Abstract
Hepatocellular carcinoma (HCC) is a major global health burden. Despite recent advances in immunotherapy, tyrosine kinase inhibitors (TKIs) treatment or combined therapies, therapeutic resistance and disease progression remain significant challenges. SOX4, a transcription factor frequently overexpressed in HCC and other cancers, has been linked to drug resistance and poor prognosis; however, the underlying molecular mechanisms remain unexplored. In this study, we identify STAT6 as a novel transcriptional target and interacting partner of SOX4 in HCC cells. Genetic ablation or knockdown of SOX4 induced hypermethylation of the STAT6 promoter, suppressing its expression, while treatment with the DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine restored STAT6 levels, indicating an epigenetic mechanism of regulation. In addition, SOX4 is physically associated with STAT6, as confirmed by co-immunoprecipitation and immunofluorescence. SOX4 depletion impaired interleukin-4 (IL-4)-induced phosphorylation of STAT6 at tyrosine residue 641 (Y641), implicating SOX4 in IL-4-mediated STAT6 activation. Chromatin immunoprecipitation (ChIP) assays demonstrated that SOX4 and STAT6 co-occupy the promoter of MTHFD2, a key enzyme in folate metabolism, regulating NADH/NADPH production and nucleotide biosynthesis. Knockdown of SOX4 or STAT6, or mutation of their binding sites within the MTHFD2 promoter, reduced MTHFD2 expression, NADPH levels, and nucleotide synthesis. Transcriptomic analyses from TCGA-LIHC and our independent cohort revealed a strong positive correlation between SOX4, STAT6, and MTHFD2, with MTHFD2 overexpression linked to poor overall survival. Clinically, elevated SOX4/STAT6/MTHFD2 axis activity was associated with resistance to immunotherapy or TKIs, either in our enrolled cohort or transcriptome data obtained from GSE109211. Metabolomic profiling further revealed increased NADPH and nucleotide biosynthesis in tumors with high SOX4/STAT6/MTHFD2 expression. Targeting STAT6 or MTHFD2 suppressed tumor growth in TKIs-resistant patient-derived xenograft models. Collectively, our findings identify the SOX4–STAT6–MTHFD2 axis as a critical driver of HCC progression and therapeutic resistance, offering a promising target for intervention in refractory HCC.
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Data availability
All supporting data are provided within the main text and supplementary materials. The high-throughput datasets generated in this study—including DNA methylation, ChIP-seq, and RNA-seq profiles—have been deposited in the GEO under accession numbers GSE286327, GSE277540, and GSE277728, respectively.
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Acknowledgements
The authors deeply appreciated Dr. Jean-Marc Egly (Directeur de Recherche Inserm, Membre de l’Institut-Academie des Sciences) giving critical comments on this manuscript. The authors are also grateful for the valuable support provided by the Microscopy, Genomic Medicine Core Laboratory, Clinical Proteomics Core Laboratory, and Laboratory Animal Center of Chang-Gung Memorial Hospital, Linkou branch. Additionally, they acknowledge the technical assistance of the Metabolomics Core Laboratory of the Healthy Aging Research Center, Chang-Gung University, Taiwan. Special thanks to Miss Yi-Ping Liu for her assistance with data retrieval and processing, as well as to the colleagues from the Research Specimen Processing Laboratory and the New Taipei Municipal Tu-Cheng Hospital for their support. Authors would like to thank Editage (www.editage.com.tw) for English language editing.
Funding
Financial Support by Chang Gung Medical Foundation in Taiwan (CMRPG3M1291/2/3 for CLT; CMRPVVM0093 for MCY, CMRPD1M0611/2 for CNT), and National Science and Technology Council (114-2320-B-182-014 for CNT; 111-2314-B-182A-035-MY3 and 114-2314-B-182A-121 for CLT, 110-2314-B-182A-059-MY3 and 113-2314-B-182A-068 for MCY).
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CL Tsai, MC Yu, and CN Tsai conceptualized and wrote the manuscript. YS Lee conducted the statistical analysis of TCGA-LIHC, ChIP, and RNA sequencing data. Hsu CL developed the HCC-PDx model and performed animal experiments. Chi LM contributed to proteomic processing and data analysis. SE Lin, Hsu HY, and MC Yu facilitated clinical specimen collection and conducted pathological analyses. HY Tang and ML Cheng assisted with LC/MS metabolite analysis.
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Tsai, CL., Yu, MC., Hsu, CL. et al. SOX4-STAT6-MTHFD2 axis drives hepatocellular carcinoma progression and treatment resistance. Cell Death Dis (2026). https://doi.org/10.1038/s41419-025-08394-2
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DOI: https://doi.org/10.1038/s41419-025-08394-2
