Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

This collection of studies begins by exploring the development of recognition memory in a group of healthy children and adolescents using experimental memory tests developed as part of this thesis. Various versions of these recognition memory tests were trialled in order to establish age appropriate tests for children aged 6-14 years. In keeping with previous literature in this area, these tests showed relatively stable familiarity memory throughout childhood compared to a steep developmental course for recollection memory. Paediatric brain tumour survivors are known to suffer from significant memory deficits following treatment. However, a clear description of this clinical group’s deficits, in terms of recognition and recall (and therefore also familiarity and recollection), has not previously been established. Using standard clinical memory assessments, the current body of work contributes to this area by characterising this population’s memory deficits as primarily recall-based, particularly when recalling information presented as prose. A sex difference is also noted; with female brain tumour survivors being significantly more impaired than their age-matched male counterparts. This finding is discussed with respect to the differing neural development of males and females. The experimental memory tests developed with normal children were also administered to a group of paediatric brain tumour patients. They were found to have a varied pattern of performance, including auditory recognition impairments but intact visual recognition, even when the test format incorporated similar foils. Associative memory tests revealed impairments in recollection-based recognition; this effect was dependant on the type of information being associated and the length of the encoding-test delay. A learning intervention was developed (and trialled with healthy children), using a method known as the ‘testing effect’, in an attempt to enhance recall of prose at long delays in a group of paediatric brain tumour survivors. Structured repeated retrieval was compared to repeated study for prose passages. This was found, with some patients, to be a successful method of improving recall after a delay of one week. Taken together, the work described in this thesis provides further understanding of recognition memory development in healthy children, novel insights into the residual memory function of paediatric brain tumour survivors and an exciting foundation on which to build a rehabilitation programme for this vulnerable group.

No description available.

Identifying exploitable differences between cancer cells and normal cells has been ongoing since the dawn of cancer therapeutics. This task has proven difficult due to the complex genetic makeup of cancers. Tumours, however, share a low oxygen (hypoxic) microenvironment that selects for malignant cancer cells. It has recently been shown that cells switch from eIF4E to eIF4E2-mediated protein synthesis during periods of hypoxia, similar to those found in tumour cores. We hypothesize that this hypoxic translation complex is required for cell survival in hypoxia and can be targeted by inhibiting the eIF4E2 cap-binding protein. Here, we show that genetically diverse cancer cells require the cap-binding protein eIF4E2 for their growth, proliferation, and resistance to apoptosis in hypoxia, but not in normoxia. Furthermore, in vitro and in vivo eIF4E2-depleted tumour models cannot grow or sustain hypoxic regions without the reintroduction of exogenous eIF4E2. Thus, tumour cells could be targeted over somatic cells by selectively inhibiting their protein synthesis machinery, much like the function of antibiotics that revolutionized medicine.

Developing and applying pre-clinical tumour models in order to determine the mechanistic action of applied therapies is essential if we aim to improve antitumour strategies in the clinical setting. The chaotic nature of tumour vasculature impacts directly on the effectiveness of combined chemo-radiotherapy and antiangiogenic (AA) strategies and as such warrants closer study. This work looked at the effects of novel AAs combined with clinically relevant radiotherapy (RT) using both conventional murine xenograft growth delay studies and real-time imaging. The imaging methodology was the non-invasive Dorsal Window Chamber/Intra Vital Microscopy (DWC/IVM) model which allows the study of real-time vascular responses to these therapies. The DWC/IVM model was applied to determine whether the DNA repair inhibitors Nicotinamide, AG14361 and AGO14699 had additional modes of action which could contribute to tumour radioresistance/radiosensitivity. Using the DWC/IVM model a secondary quantifiable mechanistic function was determined where these drugs also increased tumour vessel permeability. The DWC/IVM model was also used to investigate the effects of AZD2171 and AZD6244 combined with radiotherapy. These agents can inhibit angiogenic signalling pathways and it was demonstrated that both drugs worked by reducing tumour microvascular density when used in combination with radiation. In studies looking at the influence of hypoxia inducible factor-1(HIF-1) on tumour response to radiation the DWC/IVM model provided measurable differences in the microvascular density between HIF-1 deficient and HIF-1 competent tumours. The DWC/IVM model allowed the direct visualisation and quantification of the less well developed vasculature in HIF-1 deficient tumours compared to that found in HIF-1 competent tumours. The results provide a mechanistic basis for understanding the improved response to radiotherapy of HIF-1 deficient tumours. By applying the DWC/IVM model to conventional murine xenograft models the DWC/IVM proved itself as a useful research tool where continuous real-time non-invasive measurements could be made without the need for large numbers of time points or laborious histological analysis.

>Magister Scientiae - MSc / P53 is one of the most important tumour suppressor proteins in the body which protects the cell against the tumourigenic effects of DNA damage by initiating processes such as apoptosis, senescence and cell cycle arrest. Regulation of p53 is key — so that the abovementioned processes are not initiated inappropriately. The principle negative regulator of p53 is Murine Double Minute 2 (MDM2), a RING finger-containing protein which catalyses the attachment of lysine48-linked poly-ubiquitin chains, targeting it for degradation by the 26S proteasome. It has been found to work in conjunction with the MDM2 homologue MDMX. Retinoblastoma Binding Protein 6 (RBBP6) is a RING finger-containing protein known to play a role in mRNA 3’-end processing, as well as interacting with p53 and another crucial tumour suppressor, pRb. It has previously been shown to cooperate with MDM2 in the ubiquitination and degradation of p53 in vivo and acts as a scaffold. The objectives of this project are to investigate the proposed role of RBBP6 in the MDM2-catalysed ubiquitination of p53 using a fully in vitro ubiquitination system. Due to the difficulty of expressing full length RBBP6 in bacteria, a shortened version, dubbed "R3" was used which includes the RING finger domain but excludes the domain identified in earlier studies as the p53-binding domain. Proteins required to set up the fully in vitro p53 ubiquitination assays – including E1 and E2 enzymes, MDM2, R3, p53 and ubiquitin - were all successfully expressed in bacteria. The active 26S proteasome was successfully purified out of human cell lysates using antibodies targeting the α2-subunit. Cloning, expression and purification results showed that p53, MDM2 and R3 were not very stable proteins to work with — with degradation being initiated almost immediately after expression and purification which progressed during the downstream processing of the proteins. Although levels of intact protein were not always high, they were sufficient for in vitro assays. MDM2 and GST-R3 were both capable of poly-ubiquitinating p53 independently in "partially in vitro" assays using human cell lysate. The fully in vitro ubiquitination of p53 using MDM2 and R3 was established based on the well-known MDM2/MDMX system. When acting together R3 and MDM2 was shown to produce poly-ubiquitination which is lysine-48 linked and recognised by the 26S proteasome leading to degradation. When the proteasome inhibitor MG132 was added, the poly-ubiquitinated p53 was rescued from degradation. R3 was also shown to successfully poly-ubiquitinate p53 independently of MDM2 and also interact with p53 in vitro. These results suggest R3 to be of the same order of importance as that of MDM2 — which is known to be the most important regulator of p53. It would also rule out the proposed model of RBBP6 functioning as a scaffold as it is able to poly-ubiquitinate p53 independent of MDM2. These results allow us to better understand the mechanism in which p53 is down-regulated by E3s. / National Research Foundation (NRF)

Magister Chirurgiae Dentium (MChD) / TI and T2 squamous cell carcinomas of the head and neck have an unpredictable prognosis that often pose therapeutic problems. Sophisticated methods such as cytometric DNA analysis, immunocytochemistry and detection of cellular growth factors, have been applied with varying success rates for predicting recurrences, metastatic rates and overall prognoses. However, with the general lack of resources in Africa, devising a simple, reliable, reproducible and cost-effective method of predicting tumour behaviour to aid optimal treatment planning is imperative. Surgical excision specimens of forty-eight primary Tl and T2 squamous cell carcinomas of the floor of the mouth and tongue were histologically evaluated by two individual pathologists (double-blinded study) who had no prior knowledge of clinical course or outcome.

Cancer is a public health burden which continues to cause many deaths and an economic burden worldwide. New and improved ways of thinking about anti-cancer drug design and development are needed now and in future. Recent reports demonstrate the key role played by the tumour microenvironment (TME) in tumour progression and the development of drug resistance. This study investigated the interactions between cancer cells and the stroma within the TME, specifically fibroblasts, mesenchymal stem cells (MSC), cancer stem cells (CSCs) as well as the extracellular matrix (ECM), with the goal to develop an in vitro model that mimics solid tumours in terms of cellular characteristics and drug response. Mesenchymal stem cells were investigated as potential sources of cancer-associated fibroblasts (CAFs) in solid tumours. The expression of CAFs markers, α-SMA and vimentin, increased significantly in MSCs co-cultured with oesophageal and breast cancer cells indicating conversion of MSCs into cell-like CAFs. WHCO1 (oesophageal) and MDA MB 231 (breast) cancer cells co-cultured with MSCs survived paclitaxel and cisplatin treatments better than cancer cells alone. To assess the prognostic value of CSCs, the expression and malignant behaviour of CSC markers were also examined in clinicopathologically-confirmed oesophageal cancer biopsies and in vitro. Oesophageal cancer biopsies stained strongly for the cancer stem cell markers, CD44 and ALDH1A1, demonstrating the presence of CSCs in these tumours. FACS-isolated side population cells exhibited high levels of cancer stem cell markers, self-renewal markers and drug resistance proteins and were associated with increased drug resistance versus cancer cells. In order to measure how ECM proteins affect oesophageal cancer cell response to chemotherapeutic drugs, 3D cell-derived ECMs was used as a model. The analysis of ECM proteins using qRT-PCR in oesophageal cancer biopsies showed that collagens, fibronectin, and laminins were overexpressed in tumour tissue compared with adjacent normal tissues. The culture of cancer cells on decellularised ECMs reduced the effect of drugs on cancer cells compared to those plated on plastic (control). The reduction of the effects of drugs was associated with significant activation of survival signalling pathways. Knockdown of collagen and fibronectin with siRNA combined with drugs resulted in increased sensitivity of cancer cells to drugs and lower colony formation and cancer cell migration. Lastly, this study utilized multi-cell tumour spheroids (MCTS) from WHCO1 and MDA MB 231 cells co-cultured with WI38 and CT1 fibroblasts to mimic tumour cell-stromal cell interactions as observed within the in vivo tumour microenvironment. The data show that spheroids were more resistant to drugs than monolayer cultures of the same cells. MCTS displayed characteristics similar to in vivo tumours in terms of response to drugs. Associated with these findings were increased levels of CSCs in MCTS compared to monolayer. This study demonstrated that MSCs are a possible source of ‘CAFs' in vivo and can support cancer cell growth. This study also demonstrated the presence of CSCs in tumours and that the targeting of these cells can shrink tumours and prevent potential metastasis and relapse of tumours. This study revealed that ECM proteins play major roles in the response of cancer cells to chemotherapy and suggest that targeting ECM proteins, especially type I collagen and fibronectin, can be an effective therapeutic strategy against chemoresistant tumours. MCTS, as shown in this study, is a valuable tool for the evaluation of the therapeutic effect of drugs. Overall, this study demonstrates the critical role played by the tumour microenvironment in tumour growth and metastasis and provides new insights into cancer treatment.

Drosophila melanogaster is a valuable model for studying various aspects of human cancer, including proliferative capacity, invasiveness and metabolic adaptation typical of tumour cells to support cell growth. One of the major players in this process is Myc, which can promote tumorigenesis by triggering a metabolic reprogramming that allows cells to produce macromolecules, by modulating glycolytic flux, glutaminolysis, lipidogenesis, and autophagy. The process by which hyperproliferative cells undergo metabolic reprogramming to sustain growth can be recapitulated in the epithelial cells from Drosophila imaginal discs, where different levels of Myc induce cell competition. This process is a mechanism for selection of cells expressing higher level of Myc that acquire a super-competitor condition, with the ability to non-autonomously kill the neighbouring slow-growing cells. The direct connection between Myc, glutamine metabolism and autophagy and their role in competitive events between cancerous cells and wild type cells have not been clearly explained; therefore, the main purpose of this project is to determine a plausible link between Myc and autophagy, by examining the dependency of Myc-induced autophagy on glutaminase and major regulators of autophagy, such as TOR, Atg1, Atg5 and ammonia, a by-product of glutamine catabolism, by dissecting these mechanisms both in normal epithelial clones and hyperproliferating RasV12 -expressing cells. Our results show that Myc promotes the transcription of glutamine-related genes and the production of ammonia, and that glutaminase is necessary for Myc-induced autophagy in epithelial cells of clones of the wing imaginal discs, with a mechanism independent from TOR and Atg1. Conversely, the effect of Myc on autophagy induction is mediated by Atg5. We then investigated the contribute of Myc in autophagy in RasV12-transformed cells, that upregulate Myc to sustain growth and hyperproliferation. Intriguingly, our data report that autophagy is increased non-autonomously in neighbouring wild type cells, and that this non-autonomous RasV12-driven autophagic flux depends on Myc activity. Moreover, downregulation of glutaminase in RasV12-expressing cells significantly reduces non-autonomous autophagy. Collectively, our results give new insights on how glutamine metabolism can contribute to Myc-induced autophagy and how this enhances cancerous cell fitness.

<p> Oncolytic viruses are able to selectively replicate in tumour cells and are an attractive new avenue of cancer therapy that lacks the toxic side effects of current treatment modalities. HSV-1 mutants lacking ICPO are promising oncolytic vectors, however, the mechanisms behind viral oncolysis remain unclear. Since PML contributes to the repression of HSV-1 and also is downregulated in various types of cancer, but particularly in prostate cancer, PML has been implicated as a factor influencing the permissiveness of tumour cells to I CPO-null HSV-1 oncolysis. By screening a series of immortalized patient matched normal and tumour prostate epithelial cells for sensitivity to ICPO-null HSV-1 oncolysis and evaluating the levels of PML in each cell line, we were unable to establish a link between PML status and permissiveness to ICPO-null HSV-1 oncolytic vectors. Also, since a large proportion of the population possesses pre-existing immunity to HSV -1, which may hinder systemic administration of HSV-1 vectors, we sought to determine if BHV-1 could be an alternative oncolytic herpesvirus. BHV-1 was cytotoxic to various human immortalized and transformed cell lines in vitro, but was generally more restricted from normal human cells, suggesting that BHV -1 may have potential as an oncolytic virus. However, the sensitivity of human cells to BHV -1 infection did not correlate with type I IFN signaling, as has been demonstrated for other oncolytic viruses. Furthermore, neutralizing antibodies against HSV-1 were unable to cross-react with BHV -1 in vitro suggesting that pre-existing immunity to HSV -1 in humans may not hinder BHV -1 infection. It is hoped that these results will contribute to the understanding of viral mediated oncolysis and also provide some evidence that BHV-1 may be a new alternative oncolytic herpesvirus, however, in vivo studies are necessary to evaluate the oncolytic efficacy of BHV -1. </p> / Thesis / Master of Science (MSc)