Limits...
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.

Show MeSH

Related in: MedlinePlus

WWP2 is down-regulated in ovarian carcinomas.(A) Analysis of DNA copy number at the WWP2 locus and WWP2 transcript expression levels in TCGA's ovarian HGSC dataset. Among 554 HGSCs, heterozygous allele loss (hetloss) was observed in 410 tumors and homozygous deletion in 18 cases (homdel). (B) WWP2 and Notch3 protein expression in ovarian cancer cell lines (HGSC) and in non-transformed epithelial cell cultures established from human gynecologic organs. These include endometrial epithelial cells (EME), ovarian surface epithelial cells (OSE), and fallopian tube epithelial cells (FTE). GAPDH is included as the loading control. (C) Band intensities of WWP2 and Notch3 from immunoblotting were individually quantified and normalized to GAPDH for each sample. The ratio of Notch3 and WWP2 protein expression was determined. The ratio is higher in ovarian cancer groups (HGSC) than in normal epithelial cell group. Two-tailed Man-Whitney U test was performed to determine the level of significance. (D and E) Correlations of protein expression between WWP2 and Notch3 in 51 ovarian cancer tissue samples (D) and in 17 primary cultures of ovarian cancer cells (E). There is an inverse correlation between Notch3 expression and WWP2 expression with a Pearson's correlation coefficient of −0.36 and p<0.01 for tumor tissue samples and a Pearson's correlation coefficient of −0.45 and p<0.05 for primary cultures.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214668&req=5

pgen-1004751-g005: WWP2 is down-regulated in ovarian carcinomas.(A) Analysis of DNA copy number at the WWP2 locus and WWP2 transcript expression levels in TCGA's ovarian HGSC dataset. Among 554 HGSCs, heterozygous allele loss (hetloss) was observed in 410 tumors and homozygous deletion in 18 cases (homdel). (B) WWP2 and Notch3 protein expression in ovarian cancer cell lines (HGSC) and in non-transformed epithelial cell cultures established from human gynecologic organs. These include endometrial epithelial cells (EME), ovarian surface epithelial cells (OSE), and fallopian tube epithelial cells (FTE). GAPDH is included as the loading control. (C) Band intensities of WWP2 and Notch3 from immunoblotting were individually quantified and normalized to GAPDH for each sample. The ratio of Notch3 and WWP2 protein expression was determined. The ratio is higher in ovarian cancer groups (HGSC) than in normal epithelial cell group. Two-tailed Man-Whitney U test was performed to determine the level of significance. (D and E) Correlations of protein expression between WWP2 and Notch3 in 51 ovarian cancer tissue samples (D) and in 17 primary cultures of ovarian cancer cells (E). There is an inverse correlation between Notch3 expression and WWP2 expression with a Pearson's correlation coefficient of −0.36 and p<0.01 for tumor tissue samples and a Pearson's correlation coefficient of −0.45 and p<0.05 for primary cultures.

Mentions: We have previously demonstrated Notch3 gene amplification and over-expression in ovarian high-grade serous carcinoma (HGSC) [4]. If WWP2 is a negative regulator of Notch3, its expression is expected to be down-regulated in ovarian cancer as compared to normal tissues. To test this hypothesis, we examined the copy number alteration and expression pattern of WWP2 in ovarian HGSCs using a large summarized TCGA dataset [14]. In the 554 ovarian HGSCs that have available copy number data, the majority (77.3%) of HGSCs harbored deletions at the WWP2 locus, including 410 hemizygous and 18 homozygous deletions (Fig. 5A). In addition, based on a GISTIC copy number analysis in ovarian HGSC samples, the WWP2 locus is in a significantly deleted region (q-value = 10−13, Fig. S3) [15]. To define the minimal region of homozygous deletion- a strategy commonly used to identify potential deleted tumor suppressor gene, we aligned the homozygously deleted regions of the 18 HGSC samples and found that the region spans from 69,942,965 bp to 69,976,479 bp on chromosome 16 (hg19), which only encompasses WWP2 gene. To determine if loss of WWP2 DNA copy number affects WWP2 transcription, we determined the relationship between the gDNA and mRNA copy numbers of WWP2 using all HGSCs available in the TCGA. The result demonstrated a significant positive correlation between WWP2 gDNA copy number and WWP2 mRNA expression levels in HGSCs (Fig. S4, Pearson's r = 0.6863, p<0.0001).


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 is down-regulated in ovarian carcinomas.(A) Analysis of DNA copy number at the WWP2 locus and WWP2 transcript expression levels in TCGA's ovarian HGSC dataset. Among 554 HGSCs, heterozygous allele loss (hetloss) was observed in 410 tumors and homozygous deletion in 18 cases (homdel). (B) WWP2 and Notch3 protein expression in ovarian cancer cell lines (HGSC) and in non-transformed epithelial cell cultures established from human gynecologic organs. These include endometrial epithelial cells (EME), ovarian surface epithelial cells (OSE), and fallopian tube epithelial cells (FTE). GAPDH is included as the loading control. (C) Band intensities of WWP2 and Notch3 from immunoblotting were individually quantified and normalized to GAPDH for each sample. The ratio of Notch3 and WWP2 protein expression was determined. The ratio is higher in ovarian cancer groups (HGSC) than in normal epithelial cell group. Two-tailed Man-Whitney U test was performed to determine the level of significance. (D and E) Correlations of protein expression between WWP2 and Notch3 in 51 ovarian cancer tissue samples (D) and in 17 primary cultures of ovarian cancer cells (E). There is an inverse correlation between Notch3 expression and WWP2 expression with a Pearson's correlation coefficient of −0.36 and p<0.01 for tumor tissue samples and a Pearson's correlation coefficient of −0.45 and p<0.05 for primary cultures.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004751-g005: WWP2 is down-regulated in ovarian carcinomas.(A) Analysis of DNA copy number at the WWP2 locus and WWP2 transcript expression levels in TCGA's ovarian HGSC dataset. Among 554 HGSCs, heterozygous allele loss (hetloss) was observed in 410 tumors and homozygous deletion in 18 cases (homdel). (B) WWP2 and Notch3 protein expression in ovarian cancer cell lines (HGSC) and in non-transformed epithelial cell cultures established from human gynecologic organs. These include endometrial epithelial cells (EME), ovarian surface epithelial cells (OSE), and fallopian tube epithelial cells (FTE). GAPDH is included as the loading control. (C) Band intensities of WWP2 and Notch3 from immunoblotting were individually quantified and normalized to GAPDH for each sample. The ratio of Notch3 and WWP2 protein expression was determined. The ratio is higher in ovarian cancer groups (HGSC) than in normal epithelial cell group. Two-tailed Man-Whitney U test was performed to determine the level of significance. (D and E) Correlations of protein expression between WWP2 and Notch3 in 51 ovarian cancer tissue samples (D) and in 17 primary cultures of ovarian cancer cells (E). There is an inverse correlation between Notch3 expression and WWP2 expression with a Pearson's correlation coefficient of −0.36 and p<0.01 for tumor tissue samples and a Pearson's correlation coefficient of −0.45 and p<0.05 for primary cultures.
Mentions: We have previously demonstrated Notch3 gene amplification and over-expression in ovarian high-grade serous carcinoma (HGSC) [4]. If WWP2 is a negative regulator of Notch3, its expression is expected to be down-regulated in ovarian cancer as compared to normal tissues. To test this hypothesis, we examined the copy number alteration and expression pattern of WWP2 in ovarian HGSCs using a large summarized TCGA dataset [14]. In the 554 ovarian HGSCs that have available copy number data, the majority (77.3%) of HGSCs harbored deletions at the WWP2 locus, including 410 hemizygous and 18 homozygous deletions (Fig. 5A). In addition, based on a GISTIC copy number analysis in ovarian HGSC samples, the WWP2 locus is in a significantly deleted region (q-value = 10−13, Fig. S3) [15]. To define the minimal region of homozygous deletion- a strategy commonly used to identify potential deleted tumor suppressor gene, we aligned the homozygously deleted regions of the 18 HGSC samples and found that the region spans from 69,942,965 bp to 69,976,479 bp on chromosome 16 (hg19), which only encompasses WWP2 gene. To determine if loss of WWP2 DNA copy number affects WWP2 transcription, we determined the relationship between the gDNA and mRNA copy numbers of WWP2 using all HGSCs available in the TCGA. The result demonstrated a significant positive correlation between WWP2 gDNA copy number and WWP2 mRNA expression levels in HGSCs (Fig. S4, Pearson's r = 0.6863, p<0.0001).

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