Notch3 Interactome Analysis Identified WWP2 as a Negative Regulator of Notch3 Signaling in Ovarian Cancer

Abstract

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Author Summary

Notch pathway is important for many cellular activities, and its dysregulation leads to several diseases in humans, including cancer. Although Notch hyperactivity has been observed in many types of cancers, the interactome of Notch receptor remains largely unknown, especially for Notch3, which is involved in ovarian cancer pathogenesis. This article is the first study, to our knowledge, that delineates the Notch3 interacting network, and demonstrates that one of the Notch3 interacting proteins, WWP2, an E3 ubiquitin-protein ligase, plays a major role in negative regulation of Notch3 signaling in cancer cells. WWP2 locus was found to be deleted, and its mRNA down-regulated in a significant fraction of ovarian carcinomas. Ectopic expression of WWP2 reduced tumorigenicity of ovarian cancer cells, and counteracted Notch3-mediated phenotypes, including promotion of cancer stem-like cell phenotype and platinum resistance, further supporting its tumor suppressor role. The results from this study provide new insights into how Notch3 signaling contributes to cancer development, and should have implications for the design of Notch3-based cancer therapy.