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Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha–positive (ER+) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER+/AR+ breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER+/AR+ patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER+/AR+ primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. Implications: This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers. Mol Cancer Res; 14(11); 1054–67. ©2016 AACR.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype for which there are no approved targeted therapies. Preclinical and early clinical trials indicate that up to 50% of TNBC express androgen receptor (AR) and are potentially responsive to anti-androgens. However, the function of AR in TNBC and the mechanisms by which AR-targeted therapy reduces tumor burden are largely unknown. We hypothesized that AR maintains a cancer stem cell (CSC)-like tumor initiating population and serves as an anti-apoptotic factor that facilitates anchorage independence and metastasis. Anchorage-independent growth was assessed in TNBC cells cultured in forced suspension and apoptosis was measured with cleaved-caspase 3 antibody. CSC-like populations were assessed in vitro using ultra low attachment plates, CD44/CD24 staining, the ALDEFLUOR assay, and single cell mammosphere formation efficiency (MFE) assays. Tumor-initiating capacity was assessed in vivo using a limiting dilution assay. Lastly, the ability of a combination of enzalutamide (Enza) and paclitaxel to inhibit TNBC tumor growth was assessed in vivo. AR increased in TNBC cells in forced suspension culture compared to attached conditions. Cells that expressed AR resisted detachment-induced apoptosis. The CSC-like population increased in suspension culture and decreased following AR inhibition. Pre-treatment with Enza decreased the tumor-initiating capacity of TNBC cells. Enza decreased tumor volume and viability when administered simultaneously or subsequent to chemotherapy, but simultaneous treatment more effectively suppressed tumor recurrence. AR-targeted therapies may enhance the efficacy of chemotherapy even in TNBC with low AR expression by targeting a CSC-like cell population with anchorage independent, invasive potential.

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day 14 (P14) and lactation day 2 (L2) revealed that the majority of differentially expressed microRNA declined precipitously between late pregnancy and lactation. The decline in miR-150, which exhibited the greatest fold decrease, was verified quantitatively and qualitatively. To test the hypothesis that the decline in miR-150 is critical for lactation, MEC-specific constitutive miR-150 was achieved by crossing ROSA26-lox-STOP-lox-miR-150 mice with WAP-driven Cre recombinase mice. Both biological and foster pups nursed by bitransgenic dams exhibited a dramatic decrease in survival compared to offspring nursed by littermate control dams. Protein products of predicted miR-150 targets Fasn, Olah, Acaca, and Stat5B were significantly suppressed in MECs of bitransgenic mice with constitutive miR-150 expression as compared to control mice at L2. Lipid profiling revealed significant reduction in fatty acids synthesized by the de novo pathway in L2 MECs of bitransgenic versus control mice. Collectively, these data support the hypothesis that a synchronized decrease in miRNAs, such as miR-150, at late pregnancy serves to allow translation of targets critical for lactation.