There are two chief molecular pathways to sporadic colorectal cancer (CRC), the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. A third pathway, the pure microsatellite instability pathway, is important in inherited CRC specifically hereditary non-polyposis colorectal cancer. The CIN pathway is characterized by an adenomatous pathological precursor, aneuploidy and microsatellite stability. CIMP pathway cancers, however, are frequently proximal, develop from serrated rather than adenomatous polyps and are strongly associated with BRAF mutation. The CIMP pathway is driven primarily by epigenetic rather than genetic instability. These pathway-specific molecular traits are evident within the pathological precursors to these cancers and thus pathway divergence must occur at the beginning of carcinogenesis or even before. Although DNA methylation is recognized as a key mechanism in colorectal carcinogenesis, relatively little is known about its pattern, regulation and relevance in normal colorectal mucosa. This PhD thesis characterized the profile of DNA methylation in the normal human colorectum and explored its associations with luminal, environmental, dietary and pathological factors. The genes methylated in CRC are characterized as “type A” (Age-related) genes and “type C” (Cancer-specific) genes. Generally, “type A” genes are methylated in both normal and neoplastic tissue with the degree of methylation proportional to the age of the tissue. The methylation of “type C” genes, however, is more specific for neoplastic tissue. The primary study recruited 166 patients undergoing colonoscopy. At colonoscopy, mucosal biopsies were taken from the caecum, transverse colon, sigmoid colon and rectum. DNA methylation was analysed by MethyLight at “type A” (ESR1, GATA5, HIC1, HPP1, SFRP1) and “type C” methylation markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3). LINE-1 methylation was quantified by pyrosequencing. The last 5 “type C” markers comprise a CIMP panel used to identify CIMP cancers. Mean “type A” and CIMP panel methylation Z-scores were calculated. The PMR for each of these CpG island loci was compared to patient age, gender, previous colorectal polyps, smoking history and the presence of concomitant pathology. Most “type A” genes demonstrated strong and direct correlations between methylation and patient age (e.g. ESR1, ρ=0.66, p<0.0001) and had greater methylation within the distal compared to the proximal colorectum (e.g. ESR1, p<0.0001). On multivariate analysis, the mucosal “type A” methylation Z-score had a strong, independent, inverse association with the diagnosis of colorectal adenomas (OR=0.23, p<0.001), the precursor to CIN cancers. The mean CIMP methylation Z-score in normal mucosa, however, was significantly and independently associated with advanced proximal serrated polyps (OR=5.1, p=0.009), the precursor to CIMP cancers. The luminal and epithelial associations with colorectal methylation were explored by a randomized, double-blind, placebo-controlled trial. This experiment was undertaken to determine whether dietary supplementation could modulate epithelial DNA methylation. In addition, the study was designed to evaluate intra-individual reproducibility of the MethyLight technique. The study consisted of a 4 week cross-over trial of resistant starch and Bifidobacterium lactis either alone or as a combined synbiotic preparation, in 20 human volunteers. Rectal biopsies, faeces and serum were collected. Rectal mucosal endpoints included DNA methylation at the CpG island loci and LINE-1, epithelial proliferation (Ki67 immunohistochemistry) and crypt cellularity. Faecal short-chain fatty acid concentrations, pH, ammonia and microbiological profiles (by DGGE and sequencing) were examined. The synbiotic intervention fostered a significantly different faecal stream bacterial community than either the prebiotic or the probiotic interventions alone, but did not show any significant associations with the epithelial or luminal parameters. To explore possible associations between luminal and epithelial parameters and mucosal DNA methylation, the baseline indices were further analysed. There was a strong positive correlation between baseline epithelial proliferation and “type A” marker methylation (ρ = 0.7, p = 0.0001). Thus, “type A” methylation may reflect the cellular age or mitotic burden of a tissue, which is a function of both time and cell turnover. There were consistent inverse trends evident between faecal short-chain fatty acid levels and rectal mucosal DNA methylation. This PhD project found that DNA methylation within the normal colorectal mucosa varied with patient age and region and was strongly associated with the development of pathway-specific pathology, suggesting that the background colorectal field may predict both the at-risk patients and at-risk pathways. Diet and the luminal environment more broadly may influence levels of DNA methylation in the colorectal mucosa and could help to explain regional patterns of colorectal DNA methylation.
| Identifer | oai:union.ndltd.org:ADTP/283965 |
| Creators | Daniel Worthley |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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