Molecular Mechanisms of Mammary Gland Development

Team Publications

Year of publication 2016

Stéphanie Cagnet, Marina A Glukhova, Karine Raymond (2016 Nov 1)

Contractility Assay for Established Myoepithelial Cell Lines.

Methods in molecular biology (Clifton, N.J.) : 189-198 Learn more
Summary

The capacity of mammary myoepithelial cells to contract in response to suckling stimuli is essential for lactation. We describe here a protocol for studying the contractile activity of myoepithelial cells in vitro. This protocol includes the establishment of stable myoepithelial cell lines from mouse mammary glands and quantitative evaluation of the contraction and subsequent relaxation of cultured myoepithelial cells in response to oxytocin. It can be used for analyses of mouse mutants with gene deletions or overexpression altering myoepithelial cell function.

Fold up
A Chiche, M Moumen, M Romagnoli, V Petit, H Lasla, P Jézéquel, P de la Grange, J Jonkers, M-A Deugnier, M A Glukhova, M M Faraldo (2016 Oct 25)

p53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

Oncogene : DOI : 10.1038/onc.2016.396 Learn more
Summary

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/β-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/β-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNβcat mice displaying a constitutive activation of Wnt/β-catenin signaling in basal cells. K5ΔNβcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNβcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and β-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.Oncogene advance online publication, 24 October 2016; doi:10.1038/onc.2016.396.

Fold up

Year of publication 2015

Marisa M Faraldo, Marina A Glukhova (2015 Nov 25)

Regulating the regulator: Numb acts upstream of p53 to control mammary stem and progenitor cell.

The Journal of cell biology : 737-9 : DOI : 10.1083/jcb.201510104 Learn more
Summary

In this issue, Tosoni et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505037) report that cell fate determinant and tumor suppressor Numb imposes asymmetric cell divisions in mammary stem cells by regulating p53. Numb thereby restricts mammary stem cell expansion and controls the proliferation and lineage-specific characteristics of their progeny.

Fold up
Marina A Glukhova, Nancy Hynes, Maria dM Vivanco, Renée van Amerongen, Robert B Clarke, Mohamed Bentires-Alj (2015 Sep 3)

The seventh ENBDC workshop on methods in mammary gland development and cancer.

Breast cancer research : BCR : 119 : DOI : 10.1186/s13058-015-0629-5 Learn more
Summary

The seventh annual meeting of the European Network of Breast Development and Cancer Laboratories, held in Weggis, Switzerland, in April 2015, was focused on techniques for the study of normal and cancer stem cells, cell fate decisions, cancer initiation and progression.

Fold up
Marisa M Faraldo, Marina A Glukhova, Marie-Ange Deugnier (2015 Jun 5)

The transplantation of mouse mammary epithelial cells into cleared mammary fat pads.

Methods in molecular biology (Clifton, N.J.) : 161-72 : DOI : 10.1007/978-1-4939-2519-3_9 Learn more
Summary

The transplantation of mammary epithelial cells into the cleared fat pad allows their growth and differentiation in their normal physiological environment. This technique involves the grafting of tissue fragments or isolated cells into the mammary fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed. Such transplantation assays are particularly useful for the analysis of morphogenetic potential and stem cell activity in normal mammary epithelium and breast tumors. We describe here the main steps in the transplantation of epithelial fragments and isolated cells from mouse mammary glands and the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells.

Fold up
C Lodillinsky, E Infante, A Guichard, R Chaligné, L Fuhrmann, J Cyrta, M Irondelle, E Lagoutte, S Vacher, H Bonsang-Kitzis, M Glukhova, F Reyal, I Bièche, A Vincent-Salomon, P Chavrier (2015 Apr 21)

p63/MT1-MMP axis is required for in situ to invasive transition in basal-like breast cancer.

Oncogene : 344-57 : DOI : 10.1038/onc.2015.87 Learn more
Summary

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.

Fold up

Year of publication 2014

Amandine Di-Cicco, Valérie Petit, Aurélie Chiche, Laura Bresson, Mathilde Romagnoli, Véronique Orian-Rousseau, Maria dM Vivanco, Daniel Medina, Marisa M Faraldo, Marina A Glukhova, Marie-Ange Deugnier (2014 Dec 16)

Paracrine Met signaling triggers epithelial-mesenchymal transition in mammary luminal progenitors, affecting their fate.

eLife : DOI : 10.7554/eLife.06104 Learn more
Summary

HGF/Met signaling has recently been associated with basal-type breast cancers, which are thought to originate from progenitor cells residing in the luminal compartment of the mammary epithelium. We found that ICAM-1 efficiently marks mammary luminal progenitors comprising hormone receptor-positive and receptor-negative cells, presumably ductal and alveolar progenitors. Both cell populations strongly express Met, while HGF is produced by stromal and basal myoepithelial cells. We show that persistent HGF treatment stimulates the clonogenic activity of ICAM1-positive luminal progenitors, controlling their survival and proliferation, and leads to the expression of basal cell characteristics, including stem cell potential. This is accompanied by the induction of Snai1 and Snai2, two major transcription factors triggering epithelial-mesenchymal transition, the repression of the luminal-regulatory genes Elf5 and Hey1, and claudin down-regulation. Our data strongly indicate that paracrine Met signaling can control the function of luminal progenitors and modulate their fate during mammary development and tumorigenesis.

Fold up

Year of publication 2013

Michael D Prater, Valérie Petit, I Alasdair Russell, Rajshekhar R Giraddi, Mona Shehata, Suraj Menon, Reiner Schulte, Ivo Kalajzic, Nicola Rath, Michael F Olson, Daniel Metzger, Marisa M Faraldo, Marie-Ange Deugnier, Marina A Glukhova, John Stingl (2013 Sep 23)

Mammary stem cells have myoepithelial cell properties.

Nature cell biology : 942-50, 1-7 : DOI : 10.1038/ncb3025 Learn more
Summary

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 actin-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 two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

Fold up
Mejdi Moumen, Aurélie Chiche, Charles Decraene, Valérie Petit, Alberto Gandarillas, Marie-Ange Deugnier, Marina A Glukhova, Marisa M Faraldo (2013 Jul 25)

Myc is required for β-catenin-mediated mammary stem cell amplification and tumorigenesis.

Molecular cancer : 132 : DOI : 10.1186/1476-4598-12-132 Learn more
Summary

Basal-like breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Recent studies have linked activation of the Wnt/β-catenin pathway, and its downstream target, Myc, to basal-like breast cancer. Transgenic mice K5ΔNβcat previously generated by our team present a constitutive activation of Wnt/β-catenin signaling in the basal myoepithelial cell layer, resulting in focal mammary hyperplasias that progress to invasive carcinomas. Mammary lesions developed by K5ΔNβcat mice consist essentially of basal epithelial cells that, in contrast to mammary myoepithelium, do not express smooth muscle markers.

Fold up
S Cagnet, M M Faraldo, M Kreft, A Sonnenberg, K Raymond, M A Glukhova (2013 Jun 25)

Signaling events mediated by α3β1 integrin are essential for mammary tumorigenesis.

Oncogene : 4286-95 : DOI : 10.1038/onc.2013.391 Learn more
Summary

The constitutive activation of β-catenin signaling in the mammary basal epithelial cell layer in transgenic K5ΔNβcat mice leads to basal-type tumor development. Integrins of the β1 family and integrin-mediated signaling events have an important role in breast tumor growth and progression. We show here that the deletion of α3β1 integrin, a major laminin receptor, from the basal layer of the mammary epithelium of K5ΔNβcat mice completely prevented the tumorigenesis induced by β-catenin signaling. Moreover, the depletion of α3β1 integrin from a spontaneously transformed mouse mammary basal epithelial cell line (MEC) prevented the cells from forming colonies in soft agar and greatly reduced tumor development in orthotopic grafts. Inhibition of the integrin signaling intermediates Rac1 or PAK1 (P21-activated Kinase 1) in MEC affected tumor cell growth in soft agar, whereas the expression of activated forms of these effectors in α3-depleted cells rescued the capacity of these cells to grow in non-adherent conditions. Similarly, the tumorigenic potential of α3-depleted cells was restored by the expression of activated PAK1, as assessed by orthotopic transplantation assay. In three-dimensional Matrigel culture, MEC survival and proliferation were affected by the depletion of α3β1 integrin, which also significantly decreased the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK). Our data suggest that the activation of signaling cascades downstream from α3β1 and involving the Rac1/PAK1 pathway, MAPK and JNK, promotes prosurvival and proproliferative signals required for the malignant growth of basal mammary epithelial cells, providing further insight into the molecular mechanisms underlying breast cancer initiation and progression.

Fold up

Year of publication 2012

Aurélie Chiche, Mejdi Moumen, Valérie Petit, Jos Jonkers, Daniel Medina, Marie-Ange Deugnier, Marisa M Faraldo, Marina A Glukhova (2012 Nov 15)

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

Stem cells (Dayton, Ohio) : 1857-67 : DOI : 10.1002/stem.1429 Learn more
Summary

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.

Fold up
Mejdi Moumen, Aurélie Chiche, Marie-Ange Deugnier, Valérie Petit, Alberto Gandarillas, Marina A Glukhova, Marisa M Faraldo (2012 Mar 23)

The proto-oncogene Myc is essential for mammary stem cell function.

Stem cells (Dayton, Ohio) : 1246-54 : DOI : 10.1002/stem.1090 Learn more
Summary

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.

Fold up
Karine Raymond, Marisa M Faraldo, Marie-Ange Deugnier, Marina A Glukhova (2012 Mar 21)

Integrins in mammary development.

Seminars in cell & developmental biology : 599-605 : DOI : 10.1016/j.semcdb.2012.03.008 Learn more
Summary

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.

Fold up

Year of publication 2011

Mejdi Moumen, Aurélie Chiche, Stéphanie Cagnet, Valérie Petit, Karine Raymond, Marisa M Faraldo, Marie-Ange Deugnier, Marina A Glukhova (2011 Sep 28)

The mammary myoepithelial cell.

The International journal of developmental biology : 763-71 : DOI : 10.1387/ijdb.113385mm Learn more
Summary

Over the last few years, the discovery of basal-type mammary carcinomas and the association of the regenerative potential of the mammary epithelium with the basal myoepithelial cell population have attracted considerable attention to this second major mammary lineage. However, many questions concerning the role of basal myoepithelial cells in mammary morphogenesis, functional differentiation and disease remain unanswered. Here, we discuss the mechanisms that control the myoepithelial cell differentiation essential for their contractile function, summarize new data concerning the roles played by cell-extracellular matrix (ECM), intercellular and paracrine interactions in the regulation of various aspects of the mammary basal myoepithelial cell functional activity. Finally, we analyze the contribution of the basal myoepithelial cells to the regenerative potential of the mammary epithelium and tumorigenesis.

Fold up

Year of publication 2010

Karine Raymond, Stéphanie Cagnet, Maaike Kreft, Hans Janssen, Arnoud Sonnenberg, Marina A Glukhova (2010 Oct 12)

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

The EMBO journal : 1896-906 : DOI : 10.1038/emboj.2011.113 Learn more
Summary

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.

Fold up