Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

CpG island tumor suppressor promoter methylation in non-BRCA-associated early mammary carcinogenesis.

BACKGROUND: Only 5% of all breast cancers are the result of BRCA1/2 mutations. Methylation silencing of tumor suppressor genes is well described in sporadic breast cancer; however, its role in familial breast cancer is not known. METHODS: CpG island promoter methylation was tested in the initial random periareolar fine-needle aspiration sample from 109 asymptomatic women at high risk for breast cancer. Promoter methylation targets included RARB (M3 and M4), ESR1, INK4a/ARF, BRCA1, PRA, PRB, RASSF1A, HIN-1, and CRBP1. RESULTS: Although the overall frequency of CpG island promoter methylation events increased with age (P<0.0001), no specific methylation event was associated with age. In contrast, CpG island methylation of RARB M4 (P=0.051), INK4a/ARF (P=0.042), HIN-1 (P=0.044), and PRA (P=0.032), as well as the overall frequency of methylation events (P=0.004), was associated with abnormal Masood cytology. The association between promoter methylation and familial breast cancer was tested in 40 unaffected premenopausal women in our cohort who underwent BRCA1/2 mutation testing. Women with BRCA1/2 mutations had a low frequency of CpG island promoter methylation (15 of 15 women had

ESR1 promoter hypermethylation does not predict atypia in RPFNA nor persistent atypia after 12 months tamoxifen chemoprevention.

PURPOSE: Currently, we lack biomarkers to predict whether high-risk women with mammary atypia will respond to tamoxifen chemoprevention. EXPERIMENTAL DESIGN: Thirty-four women with cytologic mammary atypia from the Duke University High-Risk clinic were offered tamoxifen chemoprevention. We tested whether ESR1 promoter hypermethylation and/or estrogen receptor (ER) protein expression by immunohistochemistry predicted persistent atypia in 18 women who were treated with tamoxifen for 12 months and in 16 untreated controls. RESULTS: We observed a statistically significant decrease in the Masood score of women on tamoxifen chemoprevention for 12 months compared with control women. This was a significant interaction effect of time (0, 6, and 12 months) and treatment group (tamoxifen versus control) P = 0.0007. However, neither ESR1 promoter hypermethylation nor low ER expression predicted persistent atypia in Random Periareolar Fine Needle Aspiration after 12 months tamoxifen prevention. CONCLUSIONS: Results from this single institution pilot study provide evidence that, unlike for invasive breast cancer, ESR1 promoter hypermethylation and/or low ER expression is not a reliable marker of tamoxifen-resistant atypia.

Synergy between Androgen Receptor Antagonism and Inhibition of mTOR and HER2 in Breast Cancer.

The androgen receptor (AR) is widely expressed in breast cancer, and evidence suggests dependence on AR signaling for growth and survival. AR antagonists such as enzalutamide and seviteronel have shown success in preclinical models and clinical trials of prostate cancer and are currently being evaluated in breast cancer. 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 breast cancer growth. HER2+ and triple-negative breast cancer cell lines were treated with AR antagonist plus anti-HER2 mAb 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+ breast cancer 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 breast cancer therapies such as everolimus and trastuzumab through distinct mechanisms. Treatment combinations are effective in trastuzumab-resistant HER2+ breast cancer cells in vivoMol Cancer Ther; 16(7); 1389-400. ©2017 AACR.

Androgen Receptor Supports an Anchorage-Independent, Cancer Stem Cell-like Population in Triple-Negative Breast Cancer.

Preclinical and early clinical trials indicate that up to 50% of triple-negative breast cancers (TNBC) express androgen receptor (AR) and are potentially responsive to antiandrogens. 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 antiapoptotic factor, facilitating anchorage independence and metastasis. AR levels increased in TNBC cells grown in forced suspension culture compared with those in attached conditions, and cells that expressed AR resisted detachment-induced apoptosis. Culturing TNBC cells in suspension increased the CSC-like population, an effect reversed by AR inhibition. Pretreatment with enzalutamide (Enza) decreased the tumor-initiating capacity of TNBC cells and reduced tumor volume and viability when administered simultaneously or subsequent to the chemotherapeutic paclitaxel; simultaneous treatment more effectively suppressed tumor recurrence. Overall, our findings suggest that AR-targeted therapies may enhance the efficacy of chemotherapy even in TNBCs with low AR expression by targeting a CSC-like cell population with anchorage independence and invasive potential. Cancer Res; 77(13); 3455-66. ©2017 AACR.

Cooperative Dynamics of AR and ER Activity in Breast Cancer.

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.

Androgen Receptor Biology in Triple Negative Breast Cancer: a Case for Classification as AR+ or Quadruple Negative Disease.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) amplification. Due to the absence of these receptors, TNBC does not respond to traditional endocrine or HER2-targeted therapies that improve patient prognosis in other breast cancer subtypes. TNBC has a poor prognosis, and currently, there are no effective targeted therapies. Some TNBC tumors express androgen receptor (AR) and may benefit from AR-targeted therapies. Here, we review the literature on AR in TNBC and propose that TNBC be further sub-classified as either AR+ TNBC or quadruple negative breast cancer since targeting AR may represent a viable therapeutic option for a subset of TNBC.

A TDO2-AhR signaling axis facilitates anoikis resistance and metastasis in triple-negative breast cancer.

The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple-negative breast cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen-positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage-independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3-dioxygenase (TDO2), in an NF-κB-dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR), an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor-treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor-negative status, and shorter overall survival. Our results define an NF-κB-regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype.

Multiple molecular subtypes of triple-negative breast cancer critically rely on androgen receptor and respond to enzalutamide in vivo.

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.

MicroRNA regulation of epithelial plasticity in cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating multiple target genes through repression of mRNA stability or translation. miRNAs regulate a wide variety of developmental and pathological processes, and many have oncogenic or tumor suppressor activity. Carcinoma cells can undergo changes resembling a partial or transient epithelial-mesenchymal transition (EMT) as part of the metastatic cascade. Numerous miRNAs regulate EMT by targeting genes that control epithelial or mesenchymal characteristics. The involvement of miRNAs in feedback loops with EMT-regulating transcription factors reveals a critical role in governing epithelial plasticity. In this review we summarize miRNAs involved in carcinoma cell plasticity.

Evidence for phenotypic plasticity in aggressive triple-negative breast cancer: human biology is recapitulated by a novel model system.

Breast cancers with a basal-like gene signature are primarily triple-negative, frequently metastatic, and carry a poor prognosis. Basal-like breast cancers are enriched for markers of breast cancer stem cells as well as markers of epithelial-mesenchymal transition (EMT). While EMT is generally thought to be important in the process of metastasis, in vivo evidence of EMT in human disease remains rare. Here we report a novel model of human triple-negative breast cancer, the DKAT cell line, which was isolated from an aggressive, treatment-resistant triple-negative breast cancer that demonstrated morphological and biochemical evidence suggestive of phenotypic plasticity in the patient. The DKAT cell line displays a basal-like phenotype in vitro when cultured in serum-free media, and undergoes phenotypic changes consistent with EMT/MET in response to serum-containing media, a unique property among the breast cancer cell lines we tested. This EMT is marked by increased expression of the transcription factor Zeb1, and Zeb1 is required for the enhanced migratory ability of DKAT cells in the mesenchymal state. DKAT cells also express progenitor-cell markers, and single DKAT cells are able to generate tumorspheres containing both epithelial and mesenchymal cell types. In vivo, as few as ten DKAT cells are capable of forming xenograft tumors which display a range of epithelial and mesenchymal phenotypes. The DKAT model provides a novel model to study the molecular mechanisms regulating phenotypic plasticity and the aggressive biology of triple-negative breast cancers.