Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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WWOX interacts with SMAD3 and modulates its transcriptional activity in breast cells

ABSTRACT: WWOX expression is lost during tumor progression in many human malignancies including breast cancer. To understand the effects of loss of WWOX expression we analyzed the consequences of its silencing in normal human breast cells (MCF10F). WWOX silencing led to the formation of larger cell colonies, increased cell motility and decreased cell attachment. WWOX silenced cells demonstrated deregulated expression on genes involved in cell cycle, DNA damage response and cell motility. We detected an enrichment of targets activated by the SMAD3 transcription factor. Most notably expression of ANGPTL4, FST, PTHLH and SERPINE1 were all significantly increased upon WWOX silencing. Upregulation of these genes can be reversed by re-expressing WWOX in the previously silenced cells thus suggesting an inverse correlation between WWOX protein expression and SMAD3 transcriptional activity. Importantly, we demonstrate that WWOX physically interacts with SMAD3 protein via WW domain 1, that WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFM-NM-2 responsive reporter (3TP-LUX). Furthermore, WWOX expression leads to intracellular redistribution of SMAD3 protein levels redirecting protein availability from the nuclear to the cytoplasmic compartment. Interestingly, meta-analysis of gene expression breast cancer datasets indicate that WWOX and ANGPTL4 expression, encoding a secreted protein of key relevance in breast cancer lung metastatic cells, are inversely correlated and the WWOXlo/ANGPTL4hi cluster of tumors are enriched in triple-negative and basal-like sub-types. In summary, we demonstrate that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFM-NM-2 signaling in advanced breast cancer. We compared two independent shRNAs: shWWOX-A and shWWOX-B with 3 biological replicates each one, targeting different regions of the WWOX transcript as a means of ruling out any potential off-target effects.

ORGANISM(S): Homo sapiens

SUBMITTER: C. Marcelo Aldaz

PROVIDER: E-GEOD-47371 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:WWOX expression is lost during tumor progression in many human malignancies including breast cancer. To understand the effects of loss of WWOX expression we analyzed the consequences of its silencing in normal human breast cells (MCF10F). WWOX silencing led to the formation of larger cell colonies, increased cell motility and decreased cell attachment. WWOX silenced cells demonstrated deregulated expression on genes involved in cell cycle, DNA damage response and cell motility. We detected an enrichment of targets activated by the SMAD3 transcription factor. Most notably expression of ANGPTL4, FST, PTHLH and SERPINE1 were all significantly increased upon WWOX silencing. Upregulation of these genes can be reversed by re-expressing WWOX in the previously silenced cells thus suggesting an inverse correlation between WWOX protein expression and SMAD3 transcriptional activity. Importantly, we demonstrate that WWOX physically interacts with SMAD3 protein via WW domain 1, that WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter (3TP-LUX). Furthermore, WWOX expression leads to intracellular redistribution of SMAD3 protein levels redirecting protein availability from the nuclear to the cytoplasmic compartment. Interestingly, meta-analysis of gene expression breast cancer datasets indicate that WWOX and ANGPTL4 expression, encoding a secreted protein of key relevance in breast cancer lung metastatic cells, are inversely correlated and the WWOXlo/ANGPTL4hi cluster of tumors are enriched in triple-negative and basal-like sub-types. In summary, we demonstrate that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling in advanced breast cancer.

Project description:Although numerous genetic loci have been associated with coronary artery disease (CAD) with genome wide association studies (GWAS), efforts are needed to identify the causal genes in these loci and link them into fundamental signaling pathways. Toward that end, experiments reported here extend our investigation of the disease mechanism of CAD associated gene SMAD3, a central transcriptional intermediate in the TGFb pathway, investigating its role in smooth muscle biology. In vitro studies in human coronary artery smooth muscle cells (HCASMC) revealed that SMAD3 modulates cell state decisions in this cell type, promoting expression of differentiation marker genes and migration while inhibiting proliferation. RNA and chromatin immunoprecipitation sequencing (ChIPseq) studies in HCASMC identified downstream genes that reside in pathways which mediate vascular development and disease processes, including those related to atherosclerosis pathophysiology, expanding understanding of the TGFb canonical pathway in this cell type. ChIPseq studies also found colocalization of SMAD3 binding in loci targeted by TCF21, a CAD associated transcription factor that has been shown to produce a CAD protective de-differentiation program in HCASMC. In loci where these factors are juxtaposed on DNA, SMAD3 binding was anti-correlated with TCF21, and increased in cells depleted of TCF21, as shown by ChIPseq and ChIP experiments. Further, reporter gene studies revealed that while SMAD3 increased transcription at a SERPINE1 enhancer, and this effect was blocked by TCF21. Together, these data suggest that SMAD3 regulation of gene expression is modulated by TCF21, through independent regulation of jointly occupied genes and through epigenetic and possibly direct protein-protein interactions. Finally, eQTL studies in HCASMC indicated that SMAD3 expression is directly associated with increased disease risk, opposing the known protective effect of TCF21. We propose that the pro-differentiation function of SMAD3 inhibits HCASMC dedifferentiation of these cells as they respond to vascular stresses and expand and migrate to stabilize the plaque, and that SMAD3 function is directly opposed at the transcriptional level by the disease protective expression of TCF21, which promotes dedifferentiation and phenotypic modulation.

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WWOX interacts with SMAD3 and modulates its transcriptional activity in breast cells

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