Many anticancer therapies, including radiotherapy, act by damaging the deoxyribosenucleic acid (DNA) that is fundamental to cell function and proliferation. H2AX is a histone protein associated with DNA that is phosphorylated to produce γH2AX in response to DNA double strand breaks (DSBs), the most lethal lesions caused in cancer cells. This thesis examines the translation of γH2AX detection assays to clinical situations in order to provide biomarkers of response that might help to guide the treatment of cancer patients. γH2AX immunohistochemistry was developed in preclinical xenograft models, and validated over a range of radiation doses and over time after irradiation. The method was prepared for translation to archived clinical biopsy and surgical specimens. The DSB Biomarkers Pilot Study was established in order to develop a method for γH2AX quantification in direct tumour cell specimens obtained using the clinical technique of fine needle aspiration (FNA) cytology. Eleven patients undergoing anticancer therapy were recruited to the study, and the method evaluated. The coefficient of variation of the measure was 49%. Non-invasive imaging for γH2AX would allow DNA damage to be quantified in all tumour sites, and on multiple occasions. An antibody-based nuclear medicine imaging agent was re-engineered using Fab fragments of the antibody. The novel agent demonstrated improved pharmacokinetics when compared to the whole antibody agent, but reduced target specificity. The findings further develop the potential to exploit DNA damage biomarker measurements in clinical oncology.

The skin is the site where two of the most common types of epithelial cancer, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), arise. In this work, we have investigated how ASPP2, a member of a family of proteins that interact with the p53 family, can affect skin tumourigenesis. ASPP2 is expressed in the squamous epithelia of various organs, localising exclusively in the upper and most differentiated layers. We show here that Balb/c ASPP2-null and heterozygous mice develop spontaneous SCCs. To investigate how the absence of ASPP2 from the epithelial compartment could lead to tumour formation, we analysed ASPP2’s relationship with pathways involved in the normal homeostasis of the epithelium, such as p63 and Notch. ΔNp63 is the main p63 isoform expressed in the adult epidermis, and its function is to drive the proliferation of the basal keratinocytes. Aberrant or misplaced activation of ΔNp63 in the epithelium is a known initiating cause for SCC. Consistent with this, ΔNp63 was found to be highly expressed in tumours derived from ASPP2-deficient mice. Our results indicate that ASPP2 is important in limiting ΔNp63 expression in the differentiated epithelium, preventing cell proliferation in the upper layers of the skin. This is achieved by antagonising ΔNp63 transcript and protein expression, resulting in a mutually exclusive expression pattern during differentiation of keratinocytes, as well as in epithelial cancer. ASPP2 expression was found reduced or lost in human SCC cell lines and during head and neck cancer progression, reflecting what was observed in ASPP2-deficient mice. Overall, our results indicate a possible mechanism by which p63 expression can be regulated in the skin, and provide a new model for the spontaneous formation of SCC in vivo. Additionally, we found that ASPP2 can cooperate with and enhance the activity of skin pro-differentiation pathways, such as Notch. In contrast to p63, ASPP2 and Notch1 are co-expressed in the differentiated layers of the squamous epithelium. Moreover, ASPP2 can interact with components of Notch nuclear transcriptional machinery, and it is shuttled into the nuclear compartment upon activation of Notch pathway. This recruitment results in modulation of Notch transcriptional activity on specific target genes with a differential pattern of binding sites, providing new insights into the understanding of Notch transcriptional regulation.

Magnetic resonance imaging plays an important yet underutilized role in determining the natural history and prognosis of oral carcinoma. Depth of tumour invasion is an emergent factor in the oral cancer literature. However, problems exist with the definition of cut-points suitable for inclusion in TNM staging criteria. Statistical methodology represents a possible explanation but is underexplored. In this work, a review of the depth of invasion literature is conducted with emphasis on statistical technique. As well, statistical simulation is used to explore the implications of the of the minimum p-value method. The results demonstrate that the use of continuous variable categorization and multiple testing is widespread, and contributes to cut-point variability and false-positive tests. Depth, as a predictor of OCLNM and survival, must be questioned. The volume of tumour invasion is a promising prognostic factor that has not been fully investigated in the oral carcinoma literature. In this work, the volume of tumour invasion is measured on MRI and compared to thickness and maximum diameter in its capacity to predict 2-year all-cause, disease-related and disease-free survival, as well as occult cervical lymph node metastasis prediction. As part of a comprehensive approach, morphometric factors are incorporated into multifactor predictive models using regression, artificial neural networks and recursive partitioning. It is evident that MRI-based volume is superior all other linear measurements for both occult cervical lymph node metastasis and survival prediction. Artificial neural networks wee superior to all other techniques for survival prediction. There is a case for a unified artificial neural networks model for survival prediction that uses volume, midline invasion and N-stage to determine prognosis. This model can be used to determine individualized probabilities of 2-year survival. The lateral extrinsic muscles of the tongue lie just beneath the surface of the lateral tongue, yet their invasion is a criterion for T4 classification using the TNM staging system. In this work, the Visible Human Female is used to conduct an anatomic study of the extrinsic muscles of the tongue. Linear measurement is used to quantify the distance from the surface mucosa to the most superficial muscle fibres of the styloglossus and genioglossus. Further, the lateral extrinsic muscles are poorly demonstrated on MRI. An anatomic atlas of the tongue is fused with MRI images of oral carcinoma to demonstrate lateral muscle invasion. The results demonstrate that the styloglossus and hyoglossus lie very close to the surface of the lateral tongue, in some cases passing within 1 mm of the surface mucosa. These extrinsic muscles are readily invaded by even small tumours of the lateral tongue. Strict application of the TNM T4a criteria leads to unnecessary upstaging as these carcinomas do not warrant the prognosis and aggressive treatment of Stage IV disease. Extrinsic muscle invasion should be removed as a T4a criterion for the oral cavity. A separate category, T4a (oral tongue) specifying invasion of the genioglossus is also recommended. This work presented in this thesis is an original contribution to the field of oral cavity cancer research and has determined that there is capacity for improvement in current efforts to determine the natural history and prognosis of oral cavity squamous cell carcinoma. This thesis is the first to examine the role of statistical methodology in oral carcinoma depth of invasion cut-point variability. Further, this work presents an original approach to the prediction of regional metastasis and survival using advanced multivariate modeling techniques. No other work explored MRI-measured volume using the substantial sample size gathered in this thesis. Finally, this work is the first to demonstrate that lateral extrinsic muscle invasion is an unnecessary component of the T4a (oral cavity) classification criteria and should be reconsidered.

Pathogenic gain-of-function mutations associated with increased paternal age, albeit harmful to embryonic development, are paradoxically enriched in the normal testis. Evidence from previous studies indicates that these so-called paternal age-effect mutations confer a proliferative advantage to the spermatogonia in which they arise, leading to clonal expansion within the normal testis over time. Recently, spermatocytic seminoma (SS; a rare testicular germ cell tumour that occurs mainly in older men) has emerged as a key link between the processes of somatic and germline mutation (Goriely et al, Nat Genet. 41:1247-52, 2009), suggesting that the proposed clonal expansion events can in some cases lead to testicular tumourigenesis. In this thesis, I have used immunohistochemistry to seek evidence for putative clones of cells in the normal adult testis. To address this, a screening approach was developed using markers chosen from analysis of normal testicular tissues and SS. The ontogeny of OCT2 and SSX expression in human testis, from embryonic development to adulthood, identified distinct subpopulations of spermatogonia at different maturation stages. Together, these data reveal the potential of OCT2 as a novel marker of A_dark spermatogonia (human reserve spermatogonial stem cells). In parallel with these observations, two distinct types of SS characterised by differential OCT2 and SSX immunoexpression were identified, providing new evidence for heterogeneity of this tumour. This work provided the backdrop to the detailed immunohistochemical study of normal adult testis by characterising in serial sections the expression of five spermatogonial markers, MAGEA4, SSX, FGFR3, OCT2 and SAGE1, and a proliferation marker, Ki67. Independent sections were screened with predetermined criteria set to identify unusual positively-stained cellular clusters within the seminiferous tubules. Several antigenic combinations previously described in SS were observed in a subset of these clones, suggesting differing genetic origins and a possible link with early events of testicular tumourigenesis. The size (minimum number of cells) of each clonal event was estimated and its correlation with the staining pattern of the molecular markers was investigated. In summary, the data presented in this thesis convincingly identify for the first time the previously hypothesised clonal events in the testis using immunohistochemical markers. My research will pave the way for future work involving genetic analysis of microdissected cells from these putative clones, aimed at identifying the underlying mutational events thought to be present.

The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.

Cancer cells survive the harsh oxygen and nutrient deprivation of the tumour microenvironment through the selection of apoptosis-resistant and glycolytic clones (Cairns et al., 2011; Graeber et al., 1996). In particular, the integrated stress response (ISR) has been shown to be pivotal in cancer cell survival in vivo and the resistance of cancer cells to therapy (Harding et al., 2003). In recent years, it has become apparent that increased autophagy is one mechanism by which the ISR can confer resistance to stress (Kroemer et al., 2010). ATF4 is a major transcriptional effector of the integrated stress response in severe hypoxia (<0.01% O₂). ATF4 is a well-established regulator of genes involved in oxidative stress, amino acid synthesis and uptake, lipid metabolism, protein folding, metastasis, and angiogenesis. Recent work has demonstrated an important role of ATF4 in promoting resistance to severe hypoxia through the transcriptional upregulation of MAP1LC3B and ATG5, essential components of the autophagy machinery (Rouschop et al., 2009b; Rzyski et al., 2010). In this work, the author describes several novel ATF4 target genes, and examines their role in the regulation of autophagy and the resistance of cancer cells to severe hypoxia. In the first part of this thesis, the author shows that three BH3-only members of the BCL-2 family of proteins--HRK, PUMA, and NOXA--are upregulated in response to severe hypoxia in an ATF4-dependent manner. In particular, the author shows that the poorly described BH3-only protein HRK is a direct target of transcriptional activation by ATF4, and that HRK induces autophagy in severe hypoxia, thereby providing the first evidence that the integrated stress response can transcriptionally trigger the autophagy process. In contrast to the previously described role of HRK in apoptosis, this thesis demonstrates that HRK can play a pro-survival role in the context of breast cancer cells. In the latter part of this thesis, the author identifies the essential autophagy gene ULK1 as an ISR target. The author shows that ULK1 expression in severe hypoxia is transcriptionally upregulated through direct activation by ATF4. The author identifies ULK1 as a crucial regulator of autophagy and mitophagy in both normoxia and severe hypoxia and shows that ULK1 plays a pivotal role in cancer cell survival. Furthermore, it is shown that human breast cancer patients with high levels of ULK1 relapse earlier than those with low levels of ULK1, thereby identifying ULK1 as a potential target for cancer therapy.

Angiogenesis, the process by which new vessels form from existing ones, is a key event in the development of a large and malignant vascularised tumour. A rapid expansion in in vivo and in vitro angiogenesis research in recent years has led to increased knowledge about the processes underlying angiogenesis and to promising steps forward in the development of anti-angiogenic therapies for the treatment of various cancers. However, substantial gaps in knowledge persist and the development of effective treatments remains a major challenge. In this thesis we study tumour-induced angiogenesis within the context of a highly controllable experimental environment: the cornea micropocket assay. Using a multidisciplinary approach that combines experiments, image processing and analysis, and mathematical and computational modelling, we aim to provide mechanistic insight into the action of two angiogenic factors which are known to play central roles during tumour-induced angiogenesis: vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF). Image analysis techniques are used to extract quantitative data, which are both spatially and temporally resolved, from experimental images. These data are then used to develop and parametrise mathematical models describing the evolution of the corneal vasculature in response to both VEGF-A and bFGF. The first models developed in this thesis are one-dimensional continuum models of angiogenesis in which VEGF-A and/or bFGF are released from a pellet implanted into a mouse cornea. We also use an object-oriented framework, designed to facilitate the re-use and extensibility of hybrid multiscale models of angiogenesis and vascular tumour growth, to develop a complementary three-dimensional hybrid model of the same system. The hybrid model incorporates a new non-local cell sensing model which facilitates the formation of well-perfused and functional vascular networks in three dimensions. The continuum models are used to assess the utility of the cornea micropocket assay as a quantitative assay for angiogenesis, to characterise proposed synergies between VEGF-A and bFGF, and to generate experimentally testable predictions regarding the effect of anti-VEGF-A therapies on bFGF-induced angiogenesis. Meanwhile, the hybrid model is used to provide context for the comparison that is drawn between the continuum models and the data, to study the relative distributions of perfused and unperfused vessels in the evolving neovasculature, and to investigate the impact of tip cell sensing dysregulation on the angiogenic response in the cornea. We have found that by exploiting a close link with quantitative data we have been able to extend the predictive and hypothesis-testing capabilities of our models. As such, this thesis demonstrates the potential for integrating mathematical modelling with image analysis techniques to increase insight into the mechanisms underlying angiogenesis.

Prostate cancer (PCa) cells predominantly metastasize to bone and the complex crosstalk between PCa cells and osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells) leads to increased tumour growth and worsening of bone disease. Understanding the mechanisms of PCa bone metastasis can identify the aggressive fraction of PCa resulting in earlier intervention. The ability of PCa cells to express bone cell-specific features, termed osteomimicry, could potentially explain the osteotropic nature of PCa cells. The aim of this study was to determine the role of osteomimicry in the regulation of epithelial-mesenchymal transition (EMT) in bone metastatic prostate cancer cells. It was demonstrated that the osteoblast-specific marker alkaline phosphatase (bone/liver/kidney) (ALPL) was overexpressed in bone metastatic (ARCaPM), compared to non-metastatic (ARCaPE), human PCa cells. Knockdown of ALPL resulted in decreased cell viability, increased cell death and a change from mesenchymal to epithelial morphology in ARCaPM and PC3 cells, and increased CDH1 expression along with decreased migration in ARCaPM cells. Treatment with extracellular ATP also resulted in decreased viability, increased expression of epithelial markers (CDH1, KRT14) and decreased expression of mesenchymal markers (VIM, ZEB1), and reduced expression of ALPL in ARCaPM cells. Small RNA-sequencing identified microRNAs differentially expressed between ARCaPE and ARCaPM PCa cell lines: miR-373 expression was lower in ARCaPM compared to ARCaPE cells and its overexpression in ARCaPM cells resulted in a change to epithelial morphology, increased expression of the epithelial marker CDH1 and decreased expression of the mesenchymal markers VIM and ZEB1. Finally, the development of a high-throughput screening method to identify novel microRNA regulators of osteomimicry was described, which identified two microRNAs miR-199a-5p and miR-212 as positive regulators of ALP activity. Taken together, this thesis describes the identification of ALPL and ATP as novel regulators of epithelial-mesenchymal transition in PCa cells and high-throughput ALP-activity screening as a powerful tool to identify novel microRNA regulators of ALP expression.

Epidemiology of primary paediatric brain tumours has been studied extensively in developed countries of the west. Such studies are lacking in developing countries especially sub-Saharan Africa. This study seeks to establish the epidemiology of primary brain tumours seen among children that were treated at Chris Hani Baragwanath and Johannesburg Hospitals from April 1995 to April 2005. The records of 252 patients who presented with this condition during the study period were reviewed, for the following details: ● Demographic details such as age, gender and race ● Diagnosis and the date when it was made ● The follow-up period at the hospital(s)/clinic(s) ● The anatomical location of the tumours; supratentorial or infratentorial ● The treatment that was given which included mainly surgery for tumour removal or biopsy, radiotherapy, chemotherapy and others which included ventriculoperitoneal shunt, external ventricular drain insertion. ● The outcome of treatment included: - alive - dead - presumed alive - lost to follow-up It was found: ● That 225 patients had full demographic details of race, gender and age. ● That there was a slight male predominance among children with primary brain tumour. ● That the majority of children with brain tumours were black, followed by whites which is in keeping with the country’s demographics. ● The three most common tumours were astrocytomas, medulloblastomas and brainstem gliomas in the descending order of frequency. ● Medulloblastomas were the commonest tumours in the infratentorial region while craniopharyngiomas were commonest tumours in the supratentorial region. ● More children had infratentorial tumours ● Younger children were more likely to have infratentorial tumours. ● Majority of patients had surgery either for diagnosis or for diagnosis and treatment. ● Few patients were presumptively diagnosed clinically and by imaging modalities ● Combination therapy of surgery, chemotherapy and radiotherapy had the best survival outcome while chemotherapy as the only form of treatment had the worst outcome. ● The overall 5 year survival rate for all the study participants was much lower than that of their counterparts in the literature. ● Children who had craniopharyngiomas and astrocytomas had better survival. ● Mortality incidence was slightly higher for whites than blacks but that could have been skewed by a high number of blacks that was lost to follow-up. ● A higher infratentorial tumour prevalence than in the literature was noted. It was noted that racial prevalence of primary paediatric brain tumours follows population demographics. From the results of this study, there is a need for a better record keeping and improved patients follow-up. There is also a need for a larger epidemiological study in the two hospitals. There is need to establish a specialized paediatric unit which will help start a paediatric team comprising of a paediatric neurosurgeon, paediatric oncologist, paediatric intensivist and neuroradiologist with dedicated neuropaediatric ICU. Such a team given resources will improve survival outcome of children with brain tumours.

A proteína Akt modula a função de numerosos substratos envolvidos na regulação da sobrevivência celular, progressão do ciclo celular e crescimento celular. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Akt em adenoma pleomórfico, mioepitelioma e carcinoma adenóide cístico. O objetivo deste estudo foi analisar a via da proteína Akt através da avaliação da expressão das proteínas NFkB e PTEN em neoplasias benignas e malignas de glândulas salivares através das técnicas de imunohistoquímica, western blotting e imunofluorescência, e a possível interação protéica direta entre p-Akt/Mdm2 e p-Akt/PTEN em linhagem de carcinoma adenóide cístico. A superexpressão nuclear na proteína PTEN foi encontrada nas duas neoplasias malignas estudadas. Além disso, não foi observada interação direta entre as proteínas p-Akt/Mdm2 e p-Akt/PTEN, as quais apresentam localização nuclear em neoplasias de glândulas salivares. / The Akt protein modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cell growth. Previous studies from our laboratories showed overexpression of Akt and Mdm2 followed by the lack of p53 expression in pleomorphic adenoma, myoepithelioma and adenoid cystic carcinoma. The aim of this study was to analyze the Akt pathway through evaluation of expression of NFkB and PTEN proteins in pleomorphic adenoma, carcinoma ex pleomophic adenoma and adenoid cystic carcinoma by western blotting, immunofluorescence and immunohistochemical techniques, and we have intended to analyse a possible direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein in salivary gland tumours. Overexpression of nuclear PTEN was present in both carcinomas studied. In addition, there was no direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein, which presents a nuclear localization in salivary gland tumours.