T1176 Role of Recombinase (RAD51) in Ongoing Genomic Instability and Proliferation in Barrett's Adenocarcinoma Cells

Abstract

activity. Exposure to 15mmHg increased extracellular pressure stimulated serine phosphorylation of FAK (p-FAK) in Caco-2 and primary human colon cancer cells isolated from surgical specimens. This pressure-induced increase in serine p-FAK was blocked by Akt inhibitor and by siRNA silencing of Akt1 but not by silencing Akt2. Co-precipitation demonstrated that Akt associates directly with FAK. Akt-FAK association was increased by pressure and this increased association was blocked by inhibiting FAK or silencing Akt1 but not Akt2. Scanning the FAK sequence with Scansite software revealed three serine-containing consensus sequences for AKT phosphorylation in the FAK sequence. We therefore constructed a FAK non-phosphorylatable mutant with point mutations (S®A) at these three putative serine phosphorylation sites (S517/601/695) of FAK by Akt to investigate their relevance to pressurestimulated cell adhesion and tyrosine p-FAK. Indeed, overexpression of the triple mutant of FAK (S517/601/695®A) in Caco-2 cells, in contrast to wild type FAK, prevented the increase in p-FAK at Y397 and cancer cell adhesion induced by extracellular pressure. These results suggest that Akt regulates pressure-induced cancer cell adhesion by binding directly to and phosphorylating FAK at S509/601/695. This serine phosphorylation, in turn, permits the pressure-dependent tyrosine autophosphorylation of p-FAK at Y397, the conventional initiator of FAK activation. Although Akt is therefore required for FAK activation in response to pressure, further studies demonstrated that FAK also potentiates Akt activation. Blocking or silencing FAK by three different FAK-specific siRNA sequences prevented the increases in serine p-Akt (S473) and tyrosine p-FAK(Y397) induced by increased pressure. Thus, FAK and Akt bind directly and potentiate each other's activation. This novel mechanism of FAKAkt interaction suggests that FAK and Akt1 may be important dual therapeutic targets for preventing cancer cell adhesion, and eventually cancer metastasis.