Abstract 1346: Osteopontin-b and Osteopontin-c splicing isofoms activate prostate cancer cells prosurvival features

Abstract

Abstract Alternative splicing of the osteopontin (OPN) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb and OPNc. We previously demonstrated that both OPNb e OPNc are able to activate typical features of prostate cancer (PCa) progression. These data suggested that some of these tumorigenic roles are mediated by the activation of pro-survival pathways in PCa cells. The aim of this study was to delineate molecular and signaling pathways by which these two isoforms are able to mediate PCa cell survival and growth. PC3 cells overexpressing the three OPN-SI were treated with 1x10-6μg/mL of docetaxel (DXT), which was used as an in vitro model to induce cell death. Cell survival was analyzed by phase-contrast microscopy, crystal violet staining and MTT assays, in the presence or not of PI3K/Akt specific inhibitor (LY294002). Cell death has been investigated by immunoblot and flow citometry. Quantitative real time PCR assays (qRT-PCR) and immunofluorescence evaluated the involvement of epithelial mesenchymal transition (EMT) on PC3 cell survival in response to DXT treatment. PC3 cells overexpressing OPNb or OPNc treated with DXT have a higher cell density and phosphorylated AKT (Ser473) expression level compared to control cells. An additional increase in Akt (Ser473) phosphorylation was also observed after DXT treatment. PC3 cells overexpressing OPNb or OPNc isoforms also promoted additional resistance to cell death induced by DXT treatment, as compared to cells overexpressing OPNa. Combined treatment with LY294002 and DXT significantly inhibited PC3 cell survival mediated by the overexpression of OPNb and OPNc isoforms. Cells overexpressing the 3 OPN-SI similarly express Bcl-2, activated caspase 3 and 7 and also similar annexin V staining index. However, PC3 overexpressing OPNb or OPNc treated with DXT differentially express Mcl-1 and Bim. Immunofluorescence and qRT-PCR assays demonstrated that PC3 cells overexpressing these two splice variants significantly down-regulate the epithelial and upregulate the mesenchymal EMT markers, indicating that activation of this pathway is one of the mechanisms modulating PC3 cell survival in response to DXT treatment. As a whole, our data indicate important roles for PI3K/Akt and EMT signaling, besides BL2-family members differential expression on modulating PC3 pro-survival features in response to OPNb e OPNc overexpression. Citation Format: Kivvi Duarte Mello, Tatiana Martins Tilli, Ana Carolina S. Ferreira, Claudete Esteves Klumb, Luiz Eurico Nasciutti, Etel Rodrigues Pereira Gimba. Osteopontin-b and Osteopontin-c splicing isofoms activate prostate cancer cells prosurvival features. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1346. doi:10.1158/1538-7445.AM2014-1346