MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR-182 and miR-203 in our previously described prostate cell model. MiR-182 and miR-203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re-expression of miR-182 or miR-203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR-182 and miR-203 provided EPT1 cells with the ability to self-sufficiency of growth signals, a well-recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR-182 and miR-203. SNAI2 was identified as a common target of miR-182 and miR-203. Knock-down of SNAI2 in EPT1 cells phenocopied reexpression of either miR-182 or miR-203 regarding both MET and self-sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re-expression of miR-182/203 and knock-down of SNAI2. Finally, P-cadherin was identified as a direct target of SNAI2. We conclude that miR-182 and miR-203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR-182/203 in cancer related processes.The breakdown of epithelial cell homeostasis with the loss of epithelial characteristics and the acquisition of a migratory phenotype, referred to as epithelial to mesenchymal transition (EMT) is a crucial event in tumor progression and endows cancer cells with invasive and metastatic competence.1 Prostate cancer is one of the most common male cancers in Western countries. In an attempt to transform benign prostate cells to malignant ones, we have observed typical EMT features in primary prostate basal EP156T cells.2 However, both EP156T and the progeny EPT1 cells lacked malignant features such as anchorage independent growth, resistance to apoptosis and independence of external growth factors. 2,3 This unique EMT cell model provides a good opportunity to understand the mechanisms of EMT and malignant transformation in vitro.MicroRNAs (miRNAs/miRs) are a large class of endogenous tiny regulatory RNAs that extensively regulate gene expression. MiRNAs function post-transcriptionally through imperfect base pairing with specific sequences in the 3 0 untranslated regions (UTRs) of target mRNAs leading to transcript degradation or translational inhibition. 4 The role of miRNAs depends not only on their specific mRNA targets but also upon their context, such as cell type, tissue or stage of development. MiRNAs are widely recognized as important in tumor progression, it has been considered that alterations in the expression of miRNAs might contribute to the pathogenesis of most human malignancies.5 However, most of these studies utilized cancer cells, examination of the function of miRNAs in premalignant cells is thus far limited.Exploiting our unique premalignant EPT1 cell model that ...

The transcription factor p63 is central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT. Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal type cells of the model led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses showed that ΔNp63α induced gene modules involved in both cell-to-cell and cell-to-extracellular-matrix junctions in mesenchymal type cells. Genome-wide analysis of p63 binding sites using ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. DH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with ΔNp63α to reverse completely the mesenchymal phenotype. The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of ΔNp63α expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.

BackgroundExpression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.MethodsImmortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.ResultsA variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).ConclusionsRegulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users.

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.

Background: p63 is a transcription factor that is central for epithelial homeostasis and development. In our cell culture model of epithelial to mesenchymal transition (EMT) in a human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. This led us to investigate the role of p63 in EMT by a gain and loss of function approach. Results: Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal EPT1 and EPT2 cells led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). When p63 was knocked down in epithelial EP156T cells complementary results were observed. Global gene expression analyses found that ΔNp63α induced gene modules involving cell adhesion genes in mesenchymal like cells. ChIP-seq analyses confirmed the enriched binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. CDH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knockdown of these factors, respectively, were not sufficient alone or in combination with ΔNp63α for full reversal of the mesenchymal phenotype in EPT cells. Conclusion: The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The potential of reduced EMP in order to counteract metastasis and the utility of ΔNp63α expression in this regard may provide a novel strategy to counteract the metastatic process. Citation Format: Jan Roger Olsen, Anne Margrete Øyan, Kari Rostad, Margrete R. Hellem, Jie Liu, Lisha Li, David Micklem, Hallvard Haugen, James B. Lorens, Xisong Ke, Varda Rotter, Biaoyang Lin, Karl-Henning Kalland. TP63 regulation of epithelial to mesenchymal transition in an experimental prostate cell model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1820. doi:10.1158/1538-7445.AM2013-1820