P1.07-028 Dual Role of the Focal Adhesion Kinase in Small-Cell Lung Cancer

Abstract

Small cell-lung cancer (SCLC) is a devastating illness with five-year overall survival as low as 5%. The molecular steps leading to SCLC development and progression are still poorly understood and this has translated into the absence of targeted therapies. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase which regulates integrin and growth factor signaling pathways involved in cell proliferation, survival, migration, and invasion. FAK is overexpressed and/or activated in many cancers, including SCLC. We hypothesized that FAK overexpression/activation in SCLC contributes to its aggressive behavior and that FAK may represent a therapeutic target in SCLC. Two SCLC cell lines growing in suspension (NCI-H82, NCI-H146), and adherent SCLC cell lines (NCI-H196, NCI-H446) were treated with PF-228. NCI-H446 and H82 cells were stably transfected with FAK shRNA and/or FRNK using lentivirus vector. Cell proliferation was evaluated by WST-1 assay; cell cycle by flow cytometry with propidium iodide and bromodeoxyuridine; apoptosis by caspase 3 staining in flow cytometry and by cleaved PARPp85 Western blotting (WB); motility by wound healing assay; and invasion by Boyden chambers. FAK expression/activity was evaluated by WB. While PF-228 did not modify total FAK expression, it decreased FAK phosphorylation (Y397). Inhibition of FAK activity by PF-228 decreased cell proliferation, DNA synthesis, induced cell cycle arrest in G2/M phases, and increased apoptosis in dose-dependently. PF-228 also decreased motility in the adherent H196-H446 cells. To confirm the specificity of the antitumoral effects of PF-228, we stably transfected SCLC cells with FAK shRNA and FRNK and then analyzed the phenotypic changes induced by these approaches. Knockdown of total FAK protein by transfection of FAK shRNA inhibited FAK activity, but did not have any effect on cell proliferation, DNA synthesis, and cell cycle. However, reintroduction of FRNK in cells stably transfected with FAK shRNA inhibited cell proliferation and DNA synthesis. Expression of FRNK decreased cell proliferation and DNA synthesis in SCLC cells. Inhibition of FAK activity by PF-228 in SCLC cell lines demonstrates that FAK activity is required for cell proliferation, cycle progression, survival, and motility, suggesting that FAK inhibition may represent a suitable therapeutic target for SCLC. Inhibition of FAK by a genomic approach suggests that FAK has a dual role in SCLC biology, namely (i) a pro-tumoral effect related to the kinase domain, which induces downstream signals (ii) an anti-tumoral effect mediated by the non-kinase C-terminal domain (FRNK domain), which keeps inactive other pro-tumoral effectors.