The BDCA antibodies allowed reliable measurement of dendritic cell (DC) subsets and B cell numbers in the blood of normal subjects, and patients with MM throughout the disease course. The numbers of blood myeloid DC (BmDC) and blood plasmacytoid DC (BpDC) are low throughout the course of the disease, and only improve for a short period of time following autologous HSCT. Thalidomide therapy of patients with relapsed disease was associated with an increase in BmDC1 and BpDC numbers. Monocytes, mobilised at the time of stem cell collection, were used to produce mature DC (matDC) from MM patients and normal donors (ND). The matDC produced from MM patients were of poorer quality as compared to those from ND, despite using combinations of GM/IL-4, GM/IL-13, X4 and MIMIC in the production process. The combinations that contained the X4 maturation cocktail produced the best quality matDC. The DC/T cell system is abnormal in MM patients. Despite this, it is possible to produce antigen loaded mature MoDC from MM patients. When combined with T cell pre-stimulation and IL-2 expansion, these DC are capable of inducing anti-MM cytotoxic T cells, which exhibit considerable anti-MM cytolytic activity. However, the DC from MM patients still display abnormal chemokine receptor expression, which may inhibit their capability to migrate to lymph nodes in-vivo in order to generate these cytotoxic T cell responses. These observations will aid in the optimisation of DC based immune therapies for MM, and suggest that a combined immunotherapy approach using pre-stimulated T cells, MM Ag primed DC and IL-2 may produce better clinical responses in MM patients.

Autophagy is an intracellular catabolic process that involves the sequestration of proteins and whole organelles into specialized cargo vesicles (autophagosomes) and their delivery to lysosomes with subsequent degradation. Autophagy is active at low levels at any time in virtually all cells and can be induced upon a variety of different stimuli. The core function of autophagy is the degradation and recycling of intracellular material. However, how this impacts on cellular survival likely depends on the biological context. The role of autophagy in cancer is very complex and incompletely understood. It is therefore very surprising that few studies exists that employ genetically modified mouse models of human cancer to examine the role of autophagy in this context. This is even more true when considering, that pharmacological inhibition of autophagy is currently being used in several clinical trials to treat cancer of various origins. The goal of this study was to examine the role of autophagy in a mouse model of pancreatic cancer. To achieve this several mouse strains were crossed: a) Pdx1-Cre LSLKRasG12D/wt mice that develop Pancreatic Ductal Adenocarcinoma (PDAC) similar to humans initiated by oncogenic Ras and b) Atg5flox/flox or Atg7flox/flox mice that permit Cre-induced deletion of either one of the essential autophagy regulating genes 5 and 7 (Atg5, Atg7). Offspring allowed us to examine the role of autophagy in pancreatic function. Loss of autophagy in the pancreas leads to exocrine and endocrine tissue destruction and reduces survival in approx. 60% of animals. The early death in autophagy-deficient mice can be delayed by additional deletion of p53; the mortality rate however remains unchanged. Moribund mice show a diabetic phenotype with elevated blood glucose and fructosamine levels. In the absence of oncogenic Ras autophagy deletion does not lead to cancer formation or occurrence of pre-malignant lesions in mice aged up to 700d. In mice that express oncogenic Ras in the pancreas (Pdx1-CreKRasG12D/wt) additional, genetic deletion of autophagy leads to accumulation of pre-malignant Pancreatic Intraepithelial Neoplasias (PanINs) that unlike their autophagy proficient counterparts never progress to cancer. In this genetic context autophagy therefore serves as a tumour promotor. In stark contrast in mice expressing oncogenic Ras and lacking both copies of p53 (Pdx1-KRasG12D/wt p53-/-) inhibition of autophagy, either genetically by deletion of Atg5, Atg7 or pharmacologically by chloroquine, tumour onset is accelerated. Therefore in a p53-deficient situation autophagy is now a tumour suppressor. Tumours that developed from a p53-proficient background have increased autophagy compared to tumours that developed from a p53-null background. Furthermore p53-/- Atg7-/- tumours have increased glycolysis in vitro and in vivo and enhanced intracellular metabolites of the anabolic Pentose Phosphate Pathway (PPP) compared to p53-/- Atg7+/+ tumours. In summary it is the p53 status that determines the role of autophagy in PDAC development. In tumours developing from a p53-proficient background loss of autophagy completely prevents cancer development; whereas in tumours arising from p53-deficient tissue loss of autophagy accelerates tumour formation.

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.

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.

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