Studies have shown that the androgen receptor (AR) and progesterone receptor (PR) act as tumor suppressors in ER+ breast cancers [1, 2]. However, the critical downstream target genes of AR and PR that mediate their tumour suppressor activity, and whether there is an overlap between AR and PR regulation of these genes, are unknown.
We analysed ChIP-seq and RNA-seq data from ER+ breast cancer models treated with estrogen, androgen, or progesterone and identified ZBTB16 as a target gene regulated by both AR and PR that was present in gene signatures predicting better outcomes for ER+ breast cancer. We identified a hormone regulatory element (HRE) within intron 3 of ZBTB16, bound by activated AR and PR and associated with strong H3K27ac signals, indicative of an active regulatory element. CRISPR-Cas9 was used to delete a ~182bp region encompassing this HRE, creating multiple T-47D HRE-knockout (KO) clonal cell lines.
ChIP-PCR confirmed loss of AR and PR binding in HRE-KO-clonal lines. This deletion diminished androgen-induced, but not progesterone-induced, ZBTB16 expression at mRNA and protein levels. Further analysis revealed PR could regulate ZBTB16 via an alternative regulatory element not bound by AR. Proliferation assays showed greater estrogen-induced proliferation in HRE-KO-clones compared to wild-type-clones. AR-mediated growth suppression was lost in HRE-KO clones, while PR-mediated suppression remained intact. RT-PCR indicated that AR-mediated regulation of other AR target genes (e.g. SEC14L2, FKBP5) was also reduced in HRE-KO-clones. RNA-seq and GSEA confirmed a negative impact on AR signalling and growth-inhibitory pathways in HRE-KO cells.
These findings suggest ZBTB16 mediates AR and PR tumour suppressor activity in ER+ breast cancer and may serve as a potential biomarker for AR or PR agonist drug response. While both receptors regulate this gene, our data shows that the ZBTB16 intron 3 HRE is more important for AR but not PR tumour suppressive action.