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

FERGUSON BW; GAO X; ZELAZOWSKI MJ; LEE J; JETER CR; ABBA MC; ALDAZ CM

BMC CANCER

BIOMED CENTRAL LTD

Lugar: Londres; Año: 2013 vol. 13 p. 1 - 11

1471-2407

Background: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor inbreast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us toinvestigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells.Methods: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX(shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptionalregulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate adirect interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyseswere employed to determine the effect of WWOX silencing on TGFβ-signaling.Results: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genesinvolved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated bythe SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts.Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1.Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promotersand significantly quenches activation of a TGFβ responsive reporter. Additionally, WWOX expression leads toredistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFβ target ANGPTL4 plays akey role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX inmicroarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX andANGPTL4. Furthermore, the WWOXlo/ANGPTL4hi cluster of breast tumors is enriched in triple-negative and basal-likesub-types. Tumors with this gene expression signature could represent candidates for anti-TGFβ targeted therapies.Conclusions: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via directWW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOXexpression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activitythese observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling inadvanced breast cancer.