The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.

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.

Background Soy isoflavone consumption may protect against breast cancer development. We conducted a Phase IIB trial of soy isoflavone supplementation, to examine its effect on breast epithelial proliferation and other biomarkers in the healthy high risk breast. Methods 126 consented women underwent a random fine needle aspiration (rFNA); those with ≥ 4000 epithelial cells were randomized to a double-blind six-month intervention of mixed soy isoflavones (PTIG-2535) or placebo, followed by repeat rFNA. Cells were examined for Ki-67 labeling index (Ki-67 LI), and atypia. Expression of 28 genes related to proliferation, apoptosis and estrogenic effect was measured using quantitative RT-PCR. Hormone and protein levels were measured in nipple aspirate fluid (NAF). All statistical tests were 2-sided. Results 98 women were evaluable for Ki-67 LI. In 49 treated women, the median Ki-67 LI was 1.18 at entry and 1.12 post-intervention, whereas in 49 placebo subjects it was 0.97 and 0.92 (p for between-group change 0.32). Menopausal stratification yielded similar results between groups, but within premenopausal soy-treated women, Ki-67 LI increased from 1.71 to 2.18 (p=0.04). We saw no treatment effect on cytologic atypia or NAF parameters. There were significant increases in the expression of 14/28 genes within the soy, but not the control group, without significant between-group differences. Plasma genistein values demonstrated excellent compliance. Conclusions A six-month intervention of mixed soy isoflavones in healthy, high risk adult western women did not reduce breast epithelial proliferation, suggesting a lack of efficacy for breast cancer prevention, and a possible adverse effect in premenopausal women.