PO-106 Unique and overlapping role of HIF proteins in radiation response and tumour cell metabolism

Abstract

Introduction Hypoxia is a common feature of human solid tumours and caused by limited diffusion and structural aberrations of tumour vasculature. Tumour hypoxia in cancer is linked to worse outcome and treatment resistance. Hypoxic cells are also characterised by changes in their energy requirements and radiation response. Many of these adaptations are driven by the hypoxia-inducible transcription factors or HIFs. Mammalian cells encode for three HIF1–3 proteins which are O2 regulated. The similarities and differences between HIF proteins in regulating tumour cell metabolism and radiation response are still understudied. Material and methods Using gene-editing with the CRISPR/CAS9 system we generated isogenic series of cells lacking HIF1, HIF2 or both HIF1,2 in the H1299 non-small cell lung cancer cell line. We analysed the differences in hypoxia response on downstream target gene activation. In parallel, we analysed the upregulation of HIF protein expression under hypoxic conditions for each cellular model. Proliferative capacity and hypoxic tolerance were assessed for each cell line. We determined the metabolic consequences of silencing HIF by measuring oxygen consumption rate, extracellular acidification rate, extracellular pH and lipid droplet accumulation. We determined the radiation response of each cellular model under both normoxic and hypoxic conditions using clonogenic survival assays, gamma-H2AX staining and cell cycle analysis. Results and discussions We found that HIF2 compensates for the depletion of HIF1, but HIF1 is not upregulated upon HIF2 depletion. Both HIF1 and HIF2 proteins appear to be crucial for the upregulation of downstream HIF target genes such as CAIX, GLUT1 or TWIST. HIF depleted cells appear to be less glycolytic and at the same time trigger mitochondrial activity. The radiation response of the different cellular models shows a specific profile in terms of clonogenic survival and double-strand break formation in both normoxic and hypoxic conditions. Conclusion Taken together these genetically-modified cell models may help us to further define the radiation response of hypoxic tumour cells for therapeutic interventions.