A functional role for an intronic enhancer of ZBTB16 in AR-mediated tumour suppression in ER+ breast cancer

Abstract The androgen receptor (AR) is a tumour suppressor in oestrogen receptor-positive (ER⁺) breast cancer, repressing cell cycle genes and restraining proliferation. We previously identified ZBTB16 as a candidate AR-regulated tumour-suppressor gene. Here, we validated ZBTB16 as a direct AR target and tested whether it mediates AR tumour-suppressor activity. In clinical cohorts, ZBTB16 mRNA expression was reduced in breast tumours relative to normal tissue, and higher expression correlated with improved survival in ER + disease. Androgen treatment increased ZBTB16 protein in ER + breast cancer cell lines, but ZBTB16 knockdown or overexpression did not detectably alter AR anti-proliferative effects in the models tested. AR bound a conserved intron 3 hormone response region (HRR INT3 ) within the ZBTB16 locus in breast cancer models and clinical specimens, with androgen-dependent H3K27ac enrichment, consistent with an AR-responsive enhancer. HRR INT3 CRISPR/Cas9 deletion prevented AR-dependent ZBTB16 induction, impaired AR-mediated growth suppression, attenuated canonical AR target-gene activation and increased baseline proliferation across breast and prostate cancer cells with upregulation of E2F targets. These findings identify HRR INT3 as a lineage-conserved enhancer contributing to AR-mediated tumour suppression across AR + cancers.