ABSTRACT
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Background/Aims
Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, and abnormal MET expression plays a crucial role in its progression. However, the specific pathogenic mechanisms of MET in HCC have yet to be fully elucidated. This study aimed to uncover the oncogenic mechanisms of MET in HCC and explore potential therapeutic implications.
-
Methods
Transcriptomic data from the HTVi MET/β-catenin HCC model and GSEA results from TCGA LIHC cohorts were analyzed to identify key genes in HCC development. In vitro assays and in vivo models were used to investigate the role of TRIB3 in HCC progression. Immunofluorescence, co-IP, qRT-PCR, and WB revealed target genes regulated by TRIB3. An AAV8-shTRIB3 construct was developed and we assessed its therapeutic potential.
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Results
MET promoted HCC development both in vitro and in vivo by upregulating the oncogenic protein TRIB3. Mechanistically, MET transcriptionally activated TRIB3 via the ERK/SP1 axis. TRIB3 then recruited the E3 ubiquitin ligase COP1, which facilitated the ubiquitination and degradation of the tumor suppressor transcription factor FOXO1. TRIB3-mediated FOXO1 ubiquitination upregulated the expression of MET, CCND1 and TWIST1. In clinical HCC samples, TRIB3 expression was correlated with MET and FOXO1 levels. Liver-specific knockdown of TRIB3 by AAV8-shTRIB3 significantly inhibited MET-driven HCC development.
-
Conclusions
Our results revealed that TRIB3 and COP1 act as key mediators in MET-driven HCC progression. Targeting the MET-TRIB3-FOXO1 regulatory axis may offer a promising therapeutic strategy to counteract oncogenic signaling and impede HCC advancement.
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Keywords: Hepatocellular carcinoma; MET; TRIB3; COP1; FOXO1
Study Highlights
• TRIB3 overexpression was consistently observed in both the MET/β-catenin HTVi HCC mouse model and human HCC cohorts, whereby elevated expression was correlated with poor prognosis.
• TRIB3 promoted tumor growth and invasiveness by facilitating the COP1-dependent degradation of the tumor suppressor transcription factor FOXO1.
• TRIB3 was upregulated via the MET-ERK-SP1 axis to play a key role in MET-driven HCC progression.
• The MET-TRIB3-FOXO1 regulatory axis may be a promising therapeutic target for HCC.
Graphical Abstract
INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide, characterized by high heterogeneity and frequent recurrence [
1-
3]. Although HCC treatment has significantly advanced over the past two decades, many targeted therapies such as tyrosine kinase inhibitors sorafenib and tivantinib, have failed to achieve optimal clinical outcomes [
4,
5]. This is largely due to the complex molecular pathogenesis of HCC and the absence of suitable biomarkers. This has led to increased interest in combination treatment strategies and novel therapeutic targets to improve efficacy [
6].
MET, a member of the receptor tyrosine kinase family [
7], is overexpressed in 30–50% of human HCCs and functions as a driver of cancer development [
8,
9]. Accumulating evidence indicates that HCC tumors exhibiting constitutive MET signaling activation are predominantly categorized within the high-proliferation molecular subclass [
10]. These tumors manifest distinct clinical and pathological features, including a strong etiological association with histological dedifferentiation, elevated serum α-fetoprotein titers [
11], heightened propensity for vascular invasion [
12], and ultimately dismal prognosis [
13]. Ligand-dependent activation occurs via hepatocyte growth factor binding, triggering transphosphorylation of the MET receptor tyrosine kinase. This initiates downstream effector cascades, including the MAPK/ERK pathway, which coordinates essential oncogenic processes such as cellular motility, invasion, and metastasis [
14-
16]. However, the precise molecular mechanisms through which MET signaling orchestrates proliferative autonomy and phenotypic aggression in HCC remain incompletely understood.
Mammalian tribbles proteins are involved in numerous cellular processes such as cell cycle regulation, signal transduction, stress response, immunity, and metabolism [
17]. Importantly, TRIB3 supports the modulation of several kinase systems. For example, TRIB3 was found to recruit PKCα to induce EGFR phosphorylation and degradation in non-small cell lung cancer [
18]. TRIB3 was also found to interact with E3 ligase MDM2 to suppress SLUG degradation, which in turn decreased MAPK phosphorylation by inhibiting the expression of the phosphate transporter SLC34A2 [
19]. Furthermore,
TRIB3 was found to be overexpressed in multiple cancer types such as colorectal cancer [
20], ovarian cancer [
21] and liver cancer [
22], where it is believed to facilitate tumor growth and metastasis. However, its relationship with MET signaling and its functional interplay with other key molecules remain unexplored.
In this study, we found that TRIB3 is a crucial downstream target of MET. MET upregulated TRIB3 expression by activating the ERK/SP1 axis. Elevated TRIB3 bound with the E3 ubiquitin ligase COP1, thereby promoting HCC progression by mediating COP1-dependent ubiquitination of FOXO1. Knockdown of TRIB3 in hepatocytes using an adeno-associated virus 8-based vector effectively disrupted the MET-TRIB3-FOXO1 axis, suppressing MET-driven HCC development and progression. These findings suggest the therapeutic potential of targeting the MET - TRIB3 - FOXO1 axis in HCC.
MATERIALS AND METHODS
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.
RESULTS
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).
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.
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).
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.
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).
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.
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-sh
TRIB3 cells increased their expression (
Fig. 4A,
Supplementary Fig. 4C).
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).
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.
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).
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-sh
TRIB3 group, although there was no significant difference in total weight between the two groups (
Fig. 7B,
7C). The AAV8-sh
TRIB3 group exhibited significantly longer survival compared to the AAV8-shC group (
Fig. 7D). WB and IHC further indicated that tumors in the AAV8-sh
TRIB3 group exhibited reduced levels of TRIB3, MET, CCND1 and TWIST1, as well as elevated levels of FOXO1 (
Fig. 7E,
7F).
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.
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.
DISCUSSION
HCC is the most prevalent form of primary liver cancer and is most often diagnosed at an advanced stage, contributing to its poor prognosis. Aberrant MET activation is a well-established oncogenic driver in HCC and has been implicated in 30–50% of cases. Moreover, dysregulation of MET is observed in nearly half of HCC cases and all liver metastases, underscoring its potential as a therapeutic target [
42]. Despite this, clinical trials evaluating nonselective kinase inhibitors with MET inhibitory activity, such as foretinib and cabozantinib, have yielded disappointing results, failing to demonstrate significant therapeutic efficacy. Even selective MET inhibitors, such as tivantinib, have shown limited success. For example, tivantinib failed to improve OS compared to placebo in patients with MET-high advanced HCC who had previously received sorafenib [
43,
44]. These failures may be attributed to compensatory survival pathways activated upon MET inhibition, which sustain tumor cell growth and undermine therapeutic efficacy. This underscores the urgent need for a deeper understanding of the molecular mechanisms through which MET promotes HCC progression, as well as the development of more refined therapeutic strategies targeting MET-associated pathways.
The Tribble protein family has been reported to mainly act as adaptors that facilitate protein degradation or sequestration, and its members are upregulated in multiple cancer types. In this study, we identified TRIB3 as a downstream effector of MET and a critical factor in HCC progression. After integrating RNA-seq analysis of liver tissues from the MET/β-catenin hydrodynamic HCC model with TCGA data, we initially hypothesized that MET regulates TRIB3. Subsequent experimental validation confirmed that MET upregulates TRIB3 levels via the ERK/SP1 signaling axis. Importantly, while our findings emphasize the key role of the MET/ERK/SP1 axis in HCC, we recognize that TRIB3 can be regulated by other signaling pathways in different cancer types or contexts. For instance, TRIB3 was found to be regulated by TGF-β/SMAD3 in ovarian cancer cells [
45], whereas JAK1/ATF4 was found to modulate TRIB3 expression conditions in astrocytes under ER stress [
46]. These observations not only illustrate the cell type- and context-specific nature of TRIB3 regulation, but also strongly support the primacy of ERK/SP1 signaling in METdriven TRIB3 overexpression in HCC. Furthermore, we demonstrated that TRIB3 mediates the carcinogenic role of MET
in vitro and
in vivo. Nonetheless, we observed substantial variability in the number of lung metastatic nodules in our orthotopic HCC models. The variability of metastatic nodule counts across samples is likely due to inherent differences in cell line behavior and individual animal responses following orthotopic implantation. Despite this variability, our results showed statistically significant differences between experimental and control groups, supporting the conclusion that TRIB3 plays a critical role in promoting HCC progression and metastasis.
Members of the Tribble protein family harbor an atypical pseudokinase domain that acts as a regulated binding platform for substrates, which are ubiquitinated by contextspecific E3 ligases. In line with this characteristic, co-IP experiments demonstrated that TRIB3 can bind to the E3 ubiquitin ligase COP1 and function as an adapter protein. Subsequently, this complex targets the tumor suppressor FOXO1 for proteasomal degradation. Furthermore, loss of FOXO1 releases the transcriptional repression of MET, CCND1 and TWIST1, establishing a positive feedback loop that amplifies MET-driven oncogenic signaling. This loop enhances the proliferative, migratory, and invasive capabilities of HCC cells.
FOXO1 is a well-documented tumor suppressor that is downregulated in multiple cancer types, including HCC. FOXO1 plays key roles in regulating apoptosis, differentiation, and proliferation [
29]. Importantly, TCGA data indicate that higher FOXO1 expression is correlated with a better prognosis in HCC patients [
32-
34], further supporting its tumor-suppressor role. Previous studies confirmed that FOXO1 deletion promotes HCC cell proliferation and migration, consistent with the effect of MET amplification. Our findings align with and extend previous studies highlighting the role of MET and FOXO1 in HCC. However, the identification of TRIB3 as a critical mediator of the MET-FOXO1 axis represents a novel contribution to the field. Unlike prior studies focusing on direct MET inhibition, our work underscores the therapeutic potential of targeting downstream effectors such as TRIB3 to circumvent compensatory mechanisms.
Notably, the inhibition of TRIB3 expression disrupted this feedback loop, restoring FOXO1 stability and suppressing the expression of MET along with downstream oncogenic targets such as CCND1 and TWIST1. This intervention effectively attenuated MET-driven HCC progression both
in vitro and
in vivo. These findings suggest that targeting TRIB3 may be a viable therapeutic approach to overcome the limitations of current MET inhibitors, which have demonstrated suboptimal efficacy due to incomplete inhibition or the activation of compensatory pathways. While TRIB3 inhibition holds promise, several challenges must be addressed to facilitate its clinical translation. First, potential off-target effects of TRIB3 inhibitors may impact normal cellular functions, given its role in stress responses [
47] and metabolism [
48]. Secondly, compensatory mechanisms, such as upregulation of alternative ubiquitin ligases or activation of parallel oncogenic pathways, may limit the therapeutic efficacy. The ERK/SP1 axis, which regulates TRIB3 expression, represents another promising therapeutic target. ERK inhibitors such as ulixertinib have shown efficacy in preclinical experiments [
49]. Further in-depth exploration of their potential in MET-driven HCC is required in the future.
In conclusion, our findings reveal a previously unrecognized mechanism, whereby MET promotes HCC progression via TRIB3-mediated COP1-dependent degradation of FOXO1. TRIB3 serves as a bridge linking MET signaling to FOXO1 destabilization, thereby establishing a positive feedback loop that drives tumor growth and metastasis. Targeting TRIB3 represents a promising strategy that may overcome the limitations of MET inhibitors and improve therapeutic outcomes in patients with MET-dysregulated HCC. Future research should assess the clinical utility of TRIB3 inhibitors in preclinical and clinical trials. Additionally, exploring the broader regulatory network of TRIB3 and its interactions with oncogenic or tumor-suppressive pathways is crucial. For example, investigating how TRIB3 influences other components of the ubiquitination machinery or interacts with immune cells in the tumor microenvironment warrants further study. Furthermore, examining the role of TRIB3 in resistance to other therapies, including immune checkpoint inhibitors, may provide deeper insights into its therapeutic significance.
FOOTNOTES
-
Authors’ contribution
W.H. and L.X. designed the whole project. T.W., D.R., C.F., Y.L., J.L., Z.S., J.J., H.L., F.F. and H.L. performed the experiments. T.W., D.R., C.F., Y.L., J.L., Z.S., J.J., H.L., F.F. and H.L. performed data analysis and interpretation. T.W. drafted the main manuscript.
-
Acknowledgements
We extend our gratitude to the Experimental Medicine Center of Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, for providing technical support.
Research was supported by grants from the National Natural Science Foundation of China No. 82173313 (W.H.), No. 82372917 (W.H.), No. U23A20451 (L.X.), No.82273310 (L.X.), the Natural Science Foundation of Hubei Province 2022CFA016 (L.X.), and the Basic Research Support Program of Huazhong University of Science and Technology 2023BR038 (L.X.).
-
Conflicts of Interest
The authors have no conflicts to disclose.
SUPPLEMENTAL MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
(A) GSEA result of TCGA LIHC data showed the PID_MET_Pathway is enriched in TRIB3 high-expression HCC patients. (B) Relative mRNA levels of TRIB3 in HCC and normal tissues or different clinical/pathology stage of HCC patients in TCGA- LIHC. (C) HCC patients were divided into two groups (TRIB3high or TRIB3low) according to the median of TRIB3 mRNA level in TCGALIHC. Kaplan-Meier analyses for overall survival (OS) and progression-free interval (PFI) for HCC patients in these two groups. (D) Western blot analysis of TRIB3 expression in HCC and ANTs specimens. *P<0.05, **P<0.01, ***P<0.001.
Supplementary Figure 2.
(A) Western blot analysis of TRIB3 expression in HCC cell lines and TRIB3 overexpression or knockdown efficacy in the indicated cells. (B) The proliferation of the indicated cells with overexpression or knockdown of TRIB3 as measured by CCK- 8 assays. (C) Representative images of the EdU incorporation assay and quantification of EdU-positive cells (n=3, Mann–Whitney test). Scale bar: 200 μm. (D) Representative images (top) and quantification (bottom) of the wound healing assay (n=3, Mann–Whitney test). Scale bar: 200 μm. (E) Representative images (left) and quantification (right) of cells that migrated or invaded in the indicated groups. Scale bar: 200 μm. (F) Representative hematoxylin and eosin (H&E) staining and IHC of Ki67 in subcutaneous xenografts (left). Quantification of staining scores of Ki67 positive cells in subcutaneous xenografts (right) (n=6, Mann–Whitney test). Scale bar: 200 μm. *P<0.05, **P<0.01, ***P<0.001.
Supplementary Figure 3.
(A) Protein-protein interaction (PPI) network of TRIB3 conducted by STRING database. (B) The peptide fragment of COP1 as determined by mass spectrometry. (C) The interaction between exogenous TRIB3, COP1 and FOXO1 was confirmed in HEK293T cells. (D) Western blot showing representative protein levels of COP1, TRIB3, and FOXO1 in control, COP1-overexpressing and COP1-overexpressing with or without TRIB3-knockdown cancer cells. (E) Impact of COP1 overexpression on the protein stability of FOXO1 with or without MG132 as compared to control group in MCF7 and SW480 cell lines. Effects of COP1 overexpression on FOXO1 stability with or without TRIB3 knockdown in MCF7 and SW480 cell lines.
Supplementary Figure 4.
(A) Relative mRNA levels of FOXO1 in HCC and normal tissues in TCGA-LIHC. (B) HCC patients were divided into two groups (Low FOXO1 Group or High FOXO1 Group) according to the median of FOXO1 mRNA level in TCGA-LIHC. Kaplan-Meier analyses for overall survival (OS) for HCC patients in these two groups. (C) Western blot analysis of FOXO1 knockdown efficacy in the sh-TRIB3-MHCC97H cells. (D) Representative images of the EdU incorporation assay. Scale bar: 200 μm. (E) Representative images of the wound healing assay. Scale bar: 200 μm. (F) Representative images of cells that migrated or invaded in the indicated groups. Scale bar: 200 μm. (G) Representative hematoxylin and eosin (H&E) staining and IHC of Ki67 in subcutaneous xenografts (left). Quantification of staining scores of Ki67 positive cells in subcutaneous xenografts (right) (n=6, Kruskal–Wallis test). Scale bar: 200 μm. *P<0.05, **P<0.01, ***P<0.001.
Supplementary Figure 5.
(A) Western blot analysis of TRIB3 knockdown efficacy in the indicated cells. (B) Representative images of the EdU incorporation assay. Scale bar: 200 μm. (C) Representative images of the wound healing assay. Scale bar: 200 μm. (D) Representative images of cells that migrated or invaded in the indicated groups. Scale bar: 200 μm.
Supplementary Figure 6.
(A) Spearman correlation analysis between MET and SP1/FOXO1 IHC score or SP1 and FOXO1 IHC score. (B) Kaplan–Meier curves showed the correlations between MET/SP1, or MET/FOXO1 expression and OS or RFS (log-rank test). (C) Kaplan– Meier curves showed the correlations between SP1/TRIB3, or SP1/FOXO1 expression and OS or RFS (log-rank test). (D) Kaplan– Meier curves for OS and RFS based on the expression of the three-marker combination (SP1, TRIB3, and FOXO1) in HCC patients (logrank test).
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.
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.
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.
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.
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.
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.
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.
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.
Abbreviations
gene set enrichment analysis
hydrodynamic tail vein injection
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, Limin Xia2
