The role of ascorbate in the regulation of HIF-1 in cancer cells

Abstract

Cancer cells within a solid tumour are often under considerable metabolic stress, which would normally signal cell death. However, cancer cells are able to adapt and survive their inhospitable microenvironment by activating the transcription factor hypoxia-inducible factor (HIF)-1. HIF-1 drives the transcription of hundreds of genes involved in various processes known to drive cell survival and tumour progression, as well as metastasis and chemo- and radio-resistance, and is an established independent prognostic indicator in many cancer types.

HIF-1 is constantly synthesised in all cells, but its activity is kept controlled by post-translational hydroxylation, causing it to ‘switch off’ when it is not needed. The hydroxylation reactions are performed by a group of enzymes known as the HIF-hydroxylases that specifically require ascorbate (vitamin C) as a co-factor for activity. This suggests that ascorbate-deficiency may result in over-activation of the HIF-1 response and thus, tumour progression. Despite this, ascorbate has received relatively little attention as a possible inhibitor of the HIF-1 response.

It was therefore the aim of this thesis to investigate whether intracellular ascorbate levels can significantly influence HIF-1 activity in cancer cells, and whether this mechanism has any clinical relevance. These questions were addressed using multiple approaches. In vitro studies were performed to monitor the optimal intracellular ascorbate concentrations required to inhibit HIF-1 induction by various means. In addition, clinical samples from two tumour types (endometrial and colorectal) were analysed for markers of HIF-1 activation, and related to the tissue ascorbate content and clinico-pathological data. Furthermore, pharmacokinetic data was obtained describing what plasma ascorbate concentrations are needed for its optimal distribution in avascular tumour tissue.

The results presented in this thesis have shown a clear and consistent relationship between low intracellular ascorbate levels and high HIF-1 activation in cancer cells. The in vitro studies showed that ascorbate was able to dramatically inhibit HIF-1, induced by various means, and that intracellular concentrations greater than 1 mM are optimal for this effect. That these results could have clinical relevance was shown by the analysis of human tumour tissues, where low levels of tissue ascorbate were associated with higher HIF-1 activation, high tumour grade, tumour necrosis and larger tumour size. These results were markedly similar between endometrial and colorectal tumours. Furthermore, poor disease-free survival in colorectal cancer patients was associated with low tumour ascorbate content. Further to this, pharmacokinetic data indicated that a constant, saturated plasma ascorbate concentration is necessary to avoid ascorbate-deficiency in avascular regions of a tumour.

There is considerable scepticism surrounding the use of ascorbate to treat cancer, with little mechanistic or clinical evidence to inform this practice. However, the results presented in this thesis support a role for ascorbate in regulating HIF-1 and tumour progression, and this mechanism is worthy of further clinical investigation.