Abstract 1987: Identification and characterization of novel Notch3 interacting proteins in ovarian cancer

Abstract

Abstract Ovarian serous carcinoma (OSC) represents one of the most aggressive neoplastic diseases because most cases present at an advanced stage when therapy is usually ineffective. We have previously reported that Notch3 gene amplification and overexpression occurs frequently in OSC. Activation of Notch3 signaling is essential for the survival of OSC cells with Notch3 overexpression, especially in the presence of chemotherapeutic agents. Notch3 pathway activation has been known to transcriptionally regulate an array of genes, including those involved in stem cell biology and oncogenesis in OSC. The current study aims at identifying the binding proteins of Notch-ICD in OSC cells using recombinant human Notch3-ICD protein and protein interaction arrays. We have identified several novel Notch3-ICD interacting proteins. Among them, we focused on characterizing WW domain containing E3 ubiquitin protein ligase 2 (WWP2), a member of the NEDD4 family of ubiquitin-protein ligases. We found that WWP2 specifically co-immunoprecipitated with Notch3-ICD but not with Notch1-ICD, and ubiquitinated Notch3-ICD, leading to its degradation in lysosomes. Overexpression of WWP2 in OVCAR3 cells reduced Notch3 steady state protein levels, suppressed Notch3 reporter activity, and inhibited cellular proliferation. On the other hand, a dominant negative isoform of WWP2, as well as knockdown WWP2 using shRNAs, prevented ubiqutination of Notch3-ICD, increased Notch3-ICD protein levels, and enhanced Notch3 reporter gene activity. Compared to their normal counterparts, including fallopian tube and ovarian surface epithelial cells, OSC cell lines and tissues had a significantly lower mRNA and protein levels of WWP2 while Notch3 protein expression was inversely correlated with WWP2 expression levels. The above results provide new evidence that WWP2 protein is a key regulator of Notch3-ICD protein expression levels in ovarian cancer cells. WWP2 modulates Notch3 signaling activity by regulating its degradation pathway and downregulation of WWP2 may represent a novel mechanism to activate the Notch3 signaling pathway in 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 1987. doi:10.1158/1538-7445.AM2011-1987