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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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Identification of Notch3 interacting proteins in protein array.(A) Ingenuity pathway analysis shows that Notch3 interaction proteins are enriched in a network involved in gene expression, cell cycle, and development. Colored symbols: Notch3-interacting proteins identified in our screen. UBC: ubiquitin C. (B) Western blot analysis of GST pull-down was performed with GST-peptide control or recombinant WWP2-GST in the presence of increasing concentrations of rhN3-ICD protein using anti-GST antibody or anti V5 antibody. Lower panel shows western blot analysis of flow through after GST pull-down. Reduced N3-ICD is seen only after pulldown with WWP2-GST but not with control GST-peptide. (C) In vitro ubiquitination experiment was performed using recombinant human Notch3 as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5B), and FLAG-tagged WWP2. Ubiquitinated species of rhN3-ICD were detected by immunoprecipitation with V5 antibody followed by western blot with anti-ubiquitin antibody (upper panel). Western blot analysis with anti-FLAG antibody or anti-V5 antibody was performed to detect total input amounts of WWP2 and N3-ICD (lower panels). Ubiquitinated rhN3-ICD was only detected when WWP2 was present.
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pgen-1004751-g001: Identification of Notch3 interacting proteins in protein array.(A) Ingenuity pathway analysis shows that Notch3 interaction proteins are enriched in a network involved in gene expression, cell cycle, and development. Colored symbols: Notch3-interacting proteins identified in our screen. UBC: ubiquitin C. (B) Western blot analysis of GST pull-down was performed with GST-peptide control or recombinant WWP2-GST in the presence of increasing concentrations of rhN3-ICD protein using anti-GST antibody or anti V5 antibody. Lower panel shows western blot analysis of flow through after GST pull-down. Reduced N3-ICD is seen only after pulldown with WWP2-GST but not with control GST-peptide. (C) In vitro ubiquitination experiment was performed using recombinant human Notch3 as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5B), and FLAG-tagged WWP2. Ubiquitinated species of rhN3-ICD were detected by immunoprecipitation with V5 antibody followed by western blot with anti-ubiquitin antibody (upper panel). Western blot analysis with anti-FLAG antibody or anti-V5 antibody was performed to detect total input amounts of WWP2 and N3-ICD (lower panels). Ubiquitinated rhN3-ICD was only detected when WWP2 was present.

Mentions: The purpose of this study was to identify and characterize proteins that interacted with the Notch3 intracellular cytoplasmic domain (N3-ICD). First, we expressed and purified human recombinant N3-ICD protein tagged with the V5 epitope (rhN3-ICD-V5). The rhN3-ICD-V5 protein was then used as a “bait” to screen for binding proteins on the human proteome microarray, comprised of 16,368 individually purified, full-length human proteins [9]. This screen identified a number of N3-ICD-interacting proteins. Among them, RBPJ (CSL), a well-known transcriptional co-factor of the Notch receptors, was at the top of the N3-ICD binding partner list and displayed a high fluorescent binding score (Table S1). This observation indicated that protein microarray is a valid approach to rapidly and reliably identify Notch3 interacting proteins. We next performed Ingenuity Pathway Analysis to search for enriched functional links among the newly identified N3-ICD interactome. The results demonstrated that the enriched functional networks include gene expression, cell cycle, cell signaling, and cellular development (Table S2). As illustrated in Fig. 1A, ubiquitin C (UBC) is a major hub in the top functional network, suggesting the involvement of ubiquitination in regulating and/or mediating Notch3 signaling.


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)

Identification of Notch3 interacting proteins in protein array.(A) Ingenuity pathway analysis shows that Notch3 interaction proteins are enriched in a network involved in gene expression, cell cycle, and development. Colored symbols: Notch3-interacting proteins identified in our screen. UBC: ubiquitin C. (B) Western blot analysis of GST pull-down was performed with GST-peptide control or recombinant WWP2-GST in the presence of increasing concentrations of rhN3-ICD protein using anti-GST antibody or anti V5 antibody. Lower panel shows western blot analysis of flow through after GST pull-down. Reduced N3-ICD is seen only after pulldown with WWP2-GST but not with control GST-peptide. (C) In vitro ubiquitination experiment was performed using recombinant human Notch3 as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5B), and FLAG-tagged WWP2. Ubiquitinated species of rhN3-ICD were detected by immunoprecipitation with V5 antibody followed by western blot with anti-ubiquitin antibody (upper panel). Western blot analysis with anti-FLAG antibody or anti-V5 antibody was performed to detect total input amounts of WWP2 and N3-ICD (lower panels). Ubiquitinated rhN3-ICD was only detected when WWP2 was present.
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pgen-1004751-g001: Identification of Notch3 interacting proteins in protein array.(A) Ingenuity pathway analysis shows that Notch3 interaction proteins are enriched in a network involved in gene expression, cell cycle, and development. Colored symbols: Notch3-interacting proteins identified in our screen. UBC: ubiquitin C. (B) Western blot analysis of GST pull-down was performed with GST-peptide control or recombinant WWP2-GST in the presence of increasing concentrations of rhN3-ICD protein using anti-GST antibody or anti V5 antibody. Lower panel shows western blot analysis of flow through after GST pull-down. Reduced N3-ICD is seen only after pulldown with WWP2-GST but not with control GST-peptide. (C) In vitro ubiquitination experiment was performed using recombinant human Notch3 as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5B), and FLAG-tagged WWP2. Ubiquitinated species of rhN3-ICD were detected by immunoprecipitation with V5 antibody followed by western blot with anti-ubiquitin antibody (upper panel). Western blot analysis with anti-FLAG antibody or anti-V5 antibody was performed to detect total input amounts of WWP2 and N3-ICD (lower panels). Ubiquitinated rhN3-ICD was only detected when WWP2 was present.
Mentions: The purpose of this study was to identify and characterize proteins that interacted with the Notch3 intracellular cytoplasmic domain (N3-ICD). First, we expressed and purified human recombinant N3-ICD protein tagged with the V5 epitope (rhN3-ICD-V5). The rhN3-ICD-V5 protein was then used as a “bait” to screen for binding proteins on the human proteome microarray, comprised of 16,368 individually purified, full-length human proteins [9]. This screen identified a number of N3-ICD-interacting proteins. Among them, RBPJ (CSL), a well-known transcriptional co-factor of the Notch receptors, was at the top of the N3-ICD binding partner list and displayed a high fluorescent binding score (Table S1). This observation indicated that protein microarray is a valid approach to rapidly and reliably identify Notch3 interacting proteins. We next performed Ingenuity Pathway Analysis to search for enriched functional links among the newly identified N3-ICD interactome. The results demonstrated that the enriched functional networks include gene expression, cell cycle, cell signaling, and cellular development (Table S2). As illustrated in Fig. 1A, ubiquitin C (UBC) is a major hub in the top functional network, suggesting the involvement of ubiquitination in regulating and/or mediating Notch3 signaling.

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