ERBB Receptors and Their Ligands in the Developing Mammary Glands of Different Species: Fifteen Characters in Search of an Author.

The ERBB tyrosine kinase receptors and their ligands belong to a complex family that has diverse biological effects and expression profiles in the developing mammary glands, where its members play an essential role in translating hormone signals into local effects. While our understanding of these processes stems mostly from mouse models, there is the potential for differences in how this family functions in the mammary glands of other species, particularly in light of their unique histomorphological features. Herein we review the postnatal distribution and function of ERBB receptors and their ligands in the mammary glands of rodents and humans, as well as for livestock and companion animals. Our analysis highlights the diverse biology for this family and its members across species, the regulation of their expression, and how their roles and functions might be modulated by varying stromal composition and hormone interactions. Given that ERBB receptors and their ligands have the potential to influence processes ranging from normal mammary development to diseased states such as cancer and/or mastitis, both in human and veterinary medicine, a more complete understanding of their biological functions should help to direct future research and the identification of new therapeutic targets.

Metabolic Stress Adaptations Underlie Mammary Gland Morphogenesis and Breast Cancer Progression.

Breast cancers display dynamic reprogrammed metabolic activities as cancers develop from premalignant lesions to primary tumors, and then metastasize. Numerous advances focus on how tumors develop pro-proliferative metabolic signaling that differs them from adjacent, non-transformed epithelial tissues. This leads to targetable oncogene-driven liabilities among breast cancer subtypes. Other advances demonstrate how microenvironments trigger stress-response at single-cell resolution. Microenvironmental heterogeneities give rise to cell regulatory states in cancer cell spheroids in three-dimensional cultures and at stratified terminal end buds during mammary gland morphogenesis, where stress and survival signaling juxtapose. The cell-state specificity in stress signaling networks recapture metabolic evolution during cancer progression. Understanding lineage-specific metabolic phenotypes in experimental models is useful for gaining a deeper understanding of subtype-selective breast cancer metabolism.

Mammary Development and Breast Cancer: a Notch Perspective.

Mammary gland development primarily occurs postnatally, and this unique process is complex and regulated by systemic hormones and local growth factors. The mammary gland is also a highly dynamic organ that undergoes profound changes at puberty and during the reproductive cycle. These changes are driven by mammary stem cells (MaSCs). Breast cancer is one of the most common causes of cancer-related death in women. Cancer stem cells (CSCs) play prominent roles in tumor initiation, drug resistance, tumor recurrence, and metastasis. The highly conserved Notch signaling pathway functions as a key regulator of the niche mediating mammary organogenesis and breast neoplasia. In this review, we discuss mechanisms by which Notch contributes to breast carcinoma pathology and suggest potentials for therapeutic targeting of Notch in breast cancer. In summary, we provide a comprehensive overview of Notch functions in regulating MaSCs, mammary development, and breast cancer.

Twelfth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The twelfth annual workshop of the European Network for Breast Development and Cancer focused on methods in mammary gland biology and breast cancer, was scheduled to take place on March 26-28, 2020, in Weggis, Switzerland. Due to the COVID-19 pandemic, the meeting was rescheduled twice and eventually happened as a virtual meeting on April 22 and 23, 2021. The main topics of the meeting were branching and development of the mammary gland, tumor microenvironment, circulating tumor cells, tumor dormancy and breast cancer metastasis. Novel and unpublished findings related to these topics were presented, with a particular focus on the methods used to obtain them. Virtual poster sessions were a success, with many constructive and fruitful interactions between researchers and covered many areas of mammary gland biology and breast cancer.

Molecular mechanisms of mammary gland remodeling: A review of the homeostatic versus bisphenol a disrupted microenvironment.

Mammary gland (MG) undergoes critical points of structural changes throughout a woman's life. During the perinatal and pubertal stages, MG develops through growth and differentiation to establish a pre-mature feature. If pregnancy and lactation occur, the epithelial compartment branches and differentiates to create a specialized structure for milk secretion and nurturing of the newborn. However, the ultimate MG modification consists of a regression process aiming to reestablish the smaller and less energy demanding structure until another production cycle happens. The unraveling of these fascinating physiologic cycles has helped the scientific community elucidate aspects of molecular regulation of proliferative and apoptotic events and remodeling of the stromal compartment. However, greater understanding of the hormonal pathways involved in MG developmental stages led to concern that endocrine disruptors such as bisphenol A (BPA), may influence these specific development/involution stages, called "windows of susceptibility". Since it is used in the manufacture of polycarbonate plastics and epoxy resins, BPA is a ubiquitous chemical present in human everyday life, exerting an estrogenic effect. Thus, descriptions of its deleterious effects on the MG, especially in terms of serum hormone concentrations, hormonal receptor expression, molecular pathways, and epigenetic alterations, have been widely published. Therefore, allied to a didactic description of the main physiological mechanisms involved in different critical points of MG development, the current review provides a summary of key mechanisms by which the endocrine disruptor BPA impacts MG homeostasis at different windows of susceptibility, causing short- and long-term effects.

Microenvironmental control of cell fate decisions in mammary gland development and cancer.

Cell fate decisions are critical for adequate tissue development, maintenance and regeneration. In the mammary gland, epithelial cell fates are tightly controlled by the microenvironment. Here, we review how cell fate decisions are regulated by components of the microenvironment during mammary gland development and how pathological changes in the microenvironment can alter cell fates, leading to malignancy. Specifically, we describe the current understanding of how mammary cell fate is controlled and directed by three elements: the extracellular matrix, the immune microenvironment, and hormones-and how these elements can converge to create microenvironments that promote a fourth element: DNA damage.

Estrogen receptor-α signaling in post-natal mammary development and breast cancers.

17ß-estradiol controls post-natal mammary gland development and exerts its effects through Estrogen Receptor ERα, a member of the nuclear receptor family. ERα is also critical for breast cancer progression and remains a central therapeutic target for hormone-dependent breast cancers. In this review, we summarize the current understanding of the complex ERα signaling pathways that involve either classical nuclear "genomic" or membrane "non-genomic" actions and regulate in concert with other hormones the different stages of mammary development. We describe the cellular and molecular features of the luminal cell lineage expressing ERα and provide an overview of the transgenic mouse models impacting ERα signaling, highlighting the pivotal role of ERα in mammary gland morphogenesis and function and its implication in the tumorigenic processes. Finally, we describe the main features of the ERα-positive luminal breast cancers and their modeling in mice.

Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids.

Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.

BAD regulates mammary gland morphogenesis by 4E-BP1-mediated control of localized translation in mouse and human models.

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.

Mammary gland development from a single cell 'omics view.

Understanding the complexity and heterogeneity of mammary cell subpopulations is vital to delineate the mechanisms behind breast cancer development, progression and prevention. Increasingly sophisticated tools for investigating these cell subtypes has led to the development of a greater understanding of these cell subtypes, complex interplay of certain subtypes and their developmental potential. Of note, increasing accessibility and affordability of single cell technologies has led to a plethora of studies being published containing data from mammary cell subtypes and their differentiation potential in both mice and human data sets. Here, we review the different types of single cell technologies and how they have been used to improve our understanding of mammary gland development.

Differential substrate use in EGF- and oncogenic KRAS-stimulated human mammary epithelial cells.

Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate and provide a model to distinguish the metabolic influences of oncogenic mutations and nononcogenic growth.

Attenuated TGFB signalling in macrophages decreases susceptibility to DMBA-induced mammary cancer in mice.

BACKGROUND: Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates mammary gland development and cancer progression through endocrine, paracrine and autocrine mechanisms. TGFB1 also plays roles in tumour development and progression, and its increased expression is associated with an increased breast cancer risk. Macrophages are key target cells for TGFB1 action, also playing crucial roles in tumourigenesis. However, the precise role of TGFB-regulated macrophages in the mammary gland is unclear. This study investigated the effect of attenuated TGFB signalling in macrophages on mammary gland development and mammary cancer susceptibility in mice. METHODS: A transgenic mouse model was generated, wherein a dominant negative TGFB receptor is activated in macrophages, in turn attenuating the TGFB signalling pathway specifically in the macrophage population. The mammary glands were assessed for morphological changes through wholemount and H&E analysis, and the abundance and phenotype of macrophages were analysed through immunohistochemistry. Another cohort of mice received carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), and tumour development was monitored weekly. Human non-neoplastic breast tissue was also immunohistochemically assessed for latent TGFB1 and macrophage marker CD68. RESULTS: Attenuation of TGFB signalling resulted in an increase in the percentage of alveolar epithelium in the mammary gland at dioestrus and an increase in macrophage abundance. The phenotype of macrophages was also altered, with inflammatory macrophage markers iNOS and CCR7 increased by 110% and 40%, respectively. A significant decrease in DMBA-induced mammary tumour incidence and prolonged tumour-free survival in mice with attenuated TGFB signalling were observed. In human non-neoplastic breast tissue, there was a significant inverse relationship between latent TGFB1 protein and CD68-positive macrophages. CONCLUSIONS: TGFB acts on macrophage populations in the mammary gland to reduce their abundance and dampen the inflammatory phenotype. TGFB signalling in macrophages increases mammary cancer susceptibility potentially through suppression of immune surveillance activities of macrophages.

Embryonic mammary gland development.

Embryonic mammary gland development involves the formation of mammary placodes, invagination of flask-shaped mammary buds and development of miniature bi-layered ductal trees. Currently there is a good understanding of the factors that contribute to ectodermal cell movements to create these appendages and of pathways that lead to mammary specification and commitment. Gene expression profiles of early bipotent mammary stem cells populations as well as cell surface proteins and transcription factors that promote the emergence of unipotent progenitors have been identified. Analyses of these populations has illuminated not only embryonic mammary development, but highlighted parallel processes in breast cancer. Here we provide an overview of the highly conserved pathways that shape the embryonic mammary gland. Understanding the dynamic signaling events that occur during normal mammary development holds considerable promise to advance attempts to eliminate cancer by restoring differentiative signals.

The molecular basis of mammary gland development and epithelial differentiation.

Our understanding of the molecular events underpinning the development of mammalian organ systems has been increasing rapidly in recent years. With the advent of new and improved next-generation sequencing methods, we are now able to dig deeper than ever before into the genomic and epigenomic events that play critical roles in determining the fates of stem and progenitor cells during the development of an embryo into an adult. In this review, we detail and discuss the genes and pathways that are involved in mammary gland development, from embryogenesis, through maturation into an adult gland, to the role of pregnancy signals in directing the terminal maturation of the mammary gland into a milk producing organ that can nurture the offspring. We also provide an overview of the latest research in the single-cell genomics of mammary gland development, which may help us to understand the lineage commitment of mammary stem cells (MaSCs) into luminal or basal epithelial cells that constitute the mammary gland. Finally, we summarize the use of 3D organoid cultures as a model system to study the molecular events during mammary gland development. Our increased investigation of the molecular requirements for normal mammary gland development will advance the discovery of targets to predict breast cancer risk and the development of new breast cancer therapies.

Prolactin: A hormone with diverse functions from mammary gland development to cancer metastasis.

Prolactin has a rich mechanistic set of actions and signaling in order to elicit developmental effects in mammals. Historically, prolactin has been appreciated as an endocrine peptide hormone that is responsible for final, functional mammary gland development and lactation. Multiple signaling pathways impacted upon by the microenvironment contribute to cell function and differentiation. Endocrine, autocrine and paracrine signaling are now apparent in not only mammary development, but also in cancer, and involve multiple cell types including those of the immune system. Multiple ligands agonists are capable of binding to the prolactin receptor, potentially expanding receptor function. Prolactin has an important role not only in tumorigenesis of the breast, but also in a number of hormonally responsive cancers such as prostate, ovarian and endometrial cancer, as well as pancreatic and lung cancer. Although pituitary and extra-pituitary sources of prolactin such as the epithelium are important, stromal sourced prolactin is now also being recognized as an important factor in tumor progression, all of which potentially signal to multiple cell types in the tumor microenvironment. While prolactin has important roles in milk production including calcium and bone homeostasis, in the disease state it can also affect bone homeostasis. Prolactin also impacts metastatic cancer of the breast to modulate the bone microenvironment and promote bone damage. Prolactin has a fascinating contribution in both physiologic and pathologic settings of mammals.

Pubertal mammary gland development is a key determinant of adult mammographic density.

Mammographic density refers to the radiological appearance of fibroglandular and adipose tissue on a mammogram of the breast. Women with relatively high mammographic density for their age and body mass index are at significantly higher risk for breast cancer. The association between mammographic density and breast cancer risk is well-established, however the molecular and cellular events that lead to the development of high mammographic density are yet to be elucidated. Puberty is a critical time for breast development, where endocrine and paracrine signalling drive development of the mammary gland epithelium, stroma, and adipose tissue. As the relative abundance of these cell types determines the radiological appearance of the adult breast, puberty should be considered as a key developmental stage in the establishment of mammographic density. Epidemiological studies have pointed to the significance of pubertal adipose tissue deposition, as well as timing of menarche and thelarche, on adult mammographic density and breast cancer risk. Activation of hypothalamic-pituitary axes during puberty combined with genetic and epigenetic molecular determinants, together with stromal fibroblasts, extracellular matrix, and immune signalling factors in the mammary gland, act in concert to drive breast development and the relative abundance of different cell types in the adult breast. Here, we discuss the key cellular and molecular mechanisms through which pubertal mammary gland development may affect adult mammographic density and cancer risk.

Fibroblasts: The grey eminence of mammary gland development.

The essential role of mammary gland stroma in the regulation of mammary epithelial development, function, and cancer has long been recognized. Only recently, though, the functions of individual stromal cell populations have begun to become more clarified. Mammary fibroblasts have emerged as master regulators and modulators of epithelial cell behavior through paracrine signaling, extracellular matrix production and remodeling, and through regulation of other stromal cell types. In this review article, we summarize the crucial studies that helped to untangle the roles of fibroblasts in mammary gland development. Furthermore, we discuss the origin, heterogeneity, and plasticity of mammary fibroblasts during mammary development and cancer progression.

Mammary development in the embryo and adult: new insights into the journey of morphogenesis and commitment.

The mammary gland is a unique tissue and the defining feature of the class Mammalia. It is a late-evolving epidermal appendage that has the primary function of providing nutrition for the young, although recent studies have highlighted additional benefits of milk including the provision of passive immunity and a microbiome and, in humans, the psychosocial benefits of breastfeeding. In this Review, we outline the various stages of mammary gland development in the mouse, with a particular focus on lineage specification and the new insights that have been gained by the application of recent technological advances in imaging in both real-time and three-dimensions, and in single cell RNA sequencing. These studies have revealed the complexity of subpopulations of cells that contribute to the mammary stem and progenitor cell hierarchy and we suggest a new terminology to distinguish these cells.

The inflammatory environment mediated by a high-fat diet inhibited the development of mammary glands and destroyed the tight junction in pregnant mice.

Long-term intake of a high-fat diet seriously affects the health of pregnant women and leads to increased levels of inflammation in the mammary gland. Therefore, to further explore the effect of a high-fat diet on mammary gland development and the tight junction (TJ) during pregnancy, we placed mice into two groups: a high-fat diet group and a control group. We detected the expression of proteins related to fat synthesis in the mammary gland by western blotting. The results showed that a high-fat diet could lead to an increase in fat synthesis in the mammary gland. Then, the inflammatory levels and acinar cell morphology in the mammary gland were detected by ELISA and H&E staining. We also measured the levels of MAPK and NF-κB signal pathway-related proteins by western blotting. The results showed that a high-fat diet activated the MAPK and NF-κB signaling pathways and promoted the expression of inflammatory factors. Finally, the development of the mammary gland and the integrity of the TJ were determined by immunohistochemistry, immunofluorescence and western blotting. The results showed that a high-fat diet inhibited the development of the mammary gland and the expression of tight junction proteins (TJs). Our study showed that a high-fat diet could promote the expression of inflammatory factors by activating the MAPK and NF-κB signaling pathways and could reshape the microenvironment through extramammary inflammation. Finally, a high-fat diet inhibited the development of the mammary gland during pregnancy and destroyed the integrity of the TJ.