IRF8 Suppresses Hepatocellular Carcinoma Progression Through LIAS‐Dependent IFN Production and Cuproptosis Activation

ABSTRACT

Background

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide. Cuproptosis is a novel form of regulated cell death closely linked to tumor progression. Although interferon regulatory factors (IRFs) exert tumor‐suppressive effects in various cancers, its precise regulatory mechanisms concerning cuproptosis in HCC remain largely elusive.

Methods

IRF8 expression was evaluated in clinical HCC specimens and cell lines. Gain‐ and loss‐of‐function assays were conducted. Underlying transcriptional mechanisms were elucidated using ChIP‐qPCR and dual‐luciferase reporter assays. Subcutaneous nude mouse xenograft models were utilized to validate in vivo phenotypes.

Results

IRF8 was significantly downregulated in HCC. IRF8 overexpression induced oxidative stress and cuproptosis, as evidenced by excessive intracellular copper accumulation, elevated reactive oxygen species (ROS), and lipid peroxidation. Mechanistically, IRF8 directly bound to the promoter region of lipoyl synthase (LIAS) to activate its transcription. Phenotypic rescue assays confirmed that LIAS is indispensable for IRF8‐induced interferon (IFN‐α/β) production, copper engorgement, and cuproptosis. Furthermore, in vivo xenograft models demonstrated that IRF8 profoundly impeded tumor growth, an effect that was effectively abrogated by LIAS silencing.

Conclusion

IRF8 inhibits HCC progression by directly upregulating LIAS to instigate lethal cuproptosis and interferon responses. The IRF8/LIAS axis may serve as a potential mechanistic basis and candidate axis for developing HCC therapeutic strategies.

1. Introduction

Hepatocellular carcinoma (HCC) ranks among the most prevalent and lethal malignant tumors worldwide, characterized by insidious onset, high invasiveness, and frequent drug resistance [1, 2, 3]. Despite advances in surgical resection, targeted therapy, and immunotherapy, the long‐term prognosis of HCC patients remains unsatisfactory due to complex molecular pathogenesis and frequent recurrence [4]. HCC is a highly heterogeneous malignancy at both the inter‐tumoral and intra‐tumoral levels. Comprehensive gene expression profiling studies have identified multiple molecular subtypes of HCC with distinct genetic alterations, epigenetic modifications, and signaling pathway activations, which correspond to different clinical outcomes and therapeutic responses [5]. Notably, transcriptomic analyses have revealed that HCC subtypes exhibit significant heterogeneity in the expression of immune‐related genes, metabolic enzymes, and cell death regulators, and this heterogeneity profoundly influences tumor immune microenvironment composition, therapeutic vulnerability, and prognosis [6]. Therefore, exploring novel regulatory molecules and signaling pathways involved in HCC progression is of great significance for identifying diagnostic biomarkers and developing therapeutic strategies.

Cuproptosis is a newly discovered type of regulated cell death that depends on intracellular copper accumulation and triggers mitochondrial metabolism disorder by promoting lipoylated protein aggregation [7, 8, 9]. Distinct from apoptosis, ferroptosis, and necroptosis, cuproptosis provides a new direction for tumor suppression research. Lipoyl synthase (LIAS) acts as a key mediator of cuproptosis by participating in the lipoylation of mitochondrial enzymes, and its abnormal expression is closely associated with tumor proliferation and death [10, 11]. However, the transcriptional regulatory network of LIAS in HCC, especially its connection with immune‐related molecules, has not been fully elucidated.

Interferon regulatory factors (IRFs) are critical transcription factors that modulate interferon production and innate immune responses [12, 13], among which IRF8 has been reported to exert tumor‐suppressive roles in several malignancies by regulating cell apoptosis and the immune microenvironment [14, 15]. Although previous studies have suggested that IRF8 may be dysregulated in liver cancer [16], its specific expression pattern, biological function, and underlying mechanism in HCC remain controversial. More importantly, whether IRF8 participates in HCC progression by regulating cuproptosis, LIAS expression, and interferon secretion has not been reported.

Based on the above gaps, this study systematically investigated the expression of IRF8 in HCC tissues and cell lines through bioinformatics analysis and clinical sample verification. Gain‐ and loss‐of‐function assays, luciferase reporter assays, ChIP‐qPCR, and rescue experiments were performed to explore the role of IRF8 in regulating copper homeostasis, oxidative stress, interferon production, and cuproptosis. In addition, nude mouse xenograft models were applied to verify the in vivo anti‐tumor effect of IRF8. This study aimed to clarify the functions and molecular mechanism of the IRF8/LIAS axis in HCC, providing a theoretical basis for targeted therapy of liver cancer.

2. Materials and Methods

2.1. Clinical Samples and Ethical Approval

Twenty paired HCC tissues and adjacent normal liver tissues were collected from patients who underwent surgical resection at Chengdu Public Health Clinical Center. All specimens were immediately frozen in liquid nitrogen after resection and stored at −80°C for subsequent RNA and protein extraction. This study was approved by the Ethics Committee of the Chengdu Public Health Clinical Center (Approval No. YJ‐K2025‐19‐01), and all patients provided written informed consent in accordance with the Declaration of Helsinki.

2.2. Bioinformatics Analysis

The transcriptional levels of interferon regulatory factor (IRF) family members (IRF1–IRF9) in HCC and adjacent normal liver tissues were retrieved from The Cancer Genome Atlas (TCGA) database. The expression data were normalized and analyzed using R software (version 4.2.1) with the limma package. The difference in IRF family expression between HCC and normal tissues was evaluated.

2.3. Cell Culture

Human HCC cell lines (Huh7, Hep3B, SK‐HEP‐1, SNU‐449, SNU‐475) and the normal human liver cell line THLE‐2 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (11 965 092, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, 10099158, Gibco, USA) and 1% penicillin–streptomycin (15 140 122, Gibco, USA) under a humidified atmosphere of 5% CO₂ at 37°C. All cell lines were authenticated by short tandem repeat (STR) profiling (GENEWIZ, Suzhou, China) prior to the experiments and were tested routinely for mycoplasma contamination using the Mycoplasma PCR Detection Kit (ab289834, Abcam, China). All cell lines were confirmed to be mycoplasma‐free. 10 μM tetrathiomolybdate (TTM, HY‐128530, MedChemExpress, Shanghai, China) and 10 nM Elesclomol (HY‐12040, MedChemExpress, Shanghai, China) were used to suppress or induce cuproptosis in Huh7 cells with overexpressing IRF8 or Hep3B cells with knockdown of IRF8 for 24 h.

2.4. Plasmid Construction and Cell Transfection

The full‐length human IRF8 cDNA was cloned into the pcDNA3.1 vector (V79020, Invitrogen, USA) to construct the IRF8 overexpression plasmid (IRF8‐OE). Short hairpin RNAs (shRNAs) targeting IRF8 (sh‐IRF8) and a non‐targeting control shRNA (sh‐NC) were designed and inserted into the pLKO.1 vector (10 878, Addgene, USA). For rescue experiments, the LIAS overexpression plasmid (LIAS‐OE) and LIAS‐targeting shRNA (sh‐LIAS) were constructed using the same vector system. Cell transfection was performed using Lipofectamine 3000 reagent (L3000015, Invitrogen, USA) in accordance with the manufacturer's instructions. Huh7 cells were transfected with IRF8‐OE or empty vector, and Hep3B cells were transfected with sh‐IRF8 or sh‐NC. For rescue experiments, Huh7 cells were co‐transfected with IRF8‐OE and sh‐LIAS, while Hep3B cells were co‐transfected with sh‐IRF8 and LIAS‐OE. Transfection efficiency was verified by RT‐PCR after transfection (Figure S1). The sequences of sh‐IRF8 and sh‐LIAS are listed in Table S1.

2.5. Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR)

Total RNA was extracted from tissues and cells using TRIzol reagent (15596018CN, Invitrogen, USA). The concentration and purity of RNA were measured using a NanoDrop 2000 spectrophotometer (ND‐2000, Thermo Fisher Scientific, USA). Reverse transcription was performed with PrimeScript RT Master Mix (RR037Q, Takara, China) to synthesize complementary DNA (cDNA). Quantitative PCR (qPCR) was carried out using SYBR Green PCR Kit (QR0100, Sigma‐Aldrich, China) on an ABI 7900HT System (Applied Biosystems, USA). GAPDH was used as the internal reference gene. The relative gene expression level was calculated using the 2^−ΔΔCt method. The primer sequences for IRF8, LIAS, and GAPDH were provided in Table S2.

2.6. Western Blot Analysis

Cells and tumor tissues were lysed in RIPA lysis buffer (P0013B, Beyotime, China) to extract total protein. For analysis of DLAT oligomerization under native conditions, cells were lysed in Native Lysis Buffer (AB156035, Abcam, China) and the soluble proteins were collected after centrifugation at 12000 × g for 15 min at 4°C. The protein concentration was determined using the BCA Protein Assay Kit (20200ES76, Yeasen, China). Equal amounts of protein (30 μg) were separated under two conditions: (1) Conventional denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE): samples were mixed with 5× SDS loading buffer (20317ES05, Yeasen, China), heated at 95°C for 5 min, and separated on 10% SDS‐PAGE. (2) Native PAGE for DLAT oligomerization detection: samples were mixed with native protein loading buffer (MA0472, MeilunBio, China) without SDS, and loaded onto 4%–12% NuPAGE Bis‐tris gel (NP0321BOX, Thermo Fisher, USA) without heat denaturation. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, Merck, Germany). Membranes were blocked with 5% BSA (for native PAGE) or 5% non‐fat milk (for denaturing SDS‐PAGE) in TBST for 1 h at room temperature, then incubated with primary antibodies against IRF8 (PA5‐82021, 1:1000 dilution, Invitrogen, USA), SLC31A1 (27499–1‐AP, 1:1000 dilution, Proteintech, China), FDX1 (82957–1‐RR, 1:1000 dilution, Proteintech, China), LIAS (11577–1‐AP, 1:1500 dilution, Proteintech, China), DLAT (DF14046, 1:1500 dilution, Affinity, China), NDUFS1 (DF4213, 1:1000 dilution, Affinity, China), SDHB (DF12732,1:1000 dilution, Affinity, China) and GAPDH (ab9485, 1:2500 dilution, Abcam, China) overnight at 4°C. After washing three times with TBST, membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody (A0208, Beyotime, China) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagent (32 106, Thermo Fisher Scientific, USA) and imaged with a FluorChem M system (Bio‐Rad Laboratories, USA). The relative protein expression level was quantified using ImageJ software.

2.7. Immunohistochemistry (IHC) Staining

Tissue samples were fixed in 4% paraformaldehyde (P0099, Beyotime, China) for 24 h, embedded in paraffin (YA0012, Solarbio, China), and cut into 4‐μm‐thick sections. After deparaffinization with xylene and rehydration through gradient ethanol, antigen retrieval was performed by boiling the sections in citrate buffer (pH 6.0) for 15 min. Sections were incubated with anti‐IRF8 primary antibody (PA5‐82021, 1:500 dilution, Invitrogen, USA) or anti‐Ki67 antibody (28074–1‐AP, 1:1000 dilution, Proteintech, China) overnight at 4°C. After washing with PBS, sections were incubated with HRP‐conjugated secondary antibody (A0208, Beyotime, China) for 1 h at room temperature. Color development was performed using 3,3′‐diaminobenzidine (DAB) substrate (36303ES01, Yeasen, China), and sections were counterstained with hematoxylin (HY‐N0116, MedChemExpress, China). Images were captured using a light microscope (IX73, Olympus Corporation, Japan).

2.8. TUNEL Assay for Cell Apoptosis

Cell apoptosis was detected using the TUNEL Apoptosis Detection Kit (C1086, Beyotime, China). Briefly, transfected cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.1% Triton X‐100 for 5 min, and then incubated with TUNEL reaction mixture at 37°C in the dark for 1 h. Nuclei were stained with DAPI (C1002, Beyotime, China) for 5 min. Images were captured using a fluorescence microscope (IX73, Olympus Corporation, Japan), and the apoptotic rate was calculated as the percentage of TUNEL‐positive cells among total cells.

2.9. Intracellular Copper Ion Concentration Measurement

Intracellular copper ion concentration was measured using the Copper Assay Kit (E‐BC‐K300‐M, Elabscience, China). Transfected cells were harvested, washed twice with cold PBS, and lysed with lysis buffer provided in the kit. The cell lysate was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. The copper concentration was determined by measuring the absorbance using a microplate reader (UV‐1780, Shimadzu, Japan) according to the kit instructions.

2.10. Reactive Oxygen Species (ROS) Detection

Intracellular ROS levels were detected using the DCFH‐DA fluorescent probe (S0033, Beyotime, China). Transfected cells were incubated with 10 μM DCFH‐DA in serum‐free DMEM at 37°C for 20 min. After washing three times with PBS, cells were observed under a fluorescence microscope (IX73, Olympus Corporation, Japan), and the fluorescence intensity was quantified using ImageJ software.

2.11. Detection of Lipid Peroxidation and Antioxidant Markers

The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in cells and tumor tissues were detected using commercial kits. MDA content was measured with the MDA Assay Kit (S0131, Beyotime, China), SOD activity was detected with the SOD Assay Kit (S0101, Beyotime, China), and GSH content was determined with the GSH Assay Kit (S0052, Beyotime, China). All operations were performed in accordance with the manufacturer's instructions, and the absorbance was measured using a microplate reader (UV‐1780, Shimadzu, Japan).

2.12. Dual‐Luciferase Reporter Assay

The JASPAR database (https://jaspar.genereg.net/) was used to predict the DNA‐binding motif of IRF8 and its potential binding sites on the LIAS promoter. Two candidate binding regions (P1 and P2) were identified. The wild‐type (WT) LIAS promoter and its mutant constructs (P1‐MUT, P2‐MUT, and P1/P2‐MUT) were amplified by PCR and cloned into the pGL3‐Basic luciferase reporter vector (E1751, Promega, USA). Huh7 and Hep3B cells were seeded into 24‐well plates and co‐transfected with the luciferase reporter vector (WT or mutant), IRF8‐OE or sh‐IRF8, and pRL‐TK renilla luciferase vector (E2241, Promega, USA) using Lipofectamine 3000. The luciferase activity was measured using the Dual‐Luciferase Reporter Assay System (E1910, Promega, USA) according to the manufacturer's protocol. The firefly luciferase activity was normalized to the renilla luciferase activity to correct for transfection efficiency.

2.13. Chromatin Immunoprecipitation (ChIP)‐qPCR Assay

ChIP assay was performed using the ChIP Assay Kit (P2078, Beyotime, China) based on the manufacturer's instructions. Briefly, cells were cross‐linked with 1% formaldehyde for 10 min at room temperature, and the cross‐linking was terminated with glycine. Cells were lysed, and chromatin was sheared into 200–500 bp fragments by sonication. The chromatin fragments were immunoprecipitated with anti‐IRF8 antibody (PA5‐82021, 1:200 dilution, Invitrogen, USA) or normal IgG (ab172730, Abcam, China) overnight at 4°C. The immunoprecipitated DNA was purified and subjected to qPCR analysis using primers specific for the P1 and P2 regions of the LIAS promoter. The relative enrichment of IRF8 on the LIAS promoter was calculated using the 2^−ΔΔCt method, with IgG as the negative control.

2.14. In Vivo Xenograft Tumor Model

BALB/c nude mice (4–6 weeks old, 18–22 g) were purchased from Cavens Experimental Animal Center (Changzhou, China). All animal experiments were approved by the Ethics Committee of the Chengdu Public Health Clinical Center (Approval No. YJ‐K2025‐19‐01) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Stably IRF8‐overexpressing Huh7 cells (IRF8‐OE) and IRF8‐overexpressing Huh7 cells co‐transfected with sh‐LIAS (IRF8‐OE + sh‐LIAS) were constructed, and empty vector‐transfected Huh7 cells were used as the control group. Cells were resuspended in PBS at a density of 5 × 10⁶ cells/100 μL, and each mouse was subcutaneously injected with 100 μL of cell suspension into the right flank (n = 5 per group). Tumor volume was measured every 7 days using a vernier caliper, and the volume was calculated using the formula: Volume = (length × width²)/2. Thirty‐five days after inoculation, mice were euthanized by cervical dislocation, and tumors were dissected, photographed, and weighed.

2.15. Detection of Interferon (IFN) Levels

The levels of IFN‐α and IFN‐β in cell culture supernatants and tumor tissue homogenates were detected using enzyme‐linked immunosorbent assay (ELISA) kits. IFN‐α ELISA Kit (E‐EL‐H6125, Elabscience, China) and IFN‐β ELISA Kit (E‐EL‐H0085, Elabscience, China) were used based on the manufacturer's instructions. The absorbance was measured using a microplate reader (UV‐1780, Shimadzu, Japan), and the concentrations of IFN‐α and IFN‐β were calculated based on the standard curve.

2.16. Statistical Analysis

All experiments were performed in triplicate, and the data were presented as the mean ± standard deviation (SD). Statistical differences between two groups were analyzed using Student's t‐test, and differences among multiple groups were analyzed using one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test. All statistical analyses were conducted using GraphPad Prism software. p < 0.05 was considered statistically significant.

3. Results

3.1. IRF8 Is Downregulated in HCC and Promotes HCC Cell Death

To explore the expression pattern of interferon regulatory factors (IRFs) family members (IRF1–IRF9) in HCC, the transcriptional levels of IRFs were analyzed using the TCGA database. Compared with adjacent normal liver tissues, the expression of IRF8 was notably downregulated in HCC tissues (Figure 1A,B). To validate this finding in clinical specimens, the RT‐PCR and immunohistochemistry assays were performed. Consistently, IRF8 mRNA expression was markedly reduced in HCC tissues relative to normal control samples (Figure 1C), and immunohistochemistry staining further confirmed the decreased protein expression of IRF8 in HCC tissue sections (Figure 1D,E). Subsequently, Western blot analysis was conducted to detect IRF8 expression in five HCC cell lines. Relative to the normal human liver cell line THLE‐2, IRF8 protein levels were decreased in all tested HCC cell lines (Figure 1F,G). Based on these results, Huh7 cells (with the lowest IRF8 expression) and Hep3B cells (with relatively high IRF8 expression) were selected for subsequent functional experiments. We then constructed an IRF8 overexpression plasmid for transfection into Huh7 cells and an IRF8‐targeting shRNA vector for knockdown in Hep3B cells, respectively. TUNEL assays revealed that IRF8 overexpression significantly promoted cell death in Huh7 cells, whereas IRF8 knockdown remarkably suppressed cell death in Hep3B cells (Figure 1H,I).

FIGURE 1.

IRF8 is downregulated in HCC and promotes HCC cell death. (A‐B) The mRNA expression levels of IRF family members (IRF1–IRF9) in HCC and normal liver tissues were analyzed using the TCGA database. (C) The mRNA expression of IRF8 in clinical HCC tissues and normal control samples was detected by RT‐PCR (N = 20). (D‐E) The expression of IRF8 in HCC tissues and normal liver tissues was determined by immunohistochemistry staining (N = 5). Scale bar = 100 μm. (F‐G) The protein expression of IRF8 in five HCC cell lines and normal human liver cell line THLE‐2 was measured by Western blot assay (N = 3). (H‐I) After overexpression of IRF8 in Huh7 cells and knockdown of IRF8 in Hep3B cells, cell death was assessed by TUNEL assay (N = 3). Scale bar = 20 μm. Data were presented as mean ± SD, **p < 0.01.

3.2. IRF8 Induces Copper Accumulation, Oxidative Stress and Cuproptosis in HCC Cells

Given the critical role of cuproptosis in tumor progression, the effects of IRF8 on copper homeostasis and oxidative stress in HCC cells were evaluated. Measurement of intracellular copper ion concentrations demonstrated that IRF8 overexpression markedly increased copper accumulation in Huh7 cells, while IRF8 depletion significantly reduced copper levels in Hep3B cells (Figure 2A). Using the DCFH‐DA fluorescent probe, the findings indicated that IRF8 overexpression enhanced intracellular ROS accumulation, and IRF8 knockdown conversely attenuated ROS production (Figure 2B,C). In addition, the levels of lipid peroxidation and antioxidant markers were examined. IRF8 overexpression in Huh7 cells led to a significant increase in MDA content and a pronounced reduction in SOD and GSH levels. In contrast, IRF8 knockdown in Hep3B cells decreased MDA production while elevating SOD and GSH levels (Figure 2D–F). Western blot analysis of cuproptosis‐related markers showed that IRF8 overexpression upregulated the protein expression of SLC31A1, FDX1, and LIAS in Huh7 cells, whereas IRF8 silencing downregulated the expression of these three proteins in Hep3B cells (Figure 2G,H). To obtain more definitive evidence of cuproptosis, the additional cuproptosis‐specific markers were further examined. Western blot analysis (Figure 2I–K) revealed that IRF8 overexpression significantly increased the level of high‐molecular‐weight DLAT oligomer aggregates in Huh7 cells, while IRF8 knockdown markedly reduced DLAT aggregation in Hep3B cells. In addition, the IRF8 protein levels were increased in IRF8‐overexpressing Huh7 cells and decreased in IRF8‐knockdown Hep3B cells, confirming the efficiency of IRF8 overexpression/knockdown. Additionally, IRF8 overexpression reduced the expression of Fe‐S cluster proteins NDUFS1 and SDHB, while IRF8 knockdown promoted these proteins (Figure 2I–K). To confirm that IRF8‐induced cell death was copper‐dependent, the Huh7 cells overexpressing IRF8 were treated with the copper chelator tetrathiomolybdate (TTM) and the Hep3B cells knocking down IRF8 were treated with the cuproptosis activator (Elesclomol). Notably, both treatments significantly reversed the effects of IRF8‐OE or sh‐IRF8, which collectively confirmed that IRF8 induces cuproptosis in HCC cells.

FIGURE 2.

IRF8 induces copper accumulation, oxidative stress and cuproptosis in HCC cells. (A) Intracellular copper ion levels in HCC cells with IRF8 overexpression or knockdown were detected using a commercial assay kit (N = 5). (B, C) Intracellular ROS accumulation in the indicated HCC cells was measured by DCFH‐DA fluorescent probe staining (N = 5). Scale bar = 50 μm. (D‐F) The levels of MDA, SOD and GSH in HCC cells with altered IRF8 expression were determined using corresponding detection kits (N = 5). (G, H) The protein expression of cuproptosis markers SLC31A1, FDX1 and LIAS in HCC cells was examined by Western blot (N = 3). (I–K) Western blot assay was applied to detect the expressions of DLAT oligomers, NDUFS1, SDHB and IRF8 in Huh7 and Hep3B cells (N = 3). Data were presented as mean ± SD, **p < 0.01, *p < 0.05.

3.3. IRF8 Transcriptionally Activates LIAS by Directly Binding to Its Promoter

To elucidate the molecular mechanism underlying IRF8‐mediated regulation of cuproptosis, the JASPAR database was applied to predict the DNA‐binding motif of IRF8 and its potential binding sites on the LIAS promoter. Two candidate binding regions, designated P1 and P2, were identified on the LIAS promoter (Figure 3A). We then generated luciferase reporter vectors carrying wild‐type (WT) LIAS promoter, as well as single mutants (P1‐MUT, P2‐MUT) and a double mutant (P1/P2‐MUT) of the predicted binding sites using site‐directed mutagenesis (Figure 3B). Dual‐luciferase reporter assays showed that IRF8 overexpression significantly enhanced the luciferase activity of the WT LIAS promoter. Mutation of either P1 or P2 partially abrogated this stimulatory effect, while simultaneous mutation of both sites completely abolished IRF8‐mediated activation of the LIAS promoter. Conversely, IRF8 knockdown suppressed the luciferase activity of the WT LIAS promoter; this inhibitory effect was partially reversed by single‐site mutation and fully reversed by double‐site mutation (Figure 3C,D). These results identified P1 and P2 as functional binding sites that mediate IRF8‐dependent transcriptional activation of LIAS. ChIP‐qPCR assay further verified the direct binding of IRF8 to the LIAS promoter in HCC cells (Figure 3E). Western blot assays additionally demonstrated that IRF8 overexpression upregulated, while IRF8 knockdown downregulated, both IRF8 and LIAS protein levels in HCC cells (Figure 3F–H).

FIGURE 3.

IRF8 transcriptionally activates LIAS by directly binding to its promoter. (A) The potential binding sites of IRF8 on the LIAS promoter were predicted using the JASPAR database. (B–D) Wild‐type and mutant (P1‐MUT, P2‐MUT, P1/P2‐MUT) LIAS promoter luciferase reporter vectors were constructed, and the transcriptional activity was detected by dual‐luciferase reporter assay (N = 3). (E) The direct binding of IRF8 to the LIAS promoter in HCC cells was verified by ChIP‐qPCR assay (N = 3). (F–H) The protein expression of IRF8 and LIAS in HCC cells with IRF8 overexpression or knockdown was detected by Western blot (N = 3). Data were presented as mean ± SD, *p < 0.05, **p < 0.01, ns = non‐significant.

3.4. LIAS Is Required for IRF8‐Mediated IFN Production and Cuproptosis in HCC

To verify whether LIAS mediates the biological functions of IRF8 in HCC cells, the rescue experiments were further performed. Detection of interferon levels revealed that IRF8 overexpression significantly promoted the production of IFN‐α and IFN‐β in Huh7 cells, and this effect was markedly reversed by LIAS knockdown. In Hep3B cells, IRF8 silencing reduced IFN‐α and IFN‐β secretion, whereas LIAS overexpression significantly restored interferon production (Figure 4A,B). TUNEL staining showed that IRF8 overexpression‐induced cell death in Huh7 cells was significantly attenuated by LIAS knockdown. Correspondingly, the anti‐cell death effect of IRF8 knockdown in Hep3B cells was notably reversed by LIAS overexpression (Figure 4C–E). Analysis of intracellular MDA and copper ion levels indicated that LIAS knockdown suppressed the IRF8 overexpression‐mediated increase in MDA and copper accumulation in Huh7 cells, while LIAS overexpression restored the reduced MDA and copper levels caused by IRF8 silencing in Hep3B cells (Figure 4F,G). Similarly, IRF8 overexpression‐enhanced ROS accumulation was mitigated by LIAS knockdown, and IRF8 depletion‐reduced ROS production was rescued by LIAS overexpression (Figure 4H–J).

FIGURE 4.

LIAS is required for IRF8‐mediated IFN production and cuproptosis in HCC. (A, B) The production of IFN‐α and IFN‐β in HCC cells under rescue experiments was measured using commercial ELISA kits (N = 5). (C–E) Cell death in the indicated HCC cells was evaluated by TUNEL staining (N = 3). Scale bar = 20 μm. (F–G) Intracellular MDA and copper ion levels in HCC cells were detected using corresponding assay kits (N = 5). (H‐J) Intracellular ROS levels in HCC cells were assessed by DCFH‐DA fluorescent probe (N = 5). Scale bar = 50 μm. Data were presented as mean ± SD, **p < 0.01, ns = non‐significant.

3.5. IRF8 Suppresses HCC Xenograft Growth in a LIAS‐Dependent Manner In Vivo

To validate the above findings in vivo, the stable IRF8‐overexpressing Huh7 cell lines were established and a nude mouse subcutaneous xenograft tumor model was constructed. Tumor volume was monitored throughout the experiment, and tumors were harvested, photographed, and weighed 35 days after inoculation. Compared with the vector control group, IRF8 overexpression significantly suppressed the growth rate and final weight of subcutaneous xenografts. Notably, simultaneous LIAS knockdown markedly reversed the tumor‐suppressive effect of IRF8 overexpression and accelerated tumor growth (Figure 5A–C). IHC staining of the proliferation marker Ki67 in tumor tissues showed that IRF8 overexpression significantly reduced Ki67 expression, while LIAS knockdown restored Ki67 levels in IRF8‐overexpressing tumors (Figure 5D,E). Biochemical analysis of tumor tissues further demonstrated that IRF8 overexpression significantly increased copper ion concentration and the levels of IFN‐α and IFN‐β in xenografts. Conversely, LIAS knockdown significantly attenuated the IRF8‐mediated elevation of copper, IFN‐α, and IFN‐β in tumor tissues (Figure 5F–H).

FIGURE 5.

IRF8 suppresses HCC xenograft growth in a LIAS‐dependent manner in vivo. (A–C) Tumor images (A), weight (B) and volume of HCC xenografts (C). (D, E) IHC staining of Ki67 in xenograft tissues (N = 5). Scale bar = 50 μm. (F–H) The levels of copper ions, IFN‐α and IFN‐β in xenograft tumor tissues were measured using corresponding commercial kits (N = 5). Data were presented as mean ± SD, **p < 0.01, ns = non‐significant.

4. Discussion

HCC remains a leading cause of cancer‐related mortality globally, owing to its high heterogeneity, late diagnosis, and frequent resistance to conventional therapies [17]. Mounting evidence has highlighted programmed cell death pathways as promising targets for HCC intervention [18], among which cuproptosis has recently emerged as a copper‐dependent cell death modality distinct from apoptosis, ferroptosis, and necroptosis. As a core regulator of mitochondrial lipoylation and copper metabolism, LIAS has been identified as a critical mediator of cuproptosis [19], yet its upstream transcriptional regulatory network in liver cancer remains largely uncharacterized. IRFs, particularly IRF8, are well‐documented modulators of immune responses and interferon production, with context‐dependent tumor‐suppressive or oncogenic roles in human malignancies [20]. However, whether IRF8 orchestrates cuproptosis, interferon secretion, and HCC progression through targeting LIAS has never been explored. Our study uncovered an IRF8/LIAS signaling axis that connects immune regulation and copper metabolism, suggesting a mechanism by which IRF8 may suppress HCC progression.

The understanding of HCC has been greatly advanced by comprehensive genomic and transcriptomic analyses, which have revealed that HCC comprises multiple molecular subtypes with distinct gene expression signatures, driver mutations, and clinical behaviors [21, 22]. These subtypes exhibit profound differences in the activation of proliferative signaling pathways, immune cell infiltration, metabolic reprogramming, and susceptibility to programmed cell death. Previous studies have reported that IRF8 is downregulated in several solid tumors and hematological malignancies, where it suppresses tumor proliferation, invasion, and immune escape [23, 24]. In liver cancer, limited studies have implied that IRF8 may participate in hepatic inflammation and tumorigenesis [25], but its detailed expression pattern and functional mechanisms remain controversial. Our data confirmed that IRF8 is significantly reduced in HCC tissues and cell lines, consistent with its putative role as a tumor suppressor. The down‐regulation of IRF8 in HCC is consistent with the gene expression heterogeneity that characterizes this malignancy. The transcriptional levels in the TCGA database have revealed that IRF family members exhibit differential expression across HCC molecular subtypes, and this heterogeneity has important implications for patient stratification. Unlike prior studies that focused on IRF8‐mediated immune regulation or apoptosis, our work extended its biological function to copper homeostasis and oxidative stress, demonstrating that IRF8 triggers intracellular copper accumulation, ROS overproduction, and lipid peroxidation, thereby inducing cuproptosis in HCC cells. This finding broadens the functional spectrum of IRF8 beyond traditional immune regulation and connects it to metal metabolism‐driven cell death.

LIAS has been established as a key executor of cuproptosis by catalyzing protein lipoylation in the tricarboxylic acid cycle, and its dysregulation is closely associated with tumor progression [26, 27]. Our study first identified that IRF8 directly binds to two functional motifs on the LIAS promoter and transcriptionally enhances its expression, revealing a novel upstream regulatory mechanism of LIAS. Rescue assays further verified that LIAS is indispensable for IRF8‐mediated interferon production, oxidative stress, and cuproptosis, suggesting that LIAS acts as a pivotal downstream effector bridging IRF8 and HCC suppression. This regulatory cascade represents a new molecular link between immune transcription factors and copper‐dependent cell death. Although our findings position IRF8/LIAS as a biologically plausible regulatory axis that could be targeted for therapy, it is important to recognize that these conclusions are based on cell culture and xenograft models. Future studies using patient‐derived organoids, additional in vivo models, and clinical correlation analyses are needed to assess the clinical utility of this axis.

In conclusion, IRF8 functions as a tumor suppressor in HCC by transcriptionally activating LIAS to promote cuproptosis and interferon production. The IRF8/LIAS axis may serve as a novel tumor‐suppressive mechanism in HCC and warrants further investigation as a candidate therapeutic target. However, these findings are preclinical and require validation in additional HCC models and clinical studies before translational implications can be considered.

Author Contributions

Conception and design of the research: Y.W., M.L.; acquisition of data: L.Z., Z.H., B.W., L.W.; analysis and interpretation of data: M.D., R.H., H.L.; statistical analysis: X.Z., Y.Z., L.L.; obtaining funding: M.L.; drafting the manuscript: L.Z., Z.H.; revision of manuscript for important intellectual content: Y.W., M.L.

Funding

This work was financially supported by the Chengdu Medical Research Project (2025584, 2025563) and National Inheritance Studio for Famous & Senior Experts of Traditional Chinese Medicine (NATCM Personnel & Education Dept. Letter [2022] No. 75).

Ethics Statement

This study was approved by the Ethics Committee of the Chengdu Public Health Clinical Center (Approval No. YJ‐K2025‐19‐01).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: shRNA sequences in this study.

Table S2: RT‐PCR primer sequences in this study.

Figure S1: Transfection efficiency was confirmed via RT‐PCR analysis using GAPDH as internal control. (A‐B) The knockdown efficiency of sh‐IRF8 and the overexpression efficiency of IRF8 overexpression plasmid (IRF8‐OE). The sh‐IRF8#1 was selected for subsequent assays. (C‐D) The knockdown efficiency of sh‐LIAS and the overexpression efficiency of LIAS overexpression plasmid (LIAS‐OE). The sh‐LIAS#1 was selected for subsequent assays. Data were presented as mean ± SD; *p < 0.05, **p < 0.01.

Zhu L., Hu Z., Wu B., et al., “ IRF8 Suppresses Hepatocellular Carcinoma Progression Through LIAS‐Dependent IFN Production and Cuproptosis Activation,” Genes, Chromosomes and Cancer 65, no. 9 (2026): e70172, 10.1002/gcc.70172.

Data Availability Statement

The data utilized and/or analyzed throughout this research are available from the corresponding author on reasonable request.