MET promotes hepatocellular carcinoma development through the promotion of TRIB3-mediated FOXO1 degradation

Patients and tissue specimens

Patient samples used in this study were obtained from the Department of Liver Surgery at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China. We utilized paraffin tissue samples from 75 patients and fresh samples from 60 patients, all of whom had undergone hepatectomy at Tongji Hospital and were pathologically diagnosed with HCC. The study protocol, in accordance with the ethical guidelines of the 1975 Declaration of Helsinki, received approval from the Ethics Committee of Tongji Hospital, and an exemption for informed consent was granted by the institutional ethics committee (TJ-IRB20220652).

A full description of the materials and methods used in this study can be found in the supplementary methods and CTAT table.

TRIB3 is upregulated in HCC and elevated TRIB3 levels predict a poor prognosis

To identify genes activated by MET, we established HCC models using hydrodynamic tail vein injection (HTVi) of plasmids DNA encoding MET/β-catenin. Mice injected with the plasmids developed HCC, whereas the control group showed no significant pathological changes (Fig. 1A, 1B). RNA-seq analysis of liver tissues from the MET/β-catenin group identified 3,034 up- and 2,317 downregulated genes (P<0.05) compared with the control group (Fig. 1B). Gene Ontology analysis demonstrated significant enrichment of protein kinase-associated signaling pathways among the differentially expressed genes (log2FC>1, P<0.05) (Fig. 1C1). Complementing these findings, gene set enrichment analysis (GSEA) of RNA-seq data further confirmed the activation of multiple protein kinase-related pathways (Fig. 1C2). Notably, TRIB3 emerged as a prominent factor linked to critical biological processes, including kinase activity modulation and MAP kinase signaling regulation. Furthermore, GSEA of the TCGA LIHC cohort further revealed marked enrichment of the MET pathway in the TRIB3 high-expression subgroup compared to the low-expression subgroup, suggesting that TRIB3 may play key roles in MET/β-catenin-driven HCC mouse models (Supplementary Fig. 1A). Furthermore, immunohistochemistry (IHC) and multiplex immunofluorescence were performed in a hydrodynamic injection mouse model. The results demonstrated significant upregulation of TRIB3 protein expression in MET/β-catenin-driven HCC compared to normal liver tissues (Fig. 1C3).

Figure 1.

TRIB3 is upregulated in HCC specimens, and high expression of TRIB3 predicts poor prognosis. (A) Schematic diagram of HTVi of oncogenic plasmids in C57BL/6. (B) Representative images of morphology and H&E staining of HTVi model (left). The volcano plot of differential genes (DEG) between MET/β-catenin and control group (right). (C1) GO analysis of differentially expressed genes from RNA-seq. (C2) GSEA analysis of RNA-seq. (C3) IHC and mIHC of TRIB3 in MET/β-catenin HTVi HCC model (n=3, Mann–Whitney test). (D) Differential expression of the TRIB3 protein in 60 paired HCC and adjacent tissue samples in cohort 1 as determined by WB (top) and RT-qPCR (right) (n=60, Wilcoxon test). Relative TRIB3 expression analyzed by WB (left). (E) Representative IHC images and quantification analysis of TRIB3 staining in 75 pairs of HCC (n=75. Wilcoxon test); scale bar: 200 μm (left). Chi-square analysis of the relevance of TRIB3 expression with alpha-fetoprotein level, tumor number, tumor size, tumor capsule and Barcelona Clinic Liver Cancer stage in HCC patients (right). (F) HCC patients were divided into two groups (TRIB3 high or low) according to their median IHC score of TRIB3 in (E). Kaplan–Meier analyses for overall survival and RFS for HCC patients in these two groups (top) (log-rank test). Forest plot of the multivariate Cox proportional hazards model for OS and RFS (bottom). GO, Gene Ontology; GSEA, gene set enrichment analysis; HCC, hepatocellular carcinoma; HTVi, hydrodynamic tail vein injection; IHC, immunohistochemistry; OS, overall survival; WB, Western blot. *P<0.05, **P<0.01, ***P<0.001.

Analysis of TCGA data showed significantly elevated TRIB3 expression in HCC tissues compared to normal liver tissues, with higher expression linked to advanced pathological stages and poor prognosis (Supplementary Fig. 1B, 1C). In the Tongji cohort, western blotting and RT-qPCR of 60 paired HCC samples confirmed TRIB3 overexpression, which was further validated by IHC in 75 paired samples (Fig. 1D, 1E, Supplementary Fig. 1D, Supplementary Table 1). TRIB3 expression was positively correlated with tumor size (Fig. 1E and Supplementary Table 2), while also being associated with reduced overall survival (OS) and recurrence-free survival (RFS) (Fig. 1F). Cox regression analysis identified high TRIB3 levels as an independent risk factor for shorter OS and RFS (Fig. 1F, Supplementary Tables 3, 4). These findings suggest that MET overexpression may upregulate TRIB3 expression, and higher TRIB3 levels are indicative of a poorer prognosis in HCC patients.

TRIB3 promoted the proliferation, migration and invasion of HCC cells both in vitro and in vivo

To investigate the role of TRIB3 in HCC, we first assessed its expression levels in seven HCC cell lines by Western blotting (Supplementary Fig. 2A). Elevated endogenous TRIB3 expression was confirmed in several HCC cell lines. We then generated stable Hep3B and HLF cell lines overexpressing TRIB3, as well as MHCC97H and HCCLM3 cell lines with TRIB3 knockdown (Supplementary Fig. 2A). Cell proliferation was evaluated using the CCK-8 and EdU assays. TRIB3 overexpression significantly increased HCC cell proliferation, while TRIB3 knockdown had the opposite effect (Fig. 2A, Supplementary Fig. 2B, 2C). We further examined the effects of TRIB3 on cell migration and invasion using wound healing and transwell assays. Increased TRIB3 expression significantly enhanced migration and invasion in Hep3B and HLF cells, while TRIB3 knockdown reduced migration and invasion in MHCC97H and HCCLM3 cells (Fig. 2B, 2C, Supplementary Fig. 2D, 2E).

Figure 2.

TRIB3 promotes the proliferation and migration of HCC cells in vitro and in vivo. (A) The proliferation of the indicated cells as measured by CCK-8 assays (up) (n=4, Kruskal–Wallis test). Representative images of the EdU incorporation assay and quantification of EdU-positive cells (bottom) (n=3, Mann–Whitney test). Scale bar: 200 μm. (B) Representative images and quantification of the wound healing assay (n=3, Mann–Whitney test). Scale bar: 200 μm. (C) Representative images and quantification of cells that migrated or invaded in the indicated groups (n=3, Mann–Whitney test). Scale bar: 200 μm. (D) Macroscopic images of the indicated cells xenografts at 21 days post-intra-tumoral injection (left). Growth curve (medium) (n=6, Mann–Whitney test) and quantification of tumor weights (right) (n=6, unpaired t-test) of subcutaneous tumors. (E) The nude mice were implanted with the indicated cells in the liver and representative bioluminescent images were shown (left). The bioluminescent signals (medium) and body weight (right) in each group (n=6, unpaired t-test). (F) Incidence of lung metastasis (up left), representative H&E staining images of lung tissues (up right) and the number of lung metastatic nodules (bottom) in the treated nude mice (n=6, Mann–Whitney test). HCC, hepatocellular carcinoma; OD, OOO. *P<0.05, **P<0.01, ***P<0.001.

Next, we explored whether TRIB3 contributes to tumor growth and metastasis in vivo. HCC cells were subcutaneously injected into nude mice to evaluate the impact of TRIB3 on tumorigenicity. Following 21 days after inoculation, the mice were sacrificed to assess tumor growth. Mice injected with TRIB3-overexpressing Hep3B cells exhibited significantly larger tumor weights and volumes compared to those injected with control cells. Conversely, mice injected with TRIB3-knockdown MHCC97H cells had smaller tumor sizes (Fig. 2D). IHC staining for Ki-67 revealed that tumor cells with elevated TRIB3 expression also exhibited increased proliferation compared to controls (Supplementary Fig. 2F). In orthotopic HCC models, mice implanted with TRIB3-overexpressing Hep3B cells exhibited significantly higher bioluminescence intensity, increased incidence of lung metastasis, and more metastatic nodules. Conversely, TRIB3 knockdown in MHCC97H cells reduced the bioluminescence signal, while decreasing the incidence of lung metastasis as well as decreasing the number of metastatic nodules (Fig. 2E, 2F). Collectively, these results suggest that TRIB3 promotes HCC growth and metastasis.

TRIB3 mediated FOXO1 degradation by E3 ubiquitin ligase COP1 in HCC cells

To investigate the mechanism through which TRIB3 promotes HCC progression, we performed co-immunoprecipitation (co-IP) to identify proteins interacting with TRIB3. Silver staining demonstrated a prominent abundance of TRIB3 in liver tissues (Fig. 3A). Mass spectrometry (MS) analysis detected 183 TRIB3-interacting proteins (Supplementary Table 5). The most enriched among them included TPM2, TUBA1A, TUBA1C, CAPZZA2, MYL12A, COP1, GNB2, SPTAN1, GNAI3 and HSP90AB. Furthermore, we analyzed potential binding partners of TRIB3 using the STRING database (Supplementary Fig. 3A). It was found that COP1 was the sole candidate among the top 10 enriched genes in MS results within this network (Supplementary Fig. 3B). Notably, the E3 ubiquitin ligase COP1 has been shown to promote the degradation of several tumor suppressors, such as p53 [23], FOXO1 [24] and C/EBPδ [25], thereby promoting the progression of various malignancies. Furthermore, the Tribbles family, which includes atypical pseudokinases, possesses a regulated binding platform for substrates that are ubiquitinated by contextspecific E3 ligases. Based on this, we propose that TRIB3 functionally participates in the COP1-mediated ubiquitination process. Co-IP confirmed the exogenous interaction between TRIB3 and COP1 in HEK293T cells (Fig. 3B). Furthermore, confocal immunofluorescence (IF) imaging demonstrated the endogenous interaction between TRIB3 and COP1, with both proteins colocalizing predominantly in the nucleus of Hep3B and MHCC97H cells (Fig. 3B).

Figure 3.

TRIB3 mediates the COP1-induced degradation of FOXO1. (A) Verification of the co-IP products by silver stain. (B) The interaction between exogenous COP1 and TRIB3 was confirmed in HEK293T cells (up). Confocal immunofluorescence of TRIB3 and COP1 (bottom). (C) The interaction between endogenous TRIB3, COP1, and FOXO1 in wild-type HCC cell lines. (D) The interaction between endogenous COP1 and FOXO1 in shC- or shTRIB3-MHCC97H. (E) Impact of COP1 overexpression on the protein stability of FOXO1 with or without MG132 as compared to control group. Effects of COP1 overexpression on FOXO1 stability with or without TRIB3 knockdown. (F) Effect of TRIB3 on the COP1-induced ubiquitination of FOXO1. HCC, hepatocellular carcinoma.

It has been reported that COP1 could facilitate FOXO1 degradation [24], while the latter functions as a tumor suppressor in HCC [26,27]. We hypothesize that TRIB3 acts as a molecular bridge, facilitating the interaction between COP1 and its substrate to mediate targeted degradation. To test this hypothesis, we first conducted Co-IP assays using a TRIB3 antibody to confirm its co-precipitation with endogenous COP1 and FOXO1 in Hep3B and MHCC97H cells. Additionally, we performed reverse Co-IP assays using anti-COP1 antibodies to validate its interaction with endogenous TRIB3 and FOXO1 within the immunoprecipitated samples. Furthermore, Co-IP assays with anti-FOXO1 antibodies confirmed its interaction with endogenous TRIB3 and COP1 (Fig. 3C). To further verify these interactions, we assessed the exogenous binding of TRIB3, COP1, and FOXO1 in HEK293T cells via Co-IP (Supplementary Fig. 3C). Consistent with our hypothesis, Co-IP assays using anti-COP1 antibodies showed that TRIB3 knockdown significantly weaken the binding between COP1 and FOXO1 (Fig. 3D).

To confirm that FOXO1 degradation is mediated by the COP1-dependent proteasomal pathway, we assessed the expression of FOXO1 under various conditions. COP1 overexpression significantly decreased the expression of FOXO1, while proteasome inhibition or TRIB3 knockdown significantly decelerated COP1-mediated FOXO1 degradation (Fig. 3E, 3F, Supplementary Fig. 3D). Furthermore, we observed that COP1 overexpression did not significantly affect the expression of TRIB3, which indicates that COP1 cannot mediate the degradation of TRIB3. Moreover, the expression of TRIB3 was reversed by the addition of MG132, suggesting the existence of a non-specific proteasome degradation pathway that is not dependent on COP1, which requires further exploration in the future. To further explore whether this mechanism was present in different cancer types, we extended our study to the human colorectal cancer cell line SW480 and the human breast cancer cell line MCF7. It was found that proteasome inhibition or TRIB3 knockdown also significantly inhibited COP1-mediated FOXO1 degradation in SW480 and MCF7 (Supplementary Fig. 3E). These findings suggest that TRIB3 mediates COP1-induced FOXO1 degradation via the proteasomal pathway, inhibiting the transcriptional effects of FOXO1.

TRIB3 promotes HCC progression by inhibiting FOXO1 expression

FOXO1, a member of the Forkhead box (FOX) family [28], is regulated by various post-translational modifications that enable it to integrate signals and control multiple cellular functions [29]. It is downregulated in several cancers, including HCC [30,31], and plays a crucial role in anti-cancer mechanisms [32-34]. TCGA data showed reduced FOXO1 expression in HCC tumor samples compared to normal liver tissues, with higher FOXO1 levels linked to a better prognosis (Supplementary Fig. 4A, 4B). FOXO1 inhibits tumor-promoting proteins such as MET, CCND1, and TWIST1 [35-37]. In our study, FOXO1 overexpression in Hep3B-TRIB3 cells suppressed the expression of MET, CCND1, and TWIST1, while FOXO1 knockdown in MHCC97H-shTRIB3 cells increased their expression (Fig. 4A, Supplementary Fig. 4C).

Figure 4.

TRIB3 promotes HCC progression through inhibiting FOXO1 expression. (A) TRIB3 upregulate genes inhibited by FOXO1. (B) The proliferation of the indicated cells as measured by CCK-8 assays (left) and the quantification of EdU-positive cells (right) (n=3, oneway ANOVA). (C) The quantification of the wound healing assay and transwell assays (n=3, one-way ANOVA). (D) Macroscopic images (left), growth curve (medium) and quantification of tumor weights (right) of the indicated cells xenografts (n=6, one-way ANOVA). (E) The nude mice were implanted with the indicated cells in the liver and representative bioluminescent images were shown (left). The bioluminescent signals (medium) and body weight (right) in each group (n=6, one-way ANOVA). (F) Incidence of lung metastasis (left), representative H&E staining images of lung tissues (medium) and the number of lung metastatic nodules (right) in the treated nude mice (n=6, Kruskal–Wallis test). HCC, hepatocellular carcinoma; OD, OOO. *P<0.05, **P<0.01, ***P<0.001.

To investigate the role of FOXO1 in TRIB3-mediated HCC progression, CCK-8, EdU, wound healing, and transwell assays were performed. Compared to the controls, FOXO1 overexpression significantly suppressed the proliferative, migratory, and invasive capabilities of HCC cells overexpressing TRIB3. Conversely, FOXO1 knockdown enhanced the proliferative, migratory, and invasive abilities of cancer cells, which was attenuated by TRIB3 downregulation (Fig. 4B, 4C, Supplementary Fig. 4D, 4F).

Furthermore, the involvement of FOXO1 in TRIB3-driven HCC progression was further confirmed in vivo. In xenograft tumor models, overexpression of FOXO1 impaired TRIB3-induced tumor growth, while IHC staining also showed that FOXO1 overexpression reversed the increase of the Ki-67⁺ cell ratio caused by TRIB3 overexpression (Fig. 4D, Supplementary Fig. 4G). In orthotopic HCC models, overexpression of FOXO1 in Hep3B-TRIB3 cells resulted in decreased bioluminescence intensity, reduced incidence of lung metastasis, and fewer metastatic nodules (Fig. 4E, 4F).

Taken together, these findings indicate that TRIB3 promotes HCC progression by affecting FOXO1 expression.

MET upregulates TRIB3 via the ERK/SP1 axis

The molecular mechanism underlying TRIB3 upregulation in HCC is not well understood. TRIB3 was significantly upregulated in our MET/β-catenin HTVi HCC models, prompting us to further investigate the mechanism behind its regulation by MET or β-catenin. The qPCR results showed that MET, and not β-catenin, upregulated the mRNA expression of TRIB3 (Fig. 5A). WB also confirmed that MET upregulates TRIB3 protein levels (Fig. 5A). Inter-estingly, MET enhanced TRIB3 promoter activity (Fig. 5B). Previous studies have shown that MET can regulate downstream targets through transcription factors such as SP1 [38], HIF1A [39], STAT3 [40], and JUN [38]. To elucidate the mechanisms through which MET modulates TRIB3 expression, we designed a series of reporter gene constructs containing truncated fragments of the TRIB3 promoter based on the predicted binding sites of these transcription factors. A de-letion spanning from –1,003 to –477 bp markedly reduced MET-induced TRIB3 promoter activity. There was one presumptive JUN and STAT3 binding site each, as well as two presumptive SP1 and HIF1A binding sites in this sequence. By further mutating these binding sites, we found that mutating the first SP1 binding site largely abolished MET-induced TRIB3 promoter activation, while mutations in the other binding sites had a minimal effect (Fig. 5C). Consistently, SP1 knockdown significantly reduced TRIB3 promoter activity (Fig. 5D).

Figure 5.

MET upregulates TRIB3 via ERK/SP1 axis. (A) RT-qPCR validation of TRIB3 in MET or β-catenin(N90)-overexpression Hep3B cells (left) (n=3, Mann–Whitney test). WB showing that TRIB3 were upregulated by MET (right). (B) Relative luciferase activity of TRIB3 promoter (n=3, Mann–Whitney test). (C) Relative luciferase activity of indicated cells and truncated or mutated luciferase constructs (n=3, Mann–Whitney test). (D) Hep3B cells were transfected with SP1 siRNA or control siRNA. TRIB3 promoter activity and expression were measured by luciferase activity assay (left), RT-qPCR (medium) and WB (right) (n=3, one-way ANOVA). (E) MET-Hep3B cells were treated with inhibitor of ERK, JNK, P38 and PI3K. WB was used to detect the expression of TRIB3. (F) ChIP assay of SP1 binding to the TRIB3 promoter (n=3, one-way ANOVA). *P<0.05, **P<0.01, ***P<0.001.

To determine the signaling pathway involved in MET-dependent TRIB3 induction, Hep3B cells were treated with inhibitors that target the ERK, JNK, p38, and PI3K pathways, respectively. The ERK inhibitor substantially reduced MET-induced TRIB3 and SP1 expression, while the other kinase inhibitors had a minimal impact (Fig. 5E). ChIP assays revealed that the ERK inhibitor reduced SP1 binding to the TRIB3 promoter, in contrast to the other kinase inhibitors (Fig. 5F). These findings indicate that MET-induced TRIB3 expression depends on the ERK/SP1 axis.

TRIB3 is vital for MET-mediated HCC progression

In HCC, abnormal MET activity is correlated with accelerated tumor growth, aggressive invasiveness, and poor patient outcomes [8]. Given the critical role of MET in the development and progression of HCC, we investigated whether TRIB3 plays a role in MET-induced HCC progression. We initially employed a lentiviral vector to achieve stable knockdown of TRIB3 in Hep3B-MET cells (Supplementary Fig. 5A). Both the CCK-8 and EdU assays demonstrated that the facilitated proliferation induced by MET overexpression was abrogated by TRIB3 knockdown in Hep3B cells (Fig. 6A, Supplementary Fig. 5B). Wound healing and transwell assays indicated that Hep3B-MET cells with reduced TRIB3 expression had suppressed migration and invasion abilities (Fig. 6B, Supplementary Fig. 5C, 5D). In line with our in vitro findings, the results of in vivo models showed that the MET-overexpressing group exhibited significantly increased tumor growth and a higher proportion of Ki-67⁺ cells in xenograft tumor compared to the control group. These MET-induced effects were diminished when TRIB3 was knocked down (Fig. 6C, 6D). Additionally, TRIB3 knockdown in Hep3B-MET cells resulted in decreased bio-luminescence intensity, reduced incidence of lung metastasis, and fewer metastatic nodules (Fig. 6E, 6F).

Figure 6.

TRIB3 is vital for MET-mediated HCC progression. (A) The proliferation of the indicated cells as measured by CCK-8 assays (left) and the quantification of EdU-positive cells (right) (n=3, one-way ANOVA). (B) Quantification of the wound healing assay (left) and transwell assays (right) (n=3, one-way ANOVA). (C) Macroscopic images (left), growth curve (medium) and quantification of tumor weights (right) of the indicated cells xenografts (n=6, one-way ANOVA). (D) Representative hematoxylin and eosin (H&E) staining and IHC of Ki67 in subcutaneous xenografts (n=6, Kruskal–Wallis test). Scale bar: 200 μm. (E) The nude mice were implanted with the indicated cells in the liver and representative bioluminescent images were shown (left). The bioluminescent signals (medium) and body weight (right) in each group (n=6, one-way ANOVA). (F) Incidence of lung metastasis (left), representative H&E staining images of lung tissues (medium) and the number of lung metastatic nodules (right) in the treated nude mice (n=6, Kruskal–Wallis test). HCC, hepatocellular carcinoma; IHC, immunohistochemistry; OD, OOO. *P<0.05, **P<0.01, ***P<0.001.

To further investigate the role of TRIB3 in MET-driven HCC development, we performed hepatocyte-specific knockdown of TRIB3 by tail vein injection of AAV8, a wellestablished vector for liver-directed gene therapy known for its ability to achieve high-level and stable transgene expression without significant toxicity [41]. The AAV8 vector was used to express a short hairpin RNA (shRNA) targeting TRIB3, which was administered 5 weeks after HTVi (Fig. 7A). Furthermore, the tumor progression and liver/body weight ratio were significantly reduced in the AAV8-shTRIB3 group, although there was no significant difference in total weight between the two groups (Fig. 7B, 7C). The AAV8-shTRIB3 group exhibited significantly longer survival compared to the AAV8-shC group (Fig. 7D). WB and IHC further indicated that tumors in the AAV8-shTRIB3 group exhibited reduced levels of TRIB3, MET, CCND1 and TWIST1, as well as elevated levels of FOXO1 (Fig. 7E, 7F).

Figure 7.

AAV8-shTRIB3 in MET/β-catenin HTVi HCC models. (A) The schematic showed the time point of AAV8-shC or AAV8-shTRIB3 injection after HTVi. (B) The morphology of whole-liver from the AAV8-shC or AAV8-shTRIB3. (C) Liver weight/body weight ratio (left) and body weight (right) in each group (n=6, Mann–Whitney test). (D) Survival rates of each group (log-rank test). (E) The protein levels of TRIB3, FOXO1 and FOXO1 target genes in the liver tumors from the AAV8-shC or AAV8-shTRIB3. (F) Representative images of H&E and IHC staining of TRIB3, FOXO1 and FOXO1 target genes in the liver tumors from the AAV8-shC or AAV8-shTRIB3 (left). IHC scores of these genes in each group (right) (n=6, Mann–Whitney test). AAV8, adeno-associated virus 8; HCC, hepatocellular carcinoma; HTVi, hydrodynamic tail vein injection; IHC, immunohistochemistry. *P<0.05, **P<0.01, ***P<0.001.

These data suggest that TRIB3 plays a crucial role in MET-mediated HCC development and progression.

TRIB3 expression was correlated with MET and FOXO1 levels in HCC specimens

Given the pivotal role of TRIB3 in HCC progression, we further explored its clinical significance by performing IHC staining and correlation analyses to evaluate its relationship with key up- and downstream targets. In the Tongji HCC patient cohort II (n=75), we observed a significant positive correlation between TRIB3, MET and SP1 expression, as well as a negative correlation between MET/SP1/TRIB3 and FOXO1 expression levels (Fig. 8A and Supplementary Fig. 6A). These findings suggest that TRIB3 may act as a mediator linking MET activation to the suppression of FOXO1 in HCC.

Figure 8.

TRIB3 expression correlates with MET and FOXO1 expression in HCC specimens. (A) Representative images of IHC staining for MET, SP1, TRIB3, and FOXO1 in HCC samples grouped according to MET IHC score (up). Spearman correlation analysis between TRIB3 and MET/SP1/FOXO1 IHC score (bottom). Scale bar: 200 μm. (B) HCC patients were divided into two groups (MET high and MET low or SP1 high and SP1 low or FOXO1 high and FOXO1 low) according to their median IHC score of MET, SP1 or FOXO1. Kaplan–Meier analyses for OS and RFS for HCC patients in these groups. (C, D) Kaplan–Meier curves showed the correlations between MET/TRIB3, or TRIB3/FOXO1 expression and OS or RFS (log-rank test). (E) Kaplan–Meier curves for OS and RFS based on the expression of the three-marker combination (MET, TRIB3, and FOXO1) in HCC patients (log-rank test). (F) A schematic model of the mechanism of METdriven HCC. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; OS, overall survival; RFS, recurrence-free survival. *P<0.05, **P<0.01, ***P<0.001.

To assess the clinical implications of these associations, we analyzed OS and RFS in HCC patients. Our results demonstrated that patients with elevated MET/SP1 or reduced FOXO1 expression in tumor tissues had significantly shorter OS and RFS compared to those with low MET/SP1 or high FOXO1 levels (Fig. 8B), emphasizing their prognostic value. Furthermore, Kaplan–Meier survival analysis revealed that patients with high expression of MET and TRIB3 in tumor tissues had the poorest prognosis, with the shortest OS and RFS (Fig. 8C). Similarly, patients with ele-vated TRIB3 and reduced FOXO1 levels experienced markedly worse survival outcomes compared to other groups (Fig. 8D). Additionally, we assessed the prognostic impact of dual -marker combinat ions (MET+SP1, MET+FOXO1, SP1+TRIB3, and SP1+FOXO1) in our cohorts (Supplementary Fig. 6B, 6C). Importantly, we extended our analysis to include a three-marker combination (MET+TRIB3+FOXO1 or SP1+TRIB3+FOXO1), which revealed that patients with high MET or SP1 and TRIB3 expression alongside low FOXO1 levels had the worst clinical outcomes, with significantly shorter OS and RFS compared to other subgroups (Fig. 8E and Supplementary Fig. 6D). These findings highlight the superior prognostic stratification achieved by integrating all markers, providing deeper insights into their combined role in HCC progression.

These results highlight the prognostic relevance of the MET-SP1-TRIB3-FOXO1 axis in HCC. High TRIB3 expression, along with dysregulated MET and FOXO1, may serve as a biomarker for predicting poor outcomes in HCC patients. Future studies with larger cohorts and additional functional assays are needed to validate these findings and explore their potential for guiding targeted therapies.