Introduction Androgen receptor (AR) expression in breast cancer growth and progression appears to be clinically relevant and disease context specific. In oestrogen receptor (ER)α-positive primary breast cancers, AR positivity correlates with lower tumour grade and a better clinical outcome. These clinical-pathological findings mirror the capability of androgens to counteract ERα-dependent proliferation in both normal mammary epithelium and ERα-positive breast cancer preclinical models in which androgen/AR-dependent pro-apoptotic effects have been also evidenced. Here we report a novel additional mechanism underlining the protective, anti-proliferative role exerted by AR signalling. This mechanism involves modulation of the expression, cellular distribution and function of BAD, a pro-apoptotic member of the Bcl-2 family proteins, whose expression is related to a significantly better disease free survival in (ER)α-positive human breast cancers. Material and methods MCF-7, TD47D, ZR-75 breast cancer cells; qReal Time PCR; western blotting (WB); immunofluorescence analysis (IF); immunoprecipitation assay (IP); DNA affinity precipitation assay; Chromatin Immunoprecipitation Assay. Results and discussions The expression of a panel of pro/anti-apoptotic proteins was investigated in cellular protein lysates from ERα/AR-positive MCF-7 cells cultured for 1, 3 and 6 days under androgen treatment. The expression of the anti-apoptotic Bcl-2 protein, or the pro-apoptotic BID and BAX remained unchanged, while a sustained increase in the expression of the pro-apoptotic BAD could be observed, reducing the Bcl-2/BAD ratio and, thus, shifting the delicate balance between inhibitors and inducers of cell death. Interestingly, androgens induced a marked BAD levels increase into the nuclear compartment in ERα/AR-positive MCF-7, T47D and ZR-75 as well as in ERα negative/AR-positive SKBR3 cells. The androgen-regulated intracellular localization of BAD involved an AR/BAD physical interaction, suggesting a nuclear role for BAD upon androgen stimulation. Indeed, androgens induced both AR and BAD recruitment at a AP-1 and at a ARE site within the cyclin D1 promoter region, contributing to explain the anti-proliferative effect of androgens in breast cancer cells. Conclusion We defined a novel mechanism by which androgens modulate BAD expression and force its ability to act as a cell cycle inhibitor through modulation of cyclin D1 gene transcriptional activity, strengthening the protective role of androgen signalling in estrogen-responsive breast cancer
