School of Mathematics and Statistics - Research Publications
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ItemNo Preview AvailableGata-3 Negatively Regulates the Tumor-Initiating Capacity of Mammary Luminal Progenitor Cells and Targets the Putative Tumor Suppressor Caspase-14(AMER SOC MICROBIOLOGY, 2011-11)The transcription factor Gata-3 is a definitive marker of luminal breast cancers and a key regulator of mammary morphogenesis. Here we have explored a role for Gata-3 in tumor initiation and the underlying cellular mechanisms using a mouse model of "luminal-like" cancer. Loss of a single Gata-3 allele markedly accelerated tumor progression in mice carrying the mouse mammary tumor virus promoter-driven polyomavirus middle T antigen (MMTV-PyMT mice), while overexpression of Gata-3 curtailed tumorigenesis. Through the identification of two distinct luminal progenitor cells in the mammary gland, we demonstrate that Gata-3 haplo-insufficiency increases the tumor-initiating capacity of these progenitors but not the stem cell-enriched population. Overexpression of a conditional Gata-3 transgene in the PyMT model promoted cellular differentiation and led to reduced tumor-initiating capacity as well as diminished angiogenesis. Transcript profiling studies identified caspase-14 as a novel downstream target of Gata-3, in keeping with its roles in differentiation and tumorigenesis. A strong association was evident between GATA-3 and caspase-14 expression in preinvasive ductal carcinoma in situ samples, where GATA-3 also displayed prognostic significance. Overall, these studies identify GATA-3 as an important regulator of tumor initiation through its ability to promote the differentiation of committed luminal progenitor cells.
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ItemCanonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids(PUBLIC LIBRARY SCIENCE, 2018-08)Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.
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ItemConstruction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling(NATURE PORTFOLIO, 2017-11-20)The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.
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ItemTranscriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways(BMC, 2010)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.
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ItemIntegration of microRNA signatures of distinct mammary epithelial cell types with their gene expression and epigenetic portraits(BMC, 2015-06-18)INTRODUCTION: MicroRNAs (miRNAs) have been implicated in governing lineage specification and differentiation in multiple organs; however, little is known about their specific roles in mammopoiesis. We have determined the global miRNA expression profiles of functionally distinct epithelial subpopulations in mouse and human mammary tissue, and compared these to their cognate transcriptomes and epigenomes. Finally, the human miRNA signatures were used to interrogate the different subtypes of breast cancer, with a view to determining miRNA networks deregulated during oncogenesis. METHODS: RNA from sorted mouse and human mammary cell subpopulations was subjected to miRNA expression analysis using the TaqMan MicroRNA Array. Differentially expressed (DE) miRNAs were correlated with gene expression and histone methylation profiles. Analysis of miRNA signatures of the intrinsic subtypes of breast cancer in The Cancer Genome Atlas (TCGA) database versus those of normal human epithelial subpopulations was performed. RESULTS: Unique miRNA signatures characterized each subset (mammary stem cell (MaSC)/basal, luminal progenitor, mature luminal, stromal), with a high degree of conservation across species. Comparison of miRNA and transcriptome profiles for the epithelial subtypes revealed an inverse relationship and pinpointed key developmental genes. Interestingly, expression of the primate-specific miRNA cluster (19q13.4) was found to be restricted to the MaSC/basal subset. Comparative analysis of miRNA signatures with H3 lysine modification maps of the different epithelial subsets revealed a tight correlation between active or repressive marks for the top DE miRNAs, including derepression of miRNAs in Ezh2-deficient cellular subsets. Interrogation of TCGA-identified miRNA profiles with the miRNA signatures of different human subsets revealed specific relationships. CONCLUSIONS: The derivation of global miRNA expression profiles for the different mammary subpopulations provides a comprehensive resource for understanding the interplay between miRNA networks and target gene expression. These data have highlighted lineage-specific miRNAs and potential miRNA-mRNA networks, some of which are disrupted in neoplasia. Furthermore, our findings suggest that key developmental miRNAs are regulated by global changes in histone modification, thus linking the mammary epigenome with genome-wide changes in the expression of genes and miRNAs. Comparative miRNA signature analyses between normal breast epithelial cells and breast tumors confirmed an important linkage between luminal progenitor cells and basal-like tumors.
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ItemA pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1(BMC, 2015-04-03)BACKGROUND: The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation. METHODS: MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells. RESULTS: Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity. CONCLUSIONS: This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.
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ItemBarcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer(NATURE PORTFOLIO, 2019-02-15)Primary triple negative breast cancers (TNBC) are prone to dissemination but sub-clonal relationships between tumors and resulting metastases are poorly understood. Here we use cellular barcoding of two treatment-naïve TNBC patient-derived xenografts (PDXs) to track the spatio-temporal fate of thousands of barcoded clones in primary tumors, and their metastases. Tumor resection had a major impact on reducing clonal diversity in secondary sites, indicating that most disseminated tumor cells lacked the capacity to 'seed', hence originated from 'shedders' that did not persist. The few clones that continued to grow after resection i.e. 'seeders', did not correlate in frequency with their parental clones in primary tumors. Cisplatin treatment of one BRCA1-mutated PDX model to non-palpable levels had a surprisingly minor impact on clonal diversity in the relapsed tumor yet purged 50% of distal clones. Therefore, clonal features of shedding, seeding and drug resistance are important factors to consider for the design of therapeutic strategies.