Colorectal cancer is a heterogeneous disease with significant variations in pathological and molecular features, combinations of which often allow grouping of tumours with similar clinical outcomes and defined responses to chemotherapy. The traditional model of colorectal tumorigenesis is based on the adenomatous precursor lesion and is characterised by tumour suppressor gene inactivation by the mechanism of chromosomal deletion. An alternate pathway is based on an underlying defect in the mismatch repair system which results in cancers exhibiting a high level of microsatellite instability (MSI-H). In contrast to the traditional model, these tumours originate in serrated or hyperplastic lesions. In addition, an intermediate form exists which shows features of both chromosomal deletion and low level MSI (MSI-L). The importance of this group is controversial and has accordingly been less well characterised. Promoter hypermethylation is also an important mechanism for gene silencing in approximately 40% of colorectal cancers, particularly those demonstrating an MSI-H phenotype. The aim of this thesis was to identify molecular events underlying the development of microsatellite unstable colorectal tumours. Three separate approaches were adopted: examination of candidate genes, molecular profiling and comparative gene expression analysis.
A putative role for the retinoblastoma protein-interacting zinc finger (RIZ1) gene in MSI-H cancers and O-6-methylguanine DNA methyltransferase (MGMT) in MSI-L cancers is explored in chapters three and four, respectively. Methylation of the RIZ1 promoter occurred in 35% of MSI-H tumours and was almost exclusive to this group. Repeat tract mutation was observed in 23% of MSI-H tumours, but was rarely correlated with methylation, suggesting monoallelic inactivation of RIZ1 may be sufficient to contribute to tumorigenesis. Promoter hypermethylation was also observed in a subset of hyperplastic polyps, indicating it may be an early event in some tumours arising via the serrated pathway. Promoter hypermethylation of MGMT (chapter four) was observed in 64% of MSI-L tumours compared with only 13% MSI-H and 26% MSS and was highly correlated with mutation of the K-ras oncogene. This was the first demonstration of a molecular defect occurring significantly more frequently in MSI-L than MSS tumours. MGMT inactivation provides a mechanism for the dinucleotide repeat instability observed in MSI-L tumours, which may arise not by mismatch repair deficiency but by over-loading a functional mismatch repair system. The data presented in chapter four support the continued classification of MSI-L tumours in order to identify important molecular events associated with this group.
A molecular profiling approach was adopted to examine the prevalence and significance of the methylator phenotype in relation to MSI (chapter five). Five genes and four anonymous markers of methylation (methylated in tumour, MINT) were assayed and compared with twelve morphological features. Whilst methylation of both genes and MINTs was more common in MSI-H tumours, a group of non-MSI-H tumours was identified which were extensively methylated and importantly, exhibited morphological mimicry of MSI-H cancers, including presence of serration. Proximal location was strongly associated with methylation and was independent of MSI-status of the tumour. This implies the existence of a serrated pathway of neoplasia which predominantly occurs in the proximal colon and is underscored by defective methylation. Interestingly, MGMT methylation was also common in tumours with little evidence of methylation arising in the distal colon. This highlights the pervasive influence of defective methylation on colorectal tumorigenesis whereby critical tumour suppressor genes may be silenced by this mechanism in the absence of a general methylator phenotype.
A more detailed method of molecular profiling involves the use of microarray chips. However, this technology is currently limited by the incomplete characterisation of the human genome. For this reason, suppressive subtractive hybridisation was utilised in chapter six for the identification of both known and novel genes down-regulated in MSI-H cancers, as compared with matched normal mucosa. Of 115 unique sequences identified, 35 matched previously characterised genes. These included genes involved in processes critical to tumour progression such as signal transduction, cell polarity, checkpoint control, apoptosis, transcription, translation and DNA repair. The remaining 62 sequences matched expressed sequence tags (ESTs) or hypothetical proteins whilst 18 sequences were novel. Of particular interest was a sequence matching an uncharacterised EST, named MSI-1, which was down-regulated in 12/13 colorectal tumours and 9/10 colorectal cancer cell lines. The data presented in chapter six support the continued characterisation of the human genome in order to identify novel gene targets involved in tumorigenesis, which will be necessary to realise the full potential of large-scale molecular profiling.
The findings synthesised in this thesis emphasise the complexity of colorectal tumorigenesis and highlight the limitations of the current models, which may mask important subgroups with different tumour biology. Further identification of the molecular events underlying the development of different classes of colorectal tumours will assist pre-symptomatic screening and choice of post-operative chemotherapy.