Journal of Mammary Gland Biology and Neoplasia

Recent advances in genome-wide sequencing technologies have provided researchers with unprecedented opportunities to discover the genomic structures of gene regulatory units in living organisms. In particular, the integration of ChIP-seq, RNA-seq, and DNase-seq techniques has facilitated the mapping of a new class of regulatory elements. These elements, called super-enhancers, can regulate cell-type-specific gene sets and even fine-tune gene expression regulation in response to external stimuli, and have become a hot topic in genome biology. However, there is scant genetic evidence demonstrating their unique biological relevance and the mechanisms underlying these biological functions. In this review, we describe a robust genome-wide strategy for mapping cell-type-specific enhancers or super-enhancers in the mammary genome. In this strategy, genome-wide screening of active enhancer clusters that are co-occupied by mammary-enriched transcription factors, co-factors, and active enhancer marks is used to identify bona fide mammary tissue-specific super-enhancers. The in vivo function of these super-enhancers and their associated regulatory elements may then be investigated in various ways using the advanced CRISPR/Cas9 genome-editing technology. Based on our experience targeting various mammary genomic sites using CRISPR/Cas9 in mice, we comprehensively discuss the molecular consequences of the different targeting methods, such as the number of gRNAs and the dependence on their simultaneous or sequential injections. We also mention the considerations that are essential for obtaining accurate results and shed light on recent progress that has been made in developing modified CRISPR/Cas9 genome-editing techniques. In the future, the coupling of advanced genome-wide sequencing and genome-editing technologies could provide new insights into the complex genetic regulatory networks involved in mammary-gland development.

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 Hedgehog pathway is critical for many developmental processes, including the formation of several epidermal appendages. In the mammary gland strict regulation of the Hedgehog pathway is required for normal development. Alterations in Hedgehog signaling result in defects in both the embryonic and postnatal mammary gland. Activation of Hedgehog signaling either by mutation or misexpression of pathway members can lead to the development and/or progression of cancers in multiple organs. This review addresses the current understanding and controversies of Hedgehog signaling in mammary gland development and its potential role in promoting breast carcinogenesis and cancer progression.

This review is focused on genetic factors that may influence the development and/or appearance of breast cancer metastases. Over the last decade there have been significant advances in the understanding of genetic predisposition to breast cancer. The first breast cancer predisposing gene to be identified was TP53, and this was followed over the next 5 years by two more genes, BRCA1 and BRCA2, which from a population perspective are much more important than TP53. Other rarer genes have subsequently been identified, but the role of more common, less penetrant genes in breast cancer susceptibility remains unknown. Recent work has shown that breast cancers occurring in women carrying germ-line BRCA1 mutations tend to have clinicopathological features that are usually associated with a poor prognosis, such as high grade, estrogen receptor negative status and somatic TP53 mutations. On the other hand, they are usually ERBB2 negative. Whether or not such tumors are more or less likely to metastasize, and hence be associated with a poor outcome, is currently uncertain and has been the subject of much debate. Here, we outline some of the clinicopathological features of hereditary breast cancer, discuss the prognostic studies that have been performed, and introduce some possible new research directions.

The thirteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) Laboratories Annual Workshop took place on the 28–30 April 2022 in Weggis, Switzerland and focused on methods in mammary gland biology and breast cancer. Sixty scientists participated in the ENBDC annual workshop which had not been held in person since 2019 due to the global COVID-19 pandemic. Topics spanned the mammary gland biology field, ranging from lactation biology and embryonic development, single cell sequencing of the human breast, and stunning cutting-edge imaging of the mouse mammary gland and human breast as well as breast cancer research topics including invasive progression of the pre-invasive DCIS stage, metabolic determinants of endocrine therapy resistance, models for lobular breast cancer, and how mutational landscapes of normal breast during age and pregnancy determine cancer risk. The latest findings from participating researchers were presented through oral presentations and poster sessions and included plenty of unpublished work.

Mastitis is a common inflammatory disease during lactation that causes reduced milk supply. A growing body of evidence challenges the central role of pathogenic bacteria in mastitis, with disease severity associated with markers of inflammation rather than infection. Inflammation in the mammary gland may be triggered by microbe-associated molecular patterns (MAMPs) as well as danger-associated molecular patterns (DAMPs) binding to pattern recognition receptors such as the toll-like receptors (TLRs) on the surface of mammary epithelial cells and local immune cell populations. Activation of the TLR4 signalling pathway and downstream nuclear factor kappa B (NFkB) is critical to mediating local mammary gland inflammation and systemic immune responses in mouse models of mastitis. However, activation of NFkB also induces epithelial cell apoptosis and reduced milk protein synthesis, suggesting that inflammatory mediators activated during mastitis promote partial involution. Perturbed milk flow, maternal stress and genetic predisposition are significant risk factors for mastitis, and could lead to a heightened TLR4-mediated inflammatory response, resulting in increased susceptibility and severity of mastitis disease in the context of low MAMP abundance. Therefore, heightened host inflammatory signalling may act in concert with pathogenic or commensal bacterial species to cause both the inflammation associated with mastitis and lactation insufficiency. Here, we present an alternate paradigm to the widely held notion that breast inflammation is driven principally by infectious bacterial pathogens, and suggest there may be other therapeutic strategies, apart from the currently utilised antimicrobial agents, that could be employed to prevent and treat mastitis in women.

Transformation of breast cells occurs through loss or mutation of tumor suppressor genes, or activation or amplification of oncogenes, leading to deregulation of signal transduction pathways, abnormal amplification of growth signals, and aberrant expression of genes that ultimately transform the cells into invasive cancer. The goal of cancer preventive therapy, or “chemoprevention,” is to eliminate premalignant cells or to block the progression of normal cells into cancer. Multiple alterations in signal pathways and transcription factors are observed in mammary gland tumorigenesis. In particular, estrogen receptor (ER) deregulation plays a critical role in breast cancer development and progress, and targeting ER with selective ER modulators (SERMs) has achieved significant reduction of breast cancer incidence in women at high risk for breast cancer. However, not all breast cancer is prevented by SERMs, because 30–40% of the tumors are ER-negative. Other receptors for retinoids, vitamin D analogs and peroxisome proliferator–activiator, along with transcription factors such as AP-1, NF-κB, and STATs (signal transducers and activators of transcription) affect breast tumorigenesis. This is also true for the signal transduction pathways, for example cyclooxygenase 2 (Cox-2), HER2/neu, mitogen-activated protein kinase (MAPK), and PI3K/Akt. Therefore, proteins in pathways that are altered during the process of mammary tumorigenesis may be promising targets of future chemopreventive drugs. Many newly-developed synthetic or natural compounds/agents are now under testing in preclinical studies and clinical trials. Receptor selective retinoids, receptor tyrosine kinase inhibitors (TKIs), SERMs, Cox-2 inhibitors, and others are some of the promising novel agents for the prevention of breast cancer. The chemopreventive activity of these agents and other novel signal transduction inhibitors are discussed in this chapter.