Cancer behavior is regulated by the differential expression of genes that promote growth, invasiveness and abrogate apoptotic signaling. A hallmark of cancer genome is the presence of aberrant methylation, a post-synthetic modification associated with de novo silencing of gene expression. In pancreatic cancer, several genes are frequently silenced including the p16INK41 and the RASSF1A tumor suppressors, while others as MAGE-A3 and MUC2 are reactivated in a process associated with promoter hypomethylation. The mechanisms through which certain genes are hypermethylated while others are demethylated within the same cell or at different stages of growth/differentiation are not fully understood. It is debatable whether transcription silencing represents the cause or the consequence of promoter hypermethylation. Evidence suggests that hypermethylation may not be the primary cause for the transcription loss, but is used by the cancer cells to maintain the silent state of genes. In one model, the DNA methyl transferase target-specificity is influenced by the promoter competitive binding to differentially expressed nuclear proteins thus maintaining the hypomethylated status of the DNA binding motifs. Because of their low abundance in the cells it has been difficult to fully characterize these DNA-binding proteins using classical biochemical methods and to evaluate their role in the DNA hypermethylation process. Novel approaches in cancer therapeutics involve methylation-reversal strategies to curb the tumor growth or to induce anti-cancer immune responses by activating antigen-encoding genes (e.g. MAGE). Despite successful reports and the development of at least two licensed demethylating drugs, further refining is needed in order to selectively activate/silence genes of interest while avoiding a genome-wide global hypomethylation that could lead to malignant transformation in itself. For this purpose, it is critical to understand the process of DNA methylation and to identify the key regulatory elements associated with aberrant methylation and tumor phenotype. In our preliminary experiments, using DNA-capture and mass-spectrometry mapping, we demonstrated the association of two prominent DNA-binding proteins with the unmethylated state of MAGE-A3 (melanoma associated antigen) gene, suggesting that these proteins could participate in maintaining the active state of MAGE promoter. It is our hypothesis that differentially expressed, DNA-binding proteins can influence the target-specificity of DNMTs actions. Such transcription/methylation effectors could present novel therapeutic targets for modulating gene transcription and proliferative signaling in pancreatic cancer. In the current proposal, we will test a new paradigm where the differential expression of unique DNA-binding proteins is involved in the process of selective DNA methylation and the associated gene silencing. We will utilize classical nuclear protein pull-down assays coupled with the latest advances in protein detection technology and mass spectrometry and methylation-specific PCR analysis (1) to identify differentially expressed, methylation-associated DNA-binding proteins and (2) to determine their role in regulating the DNA methylation process and cancer growth. Despite technological advances in medical diagnostics and anti-cancer treatment, most of the patients with pancreatic adenocarcinoma are presented at the time of diagnosis with incurable disease. It is important therefore to understand the molecular events associated with pancreatic adenocarcinoma in particular those with the potential to serve as therapeutic targets or novel early detection biomarkers. It is our hypothesis that unique nuclear proteins are associated with aberrant changes in the cancer cell chromatin and that the identification and functional assessment of these molecules could present a novel way of understanding the events that shape the pancreatic cancer phenotype and provide novel therapeutic targets for selective activation/silencing of cancer-related genes. In the current proposal, we will characterize DNA-binding effectors and will examine their role in modulating the gene expression and differential functions of pancreatic tumor DNA. ? ? ?
