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ErbB3 is required for ductal morphogenesis in the mouse mammary gland
Amy J Jackson-Fisher, Gary Bellinger, Jerrica L Breindel, Fatteneh A Tavassoli, Carmen J Booth, James K Duong, David F Stern
Breast Cancer Research , 2008, DOI: 10.1186/bcr2198
Abstract: To investigate the roles of ErbB3 in mouse mammary gland development, we transplanted mammary buds from ErbB3-/- embryos into the cleared mammary fat pads of wild-type immunocompromised mice. Effects on ductal outgrowth were analyzed at 4 weeks, 7 weeks and 20 weeks after transplantation for total ductal outgrowth, branch density, and number and area of terminal end buds. Sections of glands containing terminal end buds were analyzed for number and epithelial area of terminal end buds. Terminal end buds were also analyzed for presence of mitotic figures, apoptotic figures, BrdU incorporation, and expression of E-cadherin, P-cadherin, α-smooth muscle actin, and cleaved caspase-3.The mammary ductal trees developed from ErbB3-/- buds only partly filled the mammary fat pad. In contrast to similar experiments with ErbB2-/- mammary buds, this phenotype was maintained through adulthood, pregnancy, and parturition. In addition, and in contrast to similar work with ErbB4-/- mammary buds, lobuloalveolar development of ErbB3-/- transplanted glands was normal. The ErbB3-/- mammary outgrowth defect was associated with a decrease in the size of the terminal end buds, and with increases in branch density, in the number of terminal end buds, and in the number of luminal spaces. Proliferation rates were not affected by the lack of ErbB3, but there was an increase in apoptosis in ErbB3-/- terminal end buds.Endogenous ErbB3 regulates morphogenesis of mammary epithelium.The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which consists of EGFR, ErbB2, ErbB3, and ErbB4, is important in many normal developmental processes and is often over-expressed or mutated in human cancer (for review, see [1]). Ligand binding stimulates homodimerization and heterodimerization of EGFR family members. The dimerization facilitates cross-phosphorylation of tyrosines within the cytoplasmic domains that become binding sites for downstream effector proteins. The proto-oncogene pr
p130Cas Over-Expression Impairs Mammary Branching Morphogenesis in Response to Estrogen and EGF  [PDF]
Maria del Pilar Camacho Leal, Alessandra Pincini, Giusy Tornillo, Elisa Fiorito, Brigitte Bisaro, Elisa Di Luca, Emilia Turco, Paola Defilippi, Sara Cabodi
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049817
Abstract: p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.
Expression and Functional Role of Sprouty-2 in Breast Morphogenesis  [PDF]
Valgardur Sigurdsson, Saevar Ingthorsson, Bylgja Hilmarsdottir, Sigrun M. Gustafsdottir, Sigridur Rut Franzdottir, Ari Jon Arason, Eirikur Steingrimsson, Magnus K. Magnusson, Thorarinn Gudjonsson
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0060798
Abstract: Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.
Estrogen regulation of mammary gland development and breast cancer: amphiregulin takes center stage
Heather L LaMarca, Jeffrey M Rosen
Breast Cancer Research , 2007, DOI: 10.1186/bcr1740
Abstract: Mammary gland development is a unique, orchestrated process that primarily occurs postnatally. At the onset of puberty, circulating ovarian steroid hormones act as pivotal mediators of ductal morphogenesis. Previous studies using estrogen receptor knockout mice revealed that estrogen receptor (ER)α signaling is required for ductal elongation, which occurs through the proliferation of terminal end buds [1]. More recently, Cathrin Brisken and colleagues [2] performed a series of elegant transplantation experiments to demonstrate that estrogen facilitates epithelial proliferation and morphogenesis by a paracrine mechanism. Specifically, ERα-null LacZ+ epithelial cells were mixed with wild-type (WT) epithelial cells and transplanted into the cleared fat pads of WT mice. More than half of the resulting outgrowths were chimeric, indicating that the presence of WT epithelial cells could rescue the ERα-null phenotype. Brisken and coworkers [2] hypothesized that estrogens stimulate a subset of ductal cells, which in turn secrete paracrine factors that allow neighboring cells to proliferate and contribute to ductal outgrowth. This postulate raises the following critical question: what is the mechanism(s) by which estrogens induce ductal epithelial proliferation? The answer to this question has direct implications for our understanding of both stem cell biology and hormone-dependent/independent breast cancer proliferation.Several candidate paracrine factors have been suggested to regulate hormone-induced proliferation and morphogenesis, including Wnt-4, receptor activator of NFκB ligand (RANKL), growth hormone and insulin growth factor II (reviewed in [3]). With regard to estrogen action, however, amphiregulin (AREG) appears to be the major paracrine mediator of ductal morphogenesis [4]. AREG is a member of the epidermal growth factor receptor (EGFR) family of ligands that binds exclusively to EGFR (ErbB1). Unlike the other EGFR ligands, AREG is upregulated in the mammary glan
Modulation of Morphogenesis by Egfr during Dorsal Closure in Drosophila  [PDF]
Weiping Shen, Xi Chen, Olga Cormier, David Chung-Pei Cheng, Bruce Reed, Nicholas Harden
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0060180
Abstract: During Drosophila embryogenesis the process of dorsal closure (DC) results in continuity of the embryonic epidermis, and DC is well recognized as a model system for the analysis of epithelial morphogenesis as well as wound healing. During DC the flanking lateral epidermal sheets stretch, align, and fuse along the dorsal midline, thereby sealing a hole in the epidermis occupied by an extra-embryonic tissue known as the amnioserosa (AS). Successful DC requires the regulation of cell shape change via actomyosin contractility in both the epidermis and the AS, and this involves bidirectional communication between these two tissues. We previously demonstrated that transcriptional regulation of myosin from the zipper (zip) locus in both the epidermis and the AS involves the expression of Ack family tyrosine kinases in the AS in conjunction with Dpp secreted from the epidermis. A major function of Ack in other species, however, involves the negative regulation of Egfr. We have, therefore, asked what role Egfr might play in the regulation of DC. Our studies demonstrate that Egfr is required to negatively regulate epidermal expression of dpp during DC. Interestingly, we also find that Egfr signaling in the AS is required to repress zip expression in both the AS and the epidermis, and this may be generally restrictive to the progression of morphogenesis in these tissues. Consistent with this theme of restricting morphogenesis, it has previously been shown that programmed cell death of the AS is essential for proper DC, and we show that Egfr signaling also functions to inhibit or delay AS programmed cell death. Finally, we present evidence that Ack regulates zip expression by promoting the endocytosis of Egfr in the AS. We propose that the general role of Egfr signaling during DC is that of a braking mechanism on the overall progression of DC.
Signalling Pathways Implicated in Early Mammary Gland Morphogenesis and Breast Cancer  [PDF]
Beatrice Howard ,Alan Ashworth
PLOS Genetics , 2006, DOI: 10.1371/journal.pgen.0020112
Abstract: Specification of mammary epithelial cell fate occurs during embryogenesis as cells aggregate to form the mammary anlage. Within the embryonic mammary bud, a population of epithelial cells exists that will subsequently proliferate to form a ductal tree filling the stromal compartment, and which can produce milk upon terminal differentiation after birth. Subsequently, these structures can be remodelled and returned to a basal state after weaning before regenerating in future pregnancies. The plasticity of the mammary epithelial cell, and its responsiveness to hormone receptors, facilitates this amazing biological feat, but aberrant signalling may also result in unintended consequences in the form of frequent malignancies. Reflecting this intimate connection, a considerable number of signalling pathways have been implicated in both mammary gland morphogenesis and carcinogenesis.
Modulation of Fibroblast Growth Factor Signaling Is Essential for Mammary Epithelial Morphogenesis  [PDF]
Xiaohong Zhang, Guijuan Qiao, Pengfei Lu
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0092735
Abstract: Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that Spry2 gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that Spry2 is expressed at various stages of the developing mammary gland. Targeted removal of Spry2 function from mammary epithelium leads to accelerated epithelial invasion. Spry2 is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, Spry2 gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of Spry2 expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by Spry2 is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.
Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes
Jimmie E Fata, Zena Werb, Mina J Bissell
Breast Cancer Research , 2003, DOI: 10.1186/bcr634
Abstract: Relatively simple in its form and function, the mammary gland nevertheless requires a complex interplay of both intracellular and extracellular signals for its development into a branched glandular structure. The extracellular matrix (ECM) has long been recognized as providing morphogenic signals during mammary gland branching morphogenesis [1]. However, systematic studies to define the precise mechanism(s) by which the ECM accomplishes its role are lacking. The unanswered questions include: do interstitial ECM and basement membrane (BM) act as single entities or do their individual components have distinct effects? What ECM receptors are used to transmit these signals, and how do ECM remodeling proteinases fit into these morphogenic events? In this review, we briefly summarize a vast amount of research that touches on these areas. We mention several existing hypotheses, put forth a few more, and suggest some possible future directions for the field.A branched epithelial structure includes a network of tubes that are integral to the function of a number of glandular organs [2,3]. Lung [4], kidney [5], salivary gland [6,7], and mammary gland [8,9] are examples of organs that develop through branching morphogenesis. The latter is unique among these organs in that the majority of its branching is postembryonic. Extensive branching begins in puberty in the female and ceases after expanding to the outer limits of the mesenchymal fat pad. A number of paracrine, juxtacrine, and autocrine factors are known to affect mammary gland branching morphogenesis [10]. We have summarized these factors in Table 1; however, a detailed discussion of these factors is beyond the scope of this review. Here, we focus primarily on the studies that involve ECM, integrin and nonintegrin ECM receptors, ECM-degrading proteinases, and proteinase inhibitors in regulation of mouse mammary gland branching morphogenesis.Branched structures are first seen in the mouse mammary gland in late embryonic d
Inhibition of Proliferation by PERK Regulates Mammary Acinar Morphogenesis and Tumor Formation  [PDF]
Sharon J. Sequeira, Aparna C. Ranganathan, Alejandro P. Adam, Bibiana V. Iglesias, Eduardo F. Farias, Julio A. Aguirre-Ghiso
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0000615
Abstract: Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2α. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2α and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-ΔC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKΔC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.
Distinct Effects of EGFR Ligands on Human Mammary Epithelial Cell Differentiation  [PDF]
Chandrani Mukhopadhyay, Xiangshan Zhao, Dulce Maroni, Vimla Band, Mayumi Naramura
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075907
Abstract: Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5+K19- hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5+K19- hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5+K19- hMEC differentiation. The present data validate the utility of the K5+K19- hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.
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