Duodenal ulceration remains a significant problem. Helicobacter pylori infection is the major cause of duodenal ulcers (DU). Worldwide, around 1 in 2 people are chronically colonised by H. pylori, most of whom will not develop gastroduodenal disease, though some will develop DU whilst others will develop gastric ulcer or gastric carcinoma. The risk of disease is related to bacterial virulence factors, host and environmental factors. Disease-specific bacterial risk factors have not been established. Duodenal ulcer promoting gene (dupA) was proposed as a disease-specific bacterial virulence factor for DU. This study aims to examine the influence of dupA on clinical outcomes and whether these are epidemiologically consistent, if the dupA status of clinical isolates correlates with the key pathogenic features of DU in vivo, and if there is a biologically plausible role in disease through effects on host immune responses. Results showed that the influence of dupA status on clinical outcomes was not specific to DU, but rather that epidemiological associations link dupA with an increased risk of gastroduodenal diseases in general for some populations. The dupA status of clinical isolates did not correlate with the key pathogenic features of DU in vivo, though there was some evidence of increased gastric mucosal inflammation in association with dupA+ strains. Experimental results using a human blood immune cell model show that monocyte-derived cells mediate a more pro-inflammatory response through interaction with the dupA system. This might be the mechanism that explains the in vivo associations of dupA with inflammation and disease. In this study population, dupA did not satisfy the criteria of a true virulence factor that promotes duodenal ulceration. The assertion of this thesis is that dupA is misnamed.

Background: Colorectal cancer is a major global public health problem that has predominantly been considered a genetic disease following a precise series of molecular events. These are characterized by sequential accumulation of genetic and epigenetic alteration in several oncogenes and tumour suppressor genes. Understanding of the genetic mechanisms that explain the initiation and evolution of colorectal cancer are key to improving risk prediction, prognostication and treatment. Aims: The aim of this study was to understand the basic principles of the molecular biology of colorectal cancer based on genomic, transcriptomic, and proteomic profiles analyses. Methods: DNA extracted from 147 formalin fixed paraffin embedded (FFPE) samples from 83 patients with colorectal cancer (first cohort) including (83 primary colorectal cancer (CRC), 22 matched liver metastases, 25 matched biopsies and 17 normal colon tissue) were screened for mutation in 26 genes (Trusight tumour kit) using Targeted Next Generation Sequencing. Additionally, exonuclease domain region of POLE and POLD1 were also screened using High Resolution Melting and Sanger Sequencing methods. These data used to: Investigate mutation profiles of CRCs among 83 primary samples. Investigate the difference between chromosomal instable-CRC (46 primary sample) and chromosomal and microsatellite stable-CRC (35 primary sample). Compare mutations in 26 genes of 25 paired biopsy samples and corresponding resection specimens. Investigate genetic discrepancies between 22 primary colorectal cancers and their respective metastases. Additionally, expression of a panel of six miRNAs (miR-20a, miR-21, miR-29a, miR-31, miR-92a and miR-224) was measured using RT-qPCR and protein expression of 20 genes was measured using Reverse Phase Protein Array (RPPA). In a second cohort including 81 primary CRC and their matched normal samples, expression of the six miRNAs and mRNA of six genes (SMAD4, PTEN, BCL2, TGFBRII, KLF4 and RASA1) targeted by the six miRNAs were measured using RT-qPCR. Additionally, expression of proteins of the targeted six genes was also measured using immunohistochemistry (IHC). Cell-free DNA (cfDNA) extracted from 16 blood samples (third cohort), which were taken from 5 CRC patients at different time points (pre- and post-surgery) were screened for mutations in KRAS, TP53, PTEN, SMAD4, BRAF and PIK3CA genes. Additionally, expression of the six miRNAs was measured using RTqPCR. Results: In the first part; investigating mutation profile of the first cohort 83 CRC showed high frequency of mutation in TP53 (75%), APC (57%) and KRAS (53%). Approximately 93% CRCs have mutation in at least one of APC/TP53/KRAS/BRAF/SMAD4/PIK3CA/PTEN/FBXW7 genes. Moreover, mutations were found in the exonuclease domain regions of POLE in 9.6% and POLD1 in 2.4%. Regarding biopsy vs resection, the mutant allele frequency was 1.03-fold higher in resection specimens than biopsies and there was no mutation in the biopsy specimens that were not seen in the resection specimens. In the second part; Comparison of CIN-CRC vs MACS-CRC, which were included in the first cohort CRCs showed similar mutation frequencies of mutation in all 28 genes except KRAS (41%CIN vs 68%MACS), POLE (15%CIN vs 2%MACS), GNAS (0%CIN vs 11%MACS). Statistically there was a significant difference (each p=0.01) which was lost following multiple testing correction. In the third part; comparison of primary CRC vs matched metastasis showed that a total of 61 non-synonymous somatic variations in 12 genes were found in primary 22 specimens whereas 60 were found in metastasis cases. The mutant allele frequency was 1.01-fold higher in primary than metastasis CRCs. Evaluated expression levels of six miRNAs and protein expression of other 20 genes, did not show any significant differences between primary CRC and matched metastasis. In the fourth part; Expression of the six miRNAs and mRNA and protein of the six targeted genes were tested in the second cohort 81 samples. Statistical analysis revealed significant increase in the expression level of miR-20a (p=0.04), miR-21 (p=0.01) miR-29a (p=0.03) and miR-31 (p=0.01) and decrease in the mRNA expression level of TGFBRII and RASA1 in tumour samples compared to normal tissues. IHC staining showed low expression level of SMAD4 in 51 (63%), PTEN 67 (83%), TGFBRII 65 (80%), RASA1 61 (75%) BCL2 47 (58%) and high expression of KLF4 36 (44%). High miR-21 and miR-224 expression were associated with low expression of TGFBRII. In addition, over expression of both miR-29a and miR-31 inversely correlated with RASA1. In the fifth part; Mutation in the cfDNA was detected in 5 cases. Two of these showed a loss of the mutant signal post-operatively. Whereas the mutant signal was persistent in the rest 3 of the cases for all the samples taken post-operatively. Although miRNAs expression was fluctuated between these time points, paired test showed a non-significant difference when comparing pre- and post-surgical miRNAs level. However, level of the cfmiRNAs is changed by more than 2 folds (upregulated) in the day of surgery compared to normal plasma as follow, miR- 20a in 1/5 (20%), miR-21 in 4/5 (80%), miR-29a in 3/5 (60%), miR-92a in 4/5 (80%) and miR-224 in 2/5 (40%). Conclusion: Investigation profiles of CRCs from both cohorts indicated that, different mutated genes and upregulated miRNAs, which are involved in different signalling pathways, may have roles in CRC carcinogenesis. Significant difference was neither noticed between MACS and CIN group and nor between primary and metastasis tumour. miRNAs from tissues and cfmiRNAs from plasma, can differentiate CRC from healthy group and have potential clinical value in early CRC detection. In addition to the resection specimens, the study found that it is acceptable to use biopsy material for predictive testing and cfNAs can be used for a variety of clinical and investigational applications.

Human colorectal cancer (CRC) is the fourth most common cause of cancer-related death in the UK and worldwide. Defects in conserved signalling pathways play key roles in the development of almost all cancers and, in CRCs, over 80% of tumours show hyper-activation of the canonical Wnt signalling pathway. This pathway, through the transcriptional activity of β-catenin (and its binding partner, TCF4), maintains the stem cell compartment of colon and intestinal-crypts as well as cancer-initiating cells. Whilst the role of β-catenin/TCF4 in the development of both normal and neoplastic colon/intestinal tissues is well documented, the molecular basis of these functionally distinct nuclear-transcriptional programs is still under investigation, hence the functional elucidation of nuclear cofactors that interact with nuclear β-catenin contribute to further unravel the mechanisms involved in the β-catenin-mediated nuclear transcription. In addition to LEF/TCFs, interaction of β-catenin with a plethora of other transcriptional co-activators and/or co-repressors remains vital for gene regulation. To this end, our lab have been dedicated to identifying -catenin/TCF4 interacting partner proteins (CIPs) capable of fine-tuning the Wnt-level in CRC cells. Among other CIPs, my proposed project was focused on FLYWCH1, a totally novel protein with a FLYWCH/Zn-finger DNA-binding domain, called “FLYWCH”. Previous data in our lab demonstrated that FLYWCH1 preferentially binds the nuclear/ un-phosphorylated--catenin whilst -catenin is still bound to TCF4 (Muhammed et al., submitted). Muhammed et al., found that FLYWCH1 is able to modulate transcription of many -catenin target genes including the stem cell marker (Lgr5) and genes that are associated with migration and invasion of CRC cells. They also showed that FLYWCH1 mRNA expression is restricted to a subpopulation of tumour cells in both human CRCs and ApcMin model mouse for intestinal cancer via in-situ hybridization (ISH). However, prior to these almost nothing was known about the FLYWCH1. In my research project it was proposed, to build on these advances in FLYWCH1，Wnt and CRC, and to undertake a cell & molecular research program on the role of the FLYWCH1-transcription regulator in potential suppression of colon cancer via direct regulation of microRNAs. However, commercially available FLYWCH1-antibodies worked endogenously only for immunocytochemistry/immunofluorescent (IF), but not for immunohisto-chemistry (IHC) and Western blotting analysis. Here we provide evidences via FLYWCH1 overexpression and shRNA knockdown in cultured fibroblast (TIG119) and CRC cell lines that FLYWCH1 possess tumour suppressor functions mainly by; i) Inhibition of cell migration via modulating actin cytoskeleton re-modelling and stress fibre formation and by targeting E-cadherin suppressor ZEB1. ii) Inhibiting cancer stemness in-vitro (colonosphere assays), by modulating the Wnt/β-catenin signalling pathway and possibly in a GSK-3β dependent manner. iii) The localization of FLYWCH1 speckled nuclear with splicing factor SC-35 foci, a potential mechanism involved in regulation of miRNA expression. iv) Positively regulating the expression of let-7 miRNA expression via modulating the LIN28A and LIN28B subcellular distribution. We also showed that FLYWCH1 expression is correlated positively with let-7 miRNA expression in primary colorectal cancer samples and matched metastases from patients. While we are currently striving for obtaining substantial knowledge about FLYWCH1 function in-vivo and mechanistic insight into the regulatory circuitry of FLYWCH1/miRNAs, collectively, our data suggest that FLYWCH1 possesses tumour suppressor activity and may exert its influence on cancer cells homeostasis through miRNA regulation.

NANOG is a transcription factor that functions as a central regulator of pluripotency and determines the cell fate in embryonic stem cells (ESCs). Dr Nateri’s laboratory and others have reported the expression of embryonic NANOG in a small subpopulation of cells in several human cancers including human colorectal cancer (CRC). NANOG positive CRC cells express the normal intestinal stem cell marker LGR5, as well as the induced pluripotent stem (iPS)-linked transcription factors SOX2, c-MYC, and high β-catenin. These cells strongly resemble a small subpopulation of poorly differentiated cancer stem cells (CSCs). Recent studies have reported that, in CSCs, the self-renewal and survival signals are dominant over the differentiation signals. This suggests that NANOG is an essential modulator of drug resistance complexity and tumour heterogeneity in cancer cells. Dedifferentiation is an established hallmark of carcinogenesis and is accompanied by key signalling pathways mediated in drug resistance, such as the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase-3β (GSK-3β)/β-catenin pathways, via epithelial–mesenchymal transition (EMT). However, the gain of stemness and the concomitant loss of differentiation might affect and alter the signalling pathways exclusive to NANOG-expressing cells that are required for drug resistance, not fully studied. Therefore, we aimed to characterise the mechanism by which CSCs gain self-renewal ability and lose differentiation ability through NANOG. Hence, this study was initially focused on testing the role of NANOG in chemotherapy drug resistance in human CRC cells, using 5-fluorouracil (5-FU)–treated HCT116 cells stably overexpressing exogenous human NANOG (HCT116GFP/NANOG) and control GFP expressing cells (HCT116GFP). We show that NANOG overexpression promotes 5-FU resistance in HCT116GFP/NANOG cells versus HCT116GFP cells (Chapter 3). To define the possible downstream regulatory pathways directly or indirectly mediated by NANOG, we examined the MAPKs; JNK (Jun-N-terminal kinase), ERK1/2 (extracellular signal–regulated kinase) and Wnt/β-catenin signalling pathways through GSK-3β association with β-catenin in HCT116GFP/NANOG cells versus HCT116GFP cells, and if NANOG mediated in activation of the EMT (epithelial–mesenchymal transition). Our data show that overexpression of NANOG protein in the CRC cells mimics previously reported ESC differentiation mechanism mediated by induction of the phosphorylated ERK1/2 (p-ERK1/2) expression and phosphorylated GSK-3β (p-GSK-3β) at Ser9 (Chapter 3). Consequently, NANOG overexpression increases the β-catenin and represses the E-cadherin, while significantly increases the vimentin level, leading to EMT activation (Chapter 4). In this study, we have also demonstrated NANOG induction of the EMT signature, results increasing activity of symmetrical cell division, while reducing differentiation of HCT116 derived colonosphere formation (Chapter 5). Taken together, this study describes the mechanisms of NANOG induction of EMT and NANOG sustainment of CSC-like traits via activation of the ERK/GSK-3β/β-catenin pathways in CRC cells. These findings highlight the specific mechanism of action of the NANOG-CSC signalling pathways in human CRC tumour heterogeneity, in which it might eventually identify promising approaches to cancer treatment via selectively targeting of CSCs.

Cten (also known as Tensin4) is the fourth member of the Tensin gene family. It lacks the N terminal actin binding domain while retaining the C terminal SH2 and PTB domains. This helps to bind Cten to the intracytoplasmic tail of β1 integrin and puts it at the heart of focal adhesions. It is reported to be a tumour suppressor in kidney and prostate cancer where normal tissues show high expression. However in a number of tumours, including colorectal cancer, Cten has been labelled as an oncogene. Cten which normally is a cytoplasmic protein gives nuclear staining in colorectal metastatic deposits. It increases motility, invasion and colony formation in colorectal cancer cells. In this study we have tried toexplore the mechanism of functional activity and regulation of Cten. We looked at Cten in the nucleus in vitro and identified new downstream binding targets. In addition we investigated the role of the SH2 domain of Cten concentrating on its downstream signalling molecules and binding partners. Furthermore, we explored regulators of Cten. In this study we have forced nuclear localisation of Cten by tagging it with a nuclear localisation sequence (NLS) and found a significant increase in cell motility. In order to investigate the SH2 domain we used site directed mutagenesis to change potentially important amino acids namely Arginine at 474 to Alanine (R474A), which is important for binding tyrosine phosphorylated proteins. Moreover, we displayed that Cten underwent tyrosine phosphorylation and additionally changed three tyrosine residues i.e. Y449F, Y479F and Y530F via site directed mutagenesis. We found R474 and Y479 to be important in regulating cell motility and that known downstream targets such as ILK and FAK are dependent on an intact SH2 domain. Furthermore we have identified Cten to be physically bound to FAK in the cytoplasm and nucleus and new downstream targets identified such as Src and Paxillin. Regarding possible regulators of Cten, we found that Cten might be a possible substrate for calpain. Another regulator considered was CD24 due to its role in movement of integrins into lipid rafts and we found it was a positive regulator of Cten. In conclusion localisation of Cten into the nucleus causes an augmentation of its motility enhancing functions. Cten regulates cell motility via its SH2 domain. Arginine 474 and Tyrosine 479 are important for its function. Cten regulates levels of ILK, FAK, Src and Paxillin through its SH2 domain and binds to FAK in both cytoplasm and nucleus. Calpain and CD24 were found to possible regulators of Cten in colorectal cancer. Future studies are needed to define its role in signalling at focal adhesions and these studies should be validated in other cancer cell models as well to establish Cten as regulator of cell motility in cancer.

Cystic fibrosis (CF) is one of the commonest life-limiting genetic disorders in the Caucasian population. Management involves frequent administration of antibiotics including aminoglycosides. With improving survival, it is time to focus on various age-related and treatment-associated adverse influences. The objective of this research was to evaluate renal function in CF, determine the effects of cumulative antibiotic exposure and to identify ways to reduce associated comorbidity. A cross-sectional study showed that a small number of adults and children with CF had low glomerular filtration rate (GFR), and there was no association between GFR and cumulative antibiotic exposure. An above normal GFR was identified in one in four children with CF. Estimated GFR calculated by creatinine-based equations did not accurately predict the GFR measured by the gold standard 51Cr-EDTA (51chromium-ethylenediamine tetraacetic Acid). Pure tone audiograms identified a raised hearing threshold in one in four people with CF, which did not correlate with increasing aminoglycoside exposure. A randomised controlled study established that there is no difference in the pharmacokinetics of tobramycin when administered intravenously in the morning or evening. A Cochrane systematic review concluded that there was insufficient evidence to support a routine use of bronchoalveolar lavage in the management of pulmonary infections with Pseudomonas aeruginosa in children with CF below 5 years old. CF gene (Cystic Fibrosis Transmembrane Conductance Regulator, CFTR) is expressed in pig kidneys. Histological and molecular experiments established that there is no difference between the newborn pigs with genotypes CFTR -/- (knockout) and CFTR +/- (heterozygous) or CFTR +/+ (wild-type) pig kidneys in the renal morphology and in the expression of various renal endocytic receptor proteins. The vascular haemodynamic parameter, augmentation index ascertained in a small group of children with CF suggests a possibility that the vascular age may be advanced in people with CF right from their childhood. In summary, these studies have established a low prevalence of renal disease in CF and a lack of association between cumulative antibiotic exposure and GFR. Further research is needed to evaluate the natural history of high GFR in paediatric CF population. Kidneys from pig model of CF may provide an alternative model to investigate the renal disease in CF.

CD24 is a small (81 amino acids) GPI anchored protein which is involved in promoting cell motility and stemness and may be a part of the metastatic process. It is a heavily glycosylated molecule and contains numerous O-glycosylation sites together with two N-glycosylation sites. N-glycosylation is thought to be important in protein function, and therefore, the aim of this study is to (a) investigate the importance of N-glycosylation in the function of CD24, (b) identify other potentially functional sites in CD24 by deletion mapping, (c) define downstream targets of CD24, and (d) identify the extrinsic signals of which activate CD24. (a) Through site-directed mutagenesis, we changed the glycosylated residues N32 (ACC to CAA) and Q52 (AAT to CAG) in CD24. Mutating each of these sites individually, when compared to pCCD24WT (wild-type CD24), caused a partial reduction in ability to induce cell motility and cell invasion (cell motility p=0.0001 cell invasion p=0.0001) and, unexpectedly, resulted in significantly enhanced cell proliferation (p=0.0001). Mutation of both sites resulted in a near loss of motility induction and retained cell proliferation. (b) We mapped the functional sites of CD24 by deleting seven amino acid segments of the whole of the mature peptide. Apart from the N-glycosylation sites, no other functional domains were identified which altered cell motility or proliferation. (c) Previously, in our lab it has been shown that Cten is downstream motility-inducing target of CD24. We hypothesised that CD24 may signal through the Notch pathway since Notch1 has an important role in maintaining CSCs. Results showed that forced expression of CD24 upregulates Notch1 and Cten whilst knockdown of CD24 causes loss of Notch1 and Cten expression. However, forced expression of CD24 with simultaneous knockdown of Notch1 resulted in failure to induce Cten. (d) CD24 is reported to act as a ligand of P-selectin. We found that stimulating CD24 expressing cell lines induced with P-selectin induced cell motility (p=0.0011) and caused an increased in the protein expression of downstream targets of CD24. Stimulating cell lines expressing CD24 with mutant glycosylation sites resulted in a failure to induce motility or CD24 targets. We conclude, the removal of the N-glycosylation sites in CD24 resulted in a loss of cell migration and invasion, thereby suggesting the importance of these sites in mediating the migration and invasion functions of CD24. Unexpectedly, these mutations also appeared to stimulate cell proliferation, suggesting that wild type CD24 can functionally inhibit cell proliferation. Deletion mapping did not reveal any other functional sites on the mature CD24 suggesting that O-glycosylation is relatively affecting the glycosylation in the biology of CD24. Notch1 was to be an important downstream target of CD24 and a regulator of Cten. The binding of P-selectin with CD24 resulted in increased motility of CD24 which is also dependent on N-glycosylation.

In health, the gut provides a barrier between the external and internal environment. This selectively permeable barrier allows absorption of nutrients and water from the gastrointestinal contents, whilst preventing the transfer of noxious material such as bacteria. During episodes of inflammation, this barrier becomes compromised, allowing transfer of noxious material into the systemic circulation, leading to disease states such as inflammatory bowel disease and septic shock. There are no clinically available compounds to combat this increase in permeability directly. The endocannabinoid system is a group of endogenous lipid signalling molecules which activate membrane-bound receptors. Plant-derived and synthetic compounds also act at these receptors, generating a wide variety of secondary effects. The aims of this study were to identify compounds with action on the endocannabinoid system which could be used clinically to treat inflammation and hyperpermeability of the gastrointestinal tract, exploring mechanisms of action. Systematic review and meta-analysis of existing literature revealed 51 preclinical studies, and 2 clinical studies examining the effect of cannabinoid compounds. In preclinical studies, cannabinoid drugs reduced myeloperoxidase activity in the gastrointestinal mucosa within mouse and rodent models of colitis (standard mean difference -1.26, 95% confidence interval (CI)-1.54 to -0.97, I2=48.1%) and macroscopic disease activity scores (standard mean difference -1.36, 95% CI -1.62 to -1.09, I2=61%). Clinical trials found no overall benefit of cannabinoid drugs in Crohn’s disease (mean difference -74.97, 95% CI –229 to 0.79, I2=75%). Two compounds, cannabidiol and palmitoylethanolamide, possessing positive outcomes and preferable side effect profiles, were put forward for further study to examine potential clinical benefit. The mechanism of action of palmitoylethanolamide and cannabidiol were explored further by examination of their effects on the immune response, permeability of cultured cell monolayers, intracellular signalling pathways, expression of membrane-bound proteins governing permeability and receptors of the cannabinoid system. We found that these agents were anti-inflammatory in both cultured Caco-2 cells and explant human colonic tissue, prevented increases in permeability secondary to inflammation, and were likely to act through adenylyl cyclase, protein kinase A and extracellular signal-regulated kinases. The downstream effects of these compounds prevented down-regulation of the TRPV1 receptor, upregulation of aquaporin 3 expression, and prevention of downregulation of claudin-3. The effects of palmitoylethanolamide and cannabidiol were then examined on permeability in the human colon in vivo by means of a double blinded, randomised controlled trial. This study demonstrated that aspirin increased the permeability of the human gut, determined by increases in urinary concentrations of lactulose and D-mannitol, quantified by mass spectrometry. Groups receiving oral cannabidiol or palmitoylethanolamide demonstrated lower urinary concentrations of lactulose and D-mannitol, suggesting that these two drugs could be used clinically to prevent disease-induced hyperpermeability. In conclusion, cannabidiol and palmitoylethanolamide have shown consistent anti-inflammatory actions in colonic ex vivo and in vitro models, and also prevented increases in intestinal permeability in vitro and also in vivo in a randomised, double blind, placebo-controlled trial. Their clinical use in IBD should now be assessed in phase II clinical trials.

C-terminal tensin-like (Cten, also known as Tensin4) is the member of the tensin gene family. Cten functions as an oncogene in a variety of cancer types and its expression is commonly associated with poor prognosis and metastasis in colorectal cancer (CRC). Although several studies have shown that Cten has a critical role in the regulation of cell motility and invasion in different tumour tissues, the underlying signalling mechanisms have not been fully elucidated. This thesis investigated the biological activity of Cten in four different ways in order to further elucidate the mechanisms of Cten signalling in CRC cells. Potential downstream targets of Cten signalling involved in the regulation of epithelial-to-mesenchymal transition (EMT) induced cell motility i.e. Rho-associated protein kinase1 (ROCK1), Src and Snail were investigated. Cten expression was manipulated in different cell lines using multiple approaches including forced expression, gene knockdown and constitutive depletion (through Crispr/Cas9 gene deletion) to eliminate artefacts of methodology and cell line specific effects. Snail, Src and ROCK1 were identified as novel downstream targets of Cten signalling and additionally, Cten was shown to increase the stabilisation of both Src and Snail proteins. The functional relevance of Cten-Snail, Cten-Src and Cten-ROCK1 signalling was assessed, and the overall findings demonstrated that Cten could promote cell motility and colony formation directly through the positive regulation of the Src/ROCK1/Snail dependent axis. To gain a deeper insight into the mechanisms of Cten’s biological function, mutations, at two important residues (i.e. arginine 474 and tyrosine 479) in the Src homology 2 (SH2) domain of Cten were introduced into one construct (GFP-CtenR474A+Y479F) using site directed mutagenesis. These two residues in the SH2 domain of Cten were found to not only be important for interacting with Src, ROCK1, or Snail signalling, but also for regulating cell motility and colony formation efficiency. Numerous Cten regulatory factors have been identified, however, little is known about how Cten is activated and regulated in cancer cells. The relationship between transforming growth factor beta 1 (TGFβ1) and Cten was investigated and stimulation of cells with TGFβ1 or knockdown of TGFβ1 resulted in changes in Cten expression as well as its downstream targets of ROCK1, Src, Snail, and N-cadherin. Furthermore, this positive interaction between TGFβ1 and Cten was functionally relevant and caused changes in cell motility. and the nuclear translocation of ROCK1, Src, and Snail protein increased by TGFβ1 is probably mediated via upregulation of the Cten signalling pathway The biological function of Cten in the nucleus was further investigated and shown to increase nuclear localisation of Src, ROCK1, and Snail, further promoting the migratory capability and colony formation efficiency in CRC cells. Finally, Cten expression was shown to positively correlate with both ROCK1 and Src expression in a series of primary CRCs. This correlation was consistent with that observed following manipulation of Cten expression in CRC cell lines. In conclusion, this study has revealed a number of novel findings regarding the biological function of Cten signalling in CRC. However, further validation of the findings may enhance the understanding of the role of Cten in the invasion-metastasis cascade in the future.

Introduction: Oesophageal cancer is more common in men than women. Oestrogen, which mediates its effects via oestrogen receptors (ERs), may be responsible for the gender disparity. This thesis investigates the role of ERs in oesophageal cancer development and explores potential therapeutic possibilities. Methods: ERα and ERβ expression in oesophageal AC cell lines (OE19 and OE33) was knocked down using siRNA, and the effect of knockdown on the expression of proliferation-associated proteins (Ki67, PCNA, E-cadherin, Cyclin D) was assessed. The effect of the SERM, tamoxifen, on oesophageal cancer cell proliferation was investigated using proliferation assays and by evaluating the effect of tamoxifen on proliferation-associated proteins. Finally, a pilot study of tamoxifen in patients with oesophageal cancer was undertaken to assess feasibility of a clinical trial and to determine the short-term biological effect of tamoxifen on proliferation, assessed by a change in the immunohistochemical expression of Ki67 between paired biopsies. Results: ERα and ERβ are expressed at the mRNA (RT-PCR) and protein level (Western Blotting) in the OE19 and OE33 cell lines. ERβ mRNA knock down was achieved in the OE33 cell line (p = < 0.0001). However, reproducible significant ERβ protein knockdown was not demonstrated, and there was no change in the expression of proliferation-associated proteins. Treatment with tamoxifen significantly inhibited OE33 cell proliferation in a dose-dependent manner (p = < 0.0001). Interestingly, treatment with tamoxifen decreased the expression of E-cadherin, but failed to change the expression of the remaining proliferation-associated proteins. Eight patients (6 male with AC and 2 female with SCC) included in the pilot study completed a median on 30 days (range: 28 – 45 days) tamoxifen treatment; the mean Ki67 Labelling Index between paired biopsies increased by 0.625% (ns). Of the two women included, Ki67 expression decreased with tamoxifen treatment. A correlation was demonstrated between a reduction in Ki67 and mean ERβ expression (r= -0.2272, ns). Discussion: ERβ is the dominant ER subtype expressed in oesophageal cancer cell lines and human cancer tissue. Tamoxifen inhibits the proliferation of oesophageal cancer cell lines in-vitro. Further studies to define the role of the ERβ subtype in oesophageal cancer and a clinical trial of tamoxifen in patients with oesophageal cancer is needed.