Mammary Gland Biology and Neoplasia

Electron Tomography Revels that Milk Lipids Originate from Endoplasmic Reticulum Domains with Novel Structural Features Abstract Lipid droplets (LD) are dynamically-regulated organelles that originate from the endoplasmic reticulum (ER), and function in the storage, trafficking and metabolism of neutral lipids. In mammary epithelial cells (MEC) of lactating animals, intact LD are secreted intact into milk to form milk lipids by a novel apocrine mechanism. The secretion of intact LD and the relatively large amounts of lipid secreted by lactating MEC increase demands on the cellular processes responsible for lipid synthesis and LD formation. As yet these processes are poorly defined due to limited understanding of LD-ER interactions. To overcome these limitations, we used rapid-freezing and freeze-substitution methods in conjunction with 3D electron tomography and high resolution immunolocalization to define interactions between LD with ER in MEC of pregnant and lactating rats. Using these approaches, we identified distinct ER domains that contribute to lipid droplet formation and stabilization and which possess unique features previously unrecognized or not fully appreciated. Our results show nascent lipid droplets within the ER lumen and the association of both forming and mature droplets with structurally unique regions of ER cisternae, characterized by the presence of perilipin-2, a protein implicated in lipid droplet formation, and enzymes involved in lipid synthesis. These data demonstrate that milk lipids originate from LD-ER domains with novel structural features and suggest a mechanism for initial droplet formation in the ER lumen and subsequent maturation of the droplets in association with ER cisternae.

Circ-TFCP2L1 Promotes the Proliferation and Migration of Triple Negative Breast Cancer through Sponging miR-7 by Inhibiting PAK1 Abstract CircRNAs are essential factors that have been verified to regulate various forms of carcinogenesis. However, the role of circRNAs in triple negative breast cancer (TNBC) tumourigenesis is not well clarified. In this study, we explored the circRNA expression profiles and possible modulation mechanism of circRNAs on triple negative breast cancer tumourigenesis. We used three pairs of triple negative breast cancer tissues and adjacent noncancerous tissues to perform a human circRNA microarray for screening of circRNA expression patterns in TNBC. The results showed that circ-TFCP2L1 was significantly up-regulated in TNBC tissues and cells, tending to have a shorter disease-free survival of TNBC patients. In vitro loss-of-function experiments showed that knockdown of circ-TFCP2L1 significantly suppressed the proliferation and migration of TNBC cells. Moreover, the results showed that the proliferation and migration capabilities and PAK1 expression in TNBC cells treated with si-circ-TFCP2L1 + miR-7 mimics were significantly suppressed compared with the normal group. Therefore, circ-TFCP2L1 was identified as a sponge of miR-7 functionally targeting PAK1 and further promoting the proliferation and migration of TNBC cells. Taken together, the results from our study reveal a novel regulatory mechanism and offer novel insight into the role of circ-TFCP2L1 in progression of triple negative breast cancer.

Redirecting Normal and Cancer Stem Cells to a Mammary Epithelial Cell Fate Abstract Tissue microenvironments, also known as stem cell niches, influence not only resident cells but also cells in surrounding tissues. Physical and biochemical intercellular signals originating from resident stem cells or non-stem cells participate in the homeostasis of the tissue regulating cell proliferation, differentiation, wound healing, tissue remodeling, and tumorigenesis. In recent publications it has been demonstrated that the normal mouse mammary microenvironment can provide development and differentiation guidance to not only resident mammary cells but also cells of non-mammary origin including tumor-derived cells. When placed in reforming mammary stem cell niches the non-mammary cells proliferate and differentiate along mammary epithelial cell lineages and contribute progeny to reforming mammary gland outgrowths. The tumor-derived cells that are redirected to assume mammary epithelial phenotypes lose their cancer-forming capacity and shift their gene expression profiles from a cancer profile towards a normal mammary epithelial expression profile. This review summarizes the recent discoveries regarding the ability of the normal mouse mammary microenvironment to dictate the cell fates of non-mammary cells introduced into mammary stem cell niches.

Calcification Microstructure Reflects Breast Tissue Microenvironment Abstract Microcalcifications are important diagnostic indicators of disease in breast tissue. Tissue microenvironments differ in many aspects between normal and cancerous cells, notably extracellular pH and glycolytic respiration. Hydroxyapatite microcalcification microstructure is also found to differ between tissue pathologies, including differential ion substitutions and the presence of additional crystallographic phases. Distinguishing between tissue pathologies at an early stage is essential to improve patient experience and diagnostic accuracy, leading to better disease outcome. This study explores the hypothesis that microenvironment features may become immortalised within calcification crystallite characteristics thus becoming indicators of tissue pathology. In total, 55 breast calcifications incorporating 3 tissue pathologies (benign – B2, ductal carcinoma in-situ - B5a and invasive malignancy - B5b) from archive formalin-fixed paraffin-embedded core needle breast biopsies were analysed using X-ray diffraction. Crystallite size and strain were determined from 548 diffractograms using Williamson-Hall analysis. There was an increased crystallinity of hydroxyapatite with tissue malignancy compared to benign tissue. Coherence length was significantly correlated with pathology grade in all basis crystallographic directions (P < 0.01), with a greater difference between benign and in situ disease compared to in-situ disease and invasive malignancy. Crystallite size and non-uniform strain contributed to peak broadening in all three pathologies. Furthermore, crystallite size and non-uniform strain normal to the basal planes increased significantly with malignancy (P < 0.05). Our findings support the view that tissue microenvironments can influence differing formation mechanisms of hydroxyapatite through acidic precursors, leading to differential substitution of carbonate into the hydroxide and phosphate sites, causing significant changes in crystallite size and non-uniform strain.

Exogenous ERα Expression in the Mammary Epithelium Decreases Over Time and Does Not Contribute to p53-Deficient Mammary Tumor Formation in Mice Abstract Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

How to Choose a Mouse Model of Breast Cancer, a Genomic Perspective Abstract Human breast cancer is a heterogeneous disease with numerous subtypes that have been defined through immunohistological, histological, and gene expression patterns. The diversity of breast cancer has made the study of its various underlying causes complex. To facilitate the examination of particular facets of breast cancer, mouse models have been generated, ranging from carcinogen induced models to genetically engineered mice. While mouse models have been generated to mimic the initiating event, including p53 loss, BRCA loss, or overexpression of HER2 / Neu / erbB2, other genomic events are often not well characterized. However, these secondary genetic events are often critical to the mouse tumor evolution, subtype, and outcome, just as they are in human breast cancer. As such, these other genomic events are a critical component of what models are chosen to study specific subtypes of human breast cancer. Here we review the genomic analyses that have been completed for various genetically engineered mouse models, how they compare to human breast cancer, and detail how this information can be used in choosing a mouse model for analysis.

The Eleventh ENBDC Workshop: Advances in Technology Help to Unveil Mechanisms of Mammary Gland Development and Cancerogenesis Abstract The eleventh annual workshop of the European Network for Breast Development and Cancer, Methods in mammary gland biology and breast cancer, took place on the 16th to 18th of May 2019 in Weggis, Switzerland. The main topics of the meeting were high resolution genomics and proteomics for the study of mammary gland development and cancer, breast cancer signaling, tumor microenvironment, preclinical models of breast cancer, and tissue morphogenesis. Exciting novel findings in, or highly relevant to, mammary gland biology and breast cancer field were presented, with insights into the methods used to obtain them. Among others, the discussed methods included single-cell RNA sequencing, genetic barcoding, lineage tracing, spatial transcriptomics, optogenetics, genetic mouse models and organoids.

BRCA1 Attenuates Progesterone Effects on Proliferation and NFκB Activation in Normal Human Mammary Epithelial Cells Abstract Germline mutations in the breast cancer susceptibility gene BRCA1, encoding a tumor suppressor protein, greatly enhance the risk of breast and ovarian cancer. This tissue-specificity implicates the role of ovarian hormones. Indeed, BRCA1 has been demonstrated to regulate the signalling axis of the hormone, progesterone, and its receptor, the progesterone receptor (PR), and progesterone action has been implicated in BRCA1-related tumorigenesis. BRCA1 also plays important roles in oxidative stress and activating nuclear factor kappaB (NFκB) signalling pathways. Like wildtype BRCA1 function, PR signalling has also been shown to inhibit NFκB activation. Although PR and BRCA1 networks are known to interact, their interaction at the level of NFκB activation in the human breast is not understood. This study investigates the effect of reduced BRCA1 expression on proliferation and NFκB activation in human breast cells, and the impact of progesterone on these effects. The major findings are that: 1) Reduced BRCA1 levels inhibit cell growth in normal human mammary cells and breast cancer cells; 2) Reduced BRCA1 levels stimulated inflammatory targets and NFκB activity in normal human mammary cells; 3) Wildtype BRCA1 inhibited the pro-proliferative effects of progesterone in normal mammary epithelial cells, and; 4) Progesterone attenuated BRCA1-mediated NFκB activation in normal human mammary cells. These data have important implications for our understanding of progesterone action in BRCA1 mutation carriers, and how inhibition of this action may potentially delay tumorigenesis or impart a more favourable prognosis.

Remodeling of Murine Mammary Adipose Tissue during Pregnancy, Lactation, and Involution Abstract White adipocytes in the mammary gland stroma comprise the majority of the mammary gland mass. White adipocytes regulate numerous hormonal and metabolic processes and exhibit compositional and phenotypic plasticity. This plasticity is exemplified by the ability of mammary adipocytes to regress during lactation, when mammary epithelial cells expand to establish sufficient milk-producing alveoli. Upon weaning, the process reverses through mammary involution, during which adipocytes extensively regenerate, and alveolar epithelial cells disappear through cell death, returning the mammary gland to the non-lactating state. Despite intensive studies on the development and involution of the mammary alveolar epithelium, the fate of mammary adipocytes during pregnancy and lactation, and the origins of mammary adipocytes regenerated during mammary involution, is poorly understood. Here, we discuss the recent discoveries of the fate of mammary adipocytes during pregnancy and lactation in a number of different mouse models, and the lineage origin of mammary adipocytes regenerated during involution.

GATA3 Truncating Mutations Promote Cistromic Re-Programming In Vitro, but Not Mammary Tumor Formation in Mice Abstract Heterozygous mutations in the transcription factor GATA3 are identified in 10–15% of all breast cancer cases. Most of these are protein-truncating mutations, concentrated within or downstream of the second GATA-type zinc-finger domain. Here, we investigated the functional consequences of expression of two truncated GATA3 mutants, in vitro in breast cancer cell lines and in vivo in the mouse mammary gland. We found that the truncated GATA3 mutants display altered DNA binding activity caused by preferred tethering through FOXA1. In addition, expression of the truncated GATA3 mutants reduces E-cadherin expression and promotes anchorage-independent growth in vitro. However, we could not identify any effects of truncated GATA3 expression on mammary gland development or mammary tumor formation in mice. Together, our results demonstrate that both truncated GATA3 mutants promote cistromic re-programming of GATA3 in vitro, but these mutants are not sufficient to induce tumor formation in mice.