Abstract
Gliomas, the most common cancer of the central nervous system (CNS) in adults, are classified based on grade, with low-grade astrocytomas and oligodendrogliomas harbouring a mutation in isocitrate dehydrogenase 1/2 (IDH1/2), whereas, high-grade glioblastomas have wild-type IDH1/2. Despite multimodal treatment, gliomas are incurable, and survival is poor; 11-15 months for patients with glioblastoma.
Gliomas are highly hypoxic and the extent of this worsens with grade. The hypoxic tumour microenvironment activates the hypoxic pathway, resulting in tumour progression, angiogenesis and treatment resistance. Under normoxic conditions, the hypoxic pathway is regulated by the 2-oxoglutarate dependent dioxygenase enzymes, prolyl hydroxylases and factor inhibiting hypoxia inducible factor. These enzymes require 2-oxoglutarate and oxygen as substrates and iron and ascorbate as co-factors. Previous studies have shown an association between increased tumour ascorbate levels and decreased hypoxic pathway activity in endometrial, colorectal, breast, thyroid and renal cancers, however, the relationship between these factors in gliomas is unknown.
The brain contains one of the highest ascorbate levels in the human body, and is one of the last organs to become deplete during deficiency. One study has shown that ascorbate levels in astrocytomas were significantly lower compared to matched normal brain tissue. The sodium dependent ascorbate transporter 2 (SVCT2) is expressed by most cells, and is involved in intracellular ascorbate accumulation. The relationship between this transporter and ascorbate levels in the brain has not been studied.
The aims of this thesis were to measure ascorbate and hypoxic pathway activity in glioma. In clinical low- and high-grade glioma samples, DNA, ascorbate content and levels of hypoxic pathway proteins were related to clinicopathological data. In vitro, ascorbate uptake in glioblastoma cells was analysed and the influence of ascorbate supplementation on hypoxic pathway activity was measured. Additionally, an in vitro glioblastoma CRISPR/Cas9 knockout model was created targeting SVCT2. Subsequently, ascorbate uptake, activity of the hypoxic pathway and its response to ascorbate treatment were analysed in these genetically edited cells.
High-grade glioma samples with high tumour ascorbate had reduced hypoxic pathway activity compared to those with low ascorbate levels. The opposite relationship was seen in low-grade clinical samples. For high-grade gliomas, survival was increased with increased tumour ascorbate. In vitro, intracellular ascorbate accumulation was cell line dependent and relied on SVCT2. In SVCT2 knockout glioblastoma cells, ascorbate uptake was significantly reduced. In glioblastoma cells, the hypoxic pathway was induced using cobalt chloride, and ascorbate supplementation was able to inhibit this. Interestingly, in SVCT2 knockout cells, the cobalt chloride induced hypoxic pathway seemed to be similarly regulated by ascorbate.
Results from this thesis are the first to investigate ascorbate and the hypoxic pathway in gliomas. Clinical data indicates that IDH mutation status plays a role in the relationship between ascorbate and the hypoxic pathway, most likely due to the production of the 2-oxoglutarate analogue 2-hydroxyglutarate. This is clinically important, as this data suggests ascorbate treatment may not be beneficial to all glioma patients.
In conclusion, this thesis has shed light into the relationship between ascorbate and the hypoxic pathway in gliomas, however further research is required to fully understand this relationship, particularly in low-grade gliomas.