729 research outputs found
Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland
BACKGROUND: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene. RESULTS: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target. CONCLUSION: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells
Scribble modulates the MAPK/Fra1 pathway to disrupt luminal and ductal integrity and suppress tumour formation in the mammary gland
Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression
Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution
Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further
The future of mammary stem cell biology: the power of in vivo transplants
The recent review by Smith and Medina [1] of in vivo transplantation models and their role in investigating mammary stem cell (MaSC) biology provides comprehensive coverage of the history and complexity of the ‘gold standard ’ MaSC assay in mice. This includes a description of the pioneering studies that showed that mammary epithelial outgrowths can be generated in cleared mammary fat pads transplanted with explants or admixtures of mammary cells [2]. However, this approach clearly does not lend itself to prospective analysis of isolated subpopulations in order to identify which cells possess in vivo regenerative activity. More recently, success in obtaining complex mammary gland structures from transplanted suspensions of single cells has now made this possible [3-7]. Moreover, the regenerated structures have been shown to contain daughter cells with the same in viv
Cell fate takes a slug in BRCA1-associated breast cancer
Understanding why BRCA1 mutation carriers have a predilection for developing clinically aggressive basal-like breast tumors could inform the development of targeted treatment or prevention strategies. Analysis of both mouse and human mammary epithelial cells has identified a role for BRCA1 in orchestrating differentiation. The ability to isolate discrete epithelial subpopulations from mammary tissue has recently directed attention to luminal progenitor cells - the descendants of mammary stem cells - as the likely 'cells-of-origin' in BRCA1-associated breast cancer. A new publication has confirmed the importance of aberrant luminal cells as key culprits and provided insights on how BRCA1 haploinsufficiency biases luminal cells toward a basal-like fate through aberrant expression of the transcription factor SLUG
Mutation analysis of FANCD2, BRIP1/BACH1, LMO4 and SFN in familial breast cancer
INTRODUCTION: Mutations in known predisposition genes account for only about a third of all multiple-case breast cancer families. We hypothesized that germline mutations in FANCD2, BRIP1/BACH1, LMO4 and SFN may account for some of the unexplained multiple-case breast cancer families. METHODS: The families used in this study were ascertained through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). Denaturing high performance liquid chromatography (DHPLC) analysis of the coding regions of these four genes was conducted in the youngest affected cases of 30 to 267 non-BRCA1/2 breast cancer families. In addition, a further 399 index cases were also screened for mutations in two functionally significant regions of the FANCD2 gene and 253 index cases were screened for two previously reported mutations in BACH1 (p. P47A and p. M299I). RESULTS: DHPLC analysis of FANCD2 identified six silent exonic variants, and a large number of intronic variants, which tagged two common haplotypes. One protein truncating variant was found in BRIP1/BACH1, as well as four missense variants, a silent change and a variant in the 3' untranslated region. No missense or splice site mutations were found in LMO4 or SFN. Analysis of the missense, silent and frameshift variants of FANCD2 and BACH1 in relatives of the index cases, and in a panel of controls, found no evidence suggestive of pathogenicity. CONCLUSION: There is no evidence that highly penetrant exonic or splice site mutations in FANCD2, BRIP1/BACH1, LMO4 or SFN contribute to familial breast cancer. Large scale association studies will be necessary to determine whether any of the polymorphisms or haplotypes identified in these genes contributes to breast cancer risk
Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways
INTRODUCTION: Molecular characterization of the normal epithelial cell types that reside in the mammary gland is an important step toward understanding pathways that regulate self-renewal, lineage commitment, and differentiation along the hierarchy. Here we determined the gene expression signatures of four distinct subpopulations isolated from the mouse mammary gland. The epithelial cell signatures were used to interrogate mouse models of mammary tumorigenesis and to compare with their normal human counterpart subsets to identify conserved genes and networks. METHODS: RNA was prepared from freshly sorted mouse mammary cell subpopulations (mammary stem cell (MaSC)-enriched, committed luminal progenitor, mature luminal and stromal cell) and used for gene expression profiling analysis on the Illumina platform. Gene signatures were derived and compared with those previously reported for the analogous normal human mammary cell subpopulations. The mouse and human epithelial subset signatures were then subjected to Ingenuity Pathway Analysis (IPA) to identify conserved pathways. RESULTS: The four mouse mammary cell subpopulations exhibited distinct gene signatures. Comparison of these signatures with the molecular profiles of different mouse models of mammary tumorigenesis revealed that tumors arising in MMTV-Wnt-1 and p53-/- mice were enriched for MaSC-subset genes, whereas the gene profiles of MMTV-Neu and MMTV-PyMT tumors were most concordant with the luminal progenitor cell signature. Comparison of the mouse mammary epithelial cell signatures with their human counterparts revealed substantial conservation of genes, whereas IPA highlighted a number of conserved pathways in the three epithelial subsets. CONCLUSIONS: The conservation of genes and pathways across species further validates the use of the mouse as a model to study mammary gland development and highlights pathways that are likely to govern cell-fate decisions and differentiation. It is noteworthy that many of the conserved genes in the MaSC population have been considered as epithelial-mesenchymal transition (EMT) signature genes. Therefore, the expression of these genes in tumor cells may reflect basal epithelial cell characteristics and not necessarily cells that have undergone an EMT. Comparative analyses of normal mouse epithelial subsets with murine tumor models have implicated distinct cell types in contributing to tumorigenesis in the different models
Resident macrophages influence stem cell activity in the mammary gland
Introduction Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland in a macrophage-depleted fat pad. Methods Two different in vivo models were used to deplete macrophages from the mouse mammary fat pad, allowing us to examine the effect of macrophage deficiency on the mammary repopulating activity of MaSCs. Both the Csf1(op/op) mice and clodronate liposome-mediated ablation models entailed transplantation studies using the MaSC-enriched population. Results We show that mammary repopulating ability is severely compromised when the wild-type MaSC-enriched subpopulation is transplanted into Csf1(op/op) fat pads. In reciprocal experiments, the MaSC-enriched subpopulation from Csf1(op/op) glands had reduced regenerative capacity in a wildtype environment. Utilizing an alternative strategy for selective depletion of macrophages from the mammary gland, we demonstrate that co-implantation of the MaSC-enriched subpopulation with clodronate-liposomes leads to a marked decrease in repopulating frequency and outgrowth potential. Conclusions Our data reveal a key role for mammary gland macrophages in supporting stem/progenitor cell function and suggest that MaSCs require macrophage-derived factors to be fully functional. Macrophages may therefore constitute part of the mammary stem cell nich
From normal cell types to malignant phenotypes
The phenotypic diversity of breast cancer has been proposed to result from different target cell types undergoing oncogenic transformation and giving rise to cancer stem cells. Global gene expression profiling revealed distinct molecular phenotypes and some of these signatures were held to reflect the cell of origin, with the basal carcinomas arising from basal/progenitor cells. Recent work challenges this view by providing evidence that luminal precursor cells are involved in the pathogenesis of basal breast cancers and has made new links between normal cell populations and molecular tumor phenotypes
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