Abstract
Clear cell RCC (ccRCC) is the most common and also most aggressive RCC type harbouring alterations in the VHL tumour suppressor gene leading to uncontrolled accumulation of pro-survival hypoxia inducible transcription factors (HIF-1/HIF-2). The second most common form, papillary RCC (pRCC), has a functional VHL protein with regulated HIF levels. Protein levels and activity of hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by enzymes belonging to the family of iron- and 2-oxoglutarate-dependent dioxygenases. Hydroxylation of HIF by proline hydroxylases (PHDs) targets the protein for degradation via the VHL-ubiquitin-ligase complex, and hydroxylation by factor inhibiting HIF (FIH) leads to its inactivation. Both PHD and FIH enzymes require ascorbate as cofactor, and absence of ascorbate leads to increased HIF activation.
Previous data in endometrial, colorectal, breast and thyroid cancer patients has shown that higher tumour ascorbate levels are associated with lower HIF-1 levels and reduced markers of tumour aggression. To verify the role of ascorbate in cancer, ccRCC tumours were specifically selected as model of VHL mutation in comparison to VHL-proficient pRCCs. Humans acquire ascorbate from their diet. It is actively absorbed from the intestines via sodium-dependent vitamin C transporter 1 (SVCT1), carried in the blood and taken up into all cells of the body via SVCT2. SVCT1 is also responsible for ascorbate reuptake in the kidney. Information of the ascorbate transporters in cancer is very sparse.
The aim of this thesis was to verify that ascorbate modulates HIF proteins by supporting the activity of the regulatory hydroxylases. Markers of HIF-1 and HIF-2 activation were analysed in banked tumour samples from kidney cancer patients with matched normal renal cortex and related to tissue ascorbate levels and clinico-pathological data. In addition, the influence of ascorbate on HIF pathway activity was monitored in VHL-proficient and VHL-defective ccRCC cell lines in vitro. Furthermore, efficiency of proline hydroxylation of HIF-1a was monitored in gene-modified VHL-defective ccRCC cells after ascorbate treatment. In addition, this thesis aimed to understand the relationship between SVCTs and ascorbate in clinical RCC samples and cell lines.
In pRCC patients, HIF pathway activity was increased in ascorbate-deficient tumours, supporting previous data in other cancer types. However, the same association was not evident in ccRCC patients. Ascorbate content was not associated with patient survival in either cohort, while a higher HIF-2 pathway score was negatively associated with disease-free survival in patients with ccRCC. In vitro, ascorbate was able to dampen induction of HIF only in VHL-proficient cells. In VHL-defective cells, elevated levels of hydroxylated HIF-1a in response to increasing intracellular ascorbate was observed. In patient samples, protein levels of SVCT1 were increased compared to normal renal cortex tissue. Tumour samples also showed an increase in the (presumed) glycosylated form of SVCT2, with a decrease seen in non-glycosylated SVCT2. Although overall levels of ascorbate were higher in tumours compared to renal cortex, no association between transporter levels and ascorbate content was detected in individual patients. Protein expression and localisation was also not significantly affected by the ascorbate in vitro.
This thesis was the first study to investigate the association between ascorbate and HIF-1/HIF-2 in RCC. Data from VHL-proficient pRCC provided evidence for a universal association between ascorbate and the HIF-pathway in cancer. The comparison with VHL-deficient ccRCC tumours supports the role of ascorbate as a modulator of tumour aggression, but only in cancer types with a functional hypoxic response which is important for the design of future clinical trials. We show mechanistic proof in living cells that ascorbate can modulate the hypoxic pathway in cancer cells by increasing HIF hydroxylase activity. The investigation into SVCTs demonstrates the complexities of ascorbate uptake mechanisms in RCC.
The results from this thesis provide the first direct evidence for the requirement of ascorbate for effective HIF-hydroxylase activity in cancer cells and strongly support a role for ascorbate as a modulator of HIF activity in cancer types with a functional hypoxic response.