Oxygen‐independent expression of <scp>HIF</scp> ‐1α during the cell cycle in hepatocellular carcinoma cells controls essential metabolic pathways under normoxia

Intra-tumoral oxygen deprivation (hypoxia) promotes the activation of hypoxia-inducible factors (HIFs) that orchestrate the transcriptional adaptation of cancer cells to hypoxia. Hypoxia is prevalent in hepatocellular carcinoma (HCC), a cancer type with limited therapeutic options. In the poorly oxygenated HCC cells, the hypoxia-inducible factor 1-alpha (HIF-1α) subunit is over-expressed and correlates with poor patient outcome. To investigate the extent of HIF-1-mediated changes in HCC cells, we applied CRISPR/Cas9 to generate Huh7 cell lines that do not express endogenous HIF-1α. A similarly produced HeLa HIF1A^-/- line was used for comparison. Initial phenotypic analysis revealed that both HIF1Α^-/- cell lines were sensitive to oxygen deprivation. However, under normoxia, only HIF1A^-/- Huh7 cells showed increased death and reduced proliferation rates compared to wild-type Huh7 cells, implying that HIF-1α is essential for hepatocellular carcinoma cell survival irrespective of oxygen levels. To better understand this phenotype, we used liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis, followed by pathway enrichment and validation of the expression of different proteins. Our results show that, in normoxic Huh7 cells, HIF-1α is essential as it maintains the expression of proteins involved in glycolysis and steroid/cholesterol biosynthesis. It was also shown that only Huh7 cells exhibit a transient expression of HIF-1α under normoxia in a cell cycle-dependent manner. As analysis of publicly available patient data indicated that our normoxic HIF-1 signature is correlated with poor prognosis, our results suggest that HIF-1 is crucial in HCC cells: it mediates their metabolic phenotype under both normoxia and hypoxia, while it is also essential for sustaining cellular growth under hypoxia.