activation of the fgfr stat3 pathway in breast cancer ... · l.r. bohrer and p. chuntova...

Tumor and Stem Cell Biology

Activation of the FGFR–STAT3 Pathway in Breast CancerCells Induces a Hyaluronan-Rich Microenvironment ThatLicenses Tumor Formation

Laura R. Bohrer1, Pavlina Chuntova4, Lindsey K. Bade5, Thomas C. Beadnell1, Ronald P. Leon1,Nicholas J. Brady4, Yungil Ryu1, Jodi E. Goldberg7, Stephen C. Schmechel1,6,8, Joseph S. Koopmeiners3,James B. McCarthy1,2, and Kathryn L. Schwertfeger1,2

AbstractAberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth,

progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerouseffects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanismsthroughwhich aberrant FGFR activity contributes to breast cancer are not completely understood.We show herethat FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blockinghyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-medi-ated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression ofhyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growthand hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibitingboth FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be arelevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, thesestudies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces aprotumorigenic microenvironment. Cancer Res; 74(1); 374–86. �2013 AACR.

IntroductionRecent genomic profiling studies have demonstrated that a

number of potentially targetable pathways are aberrantlyregulated in breast cancer, including the fibroblast growthfactor receptor (FGFR) pathway (1). Members of the FGFRfamily, composed of four FGFR genes, are transmembranereceptor tyrosine kinases that are activated by FGFs (2).Aberrant FGFR activity in breast cancers can occur througha variety of potential mechanisms, including amplification ofreceptor genes, increased protein expression of both ligands

and receptors, single-nucleotide polymorphisms, gene rear-rangements, and mutations in FGFRs, all of which have beenidentified in human breast cancer cell lines and patient sam-ples (3, 4). Experimental studies have demonstrated that FGFRactivation contributes to breast cancer growth and progres-sion (5–11). Furthermore, a number of clinical trials have beeninitiated to investigate the safety and efficacy of smallmoleculeFGFR inhibitors in breast and other cancers (3, 5).

To study FGFR1 activation, we use an inducible FGFR1(iFGFR1) construct containing a dimerization domain that isactivated with the synthetic homodimerizer B/B, resulting insustained activation of FGFR1-induced signaling pathways(11). Using this inducible model, our studies have focused onthe mechanisms through which FGFR1 activation in epithelialand tumor cells contributes to tumor initiation and growth(12–16). Specifically, we have shown that aberrant FGFR1activation in mammary epithelial cells leads to alterations inthe stroma, including the generation of a localized inflamma-tory response and alterations in the extracellular matrix(11, 16). In the studies described here, we demonstrate thatactivation of FGFR signaling pathways leads to structuralmodifications of the extracellular matrix component hyalur-onan. Hyaluronan is a glycosaminoglycan that interacts withcancer cells through various receptors, including CD44 andreceptor for hyaluronan-mediated motility (RHAMM) to pro-mote proliferation and migration. Furthermore, aberrant hya-luronan synthesis has been linked to breast cancer growth and

Authors' Affiliations: 1Department of Lab Medicine and Pathology;2Masonic Cancer Center; 3Biostatistics and Bioinformatics Core, MasonicCancer Center; 4Microbiology, Immunology and Cancer Biology GraduateProgram; 5Graduate Program in Molecular, Cellular, Developmental Biol-ogy, and Genetics; 6BioNet, Academic Health Center, University of Min-nesota, Minneapolis; 7Hamline University, Biology Department, Saint Paul,Minnesota; and 8Department of Pathology, University of Washington,Seattle, Washington

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

L.R. Bohrer and P. Chuntova contributed equally to this work.

Corresponding Author: Kathryn L. Schwertfeger, 2231 6th Street South-east, University of Minnesota, Minneapolis, MN 55455. Phone: 612-626-9419; Fax: 612-624-3869; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2469

�2013 American Association for Cancer Research.

CancerResearch

Cancer Res; 74(1) January 1, 2014374

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progression (17–21). We demonstrate here that FGFR activa-tion leads to increased synthesis of hyaluronan, which con-tributes to proliferation, migration, and resistance to chemo-therapy. Thus, these studies link aberrant activation of growthfactor receptor signaling pathways in tumor cells to protu-morigenic modifications in the surrounding stroma.Because hyaluronan is often associated with an inflamma-

tory environment (22), further studies examined the contribu-tion of FGFR-induced inflammatory pathways to hyaluronansynthesis. We demonstrate that activation of FGFR leads toincreased production of proinflammatory cytokines, includingmembers of the interleukin (IL)-6 family, which activate theSTAT3 pathway. STAT3 is a proinflammatory transcriptionfactor that contributes to breast cancer cell proliferation,migration, invasion, and chemotherapeutic resistance (23–27). In these studies, we demonstrate that FGFR-inducedSTAT3 activation contributes to hyaluronan synthesis and isimportant for FGFR-driven mammary tumor growth. Thesestudies are the first to identify hyaluronan as a downstreamtarget of FGFR activation and suggest that the addition ofmicroenvironment-targeted therapies may enhance the effi-cacy of FGFR-specific therapies in cancers associatedwith highlevels of FGFR activity.

Materials and MethodsCell cultureGeneration of HC-11 cells stably expressing the iFGFR1

construct (HC-11/R1 cells) was described previously (28), andcells were obtained from Dr. Jeff Rosen (Baylor College ofMedicine, Houston, TX) and maintained as described previ-ously (28). Of note, Hs578T, MCF-7, and MDA-MB-453 cellswere obtained from the American Type Culture Collection andmaintained as suggested. For experiments, Hs578T cells weregrown on plates coated with 1.2% polyHEMA [poly(2-hydro-xyethylmethacrylate); (Sigma)]. All cell lines were used forfewer than 6 months after resuscitation. HC-11 cells andHC-11/R1 cells are not maintained for longer than 20 passagesand are tested for mycoplasma, b-casein expression, andiFGFR1 expression regularly.

Immunoblot analysisSerum-starved cells were treated with B/B (Clontech) or

basic FGF (bFGF; Invitrogen). For blocking and inhibitorstudies, gp130-blocking antibody (R&D Systems), doxorubicin(Boynton Pharmacy), Stattic (Sigma), and/or 4-methylumbel-liferone (4-MU; Sigma) were used. ON-TARGETplus SMART-pool STAT3 and nontargeting siRNA (Thermo Scientific) wereused according to the manufacturer's instructions. Cells werelysed in radioimmunoprecipitation assay (RIPA) buffer andequal amounts of protein were analyzed by SDS–PAGE. Immu-noblot analysis was performed with the following antibodies:pSTAT3Ser727 (9134), pSTAT3Tyr705 (9131), STAT3 (9132),cleaved caspase-3 (9661), and b-tubulin (2146; Cell SignalingTechnology).

ELISASerum-starved HC-11/R1 cells were treated with B/B or

ethanol as solvent control. Hs578T cells were treated with or

without bFGF. At 2, 6, and 24 hours, conditioned media wascollected and ELISAs for mouse or human LIF, IL-6, and IL-11were performed according to the manufacturer's protocol(R&D Systems). ON-TARGETplus SMARTpool-siRNA Has2 ornontargeting siRNA (Thermo Scientific) was transfected intoHC-11/R1 cells as described previously (28). Cells were pre-treatedwith 4-MU for 1 hour before addition of B/B. To analyzehyaluronan synthesis, conditioned media samples were col-lected and tumor samples were lysed in RIPA and analyzedusing the hyaluronan ELISA (R&D Systems) according to themanufacturer's protocols.

Quantitative reverse transcription PCRCells were treated as described above and quantitative

reverse transcription (qRT)-PCR was done as described pre-viously and normalized to cyclophilin B levels (29). The fol-lowing mouse primers were used: cyclophilin B 50-TGCAGG-CAAAGACACCAATG-30 and 50-GTGCTCTCCACCTCCCGTA-30, Lif 50-GCCTCCCTGACCAATATCACC-30 and 50-GACGGCA-AAGCACATTGCTG-30, Il-6 50-TAGTCCTTCCTACCCCAATTT-CC-30 and 50-TTGGTCCTTAGCCACTCCTTC-30, and Has2 50-TGTGAGAGGTTTGTATGTGTCCT-30 and 50-ACCGTACAGT-GGAAATGAGAAGT-30.

TUNEL assaysSerum-starved cells were treated with B/B or ethanol,

doxorubicin or saline, and Stattic or dimethyl sulfoxide(DMSO) for 20 hours. Cells were fixed and stained using theDeadEnd Fluorometric TUNEL (terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling) System (Pro-mega) according to the manufacturer's protocol. Five repre-sentative pictures were taken of each treatment, and cellcounting was performed in a blinded manner.

MiceThree- to 4-week-old Balb/c female mice were purchased

from Harlan Laboratories. Of note, 250,000 HC-11/R1 cells in50% Matrigel (BD Biosciences) were injected into the fourthinguinal mammary fat pads. Mice were given twice weeklyintraperitoneal injections of B/B to activate iFGFR1. Whentumors reached at least 100 mm3, mice were given Stattic orsolvent control (DMSO) by oral gavage 5 days a week for 3weeks. At least 3 mice were in each treatment group. For thestudies with transgenic mice, mouse mammary tumor virus(MMTV)–iFGFR1 mice were treated with B/B and mammaryglands were isolated as described previously (16). All animalcare and procedures were approved by the Institutional Ani-mal Care and Use Committee of the University of Minnesota(UMN; Minneapolis, MN) and were in accordance with theprocedures detailed in the Guide for Care and Use of Labora-tory Animals.

Clinical cohort and TMA constructionSpecimens and associated clinical data were obtained from

the UMN BioNet core facility (www.bionet.umn.edu) afterapproval from the UMN Institutional Review Board (IRB).Archival formalin-fixed paraffin-embedded tissues frompatients with breast cancer treated at the UMNwere collected.

FGFR-Induced STAT3 Regulates Hyaluronan Production

www.aacrjournals.org Cancer Res; 74(1) January 1, 2014 375



Areas of invasive carcinoma were verified by a pathologist.Tissue microarray (TMA) blocks consisting of quadruplicate1 mm core carcinoma samples were constructed using amanual tissue arrayer-1 (Beecher Inc). Clinical characteristicswere abstracted from pathology reports. Coded specimens anddata were provided for this study. Patient identifiers were notavailable to the authors, but rather were heldwithin the BioNetoffice per the BioNet IRB approval.

ImmunohistochemistryMammary glands from mice were fixed, sectioned, and

stained using sodium citrate antigen retrieval as describedpreviously (14, 16). Antibodies used were pStat3Tyr705 (1:200;Cell Signaling Technology; 9145), hyaluronan synthase 2 (Has2;1:50; Santa Cruz Biotechnology; sc-365263), and phosphorylatedFGFR substrate 2 (pFRS2; 1:40; R&D Systems; AF5126). As acontrol, sections were stained with the biotinylated anti-rabbitonly. For hyaluronan-binding protein (HABP) staining, sectionswere blocked with 3% bovine serum albumin and then incu-bated overnight with 2 mg/mL biotinylated HABP. Visualizationwas performed as described above. As a control, tissues wereincubated with hyaluronidase before addition of HABP. HABP–positive stromal thickness of at least 50 ducts from 3 mice pertime point was quantified using Leica LAS software.

Three-dimensional culturePrimary mammary epithelial cells were isolated from

MMTV–iFGFR1 transgenic mice and plated in Matrigel asdescribed previously (30). About 10,000 Hs578T cells wereplated per well in Matrigel. Of note, 4-MU or DMSO (solvent)was added to the cultures for 8 days. At least 50 acini weremeasured for each condition using Leica LAS software.

Proliferation assayFor HC-11/R1 cells, Thiazolyl Blue Tetrazolium Bromide

(Sigma; M2128) was used. Serum-starved HC-11/R1 cells weretreated with B/B, 4-MU, or Stattic. MTT reagent was added tothe wells and after 2 hours proliferation was determinedaccording to the manufacturer's protocol. For Hs578T cells,the CellTiter 96 Aqueous Proliferation Assay (Promega) wasused according to the manufacturer's protocol. Briefly, 1� 105

cells/mL were resuspended in serum-free media in 96-wellplates coated with 1.2% polyHEMA. The next day cells weretreated with bFGF and either Stattic or 4-MU. The absorbancewas read at 490 nm following 24 and 48 hours of treatment.

Migration assayConfluent HC-11/R1 cells were serum-starved and wound-

healing assays were performed as described previously (28) inthe presence of B/B and/or 4-MU and/or Stattic. Area of woundclosure was quantified following 18 hours using Leica LASsoftware.

Statistical analysisExperiments were performed at least three separate times.

Statistical analysis was performed using the unpaired Studentt test to compare twomeans. In all figures, error bars representthe SEM. For the human samples, the association between

pFRS2 and phosphorylated STAT3 (pSTAT3) was evaluatedusing proportional odds logistic regression with pSTAT3 as theoutcome and pFRS2 as a covariate in a univariate regressionmodel, and the association between pFRS2 and pSTAT3 wassummarized by the OR.

ResultsFGFR activation induces synthesis of hyaluronan

We have previously used the MMTV–iFGFR1 transgenicmouse model to identify mechanisms through which FGFR1activation in mammary epithelial cells contributes to protu-morigenic changes within the stroma (12, 14–16, 28, 30). It hasbeen previously noted that activation of iFGFR1 in mammaryepithelial cells results in alterations in the surrounding extra-cellular matrix (11, 16). To gain insights into these changes,previously publishedmicroarray studies (16) were examined toidentify extracellular matrix–related genes that are regulatedfollowing iFGFR1 activation. Interestingly, Has2, which stimu-lates synthesis of the extracellular matrix component hyalur-onan, was significantly induced following 8 hours of B/Btreatment. To verify these findings, mammary glands fromB/B–treated MMTV–iFGFR1 mice were analyzed for HAS2expression and hyaluronan accumulation using immunohis-tochemical analysis. Following 48 hours of iFGFR1 activation,increased expression of HAS2 was detectable within the aber-rant budding epithelial structures (Fig. 1A). Furthermore,increased accumulation of hyaluronan in the surroundingstroma was determined by measuring the thickness of hyalur-onan-positive stroma (Fig. 1A and B).

To verify that activation of iFGFR1 leads to increasedexpression of Has2 in epithelial cells, HC-11 cells that stablyexpress iFGFR1 (HC-11/R1; ref. 28) were treated with B/Bto activate iFGFR1. Has2 expression increased followingiFGFR1 activation as shown by qRT-PCR analysis (Fig. 1C).Furthermore, increased levels of hyaluronan were detectedin the media of B/B–treated HC-11/R1 cells using an ELISA-based assay (Fig. 1D). Analysis of FGF-responsive humanbreast cancer cell lines, including the estrogen receptor–pos-itive cell line MCF-7 and the triple-negative cell line Hs578T,demonstrated that bFGF treatment also led to increased levelsof hyaluronan in the media (Fig. 1E and F). To confirm thatHAS2 is the primary hyaluronan synthase that contributes tohyaluronan synthesis, Has2 knockdown was performed usingsiRNA (Fig. 1G) and resulted in a significant decrease inhyaluronan synthesis (Fig. 1H). Together, these studies dem-onstrate that FGFR activation induces expression of HAS2,which leads to increased synthesis of hyaluronan in vitro andin vivo.

Inhibition of hyaluronan synthesis leads to decreasedproliferation, migration, and chemoresistance

Because hyaluronan has been linked to tumor growth andprogression (17–19), further studieswere performed to examinethe contributions of hyaluronan to proliferation andmigration.For these studies, cells were treated with 4-MU, which inhibitshyaluronan synthases, including HAS2 (31–33). As shownin Fig. 2A, treatment of HC-11/R1 cells with 4-MU effectivelyinhibited both basal and iFGFR1-induced hyaluronan synthesis.

Bohrer et al.

Cancer Res; 74(1) January 1, 2014 Cancer Research376



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Figure 1. FGFR activation leads to increased production of hyaluronan (HA). A, MMTV–iFGFR1 transgenic mice were treated with 1 mg/kg B/B orsolvent for 48 hours. Mammary gland sections were stained with HAS2-specific antibody or HABP. Magnification bars, 50 mm. B, quantificationof hyaluronan-positive stromal thickness in mammary glands from solvent or B/B–treated mice. C, HC-11/R1 cells were treated with solvent(�B/B) or 30 nmol/L B/B and Has2 gene expression was analyzed by qRT-PCR. D, HC-11/R1 cells were treated as described in C andhyaluronan levels in conditioned media were determined by ELISA. E and F, MCF-7 (E) and Hs578T (F) cells were treated with or without 50 ng/mLbFGF for 18 hours and hyaluronan in the conditioned media was determined by ELISA. G, HC-11/R1 cells were treated with Has2 siRNA ora nontargeting control. Expression of Has2 was measured by qRT-PCR. H, amount of hyaluronan was determined by ELISA. �, P < 0.05; ��, P < 0.01;and ��, P < 0.001.





Furthermore, treatment of HC-11/R1 cells with 4-MU inhibitediFGFR1-induced migration (Fig. 2B) and proliferation (Fig. 2C).In addition, the contribution of hyaluronan to iFGFR1-inducedsurvival in response to chemotherapy was examined by ana-lyzing apoptosis following exposure of cells to doxorubicin.Activation of iFGFR1 significantly decreased doxorubicin-induced apoptosis in the HC-11/R1 cells, which was partiallyreversed by 4-MU, demonstrating that hyaluronan contributesto iFGFR1-induced chemoresistance. Similarly, treatment ofHs578T cells with bFGF and 4-MU inhibited bFGF-inducedproliferation (Fig. 2E) and restored apoptosis in response todoxorubicin treatment (Fig. 2F). Interestingly, we found thattreatment of both cell lines with 4-MU had effects on cellbehavior independently of FGFR activation, possibly due to thedecreased levels in basal hyaluronan synthesis (Fig. 2A). Theseresults suggest that there are likely to be additionalmechanismsthat regulate hyaluronan synthesis in the absence of FGFRactivation, which may contribute to the 4-MU induced inhibi-tion of FGFR-induced phenotypes.

FGFR activation leads to increased phosphorylation ofSTAT3

Next, we examined the mechanisms involved in mediatingFGFR-induced hyaluronan synthesis. Because hyaluronan isinvolved in inflammation (22, 34), our initial studies focused onexamining inflammatory mediators such as STAT3, whichregulates inflammation-related genes, including Has2 (35). Toexamine STAT3 activation in the iFGFR1 model, HC-11/R1cells were treatedwith B/B and phosphorylation of STAT3Ser727

and STAT3Tyr705 was assessed by immunoblot analysis. Similarto what has been shown previously (36), STAT3Ser727 wasphosphorylated within 15 minutes of iFGFR1 activation,although pSTAT3Tyr705 was not detected at these time points(Fig. 3A). However, analysis of later time points demonstratedthat pSTAT3Tyr705 was detectable following 2 hours of iFGFR1activation and remained elevated throughout the 24-hour timecourse (Fig. 3B).

The timing of STAT3Tyr705 phosphorylation led to thehypothesis that FGFR induces STAT3Tyr705 phosphorylation

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Figure 2. Blocking hyaluronan (HA)synthesis leads to decreasedmigration, proliferation, andchemoresistance. A, HC-11/R1cells were treated with 250 mmol/Lof the HAS2 inhibitor 4-MU orsolvent, followed by the addition of30 nmol/L B/B or solvent.Hyaluronan was detected inconditionedmedia by ELISA. B andC, HC-11/R1 cells were treatedwith B/B, 250 mmol/L 4-MU, and/orsolvent. The change in woundclosure was determined at 18hours (B) and proliferation wasmeasured by an MTT assay (C) atday 1 or 2. D, HC-11/R1 cellswere treated with B/B, 4-MU[62.5 mmol/L (þ), 125 mmol/L (þþ),and 250 mmol/L (þþþ)], and 2mmol/L doxorubicin for 24 hours.Levels of cleaved caspase-3 andthe loading control b-tubulin wereexamined by immunoblot analysis.E, Hs578T cells were treated with50 ng/mL bFGF and 25 mmol/L4-MU or solvent. Proliferation wasmeasured at either day 1 or 2relative to solvent-only treatedsamples. F, Hs578T cells weretreated with bFGF, 4-MU [125mmol/L (þ) and 250 mmol/L (þþ)],and doxorubicin and levels ofcleaved caspase-3 and b-tubulinwere examined. �, P < 0.05;��, P < 0.01; and ��, P < 0.001.

Bohrer et al.




indirectly through inducing production of soluble factors thatactivate STAT3. To address this possibility, HC-11/R1 cellswere treated with B/B for 18 hours and conditioned mediawere used to stimulate parental HC-11 cells. pSTAT3Tyr705 waselevated in theHC-11 cells as early as 5minutes after treatmentwith conditioned media (Fig. 3C), consistent with the hypoth-esis that soluble factors produced by the cells following iFGFR1activation contribute to STAT3 activation.Further studies were performed to identify soluble factors

that induce STAT3Tyr705 phosphorylation in our system. BecauseSTAT3 is a well-established downstream target of IL-6 familycytokines (37), the ability of iFGFR1 to induce expression ofgenes in the IL-6 family was assessed. Initial screening of HC-11/R1 cells demonstrated that iFGFR1 activation led to increasedgene expression of Il-6, Lif, and IL-11 (Fig. 3D and E; data notshown). ELISA analysis of conditioned media confirmed thatsoluble LIF was detectable within 2 hours of iFGFR1 activation(Fig. 3F) and soluble IL-6was detectablewithin 6 hours (Fig. 3G).However, increased IL-11was not found in themedia at any time

point (data not shown), suggesting that IL-6 and LIF are theprimary IL-6 family cytokines produced by HC-11/R1 cells inresponse to iFGFR1 activation. Initial studies using cytokine-specific blocking antibodies demonstrated that blocking a singlecytokine was unable to completely abolish STAT3 phosphory-lation (data not shown), possibly due to ligand redundancy. IL-6family cytokines use the common receptor subunit gp130 totransmit their signals (38). As shown in Fig. 3H, treatment ofHC-11/R1 cell with a gp130-blocking antibody before activationof iFGFR1 led to a dose-dependent reduction of STAT3Tyr705

phosphorylation. These results demonstrate that activation ofiFGFR1 leads to production of IL-6 family cytokines, which actthrough gp130 to induce phosphorylation of STAT3Tyr705.

FGFR activation induces expression of IL-6 familycytokines and STAT3 activation in human breast cancercells

Further studies focused on validating FGFR-inducedSTAT3Tyr705 phosphorylation in other FGF-responsive cell

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Figure 3. Activation of iFGFR1 leads to increased pSTAT3Tyr705 in a gp130-dependent manner. HC-11/R1 cells were stimulated with solvent (�B/B) or30 nmol/L B/B, followed by protein and RNA extraction or collection of conditioned media. A and B, pSTAT3S727, pSTAT3Y705, and STAT3(loading control) were examined using immunoblot analysis. C, conditioned medium (CM) samples from treated HC-11/R1 cells were collected andadded to HC-11 cells. pSTAT3Y705 and STAT3 were examined using immunoblot analysis. D and E, qRT-PCR analysis was performed to assess Lifor IL-6 gene expression. F and G, LIF and IL-6 expression in conditioned medium was assessed by ELISA. H, HC-11/R1 were treated as abovewith the addition of IgG control or gp130-blocking antibody (0.05, 0.5, and 5 mg/mL) for 6 hours. Expression of pSTAT3Y705 and STAT3 was examined byimmunoblot analysis. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.





lines, including MCF-7, MDA-MB-453, and Hs578T (12, 39, 40).Following treatment of cells with bFGF, pSTAT3Tyr705 wasobserved at later time points, including 2 and 6 hours post-treatment, in all cell types (Fig. 4A–C), similar to what wasobserved with the HC-11/R1 cells. Furthermore, bFGF treat-ment of Hs578T cells led to increased production of IL-6 familycytokines, including IL-6 (Fig. 4D) and IL-11 (Fig. 4E), althoughLIF was not induced in these cells (data not shown). Finally,treatment of cells with a gp130-blocking antibody led todecreased pSTAT3Tyr705 in a dose-dependent manner (Fig.4F). These results verify our findings from the HC-11/R1modelthat activation of endogenous FGFR induces expression ofIL-6 family cytokines, which contribute to phosphorylation ofpSTAT3Tyr705.

To determine whether the FGFR and STAT3 signaling path-ways are correlated in human breast cancers, a TMA wasgenerated (Supplementary Table S1) and stained with anti-

bodies that recognize pSTAT3Tyr705 and pFRS2, which isindicative of activated FGFR (41). As control, tissues werestained with secondary antibody only and positive stainingwas not detected (Supplementary Fig. S1A). Both sets ofsamples were scored for weak, moderate, and strong staining(Fig. 4G). Similar to previously published results (24, 38, 42),pSTAT3Tyr705 was observed at moderate to strong levels inapproximately 60% of breast cancers (Fig. 4H). pFRS2 stainingwas found to be detectable at some level in all samples, withapproximately 85% of tumors expressing moderate to highlevels of staining (Fig. 4H), suggesting that FGFR activity ispresent in a large percentage of breast cancer samples. Analysisof all samples revealed a significant association betweenpFRS2 and pSTAT3 (OR, 4.8; 95% confidence interval, 1.91–12.05; P < 0.001), demonstrating a correlation between FGFRactivation and STAT3Tyr705 phosphorylation in a proportionof breast cancers (Supplementary Table S1).

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Figure 4. FGFR activation leads to pSTAT3Tyr705 in human breast cancer cells. Hs578T (A), MCF7 (B), and MDA-MB-453 (C) were treated with or without50 ng/mL bFGF, and expression of pSTAT3Y705 and STAT3 was examined by immunoblot analysis. D and E, Hs578T cells were treated as describedabove for the indicated times, and conditioned medium samples were collected to examine protein expression of IL-6 (D) or IL-11 (E) by ELISA.F, Hs578T cells were treated as above with the addition of IgG control or gp130-blocking antibody (0.1, 1, and 10 mg/mL) for 6 hours. G and H, a humanbreast cancer TMA was stained for pSTAT3Tyr705 and pFRS2 using immunohistochemistry. G, representative images of weak, moderate, or strongstaining intensity are shown. Magnification bars, 50 mm. H, percentage of cases based on staining intensity. ��, P < 0.01; ��, P < 0.001.

Bohrer et al.




STAT3 contributes to FGFR-induced migration,proliferation, and resistance to chemotherapyTo assess the functional contributions of STAT3 activa-

tion to FGFR-induced tumorigenic phenotypes, we used thepharmacologic inhibitor Stattic (43). Inhibition of STAT3 inHC-11/R1 cells led to decreased iFGFR1-induced migration(Fig. 5A) and proliferation (Fig. 5B). Because STAT3 hasalso been linked to resistance to chemotherapy (44), thecontribution of STAT3 activation to FGFR-induced che-moresistance was examined. Stattic restored the sensitivityof HC-11/R1 cells to doxorubicin, suggesting thatSTAT3 contributes to iFGFR1-induced chemoresistance(Fig. 5C).To verify the contribution of STAT3 to proliferation and

chemotherapeutic resistance following endogenous FGFRactivation, these processes were assessed in Hs578T cells.Consistent with the results from the HC-11/R1 cells, treat-ment of Hs578T cells with bFGF in the presence of Stattic

decreased bFGF-induced proliferation and reversed resis-tance to doxorubicin-induced apoptosis (Supplementary Fig.S2). To demonstrate that these effects are due to loss ofSTAT3 activity and not due to off-target effects of Stattic,STAT3 expression was decreased using siRNA. As shownin Fig. 5E, STAT3 siRNA decreased expression of STAT3a,while leaving the STAT3b splice variant intact. Loss ofSTAT3a expression correlated with decreased bFGF-induced proliferation of Hs578T cells (Fig. 5D) and restora-tion of chemosensitivity as shown by an increase in cleavedcaspase-3 (Fig. 5E).

A novel orthotopic mammary tumor model was used toevaluate the contribution of STAT3 activation to FGFR-induced tumor growth in vivo. HC-11/R1 cells were injectedinto fat pads of Balb/c mice and the mice were administeredB/B to induce tumor growth. Average time to tumor growth inthis model is 5.5 weeks, compared with parental HC-11 cells,which do not form palpable tumors within this timeframe

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Figure 5. STAT3 promotes FGFR-induced migration, proliferation,and chemoresistance. A, HC-11/R1 cells were treated with solventor 30 nmol/LB/B in the presence of1 mmol/L Stattic or DMSO, andwoundclosurewasmeasured after18 hours. B, HC-11/R1 cells weretreated with B/B, 2 mmol/L Stattic,or solvent for 1 or 2 days.Proliferation ratewas calculatedbyMTTassay andgiven relative to thesolvent treated samples. C,apoptosis was determined byTUNEL assay for HC-11/R1 cellstreated with B/B, 4 mmol/L Stattic,or 2 mmol/L doxorubicin for 24hours. D, Hs578T cells weretreated with nontargeting orSTAT3 siRNA for 24 hours,followed by 1 or 2 days treatmentwith 50 ng/mL bFGF, andproliferation was calculatedrelative to solvent treated samples.E, Hs578T cells were treated asdescribed above, and 2 mmol/Ldoxorubicin was added to theindicated groups for 24 hours.Expression levels of cleavedcaspase-3 and b-tubulin wereexamined by immunoblotting. F,HC-11/R1 cells were injected intothe fat pads of Balb/c mice. Micewere given twice weekly injectionsof 1 mg/kg B/B. Once tumorsreached a size of 100 mm3, micereceived either DMSO or 20 mg/kgStattic and tumor growth wasassessed. �, P < 0.05; ��, P < 0.01;��, P < 0.001.





(Supplementary Fig. S3). To determine the effects of STAT3inhibition on iFGFR1-induced tumor growth, mice bearing 100mm3 tumorswere treatedwith either Stattic or solvent control.Treatment of mice with Stattic led to tumor stabilization,resulting in a significant (P < 0.001) reduction in the size ofend-stage tumors (Fig. 5F). Together, these results demon-strate that STAT3 is an important mediator of FGFR-inducedproliferation, migration, therapeutic resistance, and tumorgrowth.

FGFR activation induces hyaluronan accumulation in aSTAT3-dependent manner

Because HAS2 was previously identified as a STAT3 targetgene (35), we determined the contribution of STAT3 to iFGFR1-mediatedHas2 expression and hyaluronan synthesis. As shownin Fig. 6A, treatment of HC-11/R1 cells with Stattic led todecreased Has2 gene expression within 2 hours of B/B treat-

ment. Furthermore, inhibition of STAT3 also led to decreasedhyaluronan synthesis following iFGFR1 activation (Fig. 6B). Toverify the link between FGFR, STAT3, and hyaluronan inhuman breast cancer cells, Hs578T cells were treated withbFGF in the presence or absence of Stattic and hyaluronansynthesis was analyzed. Although Hs578T cells already expresshigh basal levels of hyaluronan (45), inhibition of STAT3activity led to a decrease in bFGF-induced hyaluronan syn-thesis (Fig. 6C).

To confirm these findings in vivo, immunohistochemicalanalysis was performed onHC-11/R1–derived tumors (Fig. 5F).Both pSTAT3Tyr705 and HAS2 staining were observed in thetumors from solvent-treated mice (Fig. 6D). Analysis of serialsections of tumors from Stattic-treated mice revealeddecreased pSTAT3Tyr705 and HAS2 staining in the same areaswithin the tumor. Furthermore, analysis of tumor-associatedhyaluronan by ELISA revealed decreased hyaluronan levels in

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Figure6. STAT3 regulates expression of HAS2 and production of hyaluronan (HA). A, HC-11/R1 cells were treated with 30 nmol/L B/B, 4 mmol/L Stattic,or solvent for 2 hours. RNA was collected to examine expression of Has2 by qRT-PCR. B, HC-11/R1 cells were treated as in A for 18 hours.Conditioned medium was collected to examine expression of hyaluronan by ELISA. C, Hs578T cells were treated with 50 ng/mL bFGF andStattic [2 mmol/L (þ) or 4 mmol/L (þþ)] or solvent for 18 hours, and levels of hyaluronan in the conditioned medium were determined by ELISA. D, serialtumor sections from mice treated with solvent or 20 mg/kg Stattic were stained for pSTAT3Tyr705 and HAS2. E, amount of hyaluronan in tumors frommice in D was assessed by ELISA. �, P < 0.05; ��, P < 0.001.

Bohrer et al.




tumors from mice treated with Stattic (Fig. 6E), consistentwith the observed decrease in HAS2 expression.

Inhibition of hyaluronan synthesis decreases acinargrowth in three-dimensional cultureFurther studies were performed to determine the effects of

inhibiting hyaluronan synthesis using three-dimensional (3D)culture models, which provide a more relevant environmentthan cells in traditional two-dimensional culture (46). We havepreviously demonstrated that primary mammary epithelialcells isolated from MMTV–iFGFR1 transgenic mice formlarge acinar structures in 3D culture upon iFGFR1 activation(30). To assess effects of hyaluronan inhibition on acinargrowth, 4-MU was added to the 3D culture at the same timeas activating iFGFR1, which led to inhibition of iFGFR1-induced acinar growth (Fig. 7A and B). Furthermore, treat-ment of established structures with 4-MU led to inhibition

of further acinar growth (Fig. 7C and D). These studiesdemonstrate that blocking hyaluronan synthesis leads toinhibition of iFGFR1-dependent growth of both developingand established acinar structures.

Further studies were performed using the Hs578T cells,which exhibit high levels of both FGFR activation (40) andhigh levels of hyaluronan synthesis (45). The cells were platedin 3D culture and structureswere treatedwith either PD173074to inhibit FGFR activation, 4-MU to inhibit hyaluronan syn-thesis or both for 6 days, and proliferation was assessed usingphospho-histone H3 staining. Surprisingly, treatment of cellswith either PD173074 or 4-MUalone did not affect proliferation(Fig. 7E and F). However, when treated with both inhibitorstogether, proliferation was significantly decreased (Fig. 7E andF). Taken together, the 3D culture studies suggest that hyalur-onan inhibition may be a relevant approach for targetingboth FGFR-driven cancers and cancers that have high levels

Figure7. Inhibition of hyaluronan (HA) synthesis leads todecreasedFGFR-inducedgrowth in 3Dculture. A, primarymammaryepithelial cellswere isolated fromMMTV–iFGFR1 transgenic mice and plated in 3D culture. Cells were treated with 30 nmol/L B/B, 10 mmol/L 4-MU, or solvent. Light microscopyimages were obtained after 10 days in culture. B, quantification of acinar area. C, structures were treated with B/B to activate iFGFR1 for 6 days, followedby treatment with solvent or 4-MU for 8 days. Images were obtained from the same structures. D, quantification of acinar area. Red arrow,addition of 4-MU. E, Hs578T cells were plated in Matrigel. The cells were treated with solvent (control), 10 mmol/L 4-MU, 1 mmol/L PD173074, or both for6 days. The cultures were stained with phospho-histone H3 and analyzed by confocal microscopy. F, quantification of phospho-histone H3–positive cells.�, P < 0.05. G, representative images of pFRS2- and HABP-stained sections of human breast cancers. Magnification bars, 50 mm.





of FGFR activation, but are not necessarily FGFR-driven. Toassess whether high levels of FGFR activation and hyaluronancoexist in human patient samples, the TMA described abovewas stained for hyaluronan. No staining was observed inhyaluronidase-treated samples, demonstrating specificity forthe staining (Supplementary Fig. S1B). Hyaluronan was foundin all samples and was therefore present in all breast cancersamples exhibiting high levels of pFRS2 (Fig. 7G). Therefore,combination therapies targeting both FGFR activity and hya-luronan synthesis may be considered for patients with highlevels of FGFR activation.

DiscussionThe results from these studies reveal a novel link between

FGFR activation and synthesis of hyaluronan by tumor cells,thus linking intracellular signaling to alterations in themicroenvironment (Supplementary Fig. S4). Hyaluronan isan important component of the extracellular matrix that isnormally involved in a number of physiologic processes,including maintenance of tissue integrity, morphogenesis,wound healing, and inflammation (22, 34). Hyaluronan is aprevalent component of the normal human breast extra-cellular matrix and alterations in hyaluronan contribute tobreast cancer growth and progression (17–19). Tumor-associated hyaluronan alterations are complex and caninclude increased hyaluronan synthesis, changes in hyalur-onan localization and increased hyaluronan fragmentation(47, 48). High levels of hyaluronan accumulate in breastcancer, in part due to increased hyaluronan synthesis byHAS enzymes, including HAS2; these high levels of hyalur-onan are associated with reduced survival and poorresponse to therapy (49, 50). Hyaluronan is typically pro-duced in a high molecular weight form and can be cleavedinto lower molecular weight fragments by hyaluronidases orreactive oxygen species under specific conditions, such asduring inflammation and within the tumor microenviron-ment (51–53). Although high molecular weight hyaluronaninhibits tumor formation (54), low molecular weight formshave been shown to stimulate cancer cell migration andinvasion possibly through differential interactions with itsreceptors, such as CD44 and RHAMM (47, 48, 55). Althoughour studies have not specifically assessed hyaluronan frag-mentation, they demonstrate that FGFR can increase thelevels of hyaluronan within the tumor microenvironmentcreating the necessary substrate for hyaluronan fragmentproduction.

Because changes in hyaluronan are often associated withinflammation, further studies focused on the contributions ofinflammatory pathways to FGFR-mediated changes in hyalur-onan, specifically the transcription factor STAT3. In our stud-ies, we found differences in the kinetics of the two STAT3phosphorylation sites, Ser727 and Tyr705, following FGFRactivation. Phosphorylation at Ser727 has been previouslyidentified as a rapid site of phosphorylation following FGFRactivation (36). However, the observation of cytokinemediatedindirect phosphorylation at Tyr705 at later time points is noveland provides a potential feed forward mechanism throughwhich FGFR activation induces inflammation in breast cancer.

Although initially thought to be secondary to phosphorylationof Tyr705 and involved in maximal STAT3 activation, recentstudies have suggested that pSTAT3Ser727 may have differentfunctions than pSTAT3Tyr705 (56). Interestingly, pSTAT3Ser727

has been identified in early-stage tumors in melanoma, where-as pSTAT3Tyr705 has been associated with later-stage cancer(56). Further studies are required to elucidate whether thesetwo phosphorylation sites have different roles in mediatingFGFR-driven tumor growth.

Further analysis of the mechanisms driving STAT3 phos-phorylation demonstrated that FGFR activation inducesvarious IL-6 family members, which contributed to phos-phorylation of STAT3Tyr705. Although these studies focusedon actions of IL-6–mediated signaling in the tumor cells, itis likely that the IL-6 family members also act on neigh-boring epithelial cells (Supplementary Fig. S4) and cellswithin the tumor reactive stroma, including infiltratinginflammatory cells, to promote tumor growth and progres-sion. Further studies are in progress to determine therelative contributions of IL-6 family cytokine-induced sig-naling in tumor cells compared with other cell types in themicroenvironment.

These studies raise the possibility that patients with highlevels of FGFR activity and tumor-associated hyaluronan maybe candidates for combinatorial therapies targeting both ofthese molecules. Consistent with this, our 3D culture studiessuggest that although inhibition of FGFR in FGF-responsivecells alone does not affect cell proliferation, combinatorialtreatment inhibiting both FGFR and hyaluronan synthesisleads to decreased proliferation. Whether this is due to coop-erative effects of decreased signaling of both pathways or dueto removal of hyaluronan surrounding the tumor cell and thusproviding access of the drug to the cells, which has beendemonstrated in other models (57), remains to be determined.Regardless of the mechanism, these findings provide an exam-ple of the potential therapeutic impact of designing therapeu-tic strategies that target both tumor cell-specific oncogenicpathways and the protumorigenic microenvironment.

In conclusion, these studies define anovel pathway involvingFGFR, STAT3, and hyaluronan synthesis that contribute totumor growth. Because all of the components of these path-ways are known to contribute to other tumor types as well asinflammation-related diseases, this pathway likely represents ageneral mechanism that can be appliedmore broadly than justbreast cancer. Finally, these results provide important insightsinto the potential need for targeting growth factor–drivensignaling pathways within the tumor cells in combination withinhibiting hyaluronan/tumor cell interaction to more effec-tively treat patients with breast cancer.

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

Authors' ContributionsConception and design: S.C. Schmechel, J.B. McCarthy, K.L. SchwertfegerDevelopment of methodology: L.R. Bohrer, P. Chuntova, T.C. Beadnell, J.E.Goldberg, S.C. Schmechel, J.B. McCarthy, K.L. SchwertfegerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.R. Bohrer, P. Chuntova, T.C. Beadnell, R.P. Leon, N.J.Brady, Y. Ryu, J.E. Goldberg, S.C. Schmechel, K.L. Schwertfeger

Bohrer et al.




Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.R. Bohrer, P. Chuntova, T.C. Beadnell, J.S. Koop-meiners, K.L. SchwertfegerWriting, review, and/or revision of the manuscript: L.R. Bohrer, P. Chun-tova, S.C. Schmechel, J.S. Koopmeiners, J.B. McCarthy, K.L. SchwertfegerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): N.J. Brady, Y. Ryu, K.L. SchwertfegerStudy supervision: K.L. Schwertfeger

AcknowledgmentsThe authors thank Sarah Bowell, Kimberly Casey, Colleen Forster, Anthony

Rizzardi, and Xingchu Shen of UMN BioNet for assistance with TMA construc-tion and tissue sectioning. The authors also thankDr. Jeff Rosen for providing theHC-11/R1 cells and theMMTV–iFGFR1 transgenicmice andDr.Mariya Farooquifor providing comments on the article.

Grant SupportBioNet is supported by NIH grants P30 CA77598, P50 CA101955, and KL2

RR033182, and by the University of Minnesota Academic Health Center.This study was also funded by NIH/NCI R01CA132827 and AmericanCancer Society RSG-09-192-01-LIB (K.L. Schwertfeger). We would like toacknowledge the use of the confocal microscope at the Masonic CancerCenter made available through an NCRR Shared Instrumentation Grant(#1 S10 RR16851).

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 indicate thisfact.

Received August 28, 2013; revised October 10, 2013; accepted October 31, 2013;published OnlineFirst November 6, 2013.

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activation of the fgfr stat3 pathway in breast cancer ... · l.r. bohrer and p. chuntova...

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