FAD synthase confers ferroptosis resistance and restrains CD8+ T cell recruitment in hepatocellular carcinoma

Abstract

Vitamin B2 (VB2) metabolism regulates numerous cellular processes, but its role in hepatocellular carcinoma (HCC) progression remains unclear. Here we show that HCC tumors are characterized by upregulation of a VB2 metabolism signature, and VB2 metabolism promotes HCC progression. Among VB2 metabolic enzymes, flavin adenine dinucleotide synthase (FADS) is the only one that is widely overexpressed in human HCC. Elevated FADS expression correlates with resistance to anti-PD-1 therapy and poor prognosis. In vivo, FADS facilitates HCC cell growth and suppresses T cell-mediated antitumor immunity. Single-cell transcriptomic analysis reveals that FADS-induced changes occur both in the tumor cells and the intra-tumoral CD8⁺ T cells. Knocking down FADS induces HCC cell death and increases CD8⁺ T cell infiltration. Mechanistically, FADS confers ferroptosis resistance on HCC cells via enzymatic function to produce FAD and non-enzymatic function to stabilize PCBP2. Moreover, FADS impairs CD8⁺ T cell recruitment by disrupting the cGAS-STING pathway. Hesperidin, a clinically approved FADS inhibitor, shows antitumor efficacy in a mouse model. Our study thus highlights the importance of VB2 metabolism in HCC and provides the proof of principle for targeting FADS as a therapeutic strategy for HCC.

Fig. 1. VB2 metabolism promotes HCC progression.

a Comparisons of VB2 metabolism scores between tumors and non-tumor livers from TCGA-LIHC, OEP000321, and ICGC-LIRI-JP datasets. Scores were calculated for each sample using the ssGSEA algorithm based on a VB2 metabolism gene signature. b Kaplan-Meier overall survival curves stratified by VB2 metabolism scores, with optimal cutoffs determined by the surv_cutpoint function. c t-SNE visualization showing 7 major cell types in tumor samples from the immunotherapy cohort (n = 8). d Proportions of the 7 major cell types across the eight patients. e Box plots with medians (red line) and interquartile ranges depicting VB2 metabolism scores in various cell types. Scores were calculated with the AUCell algorithm based on the VB2 metabolism gene signature. f UMAP plot showing 12 major cell types in the GSE202642 dataset. g Violin plot showing VB2 metabolism scores among tumor cells and normal epithelial cells in GSE202642. h Schematic diagram of the VB2 metabolic pathway with metabolites in black and enzymes in red. i Hep3B and HCCLM3 cells treated with VB2 (10 µM), FMN (10 µM), FAD (10 µM), or PBS (Ctrl) for 48 h, followed by CCK-8 assay (n = 5 biological replicates). j Subcutaneous tumors were established in Rag1^-/- mice with Hepa1-6 cells (n = 5 per group). Mice received daily intratumoral injections of VB2 (50 mg/kg) or control solvent from day 5 to day 21. Tumor volumes were measured at the indicated timepoints. Tumors were photographed and weighed at day 21. k Proliferation of Hep3B and HCCLM3 cells with lentiviral knockdown of RFK (shRFK) or FADS (shFLAD1) (n = 5 biological replicates), as determined by CCK-8 assay and colony formation assay. l Migration of HCCLM3 cells with RFK or FADS knockdown, assessed by transwell assay (n = 3 biological replicates). For bar graphs, data are presented as mean ± SD (i–k). For box plots (a, e), the box shows the median with interquartile ranges (IQR), whiskers extend to 1.5 × IQR, and outliers are shown as individual points. P-values were calculated using one-way ANOVA with Tukey’s test (i, k), two-way ANOVA with Tukey’s test (j, k), unpaired two-sided Student’s t test (j), two-sided Wilcoxon rank-sum test (a, e, g), and log-rank test (b). Source data are provided as a Source Data file.

Fig. 2. FADS promotes tumor growth and restrains T cell-mediated antitumor immunity.

a Comparison of FLAD1 expression between tumors and non-tumor liver samples in 18 HCC datasets from the HCCDB database. b FLAD1 mRNA levels in paired tumors (T) and adjacent livers (N) from Cohort-A (n = 30 pairs). c FADS protein levels in paired tumors (T) and adjacent livers (N) from Cohort-A (n = 6 pairs). Data from 3 independent experiments with similar results. d Representative images and quantification of FADS staining in paired tumors and liver samples from Cohort-B (n = 42 pairs). Quantification is based on H-scores. e Kaplan-Meier overall survival curves of TCGA-LIHC and OEP000321 cohorts, stratified by FLAD1 expression with optimal cutoffs defined by the surv_cutpoint function. f Tumor FLAD1 expression in patients with partial response or progressive disease to immunotherapy (Fudan Cohort, n = 17). g Proportions of FLAD1⁺ tumor cells in patients with partial response or stable disease (Immunotherapy Cohort, n = 8). h Subcutaneous tumors were established in C57BL/6 mice with Hepa1-6 cells (Ctrl or shFlad1, n = 5 per group). Tumor volumes were measured at the indicated timepoints. Tumors were photographed and weighed at day 21. i, j Orthotopic tumors were established in C57BL/6 mice with Hepa1-6 cells (Ctrl or shFlad1, n = 6 per group). Livers were photographed and weighed at day 21 (i). Bioluminescence imaging was used to visualize tumors at days 14 and 21 (j). k Subcutaneous tumors in Rag1^-/- mice with Hepa1-6 cells (Ctrl or shFlad1, n = 6 per group). Tumor volumes were measured at the indicated timepoints. Tumors were photographed and weighed at day 21. l, m Orthotopic tumors in Rag1^-/- mice with Hepa1-6 cells (Ctrl or shFlad1, n = 5 per group). Livers were photographed and weighed at day 21 (l). Bioluminescence imaging was used to visualize tumors at days 14 and 21 (m). Data are presented as mean ± SD (f–h, i, k, l) or median with interquartile ranges (d). P-values were calculated using two-way ANOVA with Tukey’s test (h, k), unpaired two-sided Student’s t test (f, k, l), two-sided Wilcoxon signed-rank test (d), two-sided Wilcoxon rank-sum test (g–i), and log-rank test (e). Source data are provided as a Source Data file.

Fig. 3. Single-cell RNA-seq analysis reveals the impact of FADS on tumor microenvironment.

a Workflow of single-cell RNA-seq analysis for mouse tumors (created in BioRender. Chao, J. (2025) https://BioRender.com/bpec5ea). Orthotopic tumors were established in C57BL/6 mice with Hepa1-6 cells (Ctrl or shFlad1, n = 5 per group). Tumors were harvested at day 14 for single-cell RNA-seq. b UMAP plots showing the distribution of 10 tumor samples (5 NC [control] vs. 5 KD [Fads knockdown]) and 15 major cell types. c scDist analysis ranking perturbation levels in major cell types between NC and KD groups, displayed as effect values (red dot) with 95% confidence intervals. d Violin plot showing VB2 metabolism scores in malignant cells between NC and KD groups. e KEGG pathway enrichment analysis of differentially expressed genes in tumor cells (KD vs. NC). f UMAP plot showing the distribution of 16 T & NK lymphocytes subsets. g Annotation of T cell subsets based on canonical T cell gene signatures. Bars illustrate the composition of various T cell subsets. h scDist analysis ranking perturbation levels across T & NK lymphocytes subsets between NC and KD groups, with effect values (red dot) and 95% confidence intervals. i Interferon, dysfunction, and proliferation scores (AUCell algorithm) in CD8⁺ T cells from NC and KD groups. The box shows the median with interquartile ranges (IQR), whiskers extend to 1.5 × IQR, and no outliers were plotted. j KEGG pathway enrichment analysis of upregulated genes in CD8⁺ T cells (KD vs. NC). P-values were calculated using a two-sided Wilcoxon rank-sum test (d, i).

Fig. 4. FADS inactivates the cGAS-STING pathway to impede CD8⁺ T cell recruitment.

a Gene set enrichment analysis showing upregulation of the “Chemokine receptors bind chemokines” pathway in Hep3B cells following FADS knockdown. b Correlation analyses between FLAD1 expression and CD8⁺ T cell signature scores in TCGA-LIHC and OEP000321 datasets. c, d Representative images of FADS and CD8 staining in tumors from Cohort-B (c), and quantification of CD8⁺ T cell density (n = 42 patients) (d). FADS expression level was stratified by median H-score (high vs. low). e Representative images of CD8 staining in orthotopic and subcutaneous Hepa1-6 tumors, and quantification of CD8⁺ T cell density in orthotopic tumors (n = 4 per group) and subcutaneous tumors (n = 5 per group). f Percentages of IFN-γ⁺ and PD-1⁺ cells among CD8⁺ T cells in subcutaneous Hepa1-6 tumors (n = 5 per group), as determined by flow cytometry. g Upregulation of “DNA double-strand break response” and “DNA damage checkpoint” pathways in Hep3B cells following FADS knockdown. h Schematic illustration of DNA damage-induced cGAS-STING pathway activation. i Reactive oxygen species (ROS) levels in Hep3B cells (n = 3 biological replicates). j Representative immunofluorescence (IF) images and quantitative analysis of γH2AX staining in Hep3B cells (n = 3 biological replicates). k Representative IF images and qualitative analysis of double-stranded DNA (dsDNA) staining in Hep3B cells (n = 20 biological replicates). l Western blots showing the expression of key proteins in the cGAS-STING pathway in Hep3B cells (n = 3 independent experiments). m Relative mRNA levels of interferon-stimulated genes (ISG15, IFIT1, IFNB1) and chemokines (CCL5, CXCL9, CXCL10) in Hep3B cells (n = 3 biological replicates). FAD, 20 µM for 48 h; H-151 (STING inhibitor), 1 μM for 12 h. Data are presented as mean ± SD (e, f, i, j, m) or median with interquartile ranges (d). P-values were calculated using unpaired two-sided Student’s t test (e, f, i, j, m), two-sided Wilcoxon rank-sum test (d), Fisher’s exact test (k), and Spearman’s rank correlation (b). Source data are provided as a Source Data file.

Fig. 5. FADS produce FAD to inhibit ferroptosis.

a Representative image of cell death in Hepa1-6 cells following Fads knockdown (n = 3 biological replicates). The percentage of dead cells (7-AAD⁺) were analyzed via flow cytometry. b Relative intracellular levels of VB2, FMN, and FAD in Hep3B cells. VB2 and FMN detected by untargeted metabolomics (n = 6 biological replicates); FAD measured by a fluorescence probe (n = 5 biological replicates). c KEGG enrichment analysis of differentially expressed metabolites in Hep3B cells (Ctrl vs. shFLAD1). d Relative intracellular levels of arachidonic acid, 12(S)-HETE, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) in Hep3B cells detected in untargeted metabolomics (n = 6 biological replicates). e Relative intracellular levels of glutathione (GSH) and oxidized glutathione (GSSG) in Hep3B cells, assessed by untargeted metabolomics (n = 6 biological replicates). f Lipid peroxidation in Hep3B cells, detected using BODIPY 581/591 C11 via flow cytometry (n = 5 biological replicates). g Cell viability of Hep3B cells with FADS overexpression treated with Erastin (20 μM), or with FADS knockdown treated with Ferrostatin-1 (10 μM), as determined by CCK-8 assay (n = 5 biological replicates). h Schematic illustration of FAD serving as a coenzyme to assist glutathione reductase (GR) in converting GSSG to GSH. i GR activity, GSH levels, and GSH/GSSG ratio in Hep3B cells (n = 4 biological replicates). FAD, 20 µM for 48 h. j Levels of ROS and lipid peroxidation in Hep3B cells (n = 3 biological replicates). FAD, 20 µM for 48 h. k Percentage of dead cells (7-AAD⁺) in Hep3B cells following indicated treatments (n = 3 biological replicates). FAD, 20 µM for 48 h. l Hep3B cells with FADS-knockdown treated with VB2 (10 µM), FMN (10 µM), FAD (10 µM), or PBS (Ctrl) for 48 h, followed by CCK-8 assay (n = 5 biological replicates). Data are presented as mean ± SD (a, f, g, i, j, k, l) or median with interquartile ranges (b, d, e). P-values were calculated using unpaired two-sided Student’s t test (a, b, f, g, i, j, k, l), and two-sided Wilcoxon rank-sum test (d, e, f). Source data are provided as a Source Data file.

Fig. 6. FADS stabilize PCBP2 to inhibit ferroptosis.

a Intracellular Fe²⁺ levels in Hep3B cells (n = 5 biological replicates). b Identification of FADS-binding proteins in Hep3B cells using immunoprecipitation (IP) and mass spectrometry. The top 20 candidates are ranked by sequence coverage. c Co-IP assay showing the interaction between FADS and PCBP2 in Hep3B cells (n = 3 independent experiments). d IF staining showing co-localization of FADS and PCBP2 in Hep3B cells. e Protein levels and quantification of FADS and PCBP2 in Hep3B and HCCLM3 cells (n = 3 biological replicates). f Representative images of PCBP2 staining in tumor samples from Cohort-B, and correlation analysis of FADS and PCBP2 protein levels. g Intracellular Fe²⁺ levels and percentages of dead cells (7-AAD⁺) in Hep3B cells following FADS knockdown with or without PCBP2 overexpression (OE) (n = 3 biological replicates). h Subcutaneous tumors in C57BL/6 mice (n = 5 per group) with Hepa1-6 cells (shFlad1 vs. shFlad1 + Pcbp2-OE). Tumors were photographed and weighed at day 21. i Diagram of truncated plasmids encoding FLAG-tagged FADS and His-tagged PCBP2. j, k 293T cells were transfected with truncated plasmids of FADS and PCBP2. Co-IP assays indicated that the FADS C-terminus binds to PCBP2 (j), and the PCBP2 KH1 domain binds to FADS (k) (n = 3 independent experiments). l Docking analysis revealing specific binding sites between FADS and PCBP2. m Mutation of the binding interface between FADS and PCBP2 by replacing target amino acids with alanine disrupts their interaction, as shown by Co-IP assay (n = 3 independent experiments). WT, wide-type; Mut, mutation. n Protein levels and quantification of PCBP2 in Hep3B cells with FADS knockdown or overexpression (n = 3 biological replicates). Cells were treated with cycloheximide (CHX, 50 μg/mL) for the indicated times. o Protein levels and quantification of PCBP2 in Hep3B cells treated with or without MG132 (20 μM, 12 h) (n = 3 biological replicates). p PCBP2 ubiquitination (Ub) levels in Hep3B cells with FADS knockdown or overexpression (n = 3 independent experiments). q Co-IP assay showing interactions between USP10 and FADS, and USP10 and PCBP2 (n = 3 independent experiments). r Docking model of FADS, PCBP2, and USP10. Data are presented as mean ± SD (a, e, g, h, n, o). P-values were calculated using unpaired two-sided Student’s t test (a, e, g, h, o), two-way ANOVA with Tukey’s test (n), and Spearman’s rank correlation (f). Source data are provided as a Source Data file.

Fig. 7. Hesperidin is identified as a FADS inhibitor.

a Workflow for high-throughput virtual screening of FADS-targeting compounds. b Five compounds selected for experimental validation. c Proliferation of Hep3B, HCCLM3 and Hepa1-6 cells treated with the five compounds (100 µM) respectively, as determined by CCK-8 assay (n = 5 biological replicates). d Colony formation of Hep3B, HCCLM3 and Hepa1-6 cells following Hesperidin (100 µM) treatment. e, f Subcutaneous tumors generated by Hepa1-6 cells in Rag1^-/- mice (e) and C57BL/6 mice (f) (n = 5 per group). Hesperidin (100 mg/kg) or DMSO control was administered daily by intraperitoneal injection. Tumor volumes were measured at the indicated timepoints. Tumors were photographed and weighed at day 21. g Docking analysis showing the interaction between Hesperidin and FADS. h FAD levels in Hep3B and HCCLM3 cells treated with or without Hesperidin (100 µM, 48 h) (n = 3 biological replicates). i, j ROS and lipid peroxidation in Hep3B cells treated as indicated (n = 3 biological replicates). Hesperidin, 100 μM for 48 h; FAD, 20 μM for 48 h. k Percentage of dead cells (7-AAD⁺) in Hep3B cells (n = 4 biological replicates). Hesperidin, 100 µM for 48 h; FAD 20 µM for 48 h. l Representative images and quantification of CD8⁺ T cell density in subcutaneous Hepa1-6 tumors treated with or without Hesperidin (n = 5 per group). Data are presented as mean ± SD (c, e, f, i, j, k, l) or median with interquartile ranges (h). P-values were calculated using unpaired two-sided Student’s t test (e, f, h, i, j, k, l), and two-way ANOVA with Tukey’s test (c, e, f). Source data are provided as a Source Data file.

Fig. 8. Mechanistic insights into the role of VB2 metabolism and FADS in HCC progression (created in BioRender.

Chao, J. (2025) https://BioRender.com/oomc25g).