Inflammation Induced Cytosine Deamination and the Initiation of Cancer

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PROJECT SUMMARY/ABSTRACT Cancer remains one of the leading causes of death in the United States and worldwide with more than 8.8 million cancer related deaths each year. To reduce this public health burden, investigation of the most commonly observed substitution mutation, a cytosine to thymine transition, is crucial. An example of this predominance is evident in inflammation driven colitis associated colorectal cancer (CAC). Development of CAC involves an early critical p53 mutation dominated by cytosine to thymine transitions at 5’ cytosine, 3’ guanine (CpG) dinucleotides. These mutations arise from 5-methylcytosine deamination into thymine, forming a T:G mispaired intermediate. Subsequent replication propagates the mutation to the opposing strand, forming a T:A base pair. Although mutations in p53 have been demonstrated at the time of CAC diagnosis, little has been done to detect the exact location of low frequency mutations and mispaired intermediates in noncancerous tissues. It is also unknown how long a mutation is present before cancer diagnosis or how inflammatory conditions can promote cytosine to thymine transitions. Current sequencing methodologies involving standard PCR mask the presence of rare but potentially important mutations because amplification exponentially increases the predominant sequence and reports only that sequence. To solve these unanswered questions, we aim to mechanistically determine how inflammation increases the rate of these critical mutations and develop novel methods to detect low frequency mispaired intermediates and mutations. Given the critical role of inflammation in the etiology of CAC, we hypothesize that inflammation increases the number of mutations by providing substrates for competing DNA repair pathways and that an inflammatory environment will greatly increase the number of mutations and mispaired intermediates which leads to cancer development. We will address this hypothesis by first proving our mechanistic hypothesis of competing DNA repair and then developing methods to determine the number of global mispaired intermediates at a p53 mutation hotspot, described through three Specific Aims: 1) determine a potential mechanism by which inflammatory mediators can induce characteristic cytosine to thymine transitions; 2) design a novel glycosylase-mass spectrometry assay to measure global levels of mispaired intermediates and determine how inflammation affects mispaired intermediate production; and 3) develop a novel method to scan p53 exon 7 for cytosine deamination mutations and mispaired intermediates. By measuring low frequency mispaired intermediates and mutations long before tumor formation, we will be able to estimate the number of cells in a population that contains critical mutation and predict its prior DNA damage history. The mechanistic studies will elucidate the role of inflammation on the beginning stages of tumorigenesis, and together, the results of this proposal could lead to novel therapeutic targets or diagnostic methods for CAC or other inflammatory conditions.