Steroid hormone receptors, including estrogen receptor α (ERα), tightly regulate the cross-talk between the stroma and epithelium in the prostate. These reciprocal interactions are permanently perturbed in prostate cancer, but how this occurs at the molecular level is unknown. We hypothesised that tumour stroma acquires epigenetic modifications that fundamentally alter the balance of steroid hormone action and confer tumourigenicity. Therefore, we established primary cultures of cancer-associated fibroblasts (CAFs) and matched non-malignant prostate fibroblasts (NPFs) from 17 men with localised prostate cancer and used whole genome bisulphite sequencing to assemble the first complete DNA methylation profile of tumour stroma.
Our data revealed that CAFs and NPFs exhibited more than 7500 locus-specific differentially methylated regions. Many occurred at known regulatory loci and were associated with differentially expressed genes measured using RNAseq. These changes were highly consistent when validated using an independent cohort of patient-matched NPFs and CAFs. The differentially methylated regions were enriched for genes involved in estrogen signalling, including ESR1, which encodes ERα. The promoter of ESR1 was consistently hypomethylated in CAFs and this was associated with increased expression in CAFs compared to NPFs. Accordingly, immunohistochemistry showed a significant increase in ERα staining within tumour stroma of patient tissue specimens. Furthermore, the increased expression of ERα in CAFs was correlated with the enrichment of an estrogen-regulated gene signature. The most highly over-expressed gene downstream of ERα in this signature was the potent chemokine CXCL12. Functional assays showed that CXCL12 secreted by CAFs recruited CXCR4+ mast cells in migration assays. The mast cells in turn secreted pro-tumourigenic cytokines in response to estrogen, forming a pro-tumourigenic loop in the tumour microenvironment.
This study demonstrates that changes in DNA methylation in CAFs are highly consistent between patients and that epigenetically-regulated genes, such as ESR1, have important functional roles in the progression of prostate cancer.