Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth.
ABSTRACT: BACKGROUND:Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. METHODS:We crossed AREG-null (AREG-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. RESULTS:Intriguingly, PyMT-induced lesions progress more rapidly in AREG-/- mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG-/- mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. CONCLUSIONS:Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.
Project description:The discoidin domain receptor 1 (DDR1) is overexpressed in breast carcinoma cells. Low DDR1 expression is associated with worse relapse-free survival, reflecting its controversial role in cancer progression. We detected DDR1 on luminal cells but not on myoepithelial cells of DDR1+/+ mice. We found that DDR1 loss compromises cell adhesion, consistent with data that older DDR1-/- mammary glands had more basal/myoepithelial cells. Basal cells isolated from older mice exerted higher traction forces than the luminal cells, in agreement with increased mammary branches observed in older DDR1-/- mice and higher branching by their isolated organoids. When we crossed DDR1-/- mice with MMTV-PyMT mice, the PyMT/DDR1-/- mammary tumors grew faster and had increased epithelial tension and matricellular fibrosis with a more basal phenotype and increased lung metastases. DDR1 deletion induced basal differentiation of CD90+CD24+ cancer cells, and the increase in basal cells correlated with tumor cell mitoses. K14+ basal cells, including K8+K14+ cells, were increased adjacent to necrotic fields. These data suggest that the absence of DDR1 provides a growth and adhesion advantage that favors the expansion of basal cells, potentiates fibrosis, and enhances necrosis/hypoxia and basal differentiation of transformed cells to increase their aggression and metastatic potential.
Project description: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 (TGF?) 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 TGF? 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.
Project description:Human breast cancer is a heterogeneous disease composed of different histologies and molecular subtypes, many of which are not replicated in animal models. Here, we report a mouse model of breast cancer that generates unique tumor histologies including tubular, adenosquamous, and lipid-rich carcinomas. Utilizing a nononcogenic variant of polyoma middle T oncogene (PyMT) that requires a spontaneous base-pair deletion to transform cells, in conjunction with lentiviral transduction and orthotopic transplantation of primary mammary epithelial cells, this model sporadically induces oncogene expression in both the luminal and myoepithelial cell lineages of the normal mouse mammary epithelium. Microarray and hierarchical analyses using an intrinsic subtype gene set revealed that lentiviral PyMT generates both luminal and basal-like tumors. Cumulatively, these results show that low-level expression of PyMT in a broad range of cell types significantly increases tumor heterogeneity and establishes a mouse model of several rare human breast cancer subtypes.
Project description:Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1? (HIF-1?) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1? is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1? is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer.Mammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus ?-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1?-null (KO) cells, respectively. The impact of HIF-1? deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis.Efficient deletion of HIF-1? reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1? led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1? also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133)was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133hi versus CD133neg cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells.These results demonstrate that HIF-1? plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1? regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1? directly regulates breast TIC activity in vivo.
Project description:Maintenance of mammary functional capacity during cycles of proliferation and regression depends on appropriate cell fate decisions of mammary progenitor cells to populate an epithelium consisting of secretory luminal cells and contractile myoepithelial cells. It is well established that transforming growth factor-? (TGF?) restricts mammary epithelial cell proliferation and that sensitivity to TGF? is decreased in breast cancer. We show that TGF? also exerts control of mammary progenitor self-renewal and lineage commitment decisions by stringent regulation of breast cancer associated 1 (BRCA1), which controls stem cell self-renewal and lineage commitment. Either genetic depletion of Tgfb1 or transient blockade of TGF? increased self-renewal of mammary progenitor cells in mice, cultured primary mammary epithelial cells, and also skewed lineage commitment toward the myoepithelial fate. TGF? stabilized the abundance of BRCA1 by reducing the abundance of microRNA-182 (miR-182). Ectopic expression of BRCA1 or antagonism of miR-182 in cultured TGF?-deficient mammary epithelial cells restored luminal lineage commitment. These findings reveal that TGF? modulation of BRCA1 directs mammary epithelial cell fate and, because stem or progenitor cells are thought to be the cell of origin for aggressive breast cancer subtypes, suggest that TGF? dysregulation during tumorigenesis may promote distinct breast cancer subtypes.
Project description:Bone morphogenetic proteins (BMPs) are involved in embryonic mammary gland (MG) development and can be dysregulated in breast cancer. However, the role BMPs play in the postnatal MG remains virtually unknown. BMPs are potent morphogens that are involved in cell fate determination, proliferation, apoptosis and adult tissue homeostasis. Twisted gastrulation (TWSG1) is a secreted BMP binding protein that modulates BMP ligand availability in the extracellular space. Here we investigate the consequences of TWSG1 deletion on development of the postnatal MG. At puberty, Twsg1 is expressed in the myoepithelium and in a subset of body cells of the terminal end buds. In the mature duct, Twsg1 expression is primarily restricted to the myoepithelial layer. Global deletion of Twsg1 leads to a delay in ductal elongation, reduced secondary branching, enlarged terminal end buds, and occluded lumens. This is associated with an increase in luminal epithelial cell number and a decrease in apoptosis. In the MG, pSMAD1/5/8 level and the expression of BMP target genes are reduced, consistent with a decrease in BMP signaling. GATA-3, which is required for luminal identity, is reduced in Twsg1(-/-) MGs, which may explain why K14 positive cells, which are normally restricted to the myoepithelial layer, are found within the luminal compartment and shed into the lumen. In summary, regulation of BMP signaling by TWSG1 is required for normal ductal elongation, branching of the ductal tree, lumen formation, and myoepithelial compartmentalization in the postnatal MG.
Project description:Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast.
Project description:The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or following stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumorigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance.
Project description:Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer.
Project description:Breast cancer is a multifaceted disease, exhibiting significant molecular, histological, and pathological diversity. Factors that impact this heterogeneity are poorly understood; however, transformation of distinct normal cell populations of the breast may generate different tumor phenotypes. Our previous study demonstrates that the polyomavirus middle T antigen (PyMT) oncogene can establish diverse tumor subtypes when broadly expressed within mouse mammary epithelial cells. Herein, we assess the molecular, histological, and metastatic outcomes from distinct mammary cell populations transformed with PyMT. By combining several methodologies, including lentiviral infection, cell sorting, and transplantation, we have characterized tumors arising from enriched populations of mammary epithelial cells. We have found that expression of PyMT within different cell populations influences tumor histology, molecular subtype, and metastatic potential. 32 samples, 1 from each of 32 mouse mammary tumors arising from transplanted mouse mamary epithelial cells (MMECs) transduced with PyMT-expressing lentivirus. MMECs were sorted into four different types prior to transplant: luminal CD133+ (8 samples), luminal CD133- (11 samples), stem (6 samples), and basal (7 samples). The background for the cell donor and transplant recipients mice was FVB/NJ obtained from Jackson Laboratories.