Proteomic dissection of the early genetic changes in a colorectal cancer model

Colorectal cancer (CRC) is the third most common cancer in the UK and Adenomatous Polyposis Coli (APC) mutations are the most common genetic abnormality encountered in the sporadic form of this disease. In this study, we have used an Apc fl/fl/ mouse model which has a conditionally regulated Apc gene in the intestinal epithelium. Our hypothesis was that analysis of the changes in protein expression which occur in the intestinal epithelial cells of adult mice following Apc deletion would provide insights into signalling pathways which are also involved in early human colorectal carcinogenesis. The aims of this study were to use proteomic analysis to identify potential biomarkers for the early stages of CRC and to identify key proteins or processes involved in early colorectal carcinogenesis. iTRAQ-QSTAR proteomic analysis of intestinal epithelial extracts from Apc+/+ and Apc f//f/ mice identified 125 proteins which were differentially expressed, 50 being upregulated and 75 downregulated. We focused our efforts on the upregulated proteins as the detection of a positive signal is better for a biomarker. Ingenuity Pathway Analysis identified 19 proteins that could be detected in serum/blood, of which 13 were selected for further validation based on review of the literature. Immunohistochemistry, Western blotting and qRT-PCR identified 7 of these proteins as potential serum biomarkers of colorectal carcinogenesis. Integration of our iTRAQ data with a previous cDNA microarray performed using this mouse model identified c-Myc-dependent proteins. These included the 7 proteins identified in earlier studies and 4 additional proteins that might also play roles in colon carcinogenesis. The resulting 11 potential biomarkers were HMGB1, NCL, KRT18, RPL6, DDX5, PHB, SFRS2, FABP6, NAP1 L 1, NPM-1 and CBX3. These were further validated using another transgenic mouse model with the conditional regulation of both Apc and c-Myc genes (ApcfllfiMycfllfi), for which another iTRAQ (8- plex) analysis was performed. Confirmation studies were then carried out using the ApcMin/+ mouse model which shows more resemblance to human CRC. qRT-PCR studies comparing colonic polyp tissue samples from 6 month old ApcMin/+ mice and colonic tissue samples from their Apc+/+ wild-type counterparts showed increased expression of our candidate biomarkers in polyp tissue. For one of our candidates, HMGB1, a commercial ELlSA kit was then used to assess its serum concentration in various mouse models. This showed a statistically significant increase in serum HMGB1 concentration in Apc fl/fl mice compared to Apc+/+ mice. At 6 months of age, serum HMGB1 concentration was shown to be 1.69 fold higher in ApcMin/+ than in Apc+/+ mice. Our candidate biomarkers have also subsequently been validated using human serum and colonic tissue samples. The proteomic analysis of samples from mouse models with aberrant Apc expression has therefore generated a series of candidate biomarkers which have been demonstrated to be transferable to human CRC, thus validating our overall approach.EThOS - Electronic Theses Online ServiceGBUnited Kingdo