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Updated: Sep 10, 2025
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Hypoxia-induced HIF1A impairs sorafenib sensitivity in hepatocellular carcinoma through NSUN2-mediated stabilization
Lude Wang1, Bo Zhuang2, Yiwen Jiang3
1Key Laboratory of Nutrition and Metabolism Research for Oncology, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Zhejiang, China; Central Laboratory and Precision Medicine Center, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Zhejiang, China.
Background:
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Although sorafenib is a first-line systemic therapy for advanced HCC, resistance to treatment remains a major challenge. Hypoxia is a hallmark of the tumor microenvironment and is known to promote tumor growth and progression; however, its role in modulating sorafenib response in HCC remains poorly understood.
Methods:
HCC cell lines (Hep3B and Huh7) were cultured under normoxic or hypoxic conditions and treated with sorafenib. Cell viability, apoptosis, and colony formation assays were conducted to evaluate the effect of hypoxia on sorafenib sensitivity. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to confirm HIF1A-mediated transcriptional regulation. RNA sequencing, m5C-RIP-Seq, and bioinformatics analysis were performed to identify NSUN2-mediated downstream targets. The therapeutic efficacy of GDF15 neutralization in combination with sorafenib was assessed both in vitro and in vivo.
Results:
Hypoxia significantly reduced HCC cell sensitivity to sorafenib, as evidenced by increased IC50 values and decreased apoptosis. Mechanistically, hypoxia-induced HIF1A upregulated the RNA m5C methyltransferase NSUN2, which stabilized GDF15 mRNA through m5C modification, leading to enhanced GDF15 secretion. Neutralization of GDF15 inhibited Akt/mTOR signaling and enhanced sensitivity to sorafenib in both in vitro and in vivo models.
Conclusion:
This study uncovers a novel mechanism by which hypoxia-induced HIF1A promotes sorafenib resistance in HCC via the NSUN2-mediated stabilization and upregulation of GDF15. Targeting the HIF1A/NSUN2/GDF15 axis offers a promising therapeutic strategy to overcome sorafenib resistance in HCC patients.
Insights
Hypoxia promotes sorafenib resistance in hepatocellular carcinoma (HCC) by upregulating GDF15 via HIF1A and NSUN2. Targeting this pathway may overcome treatment resistance in HCC patients.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Hepatocellular carcinoma (HCC) presents a significant global health challenge, with limited treatment options for advanced stages.
- Sorafenib resistance is a major obstacle in treating HCC, necessitating novel therapeutic strategies.
- The role of tumor hypoxia in modulating sorafenib response in HCC remains largely unexplored.
Purpose of the Study:
- To elucidate the mechanism by which tumor hypoxia influences sorafenib sensitivity in HCC.
- To identify key molecular players involved in hypoxia-driven sorafenib resistance.
- To evaluate the therapeutic potential of targeting the identified pathway against sorafenib resistance.
Main Methods:
- Utilized HCC cell lines cultured under normoxic and hypoxic conditions with sorafenib treatment.
- Performed cell viability, apoptosis, and colony formation assays to assess drug sensitivity.
- Employed luciferase reporter, ChIP, RNA sequencing, m5C-RIP-Seq, and bioinformatics to investigate molecular mechanisms.
- Assessed therapeutic efficacy of GDF15 neutralization combined with sorafenib in vitro and in vivo.
Main Results:
- Hypoxia significantly decreased sorafenib sensitivity in HCC cells, indicated by higher IC50 values and reduced apoptosis.
- Hypoxia-induced HIF1A upregulates NSUN2, which enhances GDF15 secretion by stabilizing its mRNA via m5C modification.
- GDF15 neutralization effectively inhibited Akt/mTOR signaling, thereby restoring sorafenib sensitivity.
Conclusions:
- Identified a novel hypoxia-driven pathway (HIF1A/NSUN2/GDF15) contributing to sorafenib resistance in HCC.
- Demonstrated that targeting the HIF1A/NSUN2/GDF15 axis can overcome sorafenib resistance.
- This axis represents a promising therapeutic target for improving sorafenib efficacy in HCC patients.
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