Marina A. Glukhova

Perturbation of β1‐integrin function alters the development of murine mammary gland

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the β1‐integrin chain and the extracellular domain of the T‐cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous β1‐integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the β1‐cytoplasmic domain and thus perturb β1‐integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild‐type mice. At mid‐pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild‐type glands. In lactation, the amounts of the whey acidic protein (WAP) and β‐casein gene transcripts were diminished, and the basement membrane component, laminin and the β4‐integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, β1‐integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso‐apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function.

The importance of being a myoepithelial cell

The 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.

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.

Growth defects induced by perturbation of β1-integrin function in the mammary gland epithelium result from a lack of MAPK activation via the Shc and Akt pathways

Adhesion to extracellular matrix (ECM) induces intracellular signals that modulate cell proliferation, survival and differentiation. To study signalling events triggered by cell–ECM interactions in vivo we used transgenic mice exhibiting reduced mammary epithelial cell proliferation and increased apoptosis rates during the growth phase in pregnancy and lactation due to expression of a β1‐integrin dominant‐negative mutant in the mammary gland epithelium. Here we show that ERK and JNK MAPKs were markedly less activated in lactating transgenic glands thereby accounting for the growth defects. The FAK pathway was not affected suggesting a mechanism of activation additional to the ECM signal. On the contrary, the significant decrease of Shc phosphorylation, Grb2 recruitment and the reduced phosphorylation level of Akt Thr308 and Akt substrates FKHR and Bad detected in transgenic glands show that activation of the Shc and the Akt pathways require intact cell–ECM interactions. These results provide an insight into the mechanisms of growth control by integrin‐mediated adhesion that operate in vivo.

Control of mammary myoepithelial cell contractile function by α3β1 integrin signalling.

In the functionally differentiated mammary gland, basal myoepithelial cells contract to eject the milk produced by luminal epithelial cells from the body. We report that conditional deletion of a laminin receptor, α3β1 integrin, from myoepithelial cells leads to low rates of milk ejection due to a contractility defect but does not interfere with the integrity or functional differentiation of the mammary epithelium. In lactating mammary gland, in the absence of α3β1, focal adhesion kinase phosphorylation is impaired, the Rho/Rac balance is altered and myosin light‐chain (MLC) phosphorylation is sustained. Cultured mammary myoepithelial cells depleted of α3β1 contract in response to oxytocin, but are unable to maintain the state of post‐contractile relaxation. The expression of constitutively active Rac or its effector p21‐activated kinase (PAK), or treatment with MLC kinase (MLCK) inhibitor, rescues the relaxation capacity of mutant cells, strongly suggesting that α3β1‐mediated stimulation of the Rac/PAK pathway is required for the inhibition of MLCK activity, permitting completion of the myoepithelial cell contraction/relaxation cycle and successful lactation. This is the first study highlighting the impact of α3β1 integrin signalling on mammary gland function.

Myoepithelial Cells in the Control of Mammary Development and Tumorigenesis: Data From Genetically Modified Mice

Until recently, myoepithelial cells—the second major cell population in the mammary epithelium—were not considered to play an important role in the morphogenetic events during gland development. Mouse mutants with changes in the gene expression pattern characteristic of the basal myoepithelial cell layer have been generated and used to show that these cells influence the proliferation, survival and differentiation of luminal cells, modulate stromal–epithelial interactions and actively participate in mammary morphogenesis. Various cellular and molecular mechanisms may underlie the observed phenotypes. These include an unbalanced expression of matrix degrading metalloproteinases (MMPs) and their inhibitors, leading to changes in the composition and organization of the (extracellular matrix) ECM, the production of soluble growth factors affecting stromal and epithelial cell growth and differentiation and direct signaling through cell–cell contacts between the myoepithelial and luminal cell layers.

The Proto-Oncogene Myc Is Essential for Mammary Stem Cell Function

The mammary epithelium comprises two major cell lineages: basal and luminal. Basal cells (BCs) isolated from the mammary epithelium and transplanted into the mouse mammary fat pad cleared from the endogenous epithelium regenerate the mammary gland, strongly suggesting that the basal epithelial compartment harbors a long‐lived cell population with multipotent stem cell potential. The luminal cell layer is devoid of the regenerative potential, but it contains cells with clonogenic capacity, the luminal progenitors. Mammary BCs and luminal progenitors express high levels of the transcription factor Myc. Here, we show that deletion of Myc from mammary basal epithelial cells led to impaired stem cell self‐renewal as evaluated by limiting dilution and serial transplantation assays. Luminal progenitor population was significantly diminished in mutant epithelium suggesting control by the BC layer. Colony formation assay performed with isolated BCs showed that clonogenic capacity was abolished by Myc deletion. Moreover, transplanted BCs depleted of Myc failed to produce epithelial outgrowths. Stimulation with ovarian hormones estrogen (E) and progesterone (P) partially rescued the repopulation capacity of Myc‐depleted BCs; however, the Myc‐deficient mammary epithelium developed in response to E/P treatment lacked stem and progenitor cells. This study provides the first evidence that in the mammary gland, Myc has an essential nonredundant function in the maintenance of the self‐renewing multipotent stem cell population responsible for the regenerative capacity of the mammary epithelium and is required downstream from ovarian hormones, for the control of mammary stem and progenitor cell functions. STEM CELLS2012;30:1246–1254

β‐Catenin regulates P‐cadherin expression in mammary basal epithelial cells

P‐cadherin expression is restricted to the basal layer of stratified epithelia including that of the mammary gland. Although evidence for an important role of P‐cadherin in mammary morphogenesis and tumorigenesis is increasing, the mechanisms that regulate its expression are poorly understood. We show that in basal mammary epithelial cells, β‐catenin is associated with the P‐cadherin promoter and activates its expression independently of LEF/TCF in a cell‐type specific manner. Down‐regulation of endogenous β‐catenin levels by RNA interference technique inhibited P‐cadherin promoter activity. In vivo, in skin and mammary gland of mutant mice, activation of β‐catenin signalling correlates with up‐regulation of P‐cadherin expression. These data suggest that β‐catenin‐dependent modulation of P‐cadherin expression can contribute to the establishment of the basal phenotype.

Integrins in mammary development.

Integrins are ubiquitously expressed major cell surface receptors for extracellular matrix. Integrin interaction with their extracellular ligands triggers activation of the intracellular signaling pathways that control cell shape, motility, proliferation, survival, cell-type-specific gene expression. In this review, we summarize recent studies analyzing contribution of integrins to the control of the mammary morphogenesis and differentiation, function and maintenance of mammary stem and progenitor cells and resume the data from mouse models revealing the contribution of the integrin-mediated signaling to mammary tumorigenesis.

Somatic loss of p53 leads to stem/progenitor cell amplification in both mammary epithelial compartments, basal and luminal.

Mammary epithelium comprises a layer of luminal cells and a basal myoepithelial cell layer. Both mammary epithelial compartments, basal and luminal, contain stem and progenitor cells, but only basal cells are capable of gland regeneration upon transplantation. Aberrant expansion of stem/progenitor cell populations is considered to contribute to breast tumorigenesis. Germline deletions of p53 in humans and mice confer a predisposition to tumors, and stem cell frequency is abnormally high in the mammary epithelium of p53‐deficient mice. However, it is unknown whether stem/progenitor cell amplification occurs in both, basal and luminal cell populations in p53‐deficient mammary tissue. We used a conditional gene deletion approach to study the role of p53 in stem/progenitor cells residing in the mammary luminal and basal layers. Using two‐ and three‐dimensional cell culture assays, we showed that p53 loss led to the expansion of clonogenic stem/progenitor cells in both mammary epithelial cell layers. Moreover, following p53 deletion, luminal and basal stem/progenitor cells acquired a capacity for unlimited propagation in mammosphere culture. Furthermore, limiting dilution and serial transplantation assays revealed amplification and enhanced self‐renewal in the basal regenerating cell population of p53‐deficient mammary epithelium. Our data suggest that the increase in stem/progenitor cell activity may be, at least, partially mediated by the Notch pathway. Taken together, these results strongly indicate that p53 restricts the propagation and self‐renewal of stem/progenitor cells in both layers of the mammary epithelium providing further insight into the impact of p53 loss in breast cancerogenesis. Stem Cells 2013;31:1857‐1867