Abstract 1649: The E3 ubiquitin ligase EDD regulates platinum resistance and is a novel therapeutic target for epithelial ovarian cancer

Introduction: Ovarian cancer is treated with a combination of surgery and chemotherapeutics, including taxol and platinum agents. A major difficulty in eradicating these tumors is the selection of drug resistant cells. EDD (E3-ubiquitin ligase identified by Differential Display) is a 300 kDa E3 ubiquitin ligase that is overexpressed in 84% of recurrent, platinum-resistant ovarian cancers, but is rare in benign in borderline tumors. This correlation with chemoresistance suggests that EDD may be involved in mediating platinum resistance and/or tumor survival. Experimental Procedures: EDD protein knockdown was accomplished using siRNA (small interfering RNA) and shRNA (short hairpin RNA) for transient or stable knockdown, respectively, in ovarian cancer cell lines. Apoptosis and sensitivity to cisplatin were measured using MTS assay, propidium iodide staining, and western blotting for PARP and caspase 3 cleavage. Cisplatin resistance by EDD overexpression was determined by transient transfection of GFP, FLAG-EDD or the ubiquitin ligase deficient FLAG-EDD-C2768A into COS-7 cells followed by cisplatin treatment and apoptosis measurement in transfected cells by the TACS 2 TdT-Blue Label Kit followed by microscopy. Mice with intraperitoneal xenografts of ES2 and A2780ip2 cells were treated once weekly with cisplatin and twice weekly with control or EDD siRNA. Results: EDD was overexpressed in 5 of 7 ovarian cancer cell lines compared to benign IOSE cells. EDD overexpression in COS-7 cells was sufficient to protect from cisplatin-induced apoptosis and the induced resistance was dependent upon EDD ubiquitin ligase activity (relative apoptosis: GFP = 1; EDD = 0.42, EDD-C2768A = 1.23). Stable knockdown of EDD increased cisplatin sensitivity in ES2 cells (IC50 = 37 μM for control, 9 μM for EDD shRNA). EDD siRNA alone induced cleavage of PARP and caspase 3 compared to control siRNA and enhanced cleavage of both in the presence of cisplatin. Mouse xenograft studies demonstrated that nanoliposomal delivery (DOPC) of EDD siRNA showed a trend towards reduced tumor compared to control siRNA. In vivo, mice treated with EDD siRNA by nanoliposomal delivery (DOPC) had a trend towards less tumor than those treated with control siRNA (27.7% reduction in ES2, 42.5% in A2780ip2, p = 0.23 and 0.19 respectively). Mice treated with combined EDD siRNA and cisplatin had significantly less tumor than controls (77.9% reduction in ES2, 75.9% in A2780ip2, p = 0.004 and 0.042, respectively) and cisplatin alone (60.3-64.4% reduction, p = 0.035). Conclusions: EDD overexpression is sufficient to promote cisplatin resistance, dependent upon its E3 ubiquitin ligase activity. Targeting EDD expression induces apoptosis and cisplatin. sensitivity in vitro and in vivo. Targeting EDD may be an effective treatment for platinum-resistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2011-1649