Abstract
The hypoxia-inducible transcription factors (HIF)-1/2α are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-α isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.
Highlights
Regions of oxygen deprivation are a common feature of many solid cancers and are caused by an aberrant tumor vasculature
We verified that H1299 clones did not have the Cas9 plasmid observed a prominent in hypoxia-inducible transcription factors (HIF)-2α stabilization incubation in 1A)
High levels of HIF-1α and HIF-2α positively correlate with tumor progression and poor patient outcome [19,23]
Summary
Regions of oxygen deprivation (hypoxia) are a common feature of many solid cancers and are caused by an aberrant tumor vasculature. Hypoxic tumor cells activate stress response pathways in order to adapt to these low oxygen levels [1]. Some cells react to hypoxic stress with adaptive responses that, by changing their gene expression, confer an aggressive phenotype and resistance to therapy [2]. Hypoxic tumors are more resistant to chemotherapy because they are poorly perfused, proliferate slower and upregulate drug efflux pumps. Hypoxic tumor cells are more radiation resistant. Decreased treatment sensitivity of hypoxic tumor cells leads to chemo-radiotherapy treatment failure in cancer patients [3,4]. Despite the limited success of hypoxia modification therapy strategies in several phase 3 clinical trials, hypoxia is still a promising tumor-selective therapeutic target
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