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Aberrant nucleolar tumor suppressor function in breast cancer
Background: Breast cancer is often characterized by abnormalities of the nucleolus, suggesting that deregulation of nucleolar/ribosomal genes (rDNA) is critical for breast epithelial cell transformation. To address this issue, our lab has focused on a tumor suppressor and transcriptional repressor, MTG16, which is localized in the nucleolus of normal human mammary epithelial cells (HMECs) and is lost in breast cancer cells. Interestingly, MTG16 knock down in HMECs leads to loss of epithelial polarization, a feature of early breast epithelial tumorigenesis. Hypothesis: We hypothesize that MTG16 is part of a multi-protein complex involved in the transcriptional regulation of nucleolar/ribosomal genes, and that its loss leads to transcriptional deregulation. Specific Aims: To elucidate the role of MTG16 in nucleolar gene regulation of HMECs we propose: a) a candidate approach for testing whether MTG16 is part of rDNA-specific repressor complexes and whether its loss can lead to aberrant rDNA regulation (Specific aim 1); and b) a global approach for identifying the proteins of MTG16-containing repressor complexes and their gene targets (Specific aim 2). Study Design: Specific aim 1 : We plan to test whether: a) MTG16 interacts with known rDNA repressors both by co-immunoprecipitation and co-localization studies; b) MTG16 binds the rDNA promoter and induces transcriptional repression; c) rDNA transcription is downregulated in MTG16-overexpressing HMECs and upregulated in MTG16-knock down HMECs. Specific aim 2 : We plan to a) immunoprecipitate MTG16-containing complexes and use multidimensional protein identification technology (MudPIT) to identify the proteins interacting with MTG16 in HMECs; b) identify the target genes of these protein complexes by combining chromatin immunoprecipitation (ChIP) and promoter microarrays; c) assess the transcription of candidate target genes both in MTG16-overexpressing and MTG16-knock down HMECs. Benefits of the Research: When detected early, b reast cancer is treatable. Our proposal will provide new insights on nucleolar tumor suppressor function in breast epithelial cells. One of the major outcomes of this project will be to understand whether ribosomal gene deregulation is a consequence of loss of MTG16 tumor suppressor function. Given the dramatic biological changes that we observed after MTG16 loss in HMECs we expect to identify and develop MTG16-related biomarkers for early detection of breast cancer.
Background: Breast cancer is the consequence of a series of genetic and epigenetic events that lead to malignant transformation of human mammary epithelial cells. Critical is the loss of tumor suppressor genes, which normally prevent cancer development. A novel tumor suppressor gene, called MTG16, is commonly lost in breast cancer. We found that this tumor suppressor is localized in the nucleolus of human mammary epithelial cells (HMECs). The nucleolus is the site of ribosomal genes, which contain critical information for the production of ribosomes, fundamental machines for protein synthesis. Nucleolar structural changes and altered ribosome production is common in breast cancer. Interestingly, when we knocked down MTG16 in the nucleoli of human mammary epithelial cells, we observed features of early breast transformation, similar to what observed in ductal carcinoma in situ . Hypothesis: We hypothesize that loss of nucleolar MTG16 leads to deregulation of critical genes in breast epithelial cells. Specific Aims/Study Design: To understand the role of MTG16 in HMECs, we propose: a) a candidate approach for testing whether loss of MTG16 results in the loss of interaction with critical proteins involved in the regulation of ribosomal genes and, consequently, in ribosomal genes dysregulation (Specific aim 1); and b) a global approach, for identifying unknown MTG16-containing protein complexes and their gene targets (Specific aim 2). To this end we will specifically isolate the protein complexes containing MTG16 using reagents available in our laboratory and we will identify both proteins and DNA using advanced proteomic and genomic technologies. Benefits of the Research: When detected early, b reast cancer is a controllable and treatable disease. The proposed research will reveal new molecular markers consequent to nucleolar tumor suppressor loss in HMECs. These markers will provide new strategical tools to detect nucleolar dysfunction in early breast cancer.