EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer.

The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to 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, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 messenger RNA and EDD transfection enhanced murine Mcl-1 promoter-driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were 4- 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 [1,2-dioleoyl-sn-glycero-3-phophatidylcholine (DOPC)] and 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 that EDD is a therapeutic target for ovarian cancer.

ERK activation and nuclear signaling induced by the loss of cell/matrix adhesion stimulates anchorage-independent growth of ovarian cancer cells.

Ovarian cancer metastasis involves the sloughing of epithelial cells from the ovary into the peritoneal cavity, where the cells can survive and proliferate in peritoneal ascites under anchorage-independent conditions. For normal epithelial cells and fibroblasts, cell adhesion to the extracellular matrix is required to prevent apoptosis and for proper activation and nuclear signaling of the ERK MAP kinase. The mechanisms of ERK regulation by adhesion have been determined by our lab and others. In this report, we elucidate a novel means of ERK regulation by cellular adhesion in ovarian cancer cells. We demonstrate that ERK and its activator MEK are robustly stimulated after cell detachment from a substratum in several ovarian cancer cell lines, but not a benign ovarian cell line, independent of serum and FAK or PAK activity. MEK and ERK activation was sustained for 48 h after detachment, while activation by serum or growth factors in adherent cells was transient. Re-attachment of suspended ovarian cells to fibronectin restored basal levels of MEK and ERK activity. ERK activity in suspended cells was dynamically controlled through an autocrine stimulatory pathway and prevalent phosphatase activity. Suspended cells demonstrated higher levels of ERK nuclear signaling to Elk1 compared to adherent cells. Inhibition of ERK activation with the MEK inhibitor U0126 had minor effects on adherent cell growth, but greatly decreased growth in soft agar. These data demonstrate a unique regulation of ERK by cellular adhesion and suggest a mechanism by which ERK may regulate anchorage-independent growth of metastatic ovarian cancer cells.

Allelic exclusion of the TCR alpha-chain is an active process requiring TCR-mediated signaling and c-Cbl.

Phenotypic allelic exclusion at the TCRalpha locus is developmentally regulated in thymocytes. Many immature thymocytes express two cell surface alpha-chain species. Following positive selection, the vast majority of mature thymocytes and peripheral T cells display a single cell surface alpha-chain. A posttranslational mechanism occurring at the same time as positive selection and TCR up-regulation leads to this phenotypic allelic exclusion. Different models have been proposed to explain the posttranslational regulation of the alpha-chain allelic exclusion. In this study, we report that allelic exclusion is not regulated by competition between distinct alpha-chains for a single beta-chain, as proposed by the dueling alpha-chain model, nor by limiting CD3 zeta-chain in mature TCR(high) thymocytes. Our data instead favor the selective retention model where the positive selection signal through the TCR leads to phenotypic allelic exclusion by specifically maintaining cell surface expression of the selected alpha-chain while the nonselected alpha-chain is internalized. The use of inhibitors specific for Lck and/or other Src kinases indicates a role for these protein tyrosine kinases in the signaling events leading to the down-regulation of the nonselectable alpha-chain. Loss of the ubiquitin ligase/TCR signaling adapter molecule c-Cbl, which is important in TCR down-modulation and is a negative regulator of T cell signaling, leads to increased dual alpha-chain expression on the cell surface of double-positive thymocytes. Thus, not only is there an important role for TCR signaling in causing alpha-chain allelic exclusion, but differential ubiquitination by c-Cbl may be an important factor in causing only the nonselected alpha-chain to be down-modulated.