E3 Ligase Identified by Differential Display (EDD) Enhances Cell Survival and Cisplatin Resistance in Epithelial Ovarian Cancer and Oral Squamous Cell Carcinoma
Abstract
EDD (E3-ubiquitin ligase identified by Differential Display) is an E3 ubiquitin ligase that is overexpressed in ovarian cancer, but is rare in benign and borderline tumors. EDD is also overexpressed in recurrent, platinum-resistant ovarian cancers and is associated with a two-fold increased risk of disease recurrence and death in ovarian cancer patients, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by siRNA induced apoptosis in A2780ip2, OVCAR5, and ES-2 ovarian cancer cells, correlating with a loss of the anti-apoptotic protein Mcl-1 through a GSK-β-independent mechanism. Transient knockdown of EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, indicating that EDD may regulate Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 mRNA and EDD transfection enhanced murine Mcl-1 promoter driven luciferase expression five-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and demonstrated that these cells were four- to 21-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery (DOPC) along with cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, p=0.004; A2780ip2, 75.9% reduction, p=0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, p=0.035), with a trend in A2780ip2 (60.3% reduction, p=0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest EDD is a therapeutic target for ovarian cancer. Additionally, edd is overamplified in oral squamous cell carcinoma of the tongue. Preliminary results in this carcinoma indicate similar roles of EDD in regulating cellular survival and cisplatin resistance as demonstrated in ovarian cancer.