Abstract 2329: Restriction of androgen receptor and target gene expression

Abstract

Abstract Background: The androgen receptor (AR) is activated and pathogenic in castration resistant prostate cancer (CRPC) due to a variety of escape mechanisms from anti-androgen treatment. The “cell of origin” of prostate cancer is not finally defined, but in different models prostate epithelial basal cells have been shown to develop into cancer with authentic prostate cancer features. The AR and target genes are effectively silenced in prostate epithelial basal cells. Given the importance of activated AR in prostate cancer we performed a reassessment of the basis of its silencing in prostate epithelial basal cells. Results: EP156T cells are derived from primary prostate epithelial basal cells and were propagated in monolayer cultures with features of transit amplifying cells in medium with low calcium concentration. The AR was not detectable in these cells and neither the AR nor target genes such as KLK3 (PSA), TMPRSS2 and NKX3-1 were induced following treatment with synthetic androgen (R1881) and a variety of growth factors and combinations as determined using sensitive Taqman real-time quantitative PCR assays, gene expression microarrays of cell lysates and PSA assays of cell supernatants. This was in contrast to LNCaP control cells where KLK3 was 12-fold and TMPRSS2 was 8-fold increased following R1881 induction. EP156T cells underwent epithelial to mesenchymal transition (EMT), and a series of progeny cells with an accumulating number of malignant hallmarks were derived. In these cells a spontaneous, but limited increase in AR mRNA was found. But the cells were neither androgen responsive nor androgen dependent as determined using R1881 stimulation and bicalutamide inhibitor, respectively. Shift to high calcium concentration in the medium rapidly induced morphological differentiation of basal cells, but still the AR and AR target gene expression was restricted. In 3-dimensional cultures in Matrigel structures with a distinct outer cell layer versus different inner cells were observed. A variety of methods was used to explore the molecular basis of restricted AR and target gene expression in these different culture conditions, including chromatin immunoprecipitation, indirect immunofluorescence, fluorescent promoter reporter constructs, gene expression and Western blot assays. Conclusion: In human prostate epithelial cells with basal cell traits we found strong restrictions to expression of the AR and AR target genes in monolayer cultures with a large variety of growth factors and combinations, including androgens. High calcium concentration induced morphological changes associated with epithelial differentiation, but with restricted AR and target gene expression. Growth of epithelial cells in Matrigel was associated with evidence of morphological differentiation. This model is useful for further identification of the molecular basis of restricted AR and target gene expression in prostate cells in comparison with malignant prostate cells. Citation Format: Margrete R. Hellem, Jan Roger Olsen, Yaping Hua, Yi Qu, Kristo Marvyin, Kari Rostad, Jie Liu, Lisha Li, Varda Rotter, Biaoyang Lin, Xisong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Restriction of androgen receptor and target gene expression. [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 2329. doi:10.1158/1538-7445.AM2014-2329