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

Molecular analysis of the estrogen receptor alpha pathway in human breast cancer

© 2002 Dr. Toula BourasThe recognition that estrogen is involved in breast cancer underscores the need for achieving a higher resolution view of signalling by its cellular target the estrogen receptor alpha (ER). Central to this pathway is the ER itself, numerous interacting proteins that modulate ER activity and downstream ER target genes. Although the ER has been extensively studied in breast cancer, further study is required to examine the role of dysfunction in other components of ER signalling. Upon embarking this thesis, the possibility remained that past studies may have underestimated the frequency of somatic ER mutations in breast carcinoma specimens due to the use of whole tumour homogenates and indirect mutation screening techniques. To overcome these limitations direct DNA sequencing of the entire coding region of the ER was performed on microdissected subpopulations of tumour cells of defined ER and progesterone receptor A (PR) protein expression from thirty-four breast carcinomas. No somatic ER mutations were detected indicating that previous studies had not failed to detect ER sequence alterations due to limitations in tissue selection and mutation detection. The steroid receptor coactivator, AIB1 (amplified in breast cancer 1), previously shown to be amplified in breast cancer was therefore used as a candidate to examine the role of alterations in components of the ER transcriptional machinery. In situ hyhridisation and immunohistochemistry of 93 breast carcinomas demonstrated AIB1 mRNA overexpression was associated with high tumour grade, lack of ER and PR protein expression and strong immunoreactivity for p53 and IIER2/neu. These results highlighted the potential complex interplay between components of ER signalling with other non-steroid receptor pathways in breast cancer and stressed the need to examine ER signalling more globally. The microarray analysis of global gene expression patterns in thirty-two primary breast carcinomas or varying ER status demonstrated that ER positive and ER negative tumours display distinct expression profiles. Using the transcriptional program of the ER positive MCF-7 breast cancer cell line model in response to estrogen and the estrogen antagonist ICI 182 780 as a biological framework, the major differences related to the altered expression of downstream ER target genes and genes involved in cellular turnover. The preferential expression of multiple ER target genes in ER positive tumours was attributed to the direct transcriptional effects of ER activation. Stanniocalcin 2 (STC2), a glycoprotein hormone originally found to control calcium homeostasis in bony fish was investigated further as one of the novel ER target genes whose expression closely paralleled ER expression. In situ hybridisation and immunohistochemistry on 236 unselected breast carcinomas independently confirmed the positive association between STC2 mRNA/protein expression with tumour ER and PR status. The distinct molecular profiles associated with tumour ER status appeared to also reflect the result of ER activation or lack thereof on cellular turnover. ER negative tumours expressed relatively higher levels of promitotic genes that correlated with tumour miotic index, whereas the ER positive counterparts expressed relatively higher mRNA levels of specific anti-apoptotic genes. The conclusion drawn was that the primary sequence of the ER gene is maintained functionally intact and that ER signalling is associated with distinct gene expression profiles in ER positive versus ER negative breast tumours. The alterations identified which related to tumour ER protein status included genes that make up part of the transcriptional apparatus used by the ER such as the AIB1 coactivator, downstream ER target genes and genes implicated in the regulation of cellular turnover. A unifying implication is the potential existence of a dualistic model of breast cancer development that incorporates an ER signalling driven pathway and an alternate ER negative pathway

Global Changes in the Mammary Epigenome Are Induced by Hormonal Cues and Coordinated by Ezh2

The mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland