Fibroblast Growth Factor Receptor 3 Is a Rational ... · Large Molecule Therapeutics Fibroblast...

Large Molecule Therapeutics

Fibroblast Growth Factor Receptor 3 Is a RationalTherapeutic Target in Bladder Cancer

Kilian M. Gust1, David J. McConkey2, Shannon Awrey1, Paul K. Hegarty2, Jing Qing3, Jolanta Bondaruk2,Avi Ashkenazi3, Bogdan Czerniak2, Colin P. Dinney2, and Peter C. Black1

AbstractActivating mutations of fibroblast growth factor receptor-3 (FGFR3) have been described in approximately

75% of low-grade papillary bladder tumors. In muscle-invasive disease, FGFR3mutations are found in 20% of

tumors, but overexpression of FGFR3 is observed in about half of cases. Therefore, FGFR3 is a particularly

promising target for therapy in bladder cancer. Up to now,most drugs tested for inhibition of FGFR3have been

small molecule, multityrosine kinase inhibitors. More recently, a specific inhibitory monoclonal antibody

targeting FGFR3 (R3Mab) has been described and tested preclinically. In this study, we have evaluated

mutation and expression status of FGFR3 in 19urothelial cancer cell lines and a cohort of 170American patients

with bladder cancer. We have shown inhibitory activity of R3Mab on tumor growth and corresponding cell

signaling in three different orthotopic xenografts of bladder cancer. Our results provide the preclinical proof of

principle necessary to translate FGFR3 inhibitionwithR3Mab into clinical trials inpatientswithbladder cancer.

Mol Cancer Ther; 12(7); 1245–54. �2013 AACR.

IntroductionCancer of the bladder will be newly diagnosed in an

estimated 73,510 individuals and will cause 14,880 cancerrelated deaths in 2012 in the United States (1). This makesit the fourth most common cancer in men and the ninthmost common in women. About 70% of cases are non-muscle invasive and have a high propensity to recur,giving bladder cancer the highest recurrence rate of anycancer (2). This translates clinically into intensive surveil-lance and multiple interventions over many years,explaining the high cost of managing bladder cancer(3). The other 30% of cases are muscle invasive tumors,for which the 5-year survival is approximately 50%. Bothdisease entities are marked by a paucity of new discov-eries leading to novel therapies with clinical utility.Fibroblast growth factor receptor-3 (FGFR3) is a partic-

ularly promising target for therapy in bladder cancer.Activating FGFR3 mutations have been described inapproximately 75% of low-grade papillary bladdertumors (4). Mutations are found in only 20% of muscleinvasive tumors, but FGFR3overexpression is observed in

50% of these tumors (5, 6). The most common mutation(S249C), located on exon 7, results in the substitution ofserine at codon 249 with a cysteine residue (7, 8). Thisleads to constitutive ligand-independent dimerization ofthe receptor and activation of downstream proliferativepathways (9, 10).

Recently, a number of small molecule receptor tyro-sine kinase inhibitors have been developed to targetFGFR3 (11). These small molecule inhibitors, includingBGJ398 (Novartis), TKI258/CHIR258 (Dovitinib, Novar-tis; refs. 12–14), AZD4547 (Astra Zeneca) PD173074 (Pfi-zer), and BMS-582664 (Brivanib, Bristol–Myers Squibb;ref. 15), generally target all members of the FGFR family,vascular endothelial growth factor receptor 2 (VEGFR2),and other tyrosine kinases (11, 12). This general FGF andVEGF pathway inhibition is associated with significantdiarrhea, nausea, fatigue (16, 17), dyspnea (18), andabdominal pain (16–18), as well as thrombocytopenia andthromboembolic events (16). Hyperphosphatemia-medi-ated soft tissue calcification has been a further hurdleimpeding preclinical development of pan-FGFR inhibi-tors (11). The FGFR pathway is involved in normal phos-phate and vitamin D homeostasis, particularly throughFGF23 signaling in thebone andkidney (19).Disruption ofthis pathway with pan-FGFR inhibitors leads to hyper-phosphatemia and deposit of calcium phosphorous in thesoft tissues, including the vasculature, smooth muscle,and renal tubules. These adverse effects may be avoidedwith specific FGFR3 inhibition.

The clinical development of these inhibitors has focusedon multiple myeloma, which harbors specific transloca-tions [t(4;14)(p16;q32)] that activate FGFR3 (20), and onhepatocellular carcinoma (12), but little work has been

Authors' Affiliations: 1Vancouver Prostate Centre, Department of Uro-logic Sciences, University of British Columbia, Vancouver, British Colum-bia, Canada; 2The University of Texas MD Anderson Cancer Center,Houston, Texas; and 3Department of Molecular Oncology, Genentech Inc.,South San Francisco, California

Corresponding Author: Peter C. Black, Vancouver Prostate Centre,Department of Urologic Sciences, University of British Columbia, 2660Oak Street, Vancouver, BC V6H 3Z6, Canada. Phone: 604-875-4818; Fax:604-875-5654; E-mail [email protected]

doi: 10.1158/1535-7163.MCT-12-1150

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on November 16, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 8, 2013; DOI: 10.1158/1535-7163.MCT-12-1150

http://mct.aacrjournals.org/

done inbladdercancer.Most recently, an inhibitorymono-clonal antibody targeting FGFR3 specifically (R3Mab) hasbeen developed and undergone preliminary testing inpreclinical subcutaneousmodels of bladder cancer,whereit has shown activity on both wild-type and mutatedFGFR3 (21). In this study, we have focused on specificFGFR3 inhibition with R3Mab in an orthotopic bladdercancer model. The orthotopic model provides an appro-priate organ-specificmicroenvironment that should allowbetter translation into clinical trials.

Therefore, we aimed to show the preclinical proof ofprinciple that targeting FGFR3 with R3Mab is rationalin bladder cancer. We have measured FGFR3 mutationand expression in an American cohort of patients withbladder cancer and have shown R3Mab activity in anorthotopic xenograft model of bladder cancer. Webelieve that this justifies the translation of FGFR3 inhi-bition with R3Mab into clinical trials in patients withbladder cancer.

Materials and MethodsCell lines

Nineteen human bladder cancer cell lines were kindlyprovided by the Pathology Core of the Bladder CancerSPORE at MD Anderson Cancer Center, including UM-UC1, UM-UC3, UM-UC4, UM-UC5, UM-UC6, UM-UC7,UM-UC10, UM-UC11, UM-UC12, UM-UC13, UM-UC14,UM-UC15,UM-UC16,UM-UC17, 253J-P, 253J-BV, RT4v6,and RT112. T24 was purchased from the American TypeCulture Collection. All cell lines were cultured in Mini-mum Essential Medium (MEM), supplemented with 10%fetal bovine serum (FBS), 1%nonessential amino acids, 1%L-glutamine, and 1% sodium pyruvate (all purchasedfrom Invitrogen). The cell lines were routinely grown andpassaged at 37�C in a humidified atmosphere of 5% CO2.Cell line identitieswere confirmedbyDNAfingerprintingusing the AmpFlSTR Identifiler Amplification Kit(Applied Biosystems) or the AmpFlSTR Profiler PCRAmplification Kit (Applied Biosystems) protocols. Thecell lines were monitored regularly for mycoplasmainfection.

For in vivo studies, UM-UC1, UM-UC14, and RT112 celllines underwent transduction with a lentiviral constructcarrying the luciferase firefly gene for in vivo imaging. Theluciferase plasmid contained a blasticidin-resistance geneenabling positive selection with 10 mg/mL blasticidin(Invitrogen). Cell lines were controlled for in vitro lucif-erase activity and cell number was correlated with bio-luminescence (R > 0.99; data not shown), using the Xeno-gen IVIS Spectrum (Caliper Lifesciences). These 3 celllines were chosen due to their high levels of FGFR3expression and their growth characteristics in the ortho-topic xenograft model.

AntibodiesFGFR3 polyclonal (sc-123), FRS2a (sc-8318), cyclin D1

(sc-718), cyclin E (sc-481), and vimentin (sc-5565) antibo-dies were purchased from Santa Cruz Biotechnologies.

Antibodies detecting p-FGFR (#3471), p-FRS2 (#3864),p42/44MAPK (mitogen-activated protein kinase; #4965),phospho-p42/44-MAPK (#4370), AKT (#9272), phosphor-AKT (ser 473; # 9271S), ZEB-1 (Zinc finger E-box-bindinghomeobox 1; #3396), and cleaved caspase-3 (#9661) werepurchased from Cell Signaling. Antiphospho-tyrosine4G10 was obtained from EMD Millipore and E-cadherinantibody (#610181) was obtained from BD Biosciences. Amonoclonal Ki-67 antibody for evaluation of proliferationof xenograft tumors was purchased from Thermo Scien-tific. For expressionanalysisof FGFR3on thehuman tissuemicroarray (TMA), a polyclonal FGFR3 antibody (F0425)was obtained from Sigma-Aldrich.

ImmunohistochemistryUse of all human tissue was approved by the Institu-

tional Review Board at MD Anderson Cancer Center andwritten consent was obtained from all patients. Paraffin-embedded sections of bladder were cut to 4 mm andplaced on polysine-coated microscope slides and bakedovernight at 50�C in a dry oven. Sections were thendeparaffinized in xylene for 2� 5minutes and rehydratedin alcohol for 2 � 5 minutes. Endogenous peroxidaseactivitywasquenchedbyapplying 2%hydrogenperoxidein 30% methanol for 10 minutes. Antigen retrieval wascarried out by incubation in proteinase K (40 mg/mL PBS)for 15 minutes at 37�C in a humidified chamber. Thesections were stained with primary antibody (SigmaAldrich) overnight at 4�C. The bound primary antibo-dies were visualized by avidin–biotin complex assay(DAKO Corp.) with 3,30-diaminobenzidine as a chroma-gen (DAKO) and hematoxylin as a counterstain. FGFR3staining was indicated by characteristic brown staining.Sections from 4 normal human ureters were used as anegative control. Staining intensity was assessed on ascale from 0 (no staining) to 3 (strong staining) by apathologist (B. Czerniak).

FGFR3 mutation analysisDNA was isolated from the urothelial carcinoma cells

and tumor samples using a genomic DNA extraction kit(Qiagen) according to the manufacturers’ instructions.Exons 7 and 10 were amplified by PCR using AmpliTaqGold DNA polymerase (Applied Biosystems). The fol-lowing primers were used: 50-CGGCAGTGGCGGTG-GTGGTG-30 (sense) and 50-AGCACCGCCGTCTGGTT-GGC-30 (antisense) for exon 7 (22), and 50-CCTCAA-CGCCCATGTCTTT-30 (sense) and 50-AGGCAGCTCA-GAACCTGGTA-30 (antisense) for exon 10 (Sigma-Aldrich). Five pmol/L of each primer was added to the20-mL reaction volume, and 1-mL dimethyl sulfoxide(DMSO) was added for exon 7. The following cyclingvariables were used: 95�C for 10 minutes, then 35 cyclesof 95�C for 30 seconds, 65�C (exon 7) or 58�C (exon 10) for30 seconds, and 72�C for 30 seconds, followed by a finalincubation at 72�C for 10 minutes (22). Unincorporatedprimers and deoxynucleotides were removed usingshrimp alkaline phosphatase and exonuclease I (U.S.

Gust et al.

Mol Cancer Ther; 12(7) July 2013 Molecular Cancer Therapeutics1246




Biochemical). Direct sequencing was carried out with BigDye Terminator Cycle Sequencing and the data wereanalyzedwith SequencingAnalysis 3.0 software (AppliedBiosystems). Visual inspection of the electropherogramswas conducted using Sequence Scanner Software v1.0(Applied Biosystems). Mutation analysis was verifiedindependently by Dr. Francois Radvanyi (Institut Curie,Paris).

Proliferation assayProliferationwas assessedusing the crystal violet assay,

as described previously (23). Cells were treated withvarying concentrations of R3Mab for 48 to 72 hours. Theabsorbance was determined with a microculture platereader (Epoch, BioTek) at 562 nm. Cell survival aftertreatment was calculated as the percentage of absorbancerelative to controls.

Western blot analysisTotal protein was isolatedwith radioimmunoprecipita-

tion buffer (RIPA) supplemented with proteinase andphosphatase inhibitor for 2 hours on ice. Samples werespun at 13,000 rpm for 20 minutes, supernatants weretransferred into empty Eppendorf tubes, and proteinconcentrations were measured using the BCA proteinassay (Thermo Scientific). Thirty mg of protein was thenrun on 8% to 12% SDS polyacrylamide gels and electro-phoretically transferred to Immobilon-P membranes(Millipore). Blots were blocked with Odyssey blockingbuffer for 1 hour at room temperature and incubated inprimary antibody at 4�Covernight. After washing in TBS-T (Tris-buffered saline with 0.1% Tween 20), membraneswere incubated with horseradish peroxidase-conjugatedsecondary antibody IgG (Santa Cruz Biotechnologies) at1:5,000 dilution for 1 hour. Blots were developed using anenhanced chemiluminescence (ECL) substrate system forthe detection of horseradish peroxidase, SuperSignalWest Femto Maximum Sensitivity Substrate (ThermoScientific). Immunoprecipitation was carried out usinga2mgFGFR3polyclonal antibodyon500mgof lysateusingthe ExactaCruz B system (Santa Cruz).

Real-time PCR analysisRNA was extracted from cell lines using TRIzol (Invi-

trogen; Life Technologies) followed by reverse transcrip-tion cDNA generation using the Transcriptor First-StrandcDNA Synthesis Kit (Roche). Quantitative real-timemon-itoring of FGFR3 and EMT markers expression was car-ried out in triplicates on the ABI PRISM 7900 SequenceDetection System (Applied Biosystems) using FastStartUniversal SYBR Green Master Mix with Rox (Roche)under universal cycling conditions. Relative target geneexpression was normalized to glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) levels as an internalcontrol by the comparative cycle threshold (DCt) method.Relative expression between cell lines was expressedcomparedwith epithelial cancer cell line RT4v6 for FGFR3andwithmesenchymal cell line 253J-BV for EMTmarkers.Following 50 to 30 primer pairs were used: ZEB-1 FwdGCACCTGAAGAGGACCAGAG, Rev TGCATCTGGTGT TCC ATT TT; E-cadherin Fwd AGA ACG CAT TGCCAC ATA CAC TC, Rev CAT TCT GAT CGG TTA CCGTGA TC; vimentin Fwd ACA CCC TGC AAT CTT TCAGAC A, Rev GAT TCC ACT TTG CGT TCA AGG T;FGFR3 Fwd GAG GCC ATC GGC ATT GAC, Rev TGGCAT CGT CTT TCA GCA TCT; and GAPDH Fwd GGACCT GAC CTG CCG TCT AGA A, Rev GGT GTC GCTGTT GAA GTC AGA G. FGFR3 primers were located onexon 12 and were designed using Primer Xpress software(Applied Biosystems; Life Technologies).

Orthotopic bladder cancer xenograft modelAll animal work was approved by the Institutional

Review Board of the University of British Columbia(approval #A08-0733). Six-week-old male nude mice(Harlan Laboratories) were anesthetized with isoflurane(2 Vol.%), and analgesia was provided by subcutaneousinjectionwith buprenorphine andmeloxicam (BoehringerIngelheim). After disinfection of the abdominal wall withchlorhexidine, a low transverse laparotomy was madeand the urinary bladder was extra-corporalized. Fifty mLof a cell suspension containing 2.5 � 105 or 5.0 � 105 cellswas inoculated using a 30 G needle directly into thebladder wall (24). The incision was closed with suture.

Bioluminescence was used to quantify tumor burdenand was measured on the Xenogen IVIS Spectrum imag-ing system after intraperitoneal injection of 200 mg/kgluciferin (Caliper). Images were taken at 10 and 14 min-utes after luciferin injection and the average counts wereused for statistical analysis. Bioluminescence imagingwascarried out on the 5th or 6th day, and mice were dividedinto equal treatment groups based on tumor burden (13–16 animals per group). Treatment was started the follow-ing day and imaging was repeated every 5 days. Tumoruptake rates in our orthotopic xenograftmodelwere 100%for UM-UC14 and RT112, and 96% for UM-UC1 cells.

In vivo treatmentThe FGFR3-targeting antibody R3Mab (21) was provid-

ed by Genentech. It was reconstituted in sterile water for

Table 1. Patient and tumor characteristics forTMA analysis

Male, n (%) Female, n (%)

Age (mean, range) 65 (37–85) 67 (40–81)n 112 (77) 33 (23)Tumor grade 1 1 (1) 0 (0)Tumor grade 2 35 (31) 4 (12)Tumor grade 3 76 (68) 29 (88)Papillary 47 (42) 13 (39)Nonpapillary 65 (58) 20 (61)NMIBC 40 (36) 7 (21)MIBC 72 (64) 26 (79)

Targeting FGFR3 in Bladder Cancer

www.aacrjournals.org Mol Cancer Ther; 12(7) July 2013 1247




injection at a concentration of 10 mg/mL and stored at4�C. Control treatments included phosphate-buffer saline(PBS) and an isotype control antibody (human IgG1). Allagents were prepared freshly in PBS immediately beforeeach treatment session and injected intraperitoneally.

Evaluation of proliferation and apoptosis inorthotopic xenograft tumors

Representative tumors derived from orthotopic xeno-grafts were stained for Ki-67 and caspase-3, as markers ofproliferation and apoptosis. Staining of paraffin-embed-ded tumor sections (4 mm) was conducted on a VentanaDiscovery XT autostainer platform (Ventana MedicalSystems) with an enzyme-labeled biotin streptavidin sys-

tem and a solvent-resistant 3,30-diaminobenzidine Mapkit by using primary antibodies at a concentration of 1:500for Ki-67 and 1:50 for caspase-3 at 37�C and an incubationtime of 1 hour. After application of the secondary anti-body, a hematoxylin counterstain was carried out. Theaverage count of positively stained cells in 10 high-powerfields was evaluated on each section.

StatisticsStatistical analysiswas carried out using theChi-square

testing for analysis of immunohistochemistry (IHC), inaddition t test and ANOVA were conducted for in vitroand in vivo experiments. Significance was defined atvalues of P less than 0.05. Data were expressed as mean

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Figure 1. A, TMA analysis for total FGFR3 expression. A TMA was stained for FGFR3 expression with a human-specific FGFR3 antibody. Staining wassemiquantitatively analyzed and staining intensity assessed between 0 (no staining) and 3 (intensive staining). Average staining intensity was analyzedfor tumor grade. One-sided chi-square test was conducted and revealed a significant overexpression (P ¼ 0.047) of FGFR3 in low-grade tumors comparedwith high-grade tumors with a positive staining in 36% and 22% percent of tumors, respectively. The percentages of patients with the specifiedstaining intensities add up to more than 100% because the �1 and �2 intensity groups overlap. B, Western blot analysis of FGFR3 in relation to epithelialand mesenchymal markers in a panel of urothelial cancer cell lines. C, qPCR analysis for FGFR3 in relation to epithelial and mesenchymal markers in apanel of urothelial cancer cell lines. Expression of total FGFR3 is associated with an epithelial phenotype of cell lines. Epithelial cell lines defined byexpression of E-cadherin show higher levels of FGFR3 than mesenchymal cell lines characterized by loss of E-cadherin and expression of ZEB-1and vimentin.

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� SE, if not differently noted. Statistical analysis wasconducted using GraphPad Prism 4.03 (GraphPad Soft-ware, Inc.).

ResultsImmunohistochemistryA human bladder cancer TMA containing cores of 153

tumor samples from patients with urothelial carcinomaof the bladder (Table 1) was stained for total FGFR3expression. The median age of these patients was 65(range 37–85) years and 77% were male. Tissue wasobtained both from cystectomy and transurethral resec-tion specimens. The intensity of staining was gradedfrom 0 (no staining) to 3 (strong staining). Tumors wereclassified as invasive (T1-T4 in 68%) or noninvasive (Tain 32%), and as low (28%) or high (72%) grade. Expres-sion of FGFR3 was found in approximately 70% of bothlow- and high-grade tumors, as well as equally betweeninvasive and noninvasive tumors. High levels (�2) ofFGFR3 were observed in 36% of low-grade tumorscomparedwith 22%of high-grade lesions (P¼ 0.047; Fig.1A). No significant difference was found for invasiveversus noninvasive tumors. There was no evidence ofstaining in normal urothelium.

Mutation analysisA total of 170 fresh-frozen samples from an indepen-

dent cohort of patients with urothelial carcinoma of thebladder were analyzed for mutations in the FGFR3 geneby direct sequencing. The median age of the patientswas 66 (range 41–87) years and 77% were male. Thestage and grade of tumors are reflected in Table 2.FGFR3 mutations in exon 7 or exon 10 were found in26% of all samples and 56% of low-grade tumors. Lowgrade, noninvasive tumors showed more than a 4-foldincreased rate of FGFR3 mutation compared with high-grade, invasive tumors (Table 2). Similar analysisrevealed a S249C mutation (exon 7) in 4 of 19 cell lines,and a R248C (exon 7) and Y375C (exon 10) mutation ineach one of these cell lines. The remaining cell linescontained a wild-type FGFR3 gene (Table 2).

Expression of FGFR3 in bladder cancer cell lines inrelation to epithelial/mesenchymal markers

A panel of 12 human bladder cancer cell lines wasscreened for RNA and protein expression of FGFR3 byqPCR and Western blot. Based on prior work linkinggrowth factor receptor activity to epithelial-to-mesen-chymal (EMT) differentiation and invasive potential

Table 2. FGFR3 mutation analysis of tumor samples and urothelial carcinoma cell lines

Tumor samples

Low grade High grade Total

Noninvasive 28/50 ¼ 56% 2/18 ¼ 11% 30/68 ¼ 46%Invasive 4/7 ¼ 57% 10/95 ¼ 11% 14/102 ¼ 22%Total 32/57 ¼ 56% 12/113 ¼ 11% 44/170 ¼ 26%

Exon 7 mutation: 31 (70%) Exon 10 mutation: 13 (30%)

Exon Nucleotide position/basechange

Codon/changein amino acid

n

7 746 C > G S249C 3110 1114 G > T G372C 410 1117 A > T S373C 110 1124 A > G Y375C 710 1156 T > C F386L 1

Cell lines

Cell line Exon 7 Exon 10 Cell line Exon 7 Exon 10

253J-P wt wt UM-UC7 wt wt253J-BV wt wt UM-UC10 wt wtRT4 v6 wt wt UM-UC11 wt wtRT112 wt wt UM-UC12 wt wtT24 wt wt UM-UC13 wt wtUM-UC1 wt wt UM-UC14 S249C wtUM-UC3 wt wt UM-UC15 S249C Y375CUM-UC4 wt wt UM-UC16 S249C wtUM-UC5 wt wt UM-UC17 S249C wtUM-UC6 R248C wt






of bladder cancer cell lines, the panel was divided innoninvasive/epithelial cell lines and invasive/mesen-chymal cell lines (25). Expression of FGFR3 wasassociated with detection of E-cadherin, a marker of

epithelial differentiation. Mesenchymal cell lines,defined by expression of ZEB-1 and vimentin and lossof E-cadherin, showed lower levels of FGFR3 (Fig. 1Band C).

UM-UC1 (wt) UM-UC14 (S249C)UM-UC1 (wt)B CA

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Figure 2. A, activation of FGFR3 signaling cascade in wild-type (wt) FGFR3-harboring cell lines UM-UC1 by stimulation with fibroblast growth factor (FGF)-1.Cells were cultured in regular growth medium on 6-cm culture dishes to about 50% confluence. The cells were starved in serum-free media for24 hours. Growth medium was exchanged to medium supplemented with FGF-1 at a concentration of 10 ng/mL. The cells were then harvested onice according to the time point of 2, 5, 10, 15, and 20 minutes after stimulation. The cells were lysed with RIPA buffer including proteinase andphosphatase inhibitor and 60 mg of total protein used. Indication of activation of the FGFR3 signaling cascade is shown by a time-dependent increase ofphospho-FGFR, followed by phosphorylation of FRS-2, ERK-1/2, and Akt. B and C, inhibition of FGFR3 signaling by the FGFR3-specific inhibitory antibodyR3Mab in wt-FGFR3–harboring UM-UC1 cells and the S249C mutant FGFR3-bearing cell lines UM-UC14. B, UM-UC1 cells were plated in normal growthmedia and allowed to attach for 24 hours, then washed 2 times with PBS and then starved in media without FBS for 24 hours, followed by stimulationwith FGF-1 (15 ng/mL) for 10 minutes in the presence of heparin (10 mg/mL). Either human control IgG or R3Mab was added in serum-free media atconcentrations of 10 and 50mg/mL. Phosphorylation of FGFR, FRS2, Erk1//2, and Aktwas evaluated byWestern blot and shows lower phosphorylation levelsunder treatment with R3Mab. C, a strong autophosphorylation of FGFR3 is found in UM-UC14 cells in regular growth conditions. R3Mab blocksautophosphorylation of FGFR3S249C receptor even under stimulated conditions with supplemented FGF1, whereas Erk1/2 shows increased levels ofphosphorylation under stimulation with FGF1 and at low concentrations of R3Mab (1 mg/mL). D, in vitro growth inhibition of R3Mab on urothelial cancer celllines. The cell lines were grown up to 50% confluence in regular growth medium. R3Mab was then added to growth medium and growth inhibition wasevaluated by crystal violet staining after 48 hours of treatment with FGFR3-specific inhibitory antibody. Mesenchymal cell lines with low expression of FGFR3show no specific response to treatment with R3Mab at concentrations up to 100 mg/mL, whereas epithelial cell lines with high expression of FGFR3 show agrowth inhibition of up to 50%, as observed in UM-UC1 cells, a cell line derived from a lymph node metastasis from an urothelial carcinoma.

Gust et al.





In vitro targeting of FGFR3FGFR3 pathway activity was verified in UM-UC1

urothelial cancer cells, which express a high level ofwild-type FGFR3. Stimulation of these cells with theligand FGF-1 resulted in phosphorylation of FGFR3 andactivation of the downstream signaling cascade in a time-dependent manner. Phosphorylation of the receptor wasfollowed by phosphorylation of FRS-2, and increasingactivation of both p-Erk 1/2 and p-Akt (Fig. 2A). In thesame cell lines, inhibition of FGFR3 with R3Mab abrogat-ed receptor phosphorylation, as well as phosphorylationof FRS-2 and Erk1/2 (Fig. 2B). A similar experiment wasconducted in UM-UC14, which harbors an S249C muta-tion in exon 7 of FGFR3, but here an immuneoprecipita-tion was conducted with anti-FGFR3 and subsequentblotting with antiphospho-tyrosine as an alternativemethod to show FGFR3 phosphorylation (Fig. 2C). Treat-mentwith R3Mab in regular growthmedium resulted in aconcentration-dependent growth inhibition in 3 of 4 dif-ferent tumor cell lines in a crystal violet assay (Fig. 2D).UM-UC1 displayed the most pronounced antiprolifera-tive response, whereas the highlymesenchymal and inva-sive cells UM-UC3, UM-UC13, T24, and 253J-BV, all ofwhich do not express appreciable levels of FGFR3,showedno response. RT4V6, RT112 (both high expressionof wild-type FGFR3), UM-UC16 (low expression ofS249CFGFR3), and UM-UC14 (modest expression ofS249CFGFR3) showed only modest response.

In vivo treatment of orthotopic bladder cancerxenograftsUM-UC14, RT112, and UM-UC1 cells were orthotopi-

cally injected into the bladder wall of athymic nudemice. Successful tumor inoculation was verified by bio-luminescence imaging on the 5th or 6th day, and the micewere divided into equal groups based on tumor burden.They were then treated with active agent (R3Mab) orcontrol every 72 hours and bioluminescencewas repeatedevery 5 days.Two different dose levels were tested in UM-UC14

xenografts, including 15 mg/kg and 30 mg/kg, andcompared with a saline-treated group (IgG1 controlnot available for this experiment). Tumor growth, 30days after inoculation, showed a dose-dependent inhi-bition of tumor growth with a reduction in tumorgrowth of 22% and 33%, respectively (Fig. 3A). Eval-uation of representative tumor samples harvested fromthis experiment showed inhibition of FGFR3 phosphor-ylation by immunoprecipitation of FGFR3 and sub-sequent immunoblotting with antiphospho-tyrosine(Fig. 3B).In mice-bearing orthotopic RT112 xenografts, R3Mab

treatment at adoseof 30mg/kgwas comparedwith both asaline control group and a nontargeting human IgG1-control. The IgG1 had no effect on tumor growth, whereasmice treated with R3Mab showed a reduction in tumorgrowthby 59.2% comparedwith IgGand 57.2%comparedwith saline controls (Fig. 3C).

In vivo, UM-UC1 tumors orthotopically injected into thebladder of nude mice showed a reduction in tumorgrowth of 83.9% when treated with R3Mab compared

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Figure 3. A, dose-dependent growth inhibition of orthotopic UM-UC14xenografts. Constitutively activated, mutant FGFR3S249C-harboring UM-UC14 cells were inoculated orthotopically in bladder of nude mice. Micewere subdivided into 3 treatment arms: IgG control (n ¼ 14), R3Mab15 mg/kg body weight (BW; n ¼ 13), and R3Mab 30 mg/kg BW (n ¼ 13).Systemic, intraperitoneal applied treatment with R3Mab results in adose-dependent growth inhibition compared with nonspecific IgGcontrol treatment. Tumor growth was evaluated by bioluminescentimaging. B, Western blot analysis of tumor samples, derived fromorthotopically grownUM-UC14xenografts after treatmentwithR3Mabat15mg/kgBWand 30mg/kgBW. Tumors treatedwith R3Mab show lowerphosphorylation levels indicated by reduced pTyr, whereas levels of totalFGFR3 in the different treatment and control groups are unchanged.C, tumor growth inhibition of orthotopic RT112 xenografts. Wild-typeFGFR3-harboring RT112 cells were inoculated orthotopically in bladderof nude mice. Mice received either 30 mg/kg BW R3Mab (n ¼ 15),nontargeting IgG control (n ¼ 15) or PBS (n ¼ 15) as a negative control.Intraperitoneal treatment with R3Mab results in significant inhibition oftumor growth compared with saline-treated mice as well as mice in theIgG control arm. Tumor growth was evaluated by bioluminescentimaging.






with mice injected with IgG and 84.1% compared withsaline controls (Fig. 4A and B). Evaluation of wholebladder samples by Western blotting at the end of theexperiment showed a significant inhibition of FGFR3phosphorylation and downstream signaling, includingp-FRS2, p-ERK1/2, cyclin D1, and cyclin E (Fig. 4C).Overall, this indicates that the observed tumor growth

reduction is due to highly effective FGFR3 pathway inhi-bition by R3Mab in vivo (Fig. 4D). The inhibition of tumorgrowth is related to antiproliferative effects of R3Mabexpressed in a reduced Ki-67 proliferative index (Fig.4E), whereas no difference has been observed for expres-sion of cleaved caspase-3 in xenograft tumors (data notshown) as a marker for apoptosis.

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Figure 4. A, tumor growth inhibition of orthotopic UM-UC1 xenografts.Wild-type FGFR3-harboring UM-UC1 cells were inoculated orthotopically in bladder ofnude mice. Mice were assigned to 3 groups receiving either PBS as control (n¼ 16), nontargeting human-IgG (n¼ 13), or R3Mab at a dose of 30 mg/kg BW(n ¼ 14). Intraperitoneal treatment with R3Mab resulted in significant inhibition of tumor growth compared with saline-treated mice as well as micein the IgG control arm. Tumor growth was evaluated by bioluminescent imaging. B, bioluminescent images of orthotopic UM-UC1 xenografts. Mice wereimaged on day 5 after tumor inoculation and grouped into the different treatment arms. Pictures show representative mice of the 3 treatment groups: PBS(saline), IgG controls, and R3Mab-treated mice. C, Western blot analysis of orthotopically implanted UM-UC1 xenografts. Tumors were collected at theendpoint of the experiment (day 22) and snap frozen. Random tumors of the treatment groups were lysed and total protein blotted for members of the FGFR3signaling cascade. R3Mab-treated tumors show a significantly reduced activation of FGFR3 signaling with downregulation of FRS2, lower phosphorylationlevels of p-Erk 1/2, and lower expression of cyclin D1 and cyclin E. D, orthotopic xenografts. Full section of an orthotopically implanted bladder tumor (left).Representative tumors of orthotopic UM-UC1 xenografts treated with R3Mab or control saline at day 22 after tumor inoculation (right). E, evaluation ofxenograft tumor sections for Ki-67 staining revealed a lower proliferative index for R3Mab-treatedUM-UC1 tumors than control tumors treatedwith either PBSor nontargeting human IgG.

Gust et al.





DiscussionWe have shown a high rate of FGFR3 mutation and

overexpression in an American cohort of patients. Wehave also shown that FGFR3 signaling is active in pre-clinical models of bladder cancer, and that specific inhi-bition of FGFR3 with a monoclonal antibody inducesgrowth arrest in orthotopic bladder cancer xenografts.Our data builds on prior experience with the same inhib-itor in subcutaneous models (21), yet advances this ther-apeutic strategy to meet a significantly higher bar ofefficacy in the orthotopic setting. We believe that thisprovides the proof of principle required to investigatethis agent or a similar novel FGFR3 inhibitor in specificclinical trials for bladder cancer.We see a lower rate of FGFR3 mutations in low-grade

tumors (4, 26, 27) and a higher rate of FGFR3 overexpres-sion in high-grade tumors (5) compared with priorreports. This is most likely related to patient selection,which in turn is a reflection of the highly specializedclinical setting in which these tumor samples were col-lected. There are also likely differences in IHC method-ology and assessment of staining. A slight increase inmutation rate would be expected if exon 15 had also beenassessed.A limitation of current preclinical models of bladder

cancer is the inability to model low-grade, noninvasivedisease. Although we possess bladder cancer cell lineswith FGFR3mutations, these mutations are found only inhighly invasive cell lines that are not representative ofnonmuscle invasive disease. RT4 is the only cell linethat is low grade, but even it is invasive and it expresseswild-type FGFR3. Our data therefore do not allow us todraw any conclusions about the potential efficacy ofFGFR3 inhibition in nonmuscle invasive bladder cancer(NMIBC). We have tested tyrosine kinase inhibitors tar-geting FGFR3 in RT4v6 with excellent growth inhibition(data not shown). We and others are developing primaryxenografts using fresh patient samples in an attempt toovercome our inability to model nonmuscle invasivedisease.The optimal disease state to test FGFR3 inhibition is

open to debate. While it would seem logical to target theactivatingmutations innonmuscle invasivedisease, itwillbe challenging to give systemic therapy to this patientpopulation, especially as FGFR3 mutations indicate afavorable prognosis (8, 28, 29). A trial with gefitinib [anoral epidermal growth factor receptor (EGFR) inhibitor]failed to accrue patients in Canada at least in part becausephysicians and patients did not accept systemic therapyforNMIBC (NCT00352079). The currently availablemulti-tyrosine kinase inhibitors that target FGFR3 are allmarredwith concerns regarding systemic toxicity that wouldseverely limit their use in this setting (11). R3Mab mayprove to be better tolerated, since it should avoid thehypophosphatemia and soft-tissue calcification associat-ed with pan-FGFR inhibitors. It remains to be provenwhether these agents and especially R3Mab are effica-cious when administered intravesically.

Our results suggest that R3Mab should be tested inpatients with muscle invasive bladder cancer (MIBC)whose tumors show FGFR3 overexpression. There is apressing need for new treatments in this lethal variant ofbladder cancer, and our findings would support testingFGFR3 inhibition in conjunction with systemic cytotoxicchemotherapy. Whether FGFR3mutations are relevant inthe context of MIBC remains to be shown. Our cell lineinvestigations reveal little drug activity in invasive cellswith FGFR3 mutations, with the exception of UM-UC14.We observed a reduced growth inhibitory effect in UM-UC14 compared with prior reports (21, 30). This may be adifference in the model systems (orthotopic versus het-erotopic) or potentially related to phenotypic drift indifferent strains of UM-UC14. Regardless of cell line data,any clinical trial would have to include assessment ofFGFR3 mutation status and expression in every tumor.Patient selection based on these parameters may be animportant factor in subsequent patient selection. We arealso investigating mechanisms of resistance to R3Mab inpreclinical models with the intention of potentially usingthis information for patient enrichment in clinical trials.Since FGFR3 seems to drive growth through AKT andERK1/2 (31), it is likely that there is redundancy withother growth factor receptor pathways (32) much as wehave described with EGFR and PDGFR-ß (33).

We have confirmed the results of prior reports onFGFR3 mutation and expression patterns in urothelialcarcinoma of the bladder: FGFR3 is frequently mutatedin noninvasive bladder cancer and frequently overex-pressed in both noninvasive and invasive bladder cancer.We have shown that targeting FGFR3 with a specificinhibitorymonoclonal antibody (R3Mab) effectively abro-gates FGFR3 signaling pathway activation with a resul-tant decrease in tumor growth. This inhibitor is highlyefficacious in a selection of bladder cancer cell lines andorthotopic xenografts. These are promising results thatwarrant translation into clinical trials in patients withbladder cancer.

Disclosure of Potential Conflicts of InterestD.J. McConkey has a commercial research grant from AstraZeneca;

has ownership interest (including patents) in Apocell, Inc; and is aconsultant/advisory board member for Apocell, Inc. J. Qing hasownership interest (including patents) in Genentech. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K.M. Gust, D.J. McConkey, A. Ashkenazi, C.P.Dinney, P.C. BlackDevelopment of methodology: K.M. Gust, S. Awrey, J. Bondaruk, B.Czerniak, C.P. Dinney, P.C. BlackAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.M. Gust, S. Awrey, P.K. Hegarty, P.C. BlackAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): K.M. Gust, D.J. McConkey, J. Bondaruk, B.Czerniak, P.C. BlackWriting, review, and/or revision of the manuscript: K.M. Gust, D.J.McConkey, P.K. Hegarty, J. Qing, A. Ashkenazi, C.P. Dinney, P.C. BlackAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):K.M.Gust, S. Awrey, J. Bondaruk, B.Czerniak, P.C. BlackStudy supervision: K.M. Gust, B. Czerniak, P.C. Black






AcknowledgmentsThe authors thank Eliana Beraldi for her assistance with viral trans-

duction of cell lines. The authors also thank Ladan Fazli and Estelle Li fortheir work on Ki-67 and caspase-3 expression on xenograft tumor sections.

Grant SupportThis studywas supported by aNational Cancer Center Core Grant (The

University of Texas,M.D.AndersonCancer Center). In addition P.C. Blackhas been awarded a Developmental Grant from the NIH GU SPORE in

bladder cancer at MD Anderson Cancer Center (CA91846), a NIH T32Training Grant, an AUA Foundation Grant, and a Mentored PhysicianScientist Award from Vancouver Coastal Health Research Institute.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 28, 2012; revised April 30, 2013; accepted April 30,2013; published OnlineFirst May 8, 2013.

References1. AmericanCancer Society. Cancer Facts and Figures 2012. [cited 2012

Sep 24.] Available from: http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-031941.pdf.

2. Pashos CL, Botteman MF, Laskin BL, Redaelli A. Bladder cancer: epi-demiology, diagnosis, andmanagement. Cancer Pract 2002;10:311–22.

3. Avritscher EB, Cooksley CD, Grossman HB, Sabichi AL, Hamblin L,DinneyCP,etal.Clinicalmodelof lifetimecostof treatingbladdercancerand associated complications. Urology 2006;68:549–53.

4. BillereyC,Chopin D, Aubriot-LortonMH,Ricol D,Gil Diez deMedinaS,Van Rhijn B, et al. Frequent FGFR3mutations in papillary non-invasivebladder (pTa) tumors. Am J Pathol 2001;158:1955–9.

5. Mhawech-Fauceglia P, Cheney RT, Fischer G, Beck A, Herrmann FR.FGFR3 and p53 protein expressions in patients with pTa and pT1urothelial bladder cancer. Eur J Surg Oncol 2006;32:231–7.

6. Tomlinson DC, Baldo O, Harnden P, Knowles MA. FGFR3 proteinexpression and its relationship to mutation status and prognosticvariables in bladder cancer. J Pathol 2007;213:91–8.

7. van Oers JM, Wild PJ, Burger M, Denzinger S, Stoehr R, Rosskopf E,et al. FGFR3mutations and a normal CK20 staining pattern define low-gradenoninvasiveurothelial bladder tumours. EurUrol 2007;52:760–8.

8. van Rhijn BW, Vis AN, van der Kwast TH, Kirkels WJ, Radvanyi F,Ooms EC, et al. Molecular grading of urothelial cell carcinoma withfibroblast growth factor receptor 3 and MIB-1 is superior to path-ologic grade for the prediction of clinical outcome. J Clin Oncol2003;21:1912–21.

9. Bakkar AA, Wallerand H, Radvanyi F, Lahaye JB, Pissard S, Lecerf L,et al. FGFR3 and TP53 genemutations define two distinct pathways inurothelial cell carcinomaof the bladder. Cancer Res 2003;63:8108–12.

10. Tomlinson DC, Hurst CD, Knowles MA. Knockdown by shRNA iden-tifies S249Cmutant FGFR3 as a potential therapeutic target in bladdercancer. Oncogene 2007;26:5889–99.

11. Turner N, Grose R. Fibroblast growth factor signalling: from develop-ment to cancer. Nat Rev Cancer 2010;10:116–29.

12. Trudel S, Li ZH,Wei E,WiesmannM,ChangH,ChenC, et al. CHIR-258,a novel, multitargeted tyrosine kinase inhibitor for the potentialtreatment of t(4;14) multiple myeloma. Blood 2005;105:2941–8.

13. Lamont FR, Tomlinson DC, Cooper PA, Shnyder SD, Chester JD,Knowles MA. Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo. Br J Cancer2011;104:75–82.

14. Rebouissou S, Herault A, Letouze E, Neuzillet Y, Laplanche A, Ofua-luka K, et al. CDKN2A homozygous deletion is associated with muscleinvasion in FGFR3-mutated urothelial bladder carcinoma. J Pathol2012;227:315–24.

15. Chen J, Lee BH, Williams IR, Kutok JL, Mitsiades CS, Duclos N, et al.FGFR3 as a therapeutic target of the small molecule inhibitor PKC412in hematopoietic malignancies. Oncogene 2005;24:8259–67.

16. Jonker DJ, Rosen LS, SawyerMB, deBraud F,WildingG, SweeneyCJ,et al. A phase I study to determine the safety, pharmacokinetics andpharmacodynamics of a dual VEGFR and FGFR inhibitor, brivanib, inpatients with advanced or metastatic solid tumors. Ann Oncol2011;22:1413–9.

17. Kim KB, Chesney J, Robinson D, Gardner H, Shi MM, Kirkwood JM.Phase I/II and pharmacodynamic study of dovitinib (TKI258), aninhibitor of fibroblast growth factor receptors and VEGF receptors,in patients with advanced melanoma. Clin Cancer Res 2011;17:7451–61.

18. Ratain MJ, Schwartz GK, Oza AM, Rudin CM, Kaye SB, De Jonge MJ,et al. Brivanib (BMS-582664) in advanced solid tumors (AST): Resultsof a phase II randomized discontinuation trial (RDT). J Clin Oncol2011;29:(suppl; abstr 3079).

19. Brooks AN, Kilgour E, Smith PD. Molecular pathways: fibroblastgrowth factor signaling: a new therapeutic opportunity in cancer. ClinCancer Res 2012;18:1855–62.

20. Chesi M, Nardini E, Brents LA, Schrock E, Ried T, Kuehl WM, et al.Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma isassociated with increased expression and activating mutations offibroblast growth factor receptor 3. Nat Genet 1997;16:260–4.

21. Qing J, Du X, Chen Y, Chan P, Li H, Wu P, et al. Antibody-basedtargeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiplemyeloma in mice. J Clin Invest 2009;119:1216–29.

22. Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF,Knowles MA. FGFR3 and Ras gene mutations are mutually exclu-sive genetic events in urothelial cell carcinoma. Oncogene 2005;24:5218–25.

23. Cheng B, Fu XB, Sheng ZY, Sun TZ, Sun XQ. [A comparative study ofEGFR andFGFR-2 expression in fetal and adult skin]. Zhonghua zhengxing wai ke za zhi 2003;19:91–4.

24. Dinney CP, Fishbeck R, Singh RK, Eve B, Pathak S, Brown N, et al.Isolation and characterization of metastatic variants from humantransitional cell carcinoma passaged by orthotopic implantation inathymic nude mice. J Urol 1995;154:1532–8.

25. Black PC, Brown GA, Inamoto T, Shrader M, Arora A, Siefker-RadtkeAO, et al. Sensitivity to epidermal growth factor receptor inhibitorrequires E-cadherin expression in urothelial carcinoma cells. ClinCancer Res 2008;14:1478–86.

26. vanRhijnBW,Montironi R, Zwarthoff EC, Jobsis AC, vanderKwast TH.Frequent FGFR3 mutations in urothelial papilloma. J Pathol 2002;198:245–51.

27. Neuzillet Y, Paoletti X, Ouerhani S,Mongiat-Artus P, SolimanH, de TheH, et al. A meta-analysis of the relationship between FGFR3 and TP53mutations in bladder cancer. PLoS ONE 2012;7:e48993.

28. van Rhijn BW, Lurkin I, Radvanyi F, Kirkels WJ, van der Kwast TH,Zwarthoff EC. The fibroblast growth factor receptor 3 (FGFR3) muta-tion is a strong indicator of superficial bladder cancer with low recur-rence rate. Cancer Res 2001;61:1265–8.

29. Hernandez S, Lopez-Knowles E, Lloreta J, Kogevinas M, Amoros A,Tardon A, et al. Prospective study of FGFR3mutations as a prognosticfactor in nonmuscle invasive urothelial bladder carcinomas. J ClinOncol 2006;24:3664–71.

30. Masson-Lecomte A, Vordos D, de la Taille A, Neuzillet Y, Radvanyi F,Allory Y. [Update on FGFR3 mutation andMRES phenotype in urothe-lial carcinogenesis]. Prog Urol 2013;23:96–8.

31. AskhamJM,PlattF,ChambersPA,SnowdenH,TaylorCF,KnowlesMA.AKT1 mutations in bladder cancer: identification of a novel oncogenicmutation that can co-operate with E17K. Oncogene 2010;29:150–5.

32. Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH,Wiedemeyer R, et al. Coactivation of receptor tyrosine kinases affectsthe response of tumor cells to targeted therapies. Science 2007;318:287–90.

33. Black PC, BrownGA, Dinney CP, KassoufW, Inamoto T, Arora A, et al.Receptor heterodimerization: a new mechanism for platelet-derivedgrowth factor induced resistance to anti-epidermal growth factorreceptor therapy for bladder cancer. J Urol 2011;185:693–700.

Gust et al.





2013;12:1245-1254. Published OnlineFirst May 8, 2013.Mol Cancer Ther Kilian M. Gust, David J. McConkey, Shannon Awrey, et al. Target in Bladder CancerFibroblast Growth Factor Receptor 3 Is a Rational Therapeutic

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