Research Grants Awarded
Epigenetic Determinants Of Progenitor Cell Function In Normal And Neoplastic Human Breast Epithelium
Scientific rationale: Mammary epithelial stem cells have recently received a lot of attention because breast cancer is thought to originate from normal mammary epithelial progenitors or from cells that acquired the properties of stem cells. But the cellular identity and molecular characteristics of these cells in human breast tissue are not defined. Several studies have shown that epigenetic changes, including DNA methylation and chromatin modification, are required for the regulation of the stem cell phenotype, but the epigenetic profile of normal mammary epithelial progenitor cells and stem cell-like breast cancer cells has not been analyzed. Based on our preliminary studies we determined that the gene expression and DNA methylation profiles of putative human normal mammary epithelial stem (lin-/CD24-/CD44+), luminal epithelial (CD44-/CD24+ and CD44-/MUC1+), and myoepithelial (CD10+) cells are distinct. Each cell type has a unique DNA methylation pattern that is in part conserved during tumorigenesis, because analogous cell types from normal and neoplastic breast tissue demonstrated high similarity in gene expression and DNA methylation patterns. However, the degree of similarity between normal and cancer cells was dependent on the tumor and could potentially be used to decipher the putative cell of origin of distinct breast tumor subtypes. Genes involved in stem cell function, including numerous transcription factors (e.g. HOXA10 and FOXC1), are frequently methylated and silenced in differentiated luminal epithelial and myoepithelial cells.
Hypotheses to be tested: Based on these preliminary data and because DNA methylation and chromatin modification are inter-linked processes, I hypothesize that mammary epithelial progenitor and differentiated cells have distinct chromatin patterns that play a key role in determining stem cell phenotype and lineage-specific differentiation. Furthermore, I hypothesize that the epigenetic program of progenitor-like cells from breast tumors may be distinct from that of normal cells and it may correlate with breast tumor subtypes and clinical outcome.
Research aims and design: to test these hypotheses, I propose the following specific aims: (1) To characterize the genome-wide chromatin modification profiles of human mammary epithelial progenitor and differentiated cells isolated from normal human breast tissue by performing ChIPSeq (Chromatin immuno precipitation-sequencing) experiments. I will determine the methylation and acetylation status of various histones known to be associated with the promoter and enhancer areas of active and silenced genes. The same cell types will also be analyzed for gene expression and DNA methylation profiles and the three types of genomic data will be correlated. Using this approach we will be able to determine to what degree DNA and chromatin modification regulate cellular differentiation and progenitor cell function. (2) To characterize the genome-wide chromatin modification profiles of progenitor-like and more differentiated breast cancer cells isolated from different breast tumor subtypes (e.g. luminal, HER2+, and basal-like tumors) by performing ChIPSeq experiments. Experiments will be performed as described above for normal cells, but tumor cells will also be analyzed for genetic alterations using SNP arrays. We will compare analogous cell types from normal and neoplastic breast tissue to identify alterations in epigenetic patterns associated with tumorigenesis and to determine which normal cell type may be the potential cell of origin of different tumor subtypes. Furthermore, we also compare progenitor-like cancer cells from different tumor subtypes to define to what degree the phenotype of the cells may be influenced by tumor type-specific transforming events. (3) To determine the biological and clinical relevance of the cell type-specific epigenetic patterns.
Relevance to breast cancer: The molecular characterization of progenitor-like cells from normal breast tissue and breast carcinomas will not only improve our understanding of mammary epithelial stem cell differentiation and function, but may provide new insight into the initiating steps of breast tumorigenesis that could be exploited for future cancer preventative studies. The ?epigenetic progenitor origin of cancer? hypothesis proposes that disturbation of the normal epigenetic programs in tissue specific adult stem cells is the initiating step of carcinogenesis that precedes transforming genetic events. Thus, the characterization of these epigenetic programs is important for the identification of patients with high risk of breast cancer and ?restoring? normal epigenetic programs of breast epithelial stem cells may be a new highly effective breast cancer preventative approach. Finally, the recent introduction of several chemotherapeutic agents that modify epigenetic patterns (e.g. inhibitors of DNA methyl transferases and histone deacetylases) into clinical trials presses the need of understanding the role of epigenetic modifications play in regulating the phenotype or normal stem cells and stem-like breast cancer cells in order to avoid unforeseen side effects.
Breast cancer is a leading cause of cancer-related death in women worldwide. Last year in the United States alone over 200,000 new cases were diagnosed and over 40,000 women died from breast cancer. Although through screening efforts we now identify many breast tumors at an early, curable stage before the tumor invaded the surrounding tissue, most patients are diagnosed with more advanced breast cancers where there is a greater chance that the tumor has spread beyond the breast. Our relative inability to effectively treat advanced stage tumors is in part due to the lack of targeted therapy specific for these cancer cells. The most significant problem and the main cause of mortality in breast cancer are the recurrence and metastatic spread of the tumor. Both of these processes may be due to a subpopulation of cells within tumors that have characteristics of stem cells. Furthermore, breast cancer is thought to originate from mammary epithelial stem cells or from cells that acquired the properties of stem cells. The function and differentiation of these cells are controlled by epigenetic programs including defined patterns of DNA methylation and chromatin modifications. Alteration of the normal epigenetic programs in stem cells has been proposed to be an initiating step of tumorigenesis. Thus, understanding the epigenetic programs of normal human mammary epithelial stem cells and their differentiated progeny and comparing these to analogous cell type isolated from breast tumors will improve our understanding of the initiating steps of breast tumorigenesis and may help us identify the cell or origin of distinct breast tumor subtypes. Furthermore, because epigenetic programs can be modified by various agents (dietary factors and chemical compounds) the results of this study will yield important new data that can be the basis of future breast cancer preventative or therapeutic strategies.
Large amount of data suggest that the stem cell phenotype and differentiation are epigenetically regulated. This has been demonstrated by the analysis of the role of DNA methylation in development and differentiation. Altered DNA methylation due to elimination of DNA methyltransferases increases genomic instability and leads to tumorigenesis in animal models and hereditary defects in these processes results in developmental disorders in humans. In colon cancer patients, epigenetic alterations are detectable in the normal colon several years before the cancer is diagnosed. Preliminary data in our lab characterizing the global DNA methylation profiles of putative breast epithelial stem and differentiated cells from normal and neoplastic breast tissue also supports the hypothesis that alterations in epigenetic programs regulating cellular differentiation contribute to tumorigenesis. Most importantly, human epidemiologic studies demonstrated that blood folate levels influence breast cancer risk particularly that of estrogen receptor negative tumors. Folic acid is required for DNA methylation, although associations between folate levels and DNA methylation patterns of stem and differentiated cells have not been investigated. Based on our preliminary data we propose that low folate levels may increase breast cancer risk by decreasing DNA methylation and thus inhibiting the differentiation of mammary epithelial stem cells.
Chromatin patterns specific for normal mammary progenitor stem cells and their various differentiated progeny and analogous cell types isolated from different breast tumor types that we will identify during the course of the proposed project could potentially be used for predicting breast cancer risk, identifying targets of breast cancer prevention, and markers to monitor the success of preventative therapies. Furthermore, since our preliminary data indicates that the methylation pattern of stem and differentiated cells from normal and cancer tissue are similar and correlate with breast cancer subtypes, we will also use these markers for the classification of breast tumors into clinically relevant groups. Therefore, results of this study will allow us to expand our studies into areas that have not been investigated before and we would like to translate these findings into clinical practice in the near future. Most importantly, because chemotherapeutic agents that modify epigenetic programs are currently being tested in clinical trials in breast cancer patients, it is imperative to understand the potential effects these drugs may have on normal stem and breast cancer cells. Thus, the proposed project is highly innovative and relevant to breast cancer biology and the clinical management of breast cancer patients.