Fig. 2: Androgen treatment increased AR occupancy on ECD promoter and enzalutamide treatment decreases ECD expression. | Oncogene

Fig. 2: Androgen treatment increased AR occupancy on ECD promoter and enzalutamide treatment decreases ECD expression.

From: ECD, a novel androgen receptor target promotes prostate cancer tumorigenesis by regulating glycolysis

Fig. 2

A, B AR binding site; Top three predicted AR binding sites on the ECD promoter. C, D Chromatin immunoprecipitation (ChIP) analysis. Indicated cells were cultured in steroid-free conditions for 3 days, followed by dihydrotestosterone (DHT) treatment for 3 h. Cells were cross-linked with formaldehyde and chromatin was immunoprecipitated with antibody against AR. IgG, used as a non-binding Control. Immunoprecipitated DNA was quantified with qRT-PCR for specific binding sites at ECD promoter using primers encompassing all three sites -712 to -565 from transcription start site. Student's t-test was performed to calculate statistical significance *** p < 0.001. E, F DHT increases ECD promoter activity. ECD promoter spanning approx. 1500 bp was cloned upstream of secreted gaussia luciferase, secreted alkaline phosphatase (SEAP) was used as an internal control. Cells expressing WT promoter or various AR binding sites deleted mutants were treated with 100 nM DHT for 3 h. Supernatant was collected, and luminescence was measured using secrete-pair dual luminescence assay kit. Data represents three independent experiments, each done in triplicates. Student's t-test was performed to calculate statistical significance *** p < 0.001. G Dot plot depicting ECD mRNA expression in prostate tumors pre- and post-androgen deprivation therapy 7 samples in each case (GSE48403). A Wilcoxon matched-pairs signed-rank test was used to compare expressions in the groups. H, I Indicated PC cell lines were cultured in steroid-free medium for 72 h, cells were then treated with 10 nM DHT, followed by treatment with indicated concentrations of MDV3100 (enzalutamide) for another 48 h. Lysates were collected and western blotted with indicated antibodies. β-actin was used as a loading control. J, K qRT-PCR was performed in the same samples where the RNA was isolated by standard TRIzol phenol chloroform method. 18 s rRNA was used for normalization. Bar graphs represent fold change in AR and ECD mRNA in LNCaP (J) and C4-2B cells (K) respect to vehicle treatment. Data represent mean ± SEM from three independent experiments, each done in triplicates. Student's t-test was performed to calculate statistical significance *** p < 0.001, ** p < 0.01, ns not significant.

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