Abstract 1044: Silencing p130cas in ovarian carcinoma induces autophagic cell death

Abstract

Abstract Background: p130Cas is a central node for many cancer signaling pathways. We have previously established the clinical significance of p130cas overexpression in ovarian carcinoma and the therapeutic efficacy of silencing p130cas with siRNA in an orthotopic mouse model of ovarian carcinoma. The focus of the current work is to establish the mechanism by which p130cas gene silencing decreases tumor growth. Methods: To determine the specific mechanisms by which p130cas gene silencing abrogates tumor growth, in vitro measures of cell viability (MTT), apoptosis (TUNEL by FACS), autophagy (acridine organ staining by FACS and immunoblotting), and cell signaling (immunoblotting) were performed. Results: p130Cas treatment with siRNA yielded a 21 (SKOV3TRip2, p<0.05) to 30% (SKOV3ip1; p<0.001) reduction in tumor cell viability and a 2.5 (SKOV3ip1) to 3-fold (SKOV3TRip2) decrease in the calculated IC50 of docetaxel chemotherapy. Compared to untreated cells (in SKOV3ip1), p130cas gene silencing alone resulted in a statistically significant increase in tumor cell apoptosis (Annexin V-PE: 675% increase, p=0.002) and a 175% increase in tumor cell necrosis (Annexin V-PE and 7AAD dual staining, p=0.002). Docetaxel therapy alone at IC50 concentrations (SKOV3ip1: 1nM) resulted in a 675% increase in the percent of cells in early apoptosis (p=0.002) and a 545% increase in necrotic tumor cells (p=0.002) compared to untreated cells. Compared to untreated cells, combination therapy with p130cas siRNA plus docetaxel therapy (1nM) resulted in the greatest increase in early apoptosis (929% increase, p=0.002) and tumor cell necrosis (922% increase, p=0.002). Pre-treatment with pan-caspase inhibition (ZVAD-fmk) resulted in no significant difference in percentage of Annexin V-PE positive cells and p130cas gene silencing did not result in PARP cleavage as determined by immunoblot analysis, suggesting that a caspase-independent autophagic cell death may be activated. Treatment with p130cas siRNA resulted in signaling changes consistent with induction of autophagy including decreased levels of phospho-Akt, decreased total mTOR levels, and increased phosphorylation of Bcl-2. Furthermore, p130cas gene silencing resulted in induction of autophagy as measured by increased expression of Beclin-1 and LC-3 (immunoblotting) as well as increased autophagic vacuoles by acridine orange staining (FACS). Conclusions: Silencing p130Cas with siRNA decreases tumor cell viability by induction of both apoptotic and autophagic pathways establishing p130cas as a novel therapeutic target in ovarian carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1044.