Abstract B58: OPNc and OPNb osteopontin splicing isofoms activate prostate cancer prosurvival features through PI3K/AKT/BIM signaling pathway

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 pro-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 treated with docetaxel were used as an in vitro cell model to induce cell death. We then investigated the involvement of PI3K/Akt signaling and anti e pro-apoptotic proteins on inducing PC3 cell survival on cells overexpressing each OPN-SI. PC3 cells overexpressing the three OPN-SI were treated with 1×10-6μg/mL of docetaxel and then analysed for cell proliferation and viability using crystal violet and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) MMT assays, respectively. PC3 whole protein cell extracts were prepared to evaluate protein expression of members of PI3K/Akt and cell death signaling. PI3K/Akt signaling was analysed by immunoblot tests using specific antibodies against total and phosphorylated (ser473) Akt. The expression of apoptotic proteins was investigated by using Bcl-2, Mcl-1 e Bim specific antibodies. LY294002 was used as an specific inhibitor of PI3K signaling. PC3 cells were incubated with LY294002 in combination or not with docetaxel treatment in order to evaluate cell survival pathways. Cells overexpressing OPNb and OPNc isoforms presented a significant increase of Akt (Ser473) phosphorylation, as compared to cells overexpressing OPNa and empty vector control. An additional increase in Akt (Ser473) phosphorylation was also observed after docetaxel treatment. PC3 cells overexpressing OPNb and OPNc isoforms also promoted additional resistance to cell death induced by docetaxel treatment, as compared to cells overexpressing OPNa. Combined treatment with LY294002 and docetaxel significantly inhibited PC3 cell survival mediated by the overexpression of OPNb and OPNc isoforms. The expression of Bcl-2 protein was similar among cells overexpressing the three OPN-SI, before and after docetaxel treatment. On the other hand, cells overexpressing OPNb e OPNc presented significant reduction on the expression of Mcl-1. Cells overexpressing OPNc, but not OPNb, presented a significant inhibition of pro-apoptotic protein Bim. As a whole, our data indicate an important role of PI3K/Akt signaling activation and inhibition of pro-apoptotic proteins on the survival of PC3 cells as a result of OPNb e OPNc overexpression. Citation Format: Kivvi Duarte Mello, Tatiana Martins Tilli, Ana Carolina S. Ferreira, Claudete Esteves Klumb, Etel Rodrigues Pereira Gimba. OPNc and OPNb osteopontin splicing isofoms activate prostate cancer prosurvival features through PI3K/AKT/BIM signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B58.