cooperation between c-met and focal adhesion kinase family ... · fak inhibitors as a new strategy...

Therapeutic Discovery

Cooperation between c-Met and Focal Adhesion KinaseFamily Members in Medulloblastoma and Implicationsfor Therapy

Fadila Guessous1, Yanzhi Yang1, Elizabeth Johnson1, Lukasz Marcinkiewicz1, Matthew Smith1,Ying Zhang1, Alexander Kofman1, David Schiff2,3, James Christensen4, and Roger Abounader1,2,3

AbstractWe previously showed the involvement of the tyrosine kinase receptor c-Met in medulloblastoma malig-

nancy. The nonreceptor tyrosine kinases focal adhesion kinase (FAK) and Pyk2 are key players in the

progression of different cancers. However, their role in medulloblastoma malignancy is not well understood.

In this study, using a protein array approach, we found that c-Met induces FAK and Pyk2 phosphorylation in

medulloblastoma cells. We therefore studied the interactions between c-Met and FAK/Pyk2 and their

implications for medulloblastoma therapy. We found that c-Met activates FAK and Pyk2 in several medul-

loblastoma cell lines. We also found that FAK and Pyk2 mediate the malignant effects of c-Met on medul-

loblastoma cell proliferation, migration, and invasion. On the basis of these findings, we hypothesized that

combined c-Met and FAK inhibitions would have additive effects on the inhibition of medulloblastoma

malignancy. To test this hypothesis, we assessed the effects on medulloblastoma malignancy parameters of

single or combined treatments ofmedulloblastoma cellswith c-Met and FAK small-molecule kinase inhibitors.

We found a significant increase in the inhibitory effect of both inhibitors on medulloblastoma cell migration

and cell invasion as compared with single inhibitions (P < 0.05). In addition, oral gavage treatment with c-Met

inhibitor of mice bearing medulloblastoma xenografts significantly reduced in vivo tumor growth. Therefore,

combining c-Met inhibitors with FAK inhibitors constitutes a new potential strategy for medulloblastoma

therapy. Altogether, our study describes a role for FAK and Pyk2 in medulloblastoma malignancy, uncovers

new interactions between c-Met and FAK/Pyk2, andproposes for the first time combining anti-c-Met and anti-

FAK inhibitors as a new strategy for medulloblastoma therapy. Mol Cancer Ther; 11(2); 288–97. �2011 AACR.

Introduction

Medulloblastoma is the most common brain tumor inchildrenwith an incidence of 0.6 per 100,000 patient-yearsaccording to the Central Brain Tumor Registry of theUnited States. It is an embryonal brain tumor that arisesin the cerebellum, where it is thought to originate fromprimitive pluripotent precursor cells of the ventricularzone and cerebellar external germinal layer (1). Multiplesignaling pathways have been associated with medullo-blastoma formation and growth. These include the devel-opmental pathways Hedgehog (Hh), Notch, and Wnt aswell as the receptor tyrosine kinases (RTK) erbB2, insulin-

like growth factor receptor (IGF-R), and TrkC, and theoncoprotein Myc (2).

Our laboratory recently showed the involvement of thereceptor tyrosine kinase c-Met and its ligand hepatocytegrowth factor (HGF) in medulloblastoma malignancy (3).Inappropriate activation of the HGF/c-Met signalingpathway has been shown to be involved in the etiologyof various human cancers including brain tumors, con-ferring them with invasive and metastatic properties(2, 4, 5). On the basis of the widespread and profoundinvolvement of c-Met in cancer, several c-Met pathwayinhibitors have been recently developed. These includeribozymes, HGF kringle variants/NK4, decoy receptors,HGF or c-Met neutralizing antibodies, and small-mole-cule kinase inhibitors (4, 6, 7). One such small-moleculekinase inhibitor, PF-2341066, was recently identified asa potent, orally available, ATP-competitive, and selec-tive inhibitor of the catalytic activity of the c-Met recep-tor (8). PF-2341066 strongly inhibits c-Met phosphory-lation and signal transduction, as well c-Met oncogenicphenotypes of tumor cells and endothelial cells, andexerts its cytoreductive effect through antiproliferativeand antiangiogenic mechanisms in different cancers(9).

Authors' Affiliations: Departments of 1Microbiology and 2Neurology,3Cancer Center, University of Virginia, Charlottesville, Virginia; and 4PfizerOncology Research Unit, La Jolla, California

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Roger Abounader, University of Virginia, PO Box800168, Charlottesville, VA 22908. Phone: 434-982-6634; Fax: 434-243-6843; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0490

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(2) February 2012288

on September 15, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 21, 2011; DOI: 10.1158/1535-7163.MCT-11-0490

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Thenonreceptor tyrosine kinases, focal adhesion kinase(FAK) and the proline-rich tyrosine kinase-2 (Pyk2), haveemerged as key players in the progression of differentcancers. FAK and Pyk2 are important signaling effectorslinking integrins and growth factor signaling to cell adhe-sion, invasion, proliferation, migration, survival, andapoptosis in many cancers (10). Similar to FAK, whichundergoes autophosphorylation at the tyrosine397 (Tyr397)residue, autophosphorylation of Pyk2 at Tyr402 residueleads to the recruitment of Src family kinases, activation ofextracellular signal–regulated kinase (ERK), regulation ofion channels, cell adhesion, andmotility (11). FAK expres-sion and/or phosphorylation is elevated in a variety ofcancers and frequently correlates with malignant or met-astatic disease and poor patient prognosis (12). Manystudies have shown the association between FAK expres-sion and malignancy grade, angiogenesis, invasion, andmigration in gliomas (13–15), However, their role ininvasivemedulloblastoma is notwell understood. Recent-ly, a novel small-molecule FAK inhibitor, PF-573228, wasidentified through a combination of high throughputscreening, structure-based drug design, and conventionalmedicinal chemistry approaches. Treatment of cells withPF-573228 blocked FAK phosphorylation on Tyr397 andconcomitantly reduced the phosphorylation of the well-recognized downstream effector of FAK signaling, pax-illin (16).In the present study, using aprotein array approach,we

found that c-Met stimulation by HGF phosphorylatesFAK and Pyk2 in medulloblastoma cell lines. Therefore,we hypothesized that FAK/Pyk2 cooperate with c-Met–induced medulloblastoma malignancy and studied theinteractions between them.We found that c-Met activatesFAK and Pyk2 and that FAK and Pyk2mediate the effectsof c-Met on medulloblastoma cell migration, invasion,and proliferation. We also showed that combined target-ing of c-Met and FAK could be advantageous for medul-loblastoma therapy.

Materials and Methods

Cell culture and reagentsThree human medulloblastoma cell lines were used

for this study. DAOY and ONS-76 were grown in RPMI-1640 media supplemented with 10% FBS. D425 cellswere grown in Improved Modified Eagle Medium Zincoption and 20% FBS. HGF-overexpressing DAOY cells(DAOY-HGF) were generated in our laboratory andwere cultured in Improved Modified Eagle MediumZinc option, supplemented with 10% FBS and the selec-tion antibiotic Zeocin (1 mg/mL; ref. 17). All cells weregrown at 37�C in 5% CO2. HGF was purchased fromR&D systems. Human type IV collagen was from Sig-ma-Aldrich. The scrambled short interfering RNA(siRNA)/control-siRNA, FAK-siRNA, and Pyk2-siRNAwere purchased from Santa-Cruz Biotechnology. Oligo-fectamine and SDS-PAGE were from Invitrogen.The small-molecule c-Met kinase inhibitor PF-2341066

(METi), also clinically known as crizotinib, (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxyl]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamin, was from Pfizer.The FAK inhibitor PF-573228 (FAKi), 3,4-dihydro-6-[[4-[[[3(methylsulfonyl)phenyl] methyl]amino]-5-(tri-fluoromethyl)-2-pyrimidinyl]amino](1H)quinolinone,was purchased from Tocris Bioscience. The chemicalstructures of PF-2341066 and PF-573228 are shown inFig. 1A. Panorama antibody microarray cell signalingkit, Cy3 and Cy5 fluorescence dyes, and all chemicalsand solvents were from Sigma-Aldrich.

Protein microarraysProtein arrays were conducted to identify cell signal-

ing molecules that are modulated by HGF in medullo-blastoma cells. The Panorama antibody microarray con-tains 336 antibodies representing cell signaling mole-cules in their total and active (usually phosphorylated)forms. The antibodies are printed in duplicates on 4 � 8grids. Each grid contains 7 antibody duplicates plus aCy3- and Cy5-conjugated bovine serum albumin (BSA)positive control as well a nonlabeled BSA negativecontrol resulting in a total of 512 spots. ONS-76 medul-loblastoma cells were treated with HGF (20 ng/mL) for30 minutes and untreated cells were used as a control.The cells were processed for the microarray hybridiza-tion according to the manufacturer’s instruction. Briefly,proteins were extracted by an extracting/labeling buffercontaining Benzonase, protease inhibitors, and phos-phatase inhibitors. One milligram of protein extractfromHGF-treated and nontreated cells was labeled withCy5 or Cy3, respectively, according to the manufac-turer’s instructions and used at dye/protein ratio >2.Free nonincorporated Cy3 and Cy5 dyes were separatedby applying the labeled extracts to SigmaSpin Post-Reaction Clean-Up Columns. An equal amount oflabeled protein of both extracts (10 mg/mL) was incu-bated on the Panorama Ab Microarray slide for 30minutes; all washes were done in PBS-Tween 0.05%.The slides were air-dried before scanning with the GSI4000 scanner, and images were generated with thePanorama software (GSI Lumonics). The slide scanningwas conducted using a microarray scanner (ProScanAr-ray Scanner, Perkin Elmer). The results were analyzedusing standard median normalization between bothchannels Cy3 and Cy5 using the "ScanArray ExpressSoftware." All experiments were carried out intriplicates.

ImmunoblottingImmunoblotting was conducted using antibodies spe-

cific for phosphorylated and nonphosphorylated forms ofFAK, Pyk2 (Cell Signaling), and b-actin (Santa Cruz Bio-technologies), the loading control. To assess the effects ofHGF on FAK and Pyk2 activations, D425, DAOY, andONS medulloblastoma cells were treated with 20 ng/mLHGF for various time points (5 minutes to 6 hours) andsubsequently immunoblotted for phospho-FAK on

c-Met and FAK in Medulloblastoma

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different phosphorylation sites Tyr397, Tyr576/577, Tyr925,Ser910, or phospho-Pyk2 on Tyr402 site. The cells weresubsequently lysed with radioimmunoprecipitationassay (RIPA) buffer (1% Igepal, 0.5% sodium deoxycho-late, and 0.1% SDS in PBS) and lysates were collected andcleared by centrifugation. The protein concentration of thesupernatant was determined, equal amounts of proteinwere electrophoretically separated in PAGE, and thentransblotted to a nitrocellulose membrane. The membranewas then incubated overnight with primary antibodies at4�C. The primary antibody-bound membranes werewashed 3 times with Tween-PBS before incubation withthe corresponding horseradish-conjugated secondary anti-bodies. After a final wash, the immunoreactive signalswere detected by enhanced chemiluminescence and quan-tified by densitometry on film using computer-assistedimage analysis.

siRNA silencingFAK-siRNA andPyk2-siRNAwere used to knockdown

FAK and Pyk2 expressions in DAOY andD425 cells. Cellswere transfected with either FAK-siRNA or Pyk2-siRNAusingOligofectamine transfectionreagentaccordingto themanufacturer’s instructions. Random/scrambled siRNA(cont-siRNA) was used as a control. The knockdown ofFAK and Pyk2 was confirmed by immunoblotting usingthe FAK and Pyk2 antibodies as described above.

Cell proliferation assaysDAOY and D425 cells were seeded in triplicates, in

medium containing 10%FBS and treatedwith either FAK-siRNAorPyk2-siRNA(35nmol/L). Controlswere treatedwith cont-siRNA (35 nmol/L). Twenty-four hours later,the media were changed to low serum media (FBS 0.1%),and cells were treated with HGF (20 ng/mL). The cells

Figure 1. A, chemical structures ofPF-2341066 and PF-573228. B,HGF induces the activation of FAKand Pyk2 in medulloblastoma cellsin a time-dependent manner.Immunoblots showing the time-dependent phosphorylation ofFAK and Pyk2 on varioustyrosines (Tyr) and serines (Ser) inmedulloblastoma cells treated with20 ng/mL HGF for 5 minutes to 6hours. The blots were stripped andhybridized for b-actin as a loadingcontrol.

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were trypsinized and harvested every day, for 4 days,counted with a hemocytometer, and growth curves wereestablished.

Migration assaysWound/scratch assay was conducted as previously

described (18). DAOY cells were seeded at 80% con-fluency in low serum media for 24 hours. The cells weretreated with 35 nmol/L FAK-siRNA, Pyk2-siRNA, orcont-siRNA and 24 hours later treated with 20 ng/mLHGF. The cells were assessed for migration using thescratch assay. Cells thatmigrated into the scratch 24 hoursposttreatment were photographed at �40 magnification.

Invasion assaysTranswell invasion assays were conducted to deter-

mine whether FAK and/or Pyk2 mediate the effects ofc-Met onmedulloblastoma cell invasion.Modified BoydenChambers (Becton Dickinson) were coated with humantype IV collagen (250 mg/mL). DAOY cells (2 � 105)were treated with 35 nmol/L FAK-siRNA, Pyk2-siRNA,or cont-siRNA for 8 hours and then exposed to 20 ng/mLHGF for 24 hours. The following day, the cells wereresuspended in serum-freemedia and added to each insertin the presence or absence of HGF 20 ng/mL. Six hundredmicroliters of 10% FBS medium was placed in the lowerchamber as a chemoattractant. After 8 hours of incubationat 37�C in 5% CO2, invading cells were stained with 0.1%crystal violet solution and photographed at 40�. The cellswere then counted under a microscope in 5 randomlychosen fields.

Pharmacologic inhibition of FAK and c-MetMETi was dissolved in sterile water, aliquoted, fil-

tered, and stored in the dark, at room temperature untiluse. FAKi was prepared in sterile dimethyl sulfoxide,filtered, aliquoted, and stored at�80�C until use. DAOYcells or DAOY-HGF clones were treated with eitherFAK inhibitor FAKi (1 mmol/L), c-Met inhibitor METi(100 nmol/L), a combination of both inhibitors, orcontrol vehicle for 24 hours and subsequently treatedwith 20 ng/mL HGF for 24 hours for cell migration andfor 6 hours for cell invasion. The cells were assessed formigration or invasion, as described above. FAK and c-Met inhibitions, as well as p-FAK and p-c-Met inhibi-tions, were verified by immunoblotting. Signals werequantified by densitometry and the quantificationresults were reported for each Western blot analysis.For proliferation, DAOY cells and DAOY-HGF cloneswere seeded in 6-well plates, treated with FAKi or METior combination of both inhibitors for 24 hours at thesame concentrations listed above, and subsequentlytreated with HGF (20 ng/mL) for 24 hours. Cell growthwas monitored by counting the cells for 4 days, andproliferation curves were established. The effects ofsingle treatments on cell migration, invasion, and pro-liferation were compared with the effects of combinedinhibitor treatment.

In vivo xenograft experimentsMETi was dissolved in sterile water (stock 0.5 mol/L),

filtered and, stored in the dark at room temperature.Medulloblastoma DAOY-HGF cells (2 � 106) were im-planted in the flanks of immunodeficient mice (n ¼ 10).METi treatment started 5 days posttumor implantation.The animals were treated by oral gavage of 0.1 mL of 25mmol/L METi solution (30 mg/kg body weight) everyday for 3 weeks. Control animals were treated with anequal volume of sterile distilled water. The flank tumorswere measured with a caliper every day for 2 weeks. Atthe end of the treatment, the animalswere euthanized andthe tumors were removed, weighed, and a tumor volumeprogression curve was established.

StatisticsAll experiments were carried out at least in triplicates.

Numerical datawere expressed asmean� SD. Twogroupcomparisons were analyzed by 2-sided Student t test.Multiple group comparisons were analyzed with Bonfer-roni/Dunn multiple comparisons tests. P Values weredetermined for all analyses and P < 0.05 was consideredsignificant.

Results

c-Met activates FAK and Pyk2 as revealed by proteinarrays

Weassessed the effects of c-Met activation on global cellsignaling in medulloblastoma cells using antibodymicro-arrays containing 336 cell signaling molecule probes inactive and total states.We found numerous changes in theactivation of signal transduction proteins in response toHGF. Many of these changes were known and expectedsuch as the induction of cyclin D1, cyclin A, and phospho-Akt by HGF. Few other changes showed effects of HGFthat had not been described before in medulloblastoma(Supplementary Table S1). One suchnovel and interestingeffect of HGF was the phosphorylation of FAK at Tyr577

and the phosphorylation of the FAK-related protein tyro-sine kinase (Pyk2) at Tyr579 (Supplementary Table S1).Interestingly, both proteins are related to each other andplay critical roles in cell migration and invasion in othercancers. We therefore hypothesized that c-Met–inducedtumor cell invasion and migration could be mediated byFAK and/or Pyk2.

c-Met activates FAK and Pyk2 in medulloblastomacells in a time-dependent manner

To confirm that c-Met activates FAK and/or Pyk2 asobserved on the protein arrays, the effect of HGF on FAKand Pyk2 phosphorylations inmedulloblastoma cells wasassessed by immunoblotting. D425, DAOY, and ONS-76medulloblastoma cells were treated with 20 ng/mL HGFfor various time points (5 minutes to 6 hours) and subse-quently immunoblotted for phospho-FAK Tyr397, phos-pho-FAK Tyr576/577, phospho-FAK Tyr925, phospho-FAKSer910, or phospho-Pyk2Tyr402.HGF treatment led to FAK






and Pyk2 phosphorylations at multiple sites in all medul-loblastoma cells that were examined. c-Met activationwith HGF induced a strong phosphorylation of FAK atTyr397 and Tyr925, which are important for the maximaladhesion-induced activation of FAK and signaling todownstream effectors. c-Met activation with HGF alsoled to phosphorylation of FAKat Ser910, which is involvedinmodulating binding/stability of downstream signalingproteins, andphosphorylation of FAKat Tyr576/577,whichis strongly associated withmigration. Similarly, cell treat-ment with HGF resulted in a strong and fast phosphor-ylation of Pyk2 at Tyr402 reported to promote invasion andmigration. Phosphorylations started between 5 and 15minutes after HGF treatment and lasted for several hours

(Fig. 1B). Phosphorylations of all amino acids were notdetected in all cell lines (not shown). Overall, the abovedata show that c-Met activates FAK and Pyk2 in a time-dependent manner.

FAK and Pyk2 mediate c-Met–induced cellproliferation in medulloblastoma cells

To determine whether FAK and/or Pyk2 mediatec-Met–dependent cell growth, we assessed the effectsof c-Met activation on DAOY and D425 medulloblasto-ma cell proliferation in the setting of silenced FAK orPyk2. FAK-siRNA, Pyk2-siRNA, or scrambled controlsiRNA (cont-siRNA) were transfected into the cells toinhibit FAK and Pyk2 expressions. After 24 hours, the

Figure 2. FAK and Pyk2 mediatethe effects of c-Met onmedulloblastoma cell proliferation.FAK and Pyk2 expressions weresilenced in DAOY and D425 cellsby transfectionwith FAK-siRNA (35nmol/L) or Pyk2-siRNA (35 nmol/L),respectively. Control cells weretransfected with 35 nmol/Lscrambled siRNA (Cont-siRNA).After 24 hours, the cells weretreated with 20 ng/mL HGF andcounted every day for 4 days. Theresults show that silencing of FAKor Pyk2 significantly decreasesbasal and c-Met–induced cellgrowth. D425-FAK silencing (A),D425-Pyk2 silencing (B), DAOY-FAK silencing (C), and DAOY-Pyk2silencing (D). E, immunoblots showFAK and Pyk2 knockdown in cellstreated with respective siRNA asdescribed above. Results arerepresentative of 3 differentexperiments.

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cells were treated with 20 ng/mL HGF for 24 hoursand cell proliferation was analyzed by cell countingover 4 days. FAK and Pyk2 silencing was verified byimmunoblotting.FAK knockdown led to significant inhibition of c-Met–

dependent cell proliferation in D425 medulloblastomacells. FAK knockdown decreased cell numbers at day4 from 151 � 9.29 to 36.33 � 4.48 in HGF-untreated cells(n¼ 3;P<0.05). FAKknockdowndecreasedHGF-inducedcell numbers at day 4 from 309 � 14.57 to 81.33 � 9.56(n ¼ 3; P < 0.05; Fig. 2A). Pyk2 knockdown led to signif-icant inhibition of c-Met–dependent cell proliferation in

D425 medulloblastoma cells. At day 4 of counting, Pyk2knockdown reduced cell numbers from151� 9.29 to 18.33� 3.18 in HGF-untreated cells (n ¼ 3; P < 0.05). Pyk2knockdown decreased HGF-induced cell numbers at day4 from 309� 14.57 to 103.00� 8.39 (n¼ 3; P < 0.05; Fig. 2B).FAK silencing also led to significant inhibition of c-Met–dependent cell proliferation in DAOY cells. FAK knock-down decreased cell numbers at day 4 from 105.33� 5.70to 18.08 � 3.02 in HGF-untreated cells (n ¼ 3; P < 0.05).FAK knockdown decreased HGF-induced cell growth atday 4 from 132.00 � 7.23 to 61.50 � 2.41 (n ¼ 3; P < 0.05;Fig. 2C). Pyk2 silencing led to significant inhibition of

Figure 3. FAK and Pyk2mediate theeffects of c-Met onmedulloblastomacell migration and invasion. FAK andPyk2 expressions were silenced inDAOY andD425 cells by transfectionwith FAK-siRNA (35 nmol/L) or Pyk2-siRNA (35 nmol/L), respectively.Control cells were transfected with35 nmol/L scrambled siRNA (Cont-siRNA). After 24 hours, the cells weretreated with 20 ng/mL HGF andsubsequently assessed for migration(A) and invasion (B) using the scratchassay and the Boyden chamberassay, respectively. The results showthat silencing of FAK or Pyk2significantly decreases basal and c-Met–induced migration and invasionin medulloblastoma cells. Theimmunoblots at the bottom show theknockdown of FAK and Pyk2 inDAOY cells treated as describedabove.






c-Met–dependent cell proliferation in DAOY cells. At day4 of counting, Pyk2 knockdown reduced basal cell num-bers from 105.33 � 5.70 to 15.42 � 0.87 in HGF-untreatedcells (n ¼ 3; P < 0.05). Pyk2 knockdown decreased HGF-induced cell numbers at day 4 from 132.00 � 7.23 to 52.50� 1.84 (n¼ 3; P < 0.05; Fig. 2D). These data show that FAKand Pyk2 induce cell proliferation and mediate c-Met–dependent cell proliferation in medulloblastoma cells.

FAKandPyk2mediate c-Met–inducedmigration andinvasion of medulloblastoma cells

To determine whether FAK and/or Pyk2 mediate c-Met–dependent cell migration and invasion, we assessedthe effects of c-Met activation onDAOYmedulloblastomacell proliferation in the setting of inhibited FAK or Pyk2expressions. We tested the effect of FAK and Pyk2 knock-down on basal and HGF-induced cell migration andinvasion using the scratch assay and Transwell invasionassay, respectively. DAOY cells were treated with FAK-siRNA and FAK knockdown was confirmed by immuno-blotting. The cells were then treated with 20 ng/mL HGFand invasion was quantified. HGF induced DAOY cellinvasion in control (Fig. 3A). FAK and Pyk2 silencingsignificantly decreased HGF-induced cell invasion (Fig.3A). Similarly, HGF induced DAOY cell migration incontrol but significantly less in FAK-siRNA or Pyk2-

siRNA pretreated cells (Fig. 3B). These data show thatFAK and Pyk2 mediate c-Met–dependent migration andinvasion in medulloblastoma cells.

Combined inhibition of c-Met and FAK with small-molecule kinase inhibitors inhibits medulloblastomacell migration and invasion more than singleinhibitions

On the basis of the above results showing that c-Metactivates FAK and Pyk2 and that FAK and Pyk2 mediatethe effects of c-Met in medulloblastoma cells, we hypoth-esized that combined inhibition of c-Met and FAK couldhavegreater inhibitory effects onmedulloblastomamalig-nancy than single inhibitions. To test this hypothesis, weassessed the effects of small-molecule kinase inhibitors ofc-Met (METi) and FAK (FAKi) on medulloblastoma cellmigration, invasion, and proliferation. We first verifiedthe inhibitory effects of the small molecules on c-Metand FAK activation. DAOY and DAOY-HGF cells weretreated with 100 nmol/L METi and/or 1 mmol/L FAKifor 24 hours. METi strongly inhibited HGF-induced c-Met phosphorylation in DAOY and DAOY-HGF cells(Supplementary Fig. S1). FAKi inhibited HGF-inducedFAK phosphorylation in DAOY and DAOY-HGF cells(Supplementary Fig. S1). METi did not significantly affectFAKphosphorylation suggesting that FAK is additionally

Figure 4. Combined inhibition ofFAK and c-Met by small-moleculekinase inhibitors inhibitsmedulloblastoma cell migrationmore than single inhibitions. DAOYor DAOY-HGF cells were treatedwith either 1 mmol/L FAKi, 100nmol/L METi, a combination ofboth, or control vehicle for 1 hourbefore treatment with or without 20ng/mL HGF (DAOY only), for 24hours. The cells were assessed formigration using the scratch assay.Cells that migrated into the scratchwere photographed at �40magnification. The results showthat combined inhibition of FAKand c-Met has greater inhibitoryeffects on medulloblastoma cellmigration than single inhibitions.

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activated by factors other than c-Met (Supplementary Fig.S1).We then assessed the effects of single and combined c-

Met and FAK inhibitions on cell migration, invasion, andproliferation using a scratch assay, a Transwell invasionassay, and cell counting, respectively. Single METi andFAKi treatments inhibited basal andHGF-inducedmigra-tion ofDAOYandDAOY-HGF cells. CombinedMETi andFAKi treatments had greater inhibitory effect on medul-loblastoma cell migration and invasion than single inhibi-tions (Fig. 4). FAKi reduced the number of DAOY-HGFinvading cells through the collagen IVmatrix from 80.6�10.5 to 40.33� 5.77. METi reduced the number of DAOY-HGF invading cells through the collagen IV matrix, from80.6 � 10.5 to 29 � 2.64. The combined effect of bothinhibitors significantly decreased the number of invadingcells to 10.33 � 2.08 (P < 0.01; Fig. 5). These data suggestthat combining anti-c-Met and anti-FAK approachesmight have experimental therapeutic advantage inmedulloblastoma. However, such additive effect couldnot be detected on cell growth in either DAOY or DAOY-HGF cells (Supplementary Fig. S2).

Oral delivery of METi inhibits the in vivo growth ofhuman medulloblastoma xenograftsOur original goal was to test the combined effects of

METi and a clinically applicable FAK inhibitor on in vivomedulloblastoma growth. However, the clinically appli-cable FAK inhibitor is not commercially available and wewere not able to obtain it from the manufacturing phar-maceutical company. We therefore assessed the effects ofin vivo c-Met inhibition on medulloblastoma xenograftgrowth.To test the in vivo antitumor properties of METi, we

generated medulloblastoma xenografts by implantationof DAOY-HGF medulloblastoma cells in the flanks ofimmunodeficient mice. Tumor-bearing animals weretreated by oral delivery of METi via gavage for 3 weeks.While control animals developed very large tumors aver-aging 68.082 � 9.794 mm3 in volume, the METi-treatedgroup showed a significant inhibition of tumor growthaveraging 22.806� 2.483mm3 (n¼ 10, P < 0.01; Fig. 6). Noobvious drug toxicity was observed during the treatment.For the first time, these data show that inhibition of c-Metkinase by a small-molecule inhibitor leads to inhibition ofin vivo medulloblastoma tumor growth. The data alsoshow the feasibility of oral delivery of small-moleculekinase inhibitors of c-Met and suggest that these mole-cules can be combined with FAK inhibitors for improvedfuture medulloblastoma therapies.

Discussion

Our study shows for the first time that c-Met activatesFAK and Pyk2 and that FAK and Pyk2mediate the effectsof c-Met inmedulloblastoma. The study also establishes arole for FAK and Pyk2 in medulloblastoma malignancy.Furthermore, our study shows for the first time the anti-

oncogenic effects of small-molecule kinase inhibitors of c-Met and FAK in medulloblastoma and suggests thatcombined targeting of c-Met and FAK can be advanta-geous for medulloblastoma therapy.

Medulloblastoma is the most common malignantbrain tumor of childhood arising in the cerebellum(19, 20). It has the highest rates of metastasis outsidethe nervous system (21–23) and tends to spread hema-togenously into bones, bone marrow, lymphatic nodes,liver, and lungs (24, 25). To date, surgery and radiationtherapy remain the most effective treatment formedulloblastoma. However, craniospinal radiotherapy

Figure 5. Combined inhibition of FAK and c-Met by small-molecule kinaseinhibitors inhibits medulloblastoma cell invasion more than singleinhibitions. DAOY-HGF cells were treated with either 1 mmol/L FAKi, 100nmol/L METi, a combination of both, or control for 24 hours. The cellswere assessed for invasion using a Transwell assay. Cells that invadedthrough the membrane after 6 hours were fixed and stained with crystalviolet. The wells were photographed at�40 magnification and 5 randomfields were quantified. The results show that combined inhibition of FAKand c-Met has greater inhibitory effects onmedulloblastoma cell invasionthan single inhibitions. A, representative invasion assay. B, quantificationof invasion assay. �, P < 0.05.






increase is frequently accompanied by lifelong braindamage (20, 23). Targeting molecular pathways thatgovern medulloblastoma malignancy is a promisingapproach for achieving future improved clinical out-comes. We identified c-Met and FAK as 2 such path-ways that can be simultaneously targeted for greateranti-medulloblastoma effects.

FAK activation has been associated with a variety ofcancers and frequently correlateswithmalignancy,metas-tasis, and poor patient prognosis (12). A recent study hasshown the involvement of integrinb1/FAK signaling inmigration and invasion in medulloblastoma (26). Fewsmall-molecule inhibitors of FAK have been developed.PF-562271 has shown a highly selective and potent phar-macologic inhibitory effect on FAK catalytic activity,allowing it to be a first-in-class inhibitor reaching clinicaltrial testing for the cancer therapy (27).Unfortunately, thisinhibitor is not commercially available and we wereunable to obtain it from the manufacturer. We thereforeused the commercially available PF-573228, which cannotbe used for in vivo animal experimentation (16). Our studyshows for the first time that FAK kinase activity and FAK-associated migration and invasion are inhibited by PF-573228. Therefore, FAK and Pyk2 inhibitions represent apotential novel strategy for medulloblastoma therapy.

Given the critical role of the receptor tyrosine kinase c-Met, and its ligand HGF in the progression of differenthuman cancers including brain tumors, differentapproaches to inhibitingHGF and c-Met have been devel-oped (4). Among these, the small-molecule kinase inhib-itor of c-Met, PF-2341066, has reached phase I and II ofclinical trials and therefore represents a promising ther-apeutic approach to inhibiting c-Met in cancer andmedul-loblastoma. Our study shows for the first time the use-fulness of PF-2341066 in inhibiting c-Met activation and c-Met–dependent oncogenic effects in medulloblastomacells and tumors.

Our study shows for the first time that HGF/c-Metactivates FAK and Pyk2 in medulloblastoma. A connec-

tion between c-Met and FAK has been previouslydescribed in some cancers such as ovarian cancer (28),breast cancer (29, 30) but not in medulloblastoma. Fur-thermore, to date, no connection between c-Met and Pyk2has been described in any cancer. Our findings suggesteither a direct or an indirect FAK/Pyk2 activation by c-Met kinase. We used immunoprecipitation of FAK and c-Met upon HGF stimulation of medulloblastoma cells toexplore the possibility of direct binding of FAKand c-Met.We could not detect any direct interaction between the 2proteins (data not shown). The indirect interaction couldoccur via activation of scaffolding or key adaptor proteinsknown to mediate FAK effects such as paxillin andp130CAS (31, 32). Considering that the FAK-Src complexmediates the phosphorylation of paxillin and p130CAS,and as c-Met is known to signal both upstream anddownstream from c-Src, this latter is a possible candidatefor mediating FAK activation by c-Met.

Considering that multiple molecular pathways areinvolved in growth, survival, migration, and invasion oftumor cells, antitumor activity could be improved bysimultaneous use of individual agents targeting differentpathways such as Raf/MEK/ERK or c-Met to allow ver-tical or horizontal inhibition of relevant pathways (33, 34).Such simultaneous targeting would have to be based onmolecular findings indicating cooperation, redundancy,or compensatory mechanisms involving the targetedmolecules. Our study suggests for the first time thatcombining c-Met and FAK/Pyk2 inhibitors as anotherapproach for achieving improved antitumor effects inmedulloblastoma. Our intent was to test this combinationapproach in vivo in a medulloblastoma animal model,but we were unable to obtain the only FAK inhibitorwith proven bioavailability from the manufacturer.However, we did show for the first time the in vivousefulness of a small-molecule inhibitor of c-Met onmedulloblastoma tumor growth in paving the road fora potential testing of combined anti-Met/anti-FAK ther-apy in medulloblastoma.

Figure 6. Oral delivery of METisignificantly inhibits the in vivo growthof human medulloblastomaxenografts. Human medulloblastomaxenografts were generated by flankimplantation of 2 � 106 HGF-DAOYcells in immunodeficient mice. Fivedays posttumor implantation, theanimalswere treatedwithMETi byoralgavage (30 mg/kg body weight) onceper day, for 3 weeks. The controlgroup was treated with equal volumeof sterile distilled water. At the end ofthe treatment, animalswere sacrificedand tumor size was assessed bymeasuring tumor volume. The resultsshow that METi treatmentsignificantly inhibits in vivomedulloblastoma growth (P < 0.01).

Guessous et al.





Overall, our study uncovers previously unknown inter-actions between c-Met and FAK/Pyk2 in medulloblasto-ma and suggests that simultaneous targeting of thesemolecules might be advantageous for medulloblastomatherapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interests were disclosed.

Grant Support

Theworkwas supported byNIHRO1NS045209 and R01 CA134843 (R.Abounader).

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 July 7, 2011; revised December 9, 2011; accepted December 13,2011; published OnlineFirst December 21, 2011.

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2012;11:288-297. Published OnlineFirst December 21, 2011.Mol Cancer Ther Fadila Guessous, Yanzhi Yang, Elizabeth Johnson, et al. Members in Medulloblastoma and Implications for TherapyCooperation between c-Met and Focal Adhesion Kinase Family

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