Investigating the association between BRAFV600E and methylation in sporadic colon cancer

Abstract

Aberrant methylation of CpG island promoters is a frequent observation in cancer and is known to affect many genes, including tumour suppressor genes. Genes with methylated promoters are usually repressed and inactive, and there is good evidence that most genes that become methylated in cancer are already repressed in the normal tissues from which tumours arise. However, the methylation of some genes appears to arise at previously active loci, suggesting either a stochastic epigenetic event or that these genes are somehow predisposed to becoming methylated. The DNA mismatch repair gene MLH1 is expressed in normal colonic epithelial cells but methylated and down-regulated in some sporadic mismatch repair-deficient colon tumours. These tumours are almost invariably associated with the simultaneous methylation of multiple cancer-specific loci, termed the CpG island methylator phenotype (CIMP) and an activating mutation of BRAF (V600E), raising the possibility that a hypermethylator phenotype may arise in cancer in direct association with a specific genetic alteration. The possibility that MLH1-deficiency caused BRAF mutation was discounted as genetic deficiency of MLH1 is not associated with BRAFV600E. I explored the possibility that BRAFV600E might induce MLH1 methylation but found no evidence in support of this. I then focused on factors that might mediate CIMP gene methylation, of which MLH1 methylation is known to be a part. Bioinformatic analysis of the genes methylated in BRAFV600E colon tumours indicated a significant enrichment in binding sites for the transcription factor MAZ (MYC-associated zinc finger protein). I hypothesised that loss of MAZ might lead to MLH1 down-regulation and its subsequent methylation. In this thesis I provide evidence that both MAZ and MLH1 expression are deregulated during normal colonic epithelial differentiation. The down-regulation of MAZ by RNA interference led to a reduction in MLH1 expression and methylation of its promoter. I speculate that MLH1 methylation may be associated with BRAF mutation because transformation by BRAFV600E allows progenitor cells to undergo a degree of differentiation whilst maintaining their malignant proliferation. I speculate that it is during this process of differentiation that MLH1 becomes susceptible to methylation.