In this thesis we validate the efficacy of a new adenoviral construct in prostate cancer cell lines in preparation for a gene and immunotherapy clinical trial in prostate cancer. By demonstrating the constructs ability to infect prostate cancer cells and cause them to die with the introduction of the prodrug, CB1954 as well as releasing a biologically active cytokine, GMCSF we secured GTAC approval to proceed to a phase I/II clinical trial in patients with local relapse after treatment with curative intent for prostate cancer. A tertiary endpoint in the trial is evidence of immune responses relating to treatment. To measure this we have modified an interferon gamma ELISpot assay to measure T cell mediated immune responses. We have then used this assay on 38 patients with suspected or diagnosed prostate cancer. In this study we have found the assay to be acceptable to patients and deliverable within the setting of the clinical trial. We found evidence of strong immune responses in patients with low and intermediate risk prostate cancer based on D’Amico’s classification with these responses declining in more advanced patients. We found that some interventions lead to an increase in immune responses and these observations warrant further exploration.

Nuclear receptors (NRs) are essential transcription factors that participate in a diverse number of cellular functions. Many have attractive chemotherapeutic potential due to their ability to govern pathways of cellular differentiation, growth arrest and programmed cell death. There are numerous examples of NR signaling becoming disrupted in human malignancies including the prostate. Mechanisms that give rise to impaired receptor signalling are investigated herein, including pre-receptor regulation of NR ligand and epigenetically mediated hypoacetylation. Furthermore, attempts either to overcome or circumvent their disruption are investigated. In parallel to these one member of the NR subfamily was chosen for further analysis. The vitamin D receptor and its target gene CDKN1A were examined for recruitment of VDR, nuclear corepressor (NCOR1) and ppolymerase II to response elements. Using the non-malignant prostate epithelial cell line RWPE-1, and PC-3 prostate cancer cell line to represent stages of prostate disease progression the spatio-temporal binding characteristics in response to ligand were measured. These findings identified aberrant nuclear corepressor recruitment to the transcription start site of CDKN1A in malignant disease. In addition examination of coregulation of a microRNA known to target CDKN1A revealed a mechanism of NR sensitivity that is also perturbed in prostate cancer. MiR106b also showed elevated tumour and serum expression in prostate cancer, suggesting a new biomarker of VDR responsiveness.

Cytotoxic T Lymphocyte Associate Antigen 4 (CTLA-4) is an important negative regulator of T-cell activation. The protein is expressed in activated T-cells and can also be found in regulatory T-cells. The mechanism of action of this protein remains controversial; it has typically been associated with a cell intrinsic negative signal however, there is increasing evidence that CTLA-4 may act as an effector molecule. Surprisingly, we find that blocking CTLA-4 in a model of T-cell activation driven by ligand-expressing transfectants has no effect on either proliferation or cytokine production, suggesting that CTLA-4 doesn’t inhibit in this setting. In contrast, blocking CTLA-4 in a dendritic cell based assay enhances proliferation and cytokine production, only when the amount of co-stimulation is limiting. In these experiments CTLA-4 function correlates with decreased expression of B7 ligands on dendritic cells consistent with the removal of ligands by CTLA-4. Furthermore, the addition of CTLA-4 transfected Jurkat cells acts to suppress T cell responses consistent with a role for CTLA-4 as an effector of suppression. Overall our data do not support a role for CTLA-4 in delivering a ligand-dependent cell-intrinsic negative signal and instead suggest a role for CTLA-4 as an effector molecule which inhibits co-stimulation by APC.

Epstein-Barr virus (EBV), a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this B-cell growth transformation programme depends on the activation of a viral promoter, Wp, present in each tandemly arrayed BamHI W repeat of the EBV genome. In order to examine the role of the BamHI W region in B cell infection and growth transformation, we constructed a series of recombinant EBVs carrying different numbers of BamHI W repeats and carried out B cell infection experiments. We concluded that EBV requires at least 2 copies of BamHI W repeats to be able to activate transcription and transformation in resting B cells in vitro. At least 5 copies of BamHI W were required for optimal transcription and transformation; while increasing the number beyond 5 copies had no further effect. Experiments to try to rescue the impaired virus indicated that the expression of sufficient levels of EBNA-LP and EBNA2 from Wp are the key determinants of virus-driven B cell transformation. We believe that EBV has evolved to contain multiple copies of BamHI W repeats to ensure high levels of Wp-initiated transcripts immediately post infection.

In cancer, loss of intercellular contact contributes to tumour progression and invasion. Desmosomal cadherins are essential constituents of desmosomes – intercellular junctions that confer significant adhesive strength to epithelial tissues and cardiac muscle. Although changes in desmosomal components have been noted in a variety of cancers previously, this investigation has shown for the first time altered desmocollin expression in colorectal cancer. Real-time PCR and western blotting were used to assess desmocollin expression in a series of colorectal cancer and matched normal tissue samples. Loss of desmocollin 2 expression was observed in the cancer samples. In addition, de novo expression of desmocollins 1 and 3, which are not normally expressed in the colon, was observed. Desmoglein gene expression was also altered in the cancer samples. Although classical cadherin switching is a hallmark of the epithelial-mesenchymal transition, desmocollin switching has not previously been reported. Further experiments, to investigate the effect of loss of desmocollin 2 and desmoglein 2 on the behaviour of cultured cells were performed. In addition, experiments were carried out to identify those transcription factors that regulate desmosomal cadherin gene expression in the colon. Transcription factors of the CCAAT/enhancer-binding proteins family act as transcriptional activators of desmosomal cadherin promoters in colonic cells.

This thesis summarises the author’s lifelong work to improve outcomes for patients with musculoskeletal tumours. It starts with analysing diagnostic features and steps that could be taken to improve the time to diagnosis of these rare tumours. It continues with a number of basic science projects which the author has either carried out or collaborated with. Benign bone tumours are common in children and are discussed. The author has probably one of the largest personal experiences of treating primary malignant bone and soft tissue tumours and this is evidenced by the papers on osteosarcoma, chondrosarcoma, Ewing’s sarcoma and soft tissue sarcoma. The indications for and outcomes of major ablative amputations are outlined followed by an extensive review of the outcomes of limb salvage surgery with endoprostheses and other techniques. The role of this type of surgery in metastatic disease is put in context. Finally the importance of follow up and guidelines is considered. The thesis represents an analysis of the huge improvements that have taken place in the past 25 years in musculoskeletal oncology but also reveals the many still unanswered questions in this evolving field.

Autotaxin is an extracellular enzyme that generates lysophosphatidic acid (LPA). LPA binds up to six different cell surface G protein-coupled receptors to initiate signaling resulting in cell survival, invasion and angiogenesis. For this reason autotaxin has emerged as a therapeutic target in several different malignancies. I have used immunohistochemistry to explore the expression of autotaxin and its correlation with clinico-pathological variables in bladder and renal cancer. I show that in bladder cancer, tumours from patients with muscle invasive disease were significantly more likely to show strong autotaxin expression than were those tumours from patients without evidence of muscle involvement (p=0.009). This observation is not only consistent with the known functions of autotaxin/LPA in promoting tumour invasion, but suggests that the potential use autotaxin inhibitors in preventing bladder cancer progression warrants further investigation. Although I failed to detect autotaxin expression in the tumour cells of patients with renal cancer, I did observe high-level expression of autotaxin on the tumour-associated vasculature, which in many cases was not apparent in blood vessels of matched normal renal tissues. This points to an important role for autotaxin in renal cancer-associated angiogenesis and suggests a potential role for autotaxin inhibition as an anti-angiogenesis therapy.

It is well established that adenoviruses inactivate the host cell DNA damage response to enhance viral DNA replication. This is achieved, in part, by the ability of viral E1B55K and E4ORF6 proteins to hijack host cell cullin-containing E3 ubiquitin ligase complexes and target key cellular proteins such as Mre11, p53 and DNA ligase IV for degradation via the ubiquitin-proteasome pathway. To assess the generality of this viral response, studies were undertaken using a panel of representative serotypes from adenovirus species A to E to determine how they interact with the host cell DNA damage response. Notably, serotypes from species B and D were unable to promote the degradation and/or relocalization of Mre11 and p53, although DNA ligase IV degradation was fundamentally conserved. Furthermore, species B and D serotypes induced the sustained overexpression of transcriptionally inactive p53, and induced both ATM and ATR kinase activity. As these events would typically be viewed as detrimental to virus survival, these data suggest that different adenovirus serotypes have evolved novel strategies in order to subvert the cellular DNA damage response during infection. Adenoviruses have long been utilised as useful tools for identifying, and characterizing, the function of fundamental cellular proteins such as tumour suppressors and those involved in the DNA damage response. Therefore, studies were also carried out to identify novel Ad12E1B54K-interacting proteins through mass spectrometric analysis, and subsequently to examine the functional significance of these interactions. Several putative novel Ad12E1B54K-interacting proteins were identified using this approach, one being the transcriptional intermediary factor 1γ (TIF1γ), a transcriptional regulator that has recently been identified as a tumour suppressor. Further studies determined that TIF1γ was relocalized to nuclear tracks in an E4ORF3-dependent manner early during adenovirus infection, and was subsequently degraded in a ubiquitin-mediated proteasome-dependent manner. Uniquely, TIF1γ degradation was shown to be E1B55K/E4ORF6-independent and E4ORF3-dependent. Data presented in this thesis also suggest that E4ORF3 does not utilise host cell cullin-based E3 ubiquitin ligases in order to promote TIF1γ degradation. The ability of E4ORF3 to target cellular substrates for degradation represents a novel way in which adenoviruses are able to target cellular substrates. Significantly, TIF1γ degradation was conserved during infection with Ad serotypes from species A to C which may highlight its importance for productive viral infection. It also appears that TIF1γ may have an as yet unidentified role in the DNA damage response since it was also found to interact with components of the ATR kinase pathway.

The outer membrane (OM) of Escherichia coli is an essential organelle. The OM allows E. coli to interact with its environment and has a critical function as a barrier to prevent the entry of toxic molecules into the cell. The OM is composed of phospholipids, lipoproteins, outer membrane β-barrel proteins (OMPs) and lipopolysaccharide (LPS). The correct ratio of these components is needed to ensure proper OM barrier function is maintained. Assembly of OMPs is performed by the Bam (β-barrel assembly machinery) complex, lipoproteins by the Lol (Localisation of lipoproteins) pathway and LPS by the Lpt (LPS transport) pathway. The factors responsible for the assembly of phospholipids at the OM are unknown. This study presents two key areas in understanding OM biogenesis. Firstly, a comprehensive mutagenesis screen was performed on the Bam complex member BamE. This analysis along with the structure of BamE has indicated crucial regions for BamE function. Secondly, we have performed a structure and function analysis on the previously uncharacterised protein, YraP. The structure of YraP has been solved and represents a novel fold. Additionally, we have obtained some functional evidence that suggest that YraP is involved in phospholipid biogenesis in the OM.

Thyroid cancers are the most common endocrine malignancies and their incidence continues to rise. Over-expression of the human pituitary tumor transforming gene (hPTTG) in thyroid carcinomas is a prognostic indicator of tumour recurrence. hPTTG is multifunctional with roles in mitotic control, DNA repair, genetic instability, cell transformation and apoptosis. Importantly, hPTTG transactivates expression of growth factors implicated in proliferation and angiogenesis, and represses the sodium iodide symporter (NIS), which is essential to radioiodine treatments in thyroid cancer. hPTTG interacts with a binding factor (PBF) which is an independent transforming gene and also represses iodine uptake. Work described in this thesis provides evidence for the existence of thyroidal autocrine regulatory pathways involving hPTTG and growth factors in vitro. We directly investigated the role of hPTTG in thyroid tumourigenesis through the generation and characterisation of a murine transgenic model with thyroid-targeted hPTTG over-expression (hPTTG-Tg mice). Unexpectedly, hPTTG over-expression was not sufficient for thyroid tumourigenesis. Investigations performed in hPTTG-Tg and Pttg-/- knockout mice indicated a particularly important relationship between hPTTG and the epidermal growth factor (EGF) in vivo. hPTTG and PBF were confirmed as repressors of NIS in vivo following studies in hPTTG-Tg and PBF-Tg mice respectively. The studies described in this thesis highlight the therapeutic potential of targeting hPTTG and PBF in thyroid cancer. To this effect, specific tyrosine kinase inhibitors prevented autocrine induction of hPTTG by growth factors, and siRNA depletion of PBF restored NIS function to normal levels in hPBF-Tg thyrocytes. Based on these data, hPTTG appears to play a dual role in endocrine tumourigenesis, being involved in both tumour initiation and subsequent progression towards more aggressive phenotypes.