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    Research Grants Awarded

    Polycomb-Mediated Aberrant Dna Methylation As A Biomarker For Early Breast Cancer Detection

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    In 2006 approximately 210,648 were diagnosed with breast cancer and over 41,000 died from the disease. A major challenge in the management of solid tumors is the overall lack of prognostic tools to enable health care providers to transition from universal treatments to individualized regiments tailored to address the specific molecular abnormalities that characterize a patient?s neoplasia. In recent years, a model suggesting the stem cell origin of cancer has gained strong support in the medical literature. In this model, the mechanisms of self-renewal and maintenance of stem cell identity become dysregulated via a combination of abnormal genetic and epigenetic events. The result of these changes is uncontrolled cell proliferation which could give rise to tumor formation. Thus, elucidating the molecular mechanisms that underlie the malignant transformation of stem cells into cancer stem cells could help better understand of how human neoplasias arise. Genomics data suggest that the gene expression profile of cancer stem cells can be an informative predictor of cancer therapy outcome. The transcriptome of stem cells is controlled, in part, by the Polycomb group of genes (PcG), a family of proteins that is required to maintain stemness and which is also involved in coordinating cellular differentiation. Several reports have highlighted that genes under PcG regulation may be particularly susceptible to aberrant DNA methylation, suggesting an important role for PcG targets in development and/or progression of human cancer. According to this evidence, we hypothesize that ?Aberrant DNA methylation of PcG target genes is an early event in the development of human breast cancer?. To test our hypothesis, we propose the following specific aims: To identify PcG targets which undergo DNA methylation due to differentiation in human breast stem cells; To identify PcG targets aberrantly methylated in human primary breast tumors and To characterize the identified PcG targets exclusively methylated in human primary breast tumors. Human breast epithelial stem cells will be purchased from Celprogen and grown as mammospheres generated from single cells. The mammospheres will then be cultured on collagen-coated plates for at least 10 passages to induce differentiation. Confirmation of mammary gland cell-lineage differentiation will be assessed by testing for expression of K14, K18 and ?-casein by real-time PCR (markers for myoepithelial, ductal epithelial and alveolar lineages, respectively). DNA and total RNA will be isolated from the in vitro differentiated cell cultures and from the parental stem cells. After bisulfite treatment, the DNA samples will be analyzed by Illumina on a custom-made chip containing 1,893 identified PcG target probes as described by Lee et al. Technical and biological replicates will be conducted at random in 20% of the samples assayed to assure the reproducibility of our results. Since mammary stem cell differentiation into myoepithelial, ductal epithelial and alveolar lineages may involve the differential DNA methylation of specific PcG targets, we will treat each of these differentiated cell cultures as unique. At this point, we will have generated a list of PcG target that undergo DNA methylation as a result of in vitro differentiation of mammary stem cell into 3 distinct lineages. We will then analyze 50 snap frozen primary human tumors. In order to avoid potentially confounding factors stemming from tumor grade, only specimens with a combined histologic grade<5 (Grade I) and a mitotic count<9 mitoses/10 hpf will be used in our study. Two frozen sections collected from different areas of each specimen will be stained and visually inspected to determine the amount of tumor epithelium per sample. Micro-dissection will be used as an alternative method if the amount of normal tissue contamination in the specimens is >20%. The samples will be analyzed by Illumina using the custom chip already described. After completion of this aim, all the Illumina data will be analyzed to identify those PcG targets that exhibit aberrant DNA methylation exclusively in primary breast tumors and that are not the result of normal stem cell differentiation. The PcG targets derived from aim 2 will be sorted by frequency of DNA methylation. We will limit our initial analysis to those targets methylated in >75% of the specimens analyzed. This cutoff will be lowered (>50%) if our initial criterion yields fewer than 10 candidates. For the selected candidates, binding of the PcG repressor in the primary tissues will be confirmed by chromatin IP using antibodies against SUZ12. Functional studies will be performed: we will knock down (individually) the identified candidates in mammary epithelial stem cells using RNAi to identify those genes whose lack of expression results in measurable changes in cell growth concurrent with variations in cell morphology. Finally, we will test for the presence of methylated DNA from our candidate genes (Illumina) in fresh frozen plasma collected from the same 50 patients described in Aim 2. We anticipate the completion of Specific Aims 1 and 2 within the first 12 months. Months 13-36 will be devoted to the characterization of the identified targets through various functional assays. The main goal of our study is to identify a set of markers that could be tested via minimally invasive procedures in women at risk of developing lung cancer or in those that present an atypical mammogram. Altogether, we expect our work to generate novel data that clinicians could use to select or develop more effective treatments based on the specific molecular abnormalities that make up each tumor.

    Lay Abstract:
    In 2006 over 210,000 women were diagnosed with invasive breast cancer in the United States and over 20% of them are expected to die from the disease. Although the survival rate for breast cancer patients has improved over the past decade, the diagnosis and treatment of this neoplasia still takes a major physical and psychological toll on women. Breast cancer arises from a combination of molecular changes in mammary stem cells, which, over time, can result in tumor formation. Recently, various studies have shown that a group of genes whose expression is finely regulated in mammary stem cells by a specific group of proteins, the Polycomb family of proteins, is frequently shut off via DNA methylation in breast tumors. However, the expression of these genes is needed in normal breast cells in order to maintain their differentiation and orderly cell division. Furthermore, one specific study demonstrated that Polycomb-regulated genes are 12 times more likely to be methylated and therefore abnormally shut off in tumors, compared to those genes not regulated by Polycomb proteins. Therefore, given the important role of Polycomb-regulated genes in the control of normal cell growth, the fact that these genes are key regulators in stem cell function and the current thinking that cancer arises from abnormalities in stem cells, we hypothesize that the silencing of Polycomb targets via DNA methylation is an early event in breast tumor development. In order to test our hypothesis, we will grow breast stem cells and allow them to differentiate in culture. After differentiation, we will examine which Polycomb targets become methylated by utilizing a platform that allows for the simultaneous interrogation of 1,893 genes which in a previous study have been shown to bind SUZ12, a Polycomb protein. This first experiment will identify those genes that normally become methylated as breast stem cells differentiate, indicating that their lack of expression is to be expected in normal differentiated breast cells. Then, we will analyze the DNA methylation status of the same 1,893 genes in 50 primary breast tumors. By comparing the results of both experiments, we will be able to focus specifically on those genes whose expression is needed in normal cells but that is lost in breast cancer due to abnormal DNA methylation. Once our target genes are identified, we will use computational analyses to investigate whether a sequence component can be found that may distinguish those Polycomb targets that are susceptible to aberrant DNA methylation from those that are refractory to it. More importantly, we will carry out functional studies to try to understand how the lack of expression of these genes contributes to the development, growth and aggressiveness of breast tumor. The ultimate goal of our project is to identify a set of markers that could be tested through minimally invasive procedures in women at risk of developing breast cancer or in those patients who present an atypical mammogram, for example. Altogether, we expect our study to generate new data that clinicians could use to design potentially more effective treatments based on the specific abnormalities that make up each tumor. By the same token, our study could also aid in improving the way breast tumors are classified, by distinguishing those that may respond well to conventional chemotherapy from those which may not, for example. This distinction would result in a better management of resources and may improve a patient?s quality of life by minimizing the need to try conventional treatment approaches that, given the molecular signature of that patient?s tumor, are likely to yield negative results. Conversely, our study could help guide clinicians to administer first those therapies with the highest likelihood of success, thus potentially shortening a patient?s hospital stay or simply eliminating the patient?s exposure to therapeutic agents which may afford a low chance of becoming disease-free.