title: 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.
authors.affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Jin-Gyoung
authors.initials: JG
authors.lastname: Jung
authors.affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Alexander
authors.initials: A
authors.lastname: Stoeck
authors.affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Guan
authors.affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Pathology, Chang Gung Memorial Hospital and Chang Gung University School of Medicine, Taoyuan, Taiwan.
authors.forename: Ren-Chin
authors.initials: RC
authors.lastname: Wu
authors.affiliation: Department of Pharmacology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Center for High-Throughput Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Center for High-Throughput Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Seth
authors.initials: S
authors.lastname: Blackshaw
authors.affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Ie-Ming
authors.initials: IeM
authors.lastname: Shih
authors.affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Tian-Li
authors.initials: TL
authors.lastname: Wang
mesh_terms: D000818:Animals; D063646:Carcinogenesis; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008247:Lysosomes; D051379:Mice; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D040901:Proteomics; D034741:RNA, Small Interfering; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: C499374:NOTCH3 protein, human; D034741:RNA, Small Interfering; D000071656:Receptor, Notch3; D051880:Receptors, Notch; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases