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Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

Jung JG, Stoeck A, Guan B, Wu RC, Zhu H, Blackshaw S, Shih IeM, Wang TL - PLoS Genet. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

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.

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WWP2 counteracts Notch3-induced increase in CSC population.(A) N3-NEXT overexpression (second row) increased frequencies of verapamil-sensitive SP cell in MPSC1 and OVCAR3 cells compared with pLPC vector-transfected control (first row). Ectopic expression of WWP2 reduced SP fraction in both cell lines (third row). Co-expression of WWP2 in N3-NEXT-expressing cells reduced SP fraction (fourth row) as compared to cells with only N3-NEXT overexpression (second row). (B) Bar charts represent % of SP in each experimental condition.
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pgen-1004751-g008: WWP2 counteracts Notch3-induced increase in CSC population.(A) N3-NEXT overexpression (second row) increased frequencies of verapamil-sensitive SP cell in MPSC1 and OVCAR3 cells compared with pLPC vector-transfected control (first row). Ectopic expression of WWP2 reduced SP fraction in both cell lines (third row). Co-expression of WWP2 in N3-NEXT-expressing cells reduced SP fraction (fourth row) as compared to cells with only N3-NEXT overexpression (second row). (B) Bar charts represent % of SP in each experimental condition.

Mentions: Previous studies by our group and others have shown that Notch3 upregulation is related to the recurrence of ovarian cancer and is associated with a poor prognosis [7]. In addition, Notch 3 overexpression increases the proportion of cancer stem cell-like cells (CSCs) and resistance to platinum-based therapy [7], [17]. To determine whether WWP2 can counteract these Notch3-mediated phenotypes, we performed flow cytometry and Hoechst 33342 dye efflux assay to measured side population which is enriched with CSCs. Ovarian cancer cells were transfected with empty vector, N3-NEXT, WWP2, or N3-NEXT+WWP2. The flow cytometry results demonstrated that overexpression of Notch3 increased the side population fraction but the percentage of cells in this population was reduced when WWP2 was also expressed. Thus, the presence of WWP2 counteracts Notch effect on promoting the CSC phenotype (Fig. 8). Similarly, expression of WWP2 decreased the Notch3-induced platinum resistance as evidenced by analyzing cell viability in OVCAR3 cells cultured with carboplatin (Fig. S8).


Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

Jung JG, Stoeck A, Guan B, Wu RC, Zhu H, Blackshaw S, Shih IeM, Wang TL - PLoS Genet. (2014)

WWP2 counteracts Notch3-induced increase in CSC population.(A) N3-NEXT overexpression (second row) increased frequencies of verapamil-sensitive SP cell in MPSC1 and OVCAR3 cells compared with pLPC vector-transfected control (first row). Ectopic expression of WWP2 reduced SP fraction in both cell lines (third row). Co-expression of WWP2 in N3-NEXT-expressing cells reduced SP fraction (fourth row) as compared to cells with only N3-NEXT overexpression (second row). (B) Bar charts represent % of SP in each experimental condition.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214668&req=5

pgen-1004751-g008: WWP2 counteracts Notch3-induced increase in CSC population.(A) N3-NEXT overexpression (second row) increased frequencies of verapamil-sensitive SP cell in MPSC1 and OVCAR3 cells compared with pLPC vector-transfected control (first row). Ectopic expression of WWP2 reduced SP fraction in both cell lines (third row). Co-expression of WWP2 in N3-NEXT-expressing cells reduced SP fraction (fourth row) as compared to cells with only N3-NEXT overexpression (second row). (B) Bar charts represent % of SP in each experimental condition.
Mentions: Previous studies by our group and others have shown that Notch3 upregulation is related to the recurrence of ovarian cancer and is associated with a poor prognosis [7]. In addition, Notch 3 overexpression increases the proportion of cancer stem cell-like cells (CSCs) and resistance to platinum-based therapy [7], [17]. To determine whether WWP2 can counteract these Notch3-mediated phenotypes, we performed flow cytometry and Hoechst 33342 dye efflux assay to measured side population which is enriched with CSCs. Ovarian cancer cells were transfected with empty vector, N3-NEXT, WWP2, or N3-NEXT+WWP2. The flow cytometry results demonstrated that overexpression of Notch3 increased the side population fraction but the percentage of cells in this population was reduced when WWP2 was also expressed. Thus, the presence of WWP2 counteracts Notch effect on promoting the CSC phenotype (Fig. 8). Similarly, expression of WWP2 decreased the Notch3-induced platinum resistance as evidenced by analyzing cell viability in OVCAR3 cells cultured with carboplatin (Fig. S8).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus