SUMOylation of UBE2C facilitates hepatocellular carcinoma proliferation and invasion via the MAPK pathway

Introduction

Hepatocellular carcinoma (HCC) accounts for approximately 75–85% of all primary liver malignancies and remains the third leading cause of cancer-related mortality globally, with a high incidence in Asia and sub-Saharan Africa because of the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection (1). Despite ongoing advances in systemic therapies, such as tyrosine kinase inhibitors (sorafenib and lenvatinib), immune checkpoint inhibitors (ICIs), and their combination, the 5-year survival rate for advanced HCC remains below 20% (2,3). High recurrence rates, rapid progression, and drug resistance underscore the critical need to elucidate the molecular mechanisms that govern HCC pathogenesis and therapy failure.

One such mechanism involves posttranslational modification (PTM), particularly SUMOylation, which refers to the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to lysine residues of substrate proteins. SUMOylation regulates diverse cellular processes, including transcriptional control, chromatin organization, protein stability, and signal transduction (4). In the context of cancer, SUMOylation can alter the stability or activity of oncogenic factors, contributing to tumour growth, metastasis, and therapeutic resistance (5). Recent studies have identified UBE2C, a key player in the anaphase-promoting complex/cyclosome pathway, as a potential oncogene in various cancers, including lung, breast, and prostate cancer (6). UBE2C promotes cell cycle progression by targeting mitotic checkpoint regulators for degradation. Aberrant overexpression of UBE2C is correlated with poor prognosis and has been linked to increased proliferation, epithelial-mesenchymal transition (EMT), and invasion in HCC (7). However, the upstream regulatory mechanisms and posttranslational modulation of UBE2C in HCC remain poorly defined.

Our study revealed that UBE2C is transcriptionally activated by MYBL2, a transcription factor (TF) previously implicated in DNA replication, G2/M transition, and hepatocarcinogenesis (8). Through chromatin immunoprecipitation (ChIP) and luciferase assays, we showed that MYBL2 binds directly to the UBE2C promoter, increasing its transcriptional output. Moreover, we demonstrate that the UBE2C protein undergoes SUMOylation at K18, which is mediated by the E2 conjugating enzyme UBC9, significantly increasing UBE2C protein stability without altering messenger RNA (mRNA) levels-highlighting the importance of posttranscriptional regulation in maintaining oncogenic expression.

Functionally, UBE2C facilitates HCC cell proliferation, HCC cell invasion, and the EMT. Mechanistic analysis revealed that UBE2C activates the MAPK signalling pathway by upregulating the upstream mediators PRKCB and PLCG2. This leads to increased phosphorylation of RAF, MEK, and ERK, which are central components of the canonical MAPK cascade. The MAPK pathway has been extensively studied in cancer biology and is known to drive proliferation, angiogenesis, and immune evasion (9). In HCC, hyperactivation is frequently associated with aggressive phenotypes and resistance to standard treatments (10). Importantly, our findings demonstrate that pharmacological inhibition of MAPK signalling using trametinib abrogates UBE2C-driven tumour progression, confirming the functional relevance of the pathway.

In summary, our study identified a previously uncharacterized MYBL2-UBE2C-MAPK regulatory axis, with UBE2C SUMOylation at K18 serving as a critical stabilizing modification that sustains its oncogenic function in HCC. These findings not only deepen our understanding of SUMOylation in HCC biology but also position UBE2C as a promising dual-layer therapeutic target that is amenable to both transcriptional repression and posttranslational interference. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2000/rc).

Methods

Tissue samples

Fresh HCC and adjacent nontumourous liver tissues were obtained from patients who underwent surgical resection at First Hospital of Jiaxing, with approval from the Institutional Review Board of First Hospital of Jiaxing (approval No. 2025-ky-444), and informed consent was taken from all the patients. The samples were immediately snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde for downstream RNA, protein, and histological analyses. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell culture and transfection

HepG2, SK-Hep1, LO2, and 293T cells were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were incubated at 37 ℃ in a humidified 5% CO₂ atmosphere. Plasmids for MYBL2, UBE2C, and UBC9 overexpression were cloned and inserted into pcDNA3.1. siRNAs targeting UBE2C, MYBL2, and UBC9 were synthesized by GenePharma. Transfections were performed using Lipofectamine 3000 (Invitrogen, Shanghai, China) in accordance with the manufacturer’s instructions. Cells were harvested after 48 hours for further analysis.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using RNA-easy Isolation Reagent (No. RC112-01; Vazyme, Shanghai, China). First-strand cDNA was synthesized using a HiScript III 1st Strand cDNA Synthesis Kit (No. R312-01; Vazyme), and qRT-PCR was performed using ChamQ^TM Universal SYBR^® qPCR Master Mix (No. Q712-02; Vazyme) in accordance with the manufacturer’s instructions. Polymerase chain reaction (PCR) was run on a Bio-Rad CFX96 system. The relative gene expression was analysed using the 2^−ΔΔCt method. GAPDH was used as the internal control. The primer sequences were as follows: UBE2C forward 5’-CTGGCGATAAAGGGATTTCTGCC-3’, reverse 5’-GCGAGAGCTTATACCTCAGGTC-3’; GAPDH forward 5’-GTCTCCTCTGACTTCAACAGCG-3’, reverse 5’-ACCACCCTGTTGCTGTAGCCA-3’.

Western blotting

Proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk and incubated overnight with primary antibodies against UBE2C, MYBL2, PRKCB, PLCG2, p-RAF, p-MEK, p-ERK, N-cadherin, vimentin, Snail, MMP9, and GAPDH. After being washed, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using enhanced chemiluminescence (ECL).

Migration and invasion assays

Cell migration and invasion were assessed using 24-well Transwell chambers (8 µm pore size, Corning, Shanghai, China). For invasion assays, the chambers were precoated with Matrigel. Cells were seeded into the upper chamber in serum-free medium; medium containing 10% FBS was added to the lower chamber. After 24 h, the cells that had migrated/invaded were fixed, stained with crystal violet, and counted.

ChIP assays

ChIP assays were conducted using the SimpleChIP^® Enzymatic Chromatin IP Kit (Cell Signaling Technology, Shanghai, China, #9003) in accordance with the manufacturer’s instructions, with minor modifications. Briefly, HCC cells were fixed with 1% formaldehyde for 10 min at room temperature to crosslink DNA-protein complexes, followed by quenching with 125 mM glycine. The cells were subsequently washed, harvested, and lysed to isolate the nuclei. Chromatin was digested with micrococcal nuclease at 37 ℃ for 20 min and further sheared by brief sonication. The fragmented chromatin was incubated overnight with 5 µg of anti-MYBL2 antibody (Abcam, Shanghai, China, ab76009) or control immunoglobulin G (IgG) at 4 ℃ with protein G magnetic beads. Immunoprecipitates were sequentially washed and eluted, and DNA-protein crosslinks were reversed by heating at 65 ℃ overnight. DNA was purified using the spin columns provided in the kit. Enriched DNA fragments were analysed by quantitative polymerase chain reaction (qPCR) using primers targeting the UBE2C promoter region [−400 to 0 bp relative to the transcription start site (TSS)]. Percent input was calculated to determine fold enrichment.

In vivo experiments

For the subcutaneous xenograft model, HCC cells (1×10⁶ cells per mouse) were injected into the right posterior flank of 6-week-old male BALB/c nude mice. Tumor volume was measured with calipers and calculated using the modified ellipsoidal formula: tumor volume = 1/2 × length × width².

Statistical analysis

The data are presented as the mean ± standard deviation (SD). Statistical significance was determined using an unpaired Student’s t-test or one-way analysis of variance (ANOVA) with GraphPad Prism 8. P<0.05 was considered to indicate statistical significance.

Results

High expression of UBE2C is correlated with poor prognosis of HCC

To identify mRNA expression profiles in HCC, we first analysed the high-throughput sequencing data, which included 369 HCC samples and 160 normal tissue samples from The Cancer Genome Atlas (TCGA) database (Figure 1A). As shown in Figure 1B,1C, we identified 6,072 upregulated genes and 1,726 downregulated genes in HCC. The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis confirmed that the transforming growth factor beta (TGF-β) and JAK-STAT pathways were involved in HCC progression (Figure 1D). UBE2C expression was significantly greater in HCC tissues than in normal tissues (Figure 1E). To confirm the correlation between UBE2C expression levels in tumour cells and HCC prognosis, we compared overall survival (OS) and disease-free survival (DFS) between the two groups. Kaplan-Meier survival analysis revealed that patients in the UBE2C-high group had a shorter time to recurrence and worse OS and DFS than those in the UBE2C-low group did, suggesting that UBE2C may function as an oncogene in HCC progression (Figure 1F,1G). Next, the expression of UBE2C was further confirmed by qRT-PCR and western blotting. As shown in Figure 1H,1I, UBE2C mRNA and protein expression were upregulated in HCC tissues compared with that in adjacent normal tissues, which is consistent with the high-throughput sequencing results.

Figure 1 UBE2C was upregulated in HCC. (A-C) Heatmap and volcano map from the TCGA database revealed the differentially expressed mRNAs in HCC and normal subjects (P value <0.05 and log2-fold change >1). (D) Enrichment analysis for representative KEGG pathways involved in the dysregulation of genes associated with HCC. (E) The expression of UBE2C in the TCGA cohort is shown. (F,G) The overall survival rate and disease-free survival rate of patients with high or low UBE2C expression are shown. (H,I) The expression levels of UBE2C mRNA and protein were measured in HCC tissues and adjacent normal tissues. *, P<0.05; ***, P<0.001. ECM, extracellular matrix; HCC, hepatocellular carcinoma; HR, hazard ratio; KEGG, Kyoto Encyclopedia of Genes and Genomes; LIHC, liver hepatocellular carcinoma; mRNA, messenger RNA; N, normal; T, tumor; TCGA, The Cancer Genome Atlas; TGF, transforming growth factor; TPM, transcripts per million.

Knockdown of UBE2C slows HCC cell migration and tumorigenesis

To study the role of UBE2C in HCC progression, we performed several cell function experiments. First, cell proliferation was measured by a Ki67 staining assay. As shown in Figure 2A, UBE2C knockdown markedly inhibited the proliferation of both HepG2 and SK-Hep1 cells. Moreover, the results of cell migration and invasion assays demonstrated that knockdown of UBE2C expression significantly decreased the metastasis of HCC cells (Figure 2B). Furthermore, to investigate the role of UBE2C in cell migration, the siUBE2C was transfected into cells to compare the expression of biomarker proteins related to EMT. As shown in Figure 2C, knockdown of UBE2C expression significantly decreased the expression of N-cadherin, vimentin, Snail and MMP9, suggesting that UBE2C promoted cell migration by enhancing the EMT process. Taken together, the above data suggest that UBE2C may act as a promoter of the progression of HCC.

Figure 2 UBE2C promoted HCC progression. (A) The proliferation of HepG2 and SK-Hep1 cells after UBE2C knockdown was measured by a Ki-67 staining assay (×200 magnification). (B) Transwell assays with or without Matrigel were used to measure the effect of UBE2C on cell migration and invasion (cells were stained with crystal violet). (C) The effect of UBE2C on the HCC EMT process was measured by western blotting. ***, P<0.001. EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma.

MYBL2 is a potential TF that regulates the transcription of UBE2C

To investigate potential TFs involved in the regulation of UBE2C in HCC, a comprehensive analysis was performed using HCC and normal tissue gene expression profiles available from the TCGA and JASPAR databases. As shown in Figure 3A, in total, the expression of eight TFs was positively correlated with that of UBE2C in HCC. Furthermore, the expression of MYBL2 was most strongly correlated with the expression of UBE2C (Figure 3B). The results of the Kaplan-Meier survival analysis also revealed that patients in the MYBL2-high group had worse OS and DFS than those in the MYBL2-low group (Figure 3C). Next, we predicted the binding sites of MYBL2 in the promoter of UBE2C (Figure 3D). In addition, the binding sites of the MYBL2 and UBE2C promoters were detected by ChIP-qPCR. As shown in Figure 4A, MYBL2 was significantly enriched in the region from −400 to 0 bp from the TSS of the UBE2C promoter. Moreover, the enrichment of the binding region of MYBL2 in the promoter of UBE2C was confirmed by a luciferase reporter assay (Figure 4B). To elucidate the specific binding sites of MYBL2 in the promoter of UBE2C, a UBE2C mutated luciferase reporter vector was constructed and transfected into 293T cells. As shown in Figure 4C, overexpression of MYBL2 significantly increased the luciferase activity of the wild type (WT) group but had no effect on that of the mutant (Mut) group. We also detected the effect of MYBL2 on the expression of UBE2C by qRT-PCR and western blotting. As shown in Figure 4D, the mRNA and protein levels of UBE2C significantly increased after MYBL2 overexpression but decreased after MLLT3 knockdown. Taken together, the above results indicated that MYBL2 was enriched in the region from −400 to 0 bp of the UBE2C promoter and initiated transcription.

Figure 3 MYBL2 is a potential TF of UBE2C. (A) UBE2C-positively correlated genes overlapped with those in the JASPAR database. (B) The correlation between UBE2C and 8 potential TFs is shown. (C) The overall survival rate and disease-free survival rate of patients with high or low MYBL2 expression are shown. (D) The potential binding sites of MYBL2 in the UBE2C promoter were predicted by the JASPAR database. HR, hazard ratio; TCGA, The Cancer Genome Atlas; TF, transcription factor; TPM, transcripts per million.

Figure 4 MYBL2 increased UBE2C mRNA expression via transcription. (A,B) The enrichment of MYBL2 in the promoter of UBE2C was detected by ChIP-qPCR and luciferase reporter assays. (C) The binding sites of MYBL2 were mutated and measured by a luciferase reporter assay. (D) The expression of UBE2C was measured by qRT-PCR and western blotting. ***, P<0.001. ChIP-qPCR, chromatin immunoprecipitation quantitative polymerase chain reaction; IgG, immunoglobulin G; mRNA, messenger RNA; Mut, mutant; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild-type.

UBE2C protein is modified by SUMOylation at lysine 18 (K18)

To confirm whether the UBE2C protein can be modified by Ubc9-mediated SUMOylation, pHA-tagged Ubc9 and pMyc-tagged SUMO-1 were transfected into HEK 293T cells. As shown in Figure 5A, Ubc9 significantly increased the UBE2C SUMOylation level. Similarly, endogenous SUMO1-taging UBE2C was detected by immunoprecipitation enrichment with antibodies against UBE2C and SUMO1 (Figure 5B,5C). We subsequently sought to identify the SUMO modification site(s) in UBE2C. As shown in Figure 5D, multiple lysine residues were predicted by the SUMOplot Analysis Program. Among them, K18 had the highest score and was individually mutated to arginine for SUMOylation identification. As shown in Figure 5E, an affinity pull-down assay revealed that the K18A mutation decreased the SUMO modification level of UBE2C. In addition, when the quantity of UBE2C in eluted protein from immunoprecipitated beads was the same, the level of SUMOylated UBE2C was greater after UBC9 overexpression in both LO2 and HepG2 cells (Figure 5F). Furthermore, we confirmed the effect of UBC9 on UBE2C expression by qRT-PCR and western blot assays. As shown in Figure 5G, UBC9 had no effect on the UBE2C mRNA level. Nevertheless, western blotting results demonstrated that the protein level of UBE2C increased after UBC9 overexpression but decreased after UBC9 knockdown (Figure 5H). Our results indicated that UBC9 increased UBE2C protein levels through K18 SUMOylation.

Figure 5 UBE2C protein expression is regulated by SUMOylation. (A) SUMO1-modified UBE2C was detected in HCC cells of cotransfected with the plasmids pHA-Ubc9 and pMyc-SUMO1. (B,C) Endogenous SUMOylated UBE2C was measured in HCC cells. SUMOylated UBE2C was captured by immunoprecipitation with anti-UBE2C or anti-SUMO1 antibodies. (D) The SUMOylated region of the UBE2C protein was predicted by the SUMOplot Analysis Program. (E) Immunoprecipitation of the SUMO regulator SUMO1 in K18-mutant cells. (F) SUMOylation of UBE2C was detected after UBC9 overexpression in HCC cells. (G,H) The expression of UBE2C mRNA and protein was measured by qRT-PCR and western blotting. ***, P<0.001. mRNA, messenger RNA; HCC, hepatocellular carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; SUMO, small ubiquitin-like modifier.

UBE2C expedites HCC proliferation and invasion via the MAPK signalling pathway

To clarify the underlying signalling pathways involved in the effect of UBE2C in HCC, we analysed high-throughput sequencing data from GSE137172, which contained three control samples and three UBE2C knockdown samples in the Gene Expression Omnibus (GEO) database, and identified 821 upregulated genes and 1,799 downregulated genes (log2-fold change >1, P value <0.05) (Figure 6A). KEGG enrichment analysis indicated that UBE2C may play an important role in the VEGF signalling pathway (Figure 6B). Next, by analysing the vascular endothelial growth factor (VEGF) pathway in the KEGG database, we identified PRKCB and PLCG2, which are upstream signalling molecules of the MAPK signalling pathway, as potential targets of UBE2C (Figure 6C). Next, the expression of PRKCB and PLCG2 after UBE2C overexpression and knockdown was measured by qRT-PCR and western blotting. As shown in Figure 6D,6E, the expression of both PRKCB and PLCG2 increased after UBE2C overexpression but decreased after UBE2C knockdown. Moreover, we detected the effect of UBE2C on the activation of the MAPK signalling pathway by western blotting. The activation of p-RAF, p-ERK and p-MEK was significantly decreased after UBE2C knockdown (Figure 6F). Furthermore, rescue studies were performed to determine whether the participation of UBE2C is related to its ability to activate the MAPK signalling pathway. As shown in Figure 7A, the MAPK inhibitor trametinib significantly decreased cell migration and invasion in HCC cells. Nevertheless, overexpression of UBE2C blocked the trametinib-induced decrease in cell invasion. As shown in Figure 7B, trametinib treatment decreased the expression of EMT-related proteins (N-cadherin, vimentin, Snail and MMP9), which could be impaired by UBE2C overexpression. In summary, we revealed a novel molecular mechanism through which UBE2C regulates the progression of HCC. In addition, a selective SUMO inhibitor ML792 was utilized to inhibit SUMOylation in vivo. Tumors were reduced in size after ML-792 treatment compared with normal control (Figure 7C). Meanwhile, the expression of UBE2C and p-ERK was also decreased after SUMO inhibitor treatment in vivo (Figure 7D). In conclusion, as shown in Figure 7E, the expression of UBE2C was regulated by MYBL2-mediated transcription and Ubc9-mediated SUMOylation. In addition to facilitating invasion and proliferation, UBE2C slows the development of HCC by activating the MAPK signalling pathway.

Figure 6 The MAPK signalling pathway is a downstream target of UBE2C. (A) Differential analysis was performed to identify DE-mRNAs in the GSE137172 dataset using the “limma” package. (B,C) KEGG enrichment of DE-mRNAs in the GSE137172 dataset is shown. (D,E) The expression of PRKCB and PLCG2 was measured by qRT-PCR and western blotting. (F) The inactivation of the MAPK pathway was measured by western blotting after UBE2C knockdown. ***, P<0.001. DE-mRNA, differentially expressed messenger RNA; EGFR, epidermal growth factor receptor; KEGG, Kyoto Encyclopedia of Genes and Genomes; qRT-PCR, quantitative real-time polymerase chain reaction; VEGF, vascular endothelial growth factor.

Figure 7 Overexpression of UBE2C rescues the inhibitory effect of MAPK inhibitors on cell metastasis and the EMT in HCC. (A,B) Effects of UBE2C overexpression on the metastasis and EMT of HCC cells treated with trametinib (cells were stained with crystal violet). (C) Tumors were reduced in size after ML-792 treatment compared with normal control. (D) The expression of UBE2C and p-ERK was also decreased after SUMO inhibitor treatment in vivo. (E) Integrated model indicating the molecular mechanism of UBE2C in HCC. ***, P<0.001. EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; SUMO, small ubiquitin-like modifier.

Discussion

HCC remains a global health burden, ranking as the third leading cause of cancer-related mortality worldwide, with particularly high incidence rates in East Asia and sub-Saharan Africa because of the prevalence of HBV and HCV infections (11). Although imaging modalities, surveillance strategies, and local interventions have improved, systemic therapies for advanced HCC still face significant limitations. Agents such as sorafenib and lenvatinib provide only modest survival benefits, with many patients experiencing disease progression due to acquired resistance (12). These shortcomings emphasize the limited availability of reliable biomarkers and actionable molecular targets, making the discovery of novel oncogenic drivers essential for advancing HCC diagnosis, prognosis, and therapy.

UBE2C is a key E2 ubiquitin-conjugating enzyme within the ubiquitin-proteasome system, where it cooperates with the anaphase-promoting complex/cyclosome to catalyze the ubiquitination and degradation of cell cycle regulators such as cyclins and securin. Consistent with this core role, aberrant UBE2C upregulation has been reported in a variety of malignancies, including breast, lung, and prostate cancers (13-15), where it is associated with high tumor grade, metastasis, therapeutic resistance, and poor prognosis. These findings collectively support the view that UBE2C acts as a common oncogenic hub in solid tumors, and our data extend this concept to HCC by demonstrating that UBE2C not only enhances proliferation and invasion but also facilitates EMT and activation of MAPK signalling. Compared with the relatively well-established role of ubiquitination in HCC, the contribution of SUMOylation to hepatocarcinogenesis has only begun to be elucidated. Recent studies have shown that global changes in SUMOylation and dysregulation of SUMO pathway components, such as SUMO E1/E2 enzymes and SUMO-specific proteases, participate in the control of HCC cell survival, DNA damage responses, and metabolic adaptation (16). Nevertheless, SUMOylation of specific oncogenic effectors in HCC remains poorly characterized. Prior work has mainly focused on TFs and signalling intermediates, whereas SUMO-dependent regulation of core cell cycle and ubiquitin-proteasome machinery has been largely overlooked. In this context, our identification of UBC9-mediated SUMOylation of UBE2C at K18 as a mechanism to stabilize UBE2C and amplify downstream MAPK signalling fills an important gap in the current understanding of SUMOylation in HCC. It also links two critical regulatory layers-ubiquitin-mediated proteostasis and SUMO-mediated protein stabilization within the same oncogenic axis.

This study provides a comprehensive mechanistic understanding of how SUMOylation of UBE2C promotes HCC progression. Bioinformatic analysis of TCGA datasets revealed that UBE2C expression is significantly upregulated in HCC, with high expression correlated with poor OS and DFS. These findings were confirmed experimentally using qRT-PCR and Western blot assays on clinical specimens. Functional assays demonstrated that UBE2C knockdown impaired cell proliferation, migration, and invasion in HCC cell lines, partly through inhibition of the EMT process. The transcriptional regulation of UBE2C was found to be mediated by MYBL2, as evidenced by ChIP-qPCR and luciferase reporter assays. MYBL2 binding in the −400 to 0 bp region of the UBE2C promoter significantly increased its transcriptional activity. Posttranslationally, UBE2C was stabilized by SUMOylation at lysine residue K18, which was mediated by UBC9, without altering its mRNA level. Proteomic analysis of data from the GEO database (GSE137172) revealed that UBE2C modulates the MAPK signalling pathway via PRKCB and PLCG2. Knockdown of UBE2C inhibited MAPK activation, and pharmacological inhibition using trametinib suppressed the EMT and cell invasion, which was reversed by UBE2C overexpression. These findings define a novel regulatory axis involving MYBL2, SUMOylated UBE2C, and MAPK signalling in HCC progression.

MYBL2 is emerging as a pivotal TF in multiple malignancies (17). It governs cell cycle-related gene networks and is frequently upregulated in aggressive tumours. In glioblastoma, MYBL2 promotes temozolomide resistance via E2F1 activation (18), whereas in colorectal cancer, it sustains stemness by regulating c-Myc transcription (19). These findings underscore its versatility and oncogenic potential, but its role in HCC remains unexplored. Here, we identified the MYBL2-UBE2C-MAPK axis as a critical driver of HCC proliferation and invasion. In prostate cancer, UBE2C activates ERK signalling via PRKCB phosphorylation, enhancing cell survival and androgen-independent growth (20). Similarly, UBE2C regulates the KEAP1/NRF2 signaling pathway to promote the growth of gastric cancer (21). While these mechanisms have been observed in other cancers (22,23), their role in HCC has not been previously explored. Our study is the first to demonstrate that this regulatory axis operates in HCC and is modulated through SUMOylation, revealing dual-layer control involving transcriptional activation and PTM. This convergence highlights UBE2C as a central oncogenic node and a viable therapeutic target.

PTMs, including ubiquitination and SUMOylation, are critical for regulating protein function, stability, and subcellular localization (24,25). UBE2C, a ubiquitin-conjugating enzyme, is well documented to promote oncogenesis across multiple tumour types (26). Previous studies have linked UBE2C to cell cycle progression, metastasis, and therapeutic resistance (27-29). However, the role of SUMOylation in stabilizing UBE2C and promoting HCC progression has not been previously reported. In this context, the observed upregulation of PRKCB and PLCG2 is likely mediated through UBE2C-dependent modulation of upstream regulatory factors that converge on MAPK signalling, rather than through direct transcriptional control. Similar indirect regulatory mechanisms have been reported in other malignancies, where UBE2C enhances MAPK/ERK pathway activation by altering the stability of pathway-associated proteins, ultimately leading to transcriptional activation of downstream effectors.

Despite these insights, several limitations merit consideration. First, although the in vitro and in vivo assays support the oncogenic role of UBE2C, validation in larger patient cohorts and more physiologically relevant models is still required. Second, the upstream regulatory mechanisms controlling MYBL2 activation and the broader signalling landscape influenced by UBE2C beyond MAPK were not fully explored. Third, while SUMOylation at K18 was identified as a key stabilizing modification, the precise impact of SUMOylation on UBE2C enzymatic activity and its downstream ubiquitination substrate spectrum remains to be elucidated. Notably, although pharmacological inhibition of SUMOylation using ML792 significantly suppressed tumor growth and reduced UBE2C and p-ERK levels in a nude mouse xenograft model, comprehensive identification of SUMOylation-dependent ubiquitination substrates would require mass spectrometry-based proteomic approaches that were beyond the scope of the current study. Finally, the therapeutic implications of targeting the MYBL2-UBE2C-SUMOylation axis were assessed primarily through pharmacological intervention, and rational combination strategies warrant further evaluation in preclinical models.

Conclusions

In summary, we report a novel oncogenic axis in HCC in which MYBL2 transcriptionally activates UBE2C and UBC9-mediated SUMOylation stabilizes the UBE2C protein, facilitating MAPK pathway activation through PRKCB and PLCG2. This dual-layer regulation of UBE2C underscores its potential as a multifaceted therapeutic target. UBE2C-driven MAPK activation can be effectively targeted with clinically available MEK/ERK inhibitors, while UBE2C stabilization through SUMOylation can be indirectly suppressed using emerging SUMO-pathway inhibitors such as TAK-981. In addition, upstream regulators of MYBL2, including bromodomain and extraterminal motif (BET) or cyclin-dependent kinase (CDK) inhibitors, offer alternative strategies to attenuate this oncogenic axis in a clinically feasible manner.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2000/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2000/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2000/prf

Funding: The work was funded by grants from the Science and Technology Project of Zhejiang Medical and Health (No. 2025KY1589), Key Discipline of Hepatobiliary and Pancreatic Surgery of Jiaxing City (No. 2023-zc-005), Translational Therapy Center for Hepatobiliary Pancreatic Cancer (No. 2021-YJZX-04), Science and Technology Project of Jiaxing (Nos. 2022AD30066 and 2023AD31059), Jointly Cultivate Disciplines of Jiaxing City and Zhejiang Province (No. 2023-PYXK-001), and National Clinical Key Specialty Construction Project (No. 2023-GJZK-001).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2000/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of First Hospital of Jiaxing (approval No. 2025-ky-444), and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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