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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 regulates Notch3 signaling activity in cancer cells.(A) OVCAR3 or MCF7 cancer cells were transfected with control plasmid pLPC, WWP2, catalytically-inactive WWP2 (WWP2-CA), N3-ICD, or WWP2+N3-ICD. Relative cell numbers were measured at different time points using the SYBE Green-based assay. Data are expressed as means ± SD and Student's t-test was performed to compare the relative cell number between WWP2+N3-ICD and pLPC (* p<0.05; ** p<0.01). (B) Different groups of cells were transfected with the pJH23A (4×wtCBF1Luc) luciferase reporter construct together with pLPC, WWP2, or the catalytically-inactive WWP2 plasmid (WWP2-CA). Each experiment was performed in triplicate. Results are shown as percentage of luciferase activity compared to pLPC transfected controls. Data are expressed as means ± SD. P values were calculated by comparison between either WWP2 or WWP2-CA and the control plasmid pLPC (** p<0.01; *** p<0.001). (C) Two untransformed epithelial cell lines were transfected with WWP2 siRNA or scrambled siRNA (siSCR). Notch signaling was measured by co-transfecting the cells with a luciferase reporter plasmid, pJH23A (4×wtRBPJLuc) and the data were normalized to the luciferase activity measured by co-transfection with a control plasmid, pGL3. Data are expressed as means ± SD and statistical significance was assessed by two-tailed Student's t-test.
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pgen-1004751-g006: WWP2 regulates Notch3 signaling activity in cancer cells.(A) OVCAR3 or MCF7 cancer cells were transfected with control plasmid pLPC, WWP2, catalytically-inactive WWP2 (WWP2-CA), N3-ICD, or WWP2+N3-ICD. Relative cell numbers were measured at different time points using the SYBE Green-based assay. Data are expressed as means ± SD and Student's t-test was performed to compare the relative cell number between WWP2+N3-ICD and pLPC (* p<0.05; ** p<0.01). (B) Different groups of cells were transfected with the pJH23A (4×wtCBF1Luc) luciferase reporter construct together with pLPC, WWP2, or the catalytically-inactive WWP2 plasmid (WWP2-CA). Each experiment was performed in triplicate. Results are shown as percentage of luciferase activity compared to pLPC transfected controls. Data are expressed as means ± SD. P values were calculated by comparison between either WWP2 or WWP2-CA and the control plasmid pLPC (** p<0.01; *** p<0.001). (C) Two untransformed epithelial cell lines were transfected with WWP2 siRNA or scrambled siRNA (siSCR). Notch signaling was measured by co-transfecting the cells with a luciferase reporter plasmid, pJH23A (4×wtRBPJLuc) and the data were normalized to the luciferase activity measured by co-transfection with a control plasmid, pGL3. Data are expressed as means ± SD and statistical significance was assessed by two-tailed Student's t-test.

Mentions: To determine whether reconstitution of WWP2 expression in cancer cells resulted in reducing Notch3 signaling activity and subsequently cellular proliferation, we performed proliferation assays in OVCAR3 and MCF7 cells transduced with pLPC control plasmid, N3-ICD, WWP2, or catalytically inactive mutant WWP2-C838A (WWP2-CA). Proliferation significantly decreased in both cell lines when WWP2 was ectopically expressed as compared to cells transfected with the control plasmid or cells expressing the WWP2-CA (Fig. 6A). We also determined if N3-ICD could reverse the anti-proliferative effect imposed by WWP2 by co-transfecting cells with N3-ICD and WWP2 expression plasmids. Our data demonstrated that N3-ICD counteracted the growth-inhibitory effect of WWP2, so the growth curve of cells co-transfected with N3-ICD and WWP2 was comparable to the curve of cells transfected with control plasmid (Fig. 6A). Using a Notch signaling reporter assay, we also observed reduced endogenous Notch signaling activity when WWP2 was expressed in MCF7 and OVCAR3 cells (Fig. 6B). Expression of the enzymatically inactive mutant WWP2-C838A in MCF7 and OVCAR3 cells also reduced Notch signaling activity, however to a lesser extent. To confirm the phenotypes observed in cancer cell lines, we performed experiments in primary ovarian tumor cultures. WWP2 cDNA or control vector was co-transfected with a Notch signaling reporter, pJH23A, into primary cell cultures and Notch signaling activity of WWP2 cDNA -transfected group was measured and the data was normalized to the data obtained from the control vector-transfected group. The normalized data were plotted against Notch3 expression levels measured in the same tumor samples (Fig. S5A). The result demonstrated that ectopic expression of WWP2 potently suppressed Notch signaling in cells with high levels of Notch3 (reflecting by greater reduction of Notch signaling activity in the Notch3-high cells) (Fig. S5B).


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 regulates Notch3 signaling activity in cancer cells.(A) OVCAR3 or MCF7 cancer cells were transfected with control plasmid pLPC, WWP2, catalytically-inactive WWP2 (WWP2-CA), N3-ICD, or WWP2+N3-ICD. Relative cell numbers were measured at different time points using the SYBE Green-based assay. Data are expressed as means ± SD and Student's t-test was performed to compare the relative cell number between WWP2+N3-ICD and pLPC (* p<0.05; ** p<0.01). (B) Different groups of cells were transfected with the pJH23A (4×wtCBF1Luc) luciferase reporter construct together with pLPC, WWP2, or the catalytically-inactive WWP2 plasmid (WWP2-CA). Each experiment was performed in triplicate. Results are shown as percentage of luciferase activity compared to pLPC transfected controls. Data are expressed as means ± SD. P values were calculated by comparison between either WWP2 or WWP2-CA and the control plasmid pLPC (** p<0.01; *** p<0.001). (C) Two untransformed epithelial cell lines were transfected with WWP2 siRNA or scrambled siRNA (siSCR). Notch signaling was measured by co-transfecting the cells with a luciferase reporter plasmid, pJH23A (4×wtRBPJLuc) and the data were normalized to the luciferase activity measured by co-transfection with a control plasmid, pGL3. Data are expressed as means ± SD and statistical significance was assessed by two-tailed Student's t-test.
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Related In: Results  -  Collection

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pgen-1004751-g006: WWP2 regulates Notch3 signaling activity in cancer cells.(A) OVCAR3 or MCF7 cancer cells were transfected with control plasmid pLPC, WWP2, catalytically-inactive WWP2 (WWP2-CA), N3-ICD, or WWP2+N3-ICD. Relative cell numbers were measured at different time points using the SYBE Green-based assay. Data are expressed as means ± SD and Student's t-test was performed to compare the relative cell number between WWP2+N3-ICD and pLPC (* p<0.05; ** p<0.01). (B) Different groups of cells were transfected with the pJH23A (4×wtCBF1Luc) luciferase reporter construct together with pLPC, WWP2, or the catalytically-inactive WWP2 plasmid (WWP2-CA). Each experiment was performed in triplicate. Results are shown as percentage of luciferase activity compared to pLPC transfected controls. Data are expressed as means ± SD. P values were calculated by comparison between either WWP2 or WWP2-CA and the control plasmid pLPC (** p<0.01; *** p<0.001). (C) Two untransformed epithelial cell lines were transfected with WWP2 siRNA or scrambled siRNA (siSCR). Notch signaling was measured by co-transfecting the cells with a luciferase reporter plasmid, pJH23A (4×wtRBPJLuc) and the data were normalized to the luciferase activity measured by co-transfection with a control plasmid, pGL3. Data are expressed as means ± SD and statistical significance was assessed by two-tailed Student's t-test.
Mentions: To determine whether reconstitution of WWP2 expression in cancer cells resulted in reducing Notch3 signaling activity and subsequently cellular proliferation, we performed proliferation assays in OVCAR3 and MCF7 cells transduced with pLPC control plasmid, N3-ICD, WWP2, or catalytically inactive mutant WWP2-C838A (WWP2-CA). Proliferation significantly decreased in both cell lines when WWP2 was ectopically expressed as compared to cells transfected with the control plasmid or cells expressing the WWP2-CA (Fig. 6A). We also determined if N3-ICD could reverse the anti-proliferative effect imposed by WWP2 by co-transfecting cells with N3-ICD and WWP2 expression plasmids. Our data demonstrated that N3-ICD counteracted the growth-inhibitory effect of WWP2, so the growth curve of cells co-transfected with N3-ICD and WWP2 was comparable to the curve of cells transfected with control plasmid (Fig. 6A). Using a Notch signaling reporter assay, we also observed reduced endogenous Notch signaling activity when WWP2 was expressed in MCF7 and OVCAR3 cells (Fig. 6B). Expression of the enzymatically inactive mutant WWP2-C838A in MCF7 and OVCAR3 cells also reduced Notch signaling activity, however to a lesser extent. To confirm the phenotypes observed in cancer cell lines, we performed experiments in primary ovarian tumor cultures. WWP2 cDNA or control vector was co-transfected with a Notch signaling reporter, pJH23A, into primary cell cultures and Notch signaling activity of WWP2 cDNA -transfected group was measured and the data was normalized to the data obtained from the control vector-transfected group. The normalized data were plotted against Notch3 expression levels measured in the same tumor samples (Fig. S5A). The result demonstrated that ectopic expression of WWP2 potently suppressed Notch signaling in cells with high levels of Notch3 (reflecting by greater reduction of Notch signaling activity in the Notch3-high cells) (Fig. S5B).

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