Saem Lee

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

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 others 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.

T1155 Anticancer Activity of a Broccoli Derivative, Sulforaphane, in Barrett's Adenocarcinoma: Potential use in Chemoprevention and as Adjuvant in Chemotherapy

INTRODUCTION The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. METHODS BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was evaluated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To evaluate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. CONCLUSIONS These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.