Abstract 4483: TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells

Abstract

Total osteopontin (OPN) has been described as a master regulator of epithelial-mesenchymal transition (EMT), including in prostate cancer (PCa). However, the contribution of each OPN splicing isoform (OPN-SI), named OPNa, OPNb, and OPNc in the epithelial plasticity dynamic process is currently unknown. Besides, the contribution of alternative splicing regulation in this scenario is poorly understood. Here we aimed to investigate the expression of OPN-SI and the associated splicing pattern regulation in response to metastatic prostate cancer (PCa) cells treatment with TGF-β . Further, we aimed to investigate the resulting cell phenotypic features. To achieve this, we treated PC3 cells with TGF-β 10ng/ml for 48 h as a model to induce epithelial plasticity in these cells. Total RNA was extracted and the corresponding cDNA was tested for transcriptional levels of OPN-SI, SR and HnRNP splicing regulators as well as classical (EMT) markers by using real time PCR. EMT markers and cell morphology have also been analyzed by immunofluoresce. PC3 cell phenotype has also been evaluated by using cell migration and viabilitity assays in response to β-lapachone cytotoxic reagent. PC3 cells treated with TGF-β displayed an intermediate EMT phenotype, as evidenced by a significant downregulation of both epithelial (E-cadherin, claudin-3 andcytokeratin 18) and mesenchymal markers (vimentin, N-cadherin, snailandslug). These cells presented a higher cytoplasmic E-cadherin expression, as well as a lower vimentin cytoplasmic staining. A major cortical distribution of actin filaments beneath the cell cortex has been observed. Further, a significant downregulation of OPNa and mainly, OPNb and OPNc isoforms, as well as most tested splicing regulators has been shown. TGF-β treatment also promoted a significant inhibition on cell migration as well as higher sensitivity to β-lapachone treatment, as compared to control cells. Altogether, our data evidence that in our in vitro experimental model, TGF-β promoted a an intermediate EMT phenotype, with a predominance of epithelial features, associated to downregulation of OPN-SI and most tested splicing regulators. We then hypothesize that TGF-β may also be an EMT-like inducer, in which metastatic PCa cells could acquire major epithelial features that facilitate cell anchorage and adhesion in distant tumor sites. OPN-SI dowregulation in this process could favour epithelial-like features, as apposed to OPN-SI overexpression during tumor progression towards metastic circulating cells typically displaying mesechymal phenotypes. Citation Format: Durval Marques, Rodrigo A. Peres, Paula P. de Freitas, Abigail C. Resende, Nataly dos Santos Melo, Waldemir Fernandes de Souza, José A. Morgado-Diaz, Etel Rodrigues Pereira Gimba. TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2017-4483