Spatial and Epigenetic Polarity of the AR Promoter During Prostate Cancer Progression: An In Silico Analysis

The androgen receptor (AR) is a key regulator of prostate cancer progression, yet the relationship between AR promoter methylation, chromatin state, and receptor expression across distinct tumor phenotypes remains incompletely understood. We performed an integrative in silico analysis of the AR promoter region spanning -2000 to +500 bp relative to the transcription start site (TSS), combining motif predictions for 24 transcription factors (TFs) with DNA methylation, TF ChIP-seq, and histone modification profiles from normal prostate tissue, primary and metastatic tumors, and cell models. Integration of these datasets revealed pronounced spatial heterogeneity in the epigenetic organization of the AR promoter. The distal segment contained predicted motifs for the selected lineage-associated TFs FOXA1, GATA2, HOXB13, and NKX3-1 and showed substantial variability in DNA methylation. Reduced methylation at selected distal sites or regions was observed in primary adenocarcinoma and in AR-low and AR-negative tumors and cell models, including tumors with a neuroendocrine phenotype. In TCGA-PRAD primary tumors, between-sample differences in AR promoter methylation explained only a small proportion of the variation in AR mRNA levels. Within the distal FOXA1 motif cluster (-751 to -678 bp), within-sample-normalized FOXA1 signal was higher in primary tumors than in matched normal tissue and was further elevated in patient-derived xenografts (PDXs) originating from metastatic castration-resistant prostate cancer (mCRPC). Exploratory analysis of matched normal-tumor pairs revealed a positive correlation between changes in distal FOXA1 signal and changes in proximal H3K27ac signal. AR-positive C4-2B cells retained proximal H3K27ac without detectable FOXA1 occupancy at the distal cluster, consistent with an alternative active promoter configuration. The proximal TSS-associated segment was generally hypomethylated but displayed heterogeneous chromatin states. The -280 to +100 bp region was a recurrent focus of H3K27ac enrichment. In VCaP cells, proximal AR and ERG ChIP-seq signals overlapped, although no AR motif meeting the screening threshold was detected in this region. H3K27me3 repressive mark profiles showed marked heterogeneity across the examined models and tissues. Among models used as normal controls, RWPE-1 and PrEC showed H3K27me3 enrichment around the AR TSS, whereas primary normal prostate tissue showed no comparable enrichment. Heterogeneity was also evident between AR-negative cell lines: PC-3 showed pronounced proximal H3K27me3 enrichment, whereas DU145 lacked comparable enrichment. The analyzed mCRPC metastases likewise did not exhibit a profile comparable to that of PC-3. In AR-negative PC-3 cells, the proximal region additionally showed enrichment of KDM2B, PCGF1, and RNF2, together with strong H2AK119ub1 enrichment. These findings support a model in which the AR promoter comprises an epigenetically plastic distal segment and a proximal TSS-associated regulatory region characterized by context-dependent TF occupancy and chromatin states. This heterogeneity, together with the limited explanatory value of promoter methylation for AR mRNA levels, is consistent with DNA methylation alone being insufficient to account for differences in AR expression across prostate cancer phenotypes.