Triple-negative breast cancer (TNBC) has the lowest 5-year survival rate of invasive breast carcinomas, and currently there are no approved targeted therapies for this aggressive form of the disease. The androgen receptor (AR) is expressed in up to one third of TNBC and we find that all AR+ TNBC primary tumors tested display nuclear localization of AR, indicative of transcriptionally active receptors. While AR is most abundant in the “luminal AR (LAR)” molecular subtype of TNBC, here, for the first time, we use both the new-generation anti-androgen enzalutamide and AR knockdown to demonstrate that the other non-LAR molecular subtypes of TNBC are critically dependent on AR protein. Indeed, AR inhibition significantly reduces baseline proliferation, anchorage-independent growth, migration, and invasion and increases apoptosis in four TNBC lines (SUM159PT, HCC1806, BT549, and MDA-MB-231), representing three non-LAR TNBC molecular subtypes (mesenchymal-like, mesenchymal stem–like, and basal-like 2). In vivo, enzalutamide significantly decreases viability of SUM159PT and HCC1806 xenografts. Furthermore, mechanistic analysis reveals that AR activation upregulates secretion of the EGFR ligand amphiregulin (AREG), an effect abrogated by enzalutamide in vitro and in vivo. Exogenous AREG partially rescues the effects of AR knockdown on proliferation, migration, and invasion, demonstrating that upregulation of AREG is one mechanism by which AR influences tumorigenicity. Together, our findings indicate that non-LAR subtypes of TNBC are AR dependent and, moreover, that enzalutamide is a promising targeted therapy for multiple molecular subtypes of AR+ TNBC.

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 to 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. Lastly, 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 pre-clinical 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, since 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.

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.

Ovarian cancer metastasizes via direct seeding, whereby cancer cells shed from the primary site, resist cell death in the peritoneal cavity, then metastasize to peritoneal organs. We sought to identify molecular mechanisms that facilitate ovarian cancer cell anchorage independent survival. Gene expression profiling was performed on ovarian cancer cells grown in attached or forced suspension culture and confirmed by RT‐qPCR. Anoikis was measured by Caspase 3/7 assay. Since the long non‐coding RNA Metastasis Associated Lung Adenocarcinoma transcript 1 (MALAT1) was among the transcripts most highly increased in forced suspension culture, modified anti‐sense oligonucleotides (ASO) were used to inhibit its expression. Knockdown of RBFOX2 and KIF1B was performed using shRNAs. Publically available datasets were analyzed for association of MALAT1 gene expression with clinicopathological variables. In multiple anoikis‐resistant ovarian cancer cell lines MALAT1 expression increased after 24 and 48 h in forced suspension culture compared to attached culture. High MALAT1 is associated with increased stage, recurrence, and reduced survival in ovarian cancer, and in a small percentage of ovarian cancers MALAT1 is amplified. MALAT1 knockdown resulted in decreased proliferation, invasion, anchorage‐independent growth, and increased anoikis. Suppression of MALAT1 also resulted in decreased expression of RBFOX2, and alternative processing of the pro‐apoptotic tumor suppressor gene KIF1B. RBFOX2 suppression resulted in preferential splicing of the pro‐apoptotic isoform of KIF1B (KIFB1B‐beta) and increased anoikis. The lncRNA MALAT1 facilitates a pro‐metastatic phenotype in ovarian cancer by promoting alternative RNA processing and differential expression of anti‐apoptosis and epithelial to mesenchymal transition (EMT)‐related genes.

The androgen receptor (AR) is widely expressed in breast cancer (BC) and evidence suggests dependence on AR signaling for growth and survival. AR antagonists such as enzalutamide and seviteronel have shown success in pre-clinical models and clinical trials of prostate cancer, and are currently being evaluated in BC. Reciprocal regulation between AR and the HER2/PI3K/mTOR pathway may contribute to resistance to HER2- and mTOR-targeted therapies; thus, dual inhibition of these pathways may synergistically inhibit BC growth. HER2+ and triple-negative BC cell lines were treated with AR antagonist plus anti-HER2 monoclonal antibody trastuzumab or mTOR inhibitor everolimus. Apoptosis, cell proliferation and drug synergy were measured in vitro. Pathway component genes and proteins were measured by qRT-PCR, western blot, and reverse phase protein array. In vivo, HER2+ BC xenografts were treated with enzalutamide, everolimus, trastuzumab, and combinations of these drugs. AR antagonists inhibited proliferation of both HER2+ and TNBC cell lines. Combining AR antagonist and either everolimus or trastuzumab resulted in synergistic inhibition of proliferation. Dihydrotestosterone caused increased phosphorylation of HER2 and/or HER3 that was attenuated by AR inhibition. Everolimus caused an increase in total AR, phosphorylation of HER2 and/or HER3, and these effects were abrogated by enzalutamide. Growth of trastuzumab-resistant HER2+ xenograft tumors was inhibited by enzalutamide, and combining enzalutamide with everolimus decreased tumor viability more than either single agent. AR antagonists synergize with FDA-approved BC therapies such as everolimus and trastuzumab through distinct mechanisms. Treatment combinations are effective in trastuzumab-resistant HER2+ BC cells in vivo.

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day (P)14 and lactation day (L)2 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 crucial 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 with 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 with control mice at L2. Lipid profiling revealed a 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 crucial for lactation.

Objective To better understand the site and mode of action of aromatase inhibitors. Setting Academic research environment. Patients and Design 5 eumenorrheic (non-PCOS), early follicular phase women of normal BMI (mean = 20.47 +/− 0.68 kg/m2) and 12 normal weight, mid-reproductive aged, early follicular phase, normal BMI (mean = 20.8 +/− 1.7 kg/m2) historical controls. Interventions 2.5mg letrozole daily for 7 days. Women were sampled with daily, first morning voided urine collections, thrice weekly blood sampling, and 4 hours of q 10 minute frequent blood sampling Main outcome measures Serum LH measured using a well-characterized immunofluorometric assay. LH pulse characteristics were compared between treated and control groups using t tests. Results Mean LH and LH pulse amplitude more than doubled in women who had taken letrozole compared to controls, but the LH pulse frequency did not differ between women taking letrozole and controls. Conclusions These results indicate that the release of negative feedback inhibition of estradiol on the hypothalamic-pituitary axis in normal women by aromatase inhibitors creates an amplitude-related increase in endogenous hypothalamic-pituitary drive. The finding that mean LH and LH pulse amplitude, but not frequency, increased after letrozole suggests a possible pituitary site of action.

<p>S1. Enza or AR knockdown decrease baseline and E2-induced proliferation of breast cancer cells in vitro. S2. E2 induces and Enza inhibits AR nuclear localization. S3. AR and ER binding in MCF7 cells. S4. Enza synergizes with anti-estrogens. S5. The combination of Enza plus tam reduces ER. S6. PT12 cells are AR+ and Enza alters expression of E2-regulated genes in PT12 xenograft tumors.</p>