her2-overexpressing breast cancers amplify fgfr signaling … · cancer therapy: preclinical...

Cancer Therapy: Preclinical

HER2-Overexpressing Breast Cancers AmplifyFGFR Signaling upon Acquisition of Resistance toDual Therapeutic Blockade of HER2Ariella B. Hanker1,2, Joan T. Garrett1, M�onica Valeria Estrada2, Preston D. Moore1,Paula Gonz�alez Ericsson2, James P. Koch1, Emma Langley3, Sharat Singh3,Phillip S. Kim3, Garrett M. Frampton4, Eric Sanford4, Philip Owens5,JenniferBecker1,M.ReidGroseclose6, StephenCastellino6,Heikki Joensuu7, JensHuober8,Jan C. Brase9, Samira Majjaj10, Sylvain Broh�ee10, David Venet10, David Brown10,Jos�e Baselga11, Martine Piccart10, Christos Sotiriou10, and Carlos L. Arteaga1,2,5

Abstract

Purpose: Dual blockade of HER2 with trastuzumab andlapatinib or pertuzumab has been shown to be superior tosingle-agent trastuzumab. However, a significant fraction ofHER2-overexpressing (HER2þ) breast cancers escape from thesedrug combinations. In this study, we sought to discover themechanisms of acquired resistance to the combination oflapatinib þ trastuzumab.

Experimental Design: HER2þ BT474 xenografts were trea-ted with lapatinib þ trastuzumab long-term until resistancedeveloped. Potential mechanisms of acquired resistancewere evaluated in lapatinib þ trastuzumab-resistant (LTR)tumors by targeted capture next-generation sequencing.In vitro experiments were performed to corroborate thesefindings, and a novel drug combination was tested againstLTR xenografts. Gene expression and copy-number analyseswere performed to corroborate our findings in clinicalsamples.

Results: LTR tumors exhibited an increase in FGF3/4/19 copynumber, together with an increase in FGFR phosphorylation,marked stromal changes in the tumor microenvironment, andreduced tumor uptake of lapatinib. Stimulation of BT474 cellswith FGF4 promoted resistance to lapatinib þ trastuzumab invitro. Treatment with FGFR tyrosine kinase inhibitors reversedthese changes and overcame resistance to lapatinib þ trastuzu-mab. High expression of FGFR1 correlated with a statisticallyshorter progression-free survival in patients with HER2þ earlybreast cancer treated with adjuvant trastuzumab. Finally, FGFR1and/or FGF3 gene amplification correlated with a lower patho-logic complete response in patients with HER2þ early breastcancer treated with neoadjuvant anti-HER2 therapy.

Conclusions: Amplification of FGFR signaling promotesresistance to HER2 inhibition, which can be diminished bythe combination of HER2 and FGFR inhibitors. Clin Cancer Res;23(15); 4323–34. �2017 AACR.

IntroductionApproximately 20% of breast cancers harbor gene amplifica-

tion of the receptor tyrosine kinase (RTK) HER2 (ERBB2; ref. 1).HER2 is a member of the ERBB family of trans-membrane RTKs.Upon ligand-induced dimerization, ERBB family receptorsundergo conformational changes that trigger kinase activationand subsequent signal transduction through oncogenic path-ways, such as PI3K-Akt and RAS/RAF/MEK/ERK. Anti-HER2therapies include trastuzumab (T), a humanized monoclonalantibody directed against the ectodomain of HER2; pertuzu-mab, an antibody that blocks dimerization of HER2 with otherERBB receptors; lapatinib (L), a reversible, ATP-competitivetyrosine kinase inhibitor (TKI) of HER2 and EGFR (2); andtrastuzumab-DM1, a conjugate of trastuzumab linked to thechemotherapeutic maytansine. Recent clinical studies haveshown that dual inhibition of HER2, such as the combinationof L þ T or T þ pertuzumab, is more effective than trastuzumab(3–5). Despite these advances, resistance to dual HER2 block-ade still occurs in patients with HER2þ breast cancer. Themechanisms of acquired resistance to dual HER2 inhibitionremain to be fully explored.

1Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt Univer-sity, Nashville, Tennessee. 2Breast Cancer Program, Vanderbilt-Ingram CancerCenter, Vanderbilt University, Nashville, Tennessee. 3Prometheus Laboratories,San Diego, California. 4Foundation Medicine, Cambridge, Massachusetts.5Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.6Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline,Research Triangle Park, North Carolina. 7Department of Oncology, HelsinkiUniversity Central Hospital, Helsinki, Finland. 8Department of Gynecology,University of Ulm, Ulm, Germany. 9Novartis Pharmaceuticals, Basel, Switzerland.10Institut Jules Bordet, Universit�e Libre de Bruxelles, Brussels, Belgium. 11Depart-ment ofMedicine,Memorial SloanKetteringCancerCenter, NewYork,NewYork.

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

A.B. Hanker and J.T. Garrett contributed equally to this article.

Current address for J.T. Garrett: Division of Pharmaceutical Sciences, Universityof Cincinnati College of Pharmacy, Cincinnati, Ohio.

Corrected online January 10, 2019.

CorrespondingAuthor: Carlos L. Arteaga, Vanderbilt University Medical Center,2220 Pierce Avenue, 777 PRB, Nashville, TN 37232. Phone: 615-936-3524; Fax:615-936-1790; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-2287

�2017 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 4323

on March 9, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 5, 2017; DOI: 10.1158/1078-0432.CCR-16-2287





http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-16-2287&domain=pdf&date_stamp=2019-1-10

http://clincancerres.aacrjournals.org/



Proposedmechanisms of resistance toHER2 inhibitors includethe presence of truncated HER2, bypass signaling by other ERBBreceptors or other RTKs, activation of compensatory survivalpathways, defects in apoptosis and cell-cycle control, and muta-tional activation of the PI3K pathway [reviewed in ref. 2]. We andothers have previously shown that activating PIK3CA mutationscan promote resistance to dual inhibition of HER2 (6–8).

In order to discover novel mechanisms of resistance to dualHER2 blockade, we generated HER2-amplified BT474 xenograftsresistant to the combination of L þ T. We found increased copy-number of the FGF3, FGF4, and FGF19 genes in L þ T-resistant(LTR) tumors. The FGF3/4/19 genes reside on chromosome11q13, a region which also harbors CCND1 and is amplified inapproximately 15% of breast cancers [The Cancer Genome Atlas(9); www.cbioportal.org (10)]. The FGF3/4/19 genes encodeligands for the FGFR family of RTKs (FGFR1-4). Aberrant FGFRsignaling drives tumor cell proliferation, survival, angiogenesis,and activation of stromal fibroblasts, and has been associatedwith resistance to anumber of targeted therapies (11). In addition,FGFR1 is amplified in approximately 10% of breast cancers and isassociated with poor patient prognosis (12, 13). We show hereinthat (1) exogenous FGF promotes resistance to HER2 inhibition,(2) inhibition of treatment with FGFR TKIs reversed or reducedresistance to L þ T, and (3) somatic alterations in the FGFRpathway correlated with lack of benefit from anti-HER2 therapiesin patientswith earlyHER2þ breast cancer. These data suggest thatFGFR pathway activation promotes resistance to L þ T andsupport the combination of FGFR inhibitors with HER2-directedtherapies in patients with HER2-overexpressing breast cancer.

Materials and MethodsCell lines and inhibitors

BT474, HCC1954, andMDA-MB 361 cells were obtained fromthe American Type Culture Collection (ATCC) between 2006 and2011 andmaintained in ATCC-recommendedmedia supplemen-tedwith 10% FBS (Gibco) and 1x antibiotic/antimycotic (Gibco).All cell lines were authenticated by the ATCC using the short

tandem repeat method in January 2017. Mycoplasma testing wasconducted for each cell line before use. All experiments wereperformed less than 2 months after thawing early passage cells.

The following drugs were used: lapatinib (GW-572016, LCLaboratories), trastuzumab (Vanderbilt University Hospital Phar-macy), lucitanib (ref. 14; provided by Clovis Oncology), andAZD4547 (ref. 15; provided by AstraZeneca Pharmaceuticals).

Drug-resistant xenografts and mouse studiesA 21-day 17b estradiol pellet (Innovative Research of America)

was inserted s.c. in the dorsumof 4- to 6-week-old female athymicmice (Harlan Sprague Dawley Inc.) 1 day before tumor cellinjection. Approximately 5 � 106 BT474 cells were injected s.c.into the right flank of mice. Once tumors reached a size of�200mm3,micewere treatedwith 20mg/kg trastuzumab dilutedin sterile PBS by i.p. injection twice a week and 100 mg/kglapatinib by orogastric gavage daily. Tumor diameters were seri-ally measured with calipers, and tumor volumes were calculatedas: volume¼width2� length/2.Upon complete tumor regressionwith L þ T, treatment was halted. Once tumors recurred(�200 mm3), treatment with lapatinib and trastuzumab wasresumed. Recurrent tumors that progressed in the presence of Lþ T were serially transplanted into tumor-na€�ve nude mice. Oncethe transplanted tumors reached a volume�200mm3,mice weretreated again with Lþ T. Tumors growing in continuously treatedmice were considered to be LTR.

For therapeutic studies, LTR tumors were transplanted intotreatment-na€�ve mice following the same protocol. Once tumorsreached a volume �200 mm3, mice were randomized to any ofthese treatment arms: vehicle (0.5% hydroxypropylmethyl cellu-lose and 0.1% Tween-80); trastuzumab (20 mg/kg i.p. twice aweek) and lapatinib (100 mg/kg by oral gavage daily); lucitanib(20 mg/kg by oral gavage daily); AZD4547 (12.5 mg/kg by oralgavage daily); or TþL plus each of the FGFR inhibitors. Tumorswere harvested 24 hours after the last dose of trastuzumab and 1hour after the last dose of lapatinib, lucitanib, or AZD4547. Allanimal experiments were approved by the Vanderbilt Institution-al Animal Care andUseCommittee (IACUCprotocolM/10/111).

FISHERBB2 gene copy-numberwas determined by FISHusingHER2

Spectrum Orange and CEP17 Spectrum Green probes (Pathvi-sion). Enumeration of HER2 and CEP17 signals was done on 20cells from different fields. FGF3 gene copy-number was deter-mined by FISH using FGF3Orange and Chromosome 11 ControlGreen probes (Empire Genomics). Enumeration of FGF3 andChromosome 11 signals was done on at least 20 cells fromdifferent fields. Slides were analyzed with a reflected light fluo-rescent microscope (Olympus BX60) at 100X. Images of repre-sentative cells were captured using Cytovision software package(Applied Imaging).

Immunoblot analysisBT474 cells were serum-starved overnight and treated the

following day for 2 hours with vehicle, 1 mmol/L lapatinib, or1 mmol/L AZD4547 followed by stimulation for 15 minutes with50 ng/mL FGF3, FGF4, or FGF19 (R&D Systems). Cells werewashed with ice-cold PBS and lysed on ice in buffer containing20 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40,1 mmol/L EDTA plus protease and phosphatase inhibitors. Snap-frozen tumor fragments were homogenized using the TissueLyser

Translational Relevance

Dual blockade of HER2, with the combinations of trastu-zumab and lapatinib or trastuzumab and pertuzumab, hasbeen shown to be superior to trastuzumab alone. However,acquired resistance to anti-HER2 therapies remains a problem.We found an increase in FGF3/4/19 gene copy-number inHER2-overexpressing xenografts with acquired resistance tolapatinib þ trastuzumab. This was accompanied by anincrease in FGFR phosphorylation, reduced levels of lapatinibin drug-resistant tumors, and changes in the tumor microen-vironment. Combining FGFR inhibitors with lapatinib þtrastuzumab led to regression of the drug-resistant tumors.Finally, high FGFR1 gene expression and amplification ofFGFR1 and FGF3 correlated with an inferior clinical responseto anti-HER2 therapies in patients with early HER2þ breastcancer. These findings support profiling genes in the FGFRpathway in tumors with acquired resistance to HER2-directedtherapies and the clinical investigation of FGFR and HER2inhibitors in these treatment-refractory HER2þ breast cancers.

Hanker et al.

Clin Cancer Res; 23(15) August 1, 2017 Clinical Cancer Research4324



www.cbioportal.org


(Qiagen) and lysed in buffer containing 50 mmol/L Tris-HCl pH8.0, 150mmol/L NaCl, 2mmol/L EDTA pH 8.0, 10mmol/L NaF,20% glycerol, 1% Nonidet P-40 plus protease, and phosphataseinhibitors. After centrifugation, protein concentration in super-natants was measured using the BCA protein assay reagent(Pierce). Proteins were separated by SDS-PAGE and transferredonto nitrocellulose membranes (Bio-Rad). Primary antibodiesincludedP-HER2Y877, P-HER2Y1221/2, P-HER2Y1248, P-HER3Y1197,P-EGFRY1068, P-AktS473, P-AktT308, Akt, P-ErkT202/Y204, Erk, P-FRS2aY436, P-S6S240/244, P-FGFR1Y653/4, P-STAT3Y705, P-ACLS455,P-BADS136, P-PRAS40T246, and b-actin (Cell Signaling Technolo-gy), and HER2/ERBB2 (Neomarkers). Immunoreactive bandswere detected by enhanced chemiluminescence following incu-bation with horseradish peroxidase–conjugated secondary anti-bodies (Promega). Membranes were cut horizontally to probewith multiple antibodies. Blots probed with phospho-antibodieswere strippedwith RestoreWestern Blot Stripping Buffer (ThermoFisher Scientific) and re-probed with antibodies to the totalprotein.

FGF4 ELISABT474 parental and LTR tumor lysates were analyzed using the

Human FGF4 ELISA Kit (RayBiotech) according to the manufac-turer's instructions.

Immunohistochemistry and immunofluorescenceTumor fragments were harvested and immediately fixed in

10% buffered neutral formalin for 24 hours at room temper-ature, then dehydrated and paraffin-embedded. Five-microm-eter sections were subjected to hematoxylin and eosin stainingand immunohistochemistry (IHC) using antibodies againstHER2, P-HER2Y1221/2 (Cell Signaling Technology), Ki67(Zymed Invitrogen), and P-FGFRY653/654 (Abcam). Alternative-ly, tumor fragments were placed immediately in optimal cut-ting temperature (OCT) compound (VWR) and frozen on dryice. Eight-micrometer frozen sections were used for IHC usingan antibody against CD31 (Abcam). Trichrome staining (Sig-ma-Aldrich) was performed according to the manufacturer'sinstructions. The Histoscore for HER2 staining was graded by anexpert breast pathologist (M�onica Valeria Estrada) using amodified scoring method previously reported by Nenutil andcolleagues (16). Briefly, total cells and cells with positivemembrane staining were counted, and the percent positivecells in each high-power field (HPF) were calculated. Fieldswere then assigned a relative staining intensity score of 1 forlow, 2 for intermediate, or 3 for high staining intensity. Theproduct of the percent positive cells and staining intensity wasthen derived to create a Histoscore of 0 to 300 for each HPF.Intratumor microvascular density (MVD) was scored by count-ing the number of endothelial cells per cluster of CD31-positivecells in 3 HPFs (17, 18). Trichrome-stained tumor sections werescored in a semi-quantitative way from 0 to 5 based on thepresence and intensity of the intratumor stromal collagen usinga modified scoring system by Walsh and colleagues (19). Threeto five HPFs were reviewed, and an average was estimated asfollows: grade 0: absence of collagen, grade 1: weak staining,grade 2: weak to moderate staining, grade 3: moderate staining,grade 4: moderate to strong staining, grade 5: strong staining.Sections were scored by an expert pathologist blinded to xeno-graft type or treatment arm. Images were obtained using theOlympus DP2 software and an Olympus light microscope.

Indirect immunofluorescence was performed using dewaxedformalin-fixed, paraffin-embedded (FFPE) sections, and epitopeswere retrieved with citrate boiling (20). Primary antibodiesagainst a-smooth muscle antigen (SMA), fibronectin (Sigma),and cytokeratin 8 (RDI-Fitzgerald) were incubated overnight andstained with highly cross-adsorbed goat-derived secondary anti-bodies conjugated to AlexFluor (Life Technologies). Image acqui-sition was performed using a Zeiss Axioplane microscope withMetamorph (Molecular Devices) for image capture.

Lapatinib levels in xenograftsParental BT474 and LTR xenografts treated with lapatinib and

trastuzumab were harvested, weighed, homogenized, extracted,and analyzed by accurate LC-MS as previously described (21). Acalibration curve covering the range of lapatinib concentrationsobserved in the tissue homogenates was prepared using a lapa-tinib standard for each sample set. Each sample was analyzed intriplicate.

Cell viability assayBT474 cells were seeded in black clear-bottom 96-well plates

(Greiner Bio-One) at a density of 2,000 cells per well. The nextday,mediawere replacedwith 100mlmedia containing inhibitorsin media containing 1% charcoal-stripped serum (CSS). After 6days, nuclei were stained with 10 mg/mLHoechst 33342 (ThermoFisher Scientific) at 37�C for 20 minutes. Fluorescent nuclei werecounted using the ImageXpress Micro XL automated microscopeimager (Molecular Devices).

Statistical analysisThe number of animals in each group was calculated to mea-

sure a 25% difference between the means of groups treated withLþ T versus Lþ Tþ FGFR inhibitor with a power of 80%, aP valueof 0.01, and an SD of 12%, using PS Power and Sample SizeCalculator software (http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize). Animal experiments were conductedin a controlled and nonblinded manner. In vitro experimentswere performed at least twice and at least in triplicate each time.

Unless otherwise indicated, the Student t test was used. ANOVAand multiple comparisons tests were conducted using GraphPadPrism (GraphPad Software).

See Supplementary Methods for details on next-generationsequencing (NGS), qRT-PCR, 3D Matrigel and apoptosis assays,gene expression analysis of the FinHER dataset, and copy-numberanalysis of the NeoALTTO dataset.

ResultsBT474 xenografts develop acquired resistance to L þ T

We treated mice bearing BT474 xenografts with the combi-nation of L þ T. This treatment led to complete regression of alltumors (Fig. 1A), as reported previously (22, 23). After 24 days,treatment was stopped, and mice were monitored twice weeklyfor tumor recurrence. Some tumors recurred as early as 9 weeksafter treatment interruption. Three recurrent tumors (#32, #33,and #44) failed to respond to retreatment with L þ T (Fig. 1B).These tumors were serially transplanted into treatment-na€�venude mice; treatment with L þ T was resumed when tumorsreached a volume �200 mm3. More than 90% of tumorsderived from these xenografts did not respond to L þ T(Supplementary Fig. S1).

FGFR Signaling Promotes Resistance to Dual HER2 Blockade

www.aacrjournals.org Clin Cancer Res; 23(15) August 1, 2017 4325



http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize

http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize


LTR tumors maintain HER2 amplification, phosphorylation,and HER2 downstream signaling

Onepossiblemechanism for acquired resistance is loss ofHER2overexpression, thus abrogating HER2 dependence and theimmune mechanisms of action of trastuzumab (24). Therefore,we performed HER2 FISH on FFPE sections from parental BT474and LTR tumors. HER2 gene amplification, measured by the ratioof HER2 to chromosome 17 centromere (CEP17) probe fluores-cent signals, did not differ between drug-sensitive BT474 and LTRtumors (P¼0.61; Fig. 1C). Accordingly,HER2protein expression,detected by IHC, was unaltered in LTR tumors (Fig. 1D).

Next, we asked if HER2 remained activated in LTR tumorsgrowing on continuous therapy. Treatment of parental tumorswith Lþ T for 96 hours strongly reduced levels of P-HER2 Y1221/1222 as measured by IHC (Fig. 2A). However, P-HER2 was nolonger reduced in LTR tumors treated with Lþ T. Maintenance ofP-HER2 in themajority of treated LTR tumorswas seen byWesternblot analysis using antibodies recognizing the Y1221 autopho-sphorylation site and the Y877 Src site in HER2 (Fig. 2B), whereasP-EGFRY1068 levels did not differ between both tumor types.Although variable, total HER2 protein levels appeared higher, onaverage, in LTR tumors by Western blot analysis. In addition,

treatment with L þ T suppressed P-Erk, P-Akt, and phosphoryla-tion of Akt substrates [PRAS40, BAD, and ATP Citrate Lyase(ACL)] in parental tumors, but the signal was maintained in themajority of L þ T-treated resistant tumors (Fig. 2B), furthersuggesting that treatment no longer inhibited HER2 signal trans-duction in drug-resistant xenografts. Finally, we performed deepsequencing of the ERBB2 gene in LTR tumors, but did not find anymutations, arguing against acquired drug-resistant somatic altera-tions in HER2 (Supplementary Table S1).

Lapatinib levels are reduced in LTR tumorsTo determine if a decrease in drug uptake may account for the

maintenance of HER2 signaling in LTR xenografts, we usedaccurate mass LC-MS to measure tumor levels of lapatinib inmice treated with L þ T for at least 3 days. LTR tumors containedsignificantly lower levels of lapatinib than drug-sensitive parentalBT474 xenografts (Fig. 2C). Lapatinib has been reported to be asubstrate of the ATP-binding cassette (ABC) efflux transporters,including ABCB2 (P-gp/MDR1) and ABCG2 (BCRP/MXR/ABCP;refs. 21, 25), raising the possibility that increased expression ofdrug efflux transporters may account for the reduced levels oflapatinib. However, expression levels of ABCB1, ABCC1, and

BT474Parental

LTR 33-4

LTR 32-1-2

BT474Parental

LTR

BT474 Parental

6.00

4.00

2.00

0.00

(n = 2)LTR

(n = 9)

ATu

mor

vol

ume

(mm

3 )

Days of treatment

B

Tum

or v

olum

e (m

m3 )

Weeks after initial L+T treatment

C

HE

R2/

CE

P17

Rat

io

DH

-Sco

re

Cytoplasmic

300HER2 CST-2242 BT-T, n = 13 LTR, n = 17

250

200

150

100

50

0Membrane

700Ctrl n = 9

Lap n = 9

Lap + trastn = 8

trast n = 8

80044

32

33600

400

200

0

0.0

1.1

2.1

3.0

12

12.9

13.7

14.7

15.9

16.9

17.9

18.9

20.9

21.9

22.9

23.9

24.9

25.9

27.0

27.9

28.9

29.9

30.9

600

500

400

300

200

100

00 3 6 10 13 17 20 24

Figure 1.

HER2 gene amplification is maintainedin LTR tumors. A, Athymic female micewere injected with BT474 cells andtreated with vehicle, 100 mg/kgof lapatinib by oral gavage daily,20 mg/kg of trastuzumab givenintraperitoneally twice weekly or thecombination (lap þ trast). Treatmentwas administered for 24 days. Eachdata point represents the meantumor volume � SEM (n ¼ 8–9). B,L þ T-treated mice from A weremonitored for tumor recurrence twiceweekly. Once recurrent tumorsreached a volume �200 mm3, L þ Twere resumed and continued until thetime of tumor harvest. C, ERBB2 genecopy-numberwas determinedbyFISH.P¼ 0.61, Student t test. D, IHC analysisof HER2 in tumor sections. Shown attop are representative images.Bottom, Quantitative comparisonof cytoplasmic and membraneHER2 histoscore.

Hanker et al.





ABCG2 mRNAs measured by qRT-PCR were not altered in LTRcompared with drug-sensitive tumors (Supplementary Fig. S2),suggesting that the decrease in lapatinib levels in LTR tumors wasnot due to changes in drug efflux.

Because HER2 activation remains high in LTR tumors, wehypothesized that more complete blockade of HER2 can reverseLTR resistance. We treated cells derived from a BT474 parentalxenograft and two LTR xenografts, grown in 3D Matrigel culture,with 20 mg/mL trastuzumab þ increasing concentrations of lapa-tinib. Although LTR cells maintained resistance to trastuzumabþlower concentrations of lapatinib (�25 nmol/L), 125 nmol/Llapatinib þ trastuzumab was able to block phoshpo-HER2 andthe growth of LTR tumor-derived cells, suggesting that morecomplete suppression of HER2 can overcome drug resistance invitro (Supplementary Fig. S3).

FGFR signaling is amplified in LTR tumorsWe next performed targeted capture NGS to identify somatic

alterations associated with acquired resistance. Specifically, wecharacterized base substitutions, short insertions and deletions

(indels), copy-number alterations, and selected fusions across287 cancer-related genes. Of note, no mutations in ERBB2 werefound. We identified an increase in copy-number of FGF3, FGF4,and FGF19 in one of five LTR tumors. The FGF3/4/19 genes resideon chromosome 11q13, along with CCND1. CCND1 copy-num-ber was elevated in both LTR and BT474 parental tumors (Sup-plementary Fig. S4). FISH, using the ratio of fluorescent signalsfrom probes against FGF3 and CEN11, confirmed that FGF3 copynumber was increased >1.5-fold in the amplified LTR tumorrelative to parental BT474 tumors (Fig. 3A). Further FISH analysisrevealed that six additional LTR tumors (total 7/16; 43.8%)harbored FGF3 amplification (defined by FGF3 copy-number>4 and FGF3/CEN11 ratio >2). Likewise, FGF4 protein levels,measured by ELISA, were elevated in LTR tumors (Fig. 3B; ELISAkits for FGF3 or FGF19 from lysates were not available). Consis-tent with higher levels of FGFs in situ, FGF-amplified LTR tumorsexhibited high P-FGFR IHC staining (Fig. 3C).

In addition to exerting a tumor cell–autonomous effect incancer cells, secreted FGF ligands can also alter the tumor micro-environment by remodeling the extracellular matrix, by paracrine

A BT474 parentalVehicle

BT474 Parental96 h L+T

LTRL+T

Mem

bran

e H

-sco

re

B Parentalvehicle

96 hL+T

LTRL+T

P-HER2

6,000

250P-HER2 Y1221/2

20015010050

BT474Vehicle(n = 8)

LTRL+T

(n = 13)

BT47496 h L+T(n = 4)

P = 0.020

0

4,000

2,000

0

Resist

ant (n

= 9)

Sensit

ive (n

= 8)

Y1221/1222

P-HER2Y877

HER2 (light)

HER2 (dark)

P-EGFRY1068

P-HER3Y1197

P-AktS473

P-AktT308

Akt

P-ErkT202/Y204

Erk

β-Actin (1)

1 2 3 4 5 6 7 8 9 10 11 12

P-ACLS455

P-PRAS40T246

P-BADS136

β-Actin (2)

C

ng L

apat

inib

/g ti

ssue

Figure 2.

LTR xenografts maintain phosphorylatedHER2 and postreceptor signaling. A, IHCanalysis of P-Y1221/2 P-HER2 in tumorsections. Groups of mice bearing parentalBT474 xenografts received vehicle or LþT for 96 hours only (96 hours L þ T),whereas those bearing LTR tumors weretreated continuously; both groups ofmicewere sacrificed 24 hours after the lasttreatment with trastuzumab and 1 hourafter the last dose of lapatinib. Shown attop are representative images. Bottom,Quantitative comparison of membrane P-HER2 histoscore. � , P < 0.05 comparedwith parental BT474-untreated tumors.B,Tumors were treated as described in A.Tumor lysates were subjected toimmunoblot analysis with the indicatedantibodies. The same lysates were run ontwo separate gels. C, Mice bearing drug-sensitive and -resistant (LTR) tumorswere treated for 72 hours or longer withlapatinib and sacrificed 1 hour after thelast lapatinib dose. Tumors wereharvested and tissue sections wereweighed, homogenized, extracted, andanalyzed by accurate mass LC-MS. Eachsample was analyzed in triplicate.Concentrations are reported in nglapatinib/gram of tissue.






activation of stromal cells such as fibroblasts, and/or by promot-ing angiogenesis (11). Trichrome staining of LTR tumor sections(n¼ 8) compared with parental tumor sections (n¼ 10) revealedmarkedupregulationof collagendeposition in tumor stroma (Fig.3D and E; P¼ 0.0008). IHC analysis of LTR tumors also revealedincreased staining of SMA and fibronectin, markers of stromalfibroblasts. Further, a trend toward an increase in CD31þ bloodvessels was observed in LTR tumors compared with parentalxenografts (Fig. 3D and F; P ¼ 0.08). These microenvironmentalchanges are consistent with an increase in FGF pathway activationin situ (26).

Exogenous FGF4 induces P-ERK and promotes cell viability inthe presence of HER2 inhibitors

Maintenance of P-Akt and P-Erk in LTR tumors treated withL þ T led us to ask whether ligand-stimulated FGFR can activatethese signaling pathways, thus bypassing targeted inhibition ofHER2. Therefore, we first examined whether stimulation ofBT474 cells with exogenous FGF3, FGF4, or FGF19 could

activate PI3K/Akt and ERK when HER2 was blocked. BT474cells were serum-starved overnight and then treated � lapatinibfor 2 hours, followed by 15-minute stimulation with FGF3,FGF4, or FGF19. Treatment with lapatinib strongly suppressedP-HER2, P-AKT, and P-ERK. T308 P-AKT was modestly stimu-lated by FGF3 and FGF4. Of the three FGF ligands tested, onlyFGF4 strongly increased P-ERK and phosphorylation of theFGFR substrate, FRS2a, in the absence or presence of lapatinib(Fig. 4A), suggesting that FGF4/FGFR signaling can bypassHER2 inhibition and activate the ERK pathway. The FGFR TKIAZD4547 (15) blocked FGF4-induced P-ERK and P-FRS2a (Fig.4B). Furthermore, exogenous FGF4 significantly attenuated thegrowth-inhibitory effects of trastuzumab alone or L þ T treat-ment in BT474 cells; the effects of FGF4 were reversed by theFGFR inhibitor lucitanib, a small molecule TKI with nmol/Lactivity against FGFR1-3 (ref. 14; Fig. 4C and D). Together,these data suggest that FGF4/FGFR signaling can activate theERK pathway and partially rescue cell viability in the presenceof L þ T.

BT474 Parental(untreated) LTR (L+T)

DBT474 Parental LTRFG

F4 (p

g/m

ol)

BT474 Parental(untreated)

LTR 32-1-5L+T

H&E

Tric

hrom

eK8

/αS

MA/

DAP

IC

D31

K8/ F

ibro

nect

in/

DAP

I

Deg

ree

of fi

bros

is

Ave

rage

pos

. clu

ster

s/H

PFF

B

A

C

E

400

300

200

100

0

BT-7BT-11BT-30

LTR 32-1-

5#LTR 32

-2-1

LTR 33-1-

2LTR (n

= 8)

LTR (n =

8)

Parental

(n =

10)

Parental

(n =

10)

0.0

0.5

1.0

1.5

2.0 30

20

10

0

Figure 3.

LTR xenografts amplify FGF ligandsand downstream signaling. A, FGF3gene copy-number in parental andLTR tumors was determined by FISH.B, Lysates from tumors treated withvehicle (BT) or L þ T (LTR) weresubjected to ELISA for FGF4.(� , P < 0.05; �� , P < 0.01, relative toBT-T parental tumors, one-wayANOVA with Tukey multiplecomparison test). Data represent theaverage � SD of duplicate reactions.C, IHC analysis of P-FGFRY653/654

in tumor sections. D, FFPE sectionsfrom parental BT474 (n¼ 10) and LTR(n ¼ 8) xenografts were stained orsubjected to IHC with the indicatedantibodies. Scale bar, 200 mm. E,Trichrome staining was quantified asdescribed in Materials and Methods.Each bar represents the average� SEM (�� , P < 0.001, Student t test).F, CD31 staining was quantified asdescribed in Materials and Methods.Each bar represents the average� SEM (P ¼ 0.08, Student t test).

Hanker et al.





FGF3/4/19-amplified HER2þ cell lines are sensitive to thecombination of HER2 and FGFR inhibitors

Next, we examined whether FGFR inhibition enhanced thesensitivity of HER2þ/11q13-amplified HCC1954 and MDA-MB361 human breast cancer cell lines to Lþ T. Treatment with Lþ Tfor 24 hours led to a >twofold increase in caspase 3/7 activity inboth cell lines (Supplementary Fig. S5). Lucitanib did not induceapoptosis by itself, but significantly enhanced L þ T-inducedapoptosis in both cell lines, suggesting that FGFR inhibitorswould synergize with HER2 inhibitors in HER2þ/11q13-ampli-fied breast cancers.

Inhibition of FGFR in vivo reverses resistance to lapatinib þtrastuzumab

Wenext examinedwhether blockade of FGFR signaling with anFGFR inhibitor would restore the antitumor action of L þ Tagainst LTR tumors. For this purpose, we used lucitanib, currentlyin early phase clinical trials for cancers with aberrant FGFRpathway activation (11, 27). We treated mice with establishedLTR tumors with vehicle, L þ T, lucitanib alone, or the combi-nation of L þ T þ lucitanib for 6 weeks. Lucitanib alone signif-icantly inhibited tumor growth, but did not induce tumor regres-sions. In contrast, the 6-week treatment with L þ T þ lucitanib

reduced tumor volume by 44% compared with that at baseline(Fig. 5A).

Treatmentwith lucitanib reducedP-FGFR, P-AKT, andP-STAT3,but did not inhibit P-ERK asmeasured by immunoblot analysis ofLTR tumor lysates (Fig. 5B). Likewise, IHC of LTR tumors alsoshowed inhibition of P-FGFR by lucitanib (Supplementary Fig.S6A). The FGFR inhibitor also modestly reduced levels of P-HER2Y877, suggesting FGFR-induced transactivation of HER2at the Src site in LTR tumors. Treatment with Lþ T did not reduceP-HER2, partially reduced P-AKT and P-ERK levels, and increasedlevels of P-STAT3 (Fig. 5B), a signal transducer previously shownto be involved in resistance to targeted therapies, includinglapatinib (28). Treatment with lucitanib further inhibited P-AKTand prevented the induction of P-STAT3 in LTR tumors treatedwith L þ T (Fig. 5B).

Trichrome staining of FFPE sections from LTR tumorsshowed that the addition of lucitanib to L þ T reversed theenhanced collagen deposition seen in LTR tumors (Supple-mentary Fig. S6B). Similarly, the addition of lucitanib stronglyreduced CD31 staining and MVD (Supplementary Fig. S6B andS6C). These data suggest that the microenvironmental changesobserved in the LTR tumors (Fig. 3D) are at least in partdependent on FGFR activity.

A B

C D

# N

ucle

i/wel

l(%

of v

ehic

le c

ontr

ol)

ControlFGF4FGF4+lucitanib

# N

ucle

i/wel

l

[Lapatinib] (nmol/L)

6,000200

150

100

50

0

Vehicl

e

Tras

tuzu

mab

4,000

2,000

00.1 1 10 100 1,000

+ 20 µg/mL trastuzumab

ControlFGF4FGF4+ lucitanib

P-HER2Y877

P-HER2Y1221/2

HER2

P-FRS2αY436

P-AKTS473

P-AKTT308

AKT

P-ERKT202/Y204

ERK

β-Actin

Veh

icle

FGF3

FGF4

FGF1

9

Lap

Lap+

FGF3

Lap+

FGF4

Lap+

FGF1

0

P-FRS2αY436

P-AKTS473

AKT

P-ERKT202/Y204

ERK

β-Actin

P-S6S240/244

L+T+

FGF4

+AZD

AZD

4547

FGF4

FGF4

+AZD

L+T

L+T+

FGF4

L+T+

AZD

Vehi

cle

Figure 4.

Exogenous FGF4 activates ERKand promotes resistance to HER2inhibition. A, Parental BT474 cellsgrown in vitro were serum-starvedovernight and treated the followingday for 2 hours with vehicle or1 mmol/L lapatinib followed bystimulation for 15 minutes with50 ng/mL FGF3, FGF4, or FGF19.Whole cell lysates were subjectedto immunoblot analysis with theindicated antibodies. B, BT474 cellswere serum-starved overnight andtreated the following day for 2 hourswith vehicle, 20 mg/mL trastuzumab þ1 mmol/L lapatinib, or 1 mmol/LAZD4547, and then stimulated for15 minutes with 50 ng/mL FGF4.Whole cell lysates were subjectedto immunoblot analysis with theindicated antibodies. C, BT474 cellsgrowing in low serum (1% CSS) weretreated with vehicle or 50 ng/mL FGF4� 2 mmol/L lucitanib and � 20 mg/mLtrastuzumab. After 6 days, nuclei werestained with Hoechst and scored usingthe ImageXpress system. Datarepresent the average � SD of4 replicate wells (� , P < 0.05;�� , P < 0.001; �� , P < 0.0001,two-way ANOVA followed byBonferroni multiple comparisons test).D, BT474 cells growing in 1% CSS weretreated with increasing concentrationsof lapatinib (0.5–300 nmol/L) þ 20mg/mL trastuzumab � 50 ng/mL FGF4� 2 mmol/L lucitanib for 6 days.Hoechst-stained nuclei were scoredusing the ImageXpress system. Datarepresent the average � SD of 4replicate wells.






In addition to FGFR, lucitanib also inhibits the kinase activity ofVEGFR1-3 (14). Therefore, to determine whether the tumorregression we observed with the combination of L þ T andlucitanib was specific for FGFR, we also treated LTR tumors witha more specific inhibitor of FGFR1-3, AZD4547 (15). AlthoughAZD4547 alone did not affect tumor growth, the combination ofL þ T þ AZD4547 significantly inhibited the growth of LTRtumors (Supplementary Fig. S7A), further suggesting that FGFRinhibition can attenuate resistance to L þ T. Like lucitanib,AZD4547 inhibited FGFR phosphorylation in tumor lysates(Supplementary Fig. S7B), but not to the same degree as lucitanib(Fig. 5B). This result correlates in part with the lack of efficacy ofsingle-agent AZD4547 against LTR xenografts.

Amplification of ERBB2 and FGFR pathway genes overlaps inhuman breast cancer

Analysis of the TCGA breast cancer data revealed that nearly aquarter (23%) of ERBB2-amplified breast cancers also harbor

amplification of FGF3/4/19 (Supplementary Fig. S8). This rate ishigher than the approximately 15% of all breast cancers thatharbor 11q13 amplification. Furthermore, FGFR1 is amplified in12% of the ERBB2-amplified tumors, whereas amplifications ofFGFR2, FGFR3, and FGFR4 are rare. These data suggest that in asignificant cohort of breast cancers, the FGFR andHER2 pathwayscan cooperate to promote tumor progression. Further, aberrantFGF signaling as a result of somatic alterations in the FGFRpathway may play a role in resistance to therapeutic inhibitorsof HER2 in HER2-amplified breast cancers.

High FGFR1 expression correlates with a reduced benefit toadjuvant trastuzumab

Next, we asked whether amplification of FGFR pathway com-ponents correlates with resistance to HER2 inhibitors. We ana-lyzed gene expression of FGFR1 in HER2þ breast cancers frompatients enrolled in the FinHer clinical trial, where patients wererandomized after surgery to receive adjuvant chemotherapy �trastuzumab (Supplementary Fig. S9; ref. 29). Patients withFGFR1 expression in the bottom 80% percentile had a signifi-cantly reduced risk of disease recurrence when treated withtrastuzumab (P¼ 0.018). However, trastuzumab failed to reducethe risk of recurrence in patients with cancers in the top 20% ofFGFR1 expression (Fig. 6A).

FGFR1 and FGF3 gene amplifications associate with lowerpathologic complete response to neoadjuvant anti-HER2therapies

Finally, we interrogated whether 11q (FGF3/4/19) and/orFGFR1 amplification in pretreatment biopsies of patients enrolledin the NeoALTTO trial (3) correlated with the rate of pathologicalcomplete response (pCR) after chemotherapy plus trastuzumab,lapatinib, or L þ T. Importantly, pCR rates correlate with breastcancer survival (30). A total of 134 tumorswere available for copy-number analysis (Supplementary Fig. S10). FGFR1 and FGF3amplification, particularly in ERþ tumors, correlated with a sta-tistically lower rate of pCR (Fig. 6B). Taken together, these resultssuggest a causal association of alterations in the FGFR pathwaywith clinical resistance to anti-HER2 therapies.

DiscussionDe novo and acquired resistance toHER2 inhibitors represents a

major hurdle in the eradication of HER2þ breast cancer. Herein,we used HER2-dependent BT474 breast cancer xenografts tointerrogate mechanisms of acquired resistance to dual HER2blockade. Drug-resistant tumors retained HER2 gene amplifica-tionwithout acquiredmutations inHER2andno longer exhibiteda reduction in HER2 phosphorylation upon treatment with dualanti-HER2 therapy. They also showed markedly reduced in situlevels of lapatinib compared with parental BT474 xenografts. Wefound copy-number gains in FGF3/4/19 and evidence of increasedFGFR signaling in LTR tumors. Treatment with FGF4 attenuatedthe growth-inhibitory effect of Lþ T in vitro, and use of two FGFRTKIs overcame or reduced resistance to L þ T in vivo. Finally,alterations in the FGFR pathway correlated with a worse outcomein patients with early HER2þ breast cancer treated with adjuvantand neoadjuvant anti-HER2 therapies.

Several studies have implicated FGF/FGFR signaling in atten-uating the response to EGFR and HER2 inhibition. ExogenousFGF was previously shown to rescue a subset of HER2þ cell lines

A

B

P-HER2Y1248

P-HER2Y877

HER2

P-AktS473

P-AktT308

Akt

P-ErkT202/Y204

Erk

β-Actin

pS6S240/S244

VehicleLap +Trast Luc.

L+T+Luc

P-FGFRY653/654

pSTAT3Y705

Days of treatment

2,048

1,024

512

Tum

or v

olum

e (m

m3 )

256

128

640 10 20 30 40 50

Vehicle (n = 7)

Lap + Trast (n = 9)

Lucitanib (n = 9)

Lap + Trast + Luc (n = 8)

Figure 5.

FGFR inhibition abrogates resistance to lapatinib þ trastuzumab. A, Micebearing LTR tumors measuring �200 mm3 were treated with (1) vehicle, (2)L þ T, (3) lucitanib, or (4) L þ T þ lucitanib for 6 weeks. Each data pointrepresents the mean tumor volume � SEM. The Y axis is shown on a Log2 scale(� , P < 0.05; �� , P < 0.01, Student t test). B, Tumors were harvested at thecompletion of treatment (6 weeks). Whole tumor lysates were prepared asindicated in Materials and Methods and subjected to immunoblot analysis withthe indicated antibodies.

Hanker et al.





from lapatinib (31). The combination of lapatinib and the FGFRinhibitor dovitinib was found to reverse lapatinib resistance inSKBR3 cells in a large-scale drug combination screen (32). Dovi-tinib also reversed resistance to EGFRor ALK inhibitors in a subsetof patient-derived non–small cell lung cancer (NSCLC) cell lines(32). FGFR2 amplification was found in a lapatinib-resistantHER2þ breast cancer cell line. In this same study, high FGFR2protein levels were associated with decreased progression-freesurvival in a small cohort of breast cancer patients treated withlapatinib (33). Furthermore, combined inhibition of FGFR andHER2 reduced the growth of 4T1 mouse tumors more potentlythan either drug alone (34). In another study, combined inhibi-tion of EGFR and FGFR reduced tumor stroma and delayedrecurrence of FGFR-driven transgenic mammary tumors follow-ing FGFR inhibitor therapy (35). FGFR2 and FGFR3 have beenimplicated in resistance to EGFR inhibition in NSCLC (36) andtrastuzumab resistance in gastric cancer (37). Further, FGFRsignaling was shown to promote resistance to the KIT/PDGFRinhibitor imatinib in gastrointestinal stromal tumors throughreactivationofMAPK signaling (38). These studies further supportour conclusion that FGF3/4/19 amplification can drive resistanceto dual blockade of HER2 through amplification of FGFR signal-ing. Our study builds on these previous findings and is the first toimplicate FGF3/4/19 copy-number gain in clinical resistance toHER2 inhibitors and also to report a correlation between FGFR1and/or FGF3 amplification with a lesser response to HER2 inhi-bitors in patients with early breast cancer.

We found that stimulation of BT474 cells with FGF4was able toinduce P-FRS2 and P-ERK, whereas FGF3 and FGF19 had no effect(Fig. 4). One possibility for this difference is that FGF19 requiresthe b-Klotho coreceptor for signaling (39). Whether FGF4 is theprimary FGF ligand that activates the FGFRpathway in FGF3/4/19-amplified tumors requires additional investigation. These datasuggest that FGF4 may promote resistance to HER2 inhibitionthrough bypass activation of the ERK pathway. However, wenoted a discordance in that exogenous FGF4 treatment onlyinduced partial resistance to Lþ T in vitro (Fig. 4C andD), whereasdrug resistancewasmuchmore pronounced in LTR tumors in vivo.One possibility is that the combination of FGF3, FGF4, andFGF19 together are more effective at promoting resistance thanFGF4 alone. However, neither FGF3 nor FGF19 alone at the testedconcentrations were capable of inducing P-ERK. Another possi-bility is that increased FGF ligandsmay further promote resistancein vivo by altering the tumor microenvironment via paracrineactivation of fibroblasts and endothelial cells, consistent with thealtered tumor stroma we observed in LTR tumors (Fig. 3D). Thecombination of the FGFR TKI lucitanib with L þ T was able toreverse these changes and reduce resistance (Supplementary Fig.S6B and S6C and Fig. 5). Of note, lucitanib is currently in phase IItrials in patients with breast cancer and has shown clear clinicalactivity against tumors harboring FGFR1 and/or 11q13 amplifica-tions (27).

We note that lucitanib showed single-agent activity against LTRtumors, whereas AZD4547 did not. The difference in antitumor

Figure 6.

Alterations in FGFR1 and/or FGF3correlate with a lower clinical responseto anti-HER2 therapies. A, FGFR1mRNA expression was measured bygene expression microarrays in RNAextracted from FFPE tumor sections(n ¼ 202) collected pretreatment aspart of the FinHer trial. Kaplan–Meiercurves according to FGFR1 expressionwere generated by dichotomizingpatients at the top expression levelquintile (�20%) or bottom (80%). B,The copy-number status of FGFR1 andFGF3 was assessed in pretreatmentbiopsies from 134 HER2þ breastcancers from patients treated withneoadjuvant chemotherapyþ trastuzumab, lapatinib, or thecombination in the NeoALTTO trial.Logistic regression of the pCR inFGFR1- or FGF3-amplified patients isshown. Both, FGF3þFGFR1 amplified;CI, confidence interval; FDR, falsediscovery rate.






activitymay be due to other targets of lucitanib, such as VEGFR1-3(14), and/or due to the more potent inhibition of P-FGFR bylucitanib in vivo (Fig. 5B and Supplementary Fig. S7B). Whetherlucitanib alone or in combination with dual HER2 blockade alsoinhibits growth of BT474 parental tumors is not known. Regard-less, we show that combining L þ T with two different FGFRinhibitors diminishes resistance in LTR tumors.

In addition to discovering FGF3/4/19 copy-number gain in asubset of LTR tumors, we also observed an increase in FGF4protein levels and increased FGFR phosphorylation in LTRtumors (Fig. 3B and C). Furthermore, we found that highexpression and/or copy-number amplification of FGFR1 corre-lated with a worse outcome in patients treated with anti-HER2therapies, suggesting that multiple ways to hyperactivate theFGFR pathway (i.e., FGF4 protein overexpression or FGFR1amplification/overexpression) may contribute to resistance toanti-HER2 therapy. Additional FGFR-dependent or -indepen-dent mechanisms of resistance to dual HER2 inhibition remainto be identified.

HER2 phosphorylation levels remained high in LTR tumors,suggesting that these tumors are still dependent on HER2signaling, raising the possibility that improved inhibition ofHER2 kinase activity could overcome resistance. Indeed, wefound that higher doses of lapatinib þ trastuzumab effectivelyblocked the growth of LTR tumor-derived cells in vitro, suggest-ing that more complete suppression of HER2 can overcomeresistance (Supplementary Fig. S3). Although higher doses oflapatinib are not feasible in the clinic, more potent HER2inhibitors, such as neratinib, may be effective in LTR tumors.Alternatively, enhancing the tumor uptake of lapatinib mayalso restore drug sensitivity.

Although lapatinib uptake was significantly reduced in LTRtumors, we did not observe differences between levels of drugtransporters ABCB1, ABCC1, or ABCG2 in BT-474 parental andLTR xenografts (Supplementary Fig. S2). Lapatinib is a substratefor these drug transporters. This suggests that the reduction oflapatinib in LTR versus parental BT474 tumors is not due toaltered drug transport but may be the result of FGF-inducedchanges in the overall tumor microenvironment. Consistentwith this, LTR xenografts demonstrated marked upregulation ofcollagen deposition, increased staining of SMA and fibronectin,markers of stromal fibroblasts, and larger blood vessels.Changes in stromal cell proliferation and the deposition ofcollagen can result in changes in overall tissue heterogeneityand elasticity, as well as accompanying interstitial fluid pressure(40). Increased tissue rigidity and associated activation ofintegrin/focal adhesion signaling have been implicated inresistance to cancer therapies, including lapatinib (41–43). Wespeculate that these tumor microenvironment changes result ina reduction in lapatinib uptake in the LTR tumors as weobserved.

Over 30% of HER2þ breast cancers in the TCGA harboramplifications of FGF3/4/19, FGFR1, or both (SupplementaryFig. S8). Therefore, our findings that FGFR pathway activationcan promote resistance to HER2 inhibitors are potentiallyapplicable to a significant cohort of patients with HER2þ breastcancer. We showed that high FGFR1 expression correlated withde novo resistance to trastuzumab in the FinHER trial. However,we note that the number of patients in this cohort with highFGFR1 expression is small; therefore, this correlation warrantsconfirmation in a larger cohort. Further, amplification of FGFR1

and FGF3 correlated with lower pCR rates following neoadju-vant anti-HER2 therapy in the NeoALTTO trial. We note thatthis correlation was only significant in ERþ patients (Fig. 6B).Although this could be due to the relatively small size of theclinical cohort, this result is not inconsistent with our findingsin ERþ/HER2þ BT474 xenografts. In addition, amplification ofboth FGFR1 and 11q13, where the FGF3/4/19 genes reside, ismore prevalent in ERþ tumors and associated with endocrineresistance (13, 44, 45). These studies suggest that aberrantactivation of the FGFR pathway is associated with primary/intrinsic resistance to therapeutic inhibition of HER2. Futurestudies should examine whether FGF/FGFR signaling is alsoaltered in tumors that have acquired resistance to HER2 inhi-bitors. On this basis, we propose that the combination of HER2and FGFR inhibitors should be prospectively investigated inpatients with HER2þ breast cancer harboring somatic altera-tions in the FGFR pathway.

Disclosure of Potential Conflicts of InterestG.M. Frampton and E. Sanford have ownership interest (including patents) in

Foundation Medicine. H. Joensuu is a consultant/advisory board member forAriad, BluePrintMedicines, andOrion Pharma. J.Huober is a consultant/advisoryboardmember for Novartis and Roche. C.L. Arteaga is a consultant forGenentech.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.B. Hanker, J.T. Garrett, J. Baselga, C.L. ArteagaDevelopment of methodology: A.B. Hanker, J.T. Garrett, E. Langley, S. Singh,P.S. Kim, P. Owens, S. Castellino, S. Broh�eeAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): A.B. Hanker, J.T. Garrett, P.D. Moore, J.P. Koch, E. Langley,S. Singh, P.S. Kim, G.M. Frampton, P. Owens, J. Becker, M.R. Groseclose,S. Castellino, H. Joensuu, J. Huober, J.C. Brase, C. Sotiriou, C.L. ArteagaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.B. Hanker, J.T. Garrett, M.V. Estrada, P.G. Ericsson,S. Singh, P.S. Kim, G.M. Frampton, E. Sanford, P. Owens, J. Becker,M.R. Groseclose, S. Castellino, D. Venet, D. Brown, C. Sotiriou, C.L. ArteagaWriting, review, and/or revision of the manuscript: A.B. Hanker, J.T. Garrett,M.V. Estrada, E. Langley, G.M. Frampton, S. Castellino, H. Joensuu, J. Huober,J.C. Brase, D. Venet, M. Piccart, C. Sotiriou, C.L. ArteagaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):A.B.Hanker, P.D.Moore, P.G. Ericsson, S. Singh,G.M. Frampton, J. BeckerStudy supervision: J. Baselga, C.L. ArteagaOther (manipulation contribution): S. Majjaj

AcknowledgmentsWe thank Teresa Dugger and Violeta S�anchez for expert technical assistance

and Luigi Formisano and Brent Rexer for helpful discussions.

Grant SupportThisworkwas supported byAmericanCancer Society 118813-PF-10-070-01-

TBG (J.T. Garrett) and Department of Defense BC093376 (J.T. Garrett) Post-doctoral Fellowship Awards; Department of Defense Postdoctoral FellowshipAward BC103785 (A.B. Hanker); NIH/NCI K12 Award CA090625 (A.B. Han-ker); NCI R01 grant CA080195 (C.L. Arteaga); NIH Breast Cancer SpecializedProgram of Research Excellence (SPORE) grant P50 CA98131; and Vanderbilt-Ingram Cancer Center Support Grant P30 CA68485.

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 September 21, 2016; revised November 11, 2016; accepted March31, 2017; published OnlineFirst April 5, 2017.

Hanker et al.





References1. SlamonDJ, Clark GM,Wong SG, LevinWJ, Ullrich A,McGuireWL. Human

breast cancer: Correlation of relapse and survival with amplification of theHER-2/neu oncogene. Science 1987;235:177–82.

2. Arteaga CL, Engelman JA. ERBB receptors: From oncogene discovery tobasic science to mechanism-based cancer therapeutics. Cancer Cell2014;25:282–303.

3. Baselga J, Bradbury I, Eidtmann H, Di Cosimo S, de Azambuja E, Aura C,et al. Lapatinib with trastuzumab for HER2-positive early breast cancer(NeoALTTO): A randomised, open-label, multicentre, phase 3 trial. Lancet2012;379:633–40.

4. Swain SM, Kim SB, Cortes J, Ro J, Semiglazov V, Campone M, et al.Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastaticbreast cancer (CLEOPATRA study): Overall survival results from a rando-mised, double-blind, placebo-controlled, phase 3 study. Lancet Oncol2013;14:461–71.

5. Gianni L, Pienkowski T, Im YH, Roman L, Tseng LM, Liu MC, et al. Efficacyand safety of neoadjuvant pertuzumab and trastuzumab in women withlocally advanced, inflammatory, or early HER2-positive breast cancer(NeoSphere): A randomised multicentre, open-label, phase 2 trial. LancetOncol 2012;13:25–32.

6. Hanker AB, Pfefferle AD, Balko JM, Kuba MG, Young CD, Sanchez V, et al.Mutant PIK3CA accelerates HER2-driven transgenicmammary tumors andinduces resistance to combinations of anti-HER2 therapies. Proc Natl AcadSci U S A 2013;110:14372–7.

7. Rexer BN, Chanthaphaychith S, Dahlman K, Arteaga CL. Direct inhibi-tion of PI3K in combination with dual HER2 inhibitors is required foroptimal antitumor activity in HER2þ breast cancer cells. Breast CancerRes 2014;16:R9.

8. Loibl S, von Minckwitz G, Schneeweiss A, Paepke S, Lehmann A, Rezai M,et al. PIK3CA mutations are associated with lower rates of pathologiccomplete response to anti-human epidermal growth factor receptor 2(her2) therapy in primaryHER2-overexpressing breast cancer. J ClinOncol2014;32:3212–20.

9. Koboldt DC, Fulton RS, McLellan MD, Schmidt H, Kalicki-Veizer J, McMi-chael JF, et al. Comprehensive molecular portraits of human breasttumours. Nature 2012;490:61–70.

10. Cerami E,Gao J,DogrusozU,Gross BE, Sumer SO, Aksoy BA, et al. The cBiocancer genomics portal: An open platform for exploringmultidimensionalcancer genomics data. Cancer Discov 2012;2:401–4.

11. Dieci MV, Arnedos M, Andre F, Soria JC. Fibroblast growth factor receptorinhibitors as a cancer treatment: From a biologic rationale to medicalperspectives. Cancer Discov 2013;3:264–79.

12. Elbauomy Elsheikh S, Green AR, Lambros MB, Turner NC, Grainge MJ,PoweD, et al. FGFR1 amplification in breast carcinomas: A chromogenic insitu hybridisation analysis. Breast Cancer Res 2007;9:R23.

13. TurnerN, PearsonA, SharpeR, LambrosM,Geyer F, Lopez-GarciaMA, et al.FGFR1 amplification drives endocrine therapy resistance and is a thera-peutic target in breast cancer. Cancer Res 2010;70:2085–94.

14. Bello E, ColellaG, Scarlato V,Oliva P, Berndt A, ValbusaG, et al. E-3810 is apotent dual inhibitor of VEGFR and FGFR that exerts antitumor activity inmultiple preclinical models. Cancer Res 2011;71:1396–405.

15. Gavine PR, Mooney L, Kilgour E, Thomas AP, Al-Kadhimi K, Beck S, et al.AZD4547: an orally bioavailable, potent, and selective inhibitor of thefibroblast growth factor receptor tyrosine kinase family. Cancer Res 2012;72:2045–56.

16. Nenutil R, Smardova J, Pavlova S, Hanzelkova Z, Muller P, Fabian P, et al.Discriminating functional and non-functional p53 in human tumours byp53 and MDM2 immunohistochemistry. J Pathol 2005;207:251–9.

17. Wang D, Stockard CR, Harkins L, Lott P, Salih C, Yuan K, et al. Immuno-histochemistry in the evaluation of neovascularization in tumor xeno-grafts. Biotech Histochem 2008;83:179–89.

18. Weidner N. Current pathologic methods for measuring intratumoralmicrovessel densitywithin breast carcinoma andother solid tumors. BreastCancer Res Treat 1995;36:169–80.

19. Walsh AJ, Cook RS, Lee JH, Arteaga CL, Skala MC. Collagen density andalignment in responsive and resistant trastuzumab-treated breast cancerxenografts. J Biomed Opt 2015;20:26004.

20. Owens P, PickupMW, Novitskiy SV, Giltnane JM, Gorska AE, Hopkins CR,et al. Inhibition of BMP signaling suppresses metastasis in mammarycancer. Oncogene 2015;34:2437–49.

21. Polli JW, Humphreys JE, Harmon KA, Castellino S, O'Mara MJ, Olson KL,et al. The role of efflux and uptake transporters in [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino }methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) dispositionand drug interactions. Drug Metab Dispos 2008;36:695–701.

22. Garrett JT, Sutton CR, Kuba MG, Cook RS, Arteaga CL. Dual blockade ofHER2 in HER2-overexpressing tumor cells does not completely eliminateHER3 function. Clin Cancer Res 2013;19:610–9.

23. Garrett JT, Sutton CR, Kurupi R, Bialucha CU, Ettenberg SA, Collins SD,et al. Combination of antibody that inhibits ligand-independent HER3dimerization and a p110alpha inhibitor potently blocks PI3K signalingand growth of HER2þ breast cancers. Cancer Res 2013;73:6013–23.

24. Mittendorf EA, Wu Y, Scaltriti M, Meric-Bernstam F, Hunt KK, Dawood S,et al. Loss of HER2 amplification following trastuzumab-based neoadju-vant systemic therapy and survival outcomes. Clin Cancer Res 2009;15:7381–8.

25. Polli JW, Olson KL, Chism JP, John-Williams LS, Yeager RL, Woodard SM,et al. An unexpected synergist role of P-glycoprotein and breast cancerresistance protein on the central nervous systempenetration of the tyrosinekinase inhibitor lapatinib (N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino }methyl)-2-furyl]-4-quinazolina-mine; GW572016). Drug Metab Dispos 2009;37:439–42.

26. Turner N, Grose R. Fibroblast growth factor signalling: From developmentto cancer. Nat Rev Cancer 2010;10:116–29.

27. Soria JC, DeBraud F, Bahleda R, Adamo B, Andre F, Dientsmann R, et al.Phase I/IIa study evaluating the safety, efficacy, pharmacokinetics, andpharmacodynamics of lucitanib in advanced solid tumors. Ann Oncol2014;25:2244–51.

28. Lee HJ, Zhuang G, Cao Y, Du P, Kim HJ, Settleman J. Drug resistance viafeedback activation of Stat3 in oncogene-addicted cancer cells. Cancer Cell2014;26:207–21.

29. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, Alanko T, Kataja V, Asola R,et al. Adjuvant docetaxel or vinorelbine with or without trastuzumab forbreast cancer. N Engl J Med 2006;354:809–20.

30. Cortazar P, Zhang L, Untch M, Mehta K, Costantino JP, Wolmark N, et al.Pathological complete response and long-term clinical benefit in breastcancer: The CTNeoBC pooled analysis. Lancet 2014;384:164–72.

31. Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, et al.Widespreadpotential for growth-factor-driven resistance to anticancer kinase inhibi-tors. Nature 2012;487:505–9.

32. Crystal AS, Shaw AT, Sequist LV, Friboulet L, Niederst MJ, Lockerman EL,et al. Patient-derived models of acquired resistance can identify effectivedrug combinations for cancer. Science 2014;346:1480–6.

33. Azuma K, Tsurutani J, Sakai K, Kaneda H, Fujisaka Y, Takeda M, et al.Switching addictions between HER2 and FGFR2 in HER2-positive breasttumor cells: FGFR2 as a potential target for salvage after lapatinib failure.Biochem Biophys Res Commun 2011;407:219–24.

34. Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, et al.Combinatorial targeting of FGF and ErbB receptors blocks growth andmetastatic spread of breast cancer models. Breast Cancer Res 2013;15:R8.

35. Holdman XB, Welte T, Rajapakshe K, Pond A, Coarfa C, Mo Q, et al.Upregulation of EGFR signaling is correlated with tumor stroma remodel-ing and tumor recurrence in FGFR1-driven breast cancer. Breast Cancer Res2015;17:141.

36. Ware KE, Marshall ME, Heasley LR, Marek L, Hinz TK, Hercule P, et al.Rapidly acquired resistance to EGFR tyrosine kinase inhibitors in NSCLCcell lines throughde-repressionof FGFR2andFGFR3 expression. PLoSOne2010;5:e14117.

37. Piro G, Carbone C, Cataldo I, Di Nicolantonio F, Giacopuzzi S, Aprile G,et al. An FGFR3 autocrine loop sustains acquired resistance to trastuzumabin gastric cancer patients. Clin Cancer Res 2016;22:6164–75.

38. Li F, Huynh H, Li X, Ruddy DA, Wang Y, Ong R, et al. FGFR-mediatedreactivation of MAPK signaling attenuates antitumor effects of imatinib ingastrointestinal stromal tumors. Cancer Discov 2015;5:438–51.

39. Kurosu H, Choi M, Ogawa Y, Dickson AS, Goetz R, Eliseenkova AV, et al.Tissue-specific expression of betaKlotho andfibroblast growth factor (FGF)receptor isoforms determines metabolic activity of FGF19 and FGF21. JBiol Chem 2007;282:26687–95.

40. Lu P, Weaver VM, Werb Z. The extracellular matrix: A dynamic niche incancer progression. J Cell Biol 2012;196:395–406.






41. Egeblad M, Rasch MG, Weaver VM. Dynamic interplay between thecollagen scaffold and tumor evolution. Curr Opin Cell Biol 2010;22:697–706.

42. Lin C, Pelissier FA, Zhang H, Lakins J, Weaver VM, Park C, et al. Micro-environment rigidity modulates responses to the HER2 receptor tyrosinekinase inhibitor lapatinib via YAP and TAZ transcription factors. Mol BiolCell 2015;26:3946–53.

43. Hirata E, Girotti MR, Viros A, Hooper S, Spencer-Dene B, Matsuda M, et al.Intravital imaging reveals how BRAF inhibition generates drug-tolerant

microenvironments with high integrin beta1/FAK signaling. Cancer Cell2015;27:574–88.

44. Elsheikh S, Green AR, Aleskandarany MA, Grainge M, Paish CE, LambrosMB, et al. CCND1 amplification and cyclin D1 expression in breast cancerand their relation with proteomic subgroups and patient outcome. BreastCancer Res Treat 2008;109:325–35.

45. Roy PG, Pratt N, Purdie CA, Baker L, Ashfield A, Quinlan P, et al. HighCCND1 amplification identifies a group of poor prognosis women withestrogen receptor positive breast cancer. Int J Cancer 2010;127:355–60.


Hanker et al.




Correction

Correction: HER2-Overexpressing BreastCancers Amplify FGFR Signaling uponAcquisition of Resistance to Dual TherapeuticBlockade of HER2Ariella B. Hanker, Joan T. Garrett, M�onica Valeria Estrada,Preston D. Moore, Paula Gonz�alez Ericsson, James P. Koch,EmmaLangley, SharatSingh, PhillipS.Kim,GarrettM.Frampton,Eric Sanford, Philip Owens, Jennifer Becker, M. Reid Groseclose,Stephen Castellino, Heikki Joensuu, Jens Huober, Jan C. Brase,Samira Majjaj, Sylvain Broh�ee, David Venet, David Brown,Jos�e Baselga, Martine Piccart, Christos Sotiriou, andCarlos L. Arteaga

In the original version of this article (1), the stated disclosure of the author Carlos L.Arteaga is incorrect. The error has been corrected in the latest online HTML and PDFversions of the article. The authors regret this error.

Reference1. Hanker AB, Garrett JT, Estrada MV, Moore PD, Ericsson PG, Koch JP, et al. HER2-overexpressing

breast cancers amplify FGFR signaling upon acquisition of resistance todual therapeutic blockadeofHER2. Clin Cancer Res 2017;23:4323–34.

Published first February 15, 2019.doi: 10.1158/1078-0432.CCR-18-4267�2019 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 25(4) February 15, 20191434

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-18-4267&domain=pdf&date_stamp=2019-2-1

2017;23:4323-4334. Published OnlineFirst April 5, 2017.Clin Cancer Res Ariella B. Hanker, Joan T. Garrett, Mónica Valeria Estrada, et al. HER2upon Acquisition of Resistance to Dual Therapeutic Blockade of HER2-Overexpressing Breast Cancers Amplify FGFR Signaling

Updated version

10.1158/1078-0432.CCR-16-2287doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/04/05/1078-0432.CCR-16-2287.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/23/15/4323.full#ref-list-1

This article cites 45 articles, 19 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/23/15/4323.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/23/15/4323To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-16-2287

http://clincancerres.aacrjournals.org/content/suppl/2017/04/05/1078-0432.CCR-16-2287.DC1

http://clincancerres.aacrjournals.org/content/23/15/4323.full#ref-list-1

http://clincancerres.aacrjournals.org/content/23/15/4323.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/23/15/4323


her2-overexpressing breast cancers amplify fgfr signaling … · cancer therapy: preclinical...

Documents