Oesophageal adenocarcinoma (OAC), a cancer with dismal prognosis, has been increasing rapidly in incidence over the last 30 years, nowhere more so than in the UK. Intriguingly, it is a disease predominantly among white males, but there is a paucity of data from England. In performing a range of epidemiological studies, it has been confirmed that OAC has risen five-fold in the West Midlands, UK, five times more common among men, and predominantly a disease among Caucasians. A reduced incidence of OAC was identified among subjects with prostate cancer, suggesting a protective effect of anti-androgen therapy. Examination of a general practice database revealed a negative association with aspirin, non-steroidal anti-inflammatories and statins with OAC, and a positive association with inhaled steroids, increasing number of drugs with a side effect of reducing the lower oesophageal sphincter, and drugs used for asthma/COPD. Finally, a region wide case-control study, confirmed the positive association seen with increasing body mass index, waist circumference, smoking and reflux symptoms, with negative associations seen with a diet high in fruit and vegetables. This work has identified potentially modifiable risk factors that may be employed to reduce the incidence of oesophageal adenocarcinoma, and better help stratify those most likely to benefit from endoscopic surveillance.

Human papillomavirus (HPV) E2 is essential for transcriptional regulation of viral oncoprotein expression and the replication and persistence of episomal HPV genomes. Episomal persistence is mediated by tethering of viral genomes to host cell chromosomes during mitosis. Previous work demonstrated that interaction of E2 with the cellular DNA helicase ChlR1 is necessary for viral genome tethering. Therefore, disruption of this interaction is a potential therapeutic target for persistent HPV infections. To investigate the use of fragment-based drug discovery in the development of novel inhibitors of the E2-ChlR1 interaction, a fragment library was screened to identify those that bind E2 and several hits were identified. Concurrently, the interaction between HPV16 E2 and ChlR1 was characterised and shown to be a direct protein-protein interaction. The binding sites within E2 and ChlR1 were mapped and this information was used to identify a mutant E2 protein unable to bind ChlR1 (E2-Y131A). E2-Y131A was functionally characterised. HPV16 genomes encoding E2 wild type and Y131A were transfected into primary human keratinocytes to study the differentiation-dependent virus life cycle. Mutant genomes failed to establish genome maintenance, providing strong evidence that the interaction between HPV16 E2 and ChlR1 is necessary for the persistence of HPV infection.

Introduction: Paediatric low grade brain tumours present diagnostic and prognostic challenges, providing a need for better non-invasive imaging characterization. The value of \(^1\)H Magnetic Resonance Spectroscopy (MRS) performed on 5 scanners in the diagnosis and prognostication of an extensive bi-centre cohort of low-grade gliomas is investigated. Methods: Single voxel MRS was performed routinely in children with brain tumours at the Birmingham Children’s Hospital and Queen’s Medical Centre. Histopathological features were semi-quantified and in vitro \(^1\)H NMR used to study pilocytic astrocytoma cell lines. Magnetic Resonance Spectroscopic Imaging (MRSI) and texture analysis of MR images were performed. Results: MRS detects differences between subgroups of low grade brain tumours in children and between tumours of the same histology. High myo-inositol and glycerophosphocholine and low phosphocholine are markers of good prognosis. Histological correlates for MRS metabolites have been identified and paediatric pilocytic astrocytoma cell lines (‘typical’, metastatic and recurrence) have been discriminated. The value of MRSI in answering clinical questions has been demonstrated. Texture analysis achieved high accuracy in the diagnosis of paediatric posterior fossa tumours. Conclusion: Advanced MR techniques have a significant role in the study of paediatric brain tumours, and promising results from MRS, MRSI and texture analysis are reported here.

Heterogeneous blood vessels are created by angiogenesis and vasculogenesis in colorectal cancer (CRC). Assessing endothelial activities had promising applications. However no marker has translated to clinical care. Hence a multifactorial assessment or ‘endotheliome’, including angiogenic activity, the ‘angiome’, was proposed. I tested that circulating cellular and plasma biomarkers determined outcome(s). Flow cytometry quantified circulating endothelial cells (CECs, displaced from blood vessels) and endothelial progenitor cells (EPCs, for vasculogenesis). Plasma markers measured by ELISA were: von Willebrand factor (vWf, for endothelial damage/turnover), soluble E-selectin (adhesion in tumour migration), vascular endothelial growth factor (VEGF) and angiogenin (for the ‘angiogenic switch’). All markers were prospectively quantified in 154 CRC participants before treatment and compared to non-cancer controls. They were tested against the tumour’s histopathology and repeated after surgery +/- adjuvant therapy. CECs and EPCs were highest in CRC and correlated to VEGF only. Angiogenin was diagnostic of CRC and vWf predicted metastasis. All markers fell after surgery but inconsistently after adjuvant treatment. Lower CD34+CD45- cells identified responders to anti-angiogenic therapy. Models incorporating CEC, EPC, angiogenin and CRC stage predicted progression within 2 years better than CRC stage alone. In summary, the endotheliome and angiome are determinants of outcomes and may aid decisions on therapeutic strategies.

Nasopharyngeal carcinoma (NPC) is endemic in Southern China and South East Asia, causally linked to Epstein-Barr virus (EBV) infection, and frequently shows dysregulation in a number of stem cell maintenance signalling pathways. This thesis has endeavoured to investigate the status of one of these pathways; the Hedgehog (HH) signalling pathway, in NPC tumours, and reveals the novel finding that EBV is able to active the HH signalling pathway through autocrine induction of the SHH ligand in the C666.1 authentic EBV-positive NPC-derived cell line and latently infected epithelial carcinoma cell lines. This study demonstrates that constitutive engagement of the HH pathway in EBV-infected epithelial cells in vitro induces the expression of a number of stemness-associated genes and imposes stem-like characteristics. Using epithelial cells expressing individual EBV latent genes, this study also investigates the viral protein responsible for HH dysregulation demonstrating that EBNA1, LMP1 and LMP2A are all capable of inducing SHH ligand and activating the HH pathway, but only LMP1 and LMP2A are able to induce expression of stemness-associated marker genes. These findings not only identify a role for dysregulated HH signalling in NPC oncogenesis but also provide a novel rationale for therapeutic intervention.

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that regulates mitotic progression through targeting substrates for degradation by the 26S proteasome. In order to assess APC/C post-translational modification status, and identify novel APC/C substrates and regulators, a comprehensive analysis of the APC/C and APC/C-interacting proteins by mass spectrometry was undertaken. RNA polymerase I was identified as an APC/C-interacting complex, and the interaction was validated by reciprocal co-immunoprecipitation, GST pull-down and immunofluorescent confocal microscopy. Both RPA194 protein levels and RNA Polymerase I transcription were shown to be dependent upon APC/C activity. Ablation of APC/C function by RNAi interference increased RPA194 protein levels, and elevated RNA polymerase I activity significantly, as quantified by 5’-Fluorouridine incorporation into nascent pre-rRNA, and the increase in absolute levels of 45S, 28S and 18S rRNA transcripts, relative to non-silencing controls. A number of other potential APC/C substrates and regulators were identified by mass spectrometry. Many of these interacting proteins contained APC/C consensus degron motifs. The APC/C was also shown to be a major substrate for acetylation; a number of APC/C subunits were identified as being acetylated in vivo. In this regard, APC3 was shown to be a substrate for both CBP and p300 acetyltransferases.

In this study, I used the serum-free MCDB 153 culture system to investigate calcium-induced keratinocyte differentiation. Treating normal human keratinocytes with high extracellular calcium concentrations (1mM) increased the proportion of cells expressing differentiation-specific proteins. I showed that this was not caused by calcium-induced cell-cycle arrest, nor was it a consequence of stratification. However, the expression of differentiation-specific proteins was preceded by the formation of cadherin-mediated cell-cell adhesions. The likely importance of the cadherin-mediated adhesions in initiation the differentiation program was confirmed in two ways. Firstly, clustering cell-surface E-cadherin in low extracellular calcium using monoclonal antibodies increased the proportion of keratinocytes expressing differentiation-specific proteins. Secondly, suppressing the formation of cadherin-mediated cell-cell adhesions using synthetic peptide analogous to the cadherin recognition domain attenuated the calcium-induced expression of differentiation-specific proteins. These data are consistent with a role for the cadherin-mediated cell-cell adhesions in initiating the keratinocyte differentiation program in response to calcium in vitro. A second aspect of this project involved an investigation of the role played by the Src-family of protein tyrosine kinases at calcium-induced cadherin-mediated adherens junctions. The ubiquitously expressed members of this family, c-Src, Fyn and c-Yes were localised to the cadherin-mediated adhesions formed in response to high extracellular calcium. Treating adherent keratinocytes maintained in low extracellular calcium with a specific Src kinase inhibitor, PD162531, induced the assembly of cadherin-mediated cell-cell adhesions leading to the formation of contiguous groups of cells similar to those seen in response to high extracellular calcium. The data presented are consistent with a role for the Src kinases in regulating adherens junction turnover but do not exclude a role also in modulating aspects of differentiation.

Tyrosine kinase inhibitors (TKIs) have revolutionised the treatment of chronic myeloid leukemia (CML), however, fail to cure the disease due to the persistence of a refractory fraction of stem/progenitor cells. Autophagy is a recycling mechanism utilised by the cell as a survival mechanism under stressful conditions, and its induction has been suggested to have a cytoprotective role in cancer cells. In this study we demonstrate that autophagy is triggered in CML upon TKI-mediated inhibition of BCR-ABL, and protects from cell death. In order to evaluate if specific autophagy inhibition enhances TKI effects, we stably transduced primary CML stem/progenitor cells with a vector carrying a short-hairpin against the key autophagy gene ATG7. Knock-down of basal ATG7/autophagy levels in CML stem/progenitor cells inhibited by approximately 50% the survival of the cells in a clonogenic assay, and reduced by 75% their erythroid differentiation potential. Furthermore, ATG7 knock-down enhanced the effects of TKIs imatinib (IM; 1st), dasatinib (DAS; 2nd), nilotinib (NIL; 2nd) and ponatinib (PON; 3rd generation), reducing by 92-98% the survival of these cells in a clonogenic assay. In contrast, ATG7 knock-down in normal stem cells, with or without TKI treatment, did not have a significant effect on survival and proliferation. ATG7 was also knocked-down in final disease stage, blast crisis (BC), patient-derived K562 and KCL22 cell lines. Both cell lines appeared to depend significantly on autophagy for survival as indicated by high apoptosis levels (70-100%) after ATG7 knock-down. Interestingly, ATG7 knock-down cells appeared to be more differentiated compared to the control (scrambled shRNA). Our findings suggest a role for basal autophagy in the survival, differentiation decisions and clonogenicity of CML cells, and support the combined use of autophagy inhibition with TKIs for the eradication of CML stem/progenitor cells. This could be partially attributed to a bypass of the differentiation block upon autophagy inhibition, which facilitates TKI-targeting. We underline the necessity for the development of specific autophagy inhibitors that in combination with TKIs could potentially eradicate the fraction of persistent CML stem/progenitor cells and offer a curative option for CML patients.

In spite of extensive research there is little information about apoptosis or programmed cell death in the genesis and progression of cancers of the lung. In our project we have investigated the role of apoptosis and two of the genes controlling apoptosis (bcl2 and C040) in squamous cell carcinoma of the lung. Also we have tried to formulate an accurate way of measuring apoptotic rate in tumour specimens. We counted apoptotic cells in Haematoxylin and Eosin stained histological sections of squamous cell carcinoma of the lung. The apoptotic indices we obtained were very reliable showing remarkable reproducibility and strong correlation with apoptosis measured by monoclonal antibody to apoptosis specific protein (ASP). In our series apoptotic index did not correlate with survival, disease stage, differentiation, AgNOR or DNA ploidy. Histological sections were stained with monoclonal antibodies to Bc12 and CD40. In all, 32% of squamous cell carcinoma of lung were Bc12 positive (i.e. more than 50% of the tumour cells contained Bc12 protein) and 22% were positive for C040. The expression of Bc12 correlated positively with the apoptotic indices. Patients with Bcl2 positive squamous cell lung cancers survived significantly longer although Bcl2 expression did not correlate with any marker of disease severity e.g. stage, grade, AgNOR or DNA ploidy. C040 expression in squamous cell lung cancer had no effect on apoptosis, survival or any of the other previously mentioned markers of disease severity. The expression of C040 in our series showed a tendency to correlate inversely with the expression of Bcl2. We devised a way to measure apoptotie rate in prinwy cultures of tumour cells by serial estimation of sub-diploid fractions in cell suspensions double stained with PI and BerEP4-FITC. We believe that the apoptotic rate measured in this fashion is biologically more relevant, and therefore could be more useful in predicting prognosis, than apoptosis measured from histological sections.

In the UK, more than two million people are alive following a cancer diagnosis and people with cancer live an average six times longer than they did forty years ago. There have been dramatic survival improvements in some cancers with six now having median survival expectations of over ten years. This is remarkable but cancer consists of more than two hundred ‘types’ and, for some types, predicted survival is still only weeks. Furthermore, some issues related to long term survival are only just emerging, many remain underresearched and studies that exist have been criticised for being drawn from limited cancer sites and ignoring the coping strategies and social contexts of those diagnosed with cancer. The aim of this work is to explore the experience of living with and beyond the diagnosis of a ‘poor prognostic’ cancer in contemporary society and from a sociological perspective. The work is informed by a literature review which explores lay understanding of cancer, a theoretically driven investigation designed to produce a sociological understanding of what it is like to live with cancer, a feasibility study and a full empirical study, which were both supported by users. Data for the principal study was generated by ‘photovoice’; a novel participatory method in which participants created and discussed photographs to illustrate and describe their experience in depth. This study revealed that living with and beyond cancer was an ongoing disruptive experience for participants and their constant fear of recurrence impacted on future plans. Furthermore, society’s stigmatising perception of cancer bestowed certain responsibilities and obligations on the participants. Photographs added a power and richness to the data. This work adds to the very limited understanding of the experience of cancer and ‘survivorship’ for this group and will hopefully guide appropriate communication, service provision and future research.