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Anlotinib mediates intrinsic drug resistance in hepatoblastoma through the GAD1/GABA pathway

Pediatric Research (2025)Cite this article

Abstract

Background

Intrinsic resistance reduces the effectiveness of many anticancer therapies. Anlotinib, a small-molecule multi-targeted tyrosine kinase inhibitor, has shown potential in treating hepatoblastoma. This study investigates the role of γ-aminobutyric acid (GABA) in anlotinib resistance using in vivo and in vitro models.

Methods

HuH-6 hepatoblastoma cells were implanted into nude mice to assess the effects of anlotinib on tumor growth. Neurotransmitter-targeted metabolomics was performed to analyze neurotransmitter metabolism in xenograft tumor tissues. In vitro, HuH-6 and HepG2 cells were treated with anlotinib to evaluate changes in GABA synthesis, degradation, and associated protein expression.

Results

Anlotinib significantly inhibited HuH-6 tumor growth but was less effective than cisplatin. Neurotransmitter-targeted metabolomics showed tumors treated with anlotinib exhibited increased GABA levels. Anlotinib treatment also upregulated the protein expression of GAD1, a key enzyme in GABA synthesis. In vitro, anlotinib treatment enhanced GABA release and GAD1 expression in hepatoblastoma cells. Exogenous GABA stimulation promoted cell proliferation in vitro and tumor growth in vivo. Notably, GAD1 knockdown enhanced anlotinib’s inhibitory effects on hepatoblastoma in vitro and in vivo.

Conclusion

Anlotinib induces intrinsic resistance in hepatoblastoma by upregulating GAD1 and increasing GABA accumulation. Targeting GAD1 may enhance anlotinib’s therapeutic efficacy and help overcome resistance.

Impact

  • Anlotinib upregulates GAD1 to enhance GABA synthesis, driving intrinsic resistance in hepatoblastoma by activating tumor-promoting GABA signaling in the tumor microenvironment.

  • First identification of the GAD1/GABA axis as a critical mediator of anlotinib resistance, expanding understanding of neurotransmitter-driven drug tolerance in pediatric cancers.

  • GAD1 knockdown synergizes with anlotinib to overcome resistance, establishing a combinatorial strategy to enhance antitumor efficacy in preclinical models.

  • Proposes targeting GABA metabolism to optimize anlotinib-based therapies, addressing unmet needs in refractory hepatoblastoma treatment.

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Fig. 1: Effects of Anlotinib on HuH-6 Xenografts.
Fig. 2: Effects of Anlotinib on GABA levels and related metabolic proteins in HuH-6 xenograft tissues.
Fig. 3: Effects of in vitro anlotinib treatment on GABA production and GAD1 protein expression in hepatoblastoma cells.
Fig. 4: Effects of GABA on hepatoblastoma in vitro and in vivo.
Fig. 5: Effects of anlotinib and GAD1 knockdown on hepatoblastoma cells in vitro.
Fig. 6: Effects of anlotinib and GAD1 knockdown on HuH-6 subcutaneous xenografts in vivo.
Fig. 7: Effects of anlotinib and GAD1 knockdown on HuH-6 liver orthotopic xenografts in vivo.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by the Science and technology planning project of Jiangxi Province Office of Health Commission (202410049; 202410336), Jiangxi Province Office of Education Support Program (GJJ2201454), Key Laboratory of Cardiovascular and Cerebrovascular Diseases Prevention and Treatment of Ministry of Education (Rare Earth Special Project) (XN202026) and Ganzhou Guiding Science.

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Author notes

  1. These authors contributed equally: Haijin Huang, Yanping Feng, Yuhui Xu.

Authors and Affiliations

  1. Department of Pediatric Surgery, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China

    Haijin Huang, Wei Peng, Linshan Zeng, Yong Zeng, Jinping Liu & Haijin Liu

  2. Jiangxi Provincial Clinical Research Center for Vascular Anomalies, The First Affiliated Hospital of GanNan Medical University, Ganzhou, Jiangxi, China

    Haijin Huang & Haijin Liu

  3. Department of Neurological Surgery, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China

    Yanping Feng

  4. Department of Respiratory Medicine, Ganzhou People’s Hospital, Ganzhou, Jiangxi, China

    Yuhui Xu

  5. Department of General Surgery, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China

    Jianping Liu & Xiao He

Authors
  1. Haijin Huang
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  2. Yanping Feng
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Contributions

Haijin Huang: conceptualization, design, and manuscript preparation. Yanping Feng: project administration. Haijin Huang, Yanping Feng, Yuhui Xu, and Jianping Liu: data generation. Wei Peng and Linshan Zeng: data analysis and interpretation. Linshan Zeng and Yong Zeng: verification. Jinping Liu: visualization. Yanping Feng, Yuhui Xu: review and editing. Xiao He and Haijin Liu: funding acquisition.

Corresponding authors

Correspondence to Xiao He or Haijin Liu.

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The authors declare no competing interests.

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This study does not involve human participants, and therefore, no patient consent was required.

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Huang, H., Feng, Y., Xu, Y. et al. Anlotinib mediates intrinsic drug resistance in hepatoblastoma through the GAD1/GABA pathway. Pediatr Res (2025). https://doi.org/10.1038/s41390-025-04074-1

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