Unknown,Transcriptomics,Genomics,Proteomics

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Chromosomal Fragile Site FRA16D tumor suppressor gene Wwox contributes to aerobic metabolism and oxidative stress response

ABSTRACT: The WWOX gene spans chromosomal fragile site FRA16D, a region of DNA instability in cancer. While WWOX has some tumor suppressor characteristics, its normal role and functional contribution to cancer are unclear. Drosophila homozygous Wwox mutants are viable with no discernable phenotype. Drosophila Wwox interactors, identified by proteomics and micro-array analyses, mainly have roles in aerobic metabolism. Functional relationships between Wwox and either isocitrate dehydrogenase (IDH) or superoxide dismutase 1 (Sod1) were confirmed by phenotype modification, including Sod1 âcrinkled-wingâ, indicative of oxidative stress response. Endogenous reactive oxygen species levels reflect Wwox levels in Drosophila. WWOX mRNA levels in Drosophila and human cells correlate with IDH and Sod1 levels. Wwox therefore contributes to pathways involving glucose metabolism and oxidative stress response. Drosophila embryos were harvested between 4-8hrs and total RNA isolated to look for gene-expression differences induced by the presence of one of two altered Wwox alleles relative to wild-type. The two alleles were Wwox (a null mutation generated by homologous recombination) and Wwoxf04545 carrying a pBac insertion in exon 2 (OâKeefe et al., 2005, 2007). Embryo pools were isolated in triplicate, providing 3 biological replicates.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Gareth Price

PROVIDER: E-GEOD-22689 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

O'Keefe Louise V LV Colella Alex A Dayan Sonia S Chen Qingwen Q Choo Amanda A Jacob Reuben R Price Gareth G Venter Deon D Richards Robert I RI

Human molecular genetics 20101112 3

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a signif ...[more]

PMID: 21075834

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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: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.

Dataset Information

Chromosomal Fragile Site FRA16D tumor suppressor gene Wwox contributes to aerobic metabolism and oxidative stress response

Publications

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

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