The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro

Abstract

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Figure 1

Representative IHC results for EDD expression in (A), normal ovary (ovarian surface epithelium arrowed) and ovarian inclusion cysts (inset); (B) negative EDD staining; (C) low (<6) EDD expression; and (D) high (⩾6) EDD expression in serous ovarian carcinomas. Original magnification × 20.

Figure 2

Association of EDD expression with ovarian cancer outcome. Kaplan–Meier survival curves and log-rank P-values showing (A) recurrence-free survival stratified by EDD expression (high vs low/absent) in serous ovarian cancer patients who suffered disease recurrence following initial complete response to treatment; (B) overall survival stratified by EDD expression (high vs low/absent) in serous ovarian cancer patients who suffered disease recurrence and death following initial complete response to treatment; (C) overall survival stratified by EDD expression (high vs low/absent) in patients with progressive disease (no or partial response to treatment).

Figure 3

Knockdown of EDD expression using siRNA inhibits growth of cisplatin-resistant ovarian cancer cells. (A) Example of colony-forming assay showing inhibition of growth of ovarian A2780-cp70 cells transfected with siRNA against EDD (EDD siRNA1) as compared to siRNA against GFP (control) following treatment with cisplatin (20 μM); (B) A2780-cp70 colony number following treatment with cisplatin (20 μM) 48 h following transfection with siRNA against GFP (control) or EDD (EDD siRNA1). Results are expressed as the relative percentage of colonies as compared to untreated controls for each siRNA following adjustment for plating efficiency using untreated wells and are the means of duplicate experiments, each performed in triplicate wells. Confirmation of reduced EDD expression following siRNA transfection was determined by western blotting (below). (C) Dose–response curve of a representative experiment showing relative cisplatin sensitivity as determined by colony number of A2780-cp70 cells transfected with siRNA against EDD (EDD siRNA 1 and siRNA 2) compared to cells transfected with siRNA against GFP (control) after cisplatin treatment, as described above, and adjusted for untreated controls.