Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Hypoxia (low tissue oxygenation) is an important signalling cue for many cell types. The study of its effects has direct relevance to surgery since hypoxic gradients are generated with every cut. On a cellular level, changes in molecular oxygen are sensed by the Hypoxia-Inducible Factors (HIFs). The HIFs are a family of transcription factors that are master regulators of over 100 genes and can effect changes in multiple cellular processes including migration, survival and differentiation. The broad nature of the response to hypoxia means that study of the HIF system is also important in cancer; where many tumour cells have found ways of subverting the HIF response to ensure their continued growth and survival. This thesis explores the role of hypoxia and the HIF system in the regulation of migration, survival and differentiation in both cancer and stem cells. The first experimental chapter examines the role of hypoxia and the HIF system in the regulation of migration and three-dimensional organisation in several cancer cell lines. Using biochemical and functional assays, the HIF system is shown to exert a pleiotropic effect across a panel of cancer cell lines. In particular, HIF 1α is shown to activate proliferation in a prostate cancer cell line in findings that may be useful to inform future clinical strategies for the management of this disease. In the second experimental chapter, the first epigenetic mechanism involving histone modification for the specific regulation of HIF 2α expression is characterised. Here the family with sequence similarity 60, member A (Fam60a) protein is shown to repress expression of the HIF 2α gene through its association with the class 1 Sin3-HDAC co-repressor complex, achieving specificity by co-operation with the SP1 transcription factor. This novel mechanism is demonstrated to be important in the regulation of the basal expression of HIF 2α. Modification of HIF 2α expression through this mechanism is shown to alter cell migration, three dimensional organisation and angiogenesis in vitro. The clinical importance of these findings is demonstrated in a series of 45 patients suffering from colorectal cancer of known stage. In this cohort, the reciprocal relationship between Fam60a and HIF 2α is maintained, and both are identified as potential novel biomarkers for the development of this disease. In the final experimental chapter, the role of hypoxia in the regulation of differentiation is explored. These effects are documented in mesenchymal progenitors primarily derived from human fat. Here, hypoxia is shown to regulate differentiation in a context-dependent manner, promoting osteogenic and retarding adipose and neural differentiation in-vitro. The roles of Fam60a and HIF 2α are explored in this system. These data may be useful in optimising future surgical engraftment of these cells for regenerative purposes.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Mammalian promoters often contain DNA-elements that can be bound by a number of closely related transcription factors (TFs) that cannot bind to the same DNA-element simultaneously. It is possible that each TF responds to distinct cues, allowing the gene to be activated in response to multiple stimuli. An alternative possibility is that each TF binds sequentially, each contributing to the pre-initiation events leading up to transcription. Here, we explore the exchange of basic-Helix-Loop-Helix-Leucine-Zipper (bHLH-LZ) factors, USF1, USF2 and MITF at the TYROSINASE promoter following induction by UVB-irradiation and methotrexate-administration. We demonstrate, for the first time in human melanoma, differential induction kinetics of TYROSINASE gene in response to an initial or re-induction, a phenomenon akin to "transcription memory" previously described in yeast. We also show that USF2, specifically detected by two different antibodies targeting the N-terminal region, is largely cytoplasmic, at least in the cell lines we have investigated. We also showed that nucleo-cytoplasmic shuttling of these USF2 species is partly regulated by glucose. Using deletion mutants, we demonstrated the requirement of the amino-acids surrounding the USF-specific region and the basic domain in nuclear localisation of USF2, and that amino-acids 1−193 appear to enhance dimerization of USF2 in addition to the classical HLH-LZ dimerization domain. We will further investigate the role(s) played by MYC, MITF, HIF and USF exchange at common targets (which we identified through our ChIP-seq analysis) in gene activation and the effect on the (re)activation potential of these genes when DNA-binding by one or more of these factors are abolished, as well as when the promoter is monopolised by one of these factors through overexpression using cell lines expressing one of the bHLH-TFs under a tet-inducible promoter. In the long run, we aim to understand the potential differences in the role(s) of each bHLH-factors co-occupying E-box elements.