This thesis examines factors influencing contemporary outcomes of patients managed by the South East Wales upper GI cancer network multidisciplinary team. The hypotheses tested were: PET/CT defined tumour characteristics influence outcomes of patients with oesophagogastric cancer; Centralisation of oesophagogastric cancer services improves outcomes significantly; HER2 overexpression is a poor prognostic indicator following oesophagogastric cancer resection; An involved circumferential resection margin (CRM) following oesophagectomy is an independent predictor of survival. PET/CT N stage was an independent and significant predictor of survival (p=0.022). SUVmax correlated positively and significantly with endoluminal ultrasound-defined tumour volume (Spearman’s rho=0.339, p=0.001). Centralisation increased the proportion of patients receiving potentially curative treatment by 78% (p<0.0001), reduced serious operative morbidity by 50% (p=0.062), shortened total length of hospital stay from 16 days to 13 days (p=0.024) and improved median and 1-year survival from 8.7 months and 39% to 10.8 months and 46.8% respectively (p=0.032). Centralisation was an independent and significant predictor of survival (p=0.03). HER2 overexpression and gene amplification was a predictor of poor prognosis in patients with curable oesophageal cancer (p=0.03). CRM involvement was also an indicator of poor prognosis in these patients (p<0.001). The College of American Pathologists’ criteria differentiate a higher risk group than Royal College of Pathologists’ criteria but overlook a patient group with similar poor outcomes (p<0.001).

This study describes the translation of an assay developed for use in cell culture models to into a method of measuring patterns of DNA damage from platinum agents in human blood samples. These adduct patterns could potentially be used in future studies for the stratification of patients for response and toxicity to oxaliplatin chemotherapy. Chapters 3 and 4 of this thesis describe the steps taken to translate our DIP--‐chip assay, a tool previously used in the study of DNA repair capacity in yeast and to measure induction of platinum-DNA adducts in cell culture models, into an assay capable of reproducibly analysing chemotherapy damage in human clinical samples. These results clearly demonstrate the protocol modifications required to use the assay on human blood samples, and show the reproducibly of the assay in detecting patterns of oxaliplatin induced DNA-adducts in clinical material. Chapter 5 describes the development of novel bioinformatic tools and analysis methods for interpreting DIP--‐chip DNA--‐adduct microarray outputs. The translation of a genomic--‐scale laboratory technology into a tool for patient stratification is a technical and bioinformatics challenge. The tools developed are a significant advance on previously available bioinformatic functions, and are essential for the application of this technique as a clinically useful assay. The final results section, chapter 6, documents the successful development of functional models to experimentally confirm links between single nucleotide polymorphisms in nucleotide excision repair genes with the development of oxaliplatin induced peripheral neuropathy (OIPN). This aspect of the study utilises new information, recently derived from experiments DNA-sequencing colorectal cancer patients, to develop a functional model of OIPN in Saccharomyces cerevisiae. This model is then used to demonstrate the impact of variations in DNA repair genes on the development of OIPN ‐ a relationship that highlights the significance of DNA repair to the development of oxaliplatin toxicity.

Introduction: It has been recently shown that the WISP proteins (Wnt-inducted secreted proteins), a group of intra- and extra-cellular regulatory proteins, have been implicated in the initiation and progression of variety types of tumours including colorectal and breast cancer. However, the role of WISP proteins in gastric cancer (GC) cells and clinical implication in gastric cancer has not yet been fully elucidated. Materials and methods: The expression of the WISP transcript and proteins in a cohort of GC patients was analysed using real-time quantitative PCR and immunohistochemistry, respectively. The expression of a panel of recognised EMT (epithelial-mesenchymal transition) markers were quantified (Q-PCR) in paired tumour and normal gastric tissues. WISP-2 knockdown sublines using anti-WISP-2 ribozyme transgenes were created in GC cell lines AGS and HGC27. Using the cell models and proteins extracted from gastric tissue samples, protein microarray was used to search for potential protein partners and signalling pathways involved with WISP-2. Subsequently, the biological functions, namely, cell growth, adhesion, migration and invasion, were studied. Potential mechanisms related with EMT, extracellular matrix and MMP (Matrix metalloproteinases) and signalling pathways were investigated. Results: Expression of WISP-2 was frequently detected in GC tissues. Levels of WISP-2, not WISP-1 and WISP-3, was significantly correlated with early TNM staging and differentiation status. High levels of WISP-2 were associated with a favourable clinical outcome and survival of the patients. We also found that WISP-2 expression inversely correlated with Twist and Slug in the paired gastric samples. Knockdown of WISP-2 expression increased the rate of proliferation, migration and invasion of GC cells and influenced expression of EMT biomarkers including Twist, Slug and Ecadherin. Using an antibody based protein microarray, ERK, JNK as well as AKT proteins were found to be co-precipiated with WISP-2 protein from human gastric tissue proteins. Furthermore, WISP-2 knockdown gastric cell lines also demonstrated a change in the ERK and JNK phophorylation. Mechanistically, WISP-2 suppressed GC cell metastasis through reversing epithelial-mesenchymal transition and suppressing the expression and activity of MMP-9 and MMP-2 via JNK and ERK. Cell motility analysis indicated that WISP-2 knockdown contributed to GC cells’ motility, an effect attenuated by PLC-γ and JNK small inhibitors. Conclusions: WISP-2 transcript and protein expressions are inversely linked to disease progression and linked to the survival of patients with gastric cancer. WISP-2 has a profound influence on the migration and adhesion of gastric cancer cells and is a powerful factor to reverse the EMT process in these cells. These effects of WISP-2 are via its involvement in the ERK and JNK pathways, which in turn modulate the MMP activities. Together, WISP-2 is an important regulator of the cellular function and an important factor in the progression of gastric cancer. It acts as a potential tumour suppressor in gastric cancer.

Prognosis for lung cancer patients remains poor. For those receiving radiation therapy, local control and survival have been shown to improve with increased doses; however, deliverable dose is often limited by associated toxicity. Therefore, methods that reduce dose to normal tissues and allow isotoxic escalation are desirable. Adaptive radiation therapy seeks to improve treatment by modifying the initial plan throughout delivery, and has been shown to decrease normal tissue dose. Studies to date suggest a trend of increasing benefit with increases in replanning frequency; however, replanning is costly in terms of workload and past studies implement at most weekly adaptation. The purpose of this thesis is to quantify the benefit associated with daily replanning and characterize the tradeoff between replanning frequency and adaptive benefit. A software tool is developed to facilitate planning studies and to introduce complimentary methods for evaluating adaptive treatments. Synthetic images and contours are xii generated for each fraction of a typical fractionation schedule using principal component analysis and a novel method of sampling coefficients that preserves temporal trends in the data (e.g. tumor regression). Using the synthetic datasets, a series of adaptive schedules ranging from no adaption to daily replanning are simulated and compared to quantify adaptive benefits and characterize tradeoffs with frequency. Daily replanning resulted in significant reductions in all normal tissue planning metrics when compared to no adaptation, and incremental reductions were observed with each increase in replanning frequency while the magnitude of average reductions decreased with each step. Modest correlation between absolute change in planning target volume over the course of treatment and reductions in both mean lung dose and mean esophageal dose were observed.

Introduction The adjuvant treatment of colorectal cancer (CRC) has seen little improvement in terms of mortality of the disease in the last 40 years. There has been a resurgence in research into the use of monoclonal antibodies in the treatment of CRC. Carcinoembryonic antigen (CEA) is a useful target in cancer immunotherapy. The distribution of CEA in CRC differs from that in normal colorectal tissue. In normal colorectal tissue CEA is found only on the luminal surface of the cell which is inaccessible to intravenous antibody, whereas in CRC, CEA is found on all borders of the cell membrane and so becomes accessible to intravenous antibody. However, anti-CEA antibodies are prone to sequestration by circulating CEA. The anti-CEA antibody, PR1A3, binds only membrane-bound CEA and thus is able to overcome this problem. The aim of my research was to assess whether PR1A3 is suitable to be considered as a therapeutic agent in the treatment of CRC and what its mechanism of action might be. Methods The level of expression of CEA on a panel of cell lines was determined under different conditions using a solid-phase ELISA and FACS analysis. Humanized PR1A3 (hPR1A3) was assessed in a variety of in vitro cytotoxicity assays with colorectal cell lines expressing varying levels of CEA, using peripheral blood mononuclear cells and purified natural killer cells as sources of effector cells. The mechanism of action of PR1A3 was investigated by modifying the Fc fragment of the antibody and using antibodies to block the FcIIIA receptor on the effector cells. PR1A3 was also investigated in combination with a humanised A33 antibody. Results A panel of colorectal cell lines was found to have a range of CEA expression which could be upregulated in certain cell lines by growing the cell line beyond confluence and by treatment with the chemotherapeutic agent, 5-fluorouracil. The in vitro assays demonstrated hPR1A3 antibody-dependent and CEA-specific killing of tumour cell lines by human PBMC. The effect increased with increasing concentration of antibody and was lost by using the parent murine IgG1 PR1A3. Using 50&mu;g/ml hPR1A3, tumour cell lysis was increased by more than 3-fold above spontaneous killing (p < 0.001) in a high CEA-expressing cell line. Both antibody-dependent and antibody-independent (spontaneous) killing was blocked by using whole antibody to the Fc-&gamma;IIIA receptor, although the spontaneous killing was restored when a F(ab')2 was used instead of whole antibody. hPR1A3 and the A33 antibody showed potential synergy when used in combination against a high-CEA and a moderate-A33 expressing cell line. Conclusion The monoclonal antibody hPR1A3 causes CEA-specific lysis of human colorectal cancer-derived cell lines in the presence of human PBMCs. This lysis is dependent on the dose of the antibody, requires a compatible Fc-receptor and is inhibited by blockade of the FcγIIIA receptor. These findings show that hPR1A3 can kill tumour cells by antibody-mediated cellular cytotoxicity (ADCC) and implicate NK cells as a major contributor to this effect. The results support the development of hPR1A3 for therapy of colorectal cancer.

Melanoma is the most aggressive skin cancer with high recurrence and low survival rate. In addition to genetic mechanisms, resistance also arises from phenotypic heterogeneity in which a proportion of cells, the so-called melanoma stem or initiating cells, survive therapy. Due to a lack of reliable markers, however, there is still debate about the existence of these cells in melanoma. Consistent with phenotypic heterogeneity, previous observations in our laboratory have demonstrated that cells in melanoma can reversibly segregate in vivo into different subpopulations with different properties, such as differentiation or increased invasive capacity (potentially attributed to the existence of de-differentiated stem-like cells). To characterise these cells, a dual reporter lentiviral system was engineered, expressing fluorescent proteins under cell stage/phenotype-specific promoters. The promoters for the transcription factors POU3F2 (Brn2) (to mark de-differentiated cells) and the microphthalmia-associated transcription factor (MITF) (to mark proliferating and differentiated cells) were chosen. Lentivirally-transduced cells were used to screen a library of kinase inhibitors for their potential to affect promoter activity in vitro. The RhoA/ROCK pathway, known to contribute to invasion and metastases, was identified to play a role in Brn2 promoter activity and exhibited differential effects on both the MITF and Brn2 promoters in 501mel and SKmel28 cell lines. Through investigation of other signalling pathways involved in melanoma metastasis, we also identified the co-activator Mastermind-like 1 (MAML1), previously reported to act in the Notch pathway, as an activator of the Brn2 promoter via the transcription factor TCF3, and the MITF promoter through the lymphoid-enhancer binding factor 1 (LEF1). The effects of MAML1 on Brn2 and MITF promoter activity were potentiated by &beta;-catenin. These findings provide new opportunities for the identification of therapeutic targets to prevent metastases formation in melanoma.

The transcription factor E2F1, a critical target of the tumour suppressor pRb, is deregulated in most human cancers. Oncogenes have been shown to deregulate E2F1 through inhibition of pRB and deregulation of E2F1 is an event that occurs in most human cancers. The essential role of E2F1 in apoptosis is well documented and deregulated E2F1 can enhance drug induced death. E2F1 is induced by various chemotherapeutic drugs and this induction, in addition with oncogenic stress, contributes to increased chemosensitivity. Cells expressing the adenovirus early region 1A (E1A) oncogene have been used as a tool to identify cellular regulatory pathways that modulate chemosensitivity. E1A sensitises cells to the induction of apoptosis by diverse stimuli, including many chemotherapeutic drugs. These E1A activities are mediated through binding the RB family proteins (pRb, p107 and p130) and via the E1A N-terminal domain that interacts with different cellular protein complexes including the p300/CBP transcriptional activator and p400/TRRAP chromatin-remodeling complex. The results presented here illustrate novel mechanisms of E2F1 induction both by oncogenes and chemotherapeutic drugs. Two minimal domains of E2F1 are described that are induced following DNA damage via mechanism(s) not previously identified. In addition, data are presented which show that E1A expression not only deregulates E2F1, but also elevates E2F1 levels. E1A is dependent on interaction with RB protein to induce E2F1 levels and this elevation contributes to cell death. Using previously described protein binding deficient truncations of E1A, we demonstrate that E1A binding to the p400/TRRAP protein complex is also critical for the induction of E2F1. E1A binding to p400/TRRAP was also critical in sensitizing these cells to drug induced apoptosis. Suppression of p400 using siRNA had similar affect on E2F1 induction and caused an increase in drug sensitivity indicating that E1A inhibits p400 function. These results contribute to the understanding of how activation of the E2F1 pathway may be targeted therapeutically to enhance chemotherapy-induced tumour cell death.

Loss of expression of the human DNA mismatch repair (MMR) gene, hMLH1, is seen in a number of tumour cell lines resistant to a variety of cytotoxic drugs. The aim of this study was to identify other proteins that interact with hMLH1 to attempt to further elucidate its role in MMR and the engagement of downstream damage response pathways. A yeast two-hybrid system, an in vivo system for detecting protein-protein interactions was utilised for this purpose. Fifteen known and five unknown genes were identified as encoding proteins interacting with hMLH1. These included three known hMLH1 binding proteins, hMLH3, hPMS1 and MED1. Amongst the other genes identified was the proto-oncogene c-MYC, a gene previously implicated in genetic instability and apoptosis. Using in vitro derived mutants of c-MYC, it has been shown that hMLH1 interacts with the leucine-zipper domain of c-MYC. The effect of elevated c-MYC expression on functional MMR was examined. An inducible c-MYC expression system, Rat-1 fibroblasts expressing c-MYCERTM, a fusion of c-MYC to the hormone binding domain of the oestrogen receptor was utilised. Elevated expression of c-MYC did not effect the mismatch specific binding complex activity in these cells as measured in EMSA experiments. However c-MYC overexpression utilising the Rat-1 cMYCERTM system was shown to result in a mutator phenotype in these cells. The results suggest there may be a link between the mutator phenotype, induced through overexpression of c-MYC, and loss of MMR. Overexpression of c-MYC, which is associated with many cancers, may result in the sequestration of hMLH1 preventing functional MMR. The interaction between hMLH1 and c-MYC is proposed to act in a DNA damage response pathway which is disrupted upon aberrant c-MYC expression.

Synergistic interaction between pairs of membrane-bound receptors has been linked to signalling, cell communication and tumour progression. This study has shown that cluster of differentiation (CD) 74 and CD44 act in synergy and are susceptible to the effect of the macrophage migration inhibitory factor (MIF). MIF is a 12.5 kDa chemokine-like inflammatory mediator, whose ligand is the transmembrane receptor CD74. Recent data suggests that CD74 is involved in proinflammatory responses and tumorigenesis but detailed mechanisms are not fully understood. In normal cells CD74 functions as a chaperone of human leukocyte antigen (HLA)-DR biosynthesis and is expressed in antigen presenting cells in the absence of tumours. Notably, CD44 is also a transmembrane receptor and member of a family of cell adhesion molecules responsible for adhesion between adjacent cells (e.g. antigen presenting cells) and cells in the extracellular matrix. Western blotting and flow cytometry were employed to determine the quantitative expression of CD74, MIF and CD44 in three distinct breast tumour cell lines: CAMA-1, MDA-MB-231 and MDA-MB-435. All three cell lines showed a high expression of CD74, MIF and CD44. Modulation studies showed that IFN-γ and LPS can play a significant role in regulating the expression of CD74, proliferation and cell migration in CAMA-1 and MDA-MB-231 cells; suggesting that CD74 might be involved in controlling immunogenicity and immunoediting of breast cancer cells. To investigate the interaction of CD74 with CD44 and MIF, confocal microscopy and co-immunoprecipitation techniques were used. The three molecules form a multimeric complex in cytoplasmic compartments as measured by confocal microscopy, suggesting a mechanistic mode of action; in addition CD74, MIF and CD44 showed significant quantitative variations on all breast cancer derived cells. Knockdown of CD74 by CD74 siRNA significantly reduced CAMA-1 and MDA-MB-231 cell proliferation but increased the level of apoptotic cells. These data suggests that CD74, MIF and CD44, might facilitate signalling and hence could affect tumour progression. Measuring the co-expression levels of CD74, MIF and CD44 could potentially be used as a ‘biomarker signature’ for monitoring breast cancer tumours at different stages of the disease.

One key to understanding how cells integrate and how they respond to diverse stimuli in order to direct a transcriptional response is knowing how a transcription factor may be directed to an appropriate subset of its target genes. One mechanism with which this may be achieved is by modulation of the transcription factor’s post-translational modification status. The microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage, and it is also a lineage addiction gene in melanoma. Low or high levels of MITF expression induce a reversible cell cycle arrest. Invasive behaviour is characteristic of low MITF expression; differentiation a product of high MITF activity; and moderate levels of MITF expression promote proliferation. A major, unaddressed problem is how DNA binding by MITF may be differentially directed such that it regulates either a proliferation-associated or a differentiation-associated gene expression programme appropriate to the cellular microenvironment. This thesis explores the function and regulation of the signalling pathways controlling novel post-translational modifications of MITF. One such modification, in the DNA binding domain of MITF, defines a key switch that controls MITF’s DNA binding affinity and specificity. Moreover, a novel set of MITF target genes are revealed that extend its control beyond pigmentation and cell cycle regulation to implicate MITF as an overall regulator of cell behaviour in the melanocyte lineage.