MMP-1 and Pro-MMP-10 as Potential Urinary Pharmacodynamic ... · MMP-1 and MMP-10 in xenograft models in conjunc-tion with attenuated tumor growth. Importantly, in a phase I dose-escalation

Personalized Medicine and Imaging

MMP-1 and Pro-MMP-10 as Potential UrinaryPharmacodynamic Biomarkers of FGFR3-Targeted Therapyin Patients with Bladder Cancer

Xiangnan Du1, Benjamin C. Lin2, Qian-Rena Wang1, Hao Li1, Ellen Ingalla1, Janet Tien1, Isabelle Rooney3,Avi Ashkenazi4, Elicia Penuel2, and Jing Qing1

AbstractPurpose: The aim of this study was to identify noninvasive pharmacodynamic biomarkers of FGFR3-

targeted therapies in bladder cancer to facilitate the clinical development of experimental agent targeting

FGFR3.

ExperimentalDesign:Potential soluble pharmacodynamic biomarkers of FGFR3were identified using a

combination of transcriptional profiling and biochemical analyses in preclinical models. Two matrix

metalloproteinases (MMP),MMP-1 andMMP-10, were selected for further studies in humanbladder cancer

xenograftmodels treatedwith a specific anti-FGFR3monoclonal antibody, R3Mab. Serumandurinary levels

of MMP-1 and MMP-10 were determined in healthy donors and patients with bladder cancer. The

modulation of MMP-1 and MMP-10 by R3Mab in patients with bladder cancer was further evaluated in

a phase I dose-escalation study.

Results:MMP-1 andMMP-10mRNA and proteinwere downmodulated by FGFR3 shRNA andR3Mab in

bladder cancer cell lines. FGFR3 signaling promoted the expression and secretion of MMP-1 and pro-MMP-

10 in a MEK-dependent fashion. In bladder cancer xenograft models, R3Mab substantially blocked tumor

progression and reduced the protein levels of humanMMP-1 and pro-MMP-10 in tumor tissues as well as in

mouse serum. Furthermore, both MMP-1 and pro-MMP-10 were elevated in the urine of patients with

advanced bladder cancer. In a phase I dose-escalation trial, R3Mab administration resulted in an acute

reduction of urinary MMP-1 and pro-MMP-10 levels in patients with bladder cancer.

Conclusion: These findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10

expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic

biomarkers for R3Mab in patients with bladder cancer. Clin Cancer Res; 20(24); 6324–35. �2014 AACR.

IntroductionBladder cancer is the fifth most common cancer world-

wide, with an estimated 386,330 new cases and 150,200deaths in2008 (1).Despite radical cystectomyand intensivechemotherapy, the 5-year survival rate for patients with

localized and distant metastatic bladder cancer is approx-imately 35% and 5% to 6%, respectively (2). Thus, noveltherapeutic agents are urgently needed to improve patientoutcome.

FGFR3 belongs to a family of four structurally andfunctionally related single-transmembrane receptor tyro-sine kinases, which transduce signals frommany of the 22identified FGF polypeptides in humans (3–5). Uponcanonical FGF binding in a ternary complex with heparinsulfate proteoglycans, FGFR3 undergoes dimerization,autophosphorylation, and activation. This triggers therecruitment of adaptor proteins, such as FGFR substrate2a (FRS2a), leading to activation of several downstreamsignaling cascades, including the Ras–Raf–MAPK,PI3K–Akt–mTOR, and phospholipase C g (PLCg) path-ways. Dysregulation and activation of FGFR3 through avariety of mechanisms has been identified in a widespectrum of human cancers, including multiple myeloma(6–8), squamous cell carcinoma of the lung (9, 10),breast cancer (11, 12), glioblastoma (13, 14), testicularcancer (15), and bladder cancer (16).

1Translational Oncology, Genentech, Inc., South San Francisco, California.2Oncology Biomarker Development, Genentech, Inc., South San Fran-cisco, California. 3Oncology Clinical Science ECD, Genentech, Inc., SouthSan Francisco, California. 4Research Oncology, Genentech, Inc., SouthSan Francisco, California.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

X. Du and B.C. Lin contributed equally to this article.

Corresponding Authors: Elicia Penuel, ES Oncology Biomarker Devel-opment, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.Phone: 650-467-7214; Fax: 650-467-5470; E-mail: [email protected];and Jing Qing, Department of Translational Oncology, Genentech, Inc.,1 DNA Way, South San Francisco, CA 94080. Phone: 650-467-8266;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-3336

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(24) December 15, 20146324

on November 11, 2020. © 2014 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 17, 2014; DOI: 10.1158/1078-0432.CCR-13-3336

http://clincancerres.aacrjournals.org/

Specifically, accumulating genetic and functional evi-dence has established FGFR3 as a potential oncogenic driverand therapeutic target in bladder cancer. About 60% to 70%of non–muscle-invasive low-grade papillary and 11% to16% of muscle-invasive bladder tumors contain somaticactivating mutations in FGFR3 (17–20), the majority ofwhich are missense substitutions in the extracellular orjuxtamembrane domains of FGFR3 that lead to ligand-independent receptor dimerization and activation. Besidesactivating mutations, gene fusion between FGFR3 andTACC3 or BAIAP2L1 has been recently identified in bladdercancer cell lines as well as primary tumors, resulting inaberrant regulation and activation of FGFR3 (21, 22). Inaddition, high levels of wild-type FGFR3 is found in morethan 40% to 50%ofmuscle-invasive andmetastatic bladdercancers (19, 23). Moreover, expression of activated FGFR3transformsNIH-3T3 cells andhematopoietic cells in cultureand confers tumorigenicity in vivo (24–26). Importantly,several preclinical studies using genetic and/or pharmaco-logic approaches have demonstrated that inhibition ofFGFR3 in bladder cancer suppresses cell proliferation inculture and tumor growth in xenograft and orthotopicmodels (23, 27–31). Collectively, these data suggest thatFGFR3 could be an oncogenic driver in a subset of bladdercancers and hence that targeting this receptor may betherapeutically beneficial. Indeed, several small-moleculeinhibitors of FGFR have entered into clinical investigationsfor cancer therapy (32–34). A specific human monoclonalanti-FGFR3 antibody we developed (designated R3Mabor MFGR1877S) has been evaluated in a phase I clinicaltrial (35).

While the advances in developing FGFR3-targeted ther-apeutic agents are encouraging, identification and valida-tion of accessible, mechanism-based biomarkers to gaugetargeted pharmacodynamic modulation is urgently need-ed to facilitate clinical investigation. In the current study,combining transcriptional profiling and biochemicalapproaches, we identified MMP-1 and pro-MMP-10 asbona fide FGFR3 downstream targets and the urinary levelof these metalloproteinases could serve as potential easilyaccessible pharmacodynamic biomarkers for FGFR3-tar-geted therapy in bladder cancer.

Materials and MethodsCell culture, siRNA transfection, and reagents

Bladder cancer cell lines RT112 which contain FGFR3-TACC3 fusion endogenously (21) was purchased from theGerman Collection of Microorganisms and Cell Cultures(DSMZ, Germany). RT112 cells stably expressing doxycy-cline-inducible shRNAs targeting FGFR3 or enhanced GFP(EGFP) were described in our previous study (29). Bladdercancer cell line UMUC-14 which contains FGFR3S249C wasobtained as described (29). Bladder cancer cell line SW780which contains FGFR3-BAIAP2L1 fusion (21) was pur-chased from ATCC. All cells are stored at early passages ina central cell bank at Genentech. Cell lines were authenti-cated by short tandem repeat and genotyped upon reexpan-sions, and experiments were carried out with low-passagecultures of these stocks. The cells were maintained withDMEM supplemented with 10% FBS (Sigma), 100 U/mLpenicillin, 0.1mg/mL streptomycin, and L-glutamine underconditions of 5% CO2 at 37

�C.Rapamycin and PI3K inhibitor LY294002 were obtained

from Cell Signaling Technology. A potent and selectiveMEK1/2 inhibitor PD0325901 (Pfizer) was purchased fromSynthesis Med Chem.

All RNA interference experiments were carried out withON-TARGETplus siRNAs (25 nmol/L, Dharmacon). Cellswere transfected with Lipofectamine RNAiMax (Invitro-gen), and RNA or conditioned medium was collected at48 or 72 hours after transfection.

Gene expression array and quantitative reversetranscriptase-PCR analyses of mRNA expression level

Themicroarray studies with RT112 cells expressing doxy-cycline-inducible shRNAs targeting FGFR3 or EGFP wereconducted as described (36), and can be accessed at theGene Expression Omnibus database under the accessionnumber GSE41035. To detect transcripts of MMP-1 andMMP-10, quantitative reverse transcriptase (qRT)-PCR wasperformed with predesigned TaqMan gene expressionassays (Applied Biosystems). All reactions were performedat least in triplicates. The relative amount of all mRNAs wascalculated using the comparative Ct method after normal-ization to human RPL19.

Protein analysesCells were treated as described in the figure legends. For

total cell lysates, cells were washed twice with ice-cold PBS

Translational RelevanceDysregulation of FGFR3 has been implicated in the

pathogenesis of bladder cancer, and several investiga-tional agents targeting FGFR3 have been evaluated inearly-phase clinical studies. To facilitate the clinicaldevelopment of FGFR3-targeted therapy, we sought toidentify potential noninvasive pharmacodynamic bio-markers to gauge drug activities. Combining transcrip-tional profiling and biochemical analyses, we identifiedand confirmed two matrix metalloproteinases, MMP-1and MMP-10, as downstream targets of FGFR3. Inhibit-ing FGFR3 function with a humanized monoclonalantibody, R3Mab, led to a significant reduction ofMMP-1 and MMP-10 in xenograft models in conjunc-tion with attenuated tumor growth. Importantly, in aphase I dose-escalation trial, R3Mab caused an acutereduction of urinary MMP-1 and pro-MMP-10 levels inpatients with advanced bladder cancer. These data war-rant further investigation in larger clinical studies usingthese potentially actionable noninvasive pharmacody-namic biomarkers to stratify patients with elevatedMMP-1 and pro-MMP-10 and potentially to monitortherapeutic response.

Urinary Pharmacodynamic Markers for R3Mab in Bladder Cancer

www.aacrjournals.org Clin Cancer Res; 20(24) December 15, 2014 6325




and extracted inRIPAbuffer (Millipore) supplementedwithphosphatase inhibitor cocktail PhosSTOP and Completeprotease inhibitor cocktail (Roche Applied Science). Foranalysis of protein expression in tumor xenografts, lysatefrom tumor tissues was extracted with lysis buffer[consisting of 150 mmol/L sodium chloride, 20 mmol/LTris (pH 7.5), 2 mmol/L EDTA, 1% Triton X-100, 10mmol/L sodiumfluoride, supplementedwithprotease inhi-bitors and phosphatase inhibitors] by pulverizing the fro-zen tissues using the FastPrep-24 homogenizer as describedby the manufacturer (MP Biomedicals).

Protein lysates were analyzed by SDS-PAGE and Westernblot analysis. The primary blotting antibody for total FRS2(sc-8318) was purchased from Santa Cruz Biotechnology.The following primary antibodies were purchased fromCell Signaling Technology: pFRS2 Y196 (#3864), pMAPK(#9101), total MAPK (#4695), pAKT (#9271), and totalAKT (#9272). Anti-MMP-1 (MAB901) and anti-MMP-10(MAB910) were purchased from R&D Systems. The blotswere visualized using a chemiluminescent substrate (ECLPlus, Amersham Pharmacia Biotech).

Analyses of secretedMMP-1, pro-MMP-10, andMMP-10in conditioned medium

Secreted MMPs were measured with the following ELISAKits according to the manufacturers’ instructions: Totalhuman MMP-1 protein (ELH-MMP1) and total humanMMP-10 (ELH-MMP10-001) ELISA kits were purchasedfrom RayBiotech, and human pro-MMP-10 ELISA kit(DM100) was purchased from R&D Systems.

To collect conditioned medium: RT112 and UMUC-14cells were grown to 90% confluence in DMEM containing10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin, and100 mg/mL streptomycin. To evaluate the effects of FGFR3knockdown on MMP-1 and MMP-10 protein expression,cells were cultured in the presence or absence of doxycycline(1 mg/mL) for the indicated time, and conditionedmediumwas collected. To study the effect of R3Mab on MMP-1 andMMP-10 expression, 106 cells per dish were seeded onto 10cm dishes in complete growth medium and allowed toattach overnight. Then, serum-free DMEM containingR3Mab (15 mg/mL) or a control antibody was applied, andsupernatants were collected at the indicated time points.The conditioned medium was concentrated 10-fold usingAmicon Ultra Centrifugal filters (Millipore) for ELISA anal-ysis, and the corresponding cells were lysed in the RIPAbuffer (50mmol/L pH7.4 Tris-Cl, 1%NP40, 0.25%sodiumdeoxycholate) with complete protease inhibitor cocktail(Roche Applied Science). MMP-1 and MMP-10 amountswere normalized to total cellular proteins.

Xenograft studies and collection of serum for ELISAanalyses

All animal studies were conducted in accordance with theGuide for the Care and Use of Laboratory Animals, pub-lished by the NIH (NIH publication 8523, revised 1985).The Institutional Animal Care and Use Committee at Gen-entech reviewed and approved all animal protocols. Female

C.B-17 SCID mice, 6 to 10 weeks of age, were purchasedfrom Charles River Laboratory. Female athymic nude micewere obtained from Harlan Sprague Dawley. Mice weremaintained under specific pathogen-free conditions.Female C.B-17 SCID or athymic nudemice were inoculatedsubcutaneously with 7� 106 RT112 cells in Hank balancedsalt solution (HBSS)/Matrigel (1:1 v/v, BD Biosciences) or5 � 106 UMUC-14 cells in HBSS (0.1 to 0.2 mL/mouse),respectively. Mice with tumors reaching a mean volume of150 to 200mm3were randomly grouped into groups of 12,andwere treatedwith vehicle (PBS)orR3Mab (0.3–100mg/kg) via weekly intraperitoneal injections. Body weights andcaliper measurements were taken twice weekly. The tumorvolume (mm3) was calculated using the following formula:length � width2 � 0.5. Log2 (tumor volume) growth traceswere fitted to each treatment group with restricted cubicsplines for the fixed time effect in each group. Fitting wasdone as previously described (37) via a linear mixed effectsmodel, using the R package "nlme," version 3.1–108 in Rversion 2.15.2 (R Development Core Team 2012; R Foun-dation for Statistical Computing). Plotting was performedusing Excel 14.3.2 (Microsoft Corporation).

To analyze MMP-1 and MMP-10 levels in tumor tissuesand serum, mice with tumors reaching a mean volume of150 to 200mm3were randomly grouped into cohorts of 10,and were treated with a control human IgG1 or R3Mab(30 mg/kg) on days 1, 4, and 7. Tumors and mouse serumwere collected at indicated time points and kept frozen.Human MMP-1 and pro-MMP-10 level in mouse serumwere determined using the ELISA assays as described above.

Clinical study designThe phase IMFG4991g trial (35) was an open-label dose-

escalation study to evaluate the safety, tolerability, andpharmacokinetics of R3Mab (designated MFGR1877S) inpatients with solid tumors. The trial consisted of a doseescalation of single-agent R3Mab administered intrave-nously at 2, 4, 8, 15, and 30 mg/kg once every 28 days.Serum and urine samples were collected at designated timepoints, frozen in liquid nitrogen, and shipped to Genen-tech, Inc. for measurement of pharmacodynamic biomar-kers of R3Mab activity.

ELISA analyses of MMPs in human serum and urineThe levels of a panel of MMPs weremeasured by ELISA in

serum and urine samples from normal healthy volunteers,procured bladder cancer patients, and patients enrolled inthe phase I MFG4991g clinical trial. To this end, the humanMMP 3-Plex (i.e., MMP-1, MMP-3, and MMP-9; MesoscaleDiscovery) and human pro-MMP-10 immunoassay (R&DSystems) kits were utilized according to the manufacturer’sinstructions. For the human pro-MMP-10 immunoassaykit, serum samples were diluted 2-fold in the appropriateassay diluent. For the human MMP 3-Plex kit, serum sam-ples were diluted 10-fold. For all MMP kits, urine was usedundiluted. As a reference for quantification, a standardcurve was used for each kit according to the manufacturer’sdirections. Mann–Whitney tests were used for comparison

Du et al.

Clin Cancer Res; 20(24) December 15, 2014 Clinical Cancer Research6326




between two groups. A value of P < 0.05 was consideredstatistically significant.

ResultsFGFR3 knockdown inhibits MMP-1 and MMP-10expression and secretionUsing doxycycline-inducible shRNA targeting FGFR3, we

previously determined the transcriptional profile of RT112-derived cell lines that express endogenous or depleted levelsof FGFR3, using microarray analysis (36). To facilitateidentification of serumor urine pharmacodynamicmarkersof FGFR3-targeted therapeutics, we focused on secretedmolecules thatwere specificallymodulatedby FGFR3deple-tion. Comparing independently established stable cell linestransduced with a doxycycline-inducible control shRNAversus three independent FGFR3 shRNAs, we found thattwo matrix metalloproteinases, MMP-1 and MMP-10, wereamong the genes showing the greatest decline upon FGFR3knockdown (Supplementary Table S1). The transcriptsweredownregulated by about 16-fold and 4-fold for MMP-1 andMMP-10, respectively (Supplementary Fig. S1). Thesemicroarray results were further confirmed using qRT-PCRanalysis of the mRNA abundance of these two genes(Fig. 1A). In addition, the FGFR3-dependent regulation of

these genes was also verified in UMUC-14 bladder cancercells subjected to siRNA-mediated FGFR3knockdown (Sup-plementary Fig. S2A), or treated with a specific anti-FGFR3humanmonoclonal antibody, R3Mab (Supplementary Fig.S2B). Together, these data suggest that FGFR3 signalingpromotes transcription of MMP-1 and MMP-10 in bladdercancer cells.

Next,we examined the effect of FGFR3knockdownon theexpression and secretion of MMP-1 and MMP-10 proteins.RT112 cells were treated with or without doxycycline for 72hours to deplete FGFR3protein, and the conditionedmediawere collected at specific time points thereafter. FGFR3shRNAs substantially diminished the secretion of MMP-1and pro-MMP-10 as early as 24 hours after induction andthe decrease was sustained at 48 and 72 hours, whereas thecontrol shRNA had no effect (Fig. 1B). MMP-10 was down-regulated in a similar manner by FGFR3 shRNAs (Supple-mentary Fig. S3). Consistently, pretreatment with R3Mabalso blocked the production and secretion of MMP-1, pro-MMP-10, andMMP-10 proteins into conditionedmedia byboth RT112 and UMUC-14 cells (Fig. 1C and Supplemen-tary Fig. S4). Together, these results suggest that FGFR3signaling promotes the expression and secretion of MMP-1and MMP-10 proteins by these bladder cancer cell lines. As

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Figure 1. Inhibition of FGFR3reduces the mRNA and proteinexpression of MMP-1 and MMP-10 in RT112 bladder cancer cells.A, doxycycline-inducedknockdown of FGFR3 reducesmRNA levels of MMP-1 andMMP-10. B, doxycycline-inducedknockdown of FGFR3 reducestotal MMP-1 and pro-MMP-10secreted in the conditionedmedium. C, a specific FGFR3-blocking antibody, R3Mab,reduces total MMP-1, pro-MMP-10, and total MMP-10 secreted inthe conditioned medium by RT112cells.






the ELISA detection of pro-MMP-10 is more sensitive thanthat of MMP-10, and both pro- and enzymes show similarmodulation by FGFR3 inhibition, we focused on pro-MMP-10 protein levels in subsequent studies.

FGFR3 signaling promotes MMP-1 and MMP-10expression mainly through MEK

To study the molecular mechanisms underlying FGFR3regulation of MMP-1 andMMP-10 expression, we activatedFGFR3 signaling in bladder cancer cells with FGF1. We firstanalyzed themRNA levels of the twoMMPs. FGF1 treatmentof RT112 cells induces robust FGFR3 activation in a dose-and time-dependent fashion (36), and higher mRNA levelsof both MMP-1 and MMP-10 were detected after 6 or12-hour incubation (Fig. 2A). Consistent with thesechanges, secreted MMP-1 and pro-MMP-10 proteinsdetected in conditioned media were higher after a 24-hourincubation with FGF1 (Fig. 2B), and accumulated further at48 and 72 hours (data not shown).

To determine whether the induction of MMP-1 andMMP-10 by FGF1 depends on specific FGFR3 signalingmediators, we blocked the two major signaling branchesdownstream of FGFR3 using the PI3K inhibitor Ly294002and the MEK1/2 inhibitor PD325901. In RT112 bladdercancer cells, each inhibitor blocked FGF1-induced activa-

tion of its corresponding target, as assessed by the phos-phorylation of AKT or MAPK, respectively (Fig. 2C). WhilePI3K inhibition elicited minimal effect on the expressionand secretion of MMP-1 and MMP-10 proteins, MEK inhi-bition significantly diminished both basal and FGF1-induced expression of these two proteins (Fig. 2D). Thus,FGFR3 promotesMMP-1 andMMP-10 expression in RT112bladder cancer cells mainly through the MEK-MAPK axis.

R3Mab blocks FGFR3 signaling and reduces humanMMP-1 and MMP-10 protein levels in both bladdertumor xenograft tissues and mouse serum

To evaluate whether MMP-1 and MMP-10 could serveas potential pharmacodynamic markers for FGFR3-tar-geted therapeutics, we studied the effect of R3Mab on theexpression and secretion of these enzymes in vivo. TwoR3Mab-responsive tumor xenograft models, RT112 andUMUC-14, were selected for this study. Weekly intraper-itoneal administration of R3Mab suppressed xenograftgrowth of these models in a dose-dependent manner(Fig. 3A and Supplementary Fig. S5). First, we evaluatedFGFR3 signaling and MMP-1 and MMP-10 protein levelsin tumor xenograft tissues. Compared with the controlantibody, R3Mab markedly reduced the phosphorylationand activation of FRS2 and MAPK, two of the essential

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Figure 2. FGF1 signaling stimulatesMMP-1 and MMP-10 expressionthrough MEK. A, FGF1 inducesMMP-1 and MMP-10 mRNAexpression inRT112cells. B, FGF1treatment stimulates MMP-1 andpro-MMP-10 protein expressionand secretion into conditionedmedium by RT112 cells. C,pharmacologic inhibition of FGF1signaling in RT112 cells. RT112cells were treated with PD325901(PD901) and Ly294002 (Ly294) atindicated concentrations for3 hours, followed by FGF1(25 ng/mL) stimulation for 10minutes. Cell lysates weresubjected to Western blotanalysis. D, MEK inhibitor, butnot PI3K inhibitor, blockedFGF1-induced expression andsecretion of MMP-1 andpro-MMP-10 by RT112 cells.

Du et al.





downstream mediators of FGFR3 signaling (Fig. 3B).Concomitantly, both total MMP-1 and MMP-10 proteinlevels in the tumor lysates were decreased significantly inresponse to R3Mab (Fig. 3B).We next evaluated the effect of R3Mab on human

MMP-1 and MMP-10 protein levels in the circulatingblood from xenograft models expressing human bladdertumors. Nude mice with pre-established RT112 tumorswere treated with a low or a high dose of R3Mab (3 or30 mg/kg, respectively), and mouse serum was collectedat 48 or 96 hours after treatment. Human pro-MMP-10protein levels in mouse serum were reduced in a dose-dependent manner in R3Mab-treated group at 48 and 96hours, and the effect was more pronounced in the high-dose group at 96 hours (Supplementary Fig. S6). Weextended the analysis to days 4, 6, and 10 after treatment.Compared with the control antibody, R3Mab substan-tially and significantly suppressed the serum levels ofhuman MMP-1 and pro-MMP-10 throughout the timecourse (Fig. 3C and D). A similar reduction of serumlevels of these two proteins was induced by R3Mabtreatment of mice bearing UMUC-14 tumors (Supple-mentary Fig. S7A and S7B).

To assess whether the modulation of MMPs by R3Mabcorrelates with efficacy, we also analyzed pro-MMP-10levels in three xenograft models whose growth is not affect-ed by R3Mab, namely HT1376, UMUC-4, and SW780(Supplementary Fig. S8A and data not shown). Levels ofpro-MMP-10 were too low to be detected in serum frommice bearing UMUC-4 or HT1376 xenograft tumors (datanot shown).While pro-MMP-10was easily detected inmicewith SW780 tumors, levels of pro-MMP-10 were not affect-ed upon R3Mab treatment as observed in responsive mod-els (Supplementary Fig. S8B).

Together, these data demonstrated that R3Mab-basedinhibition of FGFR3 signaling and tumor growth occurs inconjunction with a substantial and sustained reduction ofMMP-1 and MMP-10 protein expression and secretion,suggesting that these two metalloproteinases could beexplored as potential pharmacodynamic markers of FGFR3pathway inhibition.

MMP-1 and pro-MMP-10 proteins are elevated in theurine of bladder cancer patients

Next, we evaluated MMP-1 and pro-MMP-10 proteinlevels in urine and serum specimens from healthy donors

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Figure 3. R3Mab attenuates RT112tumor growth, blocks FGFR3signaling and reduces humanMMP-1 and MMP-10 levels inxenograft tumor tissues andmouse serum. A, effect of R3Mabon pre-established RT112 bladderxenograft tumor growth. R3Mabwas administered intraperitoneallyweekly (arrows). n¼ 12 per group.B, effect of R3Mab on FGFR3signaling andMMP levels in tumorxenograft tissues. Mice with pre-established RT112 tumors weregrouped into cohorts of 5 fortreatment with either a controlantibody (Ctrl Ab) or R3Mab(30 mg/kg). Tumors wereharvested at day 7, and lysatesextracted from xenograft tumortissues were subjected toWesternblot analysis. C and D, the kineticsof R3Mab effect on serum levelsof human MMP-1 (C) andpro-MMP-10 (D) in mice bearingRT112 xenograft tumors. n ¼ 10per group.






and patients with bladder cancer (acquired from commer-cial sources). In urine samples from healthy donors (n ¼25), MMP-1 was mostly undetectable or below the lowerlimit of quantitation (LLOQ), but pro-MMP-10 was con-sistently detected above the LLOQ, albeit within a low andnarrow range (Fig. 4A). In contrast, both MMP-1 and pro-MMP-10proteinswere detected at significantly higher levelsin urine frompatients with bladder cancer (n¼ 35; Fig. 4A).A different pattern was observed in serum: both metallo-proteinases were detected above the LLOQ in all serumsamples from the same patients; however, there was nodifference in levels detected in healthy donors versuspatients with bladder cancer (Fig. 4B).

As R3Mab has been evaluated in a phase I dose-escalationclinical trial (MFG4991g) conducted in relapsed or refrac-tory, locally advanced, or metastatic solid tumors (Supple-mentary Table S2; ref. 35), analogous measurements ofMMP1andpro-MMP-10weremade in clinical pretreatmenturine and serum specimens (n ¼ 26). All patients withbladder cancer (n¼ 10) enrolled in this study have relapsedor refractory metastatic lesions, and 8 of 10 patients dis-played significantly higher urinary MMP-1 levels, with onlytwo patients having MMP-1 below the LLOQ. In contrast,urinary MMP-1 was undetectable or below the LLOQ in amajority of samples from patients with cancer with othersolid tumors, including colorectal, squamous cell carcino-ma of the head and neck, ovarian, thyroid, esophageal,adrenal, adenoid cystic carcinoma, or carcinoid tumor

(n ¼ 15; Fig. 4A). Similarly, elevated levels of pro-MMP-10 were observed in urine samples from patients withbladder cancer relative to patients with other solid tumors(Fig. 4A). In agreement with observations in the commer-cially procured specimens, there was no difference in serumMMP-1 and pro-MMP-10 levels between patients withbladder cancer or other solid tumors (Fig. 4B). Together,these data demonstrate that these two metalloproteinasesare significantly elevated in bladder cancer patient–derivedurine relative to analogous samples from healthy donors orfrom patients with several other solid tumors. However, wedid not observe a correlation between baseline MMP levelsand either FGFR3 protein expression level (as measuredretrospectively by immunohistochemistry) or clinicalresponse in the MFG4991g phase I trial (38).

R3Mab reduces urinary MMP-1 and pro-MMP-10 levelsin patients with bladder cancer

To examine whether MMP-1 and/or pro-MMP-10 couldbe used as pharmacodynamic biomarkers, patterns oftheir expression were also evaluated in urine samples fromthe MFG4991g phase I clinical trial collected predose, or atcycle 1, day 2 (C1D2), cycle 1, day 4 or 5 (C1D4D5) and,in some cases, at later time points depending on how longthe patient stayed on study and on availability of samples(Fig. 5). In all bladder cancer patients (based on 8 evaluableurine sample pairs), exposure to R3Mab reduced MMP-1levels by >50% (Fig. 5A and B). Assessment of

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Figure 4. MMP-1 and pro-MMP-10are elevated in bladder cancer(BLC) patient urine but not inserum. A, levels of MMP-1 andpro-MMP-10 in commerciallyprocured urine samples frompatients with bladder cancer(n ¼ 35) relative to healthy donors(n ¼ 25), or from the MFG4991gphase I clinical trial, includingpatients with bladder cancer(n ¼ 10) and patients with othersolid tumor types (non-BLC,n ¼ 16). Plotted n values arerepresentative of only thosesamples that were detectable(>LOD). The dotted line representsthe LLOQ. B, levels of MMP-1 andpro-MMP-10 in serum areequivalent in healthy donors,bladder cancer patients, and innon–bladder cancer patients.Note: only 5 healthy donor serumsamples were evaluated.

Du et al.





pharmacodynamic modulation in non–bladder cancerpatients was hampered by the limited number (3/16) ofsamples having sufficient MMP-1 levels at baseline. Whilemost samples were detectable, only 3 were above the lowestlevel of quantitation in the assay. However, in the cases inwhich MMP-1 could be quantified, MMP-1 levels werereduced by >50% (Fig. 5A and B).Unlike MMP-1, all urine samples had detectable pro-

MMP-10 levels for analysis. Pro-MMP-10 was reduced by>50%inhalf of thepatientswithbladder cancer treatedwithR3Mab (4/8 evaluable pairs). In contrast, there was noreduction in pro-MMP-10 in urine samples from patientswith non–bladder cancer (Fig. 5C andD). All of the patientswith bladder cancer that showed reduced levels of pro-MMP-10 were treated at the highest dose (30 mg/kg).However, two other patients in the highest dose cohort didnot show modulation of pro-MMP-10 levels. In contrast,MMP-1 was downmodulated upon antibody treatment inmost dose cohorts including the 8, 15, and 30 mg/kg dosecohorts.A number of MMPs have been postulated as potential

diagnostic and/or prognostic biomarkers in bladder cancer,but the role forMMPs as pharmacodynamic biomarkers haslargely been unexplored (39). To extend our preliminaryobservations, additional analysis was performed to evaluatetwo other MMPs, MMP-3, andMMP-9, which also harbor agroup 1 promoter element similar to MMP-1 and MMP-10(40). Statistical analysis (Mann–Whitney test) indicatedthat MMP-3 (P¼ 0.0251), but not MMP-9 was significantlyhigher in urine frompatients with bladder cancer relative tonon–bladder cancer patients in the MFG4991g phase I

study (Fig. 6A). Similar to pro-MMP-10 and MMP-1 data,no significant difference was detected in serum (Fig. 6B).Pharmacodynamic monitoring of these MMPs showed thatin most but not all cases MMP-3 and MMP-9 were down-modulated by R3Mab similarly to pro-MMP-10 andMMP-1in patient samples where all four MMPs were detectable(Fig. 6C). Together, these data suggested that anti-FGFR3therapy specifically downmodulated urinary, but notserum, MMP levels in patients with bladder cancer. It isworth noting that similar to observations at baseline, nocorrelation between pharmacodynamics measures andeither FGFR3 expression or clinical response was observed(38).

DiscussionBladder cancer is the secondmost commongenitourinary

tract malignancy, accounting for more than 73,000 newcases each year and about 15,000 deaths in 2012 in theUnited States alone (2). The receptor tyrosine kinase,FGFR3, has been postulated as an important oncogenicdriver and potential therapeutic target in this disease basedonmultiple lines of evidence, including a high frequency ofactivating mutations (17–20), aberrant regulation and acti-vation of FGFR3 by gene fusion with TACC3 or BAIAP2L1(21, 22), overexpression of FGFR3 protein in tumor tissues(19, 23), and a large body of preclinical loss-of-functionstudies (23, 27–31). Recent progress toward clinical inves-tigation of experimental agents targeting FGFR3 hasmade itimperative to identify accessible pharmacodynamic bio-markers to gauge target modulation and facilitate clinical

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Figure 5. MMP-1 and pro-MMP-10are reduced upon exposure toR3Mab in patients with bladdercancer in the MFG4991g phase Istudy. A, the overall urinary levelsof MMP-1 are reduced by R3Mabin patients with bladder cancer butnot in non–bladder cancerpatients. B, relative levels ofMMP-1 compared with predose levels.C, the overall urinary levels of pro-MMP-10 are reduced by R3Mab inpatients with bladder cancer butnot in non–bladder cancerpatients. D, relative levels of pro-MMP-10 compared with predoselevels. C1D2, cycle 1 day 2;C1D4D5, cycle 1, day 4 or day 5.






studies. Here, we report that FGFR3 stimulates MMP-1 andpro-MMP-10 expression and secretion in human bladdercancer cells through the MEK–MAPK pathway. BlockingFGFR3 signaling by a specific anti-FGFR3 human mono-clonal antibody, R3Mab, inhibited human tumor xenograftgrowth in association with a marked reduction in circulat-ing human MMP-1 and pro-MMP-10 levels. Moreover,patients with bladder cancer had an increased level ofurinary MMP-1 and pro-MMP-10 proteins. Importantly, ina phase I dose-escalation study of R3Mab, inhibition ofFGFR3 in patients with relapsed or refractory metastaticbladder cancer resulted in an acute reduction of urinaryMMP-1 and pro-MMP-10 levels. These results unveil animportant role for FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion in bladder cancer, andsupport further evaluation of these metalloproteinases aspotential pharmacodynamic markers in future FGFR3-tar-geted clinical studies.

In our unbiased expression analysis of genes encodingsecretedmolecules, we found that upon FGFR3 knockdownin RT112 bladder cancer cells, MMP-1, and pro-MMP-10were among the most prominently downregulated genes.Our studies further demonstrate that ligand-induced FGFR3activation promotes both mRNA and protein expression ofthese two metalloproteinases, and acute pharmacologicinhibition of canonical FGFR3 signaling markedlydiminishes the level of secreted MMP-1 and pro-MMP-10

in the conditionedmedium. This inhibitory effect is mainlymediated through the FGFR3–MEK–MAPK pathway, butnot the FGFR3-PI3K axis. These results extend earlier studiesby others showing that the expression of several MMP genesis regulatedby somegrowth factors and cytokines inbladdercancer. For example, basic FGF is able to stimulate theexpression of MMP-2 and MMP-9 in HT1376 cells (41),and EGF elicits the rapid induction of MMP-1 andMMP-10mRNA in T24 bladder cancer cells through the Stat3/acti-vator protein-1 (AP-1) family of transcription factors (42).Importantly, in the current study, by selectively blockingFGFR3 function with R3Mab (i.e., a specific anti-FGFR3monoclonal antibody), wewere able to clearly demonstratethat MMP-1 and pro-MMP-10 are bona fide downstreamtargets of FGFR3 signaling in bladder cancer. It is worthnoting that the current study does not exclude the possi-bility that other signaling pathways may also regulate thesetwo MMPs through MAPK or alternative mechanisms inbladder cancers.

To assess whether MMP-1 and pro-MMP-10 could serveas accessible pharmacodynamic biomarkers for FGFR3-tar-geted therapy, we have evaluated the serum and urinarylevels of these two proteins in healthy subjects and inpatients with bladder cancer, as well as in patients withvarious solid cancers enrolled in the MFG4991g phase Istudy of R3Mab. Compared with healthy controls andpatients with non–bladder cancer malignancies, patients

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Figure 6. Levels and modulation ofMMP-3 and MMP-9 by R3Mab inthe MFG4991g phase I study.A, urinary MMP-3 is elevated inpatients with bladder cancerrelative to non–bladder cancerpatients in the MFG4991g phase Istudy. On the basis of Mann–Whitney test, P ¼ 0.0251 forMMP-3; P > 0.05 for MMP-9. B, nocorresponding difference of theseMMPs was detected in serum. C,samples (n ¼ 7) with detectablelevels of all four MMPs showedvariablemodulation in response toR3Mab administration. C1D2,cycle 1 day 2; C1D4D5, cycle 1,day 4 or day 5.

Du et al.





with advanced bladder cancer showed significantly elevatedurine levels of MMP-1 and pro-MMP-10 protein. In con-trast, the concentration of these two proteins in circulatingblooddisplayednodifference amongst the different groups.This observation is in agreement with several publishedstudies. MMP-1 and MMP-10 mRNA levels have beenreported to be significantly higher in bladder cancers com-pared with normal urothelial tissues (43, 44), and MMP-1and pro-MMP-10 immunoreactivity is evidently stronger intumor cells of both superficial and muscle-invasive bladdercancers (45, 46). In addition, patients with bladder cancerwith an increased urinary MMP-1 protein level are morelikely to have an aggressive tumor of advanced stage andgrade (47, 48). Although published data on urinary pro-MMP-10 in patients with bladder cancer is more limited, arecent study demonstrates that a higher urinary pro-MMP-10 concentration could serve as a potential diagnosticbiomarker when used in combination with a panel of sixother analytes (44). Taken together, the differential expres-sion pattern between patients with bladder cancer andhealthy subjects, the relative ease in acquisition of voidurine samples, and the robust detection of urinary MMP-1and pro-MMP-10 levels suggest that these two metallopro-teinases could serve as useful and clinically relevant non-invasive biomarkers.One important finding of our current study is that in

preclinical bladder cancer xenograft models, R3Mab treat-ment inhibited FGFR3 signaling, attenuated tumor growth,and reducedMMP-1 andpro-MMP-10protein expression intumor lysates as well as in mouse serum. Consistent withthese preclinical studies, our phase I study with 26 patientsdemonstrated that administration of R3Mab led to an acutereduction of urinary MMP-1 by more than 50% in 8 of 8evaluable patients with bladder cancer. The evaluation innon–bladder cancer patients was hampered by the fact thatvery few patients had measureable levels of MMP-1 atbaseline. In the only 3 patients (2 colorectal and 1 headand neck SCC) that were evaluable, MMP-1 was alsomarkedly reduced. It is noteworthy that although a mini-mum threshold level of MMP-1 is required for monitoringpharmacodynamic modulation, the extent of downmodu-lation was not correlated with the baseline values. A similareffect was observed for pro-MMP-10, as R3Mab resulted in adecrease of urinary pro-MMP-10, albeit in only 4 of 8patients with bladder cancer. Interestingly, no modulationof pro-MMP-10 was detected in non–bladder cancerpatients, suggesting that pro-MMP-10 might be more spe-cific for bladder cancer.Additional MMPs were monitored in the clinical study.

Similar to MMP-1 and pro-MMP-10, urinary MMP-3, andto a lesser extent, MMP-9, were also elevated in bladderrelative to non–bladder cancer patients. In a majority ofpatients, there was a similar pattern of downmodulationof these MMPs by R3Mab despite some noticeable differ-ences. This observation could be partially due to the factthat these MMPs all have a proximal AP-1 binding sitein their promoters (40); conceivably, R3Mab-mediatedinhibition of the MEK-MAPK-AP-1 axis may result in

decreased transcription of these genes. Indeed, in 4T1breast cancer cells, the pan-FGFR inhibitor TKI258 sup-presses the transcription of MMP-1, -3, -9, and -10 simul-taneously (49).

The matrix metalloproteinases degrade extracellularmatrix components and activate growth factors, contribut-ing to tumor growth, progression, and metastasis (50).Several MMPs have been postulated to be diagnostic orprognostic markers in bladder cancer (39, 50). Our currentstudy demonstrates that FGFR3 signaling directly regulatesMMP-1 and pro-MMP-10 expression and secretion in blad-der cancer through the MEK–MAPK pathway. UrinaryMMP-1 and pro-MMP-10 levels were reduced substantiallyby R3Mab treatment in both preclinical models andpatients with bladder cancer. This is the first study, to ourknowledge, that identifies urinaryMMP-1 and pro-MMP-10as potential pharmacodynamic biomarkers. While weobserved clear pharmacodynamic modulation in responseto R3Mab administration, there did not appear to be acorrelation betweenmodulation and dose or FGFR3 expres-sion level or clinical benefit (as assessed by time on study ordisease progression). The lack of correlation could be due tosmall sample size and/or a change in FGFR3 expressionlevels during disease progression or prior drug treatment.Although this phase I study was not powered to assesspredictive biomarkers, the data presented here warrantfurther investigation in larger studies using these potentiallyactionable pharmacodynamic biomarkers that can be usedin the clinic in a noninvasive manner to stratify patientswith elevated MMP-1 and pro-MMP-10 profiles and poten-tially monitor therapeutic response.

Disclosure of Potential Conflicts of InterestJ. Qing, X. Du, B.C. Lin, Q.-R. Wang, H. Li, E. Ingalla, J. Tien, I. Rooney,

A. Ashkenazi, and E. Penuel are employees of Genentech. No other potentialconflicts of interest were disclosed.

Authors' ContributionsConception and design: B.C. Lin, A. Ashkenazi, E. Penuel, J. QingDevelopment of methodology: X. Du, B.C. LinAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): X. Du, B.C. Lin, Q.-R. Wang, H. Li, E. Ingalla,J. Tien, I. RooneyAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): X. Du, B.C. Lin, Q.-R. Wang, H. Li,E. Ingalla, J. Tien, A. Ashkenazi, E. Penuel, J. QingWriting, review, and/or revision of the manuscript: B.C. Lin, J. Tien,I. Rooney, A. Ashkenazi, E. Penuel, J. QingAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B.C. Lin, J. TienStudy supervision: J. Tien, I. Rooney, J. Qing

AcknowledgmentsThe authors thank Zora Modrusan for microarray profiling and Jinfeng

Liu for the informatics analysis of the array data. The authors also thankMarkDavis and Genentech’s FGFR3 development team for designing and con-ducting the phase I clinical trial of R3Mab.

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 December 11, 2013; revised September 18, 2014; acceptedOctober 2, 2014; published OnlineFirst October 17, 2014.






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2014;20:6324-6335. Published OnlineFirst October 17, 2014.Clin Cancer Res Xiangnan Du, Benjamin C. Lin, Qian-Rena Wang, et al. CancerBiomarkers of FGFR3-Targeted Therapy in Patients with Bladder MMP-1 and Pro-MMP-10 as Potential Urinary Pharmacodynamic

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