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Title: Bacteria-induced Wnt signalling as a mechanism for malignant development in the intestinal epithelium
Author: Meehan, B.
ISNI:       0000 0004 6496 6533
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Colorectal cancer (CRC) is one of the most common cancers worldwide with over a million new cases each year, and evidence implicates chronic inflammation as a risk factor for developing CRC. Inflammatory conditions place stresses on bacteria composing the human gut microbiota and alter the composition of the gut microbiota in favour of pathobiont Escherichia coli (E. coli), with resultant expansion of these bacteria increasing their interactions with the gut mucosa. Mucosa-associated E. coli have been found in high numbers within intestinal tissue taken from IBD and CRC patients. Pro-inflammatory cytokines such as cyclooxygenase-2 (COX-2) are commonly implicated in inflammatory disease, and can be released upon tissue damage or following infection. COX-2-driven prostaglandin E2 (PGE2) increases are believed to activate the cancer-related Wnt signalling pathway, with accumulation and subsequent nuclear translocation of the key Wnt protein ß-catenin aiding proliferation, migration and metastasis. With COX-2 implicated in CRC development and progression and a cascade for COX-2 and PGE2 stimulating cancer cell growth via Wnt activation, mucosaassociated E. coli infection could lead to sustained increases in Wnt signalling. In addition, as mucosaassociated E. coli strains isolated from CRC patients can survive and replicate within human macrophages and increase COX-2 and PGE2 secretion, this could enhance their tumourigenic effects. The aim of this project was to characterise the effects of mucosa-associated E. coli isolates from CRC and IBD patients on the activation of the Wnt signalling pathway both in vitro and in vivo. We first used CRC cell lines and human macrophage cells to assess the effects of mucosa-associated E. coli infections. We show consistent increases in total and nuclear localised ß-catenin at both the gene and protein level in vitro, and have shown that this correlates with increased COX-2 gene and protein levels. Downstream Wnt transcription was shown following infection, and this was inhibited by the addition of a COX-2 inhibitor. We also showed increased release of PGE2 from macrophages infected with mucosa-associated E. coli, which could be a contributing factor in driving the Wnt response in neighbouring intestinal epithelial cells. To investigate these effects in vivo, we used colonic tissue from a germ-free Il10-/- mouse model of CRC mucosa-associated E. coli infection to determine COX-2 and Wnt pathway activation. Following mono-association of germ-free Il10-/- mice with a CRC mucosaassociated E. coli isolate, significant increasesin COX-2 and ß-catenin protein expression and increased levels of ß-catenin nuclear localisation were observed. To further investigate changes in COX-2 and Wnt signalling, we used a fosmid library of 968 clones created from the fragmented DNA of another mucosa-associated E. coli isolate from a CRC patient to confirm up-regulation of COX-2 gene expression following infection of CRC cells with fosmid clones. We identified 12 fosmid clones showing confirmed COX-2 gene expression increases, and sequenced the DNA content of all 12 fosmid clones to find common genes or operons that may be contributing towards these expression changes. Sequencing showed similarities in DNA content, with particularly common homology in two fosmids containing genes involved in growth and survival, particularly in high-stress inflammatory conditions. This data may aid identification of genes or operons that contribute to the induction of COX-2 and Wnt signalling observed in vitro and in vivo. We were also able to develop a 3D organotypic cell culture using a series of colorectal cell lines at different key stages of malignant transformation in the adenoma-carcinoma pathway. Successful 3D aggregate cultures were achieved, and data obtained from infecting these aggregates would suggest that this method of culture is suitable for studying mucosa-associated E. coli-host epithelium interactions, and confirmed data previously obtained by infection of 2D monolayer CRC cell cultures and those responses seen in an in vivo gut infection model. Our study supports the hypothesis that mucosa-associated E. coli isolates from CRC and IBD patients can activate the Wnt signalling pathway both in vitro and in vivo, and provides further evidence towards mucosa-associated E. coli isolates upregulating pro-inflammatory COX-2 and PGE2 release in the gut. This suggests translational effects that might be seen in patients, which indicates a role for mucosa-associated E. coli, in the development and progression of colorectal cancer via increased Wnt signalling.
Supervisor: Campbell, B. J. ; Rhodes, J. ; Winstanley, C. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral