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A ROS-mediated oxidation-O-GlcNAcylation cascade governs ferroptosis

Nature Cell Biology volume 27, pages 1288–1300 (2025)Cite this article

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Abstract

Reactive oxygen species (ROS) play a crucial role in lipid peroxidation and the initiation of ferroptosis, markedly affecting chemotherapeutic drug resistance. However, the mechanisms by which ROS function and are sensed remain poorly understood. In this study, we identified O-GlcNAc transferase (OGT), a key enzyme in protein O-GlcNAcylation, as a sensor for ROS during ferroptosis. The ROS-induced oxidation of OGT at C845 in its catalytic domain activates the enzyme. Once activated, OGT O-GlcNAcylates FOXK2, enhancing its interaction with importin α, which facilitates FOXK2’s nuclear translocation and binding to the SLC7A11 promoter region. This, in turn, boosts SLC7A11 transcription, thereby inhibiting ferroptosis. The elevated OGT–FOXK2–SLC7A11 axis contributes to tumorigenesis and resistance to chemoradiotherapy in hepatocellular carcinoma (HCC). Our findings elucidate a ROS-induced oxidation-O-GlcNAcylation cascade that integrates ROS signalling, O-GlcNAcylation, FOXK2-mediated SLC7A11 transcription and resistance to both ferroptosis and chemoradiotherapy.

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Fig. 1: OGT is oxidized and activated by ROS produced after ferroptosis inducers treatment.
Fig. 2: OGT regulates ferroptosis by controlling the transcription of SLC7A11.
Fig. 3: OGT O-GlcNAcylates FOXK2 and promotes the transcription of SLC7A11.
Fig. 4: OGT O-GlcNAcylates FOXK2 at Ser 424 and promotes nuclear translocation of FOXK2.
Fig. 5: FOXK2 O-GlcNAcylation is upregulated in response to ferroptosis signal.
Fig. 6: FOXK2 O-GlcNAcylation promotes tumorigenesis in HCC.
Fig. 7: An elevated OGT–FOXK2-SLC7A11 axis contributes to chemotherapy resistance in HCC.

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Data availability

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (IDs 4GYW and 7YEA). Previously published ChIP–seq data that were re-analysed here are available under accession codes GSE92123 and GSM1250376. RNA-seq data that support the findings of this study have been deposited in the Sequence Read Archive under accession code SRP534714. Mass spectrometry data have been deposited in ProteomeXchange with accession code PXD064117. Uncropped immunoblot scans and numerical source data supporting the findings of this study are provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This research was financially supported by the National Institutes of Health R01-NS121243 to H.P. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We also acknowledge the support from the Mass Spectrometry and Analytical Pharmacology Shared Resource, Histopathology and Tissue Shared Resource, and Flow Cytometry and Cell Sorting Shared Resource (P30-CA051008). Instrumentation used in this study included the BD LSRFortessa and S10OD016213.

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Author notes

  1. These authors contributed equally: Hemeng Zhang, Jialin Ma.

Authors and Affiliations

  1. Department of Radiation Oncology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China

    Hemeng Zhang, Xuehui Luo, Mengjiao Cai & Suxia Han

  2. Department of Oncology, Georgetown Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC, USA

    Hemeng Zhang, Chunyan Hou, Shiya Zhu, Yihan Peng, Ping Li, Heng Meng, Yuqi Xia, Susree Modepalli, Anju Duttargi, Gary M. Kupfer, Junfeng Ma & Huadong Pei

  3. Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, China

    Jialin Ma & Ping Li

  4. GW Cancer Center, Department of Biochemistry & Molecular Medicine, George Washington University, Washington DC, USA

    Changmin Peng

  5. Flow Cytometry and Cell Sorting Center, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC, USA

    Zhinuo Jiang

  6. Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, China

    Heng Zhang

  7. Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, Wuhan, China

    Juanjuan Li

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Contributions

H.Z., J.M., C.H., X.L., S.Z., Z.J., S.M., A.D. and M.C. performed the experiments; H.Z. performed most of the cell and animal experiments and data analysis. J.M., X.L., S.M. and A.D. contributed to the clinical sample collection, pathology and data preparation. LC–MS/MS analysis and data interpretation were carried out by C.H. H.Z. and J.M. Y.P., C.P., P.L., H.M., Y.X., G.M.K., H.Z., J.M., J.L., S.H. and H.P. joined discussions and designed the research. H.Z., J.M. and H.P. wrote the paper. S.H. and H.P. supervised and administrated the project.

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Correspondence to Heng Zhang, Junfeng Ma, Juanjuan Li, Suxia Han or Huadong Pei.

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Extended data

Extended Data Fig. 1 OGT is oxidized and activated by ROS produced during ferroptosis, related to Fig. 1.

a-d, PLC/PRF/5 cells were treated with erastin for the indicated time (a, b) and concentrations (c, d). Immunoblot analyses were conducted with indicated antibodies(a, c) and quantified by ImageJ (b, d) (n = 3 experiments). e. f, Huh7 cells were treated with the indicated concentrations of RSL3. Immunoblot analyses were conducted with indicated antibodies and quantified by ImageJ (n = 3 experiments). g, MS analysis was performed to identify the oxidation sites on OGT. HA-OGT was purified from HEK293T cells following H2O2 treatment and then analysed by mass spectrometry. h, Alignments of the oxidation motif in OGT across human and other species. i-k, Huh7 cells were treated with erastin (10 μM) for 24 h (i), RSL3 (500 nM) for 24 h (j), and H2O2 (100 μM) for 1 h (k). The intracellular ROS level was detected by flow cytometry analysis. n = 3 independent experiments. l. m, Huh7 cells were treated with the indicated concentrations of H2O2. Immunoblot analyses were conducted with indicated antibodies (l) and quantified by ImageJ (m) (n = 3 experiments). n, Endogenous OGT in Huh7 cells was depleted using shRNA, then rescued with either OGT-WT or OGT-C845S. Immunoblot analyses were conducted with indicated antibodies. b,d,f,i,j,k,m Data are presented as the mean ± SEM; two-tailed Student’s t-test.

Source data

Extended Data Fig. 2 OGT regulates ferroptosis by controlling the transcription of SLC7A11, related to Fig. 2.

a-c, The protein levels of SLC7A11 were analysed in PLC/PRF/5 cells following OGT knockdown (a), treatment with OSMI-1 for 24 h as indicated (b), and overexpression OGT (c). Immunoblotting was performed using the specified antibodies (n = 3 experiments). d. e, Endogenous OGT was depleted in PLC/PRF/5 (d) and Huh7 (e) cells using shRNA, and cystine uptake levels were subsequently measured. n = 3 independent experiments. f. g, Endogenous OGT was depleted in PLC/PRF/5 (f) and Huh7 (g) cells using shRNA, and cellular GSH levels were subsequently measured. n = 3 independent experiments. h, Endogenous OGT was depleted in Huh7 cells, treated with the indicated reagents, and lipid peroxidation levels were assessed by flow cytometry. n = 3 independent experiments. i. j, Endogenous OGT was depleted in PLC/PRF/5 (i) and Huh7 (j) cells using shRNA. The cells were treated with increasing concentrations of erastin for 24 h, and cell viability was subsequently measured. Data are presented as mean ± SEM. Two-way ANOVA was used. n = 3 independent experiments. k-m, OGT was stably depleted in PLC/PRF/5 cells by shRNA and stably transfected with SLC7A11. SLC7A11 and OGT protein levels in the indicated cells were determined by Immunoblotting (k). Cellular Cystine uptake (l). and GSH levels (m) in the indicated cells were examined. n = 3 independent repeats. d,e,f,g,h,l,m, Data are presented as the mean ± SEM; two-tailed Student’s t-test; ns, not significant.

Source data

Extended Data Fig. 3 SLC7A11 is the target gene of FOXK2, related to Fig. 3.

a, Volcano plot of differentially expressed genes in FOXK2 KO versus control samples, identified by whole-transcriptome RNA-seq in Huh7 cells. Upregulated or downregulated genes were filtered by fold change > 1.0 and P < 0.05. Statistical analysis was performed using the DESeq2 software. b, Heatmap of significantly altered genes in control versus FOXK2 KO Huh7 cells. c, FOXK2 and O-GlcNAc ChIP–seq occupancy profiles at the SLC7A11 locus were visualized using IGV software. Data are from GEO with accession numbers indicated in parentheses. d, f, g, Endogenous FOXK2 in Huh7 (d), PLC/PRF/5 (f) and Hep3B (g) was depleted by shRNA, and mRNA levels (left panel) and protein levels (right panel) of SLC7A11 were determined by qRT-PCR and immunoblotting, respectively. The independent experiments were repeated three times with similar results. e, Endogenous FOXK2 in Huh7 cells was depleted by shRNA, with erasrin (10 μM) treatment for 24 h. MRNA levels (left panel) and protein levels (right panel) of SLC7A11 were determined by qRT-PCR and immunoblotting, respectively. n = 3 independent experiments. h, Endogenous FOXK1 in Huh7 cells was depleted by shRNA, and SLC7A11 protein level was determined by immunoblotting (n = 3 experiments). i, The JASPAR website predicted FOXK2 binding sites with the SLC7A11 promoter sequence. j, HEK293T cells were subjected to ChIP assays with an anti-FOXK2-specific antibody, and the amount of precipitated DNA was quantitated by qRT-PCR. k, HEK293T cells were transfected with the indicated plasmids. The cells were lysed and then incubated with either biotin-labelled oligonucleotides or an excess of unlabelled oligonucleotides for pulldown assays. The bound proteins were pulled down and analysed by immunoblotting (n = 3 experiments). l. m, HEK293T cells were transfected with the indicated plasmids (l) and shRNA (m). The SLC7A11 promoter activity was measured using dual-luciferase reporter assay. n. o, Endogenous FOXK2 in Huh7 (n) and PLC/PRF/5 (o) cells was depleted by shRNA. Cystine uptake levels were examined. p, Endogenous FOXK2 in Huh7 cells was depleted by shRNA. Lipid peroxidation level was examined. q, Endogenous FOXK2 was depleted in Huh7 cells using shRNA. The cells were then exposed to the indicated concentrations of erastin for 24 h, followed by cell viability assay. Data are presented as mean ± SEM. Two-way ANOVA was used. For j,l,m,n,o,p,q, n = 3 independent experiments. d,e,f,g,j,l,m,n,o,p Data are presented as the mean ± SEM; two-tailed Student’s t-test.

Source data

Extended Data Fig. 4 OGT interacts with and O-GlcNAcylates FOXK2, related to Fig. 3.

a, Huh7 cells were transfected with either siCon or si-FOXO1 (left panel) and FOXO3 (right panel). Knockdown efficiency was evaluated by qRT-PCR. n = 3 independent experiments. b. HEK293T cells were transfected with the indicated plasmids, and the interaction between OGT and FOXK2 was examined by Co-IP and immunoblotting using the indicated antibodies (n = 3 experiments). c, GST, GST-FOXK2 full-length (FL), and truncated mutants (M1 and M2) proteins were expressed and purified from E. coli BL21 cells. The interaction between GST–FOXK2 or M1, M2 and OGT were examined by GST pulldown assay (n = 3 experiments). Upper panel: Schematic diagrams illustrating FOXK2 truncated mutants are presented. d, HEK293T cells were transfected with plasmids encoding full-length (FL) or truncated forms of OGT (M1 and M2). Co-IP was performed to map the region of OGT involved in binding to FOXK2 (n = 3 experiments). Upper panel: Schematic diagrams illustrating the truncated OGT mutants are presented. e, Huh7 cells were transfected with Flag-FOXK2 and treated with 30 μM OSMI-1 for 24 h. FOXK2 O-GlcNAcylation was analysed by IP and immunoblotting using the indicated antibodies (n = 3 experiments). f. g, Huh7 (f) and PLC/PRF/5 (g) cells were transfected with indicated siRNAs. Immunoblotting was conducted with indicated antibodies(n = 3 experiments). h, Huh7 cells were transfected with indicated plasmids, and the O-GlcNAcylation of FOXK2 was examined by IP and immunoblotting with indicated antibodies (n = 3 experiments). i, Huh7 cells were stably transfected with OGT shRNA and/or FOXK2 shRNA, and cell death following indicated treatment was evaluated. n = 3 independent repeats. a,i, Data are presented as the mean ± SEM; two-tailed Student’s t-test; ns, not significant.

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Extended Data Fig. 5 OGT O-GlcNAcylates FOXK2 at S424, related to Fig. 4.

a, MS analysis of FOXK2 O-GlcNAcylation sites. FOXK2 was purified from HEK293T cells and analysed by MS to identify the O-GlcNAcylation sites. b, Huh7 cells transfected with HA–FOXK2 WT or S424A were treated with DMSO or 10μm Thiamet-G for 24 h. FOXK2 O-GlcNAcylation was analysed by IP and immunoblotting (n = 3 experiments). c, HEK293T cells were transfected with either HA–FOXK2 WT or S424A. The interaction between OGT and FOXK2 was analysed by Co-IP (n = 3 experiments). d, Alignments of the OGT consensus O-GlcNAcylation motif in FOXK2 from human and other species. e, FOXK2 KO PLC/PRF/5 cells were reintroduced with either FOXK2 WT or S424A. SLC7A11 mRNA levels in the indicated PLC/PRF/5 cells were measured by RT–PCR. n = 3 independent repeats. f. g, FOXK2 KO PLC/PRF/5 cells were reintroduced with either FOXK2 WT or S424A. Cystine uptake (f) and GSH levels (g) in the indicated PLC/PRF/5 cells were measured. n = 3 independent repeats. h, PLC/PRF/5 cells were depleted of FOXK2 using CRISPR-Cas9 and then stably reconstituted with either WT or S424A mutant FOXK2. The cells were then treated with increasing concentrations of erastin for 24 h, and cell viability was evaluated. Data are presented as the mean ± SEM. Two-way ANOVA was used. n = 3 independent repeats. e,f,g, Data are presented as the mean ± SEM; two-tailed Student’s t-test; ns, not significant.

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Extended Data Fig. 6 An elevated OGT–FOXK2-SLC7A11 axis contributes to chemotherapy resistance in HCC, related to Fig. 7.

a, FOXK2 KO Huh7 cells were reintroduced with either WT or FOXK2 S424A mutant. The cells were treated with increasing concentrations of 5-FU for 72 h, and cell viability was measured. n = 3 independent replicates. b, The indicated Huh7 cells (from a) were seeded and treated with 4 Gy irradiation. Colony-formation assays were conducted. n = 3 independent repeats. c. d, Huh7, Huh7 5-FUR, and Huh7 IRR cells were treated with increasing concentrations of 5-FU (c) or varying doses of irradiation (d) for 72 h, and cell viability was assessed. The IC50 values are indicated. n = 3 independent replicates. e, Intracellular ROS levels in HepG2 and 5-FU-resistant HepG2 (HepG2 5-FUR) cells were measured by flow cytometry. n = 3 independent experiments. f, The protein levels of SLC7A11, along with total O-GlcNAcylation, were determined in HepG2 5-FUR cells by immunoblotting with the specified antibodies (n = 3 experiments). g, FOXK2 O-GlcNAcylation in HepG2 5-FUR cells was analysed by the chemoenzymatic labelling method and determined by immunoblotting. h, SLC7A11 mRNA level in HepG2 5-FUR cells was determined by qPCR. n = 3 independent repeats. i, HepG2 and HepG2 5-FUR cells were treated with increasing concentrations of 5-FU for 72 h, and cell viability was assessed. The IC50 values are indicated. n = 3 independent replicates. j, Intracellular ROS levels in PLC/PRF/5 and 5-FU-resistant PLC/PRF/5 (PLC/PRF/5 5-FUR) cells were measured by flow cytometry. n = 3 independent experiments. k, The protein levels of SLC7A11 and total O-GlcNAcylation were determined in PLC/PRF/5 5-FUR cells by immunoblotting (n = 3 experiments). l, Schematic model illustrating a ROS-induced oxidation-O-GlcNAcylation cascade specific to ferroptosis, which contributes to drug resistance in HCC. Created in BioRender. Zhang, H. (2025) https://BioRender.com/ky9usx1. a,c,d,i, Data are presented as mean ± SEM, with statistical analysis performed using two-way ANOVA. b,e,h,j, Data are presented as the mean ± SEM; two-tailed Student’s t-test; ns, not significant.

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Supplementary information

Supplementary Information

Sample of FACS gating strategies for DCFH-DA (cytoplasmic ROS) and C11-BODIPY (lipid ROS) staining.

Reporting Summary

Supplementary Tables 1–3

Supplementary Tables 1–3.

Source data

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Source Data Table 1

Statistical source data of Figs. 1–7 and Extended Data Figs. 1–6.

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Zhang, H., Ma, J., Hou, C. et al. A ROS-mediated oxidation-O-GlcNAcylation cascade governs ferroptosis. Nat Cell Biol 27, 1288–1300 (2025). https://doi.org/10.1038/s41556-025-01722-w

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