Pharmacological focal adhesion kinase (FAK) inhibition suppresses cisplatin-resistant human ovarian carcinoma growth

Abstract

Objectives: FAK is a cytoplasmic protein tyrosine kinase promoting ovarian cancer tumor growth and metastasis. Phase I and II clinical trials are testing small molecule inhibitors to FAK in different cancers. Determination of cancer cell susceptibility to FAK inhibition is an important yet undefined parameter. We test the hypothesis that elevated FAK activity contributes to the growth of aggressive cisplatin (CP)–resistant ovarian cancer. Methods: Growth of paired CP-sensitive (A2780 and OVCAR10) and CP-resistant (A2780-CP and OVCAR10-CP) human ovarian cells was evaluated in adherent, nonadherent, and methylcellulose spheroid culture in the presence or absence of 100 nM concentration of FAK inhibitor (FAKi). Quantification of cancer stemlike cells (CSCs) and cell cycle analyses was performed using flow cytometry for aldehyde dehydrogenase (ALDH) activity and propidium iodide DNA staining, respectively. Cell apoptosis was evaluated with Annexin-V and (7-aminoactinomycin D) 7-AAD staining. Changes in cellular protein expression were determined with quantitative immunoblotting. Intraperitoneal tumor growth in mice was evaluated for combined inhibitory effects of CP and FAKi administration. Results: FAK tyrosine phosphorylation and CSC protein markers were elevated in CP-resistant cells, which was reversed by FAKi addition. On immunoblotting, the levels of the transcription factor OCT4, ALDH, and the cell surface protein N-cadherin were increased in CP-resistant cells. FAKi addition selectively prevented CP-resistant cell growth as spheroids. The combination of FAKi with CP exhibited a combined effect on the CP-resistant cells, with significant decreases in spheroid and tumor growth compared with either agent alone. When comparing the percentage of CSCs with flow cytometry, FAKi addition dramatically reduced the ALDH-positive cell population in both parental and CP-resistant cells. This was associated with a G1 cell cycle growth phase arrest, decreased cyclin D protein expression, but not increased cell apoptosis. (See Fig. 1.) Conclusions: CSCs are implicated in the molecular mechanisms of cisplatin-resistance, and we find that FAK inhibition prevents CP-resistant CSC growth. Because FAKi therapy is well tolerated with limited adverse events, our results support further testing of FAKi in either primary or recurrent settings of CP-resistant ovarian cancer.