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Biological Significance of Focal Adhesion Kinase in Ovarian Cancer

Role in Migration and Invasion
      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 <0.05). FAK overexpression was also associated with shorter overall survival (P = 0.008). Multivariate analysis revealed that FAK overexpression and residual disease >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.
      • Jemal A
      • Murray T
      • Samuels A
      • Ghafoor A
      • Ward E
      • Thun MJ
      Cancer Statistics, 2003.
      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.
      • Jockusch 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.
      • Zamir E
      • Geiger B
      Molecular complexity and dynamics of cell-matrix adhesions.
      • Miranti CK
      • Brugge JS
      Sensing the environment: a historical perspective on integrin signal transduction.
      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.
      • Schaller MD
      • Borgman CA
      • Cobb BS
      • Vines RR
      • Reynolds AB
      • Parsons JT
      pp125FAK, a structurally unique protein tyrosine kinase associated with focal adhesions.
      • Schaller MD
      Biochemical signals and biological responses elicited by the focal adhesion kinase.
      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.
      • Schaller MD
      Biochemical signals and biological responses elicited by the focal adhesion kinase.
      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.
      • Schaller MD
      • Borgman CA
      • Parsons JT
      Autonomous expression of a non-catalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK.
      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.
      • Cance 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.
      • Owens 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.
      • Owens 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.
      • Tremblay 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.
      • Ayaki 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.
      There are limited data regarding the role of FAK in ovarian cancer, but FAK was reported to be overexpressed in most human ovarian cancers.
      • Judson PL
      • He X
      • Cance WG
      • Van Le L
      Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma.
      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.
      • Kiechle 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.
      • Hauptmann S
      • Denkert C
      • Koch I
      • Petersen S
      • Schluns K
      • Reles A
      • Dietel M
      • Petersen I
      Genetic alterations in epithelial hybridization.
      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.

      Materials and Methods

      Cell Culture

      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.
      • Sood AK
      • Seftor EA
      • Fletcher M
      • Gardner LMG
      • Heidger PM
      • Buller RE
      • Seftor REB
      • Hendrix MJC
      Molecular determinants of ovarian cancer plasticity.
      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.

      Invasion Assay

      The membrane invasion culture system chamber was used to measure the in vitro invasiveness of all cell lines used in this study.
      • Sood AK
      • Seftor EA
      • Fletcher M
      • Gardner LMG
      • Heidger PM
      • Buller RE
      • Seftor REB
      • Hendrix MJC
      Molecular determinants of ovarian cancer plasticity.
      • Hendrix MG
      • Seftor EA
      • Seftor RE
      • Fidler IJ
      A simple quantitative assay for studying the invasive potential of high and low human metastatic variants.
      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.
      • Sood AK
      • Seftor EA
      • Fletcher M
      • Gardner LMG
      • Heidger PM
      • Buller RE
      • Seftor REB
      • Hendrix MJC
      Molecular determinants of ovarian cancer plasticity.
      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.

      Cell Migration Assay

      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.

      Immunofluorescence Staining

      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).

      Formalin-Fixed, Paraffin-Embedded Samples for FAK Immunohistochemical Staining

      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.
      • Sood 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.
      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.

      FRNK Transfection

      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.

      Measurement of Cell Spreading

      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.

      Immunoprecipitation and Western Blot Analysis

      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.

      Clinicopathological Variable Analysis

      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.

      Statistical Analysis

      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.

      Results

      FAK Expression in Ovarian Cell Lines Is Associated with Invasive Potential

      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 invasive
      • Sood AK
      • Seftor EA
      • Fletcher M
      • Gardner LMG
      • Heidger PM
      • Buller RE
      • Seftor REB
      • Hendrix MJC
      Molecular determinants of ovarian cancer plasticity.
      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).
      Figure thumbnail gr1
      Figure 1FAK analysis in nontransformed ovarian surface epithelial cells (HI0-180 and HI0-1120) and ovarian cancer cell lines (OVCAR3, SKOV3, and 222). FAK expression was analyzed by: A: Western blot of whole cell lysates; B: immunohistochemical peroxidase staining for FAK in HI0-180 (a), SKOV3 (b), OVCAR3 (c), and 222 (d); and C: immunofluorescence staining (dual) for FAK (red) and Phalloidin (green) in HI0-180 (A), OVCAR3 (B), SKOV3 (C), 222 (D), and FRNK transfectants SKOV3-FC2 (E) and 222-FC2 (F) (arrowheads indicate focal adhesions). Original magnifications: ×400 (B); ×630 (C).

      FRNK Interferes with FAK Phosphorylation and Decreases Ovarian Cancer Invasion, Migration, and Cell Spreading

      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).
      Figure thumbnail gr2
      Figure 2Western blot analysis of ovarian cancer cells (222 and SKOV3) and FRNK transfectants (222-FC1, 222-FC2, 222-FC3, SKOV3-FC2, and SKOV3-FC4). Tyrosine phosphorylation and expression of FAK was analyzed by immunoprecipitating (IP) FAK with the anti-FAK monoclonal antibody (4.47; Upstate Biotechnology, Inc.) and then immunoblotting (IB) with either anti-FAK antibody or anti-phosphotyrosine antibody. Western blot to detect FRNK was performed using a polyclonal antibody (BC4).
      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.
      • Sood AK
      • Seftor EA
      • Fletcher M
      • Gardner LMG
      • Heidger PM
      • Buller RE
      • Seftor REB
      • Hendrix MJC
      Molecular determinants of ovarian cancer plasticity.
      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.
      Figure thumbnail gr3
      Figure 3A and B: Invasion profile of ovarian cancer cell lines (222 and SKOV3) compared to FRNK transfectants (222-FC1, 222-FC2, 222-FC3, SKOV3-FC2, and SKOV3-FC4) and sham transfectants (222-neo and SKOV3-neo), based on their ability to invade a basement membrane matrix in vitro in the presence (hatched bars) or absence (solid bars) of a chemoattractant. C and D: Migration ability of ovarian cancer cells, FRNK transfected cells, and sham transfectants. E and F: Cell spreading of ovarian cancer cells and FRNK transfectants on fibronectin matrix at 20 and 60 minutes. Error bars represent SE.
      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.

      FAK Expression in Human Ovarian Cancer Samples Correlates with Clinical Features

      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 Patients
      VariableNumber
      Age59.3 years (34 to 81 years)
      Stage
          I10
          II5
          III50
          IV14
      Menopausal
          Yes60
          No19
      Histology
          Serous52
          Other27
      Grade
          Low (I or II)34
          High (III)45
      Ascites
          Yes59
          No20
          Cytoreduction
          Optimal54
      Suboptimal25
      Node status
          Positive10
          Negative22
          Not done47
      Status
          Alive without disease21
          Alive with disease11
          Dead of disease43
          Dead of other causes4
      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).
      Figure thumbnail gr4
      Figure 4Representative immunohistochemical peroxidase staining for FAK in ovarian cancer-negative control (A), normal ovarian epithelium (B), and high-grade ovarian cancer (C) obtained from a patient. Original magnifications, ×630.
      Table 2Correlation of Clinicopathological Variables with FAK Overexpression in Invasive Ovarian Cancer Patients
      FAK overexpression
      VariableYes (n = 54)No (n = 25)P
      Stage
          Low312<0.001
          High5113
      Histology
          Serous37150.46
          Other1710
      Grade
          Low (I or II)470.01
          High (III)5018
      Ascites
          No1190.14
          Yes4316
      Cytoreduction
          Optimal37170.961
          Suboptimal178
      Node status
          Positive100<0.001
          Negative913
          Not done3512
      Distant metastasis
          Yes1100.01
          No4325
      In univariate analysis, survival was adversely affected by high-stage, high-grade, and residual disease >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.
      Figure thumbnail gr5
      Figure 5Kaplan-Meier survival of ovarian cancer patients based on FAK staining intensity using the log-rank statistic.

      Discussion

      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.
      • Schaller MD
      • Parsons JT
      Focal adhesion kinase: an integrin-linked protein tyrosine kinase.
      • Schaller MD
      • Hildebrand JD
      • Parson JT
      Complex formation with focal adhesion kinase: a mechanism to regulate activity and subcellular location of Src kinases.
      • Schaller MD
      The focal adhesion kinase.
      • Hsia DA
      • Mitra SK
      • Hauck CR
      • Streblow DN
      • Nelson JA
      • Ilic D
      • Huang S
      • Li E
      • Nemerow GR
      • Leng J
      • Spencer KS
      • Cheresh DA
      • Schlaepfer DD
      Differential regulation of cell motility and invasion by FAK.
      • Kohno M
      • Hasegawa H
      • Miyake M
      • Yamamoto T
      • Fujita S
      CD151 enhances cell motility and metastasis of cancer cells in the presence of focal adhesion kinase.
      • Sieg DJ
      • Hauck CR
      • Ilic D
      • Klingbeil CK
      • Schaefer E
      • Damsky CH
      • Schlaepfer DD
      FAK integrates growth-factor and integrin signals to promote cell migration.
      FAK has been linked to integrin-signaling pathways via interactions with integrin-associated proteins such as paxillin and talin,
      • Kornberg L
      • Earp HS
      • Parsons JT
      • Schaller M
      • Juliano RL
      Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase.
      • Klingbeil CK
      • Houck CR
      • Hsia DA
      • Jones KC
      • Reider SR
      • Schlaepfer DD
      Targeting PYK2 to β1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cell.
      • Schlaepfer DD
      • Hanks SK
      • Hunter T
      • van der Greer P
      Integrin-mediated signal transduction linked to RAS pathway to GRB2 binding to focal adhesion kinase.
      • Schlaepfer DD
      • Hauck CR
      • Sieg DJ
      Signaling through focal adhesion kinase.
      • Turner CE
      • Miller JT
      Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region.
      • Burridge K
      • Turner CE
      • Romer LH
      Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
      • Chen HC
      • Appeddu PA
      • Parsons JT
      • Hildebrand JD
      • Schaller MD
      • Guan JL
      Interaction of focal adhesion kinase with cytoskeletal protein talin.
      with resultant effects on cell migration,
      • Chen HC
      • Appeddu PA
      • Parsons JT
      • Hildebrand JD
      • Schaller MD
      • Guan JL
      Interaction of focal adhesion kinase with cytoskeletal protein talin.
      • Cary LA
      • Chang JF
      • Guan JL
      Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn.
      cytoskeletal organization,
      • Burridge K
      • Turner CE
      • Romer LH
      Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
      • Burridge K
      • Fath K
      • Kelly T
      • Nuckolls G
      • Turner C
      Focal adhesion transmembrane junctions between the extracellular matrix and cytoskeleton.
      and apoptosis.
      • Xu LH
      • Owens LV
      • Sturge GC
      • Yang X
      • Liu ET
      • Craven RJ
      • Cance WG
      Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells.
      • Xu LH
      • Yang X
      • Craven RJ
      • Cance WG
      The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells.
      • Frisch SM
      • Vuori K
      • Ruoslahti E
      • Chan-Hui PY
      Control of adhesion-dependent cell survival by focal adhesion kinase.
      FAK becomes phosphorylated and activated during integrin-mediated cell adhesion. In addition to integrins, FAK can act as a transducer of certain soluble growth factors such as epidermal growth factor.
      • Jones G
      • Machado J
      • Merlo A
      Loss of focal adhesion kinase (FAK) inhibits epidermal growth factor receptor-dependent migration and induces aggregation of NH2-terminal FAK in the nuclei of apoptotic glioblastoma cells.
      Cells derived from FAK−/− mouse embryos have been shown to exhibit reduced migration relative to wild-type cells.
      • Ilic D
      • Furuta Y
      • Kanazawa S
      • Takeda N
      • Sobue K
      • Nakatsuji N
      • Nomura S
      • Fujimoto J
      • Okada M
      • Yamamoto T
      Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
      Furthermore, overexpression of FAK in Chinese hamster ovary fibroblasts led to increased migration on fibronectin and a mutation in the major autophosphorylation site of FAK abolished its ability to stimulate cell migration.
      • Cary LA
      • Chang JF
      • Guan JL
      Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn.
      FAK has also been shown to be a survival signal for anchorage-dependent cells
      • Frisch SM
      • Vuori K
      • Ruoslahti E
      • Chan-Hui PY
      Control of adhesion-dependent cell survival by focal adhesion kinase.
      • Jones G
      • Machado J
      • Merlo A
      Loss of focal adhesion kinase (FAK) inhibits epidermal growth factor receptor-dependent migration and induces aggregation of NH2-terminal FAK in the nuclei of apoptotic glioblastoma cells.
      • Ilic D
      • Furuta Y
      • Kanazawa S
      • Takeda N
      • Sobue K
      • Nakatsuji N
      • Nomura S
      • Fujimoto J
      • Okada M
      • Yamamoto T
      Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
      • Ilic D
      • Almeida EAC
      • Schlaepfer DD
      • Dazin P
      • Aizawa S
      • Damsky CH
      Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis.
      and inhibition of FAK expression in tumor cells has been shown to result in cell death.
      • Xu LH
      • Owens LV
      • Sturge GC
      • Yang X
      • Liu ET
      • Craven RJ
      • Cance WG
      Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells.
      • Xu LH
      • Yang X
      • Craven RJ
      • Cance WG
      The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells.
      Thus, it is likely that cancer cells up-regulate FAK to maintain survival during the metastatic process.
      Other mechanisms by which FAK could potentially promote tumor cell invasion and migration include promotion of MMP-2 or MMP-9 secretion.
      • Hauck CR
      • Sieg DJ
      • Hsia DA
      • Loftus JC
      • Gaarde WA
      • Monia BP
      • Schlaepfer DD
      Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells.
      • Sein TT
      • Thant AA
      • Hiraiwa Y
      • Amin AR
      • Sohara Y
      • Liu Y
      • Matsuda S
      • Yamamoto T
      • Hamaguchi M
      A role for FAK in the concanavalin A dependent secretion of matrix metalloproteinases -2 and -9.
      Shibata and co-workers
      • Shibata K
      • Kikkawa F
      • Nawa A
      • Thant AA
      • Naruse K
      • Mizutani S
      • Hamaguchi M
      Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretation by fibronectin in ovarian cancer cells.
      have demonstrated that FAK along with endogenous Ras is required for the activation of MMP-9 secretion by fibronectin stimulation. Similarly, Hauck and colleagues
      • Hauck CR
      • Sieg DJ
      • Hsia DA
      • Loftus JC
      • Gaarde WA
      • Monia BP
      • Schlaepfer DD
      Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells.
      have demonstrated that FAK dephosphorylation in human adenocarcinoma cells resulted in reduced epidermal growth factor-stimulated JNK activation and inhibited MMP-9 secretion, and potently blocked both random and epidermal growth factor-stimulated cell motility. Aguirre Ghiso
      • Aguirre Ghiso JA
      Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo.
      has demonstrated that active FAK is an important mediator of uPAR-regulated tumorigenicity and interruption of FAK mitogenic signaling, either through down-regulation of uPAR or by expression of FRNK, can force human carcinoma cells into dormancy. Our findings provide definitive evidence that inhibition of FAK phosphorylation results in reduced ovarian cancer cell invasion and migration.
      Focal adhesions are specialized sites of cell attachment to the extracellular matrix where integrin receptors link the extracellular matrix to the actin cytoskeleton. Focal adhesions consist of several proteins that seem to serve structural roles (talin, vinculin, and paxillin) and possibly regulatory roles (protein kinase C, FAK, Src).
      • Turner CE
      • Miller JT
      Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region.
      • Burridge K
      • Turner CE
      • Romer LH
      Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
      • Chen HC
      • Appeddu PA
      • Parsons JT
      • Hildebrand JD
      • Schaller MD
      • Guan JL
      Interaction of focal adhesion kinase with cytoskeletal protein talin.
      Transformed cells have fewer and less well-developed focal adhesions than their normal counterparts.
      • Kornberg LJ
      Focal adhesion kinase and its potential involvement in tumor invasion and metastasis.
      Also, the cytoskeleton is less organized and resembles that of a rounded cell about to undergo mitogenesis.
      • Burridge K
      • Fath K
      • Kelly T
      • Nuckolls G
      • Turner C
      Focal adhesion transmembrane junctions between the extracellular matrix and cytoskeleton.
      FAK has been shown to be a survival signal for anchorage-dependent cells,
      • Frisch SM
      • Vuori K
      • Ruoslahti E
      • Chan-Hui PY
      Control of adhesion-dependent cell survival by focal adhesion kinase.
      • Ilic D
      • Almeida EAC
      • Schlaepfer DD
      • Dazin P
      • Aizawa S
      • Damsky CH
      Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis.
      • Renshaw MW
      • Price LS
      • Schwartz MA
      Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.
      and inhibition of FAK expression in tumor cells has been shown to result in cell death.
      • Xu LH
      • Owens LV
      • Sturge GC
      • Yang X
      • Liu ET
      • Craven RJ
      • Cance WG
      Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells.
      • Xu LH
      • Yang X
      • Craven RJ
      • Cance WG
      The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells.
      The role of FAK in focal adhesion assembly/disassembly continues to evolve.
      • Burridge K
      • Chrzanowska-Wodnicka M
      • Zhong C
      Focal adhesion assembly.
      It was originally thought to function in the assembly of focal adhesions; however, recent studies suggest that it might actually promote the disassembly of focal adhesions.
      • Schaller MD
      Biochemical signals and biological responses elicited by the focal adhesion kinase.
      • Fincham VJ
      • Wyke JA
      • Frame MC
      V-Src-induced degradation of focal adhesion kinase during morphological transformation of chicken embryo fibroblasts.
      FAK knockout studies have demonstrated that deletion of FAK results in an early embryonic lethal mutation and the embryos died at 8.0 to 8.5 days post coitum with severe morphogenic defects.
      • Ilic D
      • Furuta Y
      • Kanazawa S
      • Takeda N
      • Sobue K
      • Nakatsuji N
      • Nomura S
      • Fujimoto J
      • Okada M
      • Yamamoto T
      Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
      Most notable defects were in the axial mesodermal tissue and the cardiovascular system. Both vasculogenesis and angiogenesis were also severely impaired.
      • Furuta Y
      • Ilic D
      • Kanazawa S
      • Takeda N
      • Yamamoto T
      • Aizawa S
      Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK.
      Interestingly, Ilic and colleagues
      • Ilic D
      • Furuta Y
      • Kanazawa S
      • Takeda N
      • Sobue K
      • Nakatsuji N
      • Nomura S
      • Fujimoto J
      • Okada M
      • Yamamoto T
      Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
      demonstrated that FAK−/− fibroblasts exhibit larger focal adhesions than control FAK-expressing fibroblasts, but a decrease in cell migration in in vitro assays. The increase in focal adhesion number was somewhat surprising, but it suggests that FAK may regulate the cycles of focal adhesion assembly and disassembly rather than assembly alone. Ren and co-workers
      • Ren X
      • Kiosses WB
      • Sieg DJ
      • Otey CA
      • Schlaepfer DD
      • Schwartz MA
      Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover.
      have shown that FAK plays a role as a regulator of disassembly of focal adhesions. FAK may control focal adhesion disassembly by down-regulating Rho activity.
      • Ren X
      • Kiosses WB
      • Sieg DJ
      • Otey CA
      • Schlaepfer DD
      • Schwartz MA
      Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover.
      Our results are consistent with the role of FAK in disassembly of focal adhesions. The highly invasive cell line 222 had the highest FAK levels, but formed poor focal adhesions. The normal ovarian cell line HI0-180 had the lowest levels of FAK, but exhibited large focal adhesions. Interestingly, after FRNK transfection, the cell line 222 began to demonstrate better focal adhesion formation.
      FAK overexpression has been demonstrated in other cancers including colon, breast, thyroid, prostate, and brain cancers.
      • Cance 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.
      • Owens 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.
      • Owens 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.
      • Tremblay 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.
      • Kornberg LJ
      Focal adhesion kinase and its potential involvement in tumor invasion and metastasis.
      • Weiner TM
      • Lin ET
      • Craven RJ
      • Cance WG
      Expression of focal adhesion kinase gene in invasive cancer.
      • Natarajan M
      • Hecker TP
      • Gladson CL
      FAK signaling in anaplastic astrocytoma and glioblastoma tumors.
      Cance and colleagues
      • Cance 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.
      have demonstrated that FAK is only weakly expressed in most benign breast and colon epithelium; however, it is up-regulated in most of the invasive carcinomas. Interestingly, FAK expression was also up-regulated in areas of dysplastic, premalignant colon epithelium, suggesting that up-regulation occurs at an early stage in tumorigenesis. Using reverse transcriptase-polymerase chain reaction and/or Western blot analysis; FAK was shown to be overexpressed in invasive thyroid tumors
      • Owens 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.
      and invasive and metastatic prostate cancers.
      • Tremblay 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.
      Han and colleagues
      • Han NM
      • Fleming RY
      • Curley SA
      • Gallick GE
      Over expression of focal adhesion kinase (p125FAK) in human colorectal carcinoma liver metastases: independence from c-src or c-yes activation.
      have shown that FAK is overexpressed in most colorectal liver metastases, but the degree of overexpression of FAK was not a significant prognostic factor for patient survival. There are limited data regarding the role of FAK in ovarian cancer. Judson and colleagues
      • Judson PL
      • He X
      • Cance WG
      • Van Le L
      Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma.
      have reported that FAK levels were increased by fourfold in most invasive ovarian adenocarcinomas compared to normal ovarian tissues. However, these studies evaluated FAK expression in homogenates of total tumor tissues, and it is not possible to detect changes in FAK specifically associated with tumor cells with this technique. To the best of our knowledge, our study is the first to demonstrate that FAK overexpression is an independent predictor of poor outcome in ovarian cancer patients.
      In summary, we have demonstrated that FAK expression is up-regulated in most invasive ovarian cancers. We also showed that interfering with FAK function results in decreased invasion and migration of ovarian cancer cells. The dramatic up-regulation of FAK in ovarian cancers, combined with the relative lack of expression in normal tissues suggests that FAK may serve as an attractive therapeutic target.

      References

        • Jemal A
        • Murray T
        • Samuels A
        • Ghafoor A
        • Ward E
        • Thun MJ
        Cancer Statistics, 2003.
        CA Cancer J Clin. 2003; 53: 5-26
        • Jockusch 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: 379-416
        • Zamir E
        • Geiger B
        Molecular complexity and dynamics of cell-matrix adhesions.
        J Cell Sci. 2001; 114: 3583-3590
        • Miranti CK
        • Brugge JS
        Sensing the environment: a historical perspective on integrin signal transduction.
        Nat Cell Biol. 2002; 4: E83-E90
        • Schaller 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: 5192-5196
        • Schaller MD
        Biochemical signals and biological responses elicited by the focal adhesion kinase.
        Biochim Biophys Acta. 2001; 1540: 1-21
        • Schaller 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: 785-791
        • Cance 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: 2417-2423
        • Owens 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: 2752-2755
        • Owens 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: 100-105
        • Tremblay 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: 164-171
        • Ayaki 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: 3106-3112
        • Judson PL
        • He X
        • Cance WG
        • Van Le L
        Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma.
        Cancer. 1999; 86: 1551-1556
        • Kiechle 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: 534-540
        • Hauptmann S
        • Denkert C
        • Koch I
        • Petersen S
        • Schluns K
        • Reles A
        • Dietel M
        • Petersen I
        Genetic alterations in epithelial hybridization.
        Hum Pathol. 2002; 33: 632-641
        • Sood 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: 1279-1288
        • Hendrix 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: 137-147
        • Sood 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: 2924-2932
        • Schaller MD
        • Parsons JT
        Focal adhesion kinase: an integrin-linked protein tyrosine kinase.
        Trends Cell Biol. 1993; 3: 258-262
        • Schaller 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: 3489-3505
        • Schaller MD
        The focal adhesion kinase.
        J Endocrinol. 1996; 150: 1-7
        • Hsia DA
        • Mitra SK
        • Hauck CR
        • Streblow DN
        • Nelson JA
        • Ilic D
        • Huang S
        • Li E
        • Nemerow GR
        • Leng J
        • Spencer KS
        • Cheresh DA
        • Schlaepfer DD
        Differential regulation of cell motility and invasion by FAK.
        J Cell Biol. 2003; 160: 753-767
        • Kohno M
        • Hasegawa H
        • Miyake M
        • Yamamoto T
        • Fujita S
        CD151 enhances cell motility and metastasis of cancer cells in the presence of focal adhesion kinase.
        Int J Cancer. 2002; 97: 336-343
        • Sieg DJ
        • Hauck CR
        • Ilic D
        • Klingbeil CK
        • Schaefer E
        • Damsky CH
        • Schlaepfer DD
        FAK integrates growth-factor and integrin signals to promote cell migration.
        Nat Cell Biol. 2000; 2: 249-256
        • Kornberg L
        • Earp HS
        • Parsons JT
        • Schaller M
        • Juliano RL
        Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase.
        J Biol Chem. 1992; 267: 23439-23442
        • Klingbeil CK
        • Houck CR
        • Hsia DA
        • Jones KC
        • Reider SR
        • Schlaepfer DD
        Targeting PYK2 to β1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cell.
        J Cell Biol. 2001; 152: 97-110
        • Schlaepfer DD
        • Hanks SK
        • Hunter T
        • van der Greer P
        Integrin-mediated signal transduction linked to RAS pathway to GRB2 binding to focal adhesion kinase.
        Nature. 1994; 372: 786-791
        • Schlaepfer DD
        • Hauck CR
        • Sieg DJ
        Signaling through focal adhesion kinase.
        Prog Biophys Mol Biol. 1999; 71: 435-478
        • Turner CE
        • Miller JT
        Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region.
        J Cell Sci. 1994; 107: 1583-1591
        • Burridge K
        • Turner CE
        • Romer LH
        Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
        J Cell Biol. 1992; 119: 393-403
        • Chen HC
        • Appeddu PA
        • Parsons JT
        • Hildebrand JD
        • Schaller MD
        • Guan JL
        Interaction of focal adhesion kinase with cytoskeletal protein talin.
        J Biol Chem. 1995; 270: 16995-16999
        • Cary LA
        • Chang JF
        • Guan JL
        Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn.
        J Cell Sci. 1996; 109: 1787-1794
        • Burridge K
        • Fath K
        • Kelly T
        • Nuckolls G
        • Turner C
        Focal adhesion transmembrane junctions between the extracellular matrix and cytoskeleton.
        Annu Rev Cell Biol. 1998; 4: 487-525
        • Xu LH
        • Owens LV
        • Sturge GC
        • Yang X
        • Liu ET
        • Craven RJ
        • Cance WG
        Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells.
        Cell Growth Differ. 1996; 7: 413-418
        • Xu LH
        • Yang X
        • Craven RJ
        • Cance WG
        The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells.
        Cell Growth Differ. 1998; 9: 999-1005
        • Frisch SM
        • Vuori K
        • Ruoslahti E
        • Chan-Hui PY
        Control of adhesion-dependent cell survival by focal adhesion kinase.
        J Cell Biol. 1996; 134: 793-799
        • Jones G
        • Machado J
        • Merlo A
        Loss of focal adhesion kinase (FAK) inhibits epidermal growth factor receptor-dependent migration and induces aggregation of NH2-terminal FAK in the nuclei of apoptotic glioblastoma cells.
        Cancer Res. 2001; 61: 4978-4981
        • Ilic D
        • Furuta Y
        • Kanazawa S
        • Takeda N
        • Sobue K
        • Nakatsuji N
        • Nomura S
        • Fujimoto J
        • Okada M
        • Yamamoto T
        Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
        Nature. 1995; 377: 539-544
        • Ilic D
        • Almeida EAC
        • Schlaepfer DD
        • Dazin P
        • Aizawa S
        • Damsky CH
        Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis.
        J Cell Biol. 1998; 143: 547-560
        • Hauck CR
        • Sieg DJ
        • Hsia DA
        • Loftus JC
        • Gaarde WA
        • Monia BP
        • Schlaepfer DD
        Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells.
        Cancer Res. 2001; 61: 7079-7090
        • Sein TT
        • Thant AA
        • Hiraiwa Y
        • Amin AR
        • Sohara Y
        • Liu Y
        • Matsuda S
        • Yamamoto T
        • Hamaguchi M
        A role for FAK in the concanavalin A dependent secretion of matrix metalloproteinases -2 and -9.
        Oncogene. 2000; 19: 5539-5542
        • Shibata K
        • Kikkawa F
        • Nawa A
        • Thant AA
        • Naruse K
        • Mizutani S
        • Hamaguchi M
        Both focal adhesion kinase and c-Ras are required for the enhanced matrix metalloproteinase 9 secretation by fibronectin in ovarian cancer cells.
        Cancer Res. 1998; 58: 900-903
        • Aguirre Ghiso JA
        Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo.
        Oncogene. 2002; 21: 2513-2524
        • Kornberg LJ
        Focal adhesion kinase and its potential involvement in tumor invasion and metastasis.
        Head Neck. 1999; 20: 745-752
        • Renshaw MW
        • Price LS
        • Schwartz MA
        Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.
        J Cell Biol. 1999; 147: 611-618
        • Burridge K
        • Chrzanowska-Wodnicka M
        • Zhong C
        Focal adhesion assembly.
        Trends Cell Biol. 1997; 7: 342-347
        • Fincham VJ
        • Wyke JA
        • Frame MC
        V-Src-induced degradation of focal adhesion kinase during morphological transformation of chicken embryo fibroblasts.
        Oncogene. 1995; 10: 2247-2252
        • Furuta Y
        • Ilic D
        • Kanazawa S
        • Takeda N
        • Yamamoto T
        • Aizawa S
        Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK.
        Oncogene. 1996; 11: 1989-1995
        • Ren X
        • Kiosses WB
        • Sieg DJ
        • Otey CA
        • Schlaepfer DD
        • Schwartz MA
        Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover.
        J Cell Sci. 2000; 113: 3673-3678
        • Weiner TM
        • Lin ET
        • Craven RJ
        • Cance WG
        Expression of focal adhesion kinase gene in invasive cancer.
        Lancet. 1993; 342: 1024-1025
        • Natarajan M
        • Hecker TP
        • Gladson CL
        FAK signaling in anaplastic astrocytoma and glioblastoma tumors.
        Cancer J. 2003; 9: 126-133
        • Han NM
        • Fleming RY
        • Curley SA
        • Gallick GE
        Over expression of focal adhesion kinase (p125FAK) in human colorectal carcinoma liver metastases: independence from c-src or c-yes activation.
        Ann Surg Oncol. 1997; 4: 264-268

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        The American Journal of PathologyVol. 188Issue 4
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          In the article entitled, “Biological Significance of Focal Adhesion Kinase in Ovarian Cancer: Role in Migration and Invasion” (Volume 165, pages 1087–1095 of the October 2004 issue of The American Journal of Pathology; https://doi.org/10.1016/S0002-9440(10)63370-6), there is a minor amendment to the legend of Figure 2 to clarify the splicing out of lanes which was not mentioned in the original legend.
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