Targeting Focal Adhesion Kinase with Dominant-Negative FRNK or

Hsp90 Inhibitor 17-DMAG Suppresses Tumor Growth and

Metastasis of SiHa Cervical Xenografts

Joerg Schwock,1,2

Neesha Dhani,1,5

Mary Ping-Jiang Cao,1,6

Jinzi Zheng,3

Richard Clarkson,1,4

Nikolina Radulovich,1,2

Roya Navab,1,6

Lars-Christian Horn,7

and David W. Hedley1,2,3,5,6

1Ontario Cancer Institute, Departments of 2Laboratory Medicine and Pathobiology, 3Medical Biophysics, and 4Radiation Oncology,5Institute of Medical Sciences, and 6Division of Applied Molecular Oncology, University of Toronto, Toronto, Ontario, Canada and7Division of Gynecologic Pathology, Institute of Pathology, University of Leipzig, Leipzig, Germany

Abstract

Focal adhesion kinase (FAK), a nonreceptor protein tyrosinekinase and key modulator of integrin signaling, is widelyexpressed in different tissues and cell types. Recent evidenceindicates a central function of FAK in neoplasia where thekinase contributes to cell proliferation, resistance to apoptosisand anoikis, invasiveness, and metastasis. FAK, like othersignaling kinases, is dependent on the chaperone heat shockprotein 90 (Hsp90) for its stability and proper function. Thus,inhibition of Hsp90 might be a way of disrupting FAKsignaling and, consequently, tumor progression. FAK isexpressed in high-grade squamous intraepithelial lesionsand metastatic cervical carcinomas but not in nonneoplasticcervical mucosa. In SiHa, a cervical cancer cell line withcharacteristics of epithelial-to-mesenchymal transition, thestable expression of dominant-negative FAK-related non-kinase decreases anchorage independence and delays xeno-graft growth. FAK-related nonkinase as well as the Hsp90inhibitor 17-dimethylaminoethylamino-17-demethoxygelda-namycin both negatively interfere with FAK signaling andfocal adhesion turnover. Short-term 17-dimethylaminoethyla-mino-17-demethoxygeldanamycin treatment prolongs survivalin a SiHa lung metastasis model and chronic administrationsuppresses tumor growth as well as metastatic spread inorthotopic xenografts. Taken together, our data suggest thatFAK is of importance for tumor progression in cervical cancerand that disruption of FAK signaling by Hsp90 inhibitionmight be an avenue to restrain tumor growth as well asmetastatic spread. [Cancer Res 2009;69(11):4750–9]

Introduction

Focal adhesion kinase (FAK), a nonreceptor protein tyrosinekinase involved in integrin signaling, fulfills complex biologicaltasks that require its kinase activity as well as its scaffoldingfunction (1). Recent reports highlight the significance of FAK inneoplasia where it has been implicated in cell proliferation,protection from apoptosis and anoikis, migration, invasion, cellspreading, adhesion, and angiogenesis, thereby influencing eachstep of the metastatic cascade (2). This functional diversity and the

finding that FAK is overexpressed in different tumors have led to anincreasing interest in the integrin-FAK-Src signaling complex aspotential target for therapeutic intervention (3–5). The expressionof the COOH-terminal, noncatalytic domain of FAK, termed FAK-related nonkinase (FRNK) (ref. 6), has been instrumental for theexploration of the cellular functions of the kinase as well as thetherapeutic potential of FAK inhibition.

Notably, not only Src but also FAK has been described as ‘‘client’’of heat shock protein 90 (Hsp90), which is responsible formaintaining the functional conformation of a range of signalingkinases (7). Hsp90 is abundant and active intracellularly as aflexible dimer. Each monomer consists of an ATPase containing N-domain, which is connected to the M-domain by a flexible, chargedlinker and followed by the COOH-terminal dimerization domain.Substrate specificity and chaperone function are regulated by anarray of cofactors, and an increased activity of the entire complexin cancer cells has been held responsible for the relative tumorspecificity of Hsp90 inhibitors (8). The uniqueness of the NH2-terminal ATP-binding pocket is the structural cause for the bindingof benzoquinone ansamycins, which mimic the kinked conforma-tion of ATP inside this pocket (9). Geldanamycin was the firstcompound of this group and initially recognized for its cytotoxicityand ability to revert the phenotype of v-Src-transformed cells (10).Because of instability and hepatotoxicity, semisynthetic derivativeswere developed. 17-Allylamino-17-demethoxygeldanamycin andthe water-soluble 17-dimethylaminoethylamino-17-demethoxygel-danamycin (17-DMAG) have entered clinical trials. 17-Allylamino-17-demethoxygeldanamycin is most advanced in clinical testingand encouraging results have been reported (11–13). Efforts arecurrently directed toward the development of fully synthetic Hsp90inhibitors with improved solubility and side effect profile (14) andtoward resolving problems associated with resistance mechanismssuch as the heat shock response (15).

Recent studies have shown that Hsp90 contributes to cellmigration and, in malignancies, to tumor progression (16, 17).These studies focused mainly on the secreted Hsp90a isoform.However, Hsp90 also maintains the stability of intracellular kinasesin charge of cell motility. Thus, inhibitors of Hsp90 should suppressnot only tumor growth but also invasion and metastasis. Althoughcancer of the uterine cervix can be detected at early stages throughscreening, therapeutic options for metastatic disease are currentlylimited to cisplatin and radiation (18). In this study, we provide arationale for FAK targeting in cervical cancer. Using stableFRNK expression as a model, we show that interference withFAK signaling decreases tumor growth and metastatic coloniza-tion. Importantly, we provide evidence that pharmacologicinhibition of Hsp90 leads to decreased FAK signaling and,

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

Requests for reprints: DavidW. Hedley, Princess Margaret Hospital/Ontario CancerInstitute, 610 University Avenue, 5th Floor, Room 203, Toronto, Ontario, Canada M5G2M9. Phone: 416-946-2262; Fax: 416-946-6546; E-mail: david.hedley@uhn.on.ca.

I2009 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-09-0454

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consequently, displays growth-inhibitory and antimetastatic effectssimilar to FAK inhibition alone.

Materials and Methods

Patient tissue and histology. Formalin-fixed, paraffin-embedded

tissues of 20 cases each of high-grade squamous intraepithelial lesion

(HGSIL) and metastatic squamous cell carcinoma (SCC) of the uterinecervix with one paired lymph node metastasis were selected from files of

the Institute of Pathology Leipzig (1997-2007). Ethics approval was obtained

from institutional committees at the University of Leipzig (protocol

037-2007) and the University Health Network (protocol 06-0804-T).Histology was examined by a senior pathologist (L-C.H.) and a patholo-

gist-in-training (J.S.). Eighteen HGSIL, 20 SCC, and 18 metastasis contained

sufficient tissue for scoring. Nonneoplastic epithelium was present in5 HGSIL and 11 SCC. Immunohistochemistry was scored for intensity

(0-3; Supplementary Fig. S1) and percent area was estimated for each

intensity level. Both values were multiplied to give a histology score

(H-score: 0-300).Reagents, cells, and viruses. Geldanamycin (Biomol) was dissolved in

DMSO at 10 mmol/L and stored at -20jC. 17-DMAG (NSC707545) was

supplied by the National Cancer Institute, stored at -20jC, and dissolved in

water or saline before use. SiHa and ME180 cells were obtained from theAmerican Type Culture Collection and cultured as recommended. DsRed2

fluorescently labeled SiHa cells were obtained from Dr. R.P. Hill (University

of Toronto). Absence of Mycoplasma was confirmed using Hoechst33342 staining (Sigma-Aldrich) and MycoAlert Detection kit (Lonza). FRNK

cDNA, a gift from Dr. M.D. Schaller (University of North Carolina), was

subcloned into a retroviral vector (pBMN-I-GFP; Addgene plasmid 1736;

http://www.stanford.edu/group/nolan/plasmid_maps/pmaps.html) andstable cell lines were generated using an amphotrophic Phoenix-MMULV

system provided by Dr. G.P. Nolan (Stanford University). Vector-control cells

were generated using the empty construct. GFP-expressing cells were

isolated by fluorescence-activated cell sorting resulting in a f98% enrichedpopulation. FRNK expression was monitored by reverse transcription-PCR

and immunoblotting. Total RNA was isolated using RNeasy Mini Kit

(Qiagen) and reverse transcribed with SuperScript II (Invitrogen). Anequivalent of 10 ng cDNA, SYBR Green Master Mix (Applied Biosystems),

and the Mx3000P PCR system (Stratagene) were used to amplify FRNK and

RPL15 housekeeping control. For primer sequences, see Supplementary

Information.In vitro assays. Migration and invasion were assessed as described (19).

Human fibronectin (5 Ag/mL; BD Biosciences) or epidermal growth factor

(50 ng/mL; Sigma-Aldrich) served as chemoattractants. Cells were used

after 4 h serum withdrawal. 17-DMAG was added into the bottom chamberfor up to 24 h. Cells on the opposite filter surface were stained with Diff-

Quik (Dade Behring) and counted at �250 magnification in one random

field/quadrant (migration) or the entire filter (invasion). Colony formation

was tested with 100 and 250 cells per 10 cm dish (triplicates). Colonynumber and size were determined after 3 weeks. Anchorage-independent

growth was tested in soft agar, and 2� a-MEM was mixed with select agar

(Invitrogen) to form a 1% (w/v) base and a 0.4% top layer in 6-well plates.Cells (1 � 104) were embedded into the top layer. Cultures were stained

with neutral red (Sigma-Aldrich) after 3 weeks and images were taken using

a Leica MZFLIII stereomicroscope. Colony number and size were

determined using Image Pro 3D Analyzer version 6.3.0.512 (MediaCybernetics).

Immunoblotting. Immunoblotting was done as described (20). Nitro-

cellulose or polyvinylidene difluoride membranes were probed with the

antibodies listed in Supplementary Information. Signal detection aftersecondary antibody incubation (GE Healthcare/Amersham) was done with

SuperSignal West Pico (Pierce). Signal quantification was done using (a)

Typhoon 9410 (GE Healthcare) with ECL Plus and Image Quant 5.2 softwareor (b) Odyssey Infrared System (LI-COR Biosciences) with IRDye800

antibodies (Rockland).

Immunohistochemistry. Tissues were fixed in 10% neutral buffered

formalin, paraffin embedded, and processed as 4 Am sections. Mouse lungs

were in situ formalin-instilled before dissection. Archival patient tissue wasprocessed alongside SiHa and ME180 xenograft sections for internal control.

After microwave antigen retrieval in 10 mmol/L citrate buffer (pH 6.0;

10 min), antibodies were incubated overnight. For antibodies and dilutions,

see Supplementary Information. Image analysis was done with in-houseimage analysis tools based on IDL 6.3 as described (21).

Immunofluorescence. Cells were grown for 24 to 48 h on type I collagen

(100 Ag/mL; BD Biosciences)-coated coverslips before drug exposure for

24 h. After fixation with 4% formalin and permeabilization with 0.1% (v/v)Triton X-100, primary antibodies in 1% (v/v) bovine serum albumin/PBS

were incubated for 1 h. For antibodies and dilutions, see Supplementary

Information. Clone 77/FAK used for immunofluorescence was generated

with an immunogen not overlapping with FRNK (amino acids 354-533).Secondary antibody goat anti-mouse IgG Alexa Fluor 555 was incubated

together with Alexa Fluor 488-phalloidin for 30 min and coverslips were

mounted with ProLong Gold containing 4,6-diamidino-2-phenylindole(Invitrogen). Confocal images were acquired using an Olympus IX81

microscope and Fluoview FV1000 software.

Animal xenografts. Female SCID mice aged 6 to 12 weeks were bred and

housed at the Ontario Cancer Institute animal facility. Experiments weredone according to regulations of the Canadian Council on Animal Care.

Subcutaneous and orthotopic cervical xenografts were done as described

(20, 22). To determine a limited dose-response relationship, orthotopic

xenografts were established over 4 weeks and treated with four doses of 10or 30 mg/kg 17-DMAG at 12 h intervals. Mice were killed and tumors were

sampled 4 h after the last treatment. For chronic administration, 17-DMAG

was given by intraperitoneal injection three times a week at 25 mg/kg asdescribed (23) starting 4 h after implantation. Controls received saline. Mice

were killed when control tumors approached f1 cm diameter. Tumors

were removed and remaining organs were examined for DsRed2-positive

metastases using a Leica MZFLIII fluorescence stereomicroscope. Subse-quently, the retroperitoneum was dissected. Interval levels (200 Am) were

cut to confirm lymph node and organ metastasis by histology. Experimental

lung metastases were initiated by tail vein injection. Cell viability in

suspension remained >95% by ViaCount Assay (Guava Technologies) untilall injections were completed. Four doses of 30 mg/kg 17-DMAG

intraperitoneally were given at 12 h intervals, the first dose at f30 min

before tail vein inoculation. Mice were monitored for distress and isolated ifsickness occurred. Sick mice were euthanized by CO2 asphyxia if there was

no clinical improvement.

Animal computer tomography (microCT). Development of lung

disease was monitored by serial microCT. Images were acquired on a GELocus Ultra microCT (80 kVp, 50 mA, voxel size: 150 Am) and analysis was

conducted on GE Microview (GE Healthcare Bio-Sciences). Mice were

maintained under isoflurane anesthesia during image acquisition.

High-performance liquid chromatography. 17-DMAG drug tissueconcentrations were measured in xenograft tumors using a high-

performance liquid chromatography method as described previously (20).

Statistical analysis. Statistical analysis was done using Mann-Whitney

test and ANOVA. Two-tailed P values < 0.05 were considered significant.Kaplan-Meier survival data were compared using the log-rank test.

GraphPad Prism version 5.01 software was used.

Results

FAK is overexpressed in HGSIL and cervical SCC. Previousresearch indicated that FAK overexpression is an early event incancer development (24). We aimed to reproduce these results inour own cohort. Because transition from a preinvasive state towarda more aggressive lesion is often associated with loss of cell-celladhesions, we also examined E-cadherin. Adjacent squamousepithelium without intraepithelial neoplasia or signs of humanpapillomavirus infection was scored for comparison. We observedan increase of FAK expression from essentially unstained nonneo-plastic epithelium to HGSIL and metastatic cancer. HGSIL andcancer displayed mostly weak to moderate staining with some

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exceptions, for example, strong focal FAK expression at theinvasion front or in a diffusely infiltrating carcinoma with partialE-cadherin loss (Fig. 1). E-cadherin expression was strongest innonneoplastic epithelium and reduced or lost in HGSIL andmetastatic cancer. Differences in H-scores were only significantbetween nonneoplastic epithelium and all other categories,suggesting that both FAK expression and E-cadherin loss areindeed early events during tumorigenesis.FRNK decreases anchorage independence, migration, and

focal adhesion signaling. To test if FAK is active in cervical cancercells, we generated FRNK-SiHa, a cell line with stable expression ofFRNK. The SiHa cell line was selected because of low E-cadherinexpression and nuclear positivity for Snail1, suggesting a lessdifferentiated phenotype compared with other cervical lines suchas ME180 (data not shown). Both SiHa and ME180 cells, however,

retain human cytokeratin expression (AE1/AE3). Cell growth inmonolayer culture over 5 days and colony formation were notsignificantly affected in FRNK-SiHa (data not shown), butanchorage-independent growth assays showed significantly smallerFRNK-SiHa colonies (Fig. 2A).

Migration and invasion of FRNK-SiHa were tested withfibronectin as chemoattractant. Migration was decreased by40% and consistent with a diminished fibronectin response(Fig. 2B). Invasion was decreased by 40% to 60% (data not shown)but failed to reach statistical significance likely due to the weakstimulatory effect of fibronectin. Basal phosphorylation levels ofFAK Y397 (autophosphorylation) and Y861 and Y925 (Src-dependent)were reduced in FRNK-SiHa (Fig. 2C). Paxillin Y118 was 50%decreased similar to p130Cas Y165, Y249, and Y410, indicating aninhibited downstream signaling due to FRNK. Paxillin Y31 remained

Figure 1. FAK expression and E-cadherinloss are early events in cervical cancer.A, FAK increase and E-cadherin loss inHGSIL, cancer, and metastases comparedwith nonneoplastic (Normal ) epithelium.B, focus of SCC invasion with adjacentFAK-negative epithelium (asterisk) andloss (arrow) of E-cadherin membranestaining. C, poorly differentiated SCC withFAK-positive (inset 1) infiltrating cells andlack of E-cadherin (inset 2) in this poorlydifferentiated component. D, HGSIL(asterisk) and cancer invasion front (inset )with pronounced FAK positivity.

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unaffected. No difference was seen for Akt and extracellular signal-regulated kinase 1/2 phosphorylation (data not shown). Treatmentwith 10 Amol/L nocodazole for 4 h with subsequent washout wasused to examine focal adhesion dynamics (25). A f2-fold increaseof FAK Y397 and Y925 in vector-control but not in FRNK-SiHa cells(Fig. 2D) suggested a decreased focal adhesion turnover.FRNK and 17-DMAG similarly interfere with focal adhesion

turnover. To determine the effect of Hsp90 inhibition on basalphosphorylation and total protein levels of FAK, monolayercultures were treated with increasing doses of geldanamycin and17-DMAG, and 100 nmol/L of either compound caused a profounddecrease in FAK Y397 and a somewhat less noticeable decrease inFAK protein after 24 h (Fig. 3A). Paxillin and the established clientAkt responded with a profound decrease of either protein level.Hsp70 was already induced at 10 nmol/L concentration, indicating

that the heat shock response precedes client protein destabiliza-tion. Next, we addressed the question if 17-DMAG was able tocause a blunting of stimulated FAK signaling. Figure 3B shows adose-dependent suppression of the response to epidermal growthfactor. FAK activity was suppressed below baseline if the treatmentwas extended >20 h. Consequently, 17-DMAG also decreased SiHacell migration against epidermal growth factor (Fig. 3C). A similarsuppression of fibronectin stimulation was observed, albeit atlower overall phosphorylation levels (data not shown).

To examine if altered FAK signaling was reflected by changes incell and focal adhesion morphology, SiHa cells were treated with100 nmol/L geldanamycin or 17-DMAG and double-labeled foractin and either FAK, paxillin, or vinculin. Drug-treated cells werecompared with FRNK-SiHa, wild-type, and vector-control cells forcell area and focal adhesion length. FRNK-SiHa and Hsp90

Figure 2. FRNK decreases anchorageindependence, cell migration, and FAKsignaling. A, FRNK-SiHa colonies in softagar are significantly smaller thanwild-type (WT ) and vector-control (CTRL ;n = 3; columns, mean; bars, SE).B, migration of FRNK-SiHa is decreasedby 40% (n = 6; columns, mean; bars, SE)due to a decreased fibronectin (FN)response. C, quantitative PCR showsspecific amplification of FRNK inFRNK-SiHa but not in wild-type andvector-control compared with RPL15.FRNK leads to decreased phosphorylationof FAK, paxillin, and p130Cas.D, FRNK-SiHa (underlined ) fails tostimulate FAK phosphorylation afternocodazole washout compared withvector-control.

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inhibitor-treated cells displayed a spread-out morphology (Fig. 4A).Double labeling revealed pronounced stress fibers connected todistinct, elongated focal adhesions in FRNK-SiHa as well asdrug-treated cells (Fig. 4B ; Supplementary Figs. S2-S4). Vinculin

confirmed our results (data not shown). Immunofluorescence forFAK using clone77 antibody produced a diminished labeling offocal adhesions in FRNK-SiHa (Fig. 4C and D), consistent with adisplacement of endogenous FAK. Together, these results suggestthat Hsp90 inhibition suppresses focal adhesion turnover in amanner similar to FRNK.FRNK delays tumor growth and lung metastasis formation

in SiHa xenograft models. Next, we determined if the suppressedFAK activity in FRNK-SiHa would translate into changes in tumorformation and metastatic potential. To screen for a potentialdifference, FRNK-SiHa and vector-control cells were initially grownin opposite flanks of single mice. This was repeated six timesstarting from separate cultures for each injection. All mice, exceptfor one outlier, showed delayed tumor development for FRNK-SiHa.To confirm this result, wild-type, FRNK-SiHa, and vector-controlcells were inoculated into flanks of individual mice showing asignificantly delayed tumor formation with FRNK (Fig. 5A). Tofurther examine metastatic potential and growth of thesexenografts, we implanted a fragment of each dissected xenograftinto the cervix of a recipient mouse. Due to extensive necrosis inlarge wild-type and vector-control tumors and the small size ofFRNK-SiHa donors on the contrary, a high variability was seen withthe resulting orthotopic xenografts and tumor weight differenceswere not significant. However, there was a trend toward smallertumors with FRNK-SiHa (Supplementary Fig. S5A). Three distantretroperitoneal tumor deposits were found in each of the wild-typeand vector-control groups by histology (250 Am levels) but none inFRNK-SiHa (Supplementary Fig. S5B). Finally, to address the effectof FRNK on metastatic colonization, each cell line was tail veininjected into 5 mice per group. Development of lung disease wasmonitored over 3 months with four microCT imaging sessions.There were three early unscheduled deaths, one in each group. Theremaining mice showed a trend toward lower disease burden inFRNK-SiHa, with 1 of 4 FRNK-SiHa versus 3 of 4 wild-type and 4 of4 vector-control mice that developed extensive lung disease(Supplementary Fig. S5C).17-DMAG suppresses in vivo FAK signaling, tumor growth,

and metastasis. Proceeding from earlier results (20), we wished toexamine if 17-DMAG treatment can cause a detectable suppressionof FAK signaling in vivo . Mice harboring orthotopic xenografts weretreated with two different dose levels of 17-DMAG by either oralgavage or intraperitoneal injection four times in 12 h intervals.Because decreased phosphorylation often seems to precede clientdegradation (23), we focused on two FAK phosphorylation sites(Y397/Y861) as well as Akt S473 (Fig. 5B). Immunoblotting revealed astatistically significant FAK Y397 down-regulation and Hsp70 up-regulation at 30 mg/kg 17-DMAG. Drug tissue concentrations fororal versus intraperitoneal application were consistent with areported oral bioavailability of f50% (Supplementary Fig. S6A). Nodifference was noted between ME180 and SiHa xenografts at thesame dose level. Because the FAK Y861 antibody distinctly labels cellmembranes, we stained control and 30 mg/kg treated SiHaxenografts by immunohistochemistry to confirm our immunoblotresults. One control specimen was excluded due to tissuedisaggregation, which interfered with membrane staining andimage analysis (Supplementary Fig. S6B). The remaining specimensrevealed, as by immunoblotting, a 40% reduction in FAKphosphorylation (Fig. 5C).

To examine the consequences of short-term Hsp90 inhibition, weinitiated a lung metastasis model (Fig. 6A). Progression of lungdisease was monitored by microCT. We observed a significant

Figure 3. Hsp90 inhibition suppresses focal adhesion signaling. A, geldanamycin(GA) and 17-DMAG cause a dose-dependent increase of Hsp70 anddestabilization of FAK and Paxillin. Akt is shown for comparison and GAPDH servedas loading control. B, 17-DMAG causes a dose-dependent blunting of epidermalgrowth factor (EGF)-induced FAK activation. C, a decrease of SiHa cell migrationagainst epidermal growth factor (24 h; n = 3; columns, mean; bars, SE).

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survival advantage in the 17-DMAG-treated group versus control.Most mice were euthanized because of terminal lung disease.Figure 6B and Supplementary Fig. S7A and B show representativemicroCT images. By autopsy, we also detected four tumor-embolickidney infarcts (three in controls and one in 17-DMAG) and twocases of bowel ischemia (one in control and one in 17-DMAG;Supplementary Fig. S7C). Two controls developed hind-leg paresispresumably due to peripheral emboli because no brain abnormalitywas found. Eight treated mice lived to the end of the experimentwithout signs of disease.

Finally, we addressed whether chronic administration of 17-DMAG could suppress tumor growth and metastasis. DsRed2fluorescent SiHa cells were used for easier detection of metastasesfrom orthotopic xenografts. 17-DMAG was given as a total of

15 treatments and all mice were killed on day 33. There was a 64%decrease in mean tumor weight in the 17-DMAG group (Fig. 6C)but no change in body weight, suggesting that the treatment waswell tolerated (Fig. 6D). Seven of 10 controls but none of the treatedmice had detectable metastases. Histology confirmed a total of10 metastases [4 retroperitoneal deposits partially with adjacentlymph node, 3 metastases to the diaphragm, and 1 each in liver,stomach, and kidney (Supplementary Fig. S8)].

Discussion

FAK has emerged as an attractive therapeutic target, particularlyin the context of tumor invasion and metastasis (2, 5). Itsoverexpression occurs as an early event during tumorigenesis

Figure 4. FRNK and Hsp90 inhibitiondelay focal adhesion turnover.A, FRNK-SiHa displays an increased cellspreading similar to 17-DMAG orgeldanamycin-treated wild-type cells(n = 100 cells). B, paxillin-labeled focaladhesions show an increased length inFRNK-SiHa as well as wild-type treatedwith 17-DMAG or geldanamycin (n = 100focal adhesions). Whiskers, range; n.s.,not significant. C, immunofluorescence forFAK (red ) and actin (green ) shows lessprominent focal adhesions in FRNK-SiHabut a dense fringe in wild-type andvector-control (arrowheads ). Actin-richareas of cytoskeletal reorganization arediminished (arrows ). D, magnification ofthe red channel illustrates diminishedFAK-labeling in FRNK-SiHa.

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and has been described in different cancer entities including SCCof mucosal surfaces (24, 26–30). Increased FAK expression in pre-cursor lesions and frankly invasive cervical cancer is an intriguingfinding because an inverse relationship between p53 and FAK hasbeen reported, which is consistent with p53 instability due to humanpapillomavirus E6 viral protein as essential etiologic factor (31, 32).Ochel and colleagues first described FAK as a Hsp90 client (7), andrecent research has highlighted the dependence of the kinase onintact Hsp90 chaperone action in vitro (23). However, few dataon FAK inhibition in cervical cancer and the potential of Hsp90inhibitors to target FAK function in vivo are available to date. Thus,we examined the consequences of compromised FAK signalingusing FRNK and 17-DMAG in different models of cervical cancer.

FRNK has been used extensively to elucidate FAK functions(33, 34), and based on its conditional expression, van Nimwegenand colleagues (35) showed that FAK was required for the earlyphase of lung metastasis formation. Thus, transient inhibition of

FAK at specific stages of the metastatic cascade might interrupt aprocess that is the most frequent cause of death in the cancerhost. To pursue this path, we first determined the FAK expressionin our own patient cohort, which showed a significant increase ofFAK in HGSIL and cancer compared with nonneoplastic epithe-lium. Stable expression of FRNK in SiHa, a cell line with epithelial-to-mesenchymal transition characteristics, did not affect prolifer-ation in monolayers but suppressed anchorage-independentgrowth. This is in line with previous reports that found no changeof in vitro or even in vivo growth with FRNK depending on cellcontext (33, 35, 36). Decreased anchorage-independence in SiHa-FRNK is likely a consequence of altered FAK signaling as shownpreviously in classic experiments with FAK-/- cells (37). Enlargedfocal adhesions and stress fiber formation are potentially due to adecreased Rac activity downstream of p130Cas, which may shift thebalance toward Rho/ROCK (38). Changes consistent with theseobservations were also found after pharmacologic Hsp90 inhibition

Figure 5. FAK inhibition decreasesxenograft growth and can be achieved with17-DMAG in vivo. A, FRNK inhibitssubcutaneous xenograft growth (2 � 106

cells inoculated; n = 5 per group;**, P < 0.01; ***, P < 0.001) due tointerference with FAK signaling.B, immunoblots of orthotopic xenografts(n = 5 per group) treated with different17-DMAG doses orally or intraperitoneally(i.p. ) show a significant FAK Y397

suppression and Hsp70 induction in SiHaat 30 mg/kg intraperitoneal comparedwith control [values normalized to SiHacontrol (dotted lines )]. C, FAK Y861

immunohistochemistry confirms FAKdephosphorylation by 40%. Representativeimages for control and treatment.High-performance liquid chromatographyresults and sections of all specimens areshown in Supplementary Fig. S6.

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with 17-DMAG. Although essentially correlative, our data supportthe notion of FAK as an important client within the focal adhesioncomplex. Because Hsp90 function is inextricably interwoven withthe stability of a host of clients, destabilization of otherpromigratory molecules may contribute to the observed effect.However, it has been argued that the pleiotropism of Hsp90inhibitors may result in a more sustained drug response; indeed, acompromised FAK function might less likely be bypassed by anincreased Src activity in that context (39). Preclinical and clinicaltesting of small-molecule FAK inhibitors will provide furthermechanistic insights into this matter.

17-DMAG significantly decreased but did not abolish FAKphosphorylation in our xenografts. Yet, we hypothesized that ashort-term treatment during a critical phase of tumor progression,

that is, escape from anoikis and extravasation, could be effective inpreventing the metastatic colonisation of a target organ. Indeed,17-DMAG treatment resulted in a survival advantage and lowerdisease burden consistent with the proposed role of FAK during theearly phase of the metastatic process (35). This is encouragingconsidering the short plasma half-life of 17-DMAG, which mightlimit the exposure of circulating cells to effective drug concen-trations (40). Although Hsp90 and extracellular Hsp90a undoubt-edly contribute to cell migration and metastasis (16, 41), there hasrecently been some controversy due to prometastatic effects ofHsp90 inhibition observed in other models (42, 43). For 17-DMAG,there are only few reported studies that address antimetastaticactivity based on different models reflecting parts of the metastaticcascade (44, 45). Thus, we tested 17-DMAG in a spontaneously

Figure 6. 17-DMAG decreases diseaseburden in two SiHa metastasis models.A, 17-DMAG improves the survival in alung metastasis model after injection of 1� 106 cells into the tail vein. B, twomicroCT imaging sessions (days 79-85and 98-102 post-inoculation) illustrate thedelayed lung metastasis formation in onetreated compared with one control mouse.The control shows nodular metastasespredominantly in the right lung andprogression to extensive disease. Thetreated animal has no detectablemetastases during CT1 and developedthree nodules in the left posterior lungapparent on CT2 (arrowheads).Anatomically equivalent scan sections areshown from both sessions. Histologiccorrelation is shown in SupplementaryFig. S7. C, 25 mg/kg 17-DMAG threetimes a week intraperitoneally significantlydecreases orthotopic tumor growth andprevents metastases (Supplementary Fig.S8). D, no significant difference in bodyweight was noted between 17-DMAG andcontrols. An early transient decrease inbody weight (days 1-5) was due topostoperative stress.

17-DMAG Targets FAK and Tumor Progression

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metastasising SiHa orthotopic xenograft model and were able toshow suppressed primary tumor growth and abolished metastasis.

Although our results show efficacy against xenograft growth andmetastasis in SiHa, they do not strictly distinguish between thesetwo processes. However, during this work, we also generated aFRNK-expressing variant of the keratinizing cell line ME180 usingthe same expression system. Although equivalent levels of FAK andFRNK were determined in monolayer cultures of SiHa and ME180,the latter cell line did not show a delayed xenograft growth inresponse to FRNK. Also, the number of metastases from orthotopicME180 xenografts did not lower significantly after chronic 17-DMAG treatment despite a 54% decreased mean tumor size (datanot shown). A lower FAK phosphorylation (Fig. 5B, lane 1 versuslane 6), likely due to lower FAK expression in xenografts(Supplementary Fig. S9), was noted for ME180 and left us tospeculate that Hsp90-independent mechanisms may play a role inthese xenografts. Indeed, FAK has been linked to the acquisition ofepithelial-to-mesenchymal transition features (46, 47). By contrast,amoeboid invasion is likely independent from FAK (48).

While this work was in progress, the clinical development of17-DMAG by Kosan Biosciences (June 2008: Bristol-Myers Squibb)was terminated. Several fully synthetic Hsp90-inhibitory com-pounds currently under development are expected to have betterside-effect profiles (14). An enhancement of cervical cancer cellradiosensitivity by Hsp90 inhibition has been shown previously (49)and should be further pursued using those compounds. Notably,Hsp90-inhibitory and heat shock-modulating effects have recentlyalso been recognized for cisplatin (50).

In summary, we show here that FAK is a potential therapeutictarget in metastatic SCC of the uterine cervix, the function of whichcan be compromised by pharmacologic inhibition of Hsp90 bothin vitro and in xenografts. Interference with FAK function suppressedtumor growth and metastatic colonization in two different modelsystems of poorly differentiated SCC. Additional work is needed toelucidate the function(s) of Hsp90 during tumor progression and toexamine the efficacy of its inhibitors in the context of cancer withepithelial-to-mesenchymal transition features.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 2/9/09; revised 3/25/09; accepted 4/2/09; published OnlineFirst 5/19/09.Grant support: Terry Fox Program Project Grant of the National Cancer Institute

of Canada (D.W. Hedley) and Ellen Epstein Rykov Memorial Prize, William S. FenwickFellowship, and Joseph M. West Family Memorial Fund of the University of Toronto(J. Schwock).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. M.D. Schaller (University of North Carolina) for the gift of theFRNK cDNA, Drs. R.P. Hill (University of Toronto) and W.R. Geddie (TorontoGeneral Hospital) for the critical reading of the article, and James C. Ho andWen-Jiang Zhang (Ontario Cancer Institute) for performing immunohistochemistryand high-performance liquid chromatography, respectively. 17-DMAG from KosanBiosciences was supplied by the Cancer Therapy Evaluation Program (National CancerInstitute).

Cancer Research

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2009;69:4750-4759. Published OnlineFirst May 19, 2009.Cancer Res   Joerg Schwock, Neesha Dhani, Mary Ping-Jiang Cao, et al.   Growth and Metastasis of SiHa Cervical XenograftsFRNK or Hsp90 Inhibitor 17-DMAG Suppresses Tumor Targeting Focal Adhesion Kinase with Dominant-Negative

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