HIF1A基因敲除HCT116细胞

Mitochondria respond to various stresses. Nevertheless, the regulation of this response while considering coordination between mitochondrial (mtDNA)- and nuclear DNA (nDNA)-encoded gene expression has been overlooked. Our RNA-seq analysis of 18 human cell lines grown in hypoxia (0.2-2% oxygen, 16-24 h) reveals a significant and coordinated reduction of mito-nuclear oxidative phosphorylation (OXPHOS) genes' expression in most (N = 11) cell lines. mtDNA copy number assessment in U87, HCT-116, MCF-7, and HeLa cells reveals non-significant changes, suggesting that the overall reduced mito-nuclear gene expression (MNGE) in hypoxia occurs at the RNA level. Analysis of HIF1α ChIP-seq experiments from cells exposed to hypoxia reveals increased binding to upstream regulatory elements of certain regulators of mitochondrial gene expression. Furthermore, RNA-seq analysis of HIF1α knockout HCT-116 cells grown in hypoxia reveals reduced mtDNA gene expression, yet no change in nDNA OXPHOS genes, suggesting that HIF1α knockout led to departure from coordination of MNGE. Finally, nascent RNA transcripts analysis (PRO-seq) in HeLa, U87, and D407 cells grown in hypoxia shows increased intensity of pausing sites throughout the mtDNA. This finding suggests an important role for transcriptional pausing in the regulation of mtDNA gene expression. Taken together, coordinated reduction of MNGE in hypoxia underlines MNGE as a pivotal player in general mitochondrial function, and particularly in response to stress.

BACKGROUND:Colorectal cancer (CRC) frequently exhibits hypoxic regions due to poor vascularization, leading to the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α). Moreover, mutations in the tumour suppressor p53 occur in approximately half of all CRCs. While the individual roles of both transcription factors in tumour cell survival are well characterized, their interaction and its influence on the metabolic adaptation of CRC cells under hypoxic stress remain unclear. METHODS:Using HCT116 CRC cells with targeted deletions of TP53 and HIF1A, we examined the effects of p53 loss on HIF-1 signalling and the respective consequences for metabolic adaptation as well as the survival of CRC cells under moderate (1% O₂) and severe (0.1% O₂) hypoxia. RESULTS:Severe hypoxia stabilized p53 protein levels despite the transcriptional repression of TP53, possibly through posttranslational mechanisms and dependent on nutrient availability. In contrast to the assumption that p53 is transcriptionally inactive under hypoxia, we observed stable expression of p53 target genes (P21, BAX) under severe hypoxia, indicating functional transactivation. Loss of p53 impaired the early induction of HIF-1 target genes (VEGF, PHD2), although HIF-1α protein levels and DNA binding were unaffected, suggesting a coactivator role for p53. Furthermore, compared with wild-type cells, p53-deficient cells presented delayed but exaggerated expression of glycolytic genes, including Glucose Uptake Transporter 1 (GLUT1), Phosphofructokinase Liver-Type (PFKL) and Lactate Dehydrogenase A (LDHA), under hypoxia, with no impairment of glycolytic function or cell viability. Remarkably, even HIF1A knockout cells preserved glycolysis, whereas glycolytic genes were significantly downregulated, indicating HIF-1-independent metabolic compensation. CONCLUSION:Our findings position p53 as a temporal gatekeeper and key regulator of hypoxic adaptation in CRC cells, coordinating early gene induction and metabolic responses. The ability of CRC cells to maintain glycolysis despite the loss of p53, respectively, HIF-1α underscores the existence of compensatory HIF-independent pathways. Targeting these alternative circuits may represent a promising strategy in hypoxic, p53-deficient CRC.