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2010;70:4151-4162. Published OnlineFirst May 11, 2010.Cancer ResJulien H. Dey, Fabrizio Bianchi, Johannes Voshol, et al.Mammary Tumor Outgrowth and MetastasisPI3K/AKT Signaling, Induces Apoptosis, and ImpairsTargeting Fibroblast Growth Factor Receptors Blocks

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Tumor and Stem Cell Biology

Targeting Fibroblast Growth Factor Receptors Blocks

PI3K/AKT Signaling, Induces Apoptosis, and Impairs

Mammary Tumor Outgrowth and Metastasis

Julien H. Dey1, Fabrizio Bianchi4, Johannes Voshol2, Debora Bonenfant2, Edward J. Oakeley3, and Nancy E. Hynes1

Abstract

Members of the fibroblast growth factor receptor (FGFR) family have essential roles in normal physiology

and in cancer where they control diverse processes. FGFRs have been associated with breast cancer develop-

ment. Thus, models to study the role of FGFR in breast cancer and their targeting potential are important. We

present an in vitro and in vivo analysis of FGFRs in the breast cancer model cell lines 67NR and 4T1. We show

that both tumor cell lines coexpress FGFRs and ligands and display autocrine FGFR signaling activity. Fibro-

blast growth factor receptor substrate 2 (FRS2), a downstream mediator of FGFR, is constitutively tyrosine

phosphorylated and multiple signaling pathways are active. Treatment of 67NR and 4T1 cultures with TKI258,

an FGFR tyrosine kinase inhibitor (TKI), caused a rapid decrease in FRS2 phosphorylation; decreased the ac-

tivity of extracellular signalregulated kinase 1/2 (ERK1/2), AKT, and phospholipase C; and blocked prolif-

eration of both tumor lines. Furthermore, TKI258 induced 4T1 apoptotic cell death via blockade of the

phosphoinositide 3-kinase/AKT pathway. In vivo, one dose of TKI258 rapidly lowered FRS2 phosphorylation

and ERK1/2 and AKT activity in mammary tumors. Long-term TKI258 treatment of 4T1 tumor and 67NR

tumorbearing mice had a significant effect on primary tumor outgrowth and 4T1 tumor induced lung me-

tastases. A microarray analysis was carried out to identify targets with roles in TKI258 antitumor activity and

potential prognostic markers in human breast tumors. Of interest are the downregulated matrix metallopro-

teases (MMP), in particular MMP9, which is essential for metastatic spread of 4T1 tumors. Cancer Res; 70(10);

415162. 2010 AACR.

Introduction

Deregulated activity of receptor tyrosine kinases (RTK) hasbeen implicated in breast cancer development. ErbB2 over-

expression has been intensely studied and has been success-fully targeted in the clinic with antibodies and small

molecular tyrosine kinase inhibitors (TKI; ref. 1). Considering

that only 25% of patients are eligible for ErbB2-directed treat-

ments, it is essential to uncover additional therapeutic tar-

gets. The association between fibroblast growth factors(FGF) and mammary cancer was first established in mouse

mammary tumor virusinduced tumors (2) and elevated

levels of FGF8 have been found in human breast tumors

(3). FGF receptor (FGFR) amplification has been reported

in subtypes of breast cancer (4, 5) and FGFR1 levels have

been linked to poor survival rates (6). Intriguingly, genome- wide screens aimed at uncovering breast cancerassociated

genes identified single nucleotide polymorphisms in FGFR2

(7, 8). Based on the increasing evidence supporting the rele-

vance of FGFRs, we have explored the role of this receptor in

breast cancer models. We show here that 4T1 and 67N mam-

mary tumor cells (9) coexpress multiple FGFRs and ligands

and display autocrine FGFR activity. Accordingly, many tar-

gets of FGFR signaling, the docking protein fibroblast growth

factor receptor substrate 2 (FRS2), Src, phospholipase C

(PLC), Shp-2, signal transducer and activator of transcrip-

tion 3 (STAT3), and phosphoinositide 3-kinase (PI3K)/AKT

(1012), are active in both tumor cell lines.

We investigated the effects of FGFR inhibition usingTKI258, an FGFR TKI (13). Treatment of 4T1 and 67NR

cultures with TKI258 decreased the activity of numerous sig-naling proteins and blocked cell proliferation. Treatment of

tumor-bearing mice with TKI258 led to a strong reduction

of mammary tumor growth and, for the aggressive 4T1 model,a decrease in lung metastasis. Moreover, we provide evidence

that FGFR blockade downregulates key players involved in the

metastatic process, in particular matrix metalloprotease-9

(MMP9) and the transcription factor Twist, which have beenshown to be major regulators of lung metastasis (14, 15).

Authors' Affiliations: 1Friedrich Miescher Institute for BiomedicalResearch; 2Novartis Pharma AG; 3Novartis Institutes for BiomedicalResearch, Basel, Switzerland; and 4IFOM, Fondazione Istituto FIRC diOncologia Molecolare at IFOM-IEO Campus, Milan, Italy

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

Corresponding Author: Nancy E. Hynes, Growth Control Department,Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse66, CH-4058 Basel, Switzerland. Phone: 41-61-6978107; Fax: 41-61-6973976; E-mail: [email protected].

doi: 10.1158/0008-5472.CAN-09-4479

2010 American Association for Cancer Research.

Cancer

Research

www.aacrjournals.org 4151


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Materials and Methods

Kinase inhibitors. TKI258 (13) was provided by Drs.D. Graus-Porta and C. Garcia-Echeverria (Novartis Institutesfor Biomedical Research, Basel, Switzerland); NVP-BEZ235was provided by Dr. S-M. Maira (Novartis Institutes for Bio-

medical Research, Basel, Switzerland); STI571 and PTK787were from Novartis Institutes for Biomedical Research. All in-hibitors were prepared as 10 mmol/L DMSO stocks.

Proliferation, migration, and apoptosis assays. The anti- proliferative effects of TKI258 were evaluated in 96-well plates over 24 to 48 hours using a bromodeoxyuridine

(BrdUrd) ELISA kit (GE Healthcare; ref. 16). The cytotoxic ef-fects of TKI258, UO126, PD173074, and NVP-BEZ235 wereevaluated in 96-well plates 24 to 48 hours after treatmentby measuring total cell number and cell death using the YO-PRO assay (Invitrogen; ref. 17). Cell migration usingTranswell assays was performed as described (ref. 18; seeSupplementary Methods for a complete description).

In vivo treatments and analysis of tumor and metasta-

sis formation. Animal experiments were done according tothe Swiss guideline governing animal experimentation and ap-proved by the Swiss veterinary authorities. 4T1 and 67NR cells(5105 o r 5 1 06) were injected in the fourth mammary fat padof 10-week-old BALB/c mice (RCC, Basel, Switzerland). Oncepalpable, tumors were measured daily and volume was calcu-lated using the following formula: height [(diameter/2)2 ].Mice were randomly distributed into treated or control groupswhen tumors reached 50 to 100 mm3 and treated with vehicle(water), TKI258 (p.o., once daily at 20, 40, or 50 mg/kg), or ve-hicle (polyethylene glycol 300) and PTK787 (p.o., once daily at25 or 50 mg/kg) for the indicated times. For experimental me-tastasis, 2.5 105 4T1 cells were injected into tail veins; 5 dayslater, mice were randomized and treated with water or TKI258(p.o., once daily, 9 days at 40 mg/kg). At the end point, micewere sacrificed and tumors and lungs were dissected. Lungswere placed in Bouin's solution to visualize and count metasta-ses. Pictures of the lungs were taken with a Leica MacroFluo Z6and the number of nodules and the surface occupied by the

metastasis were quantified using image access software.Gene expression analysis. RNA from TKI258- or DMSO-

treated 4T1 cells (triplicate experiments) was amplified andlabeled using the Ambion MesageAMP III RNA AmplificationKit (Applied Biosystems). The same protocol was applied onRNA from three tumors from mice treated 14 days withTKI258 or water. Biotinylated, fragmented cRNA was hybrid-ized to Affymetrix Mouse Gene 1.0 ST Array (Affymetrix).

Dataanalysis and genefiltering weredone using R/Bioconduc-tor (19). Signal condensation was done using only the RMAfrom the Bioconductor Affy package. Differentially expressedgenes were identified using the empirical Bayes method (Ftest) implemented in the LIMMA package and adjusted withthe false discovery rate method (20). Hierarchical clusteringand visualization were done in R. Probe sets with a log 2 aver-age contrast signal of at least 5, an adjusted P value of 0.585 (1.5-fold in linearspace) were selected leading to the identification of 2,064 and543genes changed in cells andtumors, respectively, on TKI258

treatment. The complete microarray data are available in theGene Expression Omnibus (GSE19222).

TKI258-regulated genes were analyzed in available datasets. The GEO database was used for breast cancer cohorts:TRANSBIG (GSE7390) and ERASMUS (GSE2034) and for a tu-mor model (GSE15299). Data were processed and normalized

using MAS 5.0 (GSE7390 and GSE2034) or RMA (GSE15299)algorithms as described in the GEO database. Affymetrix

probe sets were mapped to in vitro and in vivo regulatedgenes using the NetAffx web site. Hierarchical clusteringwas done with cluster 3.0 (21, 22) on log 2 median centered

data using uncentered correlation and average linkage clus-tering algorithms. Trees were displayed by Java Tree View v1.1.1. Kaplan-Meier analysis was done using JMP IN 5.1

(SAS Institute, Inc.) and relative P values were calculated with log-rank test. P values of the overlaps were calculatedusing Fisher's exact test. Functional classification analysis was done using Ingenuity Pathway Analysis (Ingenuity Sys-tems, Inc.). Gene-set enrichment analysis was done withGSEA (23) using default settings by collapsing probe sets to

unique genes and taking the probe set median expressionvalue. Significance of the enrichment was estimated by1,000 random gene-set permutations.

Results

Constitutive FGFR signaling in 4T1 and 67NR tumor

cells. BALB/c mice develop mammary tumors followinginjection of 67NR and 4T1 tumor cell lines; 4T1 tumors aremore aggressive, forming distant lung metastases (9). Bothcell lines were examined for FGFR and FGF expression byreverse transcription-PCR (RT-PCR). 4T1 cells expressFGFR1, FGFR2, and FGFR3 (Fig. 1A), with FGFR2 expressionbeing the highest. 67NR cells express FGFR2 and FGFR3.FGF1, which activates all receptors, was found in both lines(Fig. 1A). A Western blot analysis confirmed that FGFR2and FGFR3 are expressed (Supplementary Fig. S1A). Ascoexpression of FGFRs and FGF1 was observed, we testedthe hypothesis that these tumor cell lines have autocrineFGFR activity.

The activity of signaling proteins downstream of FGFR was measured in lysates of cells grown in full mediumor serum starved (Fig. 1B). The adaptor protein FRS2 linksFGFRs to various pathways (11, 12). Probing of FRS2

immunoprecipitates (IP) from 4T1 cells revealed high levelsof phospho-tyrosine (Ptyr); the level of Ptyr196-FRS2,a docking site for growth factor receptor binding protein

2 (Grb2; ref. 11), was also elevated in both cell lines(Fig. 1B). Furthermore, Ptyr and Ptyr783 were detectablein PLC, and the levels of phosphorylated extracellularsignalregulated kinase 1/2 (P-ERK1/2), P-AKT, P-STAT3,and P-Src were also high (Fig. 1B). With the exception ofP-AKT, which was slightly lower in serum-starved cells,there was little or no change in activity of the other signal-ing proteins following serum deprivation (Fig. 1B). Takentogether, these results provide strong evidence supportingthe hypothesis that 4T1 and 67NR cells possess autocrineFGFR activity.

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Figure 1. FGFR signaling in 4T1 and 67NR cells. A, semiquantitative RT-PCR for FGFRs and FGF1, with glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) as control. B, lysates from cells in serum or serum starved were immunoblotted with the indicated antibodies. FRS2 and PLC IPs were probed

for Ptyr content. C, lysates from serum grown 4T1 cultures were treated for 1 h with 1 mol/L TKI258, then IPs of FGFR3 and Gab1 were probed for

Ptyr and IPs of FRS2 were probed for Shp-2 and Grb2. Whole-cell lysates (WCL) and IPs were reprobed as indicated. D, serum-starved cultures were

pretreated for 1 h with 1 mol/L TKI258 or DMSO before FGF2 stimulation. Whole-cell lysates were prepared and immunoblot analyses done with the

indicated antibodies. FRS2 and PLC IPs were probed for Ptyr content.

FGFR and Mammary Cancer

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TKI258 lowers FGFR activity and blocks signaling

pathways. To gain more insight into the intracellular pathways controlled by FGFR, we used TKI258, an FGFR TKI(13, 24). Initially, a global phospho-proteomic screen wasundertaken to identify proteins undergoing changes in Ptyrin response to TKI258 treatment of 4T1 cells. Lysates made

from controls and TKI258-treated cultures were subjected totryptic digestion before IP with Ptyr antibodies. Pulled downpeptides were subjected to liquid chromatography-tandemmass spectrometry (LC-MS/MS) for detection and quantifi-cation of Ptyr changes. Supplementary Table S1 lists peptides

that were significantly changed in two independent analyses.One FGFR2-specific Ptyr peptide from the kinase domainwas 1.67-fold decreased, showing that TKI258 blocked FGFR

signaling. FRS2-, Mapk3-, and Gab1-specific Ptyr peptides were also strongly decreased following FGFR inhibition.FGFR3-specific Ptyr peptides were not detected in thisanalysis; however, IPs of FGFR3 from lysates of treated cellsrevealed a loss of Ptyr (Fig. 1C, left). FGFR1 levels are low in4T1 cells; however, manual inspection of the LC-MS/MS data

revealed lower levels of one FGFR1-specific Ptyr peptide.

These results suggest that all the FGFRs in 4T1 cells areactive and blocked by TKI258.

Gab1 and FRS2 IPs [Fig. 1C (middle) and D] from lysatesof inhibitor-treated cells revealed lower Ptyr content compared with controls; Ptyr196-FRS2 levels were also stronglydecreased in TKI258-treated 4T1 and 67NR cells (Fig. 1D).

Moreover, decreased levels of Shp-2 and Grb2 were com-plexed with FRS2 in TKI258-treated 4T1 cells (Fig. 1C, right)and FRS2 IPs revealed a dramatic shift [Fig. 1C (right) andD], reflecting lower activity of mitogen-activated protein ki-nase, which phosphorylates multiple threonine residues on

FRS2 (25). P-AKT and Ptyr-PLC levels were also decreasedin inhibitor-treated cells; no effect on constitutive STAT3or Src activity was seen (Supplementary Fig. S1B). Taken

together, these results confirm the phospho-proteomic analysis and show that TKI258 has a strong effect on FGFR-mediated signaling.

TKI258 treatment blocks proliferation and migration.

Effects of FGFR inhibition on proliferation were examinedusing BrdUrd incorporation assays. We observed a dose-

dependent decrease of cells in S phase, with an 80%

Figure 2. Cellular effects of TKI258. A, cultures were treated for 48 h with the indicated concentrations of TKI258; BrdUrd was added 2 h before

end of experiment. Percentage of incorporated BrdUrd relative to controls is plotted (left). Cultures were treated for 24 h with 1 mol/L TKI258 and flow

cytometry was done after propidium iodide staining. Percent G1 cells is indicated (right). B, lysates from cells treated with 1 mol/L TKI258 for different times

were immunoblotted with the indicated antibodies; tubulin served as control. C, chemokinetic and chemotactic response of 4T1 cells to serum was

measured in Transwell assays in DMSO or TKI258. *, P < 0.001 for chemokinesis (one-way ANOVA); *, P < 0.001 for chemotaxis (t test). D, cultures were

treated for 48 h with different concentrations of TKI258; cell death was determined with the YO-PRO assay and plotted relative to vehicle-treated cells.

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decrease at the highest dose (Fig. 2A, left). Furthermore,flow cytometry of propidium iodidestained cells revealeda strong G1 accumulation (Fig. 2A, right). Cyclin D1 protein

(Fig. 2B) and RNA (data not shown) decreased rapidlyfollowing TKI258 treatment, and in 4T1 cells there was anincrease in p27 (Fig. 2B). Another inhibitor that blocksFGFR, PD173074 (26), also lowered proliferation of 4T1and 67NR cells (Supplementary Fig. S1C, left). Next, we ex-amined the effect of FGFR inhibition on the directional(chemotactic) and random (chemokinetic) migration in

Transwell assay chambers. Serum was added to the lowerchamber to determine chemotaxis and to both chambersfor chemokinesis. Both types of migration were significantly

blocked in the presence of TKI258 (Fig. 2C).

TKI258 induces apoptosis in 4T1 cells that is rescued by

expression of Myr-AKT. There was also an increase in dyingcells in TKI258-treated 4T1, but not 67NR, cultures (Fig. 2D).

The same results were seen with PD173074 (SupplementaryFig. S1C, right). The process is due to apoptosis because in-creased levels of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase were detected in the cells (Fig. 2B).The differential response of the cell lines might be due, inpart, to the ability of TKI258 to decrease P-AKT levels in4T1 cells, but not in 67NR cells, growing in full serum (Sup-

plementary Fig. S1D, panels 4 and 5 versus 7 and 9). Thismight also explain the lack of p27 induction in the 67NRcells because it was recently shown that the induction ki-

netics and final p27 level were dependent on the duration

Figure 3. PI3K/AKT signaling is required for survival. A and B, cultures were treated for 24 h with different concentrations of UO126 or NVP-BEZ235;

cell number was determined and plotted relative to control. B, bottom, number of dead cells was determined and plotted relative to control. C, cells stably

expressing vector control or Myr-AKT were treated for 24 h with different TKI258 concentrations; fold increase in cell death relative to controls was

determined using the YO-PRO assay. D, lysates prepared from vector control or Myr-AKT cells cultured in serum or serum-free conditions and treated with

1 mol/L TKI258 for different times were immunoblotted as indicated.





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Figure 4. In vivo effects of TKI258. A, 4T1 tumorbearing mice were treated once with TKI258 or vehicle. Tumors were collected at various times,

and lysates prepared and immunoblotted as indicated. B, 4T1 tumorbearing mice were treated daily with TKI258 (40 or 20 mg/kg) or water for 14 d.

*, P< 0.005 (one-way ANOVA). C, 67NR tumorbearing mice were treated with TKI258 (40 mg/kg) or water for 5 d, followed by 2 d off treatment. *, P < 0.05

(Mann-Whitney test). D, 4T1 tumors from 14-dtreated TKI258 or control group were harvested; frozen sections were stained for CD31, cleaved

caspase-3, or phospho-histone H3. CD31-positive area was measured and plotted as percent of tumor area. Cleaved caspase-3positive and total

cells were counted and plotted as percentages. Phospho-histone H3positive and total cells were counted and plotted as percentages. **, P < 0.01;

*, P < 0.05 (t test).

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of AKT inhibition when a panel of PI3K inhibitors weretested (27).

Next, we analyzed if the ERK and PI3K/AKT pathwayswere responsible for TKI258 activity on proliferation and ap-optosis. The mitogen-activated protein/ERK kinase inhibitorUO126 and the PI3K inhibitor NVP-BEZ235 (28) were used totarget the respective pathways. In UO126-treated 4T1 cul-tures, proliferation was 40% decreased at the maximal dose(Fig. 3A), with no evidence of apoptosis (data not shown).NVP-BEZ235 blocked proliferation by 50% and induced celldeath (Fig. 3B). These results suggest that active FGFR signal-ing maintains high PI3K/AKT pathway activity, which isessential for 4T1 cell survival. To explore this, a vector ex-

pressing active AKT (Myr-AKT) and a control vector (pBN)were introduced into 4T1 cells and pools were examined

for TKI258 sensitivity. Myr-AKT

expressing cells were 2-foldless sensitive than controls to TKI258 treatment (Fig. 3C)and maintained high levels of P-Myr-AKT in the presenceof TKI258 in conditions of serum depletion or full serum(Fig. 3D). Taken together, these results show the importanceof the PI3K/AKT pathway for 4T1 cell survival.

Decreased tumor growth in TKI258-treated mice. Next,the in vivo effects of TKI258 were examined. First, a single50 mg/kg dose was administered to 4T1 tumorbearing mice,then tumors were collected 2, 8, and 24 hours after dosingand lysates prepared from three tumor-bearing mice per

Figure 5. Effect of TKI258 on metastasis. A, representative pictures of lungs from 14-dtreated TKI258 or control mice; front and back views of left lobes.

Bottom, quantification of metastatic foci and lung percent covered by metastases in control and TKI258-treated groups (n = 6). *, P < 0.05, one-way

ANOVA test (left) or Kruskal-Wallis test (right). B, 4T1 cells were injected in tail veins and, 5 d later, mice were treated with TKI258 or vehicle for 9 d.

Quantification of metastatic foci and percent of lung covered by metastases in control and TKI258-treated groups (n = 3) was done as in A. *, P < 0.05

(Student's t test). C, quantitative real-time RT-PCR for different MMPs was done on RNA from control and TKI258-treated 4T1 cells. D, lysates from

control and TKI258-treated 4T1 cells were immunoblotted for MMP9; tubulin served as control.





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time point were analyzed. Vehicle-treated mice had highlevels of Ptyr196-FRS2 and active ERK1/2, AKT, and PLC(Fig. 4A). Importantly, within 2 hours of TKI258 administra-tion, there was a significant decrease in Ptyr196-FRS2 andP-ERK1/2 levels in the tumors; P-AKT and P-PLC levelsdecreased to a lesser extent (Fig. 4A). Ptyr196-FRS2 levels re-

mained low for 24 hours, whereas active ERK1/2, AKT, andPLC started to increase by 8 hours (Fig. 4A). These resultsshow that TKI258 rapidly blocks the FGFR pathway in vivo inthe tumors.

Next, we tested the long-term effects of TKI258 treatment.

Mice bearing 4T1- and 67NR-induced tumors were random-ly distributed into treated or control groups. 4T1 tumorbearing mice were dosed daily at 20 and 40mg/kg for

14 days; 67NR tumorbearing mice were dosed as indicatedwith 40 mg/kg for 16 days. Nonsignificant changes in body weight were observed in TKI258-treated animals (Supple-mentary Fig. S2A). Importantly, there was a significantreduction in tumor outgrowth and in tumor weight in the

TKI258-treated groups of 4T1 tumor and 67NR tumorbearing mice (Fig. 4B and C; Supplementary Fig. S2B), show-ing that blockade of FGFR has strong antitumor activity.

To determine the mechanisms underlying TKI258 activ-ity, 4T1-induced tumors collected on day 24 were exam-ined for vessel density, proliferation, and apoptosis.

Quantification of CD31-stained and cleaved caspase-3stained sections revealed a significant decrease in vesseldensity and a significant increase in cell death inTKI258-treated animals (Fig. 4D, top and middle). Therewas no significant change in the mitotic marker phospho-

histone H3 (Fig. 4D, bottom). Taken together, these resultssuggest that TKI258 inhibits tumor outgrowth mainly byimpairing cell survival, which might result from decreased

P-AKT (Fig. 4A) and/or decreased vessel density. Theeffects of TKI258 are rapid, showing significant differencein tumor volume in the treated groups within 3 days;however, consistent long-term tumor shrinkage was notobserved (Fig. 4B and C).

Figure 6. Meta-analysis of TKI258-regulated genes in human breast tumors. A, unsupervised hierarchical clustering of TKI258-regulated genes identified

in vitro in 4T1 cells (1,648 genes) in the TRANSBIG and ERASMUS cohorts. Red tree and box indicate cluster 1A patients and relative overexpressed

genes in the cluster, respectively. B, unsupervised hierarchical clustering of genes regulated by TKI258 in 4T1 tumors (254 genes) in the TRANSBIG

and ERASMUS cohorts. Yellow tree and box and green tree and box indicate cluster 2B and cluster 1B patients and relative overexpressed genes in

the clusters, respectively. C, Kaplan-Meier plots of cluster 1A patients (red line) compared with the others (gray line) and plots considering only patients with

basal-like tumors. P values were computed using log-rank t test. D, Kaplan-Meier plots of patients in cluster 1B (green line) and cluster 2B (orange line)

compared with others (gray line) and by considering only patients with basal-like tumors. P values were computed using log-rank t test.

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TKI258-treated animals have fewer lung metastases. Toanalyze the effects of TKI258 on metastasis, lungs of 4T1tumorbearing mice sacrificed on day 24 were removedand stained with Bouin's fixative, then metastatic nodules were quantified. In vehicle-treated animals, multiple largenodules were evident, whereas the extent of lung metastasis

was dramatically reduced in TKI258-treated mice (Fig. 5A,top). Quantification of foci number and percent covered bymetastases revealed a significant decrease in both para-meters (Fig. 5A, bottom). To assess the effects of TKI258directly in the lungs, 4T1 cells were injected through the tail

vein and, 5 days later, TKI258 was administered for 9 days(Fig. 5B). TKI258 had a slight, nonsignificant effect on focinumber (Fig. 5B, top), whereas the percent covered by metas-

tases was significantly decreased (Fig. 5B, bottom). Thus,FGFR blockade also impairs the ability of 4T1 cells to growin the lungs following tail vein injection.

Array data on TKI258-treated 4T1 cells and tumor-

bearing mice. To find genes changed by TKI258, a genome- wide transcriptome analysis was done and differentially

regulated genes were identified. From triplicates of 16-hourTKI258- or vehicle-treated 4T1 cells, 2,064 significantlychanged probe sets (1,648 annotated genes) were identified.The same analysis performed on triplicate tumors from14-day TKI258-treated versus vehicle-treated mice led to theidentification of 543 genes (254 annotated genes); 65 genesoverlapped between the data sets and 61 showed the sametrend in vitro and in vivo (Supplementary Table S2). Consistentwith a reduction in lung metastasis, genes related to cell mo-tility and invasion were identified. In particular, several MMPs(MMP1, MMP3, MMP9, MMP10, and MMP13) involved inextracellular matrix degradation (29, 30) were downregulatedby TKI258. A quantitative RT-PCR analysis showed that within8 hours of TKI258 addition, there was >80% decrease inMMP1, MMP3, MMP9, and MMP10 levels (Fig. 5C); MMP9protein was almost undetectable after 24 hours of treatment(Fig. 5D). Interestingly, all these MMPs have activator protein-1 binding sites in their promoter (31), connecting TKI258-mediated ERK inhibition to their decreased expression.

TKI258-regulated genes identify clusters of breast

tumors with increased metastatic potential. Based on the

ability of TKI258 to reduce metastases and perturb genes involved in cell motility and invasion, we tested whetherTKI258-regulated genes might be enriched for prognosticmarkers in human tumors. Two cohorts of breast cancer pa-tients available in the GEO database were used: TRANSBIG(GSE7390) with 198 patients and ERASMUS (GSE2034) with

286 patients. Analyzed were 1,648 human orthologous genesaffected byin vitro, and 254 genes affected by in vivo, TKI258treatment. Unsupervised hierarchical clustering analysis inboth cohorts identified a group of patients characterizedby overexpression of the same subset of 64 in vitro TKI258-regulated genes (cluster 1A, P = 4.270, Fisher's exact test);99.4% of these genes are negatively regulated by TKI258(Fig. 6A; Supplementary Table S3A). The clustering analysiswas also performed with genes affected byin vivo treatment, which identified two patient groups overexpressing distinctgene sets (cluster 1B, 62 merged genes; cluster 2B, 42 merged

genes; Fig. 6B) that overlapped in both cohorts (cluster 1B, 42overlapping genes, P = 4.0629, Fisher's exact test; cluster 2B,15 overlapping genes, P = 4.1411, Fisher's exact test; Supple-mentary Table S3B).

Kaplan-Meier analysis revealed that in both cohorts,cluster 1A andcluster 2B patients, hada worse prognosiscom-

pared with other patients (Fig. 6C and D, top). The differencein metastasis-free survival was even stronger in the subgroupof basal-like patients (Fig. 6C and D, bottom). Conversely, pa-tients in cluster 1B had a better prognosis compared withothers (Fig. 6D, green line). Of note is the fact that the majority

of cluster 1B genes (79%; Supplementary Table S3) were upre-gulated by the inhibitor, whereas the majority of cluster 1Aand cluster 2B genes (99.4% and 65%, respectively; Supple-

mentary Table S3A and B) were negatively regulated byTKI258. Cluster 1A and cluster 2B cohorts contain almostthe same patients (Supplementary Fig. S3A and B), whereasthe overlap between the subsets of overexpressed genescharacterizing these clusters is very low (three commongenes; Supplementary Fig. S3C). This suggests that cluster

1A and cluster 2B genes might identify distinct pathwaysand mechanisms that contribute to tumor progression. Ofnote, Ingenuity Pathway Functional Analysis revealed that cellcycle and DNA replication functions are more enriched incluster 1A genes, whereas cellular movement and inflammatoryresponse functions are more enriched in cluster 2B genes(P < 0.05, Fisher's exact test; Supplementary Fig. S4). Insummary, our analysis of genes regulated by TKI258 led tothe identification of breast cancer patients in which a fractionof TKI258-downregulated genes are highly expressed and areprognostic. Indeed, these patients tend to have a poor progno-sis, in particular in the basal-like group. This cluster of highlyexpressed genes might reflect activation of signaling pathwaysthat we identified in our analysis of FGFR in 4T1 tumors.

Discussion

Despite recent advances in breast cancer treatment, thereare patients for whom no targeted therapies are available(32). Based on evidence implicating FGFRs as breast cancerrisk factors (7, 8, 33) and the identification of FGFR amplifi-cation and overexpression in specific subgroups (4, 5, 34),further studies on the potential of targeting this receptorfamily are warranted. In the work presented here, we show

that 4T1 and 67NR breast cancer models display autocrineFGFR activity due to coexpression of receptors and ligands,and both are sensitive to FGFR inhibition. TKI258 blocks

multiple signaling pathways activated by FGFRs (35), inhibits proliferation, and causes a strong reduction in mammarytumor outgrowth and, in the 4T1 model, lung metastasis for-mation. In addition to FGFR, TKI258 inhibits vascular endo-thelial growth factor receptor (VEGFR) and platelet-derivedgrowth factor receptor (PDGFR; ref. 13), prompting us to testthe VEGFR inhibitor PTK787 (36) and the PDGFR inhibitorSTI571 (37). Neither inhibitor affected the proliferation orsurvival of 4T1 cultures (Supplementary Fig. S5A and B),nor did PTK787 affect tumor outgrowth (SupplementaryFig. S5C), suggesting that the major effects of TKI258 are





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related to FGFR blockade. Taken together, our resultssuggest that in breast tumors with active FGFR signaling,anti-FGFR therapeutics including TKIs or antibodies mightshow clinical efficacy.

The question arises about what induces the high levels ofcleaved caspase-3 in the tumors from TKI258-treated mice. A

transient loss of P-AKT, P-ERK, and P-PLC was seen in thetumors, and in vitro, we found that the AKT pathway has animportant role in protecting 4T1 cells from TKI258-inducedcell death. In addition to AKT, TKI258 treatment also low-ered tumor-induced angiogenesis, which could also contrib-

ute to tumor cell death. TKI258 has the potential to block theproangiogenic FGFR in the vessels themselves, and in the tu-mor cells, we show that TKI258 decreases the levels of

MMP9, a metalloprotease with an angiogenesis-promotingrole (Supplementary Tables S4 and S5). Both activities couldcontribute to lower vessel density (38). Despite the signifi-cant increase in apoptosis, TKI258 only slowed tumor out-growth; no regression was observed. Because there was nosignificant decrease in proliferation markers in the tumors,

this suggests that the proapoptotic activity of TKI258 was in-sufficient to overcome growth-promoting signals, likely dueto factors supplied by the tumor environment. Future workwill test if FGFR inhibition in combination with other anti-tumor agents causes tumor regression.

The metastatic process is complex, and tumor cells needto overcome many barriers to reach and grow in distant or-gans (39). We recently uncovered a novel pathway essentialfor 4T1 lung metastasis (14). The serpin protease nexin-1(PN-1), via binding its receptor LRP-1, controls MMP9expression, and PN-1silenced 4T1 cells have decreasedMMP9 levels and impaired metastatic potential (14). Inter-estingly, TKI258 decreased PN-1 levels (SupplementaryTables S4 and S5). The microarray analysis also revealedmany other TKI258-regulated genes that are known to be in-volved in metastasis, including integrins, extracellular matrix proteins, and transcription factors. For example, collagentype IV5, which was reported to be downregulated ininvasive cancers (40), was increased by TKI258 treatment.Moreover, two mediators of epithelial-mesenchymal transi-tion (EMT), E-cadherin and the Twist transcription factor,

were also identified (Supplementary Table S4 and Supple-mentary Fig. S6A and B). During development, FGFR inducesEMT (41), an event characterized by loss of epithelial prop-erties and an increased mesenchymal phenotype (42, 43).Interestingly, Twist-knockdown 4T1 cells have been shownto form primary mammary tumors with impaired metastatic

potential (15). In summary, FGFR uses multiple pathways tocontrol proliferation, survival, and metastatic spread of thisbreast cancer model.

By using a biased approach, we had the possibility oflinking cancer gene expression signatures to a molecular al-teration, being TKI258-mediated blockade of FGFR signalingin this work. Interestingly, cluster 2B, from the in vivo exper-iment, was enriched for genes encoding proteins involved ininflammatory response and cellular movement (Supplemen-tary Fig. S4), suggesting that blocking signaling pathwaysdownstream of FGFR exerts effects not only in the cancer

cells but also on the tumor microenvironment. To test this,cluster 2B genes were compared with genes uncovered in astudy of an invasive cancer model in which gene expressionin the tumor epithelium and the stroma was monitored(ref. 44; GSE15299). Remarkably, when a gene-set enrich-ment analysis was performed (GSEA), cluster 2B genes were

top-scoring in terms of enrichment and significance in thestromal component of the invasive cancer (normalized en-

richment score, 2.06; P < 0.001; Supplementary Table S6).On the other hand, cluster 1A genes, which were selectedafter in vitro TKI258 treatment, were top-scoring in the

epithelial component of the tumor (normalized enrichmentscore, 2.19; P < 0.001; Supplementary Table S6). Takentogether, these results suggest that in human tumors,

inhibition of an RTK, such as FGFR, might result in aconcomitant transcriptional reprogramming of genes thatare important for tumor-stroma interaction and cancerprogression.

Gene expression signatures have become important toolsnot only to define cancer subtypes and prognosis but also

for defining combined oncogenic pathway activity in tumors(45, 46). It has also been shown that biased approaches re-lying on experimental models are powerful in identifyingcancer signatures because they are less prone to genetic het-erogeneity (47). Therefore, we took advantage of the TKI258gene signature to study its expression in breast cancer pa-tient cohorts and identified subgroups overexpressing clus-ters of genes downregulated by TKI258 (clusters 1A and 2B),whose members have a high probability of metastatic dis-ease. The two identified genetic signatures are very stablebecause their prognostic significance was confirmed in bothindependent cohorts. The patients overexpressing cluster 1Aor 2B genes are roughly the same (Supplementary Fig. S3),and it is tempting to speculate that there might be activeFGFR signaling in these tumors. This information was notavailable in the database; however, our own analysis forFGF/FGFR genes scored FGFR4 as being significantly regulat-

ed in the two cohorts of patients (Supplementary Table S7).Future work will be directed to testing this signature in moredefined groups of breast tumors. In summary, FGFRs seem tobe a valuable target for treatment of subgroups of breast can-cer patients. We show here that many biological aspects ofthe tumor, from cell proliferation to invasion and metastasis,are dependent on FGFR signaling, and blockade of the recep-tor has a strong influence on tumor growth and metastasisformation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Drs. D. Graus-Porta, C. Garcia-Echeverria, and S-M. Maira(Novartis Institutes for Biomedical Research, Basel, Switzerland) forproviding inhibitors and helpful suggestions and Dr. B. Hemmings (FriedrichMiescher Institute, Basel, Switzerland) for the Myc-Akt cDNA. We thankDr. B. Fayard for helpful comments on the manuscript.

Dey et al.




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Grant Support

The work of J.H. Dey was partially supported by TRANSFOG FP6 IP funding(LSHC-CT-2004-503438). The laboratory of N.E. Hynes is supported by theNovartis Research Foundation.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementinaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/11/2009; revised 02/04/2010; accepted 03/04/2010; publishedOnlineFirst 05/11/2010.

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