Neuregulin 3 and Erbb Signalling Networks in Embryonic Mammary Gland Development

We review the role of Neuregulin 3 (Nrg3) and Erbb receptor signalling in embryonic mammary gland development. Neuregulins are growth factors that bind and activate its cognate Erbb receptor tyrosine kinases, which form a signalling network with established roles in breast development and breast cancer. Studies have shown that Nrg3 expression profoundly impacts early stages of embryonic mammary development. Network analysis shows how Nrg/Erbb signals could integrate with other major regulators of embryonic mammary development to elicit the morphogenetic processes and cell fate decisions that occur as the mammary lineage is established.     

Pleiotropic Functions of Fibroblast Growth Factor Signaling in Embryonic Mammary Gland Development

The mammary gland is an ectodermal appendage and a defining feature of mammals. Consistent with it being a recent evolutionary novelty, many of the molecules essential for the ontogeny and morphogenesis of various vertebrate organs, including those in the fibroblast growth factor (FGF) signaling pathway, are co-opted for induction, maintenance and morphogenesis of the mammary glands. Understanding the mechanism whereby FGF signaling regulates the fundamental cell behavior during normal mammary gland develop may facilitate determination of the consequences of its deregulation during breast cancer progression.     

A Crucial Role for Fibroblast Growth Factor Signaling in Embryonic Mammary Gland Development

The fibroblast growth factors (Fgfs) represent a large group of intercellular signaling molecules that mediate their effects by binding to a class of cell surface enzymes belonging to the receptor tyrosine kinase family (FgfRs). In vitro, Fgf signaling can induce potent mitogenic, motogenic, and angiogenic cellular responses, and has been associated with a multitude of biological processes. The development of gene targeting and transgenic strategies has provided unequivocal evidence for the key involvement of Fgf signaling in mammalian developmental processes. In this review we highlight recent findings that demonstrate a critical requirement for Fgf signaling in the induction and development of the embryonic mammary gland. Furthermore, we briefly discuss the potential of Fgfs to act as oncogenic factors in mammary neoplasia.     

Evo-Devo of the Mammary Gland

We propose a new scenario for mammary evolution based on comparative review of early mammary development among mammals. Mammary development proceeds through homologous phases across taxa, but evolutionary modifications in early development produce different final morphologies. In monotremes, the mammary placode spreads out to form a plate-like mammary bulb from which more than 100 primary sprouts descend into mesenchyme. At their distal ends, secondary sprouts develop, including pilosebaceous anlagen, resulting in a mature structure in which mammary lobules and sebaceous glands empty into the infundibula of hair follicles; these structural triads (mammolobular-pilo-sebaceous units or MPSUs) represent an ancestral condition. In marsupials a flask-like mammary bulb elongates as a sprout, but then hollows out; its secondary sprouts include hair and sebaceous anlagen (MPSUs), but the hairs are shed during nipple formation. In some eutherians (cat, horse, human) MPSUs form at the distal ends of primary sprouts; pilosebaceous components either regress or develop into mature structures. We propose that a preexisting structural triad (the apocrine-pilo-sebaceous unit) was incorporated into the evolving mammary structure, and coupled to additional developmental processes that form the mammary line, placode, bulb and primary sprout. In this scenario only mammary ductal trees and secretory tissue derive from ancestral apocrine-like glands. The mammary gland appears to have coopted signaling pathways and genes for secretory products from even earlier integumentary structures, such as odontode (tooth-like) or odontode-derived structures. We speculate that modifications in signal use (such as PTHrP and BMP4) may contribute to taxonomic differences in MPSU development.     

Nodal and Cripto-1: Embryonic Pattern Formation Genes Involved in Mammary Gland Development and Tumorigenesis

Members of the TGFbeta superfamily and EGF-CFC family, such as Nodal and Cripto, are important mediators of anterior-posterior and left-right axis specification during embryogenesis. In this paper, we review the role of Nodal and Cripto as critical morphogen-like molecules, with an emphasis on Nodal and EGF-CFC signaling during embryonic pattern formation. New evidence from gene expression and transgenic mouse studies have shown that both Nodal and Cripto-1 are expressed within the mammary duct and that modulation of these genes can disrupt normal branching morphogenesis resulting in epithelial disorganization and defective ductal architecture. We describe these new findings and propose that Cripto and Nodal are candidate mammary morphogens. Finally, the data linking overexpression of Cripto and perturbations of Cripto signaling to cell transformation and tumor formation are discussed. The fact that Cripto can modulate multiple pathways suggests it may act to deregulate growth inhibitors/homeostasis factors early in the cell transformation process and then activate prosurvival pathways dependent on MAPK and PI3K/Akt later in fully transformed phenotypes.     

Lipid Transport in the Lactating Mammary Gland

Mammalian cells depend on phospholipid (PL) and fatty acid (FA) transport to maintain membrane structure and organization, and to fuel and regulate cellular functions. In mammary glands of lactating animals, copious milk secretion, including large quantities of lipid in some species, requires adaptation and integration of PL and FA synthesis and transport processes to meet secretion demands. At present few details exist about how these processes are regulated within the mammary gland. However, recent advances in our understanding of the structural and molecular biology of membrane systems and cellular lipid trafficking provide insights into the mechanisms underlying the regulation and integration of PL and FA transport processes the lactating mammary gland. This review discusses the PL and FA transport processes required to maintain the structural integrity and organization of the mammary gland and support its secretory functions within the context of current molecular and cellular models of their regulation.     

Tight Junction Regulation in the Mammary Gland

Tight junctions form a narrow, continuous sealthat surrounds each endothelial and epithelial cell atthe apical border, and act to regulate the movement ofmaterial through the paracellular pathway. In the mammary gland, the tight junctions of thealveolar epithelial cells are impermeable duringlactation, and thus allow milk to be stored betweennursing periods without leakage of milk components from the lumen. Nonetheless mammary epithelial tightjunctions are dynamic and can be regulated by a numberof stimuli. Tight junctions of the mammary gland fromthe pregnant animal are leaky, undergoing closure around parturition to become the impermeabletight junctions of the lactating animal. Milk stasis,high doses of oxytocin, and mastitis have been shown toincrease tight junction permeability. In general changes in tight junction permeability in themammary gland appear to be the results of a state changeand not assembly and disassembly of tight junctions.Both local factors, such as intramammary pressure and TGF-beta, and systemic factors, such asprolactin, progesterone, and glucocorticoids, appear toplay a role in the regulation of mammary tightjunctions. Finally, the tight junction state appears to be closely linked to milk secretion. Anincrease in tight junction permeability is accompaniedby decrease in the milk secretion rate, and conversely,a decrease in tight junction permeability is accompanied by an increase in the milk secretionrate.     

Biology of Aromatase in the Mammary Gland

While the ovaries are the principal source of systemic estrogen in the premenopausal nonpregnant woman, other sites of estrogen biosynthesis are present throughout the body and these become the major sources of estrogen beyond menopause. These sites include the mesenchymal cells of the adipose tissue and skin, osteoblasts, and perhaps chondrocytes in bone, vascular endothelial and aortic smooth muscle cells, as well as a number of sites in the brain including the medial preoptic/anterior hypothalamus, the medial basal hypothalamus and the amygdala. These extragonadal sites of estrogen biosynthesis possess several fundamental features which differ from those of the ovaries. Principally, the estrogen synthesized within these compartments is probably only biologically active at a local tissue level in a paracrine or `intracrine' fashion. Thus the total amount of estrogen synthesized by these extragonadal sites may be small, but the local tissue concentrations achieved are probably quite high, and exert significant biological influence locally. Thus these sources of estrogen play an important but hitherto largely unrecognized, physiological and pathophysiological role.     

Mammary Gland Macrophages: Pleiotropic Functions in Mammary Development

Mammary gland development is a complex process involving epithelial cells and supporting stromal cells. Macrophages (M∅s) are an important component of the mammary gland stroma and are critical for normal mammary gland development; however, the mechanisms by which macrophages regulate these processes are not well understood. M∅s are known to interact with numerous cell types, including epithelial cells, fibroblasts, adipocytes, and endothelial cells, all of which are significant components of mammary gland development. Therefore, the purpose of this review is to describe the interactions between M∅s and these various cell types and use this knowledge to identify potential functions of M∅s in the mammary gland.     

Calcium Sensing by the Mammary Gland

Calcium is an important nutrient that is secreted into milk in quantities that put a considerable stress upon maternal calcium homeostasis. Here we summarize the evidence that two important entities, the extracellular calcium-sensing receptor (CaR) and parathyroid hormone-related protein (PTHrP) are involved in a feedback loop that regulates calcium fluxes to the mammary gland. The CaR may also play a role in regulating milk secretion, and may regulate the proliferation of normal and neoplastic mammary epithelial cells. Finally, the relationship between the CaR and PTHrP in breast cancer cells may promote the formation of osteolytic bone metastases.     

Microenvironment of the Involuting Mammary Gland Mediates Mammary Cancer Progression

Breast cancer diagnosed after a completed pregnancy has higher metastatic potential and therefore a much poorer prognosis. We hypothesize that following pregnancy the process of mammary gland involution, which returns the gland to its pre-pregnant state, co-opts some of the programs of wound healing. The pro-inflammatory milieu that results, while physiologically normal, promotes tumor progression. In this review, the similarities between mammary gland involution after cessation of milk-production and pathological tissue remodeling are discussed in light of emerging data demonstrating a role for pathological tissue remodeling in cancer.     

Integrins in Mammary Gland Development and Differentiation of Mammary Epithelium

Integrins are major extracellular matrix (ECM) receptors that can also serve for some cell-cell interactions. They have been identified as important regulators of mammary epithelial cell growth and differentiation. Their ability to promote cell anchorage, proliferation, survival, migration, and the induction of active ECM-degrading enzymes suggests that they play an essential role in normal mammary morphogenesis, but, on the other hand, reveals their potential to promote tumor progression.     

Epithelial Progenitors in the Normal Human Mammary Gland

The human mammary gland is organized developmentally as a hierarchy of progenitor cells that become progressively restricted in their proliferative abilities and lineage options. Three types of human mammary epithelial cell progenitors are now identified. The first is thought to be a luminal-restricted progenitor; in vitro under conditions that support both luminal and myoepithelial cell differentiation, this cell produces clones of differentiating daughter cells that are exclusively positive for markers characteristic of luminal cells produced in vivo (i.e., keratins 8/18 and 19, epithelial cell adhesion molecule [EpCAM] and MUC1). The second type is a bipotent progenitor. It is identified by its ability to produce mixed colonies in single cell assays. These colonies contain a central core of cells expressing luminal markers surrounded by cells with a morphology and markers (e.g., keratin 14⁺) characteristic of myoepithelial cells. Serial passage in vitro of an enriched population of bipotent progenitors promotes the expansion of a third type of progenitor that is thought to be myoepithelial-restricted because it only produces cells with myoepithelial features. Luminal-restricted and bipotent progenitors can prospectively be isolated as distinct subpopulations from freshly dissociated suspensions of normal human mammary cells. Both are distinguished from many other cell types in mammary tissue by their expression of EpCAM and CD49f (6 integrin). They are distinguished from each other by their differential expression of MUC1, which is expressed at much higher levels on the luminal progenitors. To relate the role of these progenitors to the generation of the three-dimensional tubuloalveolar structure of the mammary tree produced in vivo, we propose a model in which the commitment to the luminal versus the myoepithelial lineage may play a determining role in the generation of alveoli and ducts.     

The Mammary Gland Vasculature Revisited

Concomitant with the extensive growth and differentiation of the mammary epithelium during pregnancy and lactation, and epithelial involution after weaning, the vasculature of the mammary gland undergoes repeated cycles of expansion and regression. Vascular expansion is effected by sprouting angiogenesis, intussusception and conceivably also vasculogenesis. The capacity of the epithelial cells to stimulate vascular growth and differentiation is dependent on the constellation of systemic and local hormones and growth factors as well as the changing demands for oxygenation and nutrient supply. This results in the release of angiogenic factors which stimulate endothelial cell growth and regulate vascular architecture. In contrast to the angiogenic phase of the mammary gland cycle, little is known about the control of vascular regression although this would possibly offer new insights into therapeutic possibilities against breast cancer. In this review we summarize knowledge regarding the mechanisms regulating the vasculature of the mammary gland and delineate the importance of the vasculature in the attainment of organ function. In addition, we discuss the angiogenic mechanisms observed during mammary carcinogenesis and their consequences for breast cancer therapy.     

Cholesterol Transport and Regulation in the Mammary Gland

The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.     

Biology of Glucose Transport in the Mammary Gland

Glucose is the major precursor of lactose, which is synthesized in Golgi vesicles of mammary secretory alveolar epithelial cells during lactation. Glucose is taken up by mammary epithelial cells through a passive, facilitative process, which is driven by the downward glucose concentration gradient across the plasma membrane. This process is mediated by facilitative glucose transporters (GLUTs), of which there are 14 known isoforms. Mammary glands mainly express GLUT1 and GLUT8, and GLUT1 is the predominant isoform with a K _m of ~10 mM and transport activity for mannose and galactose in addition to glucose. Mammary glucose transport activity increases dramatically from the virgin state to the lactation state, with a concomitant increase in GLUT expression. The increased GLUT expression during lactogenesis is not stimulated by the accepted lactogenic hormones. New evidence indicates that a possible low oxygen tension resulting from increased metabolic rate and oxygen consumption may play a major role in stimulating glucose uptake and GLUT1 expression in mammary epithelial cells during lactogenesis. In addition to its primary presence on the plasma membrane, GLUT1 is also expressed on the Golgi membrane of mammary epithelial cells and is likely involved in facilitating the uptake of glucose and galactose to the site of lactose synthesis. Because lactose synthesis dictates milk volume, regulation of GLUT expression and trafficking represents potentially fruitful areas for further research in dairy production. In addition, this research will have pathological implications for the treatment of breast cancer because glucose uptake and GLUT expression are up-regulated in breast cancer cells to accommodate the increased glucose need.