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Biological Significance of Focal Adhesion Kinase in Ovarian Cancer

2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês

10.1016/s0002-9440(10)63370-6

1525-2191

Anil K. Sood, Jeremy E. Coffin, Galen B. Schneider, Mavis S. Fletcher, Barry R. DeYoung, Lynn M. Gruman, David M. Gershenson, Michael D. Schaller, Mary J.C. Hendrix,

Monoclonal and Polyclonal Antibodies Research

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is activated by integrin clustering. There are limited data regarding the functional role of FAK in ovarian cancer migration and invasion. In the current study, FAK expression was evaluated in ovarian cell lines (nontransformed and cancer), 12 benign ovarian samples, and in 79 invasive epithelial ovarian cancers. All three ovarian cancer cell lines overexpressed FAK compared to the nontransformed cells. The dominant-negative construct called FAK-related nonkinase (FRNK) was introduced into two ovarian cancer cell lines (SKOV3 and 222). FRNK promoted FAK dephosphorylation without changing total FAK levels in these cell lines. Furthermore, FRNK decreased the in vitro invasive ability of ovarian cancer cells by 56 to 85% and decreased migration by 52 to 68%. FRNK-transfected cells also displayed poor cell spreading. Immunohistochemical analysis revealed that the surface epithelium from all benign ovarian samples had weak FAK expression. In contrast, 68% of invasive ovarian cancers overexpressed FAK. FAK overexpression was significantly associated with high tumor stage, high tumor grade, positive lymph nodes and presence of distant metastasis (all P values 1 cm were independent predictors of poor survival. These data indicate that FAK is overexpressed in most invasive ovarian cancers and plays a functionally significant role in ovarian cancer migration and invasion. Thus, FAK may be an important therapeutic target in ovarian carcinoma. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is activated by integrin clustering. There are limited data regarding the functional role of FAK in ovarian cancer migration and invasion. In the current study, FAK expression was evaluated in ovarian cell lines (nontransformed and cancer), 12 benign ovarian samples, and in 79 invasive epithelial ovarian cancers. All three ovarian cancer cell lines overexpressed FAK compared to the nontransformed cells. The dominant-negative construct called FAK-related nonkinase (FRNK) was introduced into two ovarian cancer cell lines (SKOV3 and 222). FRNK promoted FAK dephosphorylation without changing total FAK levels in these cell lines. Furthermore, FRNK decreased the in vitro invasive ability of ovarian cancer cells by 56 to 85% and decreased migration by 52 to 68%. FRNK-transfected cells also displayed poor cell spreading. Immunohistochemical analysis revealed that the surface epithelium from all benign ovarian samples had weak FAK expression. In contrast, 68% of invasive ovarian cancers overexpressed FAK. FAK overexpression was significantly associated with high tumor stage, high tumor grade, positive lymph nodes and presence of distant metastasis (all P values 1 cm were independent predictors of poor survival. These data indicate that FAK is overexpressed in most invasive ovarian cancers and plays a functionally significant role in ovarian cancer migration and invasion. Thus, FAK may be an important therapeutic target in ovarian carcinoma. Ovarian cancer remains the most common cause of death from a gynecological malignancy.1Jemal A Murray T Samuels A Ghafoor A Ward E Thun MJ Cancer Statistics, 2003.CA Cancer J Clin. 2003; 53: 5-26Crossref PubMed Scopus (3376) Google Scholar The high mortality related to ovarian cancer is thought to be because of the advanced stage of disease at presentation. Tumor progression toward increasing metastatic potential is a complex, multistep process and requires the coordinated expression of metastasis-promoting genes and the down-regulation of metastasis-suppressing genes. Metastatic colonization requires disseminated cells to initiate context-dependent signaling cascades that allow them to survive, enter the cell cycle, and proliferate to become metastases. Cell migration is an important component of the metastatic process and requires repeated adhesion to and detachment from the extracellular matrix microenvironment. These events are mediated, in large part, by integrins, which on engagement with components of the extracellular matrix, reorganize to form adhesion complexes termed focal adhesions.2Jockusch BM Bubeck P Giehl K Kroemker M Moschner J Rothkegel M Rudiger M Schluer K Stanke G Winkler J The molecular architecture of focal adhesions.Annu Rev Cell Dev Biol. 1995; 11: 379-416Crossref PubMed Scopus (431) Google Scholar, 3Zamir E Geiger B Molecular complexity and dynamics of cell-matrix adhesions.J Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar, 4Miranti CK Brugge JS Sensing the environment: a historical perspective on integrin signal transduction.Nat Cell Biol. 2002; 4: E83-E90Crossref PubMed Scopus (695) Google Scholar These focal adhesions orchestrate a signal transduction cascade initiated by integrin-mediated recognition of extracellular matrix components. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that localizes to contact sites in focal adhesions.5Schaller MD Borgman CA Cobb BS Vines RR Reynolds AB Parsons JT pp125FAK, a structurally unique protein tyrosine kinase associated with focal adhesions.Proc Natl Acad Sci USA. 1992; 89: 5192-5196Crossref PubMed Scopus (1295) Google Scholar, 6Schaller MD Biochemical signals and biological responses elicited by the focal adhesion kinase.Biochim Biophys Acta. 2001; 1540: 1-21Crossref PubMed Scopus (505) Google Scholar This intracellular signaling protein is associated with the cytoplasmic domain of integrin receptors and on integrin clustering is activated and autophosphorylated on tyrosine. Many stimuli can induce tyrosine phosphorylation and activation of the catalytic activity of FAK including growth factors, neuropeptides, and mechanical stimuli.6Schaller MD Biochemical signals and biological responses elicited by the focal adhesion kinase.Biochim Biophys Acta. 2001; 1540: 1-21Crossref PubMed Scopus (505) Google Scholar However, the major mode of regulation is via integrin-dependent adhesion to the extracellular matrix in in vitro studies and FAK is an integral component of the integrin-signaling pathway. The focal adhesion complex regulates cell growth, differentiation, and fate through the promotion of tyrosine phosphorylation and subsequent regulation of downstream cell survival components such as PI3-kinase, and signaling pathways such as those associated with Grb2 and Ras. Elevated tyrosine phosphatase activity or expression of the FAK C-terminal, noncatalytic domain, termed FRNK (FAK-related nonkinase), as a dominant-negative inhibitor promotes FAK dephosphorylation and inhibits FAK function.7Schaller MD Borgman CA Parsons JT Autonomous expression of a non-catalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK.Mol Cell Biol. 1993; 13: 785-791Crossref PubMed Scopus (279) Google Scholar However, most of these findings were obtained with normal fibroblasts, and thus, it is unclear whether FAK functions in a similar manner in human tumor cells, especially ovarian carcinoma. Overexpression of FAK protein has been reported in metastatic human colorectal, breast, thyroid, and prostate cancer cells.8Cance WG Harris JE Iacocca MV Roche E Yang X Chang J Simkins S Xu L Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes.Clin Cancer Res. 2000; 6: 2417-2423PubMed Google Scholar, 9Owens LV Xu L Craven RJ Dent GA Weiner TM Kornberg L Liu ET Cance WG Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors.Cancer Res. 1995; 55: 2752-2755PubMed Google Scholar, 10Owens LV Xu L Dent GA Yang X Sturge GC Craven RJ Cance WG Focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer.Ann Surg Oncol. 1996; 3: 100-105Crossref PubMed Scopus (199) Google Scholar, 11Tremblay L Hauck W Aprikian AG Begin LR Chapdelaine A Chevalier S Focal adhesion kinase (pp125FAK) expression, activation and association with paxillin and p50CSK in human metastatic prostate carcinoma.Int J Cancer. 1996; 68: 164-171Crossref PubMed Scopus (215) Google Scholar, 12Ayaki M Komatsu K Mukai M Murata K Kameyama M Ishiguro S Miyoshi J Tatsuta M Nakamura H Reduced expression of focal adhesion kinase in liver metastases compared with matched primary human colorectal adenocarcinomas.Clin Cancer Res. 2001; 7: 3106-3112PubMed Google Scholar There are limited data regarding the role of FAK in ovarian cancer, but FAK was reported to be overexpressed in most human ovarian cancers.13Judson PL He X Cance WG Van Le L Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma.Cancer. 1999; 86: 1551-1556Crossref PubMed Scopus (173) Google Scholar However, these studies have relied primarily on blotting methods to demonstrate FAK overexpression and it is difficult to distinguish the contributions of tumor versus stromal FAK with such an approach. Also, of special interest is the observation that amplification of 8q, where FAK is located, is one of the most frequent alterations in primary ovarian cancers and is associated with poorly differentiated tumors.14Kiechle M Jacobsen A Schwarz-Boeger U Hedderich J Pfisterer J Arnold N Comparative genomic hybridization detects genetic imbalances in primary ovarian carcinomas as correlated with grade of differentiation.Cancer. 2001; 91: 534-540Crossref PubMed Scopus (95) Google Scholar, 15Hauptmann S Denkert C Koch I Petersen S Schluns K Reles A Dietel M Petersen I Genetic alterations in epithelial hybridization.Hum Pathol. 2002; 33: 632-641Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Thus, we undertook the present study with the following aims: to evaluate the clinical relevance of FAK expression in ovarian cancer and to evaluate the functional role of FAK in ovarian cancer migration and invasion. The ovarian cancer cell lines used in this study were SKOV3, EG, and 222. The derivation and sources of these cell lines have been reported previously.16Sood AK Seftor EA Fletcher M Gardner LMG Heidger PM Buller RE Seftor REB Hendrix MJC Molecular determinants of ovarian cancer plasticity.Am J Pathol. 2001; 158: 1279-1288Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar These cells were maintained and propagated in vitro by serial passage in RPMI 1640 supplemented with 15% fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA). The immortalized nontransformed human ovarian surface epithelial cell lines (HI0-180 and H10-1120) were a kind gift from Dr. Andrew Godwin at the Fox Chase Cancer Center, Philadelphia, PA. The HI0-180 cells were maintained in Medium 199/MCDB 105 supplemented with 15% fetal bovine serum and 0.1% gentamicin sulfate. All cell lines are routinely screened for mycoplasma species (GenProbe detection kit; Fisher, Itasca, IL). All experiments were performed with 70 to 80% confluent cultures. The membrane invasion culture system chamber was used to measure the in vitro invasiveness of all cell lines used in this study.16Sood AK Seftor EA Fletcher M Gardner LMG Heidger PM Buller RE Seftor REB Hendrix MJC Molecular determinants of ovarian cancer plasticity.Am J Pathol. 2001; 158: 1279-1288Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 17Hendrix MG Seftor EA Seftor RE Fidler IJ A simple quantitative assay for studying the invasive potential of high and low human metastatic variants.Cancer Lett. 1987; 38: 137-147Abstract Full Text PDF PubMed Scopus (237) Google Scholar Briefly, a polycarbonate membrane with 10-μm pores (Osmonics, Livermore, CA) was uniformly coated with a defined basement membrane matrix consisting of human laminin/type IV collagen/gelatin and used as the intervening barrier to invasion. Both upper and lower wells of the chamber were filled with serum-free RPMI containing 1× MITO+ serum supplement (Collaborative Biomedical, Bedford, MA). Single cell tumor suspensions were seeded into the upper wells at a concentration of 1 × 105 cells per well. After a 24-hour incubation in a humidified incubator at 37°C with 5% CO2, cells that had invaded through the basement membrane were collected, stained, and counted by light microscopy.16Sood AK Seftor EA Fletcher M Gardner LMG Heidger PM Buller RE Seftor REB Hendrix MJC Molecular determinants of ovarian cancer plasticity.Am J Pathol. 2001; 158: 1279-1288Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar For chemoinvasion assays, conditioned media from normal skin fibroblasts (kindly provided by Dr. Gregory Goldberg, Washington University, St. Louis, MO) was added to the lower wells. Invasiveness was calculated as the percentage of cells that had successfully invaded through the matrix-coated membrane to the lower wells compared to the total number of cells seeded into the upper wells and corrected for cell proliferation. Unstimulated motility was determined in membrane invasion culture system chambers containing polycarbonate filter (with 10 μm pores) that had been soaked in 0.1% gelatin. Tumor cells (5 × 104) were seeded in each upper well, allowed to incubate at 37°C for 5.5 hours in Dulbecco's modified Eagle's medium containing 10% nuserum, and subsequently processed as described for the invasion assay. The cells were plated on coverslips coated with fibronectin (BD Biosciences, San Jose, CA) and fixed in 3.7% paraformaldehyde for 10 minutes followed by a phosphate-buffered saline wash, and then treatment with Triton 0.5% for 6 minutes. Staining was performed with mouse anti-human FAK (dilution 1:50; BD Transduction, San Diego, CA), and Phalloidin (dilution 1:600; Molecular Probes, Eugene, OR). All samples were collected in compliance with requirements of the Institutional Review Board for the Protection of Human Subjects. Formalin-fixed, paraffin-embedded samples were sectioned at a thickness of 4 μm and stained with hematoxylin and eosin (H&E) for identification. Sections adjacent to the H&E-stained sections were used for immunohistochemical staining. All slides were deparaffinized using xylene, 100% ethanol, 95% ethanol, followed by a thorough deionized water wash. A water bath antigen recovery technique, using citrate buffer, pH 6.0, was performed on all slides. The immunohistochemical staining for FAK was performed on the DAKO Autostainer (DAKO, Carpinteria, CA) using the Vectastain Universal Elite ABC peroxidase kit (Vector Laboratories, Inc., Burlingame, CA) to detect mouse anti-human FAK, clone 4.47 (dilution 1:800; Upstate Biotechnology, Waltham, MA). After deparaffinization and antigen recovery, slides were washed in Tris-buffered saline with Tween (TBST). Three blocking steps were applied: 0.03% hydrogen peroxide (DAKO) for 20 minutes followed by a TBST wash, avidin and biotin blocks were applied for 15 minutes in each solution followed by a TBST wash after each step, finally the Protein Block Serum-Free (DAKO) was applied for 15 minutes. The primary antibody was applied to the slides and incubated for 40 minutes; slides were rinsed in TBST, followed by application of the RTU Vectastain Secondary for 12 minutes. This procedure was followed by a TBST wash and then incubation with the RTU ABC reagent for 10 minutes. Color for the RTU procedure was produced by using DAB+ (brown) substrate (DAKO) for 2 to 3 minutes. Slides were counterstained with Mayer's hematoxylin for 3 minutes. Control reactions consisted of either a preimmune IgG serum (MOPC-21; Sigma Chemical Co., St. Louis, MO), which was isotype-matched for the FAK antibody at the same concentration, or incubation of sections in the absence of primary antibody. All samples were reviewed by two board-certified pathologists (M.S.F., B,D,), who were blinded to the clinical outcome of these patients. FAK expression was determined by assessing semiquantitatively the percentage of stained tumor cells and the staining intensity, as described previously.18Sood AK Fletcher MS Gruman LM Coffin JE Jabbari S Khalkhali-Ellis Z Arbour N Seftor EA Hendrix MJC The paradoxical expression of maspin in ovarian carcinoma.Clin Cancer Res. 2002; 8: 2924-2932PubMed Google Scholar The percentage of positive cells was rated as follows: 0 points, 0 to 5%; 2 points, 6 to 50%; 3 points, >50%. The staining intensity was rated as follows: 1 point, weak intensity; 2 points, moderate intensity; 3 points, strong intensity. Points for expression and percentage of positive cells were added and an overall score (OS) was assigned. Tumors were categorized into four groups: negative (OS = 0), ≤5% cells stained, regardless of intensity; weak expression (OS = 1), 1 to 2 points; moderate expression (OS = 2), 3 to 4 points; and strong expression (OS = 3), 5 to 6 points. Cells were plated at 5 × 105 cells per well of six-well dishes. The cell lines 222 and SKOV3 were transfected with 2 μg of FRNK cDNA in pcDNA3.1 vector (Invitrogen Corp., Carlsbad, CA) using Lipofectamine 2000 reagent (Invitrogen), following the manufacturer's protocol. Forty-eight hours after the transfection, G418 containing media was added and changed every 48 to 72 hours thereafter to generate stable transfectants. Sham constructs were created by transfecting pcDNA3.1 alone into both cell lines. β-Galactosidase expression was used to determine the transfection efficiency for each cell line, which was ∼35% for the SKOV3 cells and ∼50% for the 222 cells. Cells were collected by ethylenediaminetetraacetic acid treatment and resuspended in RPMI without serum. Cells (5 × 104) were plated in triplicate in 12-well plastic dishes (coated with a defined matrix as described above) containing RPMI without serum. The cells were allowed to spread for various time intervals at 37°C. For each experiment, three random fields were counted. Unspread cells were defined as round phase-bright cells; spread cells were defined as those that had extended processes, that lacked a round morphology, and that were not phase bright. Cells were lysed with 1× modified RIPA buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, and 0.5% deoxycholate) containing 25 μg/ml leupeptin (Sigma Chemical Co.), 10 μg/ml aprotinin (Sigma Chemical Co.), 1 mmol/L sodium orthovanadate, and 2 mmol/L ethylenediaminetetraacetic acid. Cells were removed from the dishes by cell scraping and the samples were then stored at −80°C. The protein concentration of the samples was determined using a BCA protein assay reagent kit, and whole cell lysates were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie BBR-250 (Sigma Chemical Co.) to ensure equal loading. Samples were transferred to nitrocellulose (Schleicher and Schuell, Keene, NH) membranes and blots were blocked with 5% nonfat milk for 1 hour at room temperature. Blots were incubated with the monoclonal FAK antibody (1:500 dilution; Transduction Laboratories, Lexington, KY) or FRNK antibody for 1 hour at room temperature with agitation, followed by incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:5000; The Jackson Laboratory, Bar Harbor, ME). Blots were developed using an enhanced chemiluminescence detection kit (ECL; Amersham Pharmacia Biotech, Piscataway, NJ). For immunoprecipitation experiments, 300 μg of cell lysate were incubated with the FAK antibody for 1 hour at 4°C. Protein-antibody complexes were incubated for 1 hour at 4°C with protein A-Sepharose-conjugated beads (preincubated with rabbit anti-mouse IgG), collected by centrifugation, washed three times with the modified RIPA buffer, and boiled in Laemmli sample buffer. The proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and immunoblotting was performed as described above. All patients underwent surgical exploration and cytoreduction as the initial treatment. The treating gynecological oncologist determined adjuvant therapy (two patients with stage IA ovarian cancer did not receive adjuvant chemotherapy; all other patients with invasive ovarian cancer were treated with adjuvant paclitaxel and platinum chemotherapy). Diagnosis was verified by pathology review at the institutional gynecological oncology tumor board. All patients were staged according to the International Federation of Gynecology and Obstetrics surgical staging system. A gynecological pathologist reviewed the pathology for all patients. Chi-square or Fisher's exact test were used as appropriate to determine differences between variables using SPSS (SPSS Inc., Chicago, IL). Kaplan-Meier survival plots were generated and comparisons between survival curves were made with the log-rank statistic. The Cox proportional hazards model was used for multivariate analysis. A P value <0.05 was considered statistically significant. FAK expression in ovarian cell lines was assessed by Western blot, immunohistochemistry, and immunofluorescence (Figure 1B, a). The nontransformed cell lines HI0-180 and H10-1120 are known to be poorly invasive16Sood AK Seftor EA Fletcher M Gardner LMG Heidger PM Buller RE Seftor REB Hendrix MJC Molecular determinants of ovarian cancer plasticity.Am J Pathol. 2001; 158: 1279-1288Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar and demonstrated low FAK expression. All of the cancer cell lines had moderate to high levels of FAK by Western blot analysis (Figure 1A). Immunohistochemical peroxidase staining confirmed the low expression of FAK by the HI0-180 cells (Figure 1B, a). In contrast, all three ovarian cancer cell lines had high FAK expression (Figure 1B, b to d). Immunofluorescence labeling for both FAK and actin revealed that the HI0-180 cell line develops well-formed focal adhesions and well-developed actin stress fibers (Figure 1C, A). Both OVCAR3 and SKOV3 cell lines also demonstrate focal adhesion formation (Figure 1C, B and C). However, the highly invasive cell line 222 demonstrated decreased focal adhesion formations and had poorly organized actin staining (Figure 1C, D). Ovarian cancer cell migration, attachment, and invasion are key steps in the metastatic process. To test the hypothesis that ectopic expression of the FAK C-terminal, noncatalytic domain, FRNK, would interfere with ovarian cancer invasion, migration, and cell spreading, FRNK cDNA was introduced into the highly invasive 222 and SKOV3 cancer cells (Figure 2). After FRNK transfection of the 222 cell line, three stably transfected clones (222-FC1, 222-FC2, and 222-FC3) were obtained and tested for their in vitro invasive potential. Similarly, after SKOV3 transfection, two stably transfected clones (SKOV3-FC2 and SKOV3-FC4) were obtained and tested. We determined the effect of FRNK transfection on FAK levels and phosphorylation by immunoprecipitation and immunoblotting. The overall levels of FAK were not affected by FRNK transfection (Figure 2) in both cell lines, but FAK phosphorylation was markedly decreased in the FRNK-transfected cells (Figure 2). Immunofluorescence microscopy revealed that SKOV3 was still able to form focal adhesions, and FRNK-transfected 222 cells acquired the ability to form focal adhesions (Figure 1C, E and F). The invasive potential of ovarian cancer cells and transfected cells was determined using the membrane invasion culture system assay and a defined basement membrane-coated barrier filter. The baseline invasion rates of these ovarian cancer cell lines have been reported previously.16Sood AK Seftor EA Fletcher M Gardner LMG Heidger PM Buller RE Seftor REB Hendrix MJC Molecular determinants of ovarian cancer plasticity.Am J Pathol. 2001; 158: 1279-1288Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar The SKOV3 cell line is moderately invasive (7.5%), and the cell line 222 is highly invasive (11.7%). The dominant-negative FRNK decreased the invasive potential of the 222 cell line by 73 to 85% (all P values <0.05) in the three clones (Figure 3). The average invasive ability of the SKOV3 transfected cells decreased by 56 to 76% (P < 0.05) when compared with the parental SKOV3 cells. We also assessed in vitro migration using the membrane invasion culture system assay and a gelatin-soaked filter. The migratory ability of the FRNK-transfected 222 cells decreased by 52 to 61% (P < 0.05) and SKOV3 cells by 65 to 68% (P < 0.05) (Figure 3). Next, we evaluated the ability of the parental and FRNK-transfected ovarian cancer cells to spread on fibronectin-coated dishes. At 20 minutes, 53% of the SKOV3 cells and 17% of the 222 cells had spread, and by 60 minutes, 78% and 75% of the cells, respectively, had spread (Figure 3). The cell spreading was markedly impaired by FRNK transfection in the ovarian cancer cells. At 20 minutes, only 1.5 to 3% (P < 0.05) of the 222 FRNK-transfected cells had spread and 20% (P < 0.05) of the SKOV3 FRNK-transfected cells had spread. Even at 60 minutes, only 11 to 21% (P < 0.05) of the 222-transfected cells and 37 to 41% (P < 0.05) of the SKOV3-transfected cells had spread. The demographic features of the patients in this study are listed in Table 1. The mean age of patients was 59.3 years. Eighty-one percent of the patients had advanced stage (III or IV) disease and 57% had high-grade (III) disease. Sixty-eight percent of the patients underwent optimal surgical cytoreduction (<1 cm of residual disease at the end of surgery).Table 1Demographic Features of Invasive Ovarian Cancer PatientsVariableNumberAge59.3 years (34 to 81 years)Stage I10 II5 III50 IV14Menopausal Yes60 No19Histology Serous52 Other27Grade Low (I or II)34 High (III)45Ascites Yes59 No20 Cytoreduction Optimal54Suboptimal25Node status Positive10 Negative22 Not done47Status Alive without disease21 Alive with disease11 Dead of disease43 Dead of other causes4 Open table in a new tab In the 12 benign ovaries all samples demonstrated weak FAK expression in the ovarian surface epithelium (Figure 4B). FAK expression was assessed using immunohistochemistry in 79 invasive ovarian cancers and representative staining results are shown in Figure 4C. In contrast to the normal ovarian surface epithelium, FAK was markedly up-regulated in the invasive ovarian cancers. FAK was detected at varying levels in all of the invasive ovarian carcinoma specimens and was overexpressed in 54 (68%) of the tumors. The correlation of FAK overexpression and various clinical variables are listed in Table 2. There was no association between FAK overexpression and histological subtypes (serous versus other), presence of ascites, and ability to achieve optimal cytoreduction. Eighty-one percent of high-stage tumors overexpressed FAK compared to only 20% of low-stage tumors (P < 0.001). FAK overexpression was also associated with high grade (P = 0.01), higher likelihood of nodal positivity (P < 0.001), and presence of distant metastasis (P = 0.01).Table 2Correlation of Clinicopathological Variables with FAK Overexpression in Invasive Ovarian Cancer PatientsFAK overexpressionVariableYes (n = 54)No (n = 25)PStage Low312<0.001 High5113Histology Serous37150.46 Other1710Grade Low (I or II)470.01 High (III)5018Ascites No1190.14 Yes4316Cytoreduction Optimal37170.961 Suboptimal178Node status Positive100 1 cm (P < 0.05, data not shown). FAK overexpression was associated with significantly worse survival (median, 7.6 years versus 2.98 years; P = 0.008; Figure 5). In multivariate analysis using the Cox proportional hazards model that involved age, tumor stage and grade, volume of residual disease, and FAK overexpression, only volume of residual disease (P < 0.02) and FAK overexpression (P < 0.03) were significant predictors of poor survival. In this study, we addressed the functional significance of FAK in ovarian cancer invasion and migration. We also evaluated the expression and clinical relevance of FAK in human ovarian cancers. Our data provide definitive evidence, at the cellular level, that the expression of FAK is up-regulated in invasive ovarian cancer cells and is associated with aggressive tumor features and poor outcome in patients. Furthermore, inhibiting FAK phosphorylation by transfecting FRNK into highly aggressive ovarian cancer cells results in decreased invasion, migration, and cell spreading, which are key components of the metastatic process. The ability of tumor cells to migrate from the site of the primary tumor and to invade surrounding tissues is a prerequisite for metastasis. FAK is a nonreceptor protein tyrosine kinase that is a critical mediator of signaling events between cells and their extracellular matrix, thereby facilitating invasion and migration.19Schaller MD Parsons JT Focal adhesion kinase: an integrin-linked protein tyrosine kinase.Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 20Schaller MD Hildebrand JD Parson JT Complex formation with focal adhesion kinase: a mechanism to regulate activity and subcellular location of Src kinases.Mol Biol Cell. 1999; 10: 3489-3505Crossref PubMed Scopus (183) Google Scholar, 21Schaller MD The focal adhesion kinase.J Endocrinol. 1996; 150: 1-7Crossref PubMed Scopus (74) Google Scholar, 22Hsia DA Mitra SK Hauck CR Streblow DN Nelson JA Ilic D Huang S Li E Nemerow GR Leng J Spencer KS Che