Age and diet are the two most clearly recognized risk factors for common sporadic colon cancer, responsible for >90% of cases in developed countries. We will make use of an important technical advance for whole genome sequencing of single cells recently reported by co-investigator Vijg that can uniquely detect rare mutational events to define the mutational load and spectrum in each of the Lgr5+, Bmi1+ and Transit Amplifying compartments in the """"""""normal"""""""" mucosa as a function of both mouse age and diet. Further, we will determine the cause and consequences of these mutations. We make use of aging mice and a unique nutritionally driven model of sporadic colon and intestinal tumors in wild-type mice that recapitulates the stochastic development of sporadic colon cancer (1-2 tumors in 25% of the mice following exposure to major human dietary risk factors for 2/3 of the host lifespan). This model has been replicated in 3 different laboratories, including ours, and pathology reported. We reported that colon and intestinal tumorigenesis in this model encompasses inflammation, ectopic and elevated Wnt signaling, altered lineage allocation and a shift to glycolysis in the mucosa. New preliminary data show accumulation of DNA damage in intestinal crypts linked to the inflammatory response, and that progeny of intestinal Lgr5+ stem cells are retained in the crypt longer in mice fed the tumor promoting diet. These data complement reports from Vijg of increased mutational load in the intestinal mucosa with age as a consequence of DNA replication. The rationale is that stem cells in the intestinal and colonic mucosa, which are targets for tumor initiating events, should serve as harbors of genomic integrity that maintain intestinal homeostasis. We hypothesize that when this fails, accumulation of mutations in progenitor cell populations increases the probability of sporadic colon and intestinal tumorigenesis. Therefore, we will 1) determine how cells in each of the Lgr5+, Bmi1+ and transit amplifying compartments accumulate mutations as a function of both age and diet before tumors develop;2) identify the mutations and mutated loci in single cells by next generation sequencing and a bioinformatic pipeline that are state-of-the-art;3) determine causes and consequences of increased mutational load in these populations;and 4) determine which mutations are likely selected for in the sporadic tumors that eventually arise. All mouse strains are in hand and many necessary crosses are bred. Reviewers of the initial submission were enthusiastic. In response to their comments, we have: a) clarified the necessary sequencing and bioinformatic resources;b) secured matching funds from the Albert Einstein Cancer Center to permit whole genome, rather than exome, sequencing of single cells;c) increased commitment of personnel for data analysis;d) provided new preliminary data that the high risk diet causes delayed exit of Lgr5+ stem cell progeny from the intestinal crypts;e) added new experiments, based on new prelim. data, to link the inflammatory response to the accumulation of DNA damage and the retention of stem cell progeny in the crypt;f) described our data establishing dietary induced alterations of the intestinal microbiome as a potential contributor to risk for tumorigenesis;g) added 2 long-time collaborators as advisors: Sellers, an expert in histopathology;Kucherlapati, a leader in the NCI TCGA effort to define genetic alterations in human colon tumors and a key contributor to both the mouse and human genome sequencing projects. He will serve as a scientific advisor and liaison with the TCGA.
Using novel methods for single cell whole genome sequencing able to detect rare mutations, and unique mouse models, we will define how age and diet, two major risk factors for common sporadic intestinal cancer, alter the mutational load and spectrum of intestinal stem cells. We will also investigate mechanisms that modulate this genetic instability and consequences of these mutations for stem cell function, intestinal homeostasis and tumor formation. This is important in understanding how tumors develop, and for new approaches for cost effective early detection and prevention.
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