, our findings have shown that a pan-FGFR inhib-itor, AZD4547, appears to be more effective than a selectiveMET inhibitor, PHA665752, against HCC-expressing phospho-MET and phospho-FGFR. Thus, the inhibition of MET inhepatocarcinoma cells expressing both pMET and pFGFR hasnot been effective, unlike that in human gastric cancer cells.Although the most robust effects on inducing cell death in HCCcell lines were seen at concentrations at or above 1 mmol/L,which might be above clinically achievable concentrations ofAZD4547, significant effects were also seen at lower concentra-tions. Similarly, the expression of downstream signaling mole-cules decreased after exposure to AZD4547 at concentrationsabove 50 nmol/L.

Based upon these results, we confirmed that an associationexists between pMET and pFGFR, using primary cancer cellsfrom patients with hepatocarcinoma. Interestingly, two out of14 primary cancer cells, 78019A and 3226B, expressed bothpMET and pFGFR. According to some reports, MET activation isrelevant to hepatitis B virus infection in hepatocarcinoma.Hepatitis B virus X protein increases invasiveness via the pro-moter activity of MET (29). In addition, hepatitis B and C virus–infected mouse models highly express HGF (30). However, theinfectious status of these viruses might not be related to FGFRand MET signaling activation in the 14 primary HCCs (Sup-plementary Table S1). Consistent with the results from cancercell lines, treatment of primary cancer cells that express pMETand pFGFR with PHA665752 as a MET inhibitor was noteffective, whereas AZD4547 was more effective against bothprimary cancer cells, implying that FGFR kinase is more impor-tant than MET kinase for hepatocarcinoma cell survival (Fig. 6).Thus, FGFR kinase may serve as a potent target of severaltherapeutic strategies for human HCC. We therefore investigat-ed the expressions of pFGFR and pMET in other primary HCCcell lines.

One limitation of this study is that it was based on a smallsample size of primary HCC samples. The subset of HCC samples

with both FGFR and Met signaling activation likely comprises asmall proportion of the total HCC population. We analyzed theexpression of pFGFR and pMET in a TMAby IHC. TMA slides werepurchased from US Biomax (the donors were from China). Nineof 53 TMA cores (16%) were double positive for the expression ofpFGFR and pMET (Supplementary Fig. S5). In addition, otherstudies reported that MET amplification is 27% (31) and FGFRamplification is 31% (32) in HCC. Given the ethnic backgroundof the donors, we expect similar results in Korean patients, but itwould be helpful to confirm this possibility using a larger cohort.Nonetheless, our results provide the first evidence regarding therelationship of pMET and pFGFR in HCC.

In conclusion,wehave evaluated the effects of AZD4547, a pan-FGFR inhibitor dependent on MET activity/phosphorylation, inhuman hepatocarcinoma cells. Given the increased effects oninducing HCC cell death at higher concentrations, it is alsopossible that other FGFR inhibitors, which can be used at highertolerable doses, even for a shorter time than used with AZD4547,might be more efficacious in such patients. The success of per-sonalized or precision medicine using targeted therapies, basedon the particular genotype of cancer, depends on the ability toidentify patients who will benefit from specific drugs. Thus, ourfindings may provide the rationale for clinical development ofFGFR inhibitors, such as AZD4547, for the treatment of selectedpatient populations with hepatocarcinoma that express phospho-MET and phospho-FGFR.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.-C. Jo, K.-p. Kim, Y.-A. Suh, D.-H. Jin, T.W. KimDevelopment ofmethodology: J.-C. Jo, J.-S. Shin, Y.-A. Suh, D.-H. Jin, T.W. KimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-C. Jo, E.K. Choi, J.-S. Shin, S.-W. Hong, S.J. Jang,J.S. Lee, D.-H. Jin, T.W. KimAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-C. Jo, E.K. Choi, J.-S. Shin, J.-H.Moon, S.-W. Hong,H.-R. Lee, S.-A. Jung, D.-H. Lee, J.E. Kim, E.K. Choi, J.S. Lee, D.-H. Jin, T.W. KimWriting, review, and/or revision of the manuscript: J.-C. Jo, J.-S. Shin,J.-H. Moon, E.K. Choi, D.-H. Jin, T.W. KimAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.K. Choi, J.-H. Moon, S.-M. Kim, S.-A. Jung,D.-H. Lee, S.H. Jung, S.-H. Lee, Y.S. Hong, Y.-A. Suh, S.J. Jang, E.K. Choi, J.S. Lee,D.-H. Jin, T.W. KimStudy supervision: J.E. Kim, Y.-A. Suh, D.-H. Jin, T.W. Kim

AcknowledgmentsThe use of AZD4547, a pan-FGFR inhibitor, was supported by AstraZeneca

through a research material transfer agreement between AstraZeneca and theASAN Institute for Life Sciences.

Figure 6.Effects of MET or pan-FGFR inhibitor on primary cancer cells from patients with hepatocarcinoma. A, i, levels of expression of phospho-MET and phospho-FGFR were evaluated by Western blotting of primary HCC cells; b-actin was used as loading control.; A, ii, immunofluorescence analysis of primary HCC cells.78019A and 3226B cells expressed pMET, pFGFR, and AFP (B and C). Differential effects of the MET inhibitor (PHA665752) or pan-FGFR inhibitor (AZD4547)on 3226B (B) and 78019A (C) cells were determined by immunofluorescence analysis. The 3226B and 78019A cells expressed Hep-par-1 and cleaved caspase-3after AZD4547 treatment at the indicated concentrations as shown by immunofluorescence (D and E). Primary HCC cells 78019A and 3226B were treatedwith various concentrations of PHA665752 or AZD4547. These cells expressed both phospho-FGFR and phospho-MET. Cells were harvested and analyzed byWestern blotting using antibodies directed against phospho-MET and phospho-FRS2; b-actin was used as loading control. 34582 (F) and 39214 (G) cellsexpressed only phospho-MET and not phospho-FRS2. The cells were treated with PHA665752 or AZD4547 at the indicated concentrations. Cells were harvestedand analyzed by Western blotting using antibodies directed against phospho-MET and cleaved caspase-3; b-actin was used as loading control.






Grant SupportThis study was supported by grants from the Korea Health 21 R&D Project,

Ministry of Health and Welfare and Family Affairs, Republic of Korea(HI06C0868; to T.W. Kim), a National Research Foundation of Korea (NRF)grant funded by the Korean government (MEST; 2013R1A2A2A01067394; toD.H. Jin), and the Asan Institute for Life Sciences, Seoul, Republic of Korea(2014-622; T.W. Kim).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 11, 2014; revised July 13, 2015; accepted August 24,2015; published OnlineFirst September 8, 2015.

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Jo et al.




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