Genes Encoding Multiple Modulators of the Immune Response Are Methylated in the Prostate Tumor Microenvironment of African Americans

<b>Background/Objectives</b>: Prostate cancer (PCa) is diagnosed at an earlier median age, more advanced stage, and has worse clinical outcomes in African American (AA) men compared to European Americans (EA). <b>Methods</b>: To investigate the role of aberrant DNA methylation in tumor-adjacent stroma (TAS), methyl binding domain sequencing (MBD-seq) was performed on AA (<i>n</i> = 17) and EA (<i>n</i> = 15) PCa patients. This was independently confirmed using the long interspersed nuclear element-1 (LINE-1) assay. Pathway analysis was performed on statistically significantly differentially methylated genes for AA and EA TAS. DNA methylation profiles of primary cultured AA and EA carcinoma-associated fibroblasts (CAFs) were compared with AA and EA TAS. AA and EA CAFs were treated with demethylating agent 5-Azacytidine (5-AzaC). <b>Results</b>: AA TAS exhibited higher global DNA methylation than EA TAS (<i>p</i>-value < 0.001). Of the 3268 differentially methylated regions identified (DMRs, <i>p</i>-value < 0.05), 85% (2787 DMRs) showed increased DNA methylation in AA TAS, comprising 1648 genes, of which 1379 were protein-coding genes. Based on DNA methylation levels, two AA subgroups were identified. Notably, AA patients with higher DNA methylation were predominantly those with higher Gleason scores. Pathway analysis linked methylated genes in AA TAS to several key signaling pathways (<i>p</i>-value < 0.05), including immune response (e.g., IL-1, IL-15, IL-7, IL-8, IL-3, and chemokine), Wnt/β-catenin, androgen, PTEN, p53, TGF-β, and circadian clock regulation. A total of 168 concordantly methylated genes were identified, with 109 genes (65%) showing increased methylation in AA CAFs and TAS (<i>p</i>-value < 0.05). Treatment with 5-AzaC significantly reduced DNA methylation of concordant genes in AA CAFs (<i>p</i>-value < 0.001). <b>Conclusions</b>: These findings suggest a distinct stromal methylome in AA, providing a foundation for integrating demethylating agents into standard therapies. This approach targets the tumor microenvironment, potentially addressing PCa disparities in AA men.