PhD Student University of Sheffield, United Kingdom
Background: Clear cell renal cell carcinoma (ccRCC) is the most common form of renal cell carcinoma (RCC). Patients with ccRCC often have a remarkably high resistance to traditional radiotherapy and chemotherapy. Previous work suggests that increased resistance to radiotherapy/chemotherapy may be due to high levels of hypoxia-inducible factors (HIFs), which under normoxic conditions are targeted for degradation by the von-Hippel Lindau protein (VHL)/E3 ligase complex. Under hypoxia, HIF levels accumulate, allowing them to activate hypoxic response elements. VHL mutations, which are commonly seen in ccRCC patients, cause constant upregulation of HIFs. Therefore, pathways promoting cell survival are constitutively upregulated. This project seeks to understand what pathways are involved in HIF-mediated genoprotection.
Methods: DNA damage assays, such as comet assays, clonogenics and cell titre blue assays were used to compare the DNA damage response between RCC4 VHL -/- and RCC4 VHL WT cell lines. Western blot and qPCR were used to compare protein expression between cell lines, focusing on VHL, HIFs, and DNA repair proteins.
Results: The expression levels and activity of a key regulator of DNA damage response was highly upregulated in RCC4 VHL -/- cells, which correlates with high HIF expression. By knocking down HIF2a, I demonstrate that HIF2a is key for this upregulation. RCC4 VHL -/- cells show greater resistance to two DNA damaging agents: camptothecin and olaparib, both of which demonstrate anti-cancer properties. However, initial results indicate that HIF knockdown is insufficient to resensitise RCC4 VHL -/- cells to camptothecin treatment.
Conclusions: Although HIF2a expression promotes upregulation of an important DNA repair protein, this is not solely responsible for the DNA damage resistance in RCC4 VHL -/- cells. The VHL/HIF pathway interplays with multiple pathways that also need to be modulated to see a substantial impact on DNA damage response.