This study investigates overexpression of the cytoplasmic protein metallothionein (MT) as a marker of precancerous change within the murine colon by immunohistochemistry and as a method for assessing the chemopreventive potential of chemical agents. It was found that the carcinogen azoxymethane (AOM) induced crypt-restricted MT overexpression in the colon of Balb/c mice and that the putative chemopreventive agents sulindac and genistein significantly inhibited AOM-induced MT overexpression in the colonic crypts but not the incidence of aberrant crypt foci (ACF). an extensively used prognostic indicator of CRC development. In spite of this, crypt-restricted MT overexpression correlated with the incidence of ACF. MT overexpression is heritable and clonal with mutated daughter cells progressively repopulating the crypt suggesting that MT overexpression denotes crypt stem cell mutation. Further investigation using a DNA fragmentation assay and β-catenin immunohistochemistry revealed that treatment with AOM significantly reduced the incidence of apoptosis and, although not significantly higher than control animals, showed nuclear localisation of β- catenin in colonic epithelial cells. Sulindac and genistein treatment had no significant effect on the incidence of apoptosis or nuclear expression of β-catenin within the colonic crypts of AOM-exposed mice. The current study suggests that crypt-restricted MT overexpression, although not implicated in the classical pathway of colorectal tumourigenesis, has potential to be used in pre-clinical animal studies as a surrogate reporter of mutations in key genes that are undetectable at the protein expression level in early stage tumour development. MT overexpression may also prove useful in pre- clinical assessment of the chemopreventive potential of drugs and dietary agents.

Background and aims Colorectal cancer (CRC) is a serious health problem with high rate of patient mortality, mostly of tumour metastasis. Identifying new therapeutic target that could hinder metastasis initiation and progression is a high priority research topic. CD24 is a glycosyl phosphatidyl inositol (GPI) anchored membrane protein that has been identified as a marker of differentiation of hematopoietic cells. It has been found to be highly expressed in several tumours and a marker of poor prognosis. More recently, it has been proposed as a marker of cancer stem cells in a number of tumours. However, the biological functions and clinical significance of CD24 have not been fully established yet. Therefore, the aim of the current study is to thoroughly scrutinise the potential biological roles and clinical implications of CD24 in CRC. Material and Methods Following screening of 26 CRC cell lines by RT-PCR, CD24 expression levels were manipulated using a dual approach of knockdown by small interference RNA (siRNA) in cell lines with high CD24 expression and ectopic expression in cell lines with low CD24 expression. Following CD24 manipulation,. functional studies were done in vitro including cell proliferation, colony formation in soft agar, response to external apoptotic stress, cell migration, and invasion. An in vivo metastatic mouse model was also tested. In order to identify potential CD24 downstream targets, (i) a phospho-kinase array was used (and targets validated by Western blotting) and (ii) a gene expression profiling analysis was performed comparing CD24+ and CD24- CRC cells .. Finally, the immunohistochemical expression of CD24 and clinical implication were tested in cases of inflammatory bowel disease, a series of CRC (n = 462) and breast cancer (n=1036). Results CD24 expression significantly enhanced cell migration, invasion and colony formation in soft agar. However, it showed no effect on cell proliferation or resistance to external apoptotic stress. Forced CD24 expression induced an increased metastatic potential in vivo, although this did not quite reach significance (p=O.09). CD24 was shown to possibly function through the activation of AKT at residue Serine-473, and to cooperate with PI3 kinase in inducing full AKT activation. Moreover, CD24 activates focal adhesion kinase, induces cadherin switch and promotes cytoskeletal changes that favour cell migration. CD24 expression was found to be regulated mainly by Wnt signalling, and partly by STAT3. In human tissues, CD24 expression was present in the crypt bases in normal colonic but there was marked up- regulation in inflammatory bowel disease. Up-regulation was also seen in colorectal adenomas and the highest level of expression was in carcinomas. In the studied CRC series, CD24 expression had no prognostic significance and did not show a significant difference between primary CRC and their matched hepatic metastases. Interestingly, CD24 expression in breast cancer showed a trend towards identifying patients with poor prognosis, however, when combined with CD44 immunostaining, cases with CD44-CD24+ phenotype showed the worst prognostic category. Conclusions CD24 is a marker of enhanced cell motility and invasion in CRC and it can endow cells with some features of sternness. These may be important in the tissue healing in inflammatory bowel disease but may also help the development of metastasis. CD24 could provide a potential therapeutic target in early CRC, particularly if combined with therapeutic agents (as for example PI3 kinase inhibitors). CD24 could be used as a prognostic marker in breast cancer but not colorectal cancer.

There is considerable supportive evidence to suggest that increased levels of DNA damage are associated with an increased risk of developing neoplastic lesions in the human colon and rectum. Within this thesis, several different topics related to DNA damage were explored in detail, principally using the single cell gel electrophoresis assay (the comet assay) to measure DNA damage both in cell line studies and in human colorectal mucosal biopsies from patients undergoing routine endoscopic examinations of the colon and rectum.

Colorectal cancer is common with a high mortality rate despite significant advances in its management. While outcomes for rectal cancer have improved over recent years, outcomes for low rectal cancer. and colon cancer have not improved to the same degree. Studies were designed to investigate the optimal operations for both low rectal cancer and colon cancer using tissue morphometry and assessment of the surgical planes. Additionally, the utility of tumour cell density as a prognostic marker and a method of assessing the response to pre-operative treatment was investigated. Extralevator 'abdominoperineal excision for low rectal cancer was shown to be associated with better pathological outcomes and planes of surgery when compared to standard abdominoperineal excision. Careful mesocolic plane surgery for colon cancer was associated with better survival when compared to disrupted specimens. Complete mesocolic excision with central vascular ligation and Japanese D3 resection resulted in an oncologically superior specimen when compared to standard surgery. They could both be performed laparoscopically and were easily adopted by a group of highly motivated surgeons. Tumours with a low tumour cell density appeared to have an independently worse prognosis, and the same technique generated a useful measure of response to pre-operative therapy in both colon and rectal cancer. Outcomes for low rectal cancer and colon cancer could be improved by surgical education programmes backed up by pathological audit. Tumour cell density could be introduced as a new prognostic marker and could be used to define the degree of response to pre-operative treatment in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies in the western world and is the third most common cause of cancer related deaths in the UK. It has a good 5 year survival rate if the cancer is diagnosed at an early stage - 93% for Dukes' stage A; however, for late stage metastatic disease, this falls to only 7%. Improvements in treatment for metastatic disease, detection and screening are required to combat these low survival rates. Preclinical models, and in particular mouse models, are invaluable for studying the mechanisms which initiate tumour development and progression. Many models of CRC exist, including models of hereditary and sporadic CRC, including human CRC cell xenografts which support rapid tumour growth, allowing for relatively fast, short term studies and investigations into novel therapeutics. The ApcMin/+ mouse model recapitulates the early stages of human CRC development, with many polyps developing throughout the small intestine and colon. This project investigated the use of non-invasive imaging with high frequency ultrasound to provide anatomical information on the abdomen, and specifically the colon, of C57BI/6J mice, and the reproducibility of a protocol to measure the colon wall thickness in vivo. Once established, these techniques were then used to detect polyps in aged (170±34 days) and 90 day old ApcMin/+. mice, showing that this technique could be used with sensitivity and specificity of 48% and 100%, respectively. This project also investigated the feasibility of using contrast enhanced high frequency ultrasound to monitor the growth of human CRC cell xenografts and qualitatively and quantitatively assess their longitudinal vascular development in vivo. These data were then used as the basis of an intervention study, which incorporated the vascular disruptive agent Combretastatin A-4 into the CE-HFUS imaging protocols in order to assess the immediate vascular kinetic impact of CA4. This showed that CE-HFUS can detect and quantitate changes in tumour vascular kinetics in vivo following treatment with a known agent, suggesting that it may be useful in preclinical drug development. The work in this Thesis has demonstrated that C57BI/6 mouse colon wall thickness can be accurately determined in vivo, in an operator independent manner. Following on from the characterisation of colon wall thickness in wild type mice; this Thesis has shown for the first time that it is possible to identify colonic polyps in ApcMin/+ mice in vivo in both 90 day old and older age mice. Work in this Thesis has also characterised i for the first time with CE HFUS the blood flow kinetics and vascular perfusion of HCT116 CRC xenograft tumours in a longitudinal study. Building on this work, this Thesis has shown for the first time disruption in blood flow kinetics to HCT116 CRC xenograft tumours after exposure to CA4.

The infiltrative growth pattern in colorectal cancer has been demonstrated to confer a poor prognosis particularly in early stage disease. The phenotype demonstrates histological features of aggression and with reference to the contrasting pushing phenotype provides an in vivo model for invasive behaviour which has hitherto not been exploited in the context of molecular biology.

Despite advances in treatment, colorectal cancer remains a major cause of cancer related morbidity and mortality. Although the overall mortality continues to improve, a significant proportion of patients still succumb to the disease. Immunohistochemical analysis of a set of tissue microarray slides obtained from patients with colorectal cancer demonstrated elevated expression of the 0 type cyclins. Expression of cyclin 02 and 03 but not 01 at the invading margin of colon cancer was associated with vascular and lymphatic invasion and an adverse prognosis. This expression did not correlate to the expression of Ki-67 which is a marker of proliferation. In order to understand this association in vitro experiments were carried out in colorectal derived cell lines (LaVa 39 and HRT18). The functional role of the D type cyclins was evaluated by abrogation of individual transcripts using short interfering RNAs. Although a short lived reduction of proliferation was seen in both cell lines with abrogation of the cyclin 01 transcript, no such effect was noted on cell apoptosis. Significant reduction in cell invasion was noted with all three 0 type cyclins, although most notably with cyclin D2 in LaVa 39. The effect of a hypoxic microenvironment on the 0 type cyclins was evaluated to simulate the assumed metabolic conditions of the tumour margin. Prolonged hypoxia resulted in the reduced expression of all three D type cyclins. along with HIF1 and HIF2. Expression of cyclin D1 and cyclin D2 were found to be downregulated following abrogation of HIF1 and HIF2 respectively. In view of the influences of the 0 type cyclins on cell invasion and prognosis, their role in the process of epithelial mesenchymal transition (EMT) was interrogated. LlM1863 cells can be transfoll11ed from epithelial organoid form to mesenchymal like monolayers by TGF-J3, and were utilised as a model of EMT. The expression of the 0 type cyclins was increased following mesenchymal transition. No change in phenotype was noted following siRNA mediated abrogation of the 0 type cyclins in pre and post EMT celis. Unlike the effect in LoVo 39 and HRT18 cell lines, reduced 0 type cyclins had no effect on proliferation. However similar to the LoVa 39 cells, reduced 0 type cyclins were associated with a small but significant reduction in cell invasion. This led us to hypothesise that although the 0 type cyclins may not be involved in induction or maintenance of EMT, they may be a part of the EMT program and contribute to metastatic progression by facilitating cell invasion. Further work in in vivo models is required to clarify this hypothesis. However, these results indicate that the 0 type cyclins have clinical potential both as a prognostic marker and as a therapeutic target.

No description available.

Background Colorectal cancer is one of the most common cancer related causes of death in the UK. Several different pathways are known to be important in the initiation of colorectal cancer: chromosomal instability, microsatellite instability and CpG island methylation. It is unknown at what stage each of these facets becomes important, and their relative importance to each other. Aim To investigate the role and timing of genomic instability in the initiation of colorectal carcinogenesis, specifically it's role at the crypt, polyp and cancer level. Methods A variety of techniques were used to study DNA repair, microsatellite instability, methylation at the crypt level & whole genome methylation of tumours, DNA double strand break repair, tumour heterogeneity and the initiation of serrated adenomas via chromosomal instability. Results Through study of the different pathways of colorectal carcinogenesis a number of novel discoveries were made. It was found that microsatellite instability exists at a higher level than previously observed and is heterogeneously distributed in colorectal pre-malignant lesions, suggesting it's role may not be as clear-cut as previously supposed. Pre-malignant lesions were also found to be particularly heterogeneous for methylation of the DNA mismatch repair system, suggesting that this may play an important role. Study of whole genome methylation in carcinomas and adenomas as compared to normal colonic mucosa highlighted a number of pathways that may be relevant in colorectal cancer, as well as identification of a potential biomarker, GRASP. It was also found that the DNA double strand break (DSB) repair system has an important role in the progression of colorectal cancer, with tumours demonstrating faulty DSB repair having a worse prognosis. This study also demonstrated that tumour heterogeneity exists, with implications for treatment and prognosis. Finally, this study identified a gene potentially involved in the initiation of serrated adenomas, SLlT2. Conclusions This study has demonstrated the importance of genomic instability in colorectal cancer, identifying potential biomarkers and pathways involved in carcinogenesis. Further study is required based on these findings.

Colorectal cancer is the third most common cancer and the second cause of cancer death in the UK. The incidence of colorectal cancer has been shown to be decreased in populations with a high dietary fibre intake. This effect is thought be attributable in part to the cellular actions of butyrate, a short-chain fatty acid (SCF A) produced by fermentation of fibre in the human colon lumen. Butyrate has been implicated in cellular homeostasis of normal colonic mucosa and it is thought to underwrite the chemoprotective effect of fibre. Angiogenesis is essential for tumour development and is stimulated at the earliest stages of the adenomacarcinoma sequence in the colon with an increase in VEGF expression. Neuropilin-l (NRP-l) is a transmembrane receptor for VEGF and semaphorins. We demonstrate here that butyrate downregulates NRP-l and VEGF at mRNA and/or protein levels in colorectal cancer cell lines. NRP-l is a known transcriptional target of Sp 1, whose activity is regulated by butyrate. We show that NRP-l down-regulation by butyrate is associated with decreased binding affinity of Sp 1 binding elements on the NRP-l promoter. Previous studies have suggested that NRP-l expression is limited within normal tissues of the gastrointestinal tract. We show that the VEGF receptor NRP-l is lower in subjects with elevated butyrate in morphologically normal tissue distant to adenomas, but the expression is lower and not butyrate responsive in the field around adenomas. We sought to investigate whether NRP-l expression is associated with enteroendocrine cells (EEC) and how each marker corresponds to butyrate level. The EEC marker chromogranin A (CgA) positive cell number is negatively correlated with butyrate in normal tissue but this relationship is lost at the field. A colocalisation analysis reveals that only 11 % of NRP-l expressing cells are also positive for CgA. Our data suggest that NRP-l is butyrate responsive in the human colon in vitro and in vivo but the majority is not expressed in the CgA + EEC population. The number of CgA + EEC is likewise influenced by butyrate; however, field effects cause this relationship to be lost. The down-regulation of the apoptosis and angiogenesis regulator NRP-l by SCF A butyrate may contribute a novel mechanism to the chemopreventive effect of dietary fibre.