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Updated: Aug 20, 2025
Induction and Testing of Hypoxia in Cell Culture
Published on: August 12, 2011
Survival Pathways of HIF-Deficient Tumour Cells: TCA Inhibition, Peroxisomal Fatty Acid Oxidation Activation and an
Monika A Golinska1, Marion Stubbs1, Adrian L Harris2
1Cancer Research UK Cambridge Institute, Cambridge University, Li Ka Shing Centre, Cambridge CB2 0RE, UK.
Abstract:
The HIF-1 and HIF-2 (HIF1/2) hypoxia responses are frequently upregulated in cancers, and HIF1/2 inhibitors are being developed as anticancer drugs. How could cancers resist anti-HIF1/2 therapy? We studied metabolic and molecular adaptations of HIF-1β-deficient Hepa-1c4, a hepatoma model lacking HIF1/2 signalling, which mimics a cancer treated by a totally effective anti-HIF1/2 agent. [1,2-13C2]-D-glucose metabolism was measured by SiDMAP metabolic profiling, gene expression by TaqMan, and metabolite concentrations by 1H MRS. HIF-1β-deficient Hepa-1c4 responded to hypoxia by increasing glucose uptake and lactate production. They showed higher glutamate, pyruvate dehydrogenase, citrate shuttle, and malonyl-CoA fluxes than normal Hepa-1 cells, whereas pyruvate carboxylase, TCA, and anaplerotic fluxes decreased. Hypoxic HIF-1β-deficient Hepa-1c4 cells increased expression of PGC-1α, phospho-p38 MAPK, and PPARα, suggesting AMPK pathway activation to survive hypoxia. They had higher intracellular acetate, and secreted more H2O2, suggesting increased peroxisomal fatty acid β-oxidation. Simultaneously increased fatty acid synthesis and degradation would have "wasted" ATP in Hepa-1c4 cells, thus raising the [AMP]:[ATP] ratio, and further contributing to the upregulation of the AMPK pathway. Since these tumour cells can proliferate without the HIF-1/2 pathways, combinations of HIF1/2 inhibitors with PGC-1α or AMPK inhibitors should be explored.
Insights
Cancers can resist HIF1/2 inhibitors by upregulating metabolic pathways and gene expression, suggesting combined therapies targeting PGC-1α or AMPK may be effective against these tumors.
Area of Science:
- Cancer Biology
- Metabolic Regulation
- Hypoxia Signaling
Background:
- Hypoxia-inducible factors (HIF1/2) are crucial in cancer, with inhibitors in development.
- Understanding cancer resistance mechanisms to anti-HIF1/2 therapy is critical.
Purpose of the Study:
- To investigate metabolic and molecular adaptations in HIF-1β-deficient hepatoma cells (Hepa-1c4) as a model for resistance to HIF1/2 inhibitors.
- To identify potential therapeutic strategies to overcome resistance to anti-HIF1/2 cancer drugs.
Main Methods:
- Utilized [1,2-13C2]-D-glucose metabolism tracing with SiDMAP profiling.
- Analyzed gene expression via TaqMan and metabolite concentrations using 1H MRS.
- Compared metabolic and molecular profiles of HIF-1β-deficient Hepa-1c4 cells with normal Hepa-1 cells under hypoxia.
Main Results:
- HIF-1β-deficient cells increased glucose uptake and lactate production under hypoxia.
- Observed elevated fluxes in glutamate, pyruvate dehydrogenase, citrate shuttle, and malonyl-CoA, with decreased TCA and anaplerotic fluxes.
- Detected increased expression of PGC-1α, phospho-p38 MAPK, and PPARα, indicative of AMPK pathway activation.
- Noted higher intracellular acetate, increased H2O2 secretion, and enhanced peroxisomal fatty acid β-oxidation.
- Identified simultaneous fatty acid synthesis and degradation, leading to ATP depletion and AMP:ATP ratio increase, further activating AMPK.
Conclusions:
- HIF-1β-deficient hepatoma cells exhibit significant metabolic and molecular reprogramming to survive hypoxia independently of HIF1/2 signaling.
- The observed adaptations, including AMPK pathway activation and altered fatty acid metabolism, confer resistance to HIF1/2 inhibition.
- Combination therapies targeting HIF1/2 inhibitors with PGC-1α or AMPK inhibitors warrant investigation for enhanced cancer treatment efficacy.
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