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.

The Milk Protein Alpha-Casein Suppresses Triple Negative Breast Cancer Stem Cell Activity Via STAT and HIF-1alpha Signalling Pathways in Breast Cancer Cells and Fibroblasts Abstract Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

Emerging Role of SOX Proteins in Breast Cancer Development and Maintenance Abstract The SOX genes encode a family of more than 20 transcription factors that are critical regulators of embryogenesis and developmental processes and, when aberrantly expressed, have been shown to contribute to tumor development and progression in both an oncogenic and tumor suppressive role. Increasing evidence demonstrates that the SOX proteins play essential roles in multiple cellular processes that mediate or contribute to oncogenic transformation and tumor progression. In the context of breast cancer, SOX proteins function both as oncogenes and tumor suppressors and have been shown to be associated with tumor stage and grade and poor prognosis. Experimental evidence demonstrates that a subset of SOX proteins regulate critical aspects of breast cancer biology including cancer stemness and multiple signaling pathways leading to altered cell proliferation, survival, and tumor development; EMT, cell migration and metastasis; as well as other tumor associated characteristics. This review will summarize the role of SOX family members as important mediators of tumorigenesis in breast cancer, with an emphasis on the triple negative or basal-like subtype of breast cancer, as well as examine the therapeutic potential of these genes and their downstream targets.

RETRACTED ARTICLE: Inhibition of Tumor Progression by N G -Nitro-L-arginine Methyl Ester in 7,12- dimethylbenz(a)anthracene Induced Breast Cancer: Nitric Oxide Synthase Inhibition as an Antitumor Prevention

A Longitudinal Study of the Association between Mammographic Density and Gene Expression in Normal Breast Tissue Abstract High mammographic density (MD) is associated with a 4–6 times increase in breast cancer risk. For post-menopausal women, MD often decreases over time, but little is known about the underlying biological mechanisms. MD reflects breast tissue composition, and may be associated with microenvironment subtypes previously identified in tumor-adjacent normal tissue. Currently, these subtypes have not been explored in normal breast tissue. We obtained biopsies from breasts of healthy women at two different time points several years apart and performed microarray gene expression analysis. At time point 1, 65 samples with both MD and gene expression were available. At time point 2, gene expression and MD data were available from 17 women, of which 11 also had gene expression data available from the first time point. We validated findings from our previous study; negative correlation between RBL1 and MD in post-menopausal women, indicating involvement of the TGFβ pathway. We also found that breast tissue samples from women with a large decrease in MD sustained higher expression of genes in the histone family H4. In addition, we explored the previously defined active and inactive microenvironment subtypes and demonstrated that normal breast samples of the active subtype had characteristics similar to the claudin-low breast cancer subtype. Breast biopsies from healthy women are challenging to obtain, but despite a limited sample size, we have identified possible mechanisms relevant for changes in breast biology and MD over time that may be of importance for breast cancer risk and tumor initiation.

A Comparative Review of Mixed Mammary Tumors in Mammals Abstract Mixed tumors are characterized by the histological identification of two or more cell types. Commonly, a mixture of epithelial and myoepithelial cells is included in abundant stroma, which can consist of myxoid, chondroid or bony matrices. Spontaneously arising mixed tumors are rare lesions in the human breast but are common in human salivary glands and canine mammary glands. Subtle histopathological characteristics and overlapping attributes of malignant lesions with other benign lesions can lead to a diagnostic challenge. Mixed tumors can present as benign or malignant. While malignant mixed tumors are quite rare in the human breast they have a poor prognosis. Benign mixed mammary tumors occur more frequently in female dogs than in humans and are usually associated with a good prognosis. This review will provide a comprehensive overview of mixed mammary tumors, across various mammalian species.