Department of Pathology

Dr Mark Arends

Research description

Colorectal carcinogenesis involves cellular transit from normal mucosa via adenomas to carcinomas. The transitions are associated with characteristic genetic changes, such as alterations to APC (>80%), the DNA mismatch repair genes MLH1 & MSH2 (~15%), K-ras (~40%), and p53 (>60%) amongst others, and these genes also influence the regulation of proliferation, differentiation and apoptosis. Mutation of APC (which can be inherited in familial adenomatous polyposis coli syndrome or FAP) or loss of APC is seen in adenomas and this represents the major pathway of adenoma formation and predisposes to further progression to carcinoma, a process that is often associated with chromosomal instability, which we have been studying with spectral karyotyping and array-Comparative Genomic Hybridisation (array-CGH). Array-CGH can also pinpoint genes that consistently show loss or gain of copy number in colorectal tumours and using this approach we have identified BRUNOL4, PARK2 and IRS2 as new genes in colorectal cancer development and progression. Studies of DNA methylation are being performed to investigate epigenetic silencing of cancer-related genes in these tumours, including MLH1, MGMT, PTEN DNMT3B and others involved in the WNT/APC/B-CATENIN signalling pathway.

A second pathway involves the transition of hyperplastic / sessile serrated polyps or lesions to serrated adenomas then to carcinomas, with evidence of associated microsatellite instability due to DNA mismatch repair deficiency, which confers an increased mutation rate particularly in repetitive DNA sequences (the "mutator phenotype"). This accounts for the susceptibility to cancer formation in colorectum, endometrium, ovary, skin and other sites in Hereditary Non- Polyposis Colorectal Cancer (HNPCC) or Lynch Syndrome (LS) patients. A new approach to diagnosis of suspected HNPCC/Lynch Syndrome has been developed using tumour analysis for both abnormal mismatch repair protein expression by immunohistochemistry and microsatellite instability testing of tumour DNA. Defective DNA mismatch repair is found in approximately 15% sporadic colorectal cancers and our studies of these have shown genetic changes in apoptosis-related genes, lower levels of chromosomal changes and altered mismatch repair protein expression (usually MLH1 silencing due to promoter hypermethylation). MSH2 and MLH1 are the most frequently mutated mismatch repair genes in HNPCC / Lynch kindreds. The regulation of apoptosis and contribution to colorectal carcinogenesis of defective mismatch repair were studied using a model null for MSH2 and also null for both MSH2 and p53, which showed that MSH2 can signal apoptosis via p53 after sensing DNA damage of mismatch type.

The regulation by mutated K-ras of proliferation, differentiation and the apoptotic response to DNA damage was examined in embryonic stem cells expressing either wild-type or mutated K-ras. Expression of mutant K-ras suppressed differentiation and enhanced apoptosis in otherwise genetically normal embryonic stem cells and was associated with a large number of transcriptomic changes observed by cDNA microarray expression profiling. A model was constructed to analyse the effects of expression of mutated K-ras (induced specifically in the intestinal epithelium following Cre-mediated loxP recombination) on intestinal tumour formation in both pathways of adenomagenesis (APC mutation and defective mismatch repair). This showed K-ras-mediated acceleration of intestinal tumorigenesis in both adenomagenesis pathways. The roles of the K-ras 4A and 4B transcripts were investigated in human cancers, ES cells and models with knockout of the K-ras exon 4A and/or knockout of the whole K-ras gene, demonstrating alterations to susceptibility to intestinal and lung tumour formation following loss of K-ras exon 4A. These models of intestinal tumorigenesis were used to determine contributions to tumour formation by RASSF1A, GNAS, PARK2 and others. Use of knockout models validated SLX4/FANCP as a new Fanconi Anaemia gene. The Fanconi DNA repair pathway was shown to be critically important in repairing acetaldehyde-induced DNA damage. Sleeping Beauty transposition studies have been used to identify new tumour-related genes, including those that cooperate with mutated Apc in intestinal tumour formation. These and other models continue to be used to identify and investigate other potentially novel intestinal cancer-related genes, such as NRBP1.