Abstract
Vitamin C is an essential micronutrient that is involved in many biological processes in the body. Ascorbate, the most common form of vitamin C found in physiological pH is widely known for its antioxidant activity and its role as a co-factor for 2-oxoglutarate dependent dioxygenases (2-OGDDs). These enzymes play integral roles in various aspects of cell biology, but recently have been of interest in cancer because of their potential involvement in tumourigenesis and cancer progression.
One of the hallmarks of growing solid tumour is oxygen deprivation or hypoxia, marked by the elevated levels of the transcription factor hypoxia-inducible factor-1 (HIF-1). This transcription factor is responsible for mediating the response of cancer cells to this harsh environment, and enhances their pathogenesis.
The oxygen-sensitive α-subunit of HIF-1 is continuously expressed in cells, but its activity is controlled by the HIF-hydroxylases, members of 2-OGDD that act as an oxygen sensor. HIF-hydroxylases activities rely on the presence of their substrates; oxygen and 2-oxogluratate, and cofactors; ascorbate and iron. Ascorbate is proposed to protect the Fe centre in the reduced state to ensure that the enzyme is available for the subsequent enzymatic reaction cycle. In this thesis, I aimed to determine the role of ascorbate in limiting HIF-1 activation in cancer metabolism, survival, and the functional aspects of cancer invasiveness.
In most mammals except humans and other species, ascorbate is made in liver cells. In cell culture conditions, most cells are unable to synthesise ascorbate and it must be added to the cell culture medium to facilitate cellular ascorbate uptake. This comes with its own complexity, because ascorbate is readily oxidised in cell culture medium and pro-oxidant reactions can lead to generation of H2O2 that is cytotoxic. My initial investigation aimed to determine the optimal conditions for ascorbate uptake into breast cancer cells in vitro. I have shown that the doses and time of incubation for ascorbate must be managed carefully to achieve optimal intracellular ascorbate levels relevant to the physiological conditions.
I have also investigated the kinetics of the expression of the ascorbate transporter sodium-dependent vitamin C transporter 2 (SVCT2) and its intracellular localisation following ascorbate supplementation. Surprisingly, the SVCT2 was localised mainly in the subcellular compartments, suggesting that following uptake into the cells, ascorbate distribution to intracellular compartments is important. The nucleus had the highest prevalence of SVCT2 localisation, potentially suggesting that it may be required there to support the activity of DNA and histone demethylases, that are members of 2-OGDDs. SVCT2 was also found on mitochondria, possibly indicating a role for ascorbate as an antioxidant and/or as a co-factor to the 2-OGDDs in the mitochondria.
In this project I also determined the effect of ascorbate supplementation on HIF-1 pathway activation in breast cancer cell lines, induced by CoCl2 and under various hypoxic conditions. This covers the impact of HIF-1 activation on cell metabolism, cell survival and functional aspects of cancer invasiveness. I found that intracellular ascorbate was able to mitigate the impact of mild hypoxia on HIF-1 activation in breast cancer cell lines, including the cell migration and extracellular matrix (ECM) remodelling capacity. However, hypoxic exposure did not induce the expression of epithelial to mesenchymal transition (EMT) markers in the breast cancer cells and therefore the potential for ascorbate to maintain the cell as epithelial phenotype could not be observed.
I further looked at the effect of ascorbate on the gene expression in breast cancer cells under mild hypoxia. The hypoxia and glycolysis pathways were among the most upregulated, and ascorbate supplementation was found to limit but not prevent the activation of these pathways. These results could provide broader information on the genes and pathways affected by ascorbate. I also monitored the effect of ascorbate supplementation on the activity of the DNA demethylases ten-eleven translocases (TETs) that catalyses the hydroxylation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). This demethylation process reverts the transcriptional silencing that are common in many types of cancer. Ascorbate affected TET activity only after days of ascorbate supplementation. Lastly, I have undertaken an initial investigation on the effect of hypoxia on functional aspects of breast cancer invasiveness. Hypoxia is proposed to induce invasiveness by promoting phenotypic change that facilitate cell motility, and the release of matrix metalloproteases (MMPs) to mediate ECM degradation. At levels of mild hypoxia (5% O2), ascorbate slightly decreased expression of the MMPs, in a manner consistent with the decrease in HIF activation under these conditions. No changes were detectable in a migration assay. However, this was monitored only after 24 h, and further analysis at shorter time points will be required to determine if there are more subtle effects on migration.
Overall, my thesis has shown that hypoxia is a main driver of breast cancer progression and that ascorbate has the capacity to dampen the activation of the hypoxic response under mild hypoxia. Breast cancer cells accumulate high concentrations of ascorbate and turnover is rapid. It is most likely that ascorbate limits the hypoxic response by supporting the activity of the HIF-hydroxylases. This suggests that ascorbate could limit the growth of tumours, and enhance the response to therapies. In this way, ascorbate could be a useful addition to cancer treatments that may improve the prognosis of cancer patients.