HCC, a rapidly progressing malignancy, is the sixth most common cancer and the third leading cause of cancer death worldwide, with approximately 906,000 new cases and 830,000 deaths per year[2]. Surgical treatment remains the preferred treatment for patients, especially at an early stage[12]. However, most patients still have a poor prognosis and low overall survival. To improve clinical outcomes, it is important to find novel therapeutic targets.
The ubiquitin–proteasome system (UPS) is a key regulator that maintains cellular function and stability and is mainly composed of E1 activating enzymes, E2 conjugating enzymes, E3 ubiquitin ligases, the proteasome and deubiquitinating enzymes[13]. UPS participates in various cellular biological processes, such as the cell cycle, gene transcription, cellular differentiation and apoptosis, which are important for maintaining the normal physiological functions of cells[14]. Based on sequence homology and structural similarity, DUBs are classified into seven subfamilies, and USP25 belongs to the largest ubiquitin-specific protease (USP) subfamily[15]. Multiple studies have shown that USP25, an important regulator, can interact with a large number of signalling molecules and participate in the regulation of various intracellular signalling pathways, especially in the development of tumours[16]. A recent study verified that USP25 was highly expressed in colon tumours, activated the Wnt signalling pathway and involved in the phosphorylation of signalling transcription factor activator 3 (STAT3) to inhibit colon tumorigenesis[17]. Another study reported that high expression of USP25 leads to the accumulation of β-catenin, which activates the Wnt/β-catenin signalling pathway and thus inhibits tumour growth and the proliferation and migration of prostate cancer cells[18]. Moreover, USP25 has been shown to play a similar role in non-small-cell lung cancer and breast cancer[19, 20]. However, its function and mechanism in HCC have not been investigated. In our study, we first analysed the expression of USP25 in HCC based on the TCGA and ICGC databases and then showed that USP25 was highly expressed in HCC cell lines. Subsequently, according to a series of experiments, we observed that overexpression of USP25 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo, especially, HCC cells spread and transferred to the lungs obviously. However, further study is warranted to confirm the mechanism of USP25 in HCC.
The Wnt/β-catenin pathway is an evolutionarily conserved signalling pathway composed of Wnt proteins, Frizzled receptor families, the casein kinase 1 (CK1) and Dishevelled (Dvl) proteins, the β-catenin/Axin/APC/GSK3β complex and LEF/TCF transcription factors[21]. When abnormally activated, the ligand protein Wnt can bind to the receptor protein FZD, which activates Dvl, promoting the dissolution of the complex and causing the large scale accumulation of β-catenin in the nucleus. Then, β-catenin binds to the intranuclear transcription factor LEF/TCF to affect tumour formation and progression through downstream target genes such as C-myc and Cyclin D1[22, 23]. The EMT refers to the biological process in which epithelial cells are transformed into cells with a mesenchymal cell phenotype through a specific programme. Cadherin is an important feature of the EMT, and the E-cadherin/β-catenin complex plays an important role in maintaining normal epithelial morphology and cell–cell adhesion[24]. When Wnt signalling is activated, β-catenin phosphorylation influences the stability of the E-cadherin/β-catenin complex, decreasing E-cadherin-mediated adhesion and disrupting the intercellular connection, thereby resulting in the enhanced invasion and migration of tumour cells[25]. The role of the Wnt/β-catenin pathway in the progression of HCC has been established[26]. For example, several studies have shown the nuclear accumulation of β-catenin in HCC, and the abnormal expression of key genes augments Wnt/β-catenin signalling activity[27]. Our research leads to similar conclusions by showing that the expression of β-catenin, C-myc and Cyclin D1 increased, while the expression levels of Axin2, APC and GSK3β decreased in HCC cell lines overexpressing USP25. Moreover, the EMT-related protein E-cadherin was expressed at a low level, and N-cadherin was highly expressed. After the addition of the signalling pathway inhibitor XAV939, the expression of these proteins was inhibited. In addition, we carried out animal experiments to verify our findings.
To clarify the mechanism of the USP25 gene, mass spectrometry experiments were performed. We found that USP25 necessarily interacted with TRIM21 to function. TRIM21, a member of the TRIM family, is expressed in both the cytoplasm and nucleus, and the gene encoding this protein is located on chromosome 11[28]. The RING domain at the amino-terminal region of the TRIM21 protein exhibits E3 ubiquitin ligase activity[29]. Notably, ultraviolet (UV) irradiation induced apoptosis of human breast cancer cells, and the RNA-binding protein HuR activated the tumour suppressor p53 mRNA translation, while TRIM21 expression induced by UV irradiation mediated the proteasomal degradation of HuR and reduced P53 levels[30]. Additionally, during radiotherapy of nasopharyngeal carcinoma (NPC), TRIM21 was shown to inhibit TP53 expression by mediating the ubiquitination and degradation of guanine monophosphate synthase (GMPS), and overexpression of TRIM21 protected NPC cells from radiation-mediated apoptosis[31]. However, Ding et al found that TRIM21 knockdown in HCC cell lines significantly enhanced HCC cell proliferation, colony formation, migration, and antiapoptotic activity in vitro, but the mechanisms of these functions were not extensively explored[32]. Considering our experimental results, we speculated that TRIM21 recruits more ubiquitin to bind β-catenin, which leads to degraded β-catenin through the ubiquitination pathway, whereas USP25 disrupts the ubiquitination pathway, causing β-catenin accumulation in the nucleus and activating the Wnt/β-catenin signalling pathway.
In summary, in the current study, we identified the overexpression of USP25 in HCC cell lines, for the first time. The results from these assays indicated that USP25 exerts positive effects on HCC cell proliferation, migration and invasion by interacting with TRIM21 and thereby regulating the Wnt/β-catenin signalling pathway and the EMT (Fig. 8).