Mammary Gland Biology and Neoplasia

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.

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.

RETRACTED ARTICLE: Inhibition of Tumor Progression by N G -Nitro-L-arginine Methyl Ester in 7,12- dimethylbenz(a)anthracene Induced Breast Cancer: Nitric Oxide Synthase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

Metformin and Breast Cancer: Molecular Targets Abstract Metformin has been the first-line drug for the treatment of type II diabetes mellitus for decades, being presently the most widely prescribed antihyperglycemic drug. Retrospective studies associate the use of metformin with a reduction in cancer incidence and cancer-related death. However, despite extensive research about the molecular effects of metformin in cancer cells, its mode of action remains controversial. The major molecular targets of metformin include complex I of the mitochondrial electron transport chain, adenosine monophosphate (AMP)-activated protein kinase (AMPK), and mechanistic target of rapamycin complex 1 (mTORC1), but AMPK-independent effects of metformin have also been described. Breast cancer is one of the leading causes of cancer-related morbidity and mortality among women worldwide. Several studies have reinforced a link between breast cancer risk and diabetes. Moreover, metformin significantly reduces breast cancer risk, compared to patients who are not using metformin and is independent of diabetes status. In this review, we summarize the current molecular evidence to elucidate metformin’s mode of action against breast cancer cells.

Mammary Epithelial Cell Lineage Changes During Cow’s Life Abstract Milk production is highly dependent on the optimal development of the mammary epithelium. It is therefore essential to better understand mammary epithelial cell growth and maintenance from the related epithelial lineage during the animal life. Here, we characterized the epithelial lineage at puberty, lactation and dry-off in bovine using the cell surface markers CD49f, CD24, and CD10. The pubertal period was characterized by a high proportion of CD49fpos cells corresponding to various epithelial subpopulations, notably the CD24pos subpopulations. The proportion of CD49fpos cells was weaker during lactation and dry-off, and CD24pos cells were relatively few. Of note, the (sub)population profile at dry-off appeared close to that during lactation. Using a targeted gene approach, we associated specific genes with epithelial subpopulations, their expression level varying, or not, according to physiological stages. Caseinswere only expressed in the CD49fmedCD24neg subpopulation. Basal marker genes (keratin(KRT)5, KRT14 and αSMA) were found in the CD49fhighCD24neg subpopulations. Luminal gene markers (KRT7, KRT8 and KRT19, CDH1 and the PRLR) were expressed in the CD49flowCD24neg subpopulation. The CD49flowCD24pos subpopulation, only abundant at puberty, expressed luminal gene markers and KI67 at high level. In contrast to others, the CD49fhighCD24poscells accounted for a small proportion of total cells, decreasing from puberty to dry-off. They were characterized by expression of luminal and basal gene markers and low KI67 level. Interestingly, this subpopulation showed a remarkable stability of gene expression profile throughout physiological stages and bear the hallmark of quiescence that designate them as the potential bovine mammary stem cells.

Application of the D492 Cell Lines to Explore Breast Morphogenesis, EMT and Cancer Progression in 3D Culture Abstract The human female breast gland is composed of branching epithelial ducts that extend from the nipple towards the terminal duct lobular units (TDLUs), which are the functional, milk-producing units of the gland and the site of origin of most breast cancers. The epithelium of ducts and TDLUs is composed of an inner layer of polarized luminal epithelial cells and an outer layer of contractile myoepithelial cells, separated from the vascular-rich stroma by a basement membrane. The luminal- and myoepithelial cells share an origin and in recent years, there has been increasing understanding of how these cell types interact and how they contribute to breast cancer. Accumulating evidence links stem/or progenitor cells in the mammary/breast gland to breast cancer. In that regard, much knowledge has been gained from studies in mice due to specific strains that have allowed for gene knock out/in studies and lineage tracing of cellular fates. However, there is a large histologic difference between the human female breast gland and the mouse mammary gland that necessitates that research needs to be done on human material where primary cultures are important due to their close relation to the tissue of origin. However, due to difficulties of long-term cultures and lack of access to material, human cell lines are of great importance to bridge the gap between studies on mouse mammary gland and human primary breast cells. In this review, we describe D492, a breast epithelial progenitor cell line that can generate both luminal- and myoepithelial cells in culture, and in 3D culture it forms branching ducts similar to TDLUs. We have applied D492 and its daughter cell lines to explore cellular and molecular mechanisms of branching morphogenesis and cellular plasticity including EMT and MET. In addition to discussing the application of D492 in studying normal morphogenesis, we will also discuss how this cell line has been used to study breast cancer progression.

A Syngeneic ErbB2 Mammary Cancer Model for Preclinical Immunotherapy Trials Abstract In order to develop a practical model of breast cancer, with in vitro and syngeneic, immune-intact, in vivo growth capacity, we established a primary cell line derived from a mammary carcinoma in the transgenic FVB/N-Tg(MMTV-ErbB2*)NDL2-5Mul mouse, referred to as “NDLUCD”. The cell line is adapted to standard cell culture and can be transplanted into syngeneic FVB/N mice. The line maintains a stable phenotype over multiple in vitro passages and rounds of in vivo transplantation. NDLUCD tumors in FVB/N mice exhibit high expression of ErbB2 and ErbB3 and signaling molecules downstream of ErbB2. The syngeneic transplant tumors elicit an immune reaction in the adjacent stroma, detected and characterized using histology, immunophenotyping, and gene expression. NDLUCD cells also express PD-L1 in vivo and in vitro, and in vivo transplants are reactive to anti-immune checkpoint therapy with responses conducive to immunotherapy studies. This new NDLUCD cell line model is a practical alternative to the more commonly used 4T1 cells, and our previously described FVB/N-Tg(MMTV-PyVT)634Mul derived Met-1fvb2 and FVB/NTg(MMTV-PyVTY315F/Y322F) derived DB-7fvb2 cell lines. The NDLUCD cells have, so far, remained genetically and phenotypically stable over many generations, with consistent and reproducible results in immune intact preclinical cohorts.

Detection of Milk Ejection Using Bioimpedance Spectroscopy in Lactating Women during Milk Expression Using an Electric Breast Pump Abstract Milk ejection is essential for effective milk removal during breastfeeding and pumping, and for continued milk synthesis. Many women are unable to accurately sense milk ejection to determine whether their infant is receiving milk or, when pumping, to switch the pump to a more effective expression pattern. To determine if changes in bioimpedance parameters are associated with milk ejection in the lactating breast during pumping. 30 lactating women participated in 2 pumping sessions within 2 weeks of each other. During pumping the breasts were monitored with bioimpedance spectroscopy (on either the pumped or the non- pumped breast), and milk flow rate and volume were measured simultaneously. All mothers completed 24-h milk productions. Linear mixed effects models were used to determine associations between milk flow rate and bioimpedance changes. Changes in bioimpedance parameters were greater at the first milk ejection when measured on the pumped breast (median (IQR): R zero: −7 (−17, −4,) % (n = 30); R infinity: −8 (−20, −2) % (n = 29); membrane capacitance: −24 (−59, −7) % (n = 27). Changes in bioimpedance detected in the non-pumped breast were lower at the first milk ejection, R zero: −3 (−8, −2) % (n = 25); R infinity: −5 (−8, −2) % (n = 23); membrane capacitance: −9 (−17, 15) % (n = 24). Smaller less consistent decreases in the bioimpedance characteristics were detected at the second milk ejection in both breasts. Bioimpedance parameters showed a consistent decrease associated with the first milk ejection when electrodes were placed on the pumped breast. Smaller decreases were observed when the non-pumped breast was monitored for the first and second milk ejection. There was wide variation in the magnitude of changes observed, and hence further development of the methodology is needed to ensure reliability.