We have shown that LEF-1 protein is expressed in both HCT116 and SW480 colorectal cancer cell lines, consistent with its expression in colorectal cancer tissue, and, using PCR, have examined the expression of specific Tcf-1, Lef-1, Tcf-3 and Tcf-4 isoforms in these cells. this analysis showed that repressive Tcf-1 isoforms are expressed in colorectal cancer cells, whilst extensive alternative splicing produces a variety of Lef-1 and Tcf-4 isoforms. To assess the role that Tcf-4 and Lef-1 were playing in colorectal cancer, siRNA mediated knockdown of these genes was used in colorectal cancer cells to investigate effects on cell proliferation, cell morphology and downstream target genes. To reduce total Wnt signalling, beta-catenin siRNA was used, and the effects compared with specific Tcf-4 and Lef-1 siRNAs. Our results showed that Tcf-4 siRNA reduced cell proliferation, halting cells in G2 phase of the cell cycle, and reducing the expression of genes involved in the cell cycle. In contrast, Lef-1 siRNA hardly affected cell proliferation, but did affect cell morphology, causing cells to become larger and more spread out. Consistent with this finding, Lef-1 siRNA also reduced the expression of genes possibly involved in cell morphology, whilst Tcf-4 siRNA did not. In addition, it seems that alternative Lef/Tcf isoforms have varying effects on target genes, since siRNA that targeted specific isoforms had different effects on Wnt target gene expression. These results suggest that Lef-1 and Tcf-4 mediate different events in colorectal cancer, a difference which may be important for the progression of the cancer.

3',4',5',5,7-Pentamethoxyflavone (PMF) is a naturally occurring flavone found in the leaves of Murraya paniculata and the fruits of Neoraputia magnifica. PMF has been shown to possess potential promise as a colorectal cancer (CRC) chemopreventive agent as it significantly inhibited adenoma development in the ApCMinl+ mouse, a model of gastrointestinal cancer. The chemopreventive activity of PMF was superior to that of two closely related flavone analogues apigenin and tricin. The aim of the work described in this thesis was to elucidate the mechanisms by which PMF can interfere with carcinogenesis. PMF was compared to tricin and apigenin in its abilities to inhibit growth, arrest cell cycle and elicit apoptosis in APC 1 0.1 cells, derived from ApCMinl+ mice. Cells were incubated with these agents (10 IlM, 24 h) and cDNA microarray analysis was performed to delineate changes in gene expression caused by these flavones. Gene changes were validated using reverse transcriptase-PCR and Western blot. PMF was administered to ApCMinl+ mice to assess its effects on protein expression in adenomas. PMF was the most growth-inhibitory of the three flavones with an ICso of 5 IlM. It arrested the cell cycle at G, and G2 phases and induced a two-fold increase in apoptosis. In the microarray study PMF modulated several key signalling pathways, most notably those involving Wnt, PI3KJAktlGSK3p and JAKJSTAT. In cells in vitro PMF significantly altered expression of Wnt8b, Wnt3a, TCF4, p-catenin, GSK3p, survivin, pSTAT3 and MCM7. In mice in vivo PMF, at a dietary dose which significantly reduced adenoma development, altered only p-catenin and MCM7 protein levels. These differences were only observed in the adenomas and not the normal mucosa. GSK3 p, p- Catenin and MCM7 may play a role in the CRC chemopreventive activity of PMF.

Colorectal cancer (CRC) is one of the commonest malignancies in the United Kingdom and tumour cell invasion and metastasis is the main cause of death. The transcriptional repressor ZEB1 has been shown to be expressed in several epithelial malignancies and embryonic epithelial-mesenchymal transitions (EMT). The understanding of cellular signalling cascades should allow the discovery of novel targets for potential future therapeutic manipulation in the treatment of colorectal cancer and other malignancies. Here, in order to further investigate the role of ZEB1 in CRC, in vitro investigations and immunohistochemical analysis of 101 colorectal cancers and their matched lymph node and liver metastases are performed. The Wnt-Inducible Signalling Proteins and Plakophilin-3 are also investigated for their relationship with ZEB1 signalling. ZEB1 is expressed in tumour cell lines with mesenchymal characteristics and over-expression in an epithelial-phenotype cancer cell line causes down-regulation of epithelial markers such as E-cadherin and Plakophilin-3. Knockdown of ZEB1 in mesenchymal cell lines caused up-regulation of PKP-3 expression. In vitro attempts at manipulation of WISP expression were unsuccessful. In CRC tumours ZEB1 was noted to be up-regulated at the tumour invasive front, with concomitant loss of Plakophilin-3 expression. However expression of these markers did not correlate with disease stage or survival on multivariable analysis. Increased WISP-1 nuclear and cytoplasmic expression were noted at the tumour invasive front and correlated with disease stage and several pathological factors on univariable analysis. Increased nuclear WISP-1 expression at the invasive front correlated with survival on uni- and multivariable analysis. This study demonstrates that ZEB1, Plakophilin-3 and WISP-1 are expressed in CRC and may be involved in tumour cell invasion and dissemination at the invasive front. Expression of nuclear WISP-1 is an independent predictor of poor survival and is worthy of further investigation as a target of future therapeutic intervention.

Introduction: Around 20-30% of the population are thought to have some form of inherited predisposition to colorectal cancer outside of the genetic syndromes of familial adenomatous polyposis (FAP), MYH-associated polyposis (MAP) and hereditary non-polyposis colorectal cancer (HNPCC). Inherited susceptibility should be particularly suspected when colorectal cancer is diagnosed at young age, when a patient presents with synchronous or metachronous colorectal cancers or adenomas, or where there is a strong family history of colorectal cancer. The rare variant hypothesis of inherited susceptibility proposes that a number of low frequency variants in a variety of different genes, each conferring a moderate but detectable increase in relative risk of developing disease, may be responsible for non- syndromic predisposition to colorectal cancer. Rare variants may occur in many genes involved in colorectal tumorigenesis, including those with roles in Wnt signalling, transcriptional activation, mismatch repair, cell cycle regulatory mechanisms and cellular adhesion. Methods: DNA from 124 United Kingdom patients with between 3 and 100 adenomatous polyps was screened for germline variants in genes involved in Wnt signalling (APC, AXINI and CTNNBI), mismatch repair (hMLHI and hMSH2) and cell cycling (TP53). The APC variants II307K and EJ317Q were detected using amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) and alkaline-mediated differential interaction (AMDI). For all other genes, PCR primers were designed to encompass the entire coding sequence of the gene and variant detection was carried out on the Transgenomic Wave" machine. Variants were sequenced and analysed using Sequencher" software. The findings in the sample population were compared with a population of 53 Korean patients with multiple adenomas, and with a panel of 483 healthy controls. Results: 30/124 (24.9%) of the U.K. multiple adenoma patients carried potentially pathogenic germline variants in the genes tested as compared to 55 (12%) of the controls. The overall association between the rare alleles at the loci tested and the formation of multiple adenomas, as compared to controls, was highly significant with an odds ratio of 2.2 (p = 0.0001). None of the variants identified in the U.K. patients was found in the Korean patients. Discussion: The difference in rare variants between cases and controls is highly significant, suggesting that many rare variants collectively contribute to inherited susceptibility to colorectal adenomas. Each variant would effect a subtle change in protein interaction or level of gene expression, resulting in only a marginal selective disadvantage but a clearly defined increase in relative risk. Such variants are likely to be population-specific, as in the cases of APC 1307K and E1317Q. Strategies for detection of multiple rare alleles include efficient variant detection and sequencing techniques in at-risk individuals, identification of candidate genes, large-scale population studies, careful selection of controls, and targeted statistical approaches. Each potential rare variant needs to be assessed for its functional consequences. These findings give support to the hypothesis that multiple rare alleles, predominantly missense, promoter and splice site variants, are collectively responsible for inherited susceptibility to colorectal cancer in the general population. It is probable that ultimately a large number of rare alleles will contribute more to the population burden of colorectal cancer than the classically inherited Mendelian syndromes associated with colorectal tumorigenesis.

Beta-galactoside binding protein (p-GBP) is a negative cell cycle regulator and an antiproliferative cytokine. The aim of this thesis is to define the molecular mechanisms whereby f3-GBP induces apoptosis in colon cancer cells. Three colon cancer cell lines were selected: SW480, derived from a primary tumour of the SW620 a metastatic derivative of SW 480 and Lo Vo a very aggressive drug resistant cell line. The results presented in this thesis demonstrate that f3-GBP can induce an early and complete inhibition of DNA synthesis and S phase arrest, paralleled with growth inhibition, which is followed by apoptosis in the p53 defective SW480 and SW620 colon cancer cells as well as in the p53 wild type Lo Vo cells. The molecular events leading to this response were examined by investigating cell cycle regulatory machinery and survival pathways. The observations as a consequence of treatment f3-GBP are following: cyclin E was persistently expressed and cyclin A was downregulated; E2F! transcription factor was persistently expressed before cell underwent apoptosis; phosphorylation of Chk2 was increased at an early stage before apoptosis occurred. f3-GBP was found to be an inhibitor of PI3K activity, however the downstream target Akt was found not to be decreased before the induction of apoptosis. Both ERK levels and Akt levels increased before both signallings were quenched as cells entered apoptosis. These findings suggest that f3-GBP can coordinate the cell cycle regulatory and different survival mechanism to arrest growth and induce apoptosis. The persistent expression of cyclin E and downregulation of cyc1in A may contribute to S phase arrest and apoptosis. The inhibition of DNA synthesis and activation ofERK may activate DNA checkpoints. The checkpoint kinase may playa critical role as f3-GBP induced activation of Chk2 could result in persistent expression of E2F! and therefore lead to p53-independent apoptosis. The ectopically expressed E2F! in S phase may playa crucial role because of the inherently different levels of E2F! between cancer cells and normal cells. The selective induction of apoptosis may be due to the integrated response of these mechanisms.

No description available.

Mismatch repair is a vital DNA repair mechanism whose absence leads to a tolerance towards mutations and a predisposition to colon cancer. MLHl is one of the main proteins involved and is highly conserved from E. coli to human. It not only plays a role in repair, but can signal the cell to die if damage levels become too high. The mechanism by which MLHl triggers cell death in response to damage is not entirely clear, and is likely to differ between normal and cancerous cells. Previous work in the Walsh lab had generated MLH1-depleted subclones of a telomerase- immortalised normal human fibroblast cell line. These had been generated by transfecting the parental line hTERT-1604 with an shRNA vector against MLHl and selecting subclones which had reduction in MLHl to various degrees. I further characterised these MLHl knockdown cell lines and revealed that they also exhibit resistance to the methylating agent N-Methyl-N-Nitrosourea. Through the use of various assays we were able to determine that the hTERT immortalised cells did not undergo cell cycle arrest, apoptosis or senescence in response to MNU as colon cancer cell do. Instead, they undergo an MLH1-dependant form of programmed cell death mediated by PARP, but independent of caspase, P53 and ATM/ATR. In 2004, DNMTl deficiency was also implicated in causing MMR defects in mouse embryonic stem cells without a causal mechanism being identified. The effects of DNMTl deficiency are not the same in stem cells and differentiated cells. To determine if depletion of DNMTl can also cause MMR defects in normal human cells, I created DNMT1-depleted hTERT-1604 cells using the same shRNA-mediated strategy as above. Subsequent characterisation of the DNMT1-depleted subclones established that they have a reduction in DNA methylation and the most severely reduced cells are arrested at the G2/M checkpoint. Subclones with significant reductions in DNMT1 also exhibited a decrease in MLH1 expression at the protein, but not the mRNA level. This reduction in MLH1 expression was reversed when a protease inhibitor was employed. The subclone with 31% DNMT1 expression exhibited microsatellite instability, providing further evidence for an interaction between mismatch repair and DNMT1 in human cells and suggesting a mechanism by which this may occur. In conclusion, the work presented here demonstrates a novel role for the mismatch repair protein MLH1 in triggering PARP-dependent cell death in response to damage by MNU. It also shows a link between DNMT1 depletion and MMR deficiency through destabilisation of MLH1.

Every day each cell in the body is under threat from DNA damaging agents that have the potential to disrupt the passing of intact genetic information from one generation to the next. The presence of a wide variety of threats has led to the evolution of numerous DNA damage response pathways in order to deal with the damage that they may cause. The mismatch repair system is primarily responsible for the removal of incorrect base insertions and deletions occurring during replication and its importance is highlighted by its conservation from bacteria to humans. MLHl is one of the main components of this repair pathway and loss of this protein has been associated with a number of cancers, particularly hereditary colon cancer. Defects in the mismatch repair machinery have also been associated with resistance to a number of chemotherapeutic drugs and instability of the genome at repeat sequences. The present data initially examines the role of MLHl in DNA damage responses following induction of damage by a number of different treatments in telomerase- immortalised human fibroblasts stably depleted of MLHl using an integrated shRNA plasmid. The importance of a number of specific pathways, namely ATM/ATR, caspase, p53 and PARP, involved in the damage response is assessed through the use of inhibitors. A number of studies have shown that loss of the DNA methyltransferase maintenance protein DNMTl initiates p53-dependent cell death in differentiated cells. While the absence of DNMTl is tolerated in undifferentiated embryonic stem cells in terms of viability, these cells exhibit the hallmarks of mismatch repair deficiency, i.e. drug resistance and genomic instability, although transcription levels of the mismatch genes remains unaffected. Depletion of DNMTl in the colon cancer cell line RT29 resulted in induction of rapid cell death which could be ablated through the inhibition of not just p53 but also P ARP. Analysis of the mismatch repair components showed that although transcription levels were not affected, a reduction in DNMTl resulted in the depletion of a number of DNA repair proteins, suggesting that DNMTl is a key protein involved not only in DNA methylation, but also in the stability of the mismatch repair system.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Due to its pattern of increased incidence and mortality rates in Western countries, lifestyle factors, particularly diet, have been implicated in the aetiology of this disease. Recently, interest in the consumption of polyphenols, especially abundant in berries, has received a great deal of attention due to the putative anticancer activity of these compounds. In vivo, polyphenols undergo digestive conditions post consumption that alters the structure and possibly the function of these compounds. The focus of this study was firstly to produce berry extracts which had undergone in vitro simulated gastric and pancreatic digestion followed by fermentation of this colon-available extract with human faecal innocula. Secondly, in order to evaluate the putative anticancer activity of these extracts in vitro, their effect on models of colorectal cancer representing the key stages of initiation, promotion and invasion were investigated. Whole berry extracts and isolated berry components have previously been shown to modulate cellular functions involved in carcinogenesis such as cell cycle and proliferation in in vitro models of key stages of colon cancer. In the present study the bioactivity of digested and fermented extracts was demonstrated at a physiologically relevant dose range. Colon-available and fermented berry extracts significantly reduced hydrogen peroxide-induced DNA damage and relative mutation frequency in HT29 and HT29 (G 17 neo) cells respectively at non-toxic concentrations (0 - 50 ug/ml GAE). The extracts also inhibited invasion of HTl15 cells independent from migration. There was limited difference in the magnitude of effect between berry types; the exception was that only colon-available lingonberry extract was able to modulate proliferation and cell cycle progression ofHT29 cells at 50 ug/ml GAE. Also only a slight decrease in bioactivity was observed after in vitro fermentation. Due to the effectiveness of colon-available lingonberry extract in modulating several models of stages of colon cancer in HT29 cells, its effect on gene and protein expression was evaluated. These results were suggestive that this extract modulated the biological activity of cells in in vitro models of colorectal cancer through inducing transcription of genes responsible to halt the cell cycle and those involved in DNA damage repair. Preliminary work of an exploratory nature revealed that colon-available lingonberry extract caused changes in the metabolome of HT29 cells and may be of importance in modulating bioactivity of the cell although the individual metabolites were not identified. Overall this work has illustrated that colon-available and fermented berry extracts retain their biological activity in in vitro models of colorectal cancer, despite modification to their structures. The fact that breakdown products and metabolites can modulate cellular functions associated with colon cancer, similar to the parent compounds, may aid in the understanding of the mechanisms involved in modulating the pathophysiology of colorectal cancer.

The discovery of underlying mechanisms of drug resistance and the development of novel agents to target these pathways is a priority for patients with advanced colorectal cancer (CRC). The aim of this study was to identify novel targets whose knock-down is important in mediating sensitivity to 5-FU and oxaliplatin in Kras wild type and mutant CRC models. Materials and Methods. Transcriptional profiling (Almac Diagnostics Colorectal Cancer Disease Specific Array'") of pre-treatment metastatic CRC liver biopsies and oxaliplatin/5-FU resistant HCTl16 cell lines followed by Pathway Analysis and Gene Set Enrichment Analysis (GSEA) were used to identify individual genes from novel drug-sensitivity pathways for incorporation into a RNAi screen. Results. We identified panels of genes whose expression is altered (acutely and basally) between sensitive and 5-FU- or oxaliplatin-resistant models. The significant pathways involved in 5-FU/oxaliplatin resistance included Cell Cycle, Focal Adhesion, Insulin and MAPK signalling. In the MAPK pathway, we found that FGFR4 silencing potently increased apoptosis in Kras wild type and mutant CRC cells, and this was further enhanced when FGFR4 siRNA was combined with 5-FU or oxaliplatin. FGFR4 inhibition completely inhibited migration of Kras mutant HCTl16 cells and we found that FGFR4 silencing resulted in strong inhibition of ST A T3 activity in Kras mutant, but not Kras wild type, CRC cells. Conclusions. This study demonstrates the utility of microarray expression data, obtained from pre-clinical and clinical samples, and analyzed by pathway and Gene Set Enrichment Analysis to identify pathways of oxaliplatin/5-FU sensitivity in CRC. In addition FGFR4 inhibition in combination with 5-FU or oxaliplatin could represent a novel treatment strategy for Kras mutant and wild type CRC tumours. We are currently investigating FGFR4 small molecule inhibitors in preclinical in vitro and in vivo models.