Perimenopausal and Menopausal Mammary Glands In A 4-Vinylcyclohexene Diepoxide Mouse Model.

As both perimenopausal and menopausal periods are recognized critical windows of susceptibility for breast carcinogenesis, development of a physiologically relevant model has been warranted. The traditional ovariectomy model causes instant removal of the entire hormonal repertoire produced by the ovary, which does not accurately approximate human natural menopause with gradual transition. Here, we characterized the mammary glands of 4-vinylcyclohexene diepoxide (VCD)-treated animals at different time points, revealing that the model can provide the mammary glands with both perimenopausal and menopausal states. The perimenopausal gland showed moderate regression in ductal structure with no responsiveness to external hormones, while the menopausal gland showed severe regression with hypersensitivity to hormones. Leveraging the findings on the VCD model, effects of a major endocrine disruptor (polybrominated diphenyl ethers, PBDEs) on the mammary gland were examined during and after menopausal transition, with the two exposure modes; low-dose, chronic (environmental) and high-dose, subacute (experimental). All conditions of PBDE exposure did not augment or compromise the macroscopic ductal reorganization resulting from menopausal transition and/or hormonal treatments. Single-cell RNA sequencing revealed that the experimental PBDE exposure during the post-menopausal period caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts. The environmental PBDE exposure resulted in similar transcriptomic changes to a lesser extent. In summary, the VCD mouse model provides both perimenopausal and menopausal windows of susceptibility for the breast cancer research community. PBDEs, including all tested models, may affect the post-menopausal gland including impacts on the non-epithelial compartments.

Molecular features of luminal breast cancer defined through spatial and single-cell transcriptomics.

BACKGROUND: Intratumour heterogeneity is a hallmark of most solid tumours, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing on patient-derived xenografts (PDXs) to profile spatially resolved cell populations within oestrogen receptor-positive (ER+ ) breast cancer and to elucidate their importance in oestrogen-dependent tumour growth. METHODS: Two PDXs of 'ER-high' breast cancers with opposite oestrogen-mediated growth responses were investigated: oestrogen-suppressed GS3 (80-100% ER) and oestrogen-dependent SC31 (40-90% ER) models. The observation was validated via single-cell analyses on an 'ER-low' PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by a public ER+ breast cancer single-cell dataset and protein-based dual immunohistochemistry (IHC) of SC31 examining important luminal cancer markers (i.e., ER, progesterone receptor and Ki67). The translational implication of our findings was assessed by clinical outcome analyses on publicly available cohorts. RESULTS: Our space-gene-function study revealed four spatially distinct compartments within ER+ breast cancers. These compartments showed functional diversity (oestrogen-responsive, proliferative, hypoxia-induced and inflammation-related). The 'proliferative' population, rather than the 'oestrogen-responsive' compartment, was crucial for oestrogen-dependent tumour growth, leading to the acquisition of luminal B-like features. The cells expressing typical oestrogen-responsive genes like PGR were not directly linked to oestrogen-dependent proliferation. Dual IHC analyses demonstrated the distinct contribution of the Ki67+ proliferative cells toward oestrogen-mediated growth and their response to a CDK4/6 inhibitor. The gene signatures derived from the proliferative, hypoxia-induced and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the oestrogen-responsive signature showed better prognoses, suggesting that this compartment would not be directly associated with oestrogen-dependent tumour progression. CONCLUSIONS: Our study identified the gene signature in our 'proliferative' compartment as an important determinant of luminal cancer subtypes. This 'proliferative' cell population is a causative feature of luminal B breast cancer, contributing toward its aggressive behaviours.

Identification and characterization of a proliferative cell population in estrogen receptor-positive metastatic breast cancer through spatial and single-cell transcriptomics.

Background: Intratumor heterogeneity is a hallmark of most solid tumors, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing technologies to profile spatially resolved cell populations within estrogen receptor-positive (ER + ) metastatic breast cancers and elucidate their importance in estrogen-dependent tumor growth. Methods: Spatial transcriptomics and single-cell RNA-sequencing were performed on two patient-derived xenografts (PDXs) of "ER-high" metastatic breast cancers with opposite estrogen-mediated growth responses: estrogen-suppressed GS3 (80-100% ER) and estrogen-stimulated SC31 (30-75% ER) models. The analyses included samples treated with and without 17ß-estradiol. The findings were validated via scRNA-seq analyses on "ER-low" estrogen-accelerating PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by the analysis of a publicly available single cell dataset and a protein-based dual immunohistochemical (IHC) evaluation using three important clinical markers [i.e., ER, progesterone receptor (PR), and Ki67]. The translational implication of these results was assessed by clinical outcome analyses on public breast cancer cohorts. Results: Our novel space-gene-function study revealed a "proliferative" cell population in addition to three major spatially distinct compartments within ER + metastatic breast cancers. These compartments showed functional diversity (i.e., estrogen-responsive, proliferative, hypoxia-induced, and inflammation-related). The "proliferative ( MKI67 + )" population, not "estrogen-responsive" compartment, was crucial for estrogen-dependent tumor growth, leading to the acquisition of luminal B features. The cells with induction of typical estrogen-responsive genes such as PGR were not directly linked to estrogen-dependent proliferation. Additionally, the dual IHC analyses demonstrated the distinct contribution of the Ki67 + proliferative cells toward estrogen-mediated growth and their response to palbociclib, a CDK4/6 inhibitor. The gene signatures developed from the proliferative, hypoxia-induced, and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the high estrogen-responsive scores showed better prognosis, confirming that the estrogen-responsive compartment would not be directly associated with estrogen-dependent tumor progression. Conclusions: For the first time, our study elucidated a "proliferative" cell population distinctly distributed in ER + metastatic breast cancers. They contribute differently toward progression of these cancers, and the gene signature in the "proliferative" compartment is an important determinant of luminal cancer subtypes.

Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium.

Fibroblasts have been shown to be one of the essential players for mammary gland organization. Here, we identify two major types of mouse mammary gland fibroblasts through single-cell RNA sequencing analysis: Dpp4 + fibroblasts and Dpp4 - fibroblasts. Each population exhibits unique functional characteristics as well as discrete localization in normal mouse mammary glands. Remarkably, estrogen, a crucial mediator of mammary gland organization, alters the gene expression profiles of fibroblasts in a population-specific manner, without distinct activation of estrogen receptor signaling. Further integrative analysis with the inclusion of five other publicly available datasets reveals a directional differentiation among the mammary gland fibroblast populations. Moreover, the combination with the mouse mammary epithelium atlas allows us to infer multiple potential interactions between epithelial cells and fibroblasts in mammary glands. This study provides a comprehensive view of mouse mammary gland fibroblasts at the single-cell level.

White button mushroom interrupts tissue AR-mediated TMPRSS2 expression and attenuates pro-inflammatory cytokines in C57BL/6 mice.

White button mushroom (WBM) is a common edible mushroom consumed in the United States and many European and Asia-Pacific countries. We previously reported that dietary WBM antagonized dihydrotestosterone (DHT)-induced androgen receptor (AR) activation and reduced myeloid-derived suppressor cells (MDSCs) in prostate cancer animal models and patients. Transmembrane protease serine 2 (TMPRSS2), an androgen-induced protease in prostate cancer, has been implicated in influenza and coronavirus entry into the host cell, triggering host immune response. The present study on C57BL/6 mice revealed that WBM is a unique functional food that (A) interrupts AR-mediated TMPRSS2 expression in prostate, lungs, small intestine, and kidneys through its AR antagonistic activity and (B) attenuates serum pro-inflammatory cytokines and reduces MDSC counts through its immunoregulatory activity. These findings provide a scientific basis for translational studies toward clinical applications of WBM in diseases related to TMPRSS2 expression and immune dysregulation.

Exploring the Biological Activity and Mechanism of Xenoestrogens and Phytoestrogens in Cancers: Emerging Methods and Concepts.

Xenoestrogens and phytoestrogens are referred to as "foreign estrogens" that are produced outside of the human body and have been shown to exert estrogen-like activity. Xenoestrogens are synthetic industrial chemicals, whereas phytoestrogens are chemicals present in the plant. Considering that these environmental estrogen mimics potentially promote hormone-related cancers, an understanding of how they interact with estrogenic pathways in human cells is crucial to resolve their possible impacts in cancer. Here, we conducted an extensive literature evaluation on the origins of these chemicals, emerging research techniques, updated molecular mechanisms, and ongoing clinical studies of estrogen mimics in human cancers. In this review, we describe new applications of patient-derived xenograft (PDX) models and single-cell RNA sequencing (scRNA-seq) techniques in shaping the current knowledge. At the molecular and cellular levels, we provide comprehensive and up-to-date insights into the mechanism of xenoestrogens and phytoestrogens in modulating the hallmarks of cancer. At the systemic level, we bring the emerging concept of window of susceptibility (WOS) into focus. WOS is the critical timing during the female lifespan that includes the prenatal, pubertal, pregnancy, and menopausal transition periods, during which the mammary glands are more sensitive to environmental exposures. Lastly, we reviewed 18 clinical trials on the application of phytoestrogens in the prevention or treatment of different cancers, conducted from 2002 to the present, and provide evidence-based perspectives on the clinical applications of phytoestrogens in cancers. Further research with carefully thought-through concepts and advanced methods on environmental estrogens will help to improve understanding for the identification of environmental influences, as well as provide novel mechanisms to guide the development of prevention and therapeutic approaches for human cancers.

Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis.

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.

White Button Mushroom (Agaricus bisporus) Interrupts Tissue AR-TMPRSS2 Expression and Attenuates Pro-inflammatory Cytokines in C57BL/6 Mice: Implication for COVID-19 Dietary Intervention.

Transmembrane protease serine 2 (TMPRSS2), an androgen-induced protease associated with prostate cancer, is one putative receptor for coronavirus entry into host cells, where triggering aggressive inflammatory cytokine storm and possibly death in COVID-19 patients. We previously reported that dietary white button mushroom (WBM) antagonized dihydrotestosterone (DHT)-induced androgen receptor (AR) activation and reduced myeloid-derived suppressor cells (MDSCs) in prostate cancer animal models and patients. The present study on C57BL/6 mice revealed that WBM is a unique food that A ) interrupts DHT induced AR-TMPRSS2 expression in putative COVID-19 targeted organs through its AR antagonistic activity and B ) attenuates serum pro-inflammatory cytokines which have been implicated in COVID-19 pathogenesis. We hereby propose WBM intake as a potentially low-cost, efficient, and safe dietary intervention to mitigate COVID-19.

White button mushroom (Agaricus bisporus) disrupts androgen receptor signaling in human prostate cancer cells and patient-derived xenograft.

White button mushroom (WBM) (Agaricus bisporus) is a potential prostate cancer (PCa) chemo-preventative and therapeutic agent. Our clinical phase І trial of WBM powder in patients with biochemically recurrent PCa indicated that WBM intake reduced the circulating levels of prostate-specific antigen (PSA). We hypothesized that WBM exerts its effects on PCa through the androgen receptor (AR) signaling axis. Therefore, we conducted a reverse translational study with androgen-dependent PCa cell lines (LNCaP and VCaP) and patient-derived-xenografts (PDX) from a prostate tumor (TM00298). In both LNCaP and VCaP cells, western blots and qRT-PCR assays indicated that WBM extract (6-30 mg/mL) suppressed DHT-induced PSA expression and cell proliferation in a dose-dependent manner. Immunofluorescence analysis of AR revealed that WBM extract interrupted the AR nuclear-cytoplasmic distribution. PSA promotor-luciferase assay suggested that WBM extract inhibited DHT-induced luciferase activity. RNA-Seq on WBM-treated LNCaP cells confirmed that WBM treatment suppressed the androgen response pathways and cell-cycle control pathways. Our PDX showed that oral intake of WBM extract (200 mg/kg/d) suppressed tumor growth and decreased PSA levels in both tumors and serum. In the present study, we also identified a conjugated linoleic acid isomer (CLA-9Z11E) as a strong AR antagonist by performing LanthaScreen TR-FRET AR Coactivator Interaction Assays. The inhibitory effect of CLA-9Z11E (IC50: 350 nM) was nearly two times stronger than the known AR antagonist, cyproterone acetate (IC50: 672 nM). The information gained from this study improves the overall understanding of how WBM may contribute to the prevention and treatment of PCa.