Androgens Modulate Bcl-2 Agonist of Cell Death (BAD) Expression and Function in Breast Cancer Cells

Abstract

Androgen receptor (AR) expression in estrogen receptor-positive (ER+) breast cancer (BC) correlates with lower tumor grade and a better clinical outcome. Additionally, in normal mammary epithelium or ER+ BC preclinical models, androgens counteract basal/ER-dependent proliferation. Here, we report an additional mechanism, underlining the protective role exerted by AR. Specifically, the activation of intracellular AR upregulates the Bcl-2-family protein BAD, and TCGA database analyses show that in ER+ BC, BAD expression is associated with better disease-free survival. Ligand-activated AR influences its own and BAD cellular compartmentalization by enhancing levels in the nucleus, as well as in mitochondrial fractions. In both compartments, BAD exerts unconventional functions. In the nucleus, BAD and AR physically interact and, upon androgen stimulation, are recruited at the AP-1 and ARE sites within the cyclin D1 promoter region, contributing to explaining the anti-proliferative effect of androgens in BC cells. Androgens cause an enrichment in BAD and AR content in the mitochondria, correlated with a decrease in mitochondrial function. Thus, we have defined a novel mechanism by which androgens modulate BAD expression, its mitochondria localization, and nuclear content to force its ability to act as a cell cycle inhibitor, strengthening the protective role of androgen signaling in estrogen-responsive BCs.

Keywords: AR; BAD; MCF-7; androgens; breast cancer; cyclin D1; mitochondria.

Figure 1

BAD and AR relative expression in MCF-7 cells and breast cancer patients. (A) Western blotting analysis of protein lysates from MCF-7 breast cancer cells treated with vehicle (-) or 10 nM Mb for 1, 2, and 3 days. (B) Band intensities from panel A were measured and normalized to the relative GAPDH content. Histogram represents the normalized Bcl-2/BAD ratio. (C) Quantitative Real-Time RT-PCR from MCF-7 cells treated with vehicle (-) or 10 nM Mb for 24 h. BAD mRNA expression was normalized to 18S rRNA content. Data represent the mean ± S.D. of three separate experiments, each performed in triplicate. * p < 0.05 vs. vehicle. (D–F) Overall survival (OS) and relapse-free survival (RFS) were evaluated in a cohort of ER+ BC patients. Kaplan–Meier analysis was performed regardless of specific treatments. Kaplan–Meier was plotted for high (above median, in red) and low (below median, in black) AR (D), BAD (E) and concomitant AR/BAD (F) expression. Biased and outlier data were excluded from the analysis. Hazard-ratios were calculated at the best auto-selected cut-off, and p-values were calculated using the log rank test.

Figure 2

Androgens induce BAD nuclear translocation in breast cancer cells. (A) Immunofluorescence assay for BAD (green) was performed in MCF-7 cells treated with 10 nM Mb for 24 h. DAPI staining was used for nuclei detection. Bar = 20 μm (B) Western blotting analysis of cytoplasmic, nuclear, and mitochondrial protein fractions from MCF-7 breast cancer cells treated for 24 h with vehicle (-) or 10 nM Mb as indicated. Lamin B, GAPDH, actin, and VADC1 were used as a loading control.

Figure 3

Ligand-activated AR impairs mitochondria functions. (A) MCF-7 cells treated with vehicle (-) or 10 nM Mb were stained with 200 nM MitoTracker Red CMXRos (CMXRos). DAPI was used to stain nuclei. The fluorescent signal was analyzed using an FV3000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Bar = 50 μm (B) Quantitative RNA from MCF-7 cells treated with vehicle (-) or 10 nM Mb for 24 h was analyzed using Real-Time RT-PCR for DRP1 and normalized to 18S rRNA content. Data represent the mean ± S.D. of three separate experiments, each performed in triplicate. * p ≤ 0.01. (C) Western blotting analysis of OXPHOS and VDAC1 in mitochondrial protein fractions from MCF-7 breast cancer cells treated for 24 h with vehicle (-) or 10 nM Mb. Cytoplasmic proteins demonstrate no signal. Red Ponceau demonstrates equal loading.

Figure 4

BSA-conjugated testosterone induces apoptosis without influencing BAD cellular compartmentalization. (A) MCF-7 cells treated with 10 nM or 100 nM T-BSA for 6 days were subjected to TUNEL nuclear staining and observed under a fluorescence microscope. DAPI staining was used for nuclei detection. Bar = 100 µm (B) Cytosol/nuclear protein fractions from MCF-7 cells treated with vehicle (-), and T-BSA (10, 100 nM) for 24 h. Lamin B and GAPDH were used as loading control.

Figure 5

Mibolerone induces the formation of an AR/BAD complex and influences BAD recruitment at the AP-1 and ARE sites on the cyclin D1 gene promoter. (A) Cytosol and nuclear lysates from MCF-7 cells treated with 10 nM Mb for 24 h were immunoprecipitated with anti-BAD antibody and immunoblotted to detect AR protein levels. (B–C) Nuclear extract from MCF7 cells treated with 10 nM Mb or vehicle (-) for 2 h were incubated with biotinylated oligonucleotides containing the CCND1-AP-1 and -ARE sites and subjected to DAPA. Bound proteins were subjected to Western blotting analysis using anti-AR or anti-BAD antibodies. The unbound fraction was loaded as negative control; nuclear extracts were loaded as positive control. (D–E) Chromatin from MCF-7 cells treated with 10 nM Mb or vehicle (-) for 2 h was precipitated using anti-AR or anti-BAD antibodies. PCR was carried out using primers amplifying the region indicated by arrows and analyzed by agarose gel electrophoresis. IgG indicates negative control samples. DNA input indicates the loading control.