Biological significance of focal adhesion kinase in ovarian cancer: role in migration and invasion

Abstract

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.

Figure 1-4236

FAK 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).

Figure 2-4236

Western 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).

Figure 3-4236

A 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.

Figure 4-4236

Representative 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.

Figure 5-4236

Kaplan-Meier survival of ovarian cancer patients based on FAK staining intensity using the log-rank statistic.