Aim: Fifty percent of patients with squamous cell carcinoma of the Head and Neck develop loco-regional recurrence after treatment. Factors leading to this failure are most likely altered intra-tumoural glucose metabolism and increased hypoxia. Tissue glucose utilisation and the degree of hypoxia can be visualised by CTPET imaging with 18FDG and hypoxic radio-nuclides. This thesis has investigated 18FDG CT-PET guided target volume delineation methods and attempted to validate 64Cu-ATSM as a hypoxic radio-nuclide in patients with squamous cell carcinoma of the Head and Neck. Materials and Methods: Eight patients with locally advanced disease underwent 18FDG CT-PET imaging before and during curative radiotherapy or chemo-radiotherapy. Fixed (SUV cut off and percentage threshold of the SUVmax) and adaptive thresholds were investigated. The functional volumes automatically delineated by these methods and SUVmax were compared at each point, and between thresholds. Four patients with locally advanced disease, two to seven days prior to surgery, underwent 3D dynamic CT-PET imaging immediately after injection of 64Cu- ATSM. Two patients were also imaged 18 hours after injection, and two underwent a dynamic contrast-enhanced CT to evaluate intra-tumoural perfusion. All patients received pimonidazole before surgery. The pimonidazole, GLUT1, CAIX, and HIF1a immuno-histochemical hypoxic fractions were defined. Staining was correlated with the retention pattern of 64Cu-ATSM at 3 time points. Hypoxic target volumes were delineated according to tumour to muscle, blood and background ratios. Results: 18FDG primary and lymph node target volumes significantly reduced with radiation dose by the SUV cut off method and correlated with the reduction in the SUVmax within the volume. Volume reduction was also found between thresholds by the same delineation method. The volumes delineated by the other methods were not significantly reduced (except the lymph node functional volume when defined by the adaptive threshold). 64Cu-ATSM correlated with hypoxic immuno-histochemical staining but not with blood flow. Tumour ratios increased with time after injection, which influenced the delineated hypoxic target volume. Conclusion: Dose-escalated image-guided radiotherapy strategies using these CT-PET guided functional volumes have the potential to improve loco-regional control in patients with squamous cell carcinoma of the Head and Neck. CT-PET 18FDG volume delineation is intricately linked to the method and threshold of delineation and the timing of the imaging. 64Cu-ATSM is promising as a hypoxic radio-nuclide and warrants further investigation.

Epigenetic control of gene expression is vital for normal development and differentiation of cells, and is also important in the development of disease. In particular, there is a strong association between hallmark epigenetic changes and cancer – namely, genome wide hypomethylation, gene specific hypermethylation and characteristic histone modification. Almost all studies of cancer epigenetics to date have been conducted in malignant tissues or already transformed cell lines, and therefore do not take into account epigenetic changes occurring during the process of transformation. Our lab has developed a line of primary mesenchymal stem cells (MSC; thought to be the origin of various types of sarcoma) in which five oncogenic steps towards a fully transformed state are sequentially introduced including: human telomerase, necessary to extend the life span of MSC in culture, genes to inactivate the p53 and pRb tumour suppressor genes and genes to activate the oncogenes c-Myc and Ras. I hypothesized that hallmark epigenetic changes take place in this step-wise model of transformation, and aimed to investigate genome wide hypomethylation and the activity of the polycomb repressive 2 (PRC2) complex in this system. Utilizing the PCR based technique MethyLight, I show that transformed MSC are hypomethylated compared to parental MSC, with this decrease in methylation occurring on the introduction of oncogenic H-Ras in the final step. I also show that this hypomethylation is a gradual event following H-Ras expression, and transformation can take place in the absence of hypomethylation. I demonstrate that the three core components of the PRC2 complex are up-regulated during step-wise transformation and that PRC2 target genes are down-regulated. Finally, I show that MSC are able to be transformed when the PRC2 components EZH2 and SUZ12 are knocked down before the final oncogenic hit. These studies show that hallmark epigenetic changes occur during step-wise transformation and suggest that tumour-associated epigenetic changes occur following genetic aberrations. This model is valuable and relevant to further explore the mechanisms behind epigenetic alterations in cancer.

Purpose: The human epidermal growth factor receptors EGFR (erbB1) and HER2 (erbB2/neu) are involved in mediating resistance to chemotherapy and ionising radiation (IR). In vitro studies demonstrate that small molecule tyrosine kinase inhibitors (TKIs) which target these receptors can increase the effectiveness of DNA damaging agents. However, these combinations have failed to produce the clinical results anticipated and one potential explanation is that the inhibition of EGFR and HER2 cell signalling pathways by TKIs is short lived, with cells able to switch to alternative mechanisms of signalling through HER3. The purpose of this study was to examine whether the duration of exposure to TKIs modulates the induction and repair of DNA damage produced by chemotherapy or IR and describes attempts to elucidate the role of HER2 in mediating resistance to chemotherapy. Experimental design: Two HER targeting TKIs, lapatinib and gefitinib were investigated. The effect of lapatinib in combination with cisplatin and doxorubicin on the inhibition of cell proliferation and the role of schedule were examined in drug combination assays. The influence of the duration of exposure to TKIs on the induction and repair of DNA lesions induced by cisplatin, IR, doxorubicin, etoposide and m-AMSA were investigated using the alkaline and neutral Comet assays and measurement of γH2AX and RAD51 foci. DNA expression arrays were used to identify the potential mechanisms through which HER2 produces resistance to cisplatin in cells transfected with HER2. Results: Lapatinib is able to synergistically inhibit cell proliferation in combination with cisplatin or doxorubicin in a schedule dependent manner. Duration of exposure to TKIs has no effect on the induction of DNA lesions by cisplatin or IR, but significantly reduces the production of DNA double strand breaks by doxorubicin, etoposide and m-AMSA in part through the down-regulation of the expression of topoisomerase IIα (Topo IIα), increasing resistance to these drugs. Conclusions: These results indicate the scheduling of small molecule TKIs targeted against EGFR and HER2 is important and continuous exposure to these drugs induces resistance to doxorubicin, etoposide and m-AMSA, through reduced expression of their target, Topo IIα. The importance of schedule should be considered when combining TKIs with chemotherapy in clinical practice.

Antibody directed enzyme prodrug therapy (ADEPT) is a drug delivery system developed for the treatment of cancer. ADEPT uses a systemically administered antibody, tethered to an enzyme, to localize enzyme in tumour deposits. When the antibody-enzyme has cleared from the circulation, a low-toxicity prodrug is given. The prodrug is converted by the tumour-bound enzyme into an active cytotoxic drug. The system has potential to generate a highly potent cytotoxic agent at the tumour site. A clinical ADEPT system using MFECP1, a recombinant fusion protein consisting of an anti-carcinoembryonic antigen single chain Fv antibody and the bacterial enzyme carboxypeptidase G2, in combination with a bis-iodo phenol mustard prodrug (BIP) has been developed. A previous phase I/II clinical trial established the maximum tolerated dose of a single treatment cycle of this ADEPT system. In-vivo models with human tumour xenografts indicate that repeated ADEPT treatment with MFECP1/BIP led to greater efficacy without increased toxicity. This thesis aims to establish conditions required for safe and effective ADEPT when using MFECP1/BIP in man. This was achieved by conducting a phase I/II clinical trial of repeat-treatment ADEPT and comparing and combining the results with data from the single-treatment trial. The combined dataset provided mechanistic and clinical information on 43 patients. Multiple parameters were investigated to examine the likely cause of toxicity and clinical risk factors for its occurrence. Efficacy was evaluated using CT, FDG-PET and serum tumour markers. The nature of the immune response to MFECP1 was investigated and possible strategies to reduce immunogenicity were developed. Results showed that repeated therapy was feasible in man and did not increase the risk of MFECP1 infusion reactions. At the maximum tolerated total prodrug dose for 2 ADEPT treatments, one of three patients experienced tumour response on FDG-PET imaging. This MD (Res) thesis significantly increases understanding of the conditions required for safe and effective ADEPT.

In dividing cells, centrosomes act as the primary microtubule organising centre to orchestrate mitotic spindle assembly. Bipolar spindle assembly is responsible for accurate segregation of sister chromatids, such that each daughter cell receives an identical copy of the genome. Changes in centrosome number can lead to a lack of mitotic fidelity and genome instability. Chromosomes are replicated in a controlled and timely fashion and the same is true for centrosomes so that cells enter mitosis with two centrosomes. Further to their role in spindle assembly, centrosomes are also important in cell cycle regulation and DNA damage checkpoint signalling. Centrosomes are particularly important in the regulation of neuroepithelial cell division in the developing brain: all known incidences of primary microcephaly are caused by mutations in centrosome or spindle pole proteins. Xenopus laevis Cep63 is a target of DNA damage kinase, ATM, and it’s important for the formation of bipolar mitotic spindles (Smith et al., 2009). Therefore, Cep63 is an exciting candidate for maintenance of genome stability. Human Cep63 has been identified as a centrosome protein (Andersen et al., 2003), but its function was uncharacterised. In this thesis Cep63 was shown to be a constitutive centrosome protein, which plays a role in the regulation of centriole duplication. Cep152 was identified as a Cep63 interacting protein; and Cep63 and Cep152 are dependent on each other for their centrosomal localisation. Cep152 is required for centriole duplication via recruitment of essential duplication factors, Plk4 and CPAP, to the centrosome (Dzhindzhev et al., 2010b, Cizmecioglu et al., 2010, Hatch et al., 2010b). Furthermore, mouse embryonic fibroblasts in which the Cep63 gene is disrupted show decreased centriole numbers and signs of genome instability. Intriguingly, preliminary analysis of mouse embryos points to a potential link between Cep63 deficiency and microcephaly. We propose that Cep63 and Cep152 function together to ensure correct centrosomal levels of the essential centriole duplication factors Plk4 and CPAP.

Preclinical studies of vascular disruptive and anti-angiogenic agents, combined with radiation, have demonstrated the potential for enhanced anti-tumour activity. However, the optimal strategy and scheduling for combining these treatments with radiotherapy remains uncertain. In this thesis, combretastatin-A4-phosphate (CA4P) given concurrently with fractionated radiotherapy has been studied using preclinical models, in addition to assessing the impacts of adding the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (L-NNA), or the anti-EGFR monoclonal antibody, cetuximab, to this combination. As part of an ongoing phase Ib clinical trial, the combination of CA4P and radiotherapy was investigated in patients with non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where concurrent cetuximab was also given. Functional imaging techniques, such as dynamic contrast enhanced (DCE)-CT and positron emission tomography (PET), provide non-invasive biomarkers, which can be harnessed to aid diagnosis, determine response to treatment and also offer prognostic information. The use of volumetric DCE-CT parameters as biomarkers of tumour hypoxia and angiogenesis in NSCLC has been explored here, with significant negative correlations demonstrated between DCE-CT parameters and immunohistochemical staining of intra-tumoural hypoxia. This illustrates the potential ability of volumetric DCE-CT to quantify whole tumour hypoxia in NSCLC. The translational research described in this thesis, has established that the vascular disruptive effects of CA4P can be safely used in combination with fractionated radiotherapy in the clinical setting, producing demonstrable tumour vascular effects. However, despite promising preclinical tumour growth delay effects, the addition of cetuximab produced dose-limiting cardiotoxicity. In patients receiving CA4P and radiotherapy, DCE-CT and circulatory biomarkers, including cytokines (VEGF, VEGFR-1, G-CSF and SDF-1), were utilised to assess treatment-induced changes in tumour vascularity and vasculogenesis. The findings in this thesis provide further information to guide future studies combining vascular targeted therapies and radiation, highlighting the role of DCE-CT and functional imaging in such work.

Prostate cancer is the most prevalent male cancer in the United Kingdom. The etiology of prostate cancer is complex and multifactorial, with diet appearing to play a crucial role in disease prevention. Diets consumed in "Westernized" societies are rich in fats and processed foods, while diets consumed in Asia, where prostate cancer incidence is comparatively lower, are rich in plant based foods. Epidemiological evidence supports the protective role of legumes and pulses, such as soybean, against prostate carcinogenesis. A putative chemo-preventive agent present in soybean and legumes is the Bowman-Birle Inhibitor (BBI), a protease inhibitor whose anti-cancer potential has been demonstrated in in vitro and in vivo models, including prostate cancer. The aim of this thesis was to investigate the effects of soybean and chickpea protease inhibitor concentrates (PICs) in LNCaP, DU 145 and PC-3 prostate cancer cell models that represented differing stages of carcinogenesis, namely initiation, promotion and metastasis. A pilot in vivo study was also carried out in mice injected with DU 145 cells to investigate the effect on tumour mass growth. Our study highlights anti-genotoxic activity of chickpea PIC that induced a reduction in H20 2-induced DNA strand breaks assessed by the Comet assay in LNCaP cells. Both PICs proved effective against cancer promotion, decreasing cell proliferation in all three cell lines in vitro, with cell-cycle arrest apparent at the S phase in PC-3 cells The findings suggest that soybean PIC may possess anti-metastatic activity given the observed inhibition of highly invasive PC-3 cells through Matrigel and associated down-regulation of MMP-l. A beneficial modulation of genes linked to the initiation, promotion and progression stages of prostate carcinogenesis was observed. In conclusion, our study supports the role of PICs as anti-cancer agents against prostate cancer and suggests possible mechanisms of actions accounting for the observed effects.

There is a lack of robust biomarkers of CRC risk. Epigenetic changes in the WNT-related SFRP4, a gene whose expression is down-regulated early in CRC development, may be a potential CRC risk biomarker. If SFRP4 promoter methylation proved to be a useful biomarker of CRC risk, it would have potential implications for CRC screening. In addition, it could be used as a surrogate endpoint for investigations of CRC risk modifying interventions. SFRP4 methylation at several CpG sites was quantified in macroscopically normal rectal mucosal biopsies from volunteers at a relatively lower and higher CRC risk in two studies viz. the BORICC Study and the DISC Study. In the BORICC Study, the mean SFRP4 methylation of the 5 CpG sites investigated was significantly (p=0.036) higher in those in the higher risk group than in healthy controls. In the DISC Study, SFRP4 methylation was also higher at all CpG sites in the higher risk groups than in healthy controls but the differences were not statistically significant. In the BORICC Study SFRP4 methylation was also quantified in buccal cells matched to the rectal biopsies for the volunteers at a relatively lower and higher CRC risk. In contrast with the findings from the rectal mucosa, SFRP4 methylation was significantly (p<0.001) lower at all CpG sites in those in the higher risk group than in healthy controls. At CpG sites 1 and 4 only, SFRP4 methylation in the rectal biopsies and buccal cells was correlated significantly (p=0.001 and p=0.041 respectively). The healthy controls in the DISC Study were entered into a 50 day dietary intervention study and randomised to two potential chemoprevention agents; resistant starch and polydextrose in a 2  2 factorial design. SFRP4 methylation levels were quantified before and after the dietary intervention. Individually, resistant starch and polydextrose had no detectable effect on SFRP4 methylation levels. However, there was evidence of an interaction between the two intervention agents which was qualitatively similar at all CpG sites investigated. This interaction was statistically significant (p=0.008) at CpG site 2. The biological interpretation of this interaction cannot be determined until the study is unblinded. This study has provided preliminary evidence that SFRP4 methylation may be a novel epigenetic biomarker of CRC risk and that measurement in DNA from buccal cells may be a useful surrogate for invasive measurements on rectal mucosa.

Hepatocellular carcinoma (HCC) often arises on the background of chronic liver disease, and effective systemic treatments for HCC are limited. The disease is common globally and the majority of those affected survive less than 1 year. Expression of Sulfatase 2 (SULF2), an extracellular heparan sulfate 6-O-endosulfatase which modulates growth factor/receptor tyrosine kinase and Wnt signalling, has been reported to be increased at the mRNA level in advanced HCC. This thesis has explored the potential of SULF2 as a candidate for targeted anti-cancer therapy. Expression of SULF2 was compared at the mRNA and protein levels in 6 HCC cell lines. The impact of SULF2 silencing on signalling pathways and cell growth was assessed in vitro and in vivo using shRNA. Three of the 6 HCC cell lines showed high SULF2 expression at both the mRNA and protein level. The effect of SULF2 gene silencing in HCC cells was cell line-dependent, with inhibition of Wnt-3a-induced β-catenin-dependent transcriptional activity in the HuH-7 cell line and inhibition of FGF-1/2-stimulated phosphorylation of ERK, and IGF-II-stimulated phosphorylation of AKT, in the SNU-182 cell line. SULF2 suppression significantly reduced cell growth and proliferation in both cell lines. Xenograft implantation using HuH-7 cells was completely abrogated by silencing of SULF2. Microarray gene expression analysis of HuH-7 cell lines showed that SULF2 suppression dramatically upregulated catalytically active angiotensin converting enzyme 2 (ACE2) at the mRNA and protein level. The level of the ACE2 product, the hepta-peptide angiotensin 1-7 that has been reported to have anti-proliferative and anti-angiogenic activities, was also increased. Recombinant SULF2 enzyme was produced and purified, and commercially available sulfatases were characterised, for screening of potential small-molecule inhibitors of SULF2. Together, the studies described in this thesis have shown that SULF2 is an attractive and tractable target for the treatment of HCC.

Many cell signalling pathways contribute to prostate cancer (PCa) initiation and survival, but the interplay between them remains poorly understood. In this thesis, conditional transgenic models were employed to investigate the impact of mutating the proto-oncogenes β-catenin, and Kras, and the tumour suppressor gene PTEN, alone and in combination within the murine prostate. β-catenin and Pten single mutants display progression from PIN to invasive adenocarcinoma that appears similar to human Gleason pattern 3. Tumours demonstrate co-activation of the Wnt, PI3K/Akt/mTOR, and Ras/MAPK pathways. Kras single mutants develop PIN but do not progress to adenocarcinoma. Double and triple mutants develop more aggressive tumours comparable to Gleason pattern 4. Furthermore, double and triple mutants display upregulated Wnt, PI3K/Akt/mTOR and Ras/MAPK signalling. Expression of p63 negatively correlates with tumour grade, and moreover, progression from PIN to invasive adenocarcinoma is characterised by the synergistic elevation of AR and Ki67, which parallels human PCa. Malignant transformation of a normal prostate epithelial stem cell (PrSC) gives rise to the cancer stem cell (CSC), which may contribute to castrate-resistant PCa. In this study a 3D primary tissue culture assay was optimised to allow maintenance and expansion of prostate cells that display stem-like characteristics in vitro. PrSCs can be isolated by fluorescence activated cell sorting for the antigenic profile Lin-Sca1+CD49f+TropHi, and consistently form spherical structures in Matrigel. PrSCs can withstand multiple round of passage in vitro, and demonstrate the ability to maintain an undifferentiated state. PrSC spheres possess a basal phenotype, supporting a basal location of PrSCs in prostate epithelium. In summary, these mouse models of PCa demonstrate cooperation between important cell signalling pathways in prostate tumourigenesis, and provide a platform for testing novel therapeutics. In addition, the successful isolation and cultivation of cells with stem-like attributes will allow their unique biological properties to be explored further, providing a baseline standard with which to compare the function of CSCs.