title: DNA methylation &amp; its regulation in colorectal tumours
Creators: name: Ward, Joseph Christopher
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: subject: DNA methylation
subject: colorectal cancer
subject: Genetics
subject: FOS: Biological sciences
subject: bioinformatics

description: Colorectal cancer (CRC) is the fourth most common form of cancer in the UK and is associated with approximately 17,000 deaths each year. DNA methylation represents a critical epigenetic mechanism by which gene expression is controlled in cells, with aberrant DNA hyper-methylation often reported in CRC. Significant DNA hyper-methylation has been reported in a subset of CRCs and is referred to as the CpG Island Methylator Phenotype (CIMP). The precise mechanisms underlying CIMP and how DNA hyper-methylation contributes to colorectal tumorigenesis remains incompletely understood, therefore this thesis seeks to address both of these questions. Firstly, Genome-Wide Association Study (GWAS) meta-analysis data revealed an association between the 4q24 locus of chromosome 4 and CRC predisposition in Europeans. Subsequent conditional analysis, fine-mapping and in silico functional annotation identified a list of candidate causal polymorphisms with functional relevance in colonic tissues. Mendelian Randomisation and genotype-expression analysis identified down-regulation of Ten-Eleven Translocation 2 (TET2) as the candidate causal factor underpinning the GWAS association with CRC risk at this locus. The TET2 gene is involved in active DNA de-methylation, with pathogenic loss-of-function mutations thought to drive a hyper-methylated phenotype in acute myeloid leukaemia (AML). Similarly, pathogenic mutations in isocitrate dehydrogenase (IDH) 1 and 2 have also been reported in AML and present with a similar hyper-methylated phenotype. It has been hypothesised that 2-hydroxyglutarate produced by mutant IDH drives this hyper-methylation via the competitive inhibition of TET2. Mutations in TET2 and IDH are uncommon in CRC but it is possible that mutations in these genes may drive CIMP in such cancers. Methylation array data of colorectal adenocarcinomas from The Cancer Genome Atlas identified elevated DNA methylation in TET2-mutant and IDH-mutant CRCs compared to their wild-type (WT) counterparts, as well as a significant correlation between IDH mutations and CIMP+ CRC. CIMP+ CRCs presented with an enrichment of significantly hyper-methylated probes in bivalent promoter regions, with TET2-mutant and IDH-mutant cancers possibly showing an even greater enrichment at these regions than their WT CIMP+ counterparts. A number of significantly hyper-methylated probes in TET2-mutant CIMP+ cancers were also hyper-methylated in IDH-mutant CIMP+ cancers, localising around the transcription start site of a number of candidate tumour suppressor genes – indicating TET2 and IDH mutations may both drive CIMP+ CRC via the aberrant hyper-methylation and transcriptional silencing of tumour suppressor genes. 5-methylcytosine (5-mC) regularly undergoes spontaneous deamination to thymine. The methyl-CpG binding domain 4 (MBD4) gene encodes a protein involved in catalysing the repair of this deamination by excising the thymine from the resultant T:G DNA mismatch. Germline inactivation of MBD4 has been suggested to predispose affected individuals to intestinal polyposis and some forms of cancer, including uveal melanoma. This predisposition is potentially driven by pathogenic C → T mutations at CpG sites arising within the protein-coding sequence of cancer driver genes as a consequence of spontaneous deaminations of 5-mC going unrepaired. Individuals with germline pathogenic mutations in MBD4 and subsequent somatic loss of heterozygosity were shown to have increased numbers of C → T mutations at CpG sites, including a number of pathogenic mutations in cancer driver genes that have been previously reported in the literature. It was also found that CpG sites that were highly-methylated and in later-replicating regions of the genome were at the greatest risk of spontaneous deamination, thereby describing MBD4 as a cancer predisposition gene and how DNA methylation is mechanistically associated with C → T mutagenesis at CpG sites. In addition to unrepaired spontaneous deaminations, errors made by DNA polymerase epsilon (POL-ε) during DNA replication are an alternative mechanism by which C → T mutations at CpG sites may arise. CRCs with POL-ε exonuclease domain mutations (EDMs) and microsatellite unstable (MSI+ ) cancers presented with more C → T mutations at CpG sites than microsatellite stable (MSS) polymerase wild-type (POL-WT) CRCs. There was an excess of C → T mutations at CpG sites on the leading strand template in MSI+ CRCs and cancers with POL-ε EDMs, indicating that these mutations were likely the result of unrepaired DNA mismatches arising as a consequence of the erroneous incorporation of adenine opposite a template 5-mC by POL-ε propagating into C → T mutations in the next round of DNA replication. Similarly to spontaneous deaminations, C → T mutagenesis was more common at highly-methylated CpG sites, indicating that DNA methylation also influences the likelihood of replication errors. Therefore, methylation-induced DNA replication error may represent another mechanism by which DNA methylation may contribute to colorectal tumorigenesis. In conclusion, this thesis provides an in-depth investigation into the role of DNA methylation in CRC pathogenesis and suggests that DNA hyper-methylation may drive colorectal tumorigenesis via multiple mechanisms, including the direct modification of the expression of CRC driver genes or by indirectly driving C → T mutagenesis at CpG sites.