MET is overexpressed in 30–50% of patients with hepatocellular carcinoma (HCC), and its overexpression is associated with poor prognosis [
1]. However, the clinical efficacy of targeting MET with inhibitors such as tivantinib has been limited [
2]. This unfavorable outcome can be attributed to the complexity of signaling pathways, compensatory mechanisms, acquired resistance and lack of patient stratification [
3]. Therefore, understanding the intricate relationships among these factors to increase the efficacy of MET inhibitors in the treatment of HCC patients is crucial. In an issue of
Clinical and Molecular Hepatology, Wang et al. [
4] reported that Tribbles Pseudokinase 3 (TRIB3) is upregulated via the MET-ERK-SP1 axis, forming a positive feedback loop by regulating FOXO1 ubiquitination. These findings offer new mechanistic insights into the compensatory acquired resistance that may lead to the failure of MET treatment in HCC patients.
To identify new signaling pathways for MET signaling, Wang et al. employed a hydrodynamic tail vein injection (HTVi) model, injecting plasmid DNA encoding MET along with β-catenin, and compared the genetic profiles of the treated mice with those of control mice. They identified significant enrichment of protein kinase-associated pathways, in which TRIB3 was found to be preferentially upregulated. Consistent with this finding, there was marked enrichment of the MET pathway in patients with high TRIB3 expression, further confirming the role of TRIB3 in the MET/β-catenin-driven mouse HCC model. Consistent with a previous report [
5], TRIB3 was overexpressed in HCC, and its overexpression was correlated with poor prognosis. Using overexpression and knockdown approaches, TRIB3 was found to be crucial for regulating HCC cell proliferation, tumor growth, and metastasis.
TRIB3 is a member of the pseudokinase family and is characterized by its lack of functional kinase activity in the central domain. This is due to the absence of ATP-specific binding sites and a catalytic core. As a result, researchers are focusing on its role as an adaptor and scaffold protein in its C-terminal domain, which regulates various biological processes through interactions with several proteins, including EGFR [
6] and β-catenin [
7]. In this context, Wang et al. employed co-immunoprecipitation (co-IP) coupled with mass spectrometry analysis to identify several potential binding partners of TRIB3. Among these, the E3 Ubiquitin- Protein Ligase COP1 was selected for further functional characterization, as it was identified within the TRIB3 network using the STRING database. In support of this finding, a previous report revealed that COP1 is recruited by TRIB3, leading to the ubiquitination of acetyl-coenzyme A carboxylase (ACC) and thereby regulating lipid metabolism in hepatocytes [
8]. However, the interplay between TRIB3 and COP1 in the context of cancer remains unexplored. First, they confirmed the binding between TRIB3 and COP1 by co-IP. Second, they discovered that TRIB3 recruits and binds to COP1, which subsequently ubiquitinates FOXO1, a protein previously reported to be degraded by COP1 [
9]. This observation is not limited to HCC cells; similar findings were observed in colon and breast cancer cell lines, where repression of TRIB3 inhibited COP1-mediated FOXO1 degradation. Interestingly, although a protein complex of TRIB1/COP1/FOXO1 was identified, COP1 was found to degrade FOXO1 but not TRIB3. This discrepancy can be attributed to the differential physical interactions between COP1 and TRIB3 and between COP1 and FOXO1. These solid data provide evidence that TRIB3 mediates COP1-induced FOXO1 degradation via the proteasomal pathway.
On the basis of previous reports, FOXO1 inhibits the expression of several cancer-promoting proteins, such as MET [
10], CCND1 [
11], and TWIST [
12]. Researchers have reported that these proteins are downregulated in FOXO1-overexpressing HCC cells, whereas the suppression of FOXO1 results in the opposite effects. Functionally, TRIB3 was found to regulate HCC growth and progression by altering FOXO1-mediated CCND1 and TWIST regulation. Notably, the increased interaction among TRIB3, COP1, FOXO1, and MET results in the formation of a positive feedback loop in MET signaling. These findings provide new insights into acquired resistance to MET inhibition in HCC. However, the functional roles of CCND1 and TWIST in MET-driven HCC progression require further confirmation in future studies.
The molecular mechanism underlying TRIB3 upregulation in HCC is not fully understood. To date, SP2 has been identified as a factor that targets TRIB3 expression, driving HCC invasion and metastasis [
13]. Wang et al. demonstrated that MET increases TRIB3 promoter activity through SP1 regulation, a finding further confirmed by the creation of mutants in which the SP1 binding sites were deleted. The ERK inhibitor significantly reduced MET-induced TRIB3 and SP1 expression. Chromatin immunoprecipitation assays revealed that the ERK inhibitor decreased SP1 binding to the TRIB3 promoter, indicating that MET-induced TRIB3 expression relies on the ERK/SP1 signaling axis. Furthermore, the authors highlighted the crucial role of TRIB3 in MET-driven HCC proliferation and invasion in vitro, as evidenced by the abrogation of MET overexpression effects in TRIB3-repressed HCC cells. Using the AAV8 vector for specific delivery of a short hairpin RNA targeting TRIB3 to the liver following HTVi injection of MET/β-catenin for 5 weeks, a reduced liver/body weight ratio with increased survival was observed in the AAV8-shTRIB3 group. These findings underscore the role of TRIB3 in MET-driven HCC tumor development and progression in vivo. Accompanying these phenotypic changes were reduced levels of TRIB3, MET, CCND1, and TWIST1, along with elevated levels of FOXO1. These findings further verify the TRIB3/MET/CCND1/TWIST1 signaling cascade in MET-driven HCC.
In a cohort of 75 HCC patients, a significant positive correlation was detected between the expression levels of TRIB3, MET, and SP1, and a negative correlation was detected between the expression levels of MET/SP1/TRIB3 and FOXO1. These findings further validate the role of TRIB3 in linking MET activation to FOXO1 suppression, as observed in the in vitro data and in a mouse model. Finally, the prognostic value of this signaling cascade was evaluated. HCC patients with elevated MET/SP1 expression or reduced FOXO1 expression in tumor tissues had significantly shorter overall survival (OS) and recurrence-free survival (RFS) than did those with high MET/SP1 or FOXO1 expression. Interestingly, HCC patients with high expression levels of both MET and TRIB3 in tumor tissues presented the shortest OS and RFS. Further patient stratification revealed that those with high MET or SP1 and TRIB3 expression, alongside low FOXO1 levels, had the worst clinical outcomes, with significantly shorter OS and RFS than the other subgroups did. These results underscore the enhanced prognostic stratification obtained by combining all the markers, offering greater insights into their collective influence on HCC progression.
In summary, this study offers new mechanistic insights along with significant clinical and therapeutic implications regarding MET signaling in HCC. First,
TRIB3 expression was found to be elevated in both MET/β-catenin-driven HCC mouse tumors and in patient cancer tissues with high MET expression. Second, TRIB3 promotes MET-driven HCC tumor growth and invasiveness by facilitating COP1-mediated degradation of CCND1 and TWIST. Third, the TRIB3/COP1/FOXO1 complex and MET create a positive feedback loop involved in MET signaling (
Fig. 1). Fourth, TRIB3 is upregulated through the MET-ERK-SP1 axis. Finally, the MET-TRIB3-FOXO1 signaling cascade serves as a means of patient stratification for effective therapeutic targeting of the MET pathway in HCC patients. Despite the promising findings presented in this study, it has several limitations that warrant further investigation in the future. Since this pathway was identified through HTVi injection of two oncogenes, MET and β-catenin, it remains uncertain whether this pathway is commonly activated in β-catenin-driven HCC. Given that HGF/MET signaling plays a crucial role in regulating tumor plasticity and drug resistance [
14], evaluating the role of the identified pathway in cancer stemness and its impact on resistance to targeted therapies such as sorafenib and lenvatinib is important. Additionally, investigating whether this pathway is activated in the tumor microenvironment and influenced by HGF supplementation would be interesting. The authors demonstrated that TRIB3 was downregulated by the addition of MG132, suggesting the existence of an alternative proteasome degradative pathway induced by MET activation. Finally, it is important to assess whether enhanced therapeutic efficacy can be achieved by combining MET inhibitors with TRIB3 inhibition. Nevertheless, this study is crucial for identifying a new signaling pathway that could improve the efficacy of MET inhibition in HCC through targeted therapies and patient stratification.
FOOTNOTES
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Authors’ contribution
M.S.C drafted the manuscript. T.K.L reviewed and finalized the manuscript.
-
Acknowledgements
This work was supported by the Research Impact Fund (R5008-22F) and the RGC General Research Fund (15101621).
-
Conflicts of Interest
The authors have no conflicts of interest to declare.
Figure 1.Schematic representation of the role of TRIB3/COP1/ FOXO1 in MET-driven HCC progression. HCC, hepatocellular carcinoma; TRIB3, Tribbles Pseudokinase 3.
Abbreviations
hydrodynamic tail vein injection
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Citations
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- Correspondence: Response to Editorial on “Unveiling TRIB3: A New Mediator in MET-Driven Hepatocellular Carcinoma Progression”
Tiantian Wang, Wenjie Huang, Limin Xia
Clinical and Molecular Hepatology.2025;[Epub] CrossRef
