Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

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A novel method for the synthesis of nonsymmetrical 3,3'-(aryl/alkyl-methylene)bis-2-hydroxy-1,4-naphthoquinones was developed by using the Mannich adduct of naphthoquinone and the reaction with another moiety of 2-hydroxy-1,4-naphthoquinone. This novel method produces for the first time nonsymmetrical 3,3'-(aryl/alkyl-methylene)bis-2-hydroxy-1,4-naphthoquinones. In a preliminary study, these compounds (15c, 15f and 15h) were evaluated regarding their effect over the viability of PC3 metastatic prostate cancer cell using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays at 100 μM. Three of these compounds presented relevant cytotoxic effects at 72 h posttreatment.

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