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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 suppresses tumor development of ovarian cancer cells.(A) Tumorigenic SKOV3-CR cells were transfected with either the plasmid encoding WWP2 cDNA or the control plasmid, pLPC. The expression of WWP2 was determined by Western blot. (B) Cells were injected into the right flanks of athymic nu/nu mice. The tumor size was measured by a caliper every three days. Two-tailed Student's t-test was used to determine the significance level. ** p<0.01; *** p<0.001 (C) Tumor weight was measured at the end point. Two-tailed Student's t-test was employed to determine the significance level.
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pgen-1004751-g007: WWP2 suppresses tumor development of ovarian cancer cells.(A) Tumorigenic SKOV3-CR cells were transfected with either the plasmid encoding WWP2 cDNA or the control plasmid, pLPC. The expression of WWP2 was determined by Western blot. (B) Cells were injected into the right flanks of athymic nu/nu mice. The tumor size was measured by a caliper every three days. Two-tailed Student's t-test was used to determine the significance level. ** p<0.01; *** p<0.001 (C) Tumor weight was measured at the end point. Two-tailed Student's t-test was employed to determine the significance level.

Mentions: To determine whether WWP2 expression affects tumor development, we ectopically expressed WWP2 in SKOV3-CR cells which were pre-established to develop carboplatin resistance (CR) phenotype and expressed abundant Notch3. SKOV3-CR cells transfected with the empty vector, pLPC, served as a control. The presence or absence of ectopic WWP2 expression in these transfected cells was validated by Western blot (Fig. 7A). A total of 3×106 cells were injected subcutaneously into the athymic nu/nu mice and tumor growth was monitored every three days. We observed that WWP2 expression led to a significant reduction in tumor formation and in fact as compared to the control group, tumors in the WWP2 expressing group were hardly detectable (Fig. 7B). Tumor weight measured at the endpoint of the study was also significantly lower in the WWP2-expressing group than in the control group (Fig. 7C).


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 suppresses tumor development of ovarian cancer cells.(A) Tumorigenic SKOV3-CR cells were transfected with either the plasmid encoding WWP2 cDNA or the control plasmid, pLPC. The expression of WWP2 was determined by Western blot. (B) Cells were injected into the right flanks of athymic nu/nu mice. The tumor size was measured by a caliper every three days. Two-tailed Student's t-test was used to determine the significance level. ** p<0.01; *** p<0.001 (C) Tumor weight was measured at the end point. Two-tailed Student's t-test was employed to determine the significance level.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004751-g007: WWP2 suppresses tumor development of ovarian cancer cells.(A) Tumorigenic SKOV3-CR cells were transfected with either the plasmid encoding WWP2 cDNA or the control plasmid, pLPC. The expression of WWP2 was determined by Western blot. (B) Cells were injected into the right flanks of athymic nu/nu mice. The tumor size was measured by a caliper every three days. Two-tailed Student's t-test was used to determine the significance level. ** p<0.01; *** p<0.001 (C) Tumor weight was measured at the end point. Two-tailed Student's t-test was employed to determine the significance level.
Mentions: To determine whether WWP2 expression affects tumor development, we ectopically expressed WWP2 in SKOV3-CR cells which were pre-established to develop carboplatin resistance (CR) phenotype and expressed abundant Notch3. SKOV3-CR cells transfected with the empty vector, pLPC, served as a control. The presence or absence of ectopic WWP2 expression in these transfected cells was validated by Western blot (Fig. 7A). A total of 3×106 cells were injected subcutaneously into the athymic nu/nu mice and tumor growth was monitored every three days. We observed that WWP2 expression led to a significant reduction in tumor formation and in fact as compared to the control group, tumors in the WWP2 expressing group were hardly detectable (Fig. 7B). Tumor weight measured at the endpoint of the study was also significantly lower in the WWP2-expressing group than in the control group (Fig. 7C).

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