Elucidate the Biological Significance of Histone H2BG54D Mutation in Pancreatic Cancer

Description

Pancreatic cancer is a lethal disease with a median survival of 6 months and five-year survival rate less than 8%. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and accounts for more than 90% of all cases. Recent genomics studies have demonstrated that somatic copy number alterations and DNA mutations on oncogenes and tumor suppressor genes are the key drivers of PDAC. Interestingly, a significant number of mutations on genes that encode epigenetic modifiers were also identified, highlighting that epigenetics might play crucial roles in PDAC.We have previously elucidated the role of a driver mutation; histone H3K27-to-M mutation in pediatric brain cancer. The dominant negative effect of H3K27M mutant histones inhibits the di and tri-methylation of H3 at lysine 27, leading to dramatic changes in global gene expression including the epigenetic silencing of tumor suppressor p16INK4a. To address whether additional histone mutations also drives the formation of PDAC, we analyzed the “The Cancer Genome Atlas” (TCGA) database and have identified a somatic missense mutation on histone H2B; Glycine 54-to-Aspartic acid (H2B G54D) in 6.9% PDAC patients (10 out of 146). The same mutation also exists in brain and lung cancers, suggesting that the H2BG54D mutation might play a role in tumorigenesis. Glycine 54 of H2B is evolutionarily conserved from yeast to human, and the G54 residue locates in close proximity to the double strand DNA near the DNA entry/exit site of nucleosome. Intriguingly, our in vitro nucleosome stability assays demonstrated that the binding of DNA to nucleosome is weakened in nucleosomes reconstituted with the G54D H2B mutant. In addition, our nucleosome pulling assay coupled with optical tweezers showed that the mutant H2B-nucleosome is less stable than wildtype at single molecule level, indicating that the substitution of a neutral charge glycine residue with a negatively charged aspartic acid at H2B-54 affects nucleosome stability. Interestingly, pancreatic cell lines ectopically expressing the H2BG54D mutants have increased cell proliferation and migration, raising the possibility that the H2BG54D mutant might take part in the development and progression of PDAC. More importantly, Kaplan-Meier survival analysis revealed that PDAC patients with the H2BG54D mutation had poor overall survival compared to non-G54D, further supporting the idea that the H2BG54D mutation might play a crucial role in PDAC.We hypothesize that the H2BG54D is one of the driver mutations of PDAC by reducing the stability of nucleosome. It subsequently alters the biological activities on chromatin and confers oncogenic properties in pancreatic cancer cells. In this proposal we aim to define and elucidate the effects of this mutation in chromatin function and tumorigenesis of PDAC. Our objectives are to 1) investigate the effect of H2BG54D mutation in transcription, 2) define the effects of H2BG54D mutant histone in DNA replication and repair and 3) elucidate how H2BG54D contributes to oncogenic properties in pancreatic cells.By completing this project we will uncover the significance and the clinical relevance of the H2BG54D mutation and make a major breakthrough in cancer biology by linking genetic mutation and epigenetic gene regulation in the development of pancreatic cancer. Knowledge gained from this work will be translated to the development of new strategies in treating this subtype of PDAC.