The mammary gland epithelium comprises two major cell types: basal and luminal. Basal cells interact directly with the extracellular matrix (ECM) and express higher levels of the ECM receptors, integrins, than luminal cells. We show that deletion of beta1 integrin from basal cells abolishes the regenerative potential of the mammary epithelium and affects mammary gland development. The mutant epithelium was characterized by an abnormal ductal branching pattern and aberrant morphogenesis in pregnancy, although at the end of gestation, the secretory alveoli developed from beta1 integrin-positive progenitors. Lack of beta1 integrin altered the orientation of the basal-cell division axis and in mutant epithelium, in contrast to control tissue, the progeny of beta1 integrin-null basal cells, identified by a genetic marker, was found in the luminal compartment. These results reveal, for the first time, the essential role of the basal mammary epithelial cell-ECM interactions mediated by beta1 integrins in the maintenance of a functional stem cell population, mammary morphogenesis and segregation of the two major mammary cell lineages.

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt acin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using 2 independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage tracing approach we follow the progeny of α-smooth muscle actin-expressing myoepithelial cells and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

Wnt/β-catenin signaling pathway is involved in the maintenance of the progenitor cell population in the skin, intestine and other tissues, and its aberrant activation caused by stabilization of β-catenin contributes to tumorigenesis. In the mammary gland, constitutive activation of Wnt/β-catenin signaling in luminal secretory cells results in precocious lobuloalveolar differentiation and induces adenocarcinomas, whereas the impact of this signaling pathway on the function of the second major mammary epithelial cell lineage, the basal myoepithelial cells, has not been analyzed. We have used the keratin (K) 5 promoter to target the expression of stabilized N-terminally truncated β-catenin to the basal cell layer of mouse mammary epithelium. The transgenic mice presented an abnormal mammary phenotype:precocious lateral bud formation, increased proliferation and premature differentiation of luminal epithelium in pregnancy, persistent proliferation in lactation and accelerated involution. Precocious development in pregnancy was accompanied by increased Myc and cyclin D1 transcript levels, and a shift in p63 variant expression towards the ΔNp63 form. The expression of ECM-degrading proteinases and their inhibitors was altered in pregnancy and involution. Nulliparous transgenic females developed mammary hyperplasia that comprised undifferentiated basal (K5/14-positive, K8- and α-smooth muscle-actin-negative) cells. Multiparous mice, in addition, developed invasive basal-type carcinomas. Thus, activation of β-catenin signaling in basal mammary epithelial cells affects the entire process of mammary gland development and induces amplification of basal-type cells that lack lineage markers, presumably, a subpopulation of mammary progenitors able to give rise to tumors.

224 ECM = extracellular matrix; FGF2 = basic fibroblast growth factor; PTHrP = parathyroid hormone-related protein; TEB = terminal end buds. Breast Cancer Research Vol 4 No 6 Deugnier et al. IntroductionThe mammary gland consists of secretory alveoli connected by a system of branching ducts embedded in connective tissue. The epithelial cells that compose the gland are arranged in two layers, the luminal epithelial layer and the basal myoepithelial layer. The whole structure is surrounded by a basement membrane.The myoepithelial layer is organised differently in the ducts and in the lobules. In the ducts, elongated myoepithelial cells form a more or less continuous layer and are in direct contact with the basement membrane, and hence with the stroma. The interaction between ductal luminal cells and the extracellular matrix (ECM) is largely mediated by the myoepithelium, although some of the luminal cells in the mammary ducts may reach the basement membrane. Alveolar myoepithelial cells are of stellate shape and form a basket-like structure around the acini, resulting in the exposure of most of the basal surface of the luminal cell to the basement membrane. Differentiated myoepithelial cells are highly contractile and their ultrastructure is reminiscent of that of smooth muscle cells. Myoepithelial cells contain large amounts of microfilaments, dense plaques (cell-matrix adherens junctions characteristic of smooth muscle cells) and smooth muscle-specific cytoskeletal and contractile proteins. They are true epithelial cells, however, because the major components of their intermediate filament system are the cytokeratins 5 and 14 (K5 and K14), because they form desmosomes, hemidesmosomes and cadherin-mediated cell-cell junctions, and because they are permanently separated from the connective tissue by the underlying basement membrane.The contractile properties and the central role in milk ejection during lactation are the most studied aspects of mammary myoepithelial cell function. In addition, the tumour suppressor potential of mammary myoepithelial cells has recently received considerable attention. However, the role played by the myoepithelial cells in ReviewThe importance of being a myoepithelial cell AbstractThe mammary myoepithelial cell was named the 'Cinderella of mammary cell biology' in light of the earlier focus on the luminal cell. Mammary myoepithelial cells have recently been described as 'natural tumour suppressors'. We now need to understand more about their origin and to reconsider their place in the complex process of mammary morphogenesis. In the present review, we discuss the lineage segregation of mammary myoepithelial cells and their functions in mammary gland development. These functions include their effects on luminal cell growth and differentiation, their key role in the establishment of the polarised mammary epithelial bilayer and the control of stromal invasion in breast cancer.

BackgroundMorphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) “master genes”. EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.Methodology/Principal FindingsUsing a Slug–lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10–20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.Conclusions/SignificanceWe conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and provide physiological relevance to broadening EMT pathways.

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.

A mouse mammary epithelial cell line with morphogenetic properties in vivo, Comma-Dbeta, was used to isolate and to characterize mammary progenitor cells. We found that a homogeneous cell population expressing high surface levels of stem cell antigen 1 (Sca-1) was able to give rise in vivo to ductal and alveolar structures comprising luminal secretory and basal myoepithelial cells. Unlike the Sca-1(high), the Sca-1(neg/low) cell population displayed a reduced morphogenetic potential. The Sca-1(high) cells presented moderate CD24, high CD44 and alpha6 integrin surface levels, expressed basal cell markers p63, keratins 5 and 14, but no luminal and myoepithelial lineage markers. In culture, the Sca-1(high) cells generated identical daughter cells that retained their in vivo developmental potential, indicating that these cells were maintained by self-renewal. Plated at clonogenic density in Matrigel, Sca-1(high) cells formed spheroids that included luminal and myoepithelial cells. Thus, the isolated Sca-1(high) basal cells possess several features of stem/progenitor cells, including specific markers, self-renewal capacity, and the ability to generate the two major mammary lineages, luminal and myoepithelial. These data provide evidence for the existence of basal-type mouse mammary progenitors able to participate in the morphogenetic processes characteristic of mammary gland development.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.