Crk and CrkL present with different expression and significance in epithelial ovarian carcinoma

MOLECULAR CARCINOGENESIS 50:506–515 (2011)

Crk and CrkL Present With Different Expressionand Significance in Epithelial Ovarian Carcinoma

Jin Wang,1,2 Ya-ling Che,1,2 Gang Li,2,3 Bin Liu,4 Tai-min Shen,1,4 Hui Wang,1,2 and Hua Linghu1*1Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing,PR China2Experimental Research Center, The First Affiliated Hospital, Chongqing Medical University, Chongqing, PR China3Division of Cardiology, Department of Geriatrics, The First Affiliated Hospital, Chongqing Medical University,Chongqing, PR China4Department of Pathology, Basic Medical College, Chongqing Medical University, Chongqing, PR China

Adaptor protein Crk and CrkL were thought to be closely related because both consist of one SH2 and two SH3domains and share 60% homology with the highest identity within their functional domains. Their functions were mostpresumed to be in part, if not all, redundant. And both were suggested to be implicated in carcinogenesis. In this study,

both Crk and CrkL presented with much higher expression in ovarian cancer tissues than those in normal and benignovarian tissues. However, in contrast with CrkL, high Crk expression displayed close association with advanced stages andhigh-grade diseases. Furthermore, the differential binding selectivity of Crk and CrkL to their downstream partners Dock

180 and C3G was demonstrated in ovarian cancer cell line SKOV3 through coimmunoprecipitation. Additionally, Crk-knockdown cells presented with changed morphology, reduced growth, and cell invasion but remained viable. Incontrast, all CrkL-knockdown cells could not survive over time, gradually detaching from the bottom of plastic dish. In

conclusion, these two highly homologous proteins hold features that allow for the differential association with eachbinding molecules, thereby activating different signaling pathways and being involved in diverse roles in ovarian cancer.� 2011 Wiley-Liss, Inc.

Key words: Crk; CrkL; signal adaptor; epithelial ovarian carcinoma

INTRODUCTION

The mortality of epithelial ovarian cancer (EOC)ranks first among all female reproductive malignan-cies. Traditional therapy, which contains primarilyoperation and chemotherapy, could save only a few[1]. Tumor invasion and metastasis greatly limittreatment options and account for 90% of cancer-related death [2], and this is also true for ovariancancer. Most efforts to improve its prognosis endedup with little progress due to its elusive mechanism.Small GTPase, such as Rac, Ras, and Rap, was knownto play an important role in controlling many phys-iological and pathological events, which includetumor dissemination. However, the mechanismunder how small GTPase is regulated in cancer cellsremains unclear. Adaptor protein Crk and CrkL wererecognized to be involved in several GTPase regula-tion because of their downstream effectors such asDock180, C3G, and Sos, the guanine–nucleotideexchange factors (GEF) for Rac, Rap, and Ras,respectively [3–7], even though their individualroles remain not so clear.

Cancer targeted therapy seems attractive recently.The rationale for target therapy should be targetingthe genes or proteins which are situated at the keypoints of malignant growth and spreading. The tar-geting therapy should hold little common featureswith the course of normal regeneration and

embryogenesis, such that the therapy can be aimedat cancer with pinpoint accuracy. However, accord-ing to recent findings, cancer cells often co-opt nor-mal regenerative processes to aid in theirprogression. Tumor cells render themselves domi-nant over normal cells through prosurvival andproinvasive pathways [8], in which different adaptorproteins exert indispensable roles.Adaptor proteins are a kind of proteins, which

themselves have no enzymatic activity but usuallycontain at least two domains, through which vari-ous kinds of proteins in different pathways could becombined. Their finding and identification haverenewed our understanding of cellular signal path-way: the combination of proteins in different signal

Additional supporting information may be found in the onlineversion of this article.

Abbreviations: EOC, epithelial ovarian cancer; Crk, CT10 regulatorof kinase; SH2, Src homology 2; SH3, Src homoloyg 3; BOT, benignovarian tumor; IOD, optical density; siRNA, small interfering RNA

Jin Wang and Ya-ling Che contributed equally to this work.

*Correspondence to: Department of Obstetrics and Gynecology,The First Affiliated Hospital, Chongqing Medical University, Chongq-ing 400016, PR China.

Received 7 May 2010; Revised 17 December 2010; Accepted 5January 2011

DOI 10.1002/mc.20745

Published online 11 February 2011 in Wiley Online Library(wileyonlinelibrary.com).

� 2011 WILEY-LISS, INC.

pathways has made cellular events go smoothly andorderly [9].v-Crk was first found in avian sarcoma virus CT-

10, and it was named as Crk because of its capabilityof activating kinases (CT10 regulator of kinase, Crk)[10]. Its cellular homologues c-Crk (encoded by Crkgene, which is located in human chromosome17p13.3 [11]) and c-CrkL (Crk-like, encoded by CrkLgene, which is located in human chromosome22q11 [12]) were identified later, and c-Crk primar-ily contains two splicing products c-CrkI and c-CrkII. Crk family was the first identified adaptorproteins, which connect with upstream proteinsthrough their SH2 (Src homology 2) domain andwith downstream proteins through their N-terminalSH3 (Src homoloyg 3) domain, and thus transmitthe cellular signals down to their target subcellularareas [9].Both Crk and CrkL were found to link with

pYXXP (phosphor-Tyr-X-X-pro) motifs throughtheir SH2 domains. And p130Cas and paxillin havebeen testified to comprise such motifs and becomethe major binding partners of the SH2 domain ofCrk/CrkL [13] once induced by integrin receptors orgrowth factor receptors [14,15]. Upon linked to theirupstream proteins through SH2 domains, Crk andCrkL function as adaptors by associating with theirtarget molecules through their N-terminal SH3domains [16,17]. The pleiotropic function of Crkand CrkL depend upon the variety of their down-stream binding proteins [18]. Among all combiningpartners, two kinds of guanine nucleotide exchangefactors C3G and Dock180 were largely reported[3,4,19–23], except for the interaction between CrkLand Dock180, which has not yet been demonstratedbefore.Although both Crk and CrkL have been found to

hold oncogenic and transformative activity [24–30],it remains an enigma as for the exact role of Crk andCrkL, respectively. Previously, their functions incells were most presumed to be in part, if not all,redundant [9]. This mainly results not only fromtheir 60% homology in structure with the highestidentity within their functional domains, but fromthe findings as well that most Crk/CrkL SH2- andSH3-binding proteins contain several docking siteswith much sequence similarity [9]. If this is the case,concerns would arise in the gene therapy for carci-noma: possible therapeutic means that target Crkmay be compromised by the presence of CrkL andvice versa. However, no direct evidence could befound to support this conjecture.In this report, we analyzed Crk and CrkL expres-

sion in ovarian epithelial carcinoma tissues withdifferent stage and grade, and analyzed their effectupon survival. Next, the combining profile of Crk/CrkL with their well-known downstream molecules(Dock180 and C3G) was analyzed in ovarian cancercell line SKOV3, respectively. Additionally,

endogenous expression of Crk and CrkL wasknocked down, respectively, in ovarian cancer cellline SKOV3 through RNA interference, and Crk- andCrkL-knockdown cells present with differentphenotypes.

MATERIALS AND METHODS

Patient Samples

Following approval by the Institutional ReviewBoard for the Protection of Human Subjects inChongqing Medical University, formalin-fixed, par-affin-embedded blocks from 134 ovarian samples ofhuman EOC, 10 of benign ovarian tumor (BOT) and10 of normal ovarian surface epithelium sampleswere collected. Additionally, fresh ovarian tissuesfrom the other 15 EOC patients, 2 BOT patientsand one patient with normal ovaries were collectedand stored at liquid nitrogen for immunoblotting.In this study, all EOC samples were from patientswith serous adenocarcinoma, and BOT samples werefrom patients with serous adenoma. All EOCpatients underwent surgical exploration and cytor-eduction as the initial treatment. All of EOCpatients were staged according to the FIGO surgicalstaging system (briefly, stage I with malignancy ofone (Ia) or both (Ib) ovaries; without ascites; stage IIwith malignancy of one (IIa) or both (IIb) ovaries,with pelvic extension and ascites; stage III withmalignancy involving one/both ovaries, intraperi-toneal metastases outside pelvis and/or positiveretroperitoneal lymph nodes; stage IV with involve-ment of one/both ovaries withmetastases and histo-logically confirmed extension to pleural cavity orliver [31]). An experienced gynaecologic pathologistwithout knowledge of clinical data reviewed all ofthe pathology results for all of the patients.

Immunohistochemistry Staining of Crk and CrkL

For immunohistochemical analysis, paraffin-embedded tissues were sectioned (5-mm thick) andused to assess Crk and CrkL expression. Briefly,formalin-fixed and paraffin-embedded sections weredeparaffinized by sequential washing with xylene,100% ethanol, 95% ethanol, 80% ethanol, and PBS.After antigen retrieval in microwave, endogenousperoxidases were blocked with 3% H2O2 in metha-nol for 5 min. After PBS washes, slides were blockedwith 5% normal horse serum and 1% normal goatserum in PBS for 15 min at room temperature,followed by incubation with primary antibodyin blocking solution overnight at 48C. Next, theappropriate secondary antibody conjugated tohorseradish peroxidase (HRP) in blocking solutionwas added for 1 h at room temperature. HRPwas detected with 3,30-diaminobenzidine (DAB;Phoenix Biotechnologies, Huntsville, AL) substratefor 5 min, washed, and counterstained withhematoxylin.

ROLE FOR Crk AND CrkL IN OVARIAN CANCER CELLS 507

Molecular Carcinogenesis

For quantitative analysis of immunostainingintensity of Crk and CrkL, we computed the inte-grated optical density (IOD) and measured 15blinded samples for each experimental group. Digi-tally fixed images were analyzed at 200� magnifi-cation using an Olympus BX51 (Tokyo, Japan) lightmicroscope equipped with an image analyzer (Ima-ge Pro Plus, version 6.0.0.260). IOD was calculatedfor arbitrary areas, each sample analyzed with thesame size. All data are a mean value and statisticalanalysis was applied to compare the results fromgroups with different histological grade and FIGOstage.

Cell Culture

The ovarian cancer cell line SKOV3 (obtainedfrom Department of Pathology, Chongqing MedicalUniversity, PR China) was maintained and propa-gated in vitro by serial passage in RPMI-1640 supple-mented with 15% fetal bovine serum and 0.1%gentamicin sulfate (Gemini Bioproducts, Calabasas,CA). All experiments were performed at 70–80%confluence.

Antibodies, Compounds, and Plasmids

Mouse monoclonal antibody (mAb, clone 22) forCrk was purchased from Transduction Laboratories(Lexington, KY). Antibody for CrkL (C20), C3G(C19), and Dock180 (H4) was obtained from SantaCruz Biotechnology (Santa Cruz, CA). GST-PAK2-RBD was purchased from Upstate (Charlottesville,VA). Specific Crk small interfering RNA (siRNA) tar-geting sequence Crki1 (50-GAGTTTGATTCATTG-CCTG-30) and Crki2 (50-UGCCUAC GACAAGA-CAGCC-30), and three CrkL siRNA targeting sequen-ces, CrkLi1 (50-CTC GCT GCC CAA CCG CCG T-30),CrkLi2 (50-TGA TGC CGA AGA CCT GCC C-30), andCrkLi3 (50-GTG AAC GGG CGC AAA GGG C-30),were cloned into the retrovirus vector pSUPER.retro.neo (Oligo Engine, Seattle, WA). PlasmidspEGFP-actin was a gift (see Acknowledgment).

Immunoprecipitation and Immunoblotting

Cells were lysed with lysis buffer containing150 mM NaCl, 10 mM Tris–hydrochloride (pH 7.5),5 mM EDTA, 10% glycerol, 0.5% Nonidet P-40,1 mM sodium orthovanadate, 1 mMphenylmethyl-sulfonyl fluoride (PMSF), 50 mM sodium fluoride.Lysates were incubated with antibodies for 1 h at48C, followed by incubation with protein G- orprotein A-sepharose beads (Calbiochem, Darmstadt,Germany) for 1 h at 48C while rotating. Immuno-precipitates were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis andimmunoblotted onto polyvinylidene fluoride (PVDF)filter (Immobilon, Millipore Co., Bedford, MA). Thefilter was blocked with 3% bovine serum album inand then incubated with primary Abs followed by

peroxidase-conjugated secondary Abs. Positive sig-nals were detected by enhanced chemilumines-cence system (Amersham Biosciences, Piscataway,NJ) and analyzed by Chemi Doc XRS detection sys-tem (Bio-RAD, Hercules, CA).

Immunoblotting for Crk/CrkL Expression inOvarian Tissues

Ovarian tissue homogenates and lysates wereprepared with lysis buffer containing proteaseinhibitors. Equal amount of protein was separatedby sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis and immunoblotted onto PVDF, andfollowed by the steps above.

Establishment of Crk- and/or CrkL-KnockdownSKOV3 Cells (Clones)

It was described previously. In brief, Cells weretransfected with subcloned pSUPER-retro.neo-iCrkor pSUPER-retro.neo-iCrkL, followed by selectionwith neomycin sulfate for 2–3 weeks. Survivedclones were isolated and the expression levels ofCrk/CrkL were analyzed by immunoblotting.

Growth Rate Measurement

To measure growth rate, 1 � 105 cells were seededonto 60-mm diameter plates with DMEM contain-ing 10% FBS, and the numbers of cells were countedeveryday using a hemocytometer.

Colony Formation Assay

To analyze the anchorage-independent growthof cells, 1 � 104 of cells were plated in 60-mmdiameter plates with 3 ml of 0.4% noble agar inDMEM in 10% FBS, overlaying 5 ml of 0.5% bactoagar in DMEM with 10% FBS. Colonies were scored3 weeks after plating. The number of colonieslarger than 50 mm in diameter was counted undera microscope.

Serial Observation for the Viability ofCrkL-Knockdown Cells

One cluster of each CrkLi clones (confirmedby immunoblotting) were marked and observedwith phase contrast microscope continually fortheir condition over time.

GST Pull-Down Assay for Rac1 Activity

Detection of the GTP-bound form of Rac1 wasperformed as described by the Manufacturer’smanual (Upstate, Charlottesville, VA). Briefly, cellswere lysed with buffer containing 25 mM HEPES(pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA,1% NP-40, 10 mM MgCl2, 1 mg/ml aprotinin, and1 mMPMSF. Lysates were centrifuged at 12,000 rpmat 48C for 1 min, and the supernatants were

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incubated with 10 ml of GST-PAK2-RBD and gluta-thione beads. The resulting precipitants were ana-lyzed by immunoblotting with anti-Rac1.

Fluorescence Staining

Subconfluent cells grown on a glass coverslip werefixed with 3.7% formaldehyde in PBS for 15 minand permeabilized with 0.2% Triton X-100 in PBSfor 4 min. To visualize actin filaments (F-actin), cellswere stained with Alexa594-conjugated phalloidin(1 mg/ml) for 60 min and examined with confocalimmunofluorescence microscope (LEICA TCS SP2,Mannheim, Germany).

Invasion Assay

The lower surface of the filter Bio Coat MatrigelInvasion Chamber (BD Biosciences, Franklin Lakes,NJ) was coated with 1 mg of collagen. Fifty thou-sands cells were seeded in the upper chambers (24-well chambers) in 0.5 ml of serum-free mediumcontaining 0.01% BSA and 3.3 mM glucose, andthe bottom chambers contained the appropriatemedium with 1% FBS as a chemoattractant. Thenon-invading cells on the upper surface of the filterswere removed by wiping with a cotton swab. Cells atthe bottom side of the membranes were fixed withethanol, and stained with 0.5% methyl blue. Thenumber of cells invading through the matrigelmembrane was counted in microscopic fields at200� magnification. To minimize the bias, at leastthree randomly selected fields were counted. Datawere averages of triplicate determinants for eachcondition.

Statistical Analysis

All experiments were repeated at least three times.Statistical analysis was performed on relationshipbetween tumor grade and Crk/CrkL expression byANOVA using software of SPSS 10.0 (SPSS, Inc., Chi-cago, IL). Kaplan–Meier survival curve, log-rank test,and Cox hazards regression model were used in sur-vival analysis. The statistically significant differencewas set as P < 0.05 (two-sided probability).

RESULTS

Crk and CrkL Expression in EOC Tissue and TheirAssociation With Survival

Given the lack of information about the clinicalsignificance of Crk/CrkL expression in human EOC,we first examined 134 samples stained for Crk andCrkL expression (Figure 1A), with 10 samples of nor-mal ovarian tissues and 10 samples of BOT tissuesas control. Crk showed positive immunoreactivitymainly in the cytoplasms of the cells and a littlestaining in the nuclei of the cells. CrkL showedpositive immunoreactivity in the cytoplasms ofthe cells. Crk and CrkL expression were present in114 (85.08%) and 110 (82.09%) cases of EOC,

respectively, in contrast with low positivity in nor-mal and benign ovarian tissues (10% and 20% forCrk, 10% and 30% for CrkL, respectively, Figure 1B).The representative images of Crk/CrkL expression inbenign and malignant ovarian tumor tissues aredemonstrated in Figure 1A. High Crk expressionwas mainly observed in high grade (Figure 1C andSupplementary Figure 1A) and advanced stage dis-eases (Figure 1D). As compared, CrkL expressionpresented with similar association with histologicdifferentiation but with no linkage with the stageof diseases.Besides the results above, high Crk/CrkL expres-

sion levels were also demonstrated in fresh ovariantissue samples from 15 patients with EOC by usingWestern blot (Supplementary Figure 1D).To further clarify the association of Crk/CrkL

expression with carcinogenesis of EOC, follow-updata were utilized to analyze the effect of Crk/CrkLexpression upon survival. Multivariate analysisthrough Cox proportional hazards model suggeststhat FIGO stage was the only independent prognosisfactor of EOC (data not shown). To eliminate theinfluence of FIGO stage, we chose 93 patients withFIGO stage III and put them into three groups basedupon their Crk/CrkL staining IOD percentile fromlow to high (with IOD of �P33, P34–P66, and ofP67–P100, respectively). No significant differencewas found in effect of different CrkL expression levelupon survival (data not shown). As compared, thosewith the highest Crk expression seem to presentwith the best survival out of our expectation (indi-cated by Supplementary Figure 2).

Association of Crk/CrkL With Its Combining Partners

In SKOV3 cells, Crk and CrkL present with differ-ent selectivity to their two targeting proteins invivo, that is: CrkL presented with similar associationwith Dock180 (Figure 2A) to that with C3G(Figure 2B). However, as for Crk, its two splicingproducts CrkI and CrkII demonstrated differentselectivity with Dock180, that is: CrkI seems to holdmuch more affinity with Dock180 than CrkII, eventhough no obvious difference was found betweenthem in the combination with C3G. Besides this,consistent expression intensity of CrkI and Dock180was also observed through immunoblotting in freshovarian tissues (Supplementary Figure 3). Given thesimilar finding of the contribution of CrkI but notCrkII to the malignancy of glioblastoma reportedelsewhere [26], CrkI–Dock180 complex might beimplicated in the tumorigenesis in ovarian cancer.

Establishment of Crk Knockdown Cells andMorphological Observation

siRNA was utilized to reduce the endogenousexpression of Crk in SKOV3 cells. By using CrksiRNA in the pSUPER.retro.neo vector, we success-fully established two Crk knockdown SKOV3 cell



lines named as Crki1 and Crki2, respectively. Asindicated in Figure 3A, Crki1 cells presented withunchanged CrkII and reduced CrkI while Crki2 cellspresented with reduced CrkII and completelydeleted CrkI. However, during our observation,reduced CrkII expression was only demonstratedin early-passage Crki2 cells (data not shown).Endogenous protein levels of CrkL were unchangedas were those of actin.

To assess the involvement of Crk in the regulationfor the remolding of actin cytoskeleton, we per-formed actin staining and observed clear structureof lamellipodia and filopodia at the edge of controlcells but not of either Crki1 or Crki2 cells (Figure 3B-b).Furthermore, through transient transfection of theGFP fusion form of actin, we observed rigid actinstructure in Crki cells, with dense actin bundlespresent at the edge of the cytoplasm (Figure 3B-d).

Analysis of Rac Activity and Cell Invasion

As Crk is known to regulate Rac through Dock180,and CrkI other than CrkII present with consistentassociation with Dock180 in SKOV3 cells, weexamined the activity of Rac through pull-downassy. Consistent with the reduced formation oflamellipodia, Rac-GTP was decreased in Crki1 andCrki2 cells (Figure 4A). In accordance with this,great suppression of cellular invasion was also foundin Crki cells (Figure 4B).

Cell Growth Analysis

To verify the upregulative role of Crk in thegrowth of SKOV3 cells, anchorage-dependent and-independent growth were examined. Comparedwith parental cells and control cells, growth rate ofCrk knockdown SKOV3 cells was diminished when

Figure 1. Both Crk and CrkL are frequently activated in epithelialovarian carcinomas. (A) Immunohistochemical staining was per-formed with anti-Crk (upper lanes)/anti-CrkL (lower lanes) antibodyon archival tissue sections. The representative photographs of benign(left panels) and malignant ovarian tumors (right panels). Scale bar100 mm. (B) Histogram indicating immunostaining profile. Positiverate indicates the proportion of positive expression numbers to thetotal numbers examined. The expression of both Crk and CrkL wasmore frequent in EOC than in BOT and normal ovarian tissues. (C)

Histogram indicating the expression intensity of Crk/CrkL accordingto histologic differentiation. The difference of Crk/CrkL among gradegroups was analyzed statistically by ANOVA, F-value was 67.38 forCrk, �P < 0.01, and 9.910 for CrkL, ��P < 0.015, respectively. (D)Histogram indicating the expression intensity of Crk/CrkL in differentFIGO stages. The difference of Crk/CrkL between stage I/II and stageIII/IV was analyzed statistically by independent T-test, T-value was3.789 for Crk, �P < 0.05 and 0.139 for CrkL, P > 0.05, respectively.(E) Survival in patients with stage III disease by Crk expression.

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Figure 2. In vivo association of Crk/CrkL with two known target molecules Dock180 and C3G in SKOV3 cells.(A) Co-immunoprecipitation by using anti-Dock180 mAb (lane 1), followed by immunoblotting using anti-Crk(upper panel), anti-CrkL Ab (middle panel), and anti-Dock180 (lower panel). Mouse control IgG was used asnegative control (lane 2). TCl, total cell lysate (lane 3). (B) Co-immunoprecipitation by using anti-C3GAb(lane 4), followed by immunoblotting using anti-Crk (upper panel), anti-CrkL Ab (middle panel), and anti-C3G(lower panel). Rabbit control IgG was used as negative control (lane 5). TCl, total cell lysate (lane 6).

Figure 3. (A) Establishment of Crk knockdown SKOV3 cell line and presentation of representative CrkL knock-down SKOV3 clones. Total cell lysates of scramble siRNA vector transfected cells (lane 1) and two Crk siRNAtransfected cell Crki1 (lane 2) and Crki2 (lane 3) were immunoblotted with anti-Crk, anti-CrkL, or anti-actin Abs.(B) Morphology of mock control (panels a and c) and Crk knockdown SKOV3 cells (panels b and d) throughphalloidine staining (panels a and b) and GFP-actin transfected cells (panels c and d). Arrow and arrowhead indicatelamellipodia and filopodia, respectively. Scale bar 50 mm.



plated on non-coating culture plates. We nextexamined the anchorage-independent growth ofthese cells by soft agar colony formation assay.The numbers of medium sized colonies were con-siderably decreased in Crk knockdown cell lines,and large colonies were easily detected in controlcells, but not in Crk knockdown cells (Figure 5).

Establishment of CrkL Knockdown Cells and Observationfor Their Viability

In contrast with Crk knockdown cells, stableCrkL-knockdown cells could not be establishedwhatsoever effort we made. During the screeningof CrkL-knockdown cells with neomycin, 18 clonesof CrkLi1, 24 clones of CrkLi2, and 30 clones ofCrkLi3, that is, a total of 72 survived clones werepicked up and confirmed as CrkL-knockdown clonesby immunoblotting (data not shown, the represen-tative image is demonstrated in Figure 6A). How-ever, these screened clones could not furthersurvive over passage even though some clones pre-sented with only partially reduced CrkL (such asCrkLi2). Instead, gradual death and detachmentfrom the plastic plates was found when seriallyobserved, which was exemplified by one cluster ofeach CrkLi clones. The representative image is pre-sented in Figure 6B.

DISCUSSION

No report has been found to distinguish the roleof Crk fromCrkL in the carcinogenesis, even thoughboth have been reported to hold oncogenic and

transformative activity [24–30]. In our study, bothCrk and CrkL demonstrated intensive expression inEOC as compared with those in benign and normalcounterpart (Figure 1 and Supplementary Figure 1),which seems to support the involvement of bothin ovarian carcinogenesis. However, the definiteassociation of Crk with high grade and advanceddiseases indicates its implication in the ovariantumorigenesis. In contrast, the differential associ-ation of CrkL with tumor grade or FIGO stagesuggested that it acts a little differently in ovariancancer. Survival analysis seems to support the roleCrk in the carcinogenesis of EOC, even though theinverse association of Crk expression with survivalseems unexpected. Given the oncogenic role of Crkin various malignancies, tumors with high Crkexpression were assumed to be actively growing,which might possess higher responsiveness to che-motherapy theoretically. However, the linkage ofCrk with drug sensitivity needs further identifying.To approach the individual role of each, in vivo

binding of Crk/CrkL with their known combiningpartners Dock180 and C3G was assessed in one ofclassical ovarian cancer cell lines SKOV3. As com-pared with the indifferent association of CrkL withDock180 and C3G, two splicing components of Crk(CrkI and CrkII) showed different preferentiality intheir linkage with Dock180 and C3G, respectively.Predominant combination of CrkI other than CrkIIwith Dock180 was identified (Figure 2), and thisassociation was supported by the co-expressionof CrkI and Dock180 in ovarian cancer tissues(Supplementary Figure 2). Such different association

Figure 4. (A) Activity of Rac determined by pull-down assay in control and Crk knockdown cells. The GTP boundform of Rac was precipitated by GST-PAK2-RBD and probed with anti-Rac1 Ab. (B) Invasion of SKOV3 cells wereanalyzed by Matrigel transwell assay. The number of moved cells were counted and described as histograph withstandard error.

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with the same target molecules might contribute tothe different functions of Crk from CrkL in ovariancancer cells.To further address this issue, siRNA was utilized to

knock down the endogenous expression of Crk andCrkL in SKOV3 cells, respectively. Similar to theprevious results in MCAS cells [32], Crk-knockdowncells were established without any difficulty. Despitewith various degree of CrkII remained, thesetwo lines of Crk-knockdown cells presented withchanged morphology in actin cytoskeleton,reduction in Rac activity and cell invasion, and cellgrowth (Figures 3, 4, and 5). This indicates thatit maybe CrkI other than CrkII that mediatesp130Cas/paxillin and Dock180 to activate Rac,which might contribute to the malignant behaviorof ovarian cancer cell SKOV3.

As compared, CrkL-knockdown cells could not beestablished because these cells could not surviveover passage even though CrkL-knockdown cloneswere picked up and confirmed successfully. Thisoccurred in another ovarian cancer cells MCAS(unpublished). Therefore, we followed up these con-firmed CrkLi clones serially and found that thosecells with CrkL knocked down will detach fromthe bottom of the plastic dish gradually and dieeventually (Figure 6). Given the previous findingsin CrkL knockout mouse [33], these findings indi-cated that in comparison with the role of Crkinvolved in tumorigenesis, CrkL seems to be associ-ated with some vital events.Taken together, these different features of Crk from

CrkL raised the possibility that they servedifferently in ovarian tumorigenesis: Crk, especially

Figure 5. (A) Growth rates of control and Crk knockdown cells were measured for 6 days on plastic dishes.(B) Colony formation assay. Right panels demonstrated the formed colonies in soft agar. Numbers of colonieswere measured and displayed as bar graph. Black bar, colonies sized 50–100 mm in diameter; white bar,colonies 100–200 mm in diameter; gray bar, more than 200 mm in diameter. Scale bar 20 mm.



CrkI, may be critical for the malignant behavior,especially for the untoward expansion and aggressivecell motility of ovarian cancer cells. As compared,CrkL may be associated with basic cell viability, andfatal results may occur if knocked down. That is, ourfindings point to Crk (especially CrkI) rather thanCrkL as an EOC biomarker correlating with carcino-genesis. Their different functions in ovarian cancercells might result from different preferential combin-ing with known binding partners, or from someunique combining partners which should be ident-ified further in the future.

ACKNOWLEDGMENTS

We thank Prof. Shinya Tanaka in Hokkaido Uni-versity, Japan for his technical support and gener-ously providing pEGFP-actin plasmid. This workwas supported by the grant from the NationalNatural Science Foundation of China (NSFC nos.C30672432 and C30772330), and was supportedpartly by a grant for returned students abroad fromtheMinistry of Education of the People’s Republic ofChina (2007), Natural Science Foundation Project ofCQ CSTC, 2007BB5319, and the grant for Medical

Figure 6. (A) Establishment of CrkL knockdown SKOV3 cell clonesand presentation of representative CrkL knockdown SKOV3 clones.Total cell lysates of scramble siRNA vector transfected cells (lane 1)and three CrkL siRNA transfected cell Crki1 (lane 2), Crki2 (lane 3),and CrkLi3 (lane 4) were immunoblotted with anti-CrkL, anti-Crk, oranti-actin Abs. (B) Change in the viability of representative CrkL-knockdown SKOV3 clone. During the screening of CrkL-knockdown

cells with puromycin, a total of 72 survived clones were picked upand confirmed as CrkL-knockdown clones by immunoblotting. How-ever, these screened clones could not further survive over passage.As a result, three clones were marked and the change in theirmorphology and number were observed continually with phase-con-trast microscope. Scale bar 50 mm.

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Sciences from the First Hospital, ChongqingMedical University (YXJJ2009-06).

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Crk and CrkL present with different expression and significance in epithelial ovarian carcinoma