Oral cancer has caused great concern in all of the western countries over the past two decades because of its progressively increasing incidence, mainly in young males, and its consistently low 5-year survival rate. Oral premalignant lesions (dysplasias) are thought to precede the development of cancer, but no clinical or histological criteria is at present available to predict their potential for malignant transformation. Therefore, it would be of diagnostic and therapeutic relevance to understand the molecular event, involved at different stages of oral cancer. Using a unique series of primary cultures from biopsies of normal oral mucosa, dysplasias and squamous cell carcinomas we have identified molecular changes characteristic of early oral cancer progression. Our group reported previously that acquisition of the immortal phenotype is an early event in oral cancer development (McGregor et al. 1997): data obtained during the course of this thesis project indicate that about half of oral dysplasia cultures are immortal and this is associated with loss of expression of retinoic acid receptor (RAR)-b and the cell cycle inhibitor, p16INK4a, p53 mutations and increased levels of telomerase/hTERT mRNA. In contrast, increased expression of the EGF receptor (EGF-R), known to be a characteristic of oral cancer, does not occur until after the dysplasia stage in squamous cell carcinomas (SCCs). Interestingly, one atypical mortal dysplasia with a considerably extended lifespan has lost expression of RAR-b and p16INK4a, but it still expresses wild-type p53 (albeit at higher level than normal) and has not activated telomerase. Further experiments demonstrate that retroviral transduction of hTERT immortalises D17 and this is associated with telomere lengthening but p53 remains wild-type. In contrast, transduction of hTERT does not extend the lifespan of two other typical mortal dyplasia cultures (that retain RAR-b and p16INK4a expression), even though telomeres are lengthened. Thus, telomerase activation/telomere maintenance is not sufficient per se to immortalise mortal dysplasias without concomitant loss of RAR-b and/or p16INK4a, but p53 mutation is not required if telomerase is activated exogenously. However, further work is required to clarify whether the molecular changes associated with the acquisition of an immortal phenotype in culture have any clinical or prognostic significance.

An investigation was carried out to examine the production of the inflammatory cytokines IL1, IL1, IL6, TNF, and TNF in two tumour cell lines, the MCF-7 breast cell line and the T-24 bladder cell line, and in samples of breast, bladder, lung and ovarian tumours. Two methods were used to investigate cytokine production. These were the polymerase chain reaction method (PCR) to examine cytokine mRNA production and immuno-staining of frozen or paraffin-embedded tissue sections to demonstrate the presence of the cytokine polypeptide directly. In the PCR experiments, the most frequently found cytokine was IL6, followed by IL1. Only a few tumours of any type displayed TNF, and even fewer produced TNF. In the immunostaining experiments performed on frozen sections, IL1 and IL6 proteins were detected in sections of tumours which gave positive results with PCR. Cell phenotyping indicated that the IL1 and IL6 were probably being synthesised by the tumour cells themselves although there was lymphocyte infiltration in every section examined. In the immuno-histology study performed on the paraffin-embedded sections, a new collection of tumours was used. These tumours were not subjected to parallel PCR due to size of tumour samples being too small. The results obtained from these experiments conflicted with the results observed in the PCR study. IL1 was detected in all of the breast tumours used for immuno-histology but in none of the breast tumours in the PCR experiments. While the conflict could not definately be resolved, it was thought that the results of the immuno-histology experiments were more accurate as they detected expression of cytokine protein on a cellular scale. The immuno-histology experiments demonstrated that some tumour cells produced IL1 in breast and bladder carcinomas, and some produced IL6 in breast and lung carcinomas.

Aberrant methylation of CpG islands (CGIs) is associated with transcriptional silencing of key tumour suppressor genes in cancer and is a frequent epigenetic event in epithelial ovarian cancer (EOC). The methylation status of 24 CGIs in a retrospective group of 142 EOCs and 16 non-tumour adjacent tissues were analysed using methylation-specific PCR (MSP) and Combined Bisulphite Restriction Analysis (COBRA) methods. CGI methylation of at least one of these loci was a frequent event in both early (78%) and late stage (60%) disease. A group of loci were identified as being methylated in 64% of early stage tumours (CGIs linked to the OPCML, RASSFIA and HIC1genes). The HIC1 CGI was frequently methylated in matched non-tumour adjacent tissues, but not in normal ovarian surface epithelium, potentially representing an early epigenetic event in the carcinogenic process present even before apparent morphological change. Differential methylation hybridisation (DMH) of a 12K CGI microarray using ovarian cell lines identified methylation of a CGI located at the LMX1A gene. This CGI was shown by MSP to be a potential early epigenetic marker methylated in 75% of early stage ovarian tumours. 87.5% of the early stage tumours examined were methylated in at least one of four loci (LMX1A, OPCML, RASSFIA or HIC1). The clinical application of this group of methylated CGIs was examined in matched plasma from chemonaive patients with EOC for similar methylation changes. Methylation of LMX1A was detected in 43.3% of all plasma samples and in 48.2% of those patients with methylated LMXIA in their tumour. When methylation was detected in plasma, it was always detectable in the corresponding tumour. Therefore, detection of LMX1A methylation in plasma has a sensitivity of 48.2% and a specificity of 100%.

Epigenetic mechanisms have been implicated in the regulation of telomerase gene expression and here we show that specific modifications within the chromatin environment of the hTR and hTERT promoters correlate with expression of hTR and hTERT in ALT, normal and telomerase-positive tumour cell lines. Lack of expression of hTR and hTERT is associated with repressive histone modification, while, hTR and hTERT expression is associated permissive histone modifications. Methylation of lysine 20 H4 was not linked to gene expression but instead was specific to the hTR and hTERT promoters of ALT cells providing an insight into the differences between ALT and telomerase-positive cells as well as a novel marker for the ALT phenotype. Basal transcription machinery dynamics were also shown to be different between normal and cancer cells at the telomerase gene promoters. Modulation of the chromatin environment was also shown to cause re-expression or increased expression of hTR and hTERT further supporting the role of the chromatin environment in controlling telomerase gene expression. Epigenetic mechanisms are also shown to be involved in the repression of hTERT transcription in human mesenchymal stem cell (hMSCs) and modulation of the chromatin environment is shown to allow re-expression of hTERT expression, while the disruption of telomerase gene expression in human haematopoietic stem cells (hHSCs) in chronic myeloid leukaemia (CML) and the role of the chromatin environment was also studied. These data establishes how epigenetic mechanisms can contribute to transcriptional regulation of telomerase and also highlights the potential importance of epigenetics in senescence and tumourigenicity.

Radiotherapy is commonly used in the treatment of head and neck cancer. For early stage tumours, conventional radiotherapy techniques have a high cure rate and low levels of long-term complications. Patients with more advanced cancers have much lower cure rates and high levels of treatment-related complications. Intensity modulated radiotherapy (IMRT) is a new form of focussed radiation therapy. It has been used to reduce the radiation dose to normal tissue structures and increase the dose delivered to tumour bearing tissues. This potentially allows reduced side effects and increased tumour control compared to conventional radiotherapy. The rationale of this thesis was to test whether these twin goals could be achieved in head and neck cancer patients. The first part of the thesis describes improvements in patient immobilisation, optimisation of techniques for neck irradiation, and evaluation of the technique in a busy radiotherapy department. It includes pre-clinical evaluation of IMRT for different tumour sites, the development of quality assurance programs and the conduct of a national randomised controlled trial of parotid-sparing IMRT. This trial concluded that IMRT significantly reduced patient-reported xerostomia, allowed recovery of saliva production and improved quality of life. The second part of the thesis describes pre-clinical evaluation of techniques to escalate radiation dose in patients with larynx and hypopharynx tumours. A phase I/II clinical trial showed that higher doses of radiation can be delivered at the expense of an increase in acute radiation toxicity but without a measurable increase in late radiation side effects. In the larynx and hypopharynx groups, a possible increase in local control was observed. This thesis describes the process of evaluation of a new radiotherapy technology and could be used as a template for testing other new technologies in the future.

The purpose of this research was to enhance the targeted radiotherapy of two metastatic malignant diseases: neuroblastoma and prostatic carcinoma. By virtue of its high affinity for the norepinephrine transporter (NET), [131I]meta-iodobenzylguanidine ([131I]MIBG) has been used for the therapy of tumours of neuroectodermal origin for more than 25 years. Although not yet universally adopted, [131I]MIBG targeted radiotherapy remains a highly promising means of management of neuroblastoma. MIP-1095, a glutamate-urea-lysine dipeptide has high affinity for prostate-specific membrane antigen (PSMA) and has recently demonstrated exquisite specificity for PSMA-expressing, metastatic prostatic carcinoma. Preliminary imaging studies in patients, using [123I]MIP-1095, revealed tumour-selective binding and prolonged retention only in malignant sites. This indicates the therapeutic potential of this agent when labeled with Iodine-131. Our aim is to make the most effective use of [131I]MIBG and [131I]MIP-1095 for the treatment of neuroblastoma and prostatic carcinoma by combining the [131I]-labelled radiopharmaceutical with radiosensitiser drugs. The thiol-containing molecule disulfiram was selected for combination with targeted radiotherapy because of its reported inhibition of the 26S proteasome and NF-kB activity, its ability to chelate copper and its pro-oxidative effects. The copper-dependence of the cytotoxicity and radiosensitising activity of disulfiram was established in neuroblastoma cell models. Radiation dose enhancement values at the 50% toxicity level were 4.24 and 2.00 in SK-N-BE(2c) and UVW/NAT cells, respectively. The radiosensitising mechanism of disulfiram-copper was shown to involve the inhibition of cell cycle arrest in G2. The enhancement of the cytotoxicity of [131I]mIBG and of [131I]MIP-1095 by disulfiram-copper was demonstrated by delay of the growth of multicellular tumour spheroids. Finally, the screening of the enhancing effect of chemotherapeutic agents on the spheroid growth delay induced by [131I]MIP-1095 indicated that combinations with topotecan, nutlin-3, bortezomib or olaparib have good prospects for therapy of metastatic prostate carcinoma. In conclusion, it is expected that DSF:Cu will enhance the outcome of patients undergoing targeted radiotherapy due to its radiosensitising properties.

One of the major problems associated with colorectal cancer is resistance to cytotoxic chemotherapeutic agents. New strategies are therefore required to inhibit colon cancer proliferation and survival. Here I use modulators of cAMP pathways, including inhibitors of phosphodiesterase 4 (PDE4) enzymes, which are under clinical development for other disease states, to inhibit the breakdown of cAMP and to assess the effects of raising intracellular cAMP on colon cancer proliferation and survival. I found that some chemo-resistant cancer cells are addicted to keeping low cAMP in PDE4 regulated compartments, and modulation of this pool causes G1/S-phase arrest and apoptosis. I also show that PDE4 controlled cAMP negatively regulates the PI 3-Kinase/Akt pathway, which some cells are addicted to for survival. Furthermore, I investigated the expression and role of PDE4 enzymes in metastatic colon cancer cells and assessed the effects of modulating their expression on survival. Also, I used a clinically relevant analogue of forskolin, an agonist of adenylyl cyclase, to examine the general effect on growth of epithelial cancer cell lines. This work might provide new strategies for the treatment of advanced colon cancer.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world, and remains incurable with current chemotherapy. Although CLL was long-regarded as an autonomous accumulation of functionally incompetent lymphocytes that escape apoptosis, significant rates of clonal proliferation and death have now been elegantly demonstrated in CLL patients in vivo. This, coupled with the high rates of spontaneous apoptosis observed on ex-vivo culture, confirms that CLL is a dynamic disorder, in which the tumour microenvironment is central to leukemic cell survival. Recent advances in CLL cell biology implicate signalling through the B cell antigen receptor (BCR) in the pathogenesis and progression of the disease. The absence of significant somatic hypermutation of the immunoglobulin heavy chain variable region (IgVH), which largely correlates with the expression of ZAP-70, in CLL cells is a significant adverse prognostic marker. CLL cases expressing an unmutated IgVH gene generally retain the ability to signal through the BCR. Components of the BCR signalling pathway are therefore attractive novel therapeutic targets, with potential selectivity for adverse prognostic groups. The non-receptor tyrosine kinases Lyn (a Src kinase) and c-Abl are both required for effective BCR signalling. Both are over-expressed, and constitutively active in CLL, and inhibition of either induces a degree of apoptosis. Dasatinib is a Src/c-Abl tyrosine kinase inhibitor in clinical use in chronic myeloid leukaemia. The main objective of this project was to conduct translational studies to determine the anti-leukaemic effects of dasatinib on CLL cells in vitro. While the Src kinase inhibitor PP2, and the c-Abl inhibitor imatinib induced apoptosis of CLL cells at micromolar concentrations, dasatinib induced apoptosis of CLL cells at clinically achievable nanomolar concentrations, with an EC50 in the region of 10-30 nM, and plateau in effect above 100 nM. CLL cell treatment with 100 nM dasatinib for 48 hr led to a mean reduction in viability of 33.7%, but with significant inter-sample variability. No correlation was observed between dasatinib sensitivity and the established prognostic factors clinical stage, ZAP-70 status, or cytogenetic subgroup. Notably, CLL cells known to contain the 17p deletion, resulting in p53 dysfunction, responded similarly to other samples. Apoptosis induced by dasatinib involved loss of mitochondrial membrane potential and was caspase-dependent. Although dasatinib treatment alone rarely induced apoptosis of over 50% of CLL cells, synergy was observed between dasatinib and the current first-line chemotherapeutic agents fludarabine and chlorambucil. Moreover, dasatinib exhibited synergy with a novel Bcl-2 inhibitor, and the HSP90 inhibitor 17-DMAG. Recently, antigen-independent ‘tonic’ BCR signalling has been linked to the pathogenesis of B cell lymphomas. Tonic signalling is proposed to be mediated by basal activity of Lyn and Syk kinases recruited to the BCR. As Syk is also over-expressed in CLL, we hypothesised that dasatinib sensitivity may correlate with inhibition of components of tonic BCR signal transduction. Indeed, CLL cells contained constitutively phosphorylated SykY348. Furthermore, a significant inverse correlation was observed between basal SykY348 phosphorylation and dasatinib sensitivity in individual samples, suggesting its’ utility as a biomarker of response. Dasatinib consistently inhibited an increase in SykY348 phosphorylation on BCR crosslinking. In addition, dasatinib inhibited calcium flux, and prevented Akt and MAPK phosphorylation on BCR stimulation. Moreover, dasatinib prevented up-regulation of Mcl-1 and blocked the increase in CLL cell survival observed on prolonged BCR stimulation, confirming inhibition of BCR signalling as a functionally relevant treatment strategy in CLL. Although dasatinib prevented CXCR4 down-regulation following BCR stimulation of CLL cells, dasatinib also specifically inhibited PI-3K/Akt activation upon CXCR4 stimulation by SDF-1, resulting in reduced actin polymerization and migration following SDF-1 stimulation. While the full translational implications of these observations remain to be determined, these data demonstrate that the anti-leukaemic effects of dasatinib extend beyond direct inhibition of BCR signal transduction. It is well recognised that bone marrow (BM) stromal cells and blood-derived ‘nurse-like’ cells protect CLL cells from spontaneous apoptosis in vitro. More recently, proliferating CLL cells have been identified within specialised ‘proliferation centres’, admixed with appreciable numbers of T lymphocytes, predominantly activated CD4+ T cells expressing CD40 ligand (CD154), and interleukin 4 (IL-4). CD40/IL-4 stimulation of CLL cells in vitro leads to up-regulation of the anti-apoptotic Bcl-2 family proteins Bcl-xL and Mcl-1, and the pro-proliferative protein survivin, mimicking the expression profile of CLL cells isolated from patient lymph nodes (LNs). We were interested to determine whether the effects of dasatinib on CLL cells were modulated by these microenvironmental factors. To achieve this, CLL cells were co-cultured with either the murine BM stromal cell line NT-L, or NT-L cells stably transfected to express CD154, the latter with IL-4 added to the culture medium (154L/IL-4 system). The pro-apoptotic effect of dasatinib in CLL cells was abrogated by stromal cell co-culture, with or without CD154 and IL-4. Stromal cell-mediated resistance to dasatinib involved Akt and MAPK signalling, as evidenced by the ability of both the PI-3K inhibitor LY294002, and the MEK inhibitor PD98059 to restore dasatinib sensitivity of cells in NT-L co-culture. 154L/IL-4 co-culture activated multiple MAPK, associated with up-regulation of Bcl-xL, Mcl-1, and survivin, which was not inhibited by dasatinib. Dasatinib also failed to inhibit CLL cell proliferation in the 154L/IL-4 system. While dasatinib retained the ability to sensitise CLL cells to both fludarabine and chlorambucil in NT-L co-culture, the addition of CD154 and IL-4 rendered cells resistant to all drug combinations. Dasatinib did however retain the ability to sensitise CLL cells to the HSP90 inhibitor 17-DMAG in both NT-L and 154L/IL-4 co-culture. In conclusion, these studies demonstrate that dasatinib offers much as a novel therapeutic strategy for CLL, overcoming pro-survival signalling through the BCR. However, our data suggest that dasatinib may be best utilised in combination treatment strategies with agents that can target antigen-independent signalling networks within the microenvironment. Collectively, this work provides valuable information that will inform future clinical trials of Src/c-Abl inhibitors in CLL.

The permanent cell cycle arrest known as cellular senescence is a major block to tumorigenesis. Currently the effects of latent senescence signaling on disease progression, response to therapy and outcome are poorly understood. Furthermore, the role of microRNAs in the regulation of senescence remains to be fully elucidated. For immortalisation to occur replicative senescence must be bypassed usually by activating a telomere maintenance mechanism (TMM). However, the expression differences between TMMs are also poorly understood. To address these questions a combination of gene expression and miRNA microarray profiling, virtual drug and siRNA kinase screening were utilised. These findings highlight the distinct roles of secretory and damage associated senescence pathways in disease progression and in response to therapy. Examination of the differentially expressed genes between TMMs also highlighted a differentially expressed gene expression network surrounding TERT, regulated by c-Myc and TCEAL7 in TMMs. These findings give further insight into the complex regulation network surrounding senescence signaling during tumorigenesis.

Chronic myeloid leukaemia (CML) is a myeloproliferative disease of stem cell origin. It is characterised by the Philadelphia chromosome and Bcr-Abl oncoprotein which is a constitutively activated tyrosine kinase that is causative in CML. Treatment of CML was revolutionized by tyrosine kinase inhibitors (TKIs) in the last decade. TKIs target Bcr-Abl and block its tyrosine kinase activity. Despite the success of TKI in eliminating differentiated CML cells, primitive quiescent CML stem cells still resist or persist with TKI treatment. In this study, several strategies have been investigated towards elimination of TKI-insensitive primitive CML stem cells. At first, the effect of nuclear entrapment of Bcr-Abl protein in CML cells was studied. Although Bcr-Abl protein is located in the cytoplasm of CML cells, TKIs, such as imatinib mesylate (IM), can induce the nuclear translocation of Bcr-Abl. Nuclear Bcr-Abl tyrosine kinase is capable of inducing apoptosis after drug washout, and leptomycin B (LMB) can trap translocated Bcr-Abl in the nucleus. Primary CML CD34+ cells were treated with IM and LMB either continuously or sequentially. It was found that neither regimen significantly increased the anti-proliferative effect of IM in primary CML CD34+ cells. It was also observed that the majority of Bcr-Abl was still retained in the cytoplasm of CML cells treated with IM and LMB, suggesting other mechanisms apart from its tyrosine kinase activity retained Bcr-Abl protein in the cytoplasm of CML cells. Next the subcellular distribution of Bcr-Abl was investigated in TKI-insensitive CML progenitors which survived 12-Day treatment of a potent TKI dasatinib (150nM). It was demonstrated that about 50% of Bcr-Abl was still retained in the cytoplasm of TKI-insensitive primary CML progenitors, although there was a significant increase in nuclear Bcr-Abl level in these survived cells compared to the no drug control (NDC). It was hypothesised that the cytoplasmic retention of Bcr-Abl was caused by its cytoplasmic binding partners, and it was found that a proportion of Bcr-Abl protein was associated with 14-3-3 proteins in the surviving cells, indicating the cytoplasmic retention of Bcr-Abl might be due to its binding with 14-3-3 proteins. In addition, due to the rapid nature of kinase inhibition and nuclear transportation, studies are required to measure the earliest time-point of inhibition of Bcr-Abl tyrosine kinase by IM. The common method to do this is to employ p-CrkL which has been widely used as a surrogate marker of Bcr-Abl tyrosine kinase activity, but the accuracy of p-CrkL as an indicator of Bcr-Abl tyrosine kinase status at early time-points during in vitro TKI treatment has not been examined. It was demonstrated that p-CrkL was not a reliable indicator of Bcr-Abl kinase activity within 24 hours of IM treatment in vitro and indicated that the early responses to IM and dasatinib were different. It was also observed that there was a rapid and active dephosphorylation of Bcr-Abl within 1 hour of TKI treatment, driven at least in part by protein tyrosine phosphatase activity. Furthermore, a farnesyltransferase inhibitor (FTI) BMS-214662 preferentially induces apoptosis in CML stem and progenitor cells compared to their normal counterparts, but another similar FTI BMS-225975 does not. However, the mechanism of action of BMS-214662 in inducing apoptosis of CML cells is not clear. When BMS-214662 was used as a negative control in the p-CrkL study, it was unexpectedly observed that the drug accumulated Bcr-Abl protein in CML cells. Thus, it was hypothesised that BMS-214662 may function as a proteasome inhibitor, which could result in accumulation of Bcr-Abl protein and further elevation of intracellular ROS level, leading to apoptosis of CML cells. Although BMS-214662 treatment induced accumulation of total ubiquitinated proteins and specifically Bcr-Abl protein in K562 cells, BMS-225975 had very similar effects, and this might result from the common mechanism of BMS-214662 and BMS-225975, which is their FTI activity. On the other hand, BMS-214662 induced significantly higher levels of intracellular ROS in both proliferating and non-proliferating K562 cells with respect to BMS-225975, indicating the production of ROS may be involved in the non-FTI mechanism of action of BMS-214662. However it was concluded that BMS-214662 was not a proteasome inhibitor like bortezomib. Finally, the use of synthetic low density lipoprotein (sLDL) as a vehicle for drug delivery to overcome the insufficient intracellular drug concentration in CML stem cells was investigated. Uptake and internalization of unloaded sLDL particles by CML cell line K562 and for the first time by CML stem cells was observed, demonstrating the targeting potential of sLDL particles in CML when they are loaded with drugs. Overall, this study provides further understanding of CML treatment and identifies some alternative strategies to target CML stem cells that may be used in combination with TKI to enhance the eradication of this stem cell-driven disease.