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The female mammary epithelium undergoes reorganization during development, pregnancy, and menopause, linking higher risk with breast cancer development. To characterize these periods of complex remodeling, here we report integrated 50 K mouse and 24 K human mammary epithelial cell atlases obtained by single-cell RNA sequencing, which covers most lifetime stages. Our results indicate a putative trajectory that originates from embryonic mammary stem cells which differentiates into three epithelial lineages (basal, luminal hormone-sensing, and luminal alveolar), presumably arising from unipotent progenitors in postnatal glands. The lineage-specific genes infer cells of origin of breast cancer using The Cancer Genome Atlas data and single-cell RNA sequencing of human breast cancer, as well as the association of gland reorganization to different breast cancer subtypes. This comprehensive mammary cell gene expression atlas ( https://mouse-mammary-epithelium-integrated.cells.ucsc.edu ) presents insights into the impact of the internal and external stimuli on the mammary epithelium at an advanced resolution.

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Menopause is a critical window of susceptibility for its sensitivity to endocrine disrupting chemicals due to the decline of endogenous estrogen. Using a surgical menopausal (ovariectomized) mouse model, we assessed how mammary tissue was affected by both 17ß-estradiol (E2) and polybrominated diphenyl ethers (PBDEs). As flame retardants in household products, PBDEs are widely detected in human serum. During physiologically-relevant exposure to E2, PBDEs enhanced E2-mediated regrowth of mammary glands with terminal end bud-like structures. Analysis of mammary gland RNA revealed that PBDEs both augmented E2-facilitated gene expression and modulated immune regulation. Through single-cell RNA sequencing (scRNAseq) analysis, E2 was found to induce Pgr expression in both Esr1+ and Esr1- luminal epithelial cells and Ccl2 expression in Esr1+ fibroblasts. PBDEs promote the E2-AREG-EGFR-M2 macrophage pathway. Our findings support that E2 + PBDE increases the risk of developing breast cancer through the expansion of estrogen-responsive luminal epithelial cells and immune modulation.

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Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in household materials. Their environmental persistence has led to continuous human exposure and significant tissue levels. Three PBDE congeners (BDE-47, BDE-100, and BDE-153) have been frequently detected in human serum. Although these compounds appear to possess endocrine disrupting activity, studies are largely missing to determine the biological mechanisms of PBDEs in breast cancer cells. Here, we assessed PBDE bioactivities with three complementary strategies: receptor binding/activity assays; nonbiased RNA-sequencing analysis using an estrogen-dependent breast cancer cell line MCF-7aroERE; and in vivo assessments using patient-derived xenograft (PDX) models of human breast cancer. According to the results from in vitro experiments, the PBDE congeners regulate distinct nuclear receptor signaling pathways. BDE-47 acts as a weak agonist of both estrogen receptor α (ERα) and estrogen-related receptor α (ERRα); it could stimulate proliferation of MCF-7aroERE and induced expression of ER-regulated genes (including cell cycle genes). BDE-153 was found to act as a weak antagonist of ERα. BDE-100 could act as (1) an agonist of aryl hydrocarbon receptor (AhR), inducing expression of CYP1A1 and CYP1B1 and (2) as a very weak agonist/antagonist of ERα. In vivo, a mixture of the three congeners with ratios detected in human serum was tested in an ER+ PDX model. The mixture exhibited estrogenic activity through apoptosis/cell cycle regulation and increased the expression of a proliferation marker, Ki-67. These results advance our understanding of the mechanisms of PBDE exposure in breast cancer cells.

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While ER has multiple biological effects, ER-cyclin D1-CDK4/6-RB is a critical pathway for the action of estrogen on the cell cycle, especially for breast cancers that rely on estrogen for growth. The latest and most efficient CDK4/6 inhibitors target the phosphorylation of retinoblastoma (RB) tumor suppressor gene; thus, altering levels of many cell cycle molecules. Estrogen receptor (ER)+/HER2- breast cancers have shown great progression free survival when CDK4/6 inhibitors are combined with endocrine therapies. Here we report the mechanism of antiestrogen (fulvestrant) combination with CDK4/6 inhibitors is due to synergism in the suppression of ER-mediated cell cycle progression. Furthermore, we performed single cell analysis of cells from an estrogen dependent/hormone receptor-positive patient derived xenograft (PDX) tumor model treated with palbociclib. These single cells expressed various levels of ER and RB which are involved in cell cycle regulation; and the response to palbociclib treatment relies not only on the ER-cyclin D1-CDK4/6-RB pathway but it is also dependent on elevated levels of ER and/or RB. Our preclinical studies show that palbociclib response is dependent on cells with ER, which is directly involved in cell cycle progression in hormone receptor positive (HR+) breast cancer.

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The estrogen-related receptor α (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has also recently been shown to regulate an enzyme in the ß-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., small interfering RNA knockdown), we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. To our knowledge, this study is the first comprehensive analysis in discovering potential endocrine disrupters that affect the ERRα signaling pathway.

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