Abstract
Colorectal cancer (CRC) is a considerable health burden being the second highest cause of cancer deaths globally. While overall cases of CRC have been declining worldwide, there has been an increase in the incidence of the disease among patients under 50 years of age. The majority of these cancers are sporadic and the increase in incidence may reflect changing lifestyle, exposing young people to more and earlier pro-oncogenic factors.
An early event in the development of CRC is the loss of normal structure of the epithelium and key to this is the loss of cell to cell contact. E-cadherin, encoded by the CDH1 gene, is a membrane-bound protein whose extracellular domains bind to E-cadherin of neighbouring cells forming adherens junctions as the primary event in intercellular contact. Loss of E-cadherin leads to the breakdown of this organised epithelial structure and can lead to the development of cancer.
The research detailed in this thesis looked at E-cadherin expression status and mutation of the CDH1 gene in two separate cohorts. Firstly tumours from young Pakistani patients with early-onset colorectal signet-ring cell carcinomas (SRCCs), due to their histological similarity to SRCCs found in hereditary diffuse gastric cancers where mutation of CDH1 is a common causative factor and, secondly a local New Zealand cohort of early-onset CRC cases.
E-cadherin was found to be absent or weak in the colorectal SRCC samples and in a small number of cases this corresponded to germline CDH1 mutation. However, the remaining SRCC samples had low levels of CDH1 mutation despite loss of E-cadherin expression, suggesting that while E-cadherin loss is common in colorectal SRCC it is not due to CDH1 mutation in most cases. Amongst the New Zealand samples, only one tumour, the sole SRCC case in the cohort, showed loss of E-cadherin but this was not correlated with CDH1 mutation which was not a common feature of this cohort.
Additionally, APC sequencing was performed on the New Zealand cohort. APC is mutated in 60-80% of sporadic CRC tumours but with reportedly lower levels in younger patients. However 72% of the New Zealand early-onset CRC cohort was found to have a mutation in APC, a higher proportion than expected. This may reflect a greater coverage of APC by the sequencing methodology employed in this study compared to previous studies with a high proportion of mutations occurring outside the commonly studied mutation cluster region of APC. Loss of heterozygosity at the APC locus was found in only three patients, all of whom had APC mutations occurring close to codon 1300, reflective of a previous studies in older-onset CRC.
While environmental and lifestyle factors are widely considered to have roles in the development of sporadic CRC, there is growing evidence of the gut microbiota being a factor in colorectal carcinogenesis. One well-studied toxin-producing bacteria is Enterotoxigenic Bacteroides fragilis (ETBF) that causes cleavage of E-cadherin in colonic epithelial cells. Study of the effect of the B. fragilis toxin (BFT) on colonic epithelial cells has focused on the cell line HT29 which shows a rapid morphological change upon incubation with BFT. However, HT29 cells only have truncated forms of APC. In the last part of this study one mutated APC allele in HT29 was corrected by genome editing in order to study the effects of BFT on a cell line expressing full-length APC, and thereby to increase our understanding of the role of APC in colorectal carcinogenesis.
The rapid change in morphology upon exposure to BFT in HT29 cells has been attributed to the BFT-mediated cleavage of E-cadherin. However, edited HT29 cells containing full-length APC maintained their structure after 6 hours incubation with ETBF supernatant. Moreover, fluorescent immunohistochemistry showed that cell morphology was maintained despite the cleavage of E-cadherin, suggesting that the structural integrity of the edited cells was due to some internal function of APC rather than E-cadherin cleavage.
In summary, this research found that E-cadherin loss was commonly found in colorectal SRCCs but predominantly occurred independently from CDH1 mutations. Furthermore, other than in SRCCs, E-cadherin loss was not a common feature of early-onset CRC. Conversely, APC mutation was very common, with many mutations being found outside the mutation cluster region of APC, suggesting APC sequencing strategies should be targeted more widely throughout the gene. These results suggest a role for the APC protein in stabilising the cellular morphology of HT29 cells exposed to bacterial toxin, which is independent of E-cadherin. The edited HT29 cell line is likely to be a useful tool in the study of bacterial and other environmental effects on colorectal carcinogenesis.