Figure 4. Androgen Receptor (AR) directly represses Sox2 expression in castration-resistant CWR-R1 cells.

A) Western blot of a panel of non-malignant and prostate cancer cell lines for Sox2, Nanog, Oct4, and AR. β-Actin was used as a loading control. Expression of Sox2 in castration-resistant CWR cells is not accompanied by co-expression of Nanog or Oct4. LNCaP, C4-2B, LAPC-4, and MDA-PCa2B cells expressed detectable Nanog, which is presumably the NanogP8 retrogene (Jeter et al., 2011). The human embryonal carcinoma cell line NCCIT was used as a positive control for Sox2, Nanog, and Oct4 at a 1∶10 dilution. B) Decreased expression of Sox2 upon AR stimulation with physiologic levels of androgen (1 nM R1881) in castration-resistant CWR-R1 prostate cancer cells. Protein lysates from cells treated at defined intervals (3–48 hours) after androgen treatment were subjected to western blotting, and accumulation of secreted PSA expressed in the media validates increased AR signaling. C) Rapid decrease of Sox2 mRNA in CWR-R1 cells upon AR stimulation as measured by qPCR. Levels at 0.5 hrs and beyond represent a statistically significant decrease in Sox2 mRNA (p<0.05). D) AR Chromatin Immunoprecipitation (ChIP) documents direct binding of ligand-activated AR to the Sox2 enhancer region in response to AR stimulation by R1881. CWR-R1 cells were treated with vehicle control or 1 nM R1881, and enrichment of the Sox2 promoter after AR-ChIP was normalized as a percentage of total chromatin input. IgG and Histone H3 served as negative and positive controls, respectively. When compared to total input, both the positive control Histone H3 and ligand-activated AR significantly enriched for the Sox2 enhancer (p<0.05). Data represents three independent experiments.