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Abstract
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 Apcfl/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
Apcfl/fl 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, NAP1L1, NPM-1
and CBX3. These were further validated using another transgenic mouse model
with the conditional regulation of both Apc and c-Myc genes (Apcfl/flMycfl/fl), 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 ELISA kit was then used to
assess its serum concentration in various mouse models. This showed a
statistically significant increase in serum HMGB1 concentration in Apcfl/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.