Abstract 1475: Molecular mechanisms of “SNAIL” activation in invasive bladder cancer of p53/Rb double knockout mice exposed to chemical carcinogen BBN

Epithelial-mesenchymal transition (EMT) plays a central role in empowering tumor cells to acquire invasive properties. It appears that EMT is not a random process but is highly regulated by transcription regulators. Of particular interests are the transcription repressors that inhibit the expression of cell-cell junction proteins, such as E-cadherin. Recent in vitro studies indicate that increased signaling of “Snail” the transcription repressor per se is sufficient to confer an invasive phenotype on otherwise non-invasive cells. Despite these recent advances, relatively little is known about the mechanisms of Snail activation in invasive bladder cancer. In this study, using p53 and Rb double knockout mice exposed the carcinogen BBN, we examined the expression levels of “Snail” and E-cadherin protein in bladder cancer tissues. Findings from both immunohistochemical and immunofluorescence staining revealed overexpression of Snail in the stromal as well as in the tumor regions. Although a complete loss of E-cadherin was not evident in all the tumors examined, most of the sections analyzed showed a moderate to weak expression of E-cadherin. This is consistent with the findings from Western blot analysis of the protein lysate from human bladder cancer cells with varying status of p53 and Rb. To explore the mechanism of Snail activation leading invasive bladder cancer, we transfected the human bladder cancer cells with siRNAs for Snail, p53 and Rb. Our findings were consistent with the in vivo studies, in that Western blot analysis revealed only a moderate inhibition of E-cadherin in p53 or Rb knockout cells. However, the Snail knockout cells showed less invasiveness (migration) compared to the control, and was associated with a significant inhibition of E-cadherin expression. To investigate further on the possible role of TGFβ on Snail activation, we treated the bladder cancer cells with TGFβ for 12h and examined the effect on Smad 4 and 7, Snail and E-cadherin expression. Activation of Snail was associated the expression of vimentin, a typical marker of mesenchymal cells, and cytokeratins of epithelial cells suggesting TGFβ mediated Snail activation. Finally, we extended our analysis to determine the role of noncoding microRNAs that act as post-transcriptional repressors. To this end we performed microRNA profiling of J82 and HT1376 bladder cancer cells using human miRNA arrays. Findings on differential expression of potential microRNA network involved in the regulation of TGFβ, Snail and E-cadherin expression associated with urothelial tumor plasticity are discussed. Overall findings from these studies suggest possible mechanism(s) of Snail activation and E-cadherin regulation in invasive bladder cancer for the first time and may pave the way for novel therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1475.