TRIM37 interacts with PTEN to promote progression of hepatocellular carcinoma cells by modulating AKT/GSK-3β/β-Catenin pathway

Introduction

Liver cancer is the fourth most predominant malignancy worldwide and is the second leading cause of malignancy-associated deaths, with a 5-year overall survival (OS) rate <20% (1,2). Hepatocellular carcinomas (HCCs) account for 75–85% of primary liver malignancies and are predominantly associated with metabolic disorders, hepatitis B (HBV) and C (HCV) viruses, and aflatoxin exposure (3). Epidemiological evidence that nonalcoholic fatty liver disease (NAFLD) is a leading aetiology underlying many cases of HCC (4). The management of HCC is primarily guided by the tumor stage, with surgical resection and liver transplantation constituting the primary treatment options, especially for early-stage HCC. These interventions are associated with a relatively improved 5-year OS rate. However, the majority of individuals are diagnosed at progressive, non-curable stages due to the lack of specific symptoms in the early phase, which precludes the possibility of curative surgical resection. Consequently, the average 5-year OS rate for HCC patients remains low at approximately 19.6%, with rates decreasing to as low as 2.5% in cases of advanced metastatic disease (5).

The poor prognosis in advanced HCC is primarily attributed to its aggressive proliferation, high metastatic potential, and therapeutic resistance. These challenges underscore the urgent requirement to investigate the molecular mechanisms underlying HCC progression to improve OS.

The tripartite motif-containing protein (TRIM) family, comprising over 80 members in humans, constitutes a genetically conserved group involved in a broad spectrum of pathological processes, including immune dysfunction, viral infections, transcriptional regulation, developmental anomalies, neurodegenerative disorders, and various malignancies (6). Among these, tripartite motif-containing 37 protein (TRIM37) is located on chromosome 17q23 and characterized by the presence of a conserved B-box, RING finger domain, and coiled-coil region (7). The RING finger domain confers E3 ubiquitin ligase activity to TRIM37, allowing it to mediate substrate-specific protein degradation via the ubiquitin-proteasome pathway (8).

Much evidence indicates that TRIM37 has a vital function in cancer progression. Xu et al. demonstrated that TRIM37 is upregulated in gallbladder malignancy, where it triggers tumorigenesis through stimulation of the Wnt/β-catenin pathway (9). Similarly, Qu et al. found that TRIM37 overexpression is shown in T-cell acute lymphoblastic leukemia, promoting cell proliferation and reducing drug sensitivity via the PI3K/AKT pathway (10). Additionally, TRIM37 is overexpressed in HCC, enhancing cell migration, metastasis, and chemoresistance, and is related to poor prognosis (11). Despite these findings, the regulatory function of TRIM37 in the PTEN/AKT/GSK-3β/β-catenin axis in HCC remains largely underexplored.

Herein, we illustrated that TRIM37 overexpression is significantly upregulated in HCC tissues and cell lines. We examined cellular processes, including growth, invasion, and epithelial-mesenchymal transition (EMT) in Huh7 cells overexpressing TRIM37 and in HCCLM3 cells with TRIM37 knockdown. Our findings indicate that TRIM37 facilitates HCC progression.

Mechanistically, TRIM37 promotes HCC progression by inducing the ubiquitination-mediated degradation of PTEN and subsequently activating the AKT/GSK-3β/β-catenin pathway. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0447/rc).

Methods

Clinical tissue specimens

Twenty-one pairs of HCC tissues and neighboring healthy liver tissues were surgically excised from individuals at The First Hospital of Jiaxing. No patient underwent preoperative radiation or chemotherapy. The tissue samples were pathologically confirmed as HCC and subsequently preserved at −80 ℃. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Hospital of Jiaxing (China) (No. 2024-KY-514). Informed consent was obtained from all participants in this study before sample collection.

Antibodies

The subsequent primary antibodies were utilized: TRIM37 (DF14825, Affinity Biosciences, 1:200 dilution), PTEN (ab267787, Abcam, 1:1,000), β-catenin (sc-7963, Santa Cruz, 1:200), Gsk-3β (#12456, Cell Signaling Technology, 1:1,000), p-Gsk3β (#5558, Cell Signaling Technology, 1:1,000), p-AKT (#4060, Cell Signaling Technology, 1:1,000), AKT (#4691, Cell Signaling Technology, 1:1,000), E-cadherin (#3195, Cell Signaling Technology, 1:1,000), N-cadherin (#R23341, Zenbio, 1:1,000), Vimentin (#5741, Cell Signaling Technology, 1:1,000), GAPDH (#R24404, Zenbio, 1:1,000), and β-actin (#R23613, Zenbio, 1:2,000).

Cell culture

The HepG2, HCCLM3, Huh7, and Sk-Hep-1 HCC cell lines were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell culture was conducted in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, USA) enriched with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA). L02 normal human liver epithelial cell line was acquired from Shanghai, China’s Gaining Biological Technology Co., Ltd., and cultivated in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) enriched with 10% FBS. Treating the cells was conducted in a humidified environment with 5% CO₂ and incubated at 37 ℃.

Cell Counting Kit-8 (CCK-8) assay

CCK-8 (Dojindo Laboratories, Japan) was utilized to assess the cell viability. Cells were plated into 96-well plates (3×10³ cells/well), and viability was assessed at 0, 12, 24, 48, and 72 h post-plating. For cell viability assessment, 10 µL of CCK-8 solution was applied to every well, then a 1-h incubation was conducted in the dark at 37 ℃. A microplate reader was employed to assess the absorbance at 450 nm.

Cell transduction

HCCLM3 cell transduction was conducted with lentiviral particles encoding either short hairpin RNA (shRNA) targeting TRIM37 or a non-targeting scrambled control shRNA at 10 multiplicity of infection (MOI) with 8 µg/mL Polybrene (Sigma-Aldrich, St. Louis, USA). After 24 hours, the medium was substituted with new, complete DMEM augmented with 10% FBS. Puromycin (2 µg/mL; Invitrogen, Carlsbad, CA, USA) was applied 48 hours post-transduction to select stably transduced cells, with the selection maintained for 7 days. Western blot (WB) analysis validated the efficiency of knockdown using an anti-TRIM37 antibody. The shRNA sequences targeting TRIM37 were designed as follows—sense: 5'- GCTGGAATCTGACAAGAATTT-3'; antisense: 5'-AAATTCTTGTCAGATTCCAGC-3'; sense: 5'-GCAGTTTGTGATGAACTAAT-3'; antisense: 5'-ATTAGTTCATCACAAACTGC-3'. Short hairpin RNA negative control (shNC)—sense: 5'-TTCTCCGAACGTGTCACGT-3'; antisense: 5'-ACGTGACACGTTCGGAGAA-3'. The specificity of shRNA sequences was verified using National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) against the human transcriptome, and potential off-target effects were further evaluated using online small interfering RNA (siRNA)/shRNA off-target prediction tools. No notable homologous sequences in other human genes were identified, ensuring high target specificity.

Similarly, Huh7 cells were transduced with lentiviral vectors encoding either human TRIM37 or an empty vector control at an MOI of 20 with 8 µg/mL Polybrene, which enhanced the transduction efficiency. After 72 h, the cells were harvested for RNA and protein extraction. Overexpression was validated via quantitative polymerase chain reaction (qPCR) and WB analysis using an anti-TRIM37 antibody.

Transwell invasion assay

In a low-serum medium, the HCC cells were resuspended. Matrigel was pre-coated onto 24-well Transwell chambers (pore size =8 µm) (Corning, NY, USA) for the invasion assay. The bottom chamber received 600 µL of culture medium enriched with 20% FBS, while the top chamber was seeded with around 2×10⁵ cells in 200 µL of medium. Staining with 0.1% crystal violet and fixation with 4% paraformaldehyde (PFA) were applied to the cells after 48 hours. For transwell invasion assays, cell counting was conducted in a blinded fashion to avoid observer bias. To measure the number of invasive cells, five fields were picked at random for each well and counted under a light microscope.

Colony formation assay

A total of 5×10² HCC cells were placed into every well of a six-well plate and kept at a temperature of 37.5 ℃ with 5% CO₂. A 4% PFA was utilized to fix the cells for 15 min after 12 days, and 0.1% crystal violet was employed to stain them at room temperature for another 15 min. Under an inverted microscope, colonies with 50 cells or more were calculated.

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR)

We used TRIzol reagent (Invitrogen, Carlsbad, USA) to extract total RNA from the cultivated cells. The PrimeScriptTM RT Reagent Kit with gDNA Eraser of Takara Biomedical Technology (Beijing, China) was used to synthesis complementary DNA (cDNA) as per the guidelines of the manufacturer. SYBR Premix Ex Taq and the StepOnePlus Real-Time PCR System were acquired from Takara Biomedical Technology in Beijing, China, and used for the qRT-PCR assay. Initial denaturation at 95 ℃ for 30 s, followed by 40 cycles of denaturation at 95 ℃ for 5 s, annealing at 60 ℃ for 30 s. Using the comparative 2^−ΔΔCt technique, the relative messenger RNA (mRNA) levels were standardized to GAPDH. The primer sequences utilized were as follows—TRIM37: F—5'-AGGTAGCCAAACTTCGTCGG-3'; R—5'-GAAGTTTCAGGCAAGCCAGC-3'; GAPDH: F—5'-ATGGGCAGCCGTTAGGAAAG-3'; R—5'-AGGAAAAGCATCACCCGGAG-3'.

WB assay

Total protein isolation was conducted from cells via RIPA lysis buffer (Beyotime, Shanghai, China), and protein concentration was measured using the bicinchoninic acid (BCA) assay (Beyotime). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently were transferred onto polyvinylidene fluoride (PVDF) membranes. Before being treated with secondary antibodies at room temperature for 2 hours, 5% skimmed milk was utilized to block the membranes and overnight incubation was conducted with the appropriate primary antibodies at 4 ℃. Proteins bands were visualized using enhanced chemiluminescence (ECL) (Beyotime), and band intensities were quantified using ImageJ software.

Immunohistochemical (IHC) analysis of human liver tissues

The 4-µm-thick sections of human HCC tissues and adjacent normal tissues, which were preserved in formalin and embedded in paraffin, were assessed via IHC analysis. After deparaffinization, antigen retrieval was conducted in citrate buffer (pH 6.0) at 95 ℃ for 15 min. To inhibit endogenous peroxidase action, 3% hydrogen peroxide was utilized for 10 min. The tissue portions were subsequently treated with a primary antibody targeting TRIM37 (DF14825, Affinity Biosciences, 1:60) overnight at 4 ℃ after being blocked with 5% BSA for 30 min. Assays for TRIM37 detection were conducted with the use of 3,3'-diaminobenzidine (DAB) chromogen and secondary antibodies coupled with horseradish peroxidase (HRP). A counterstain of hematoxylin was applied to the tissue slides. Every staining run contained a negative control to evaluate non-specific background signal; this control did not contain the primary antibody. Protein expression was semi-quantitatively evaluated by three independent pathologists blinded to sample identity, utilizing a scoring system depending on staining strength [0–3] and percentage of positive cells [0–4]. The final immunoreactivity score was calculated as the result of the two scores [0–12].

Co-immunoprecipitation (Co-IP) assay

Huh7 cell lysis was conducted in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] enriched with protease and phosphatase inhibitors. Subsequently, a 15-min centrifugation of lysates was conducted at 12,000 ×g at 4 ℃, with 500 µg of the produced supernatant incubated overnight at 4 ℃ with 2 µg/mL anti-TRIM37 antibody or control IgG (ab172730, Abcam, 1:1,000). Then, a 2-h incubation of cells was conducted with Protein A/G magnetic beads. Immunocomplexes were rinsed 4 times with RIPA buffer, eluted in Laemmli buffer, and assessed using WB as previously described. Input lysates (5% of the total) were used as loading controls to validate protein presence.

Ubiquitination assay

Treating the HCCLM3 cells with 10 µM MG-132 (Selleckchem) was conducted for 4 h to inhibit proteasomal activity. Cell lysis was conducted in denaturing buffer (6 M urea, 1% SDS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) enriched with 10 mM N-ethylmaleimide (NEM) to preserve ubiquitin conjugates. Lysates were boiled at 95 ℃ for 5 min, sonicated, and spun at 12,000 ×g for 10 min at 4 ℃. The 1 mg of protein was diluted 10-fold in PBS and overnight incubation was conducted at 4 ℃ with anti-PTEN antibody or control IgG, then a 2 h incubation was conducted with Protein A/G magnetic beads (Thermo Fisher). Six washes of the beads were conducted with Triton X-100 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100) and eluted in 1× Laemmli buffer at 95 ℃ for 5 min. Proteins were divided by 8% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-ubiquitin (1:400, 10201-2-AP, Proteintech) and anti-PTEN antibodies. Input lysates (5%) served as loading controls. To ensure reproducibility, all trials were conducted in triplicate.

Statistical analyses

Data are reported as mean ± standard deviation (SD). GraphPad Prism 8 was utilized to conduct statistical analyses. Student’s t-test was utilized to compare between two groups, while one-way analysis of variance (ANOVA) was utilized to compare among numerous groups. For CCK8 results analysis, Two-way ANOVA followed by Tukey’s multiple comparisons test was used. A P<0.05 was believed to be significant.

Results

TRIM37 overexpression in HCC tissues and cell lines

To evaluate TRIM37 expression in HCC, the publicly available Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database was analyzed. The results illustrated that TRIM37 mRNA concentrations were significantly elevated in HCC tissues (T) compared to neighboring non-cancerous tissues (N) (Figure 1A). Moreover, elevated TRIM37 expression was associated with reduced OS in individuals with HCC (Figure 1B), yet the correlation between TRIM37 and survival needed further exploration. To validate these findings, qRT-PCR and IHC were carried out to assess TRIM37 mRNA and protein levels, respectively, in clinical HCC specimens. Notably, qRT-PCR analysis of six matched pairs of HCC and neighboring healthy tissues revealed higher TRIM37 mRNA levels in HCC tissues (Figure 1C). Similarly, IHC analysis of 15 matched pairs of HCC and neighboring healthy tissues demonstrated significantly increased TRIM37 mRNA protein levels in HCC tissues (Figure 1D,1E). In addition, WB analysis showed that TRIM37 was higher in four HCC cell lines (Huh7, HCCLM3, HepG2, and Hep3B) than in the healthy liver epithelial cell line LO2, with HCCLM3 exhibiting the highest expression and Huh7 the lowest (Figure 1F). Based on these differential expression levels, Subsequently, HCCLM3 cells were chosen for TRIM37 knockdown depending on these differential expression levels, while Huh7 cells were selected for TRIM37 overexpression.

Figure 1 TRIM37 expression is upregulated in HCC tissues and cell lines, and its upregulation is linked to poor patient survival. (A) TRIM37 mRNA expression levels were assessed using TCGA dataset via online bioinformatics tools. (B) GEPIA2 analysis showed that TRIM37 overexpression relates to reduced overall survival in HCC patients. (C) qRT-PCR analysis of TRIM37 level in six paired HCC (T) and adjacent non-cancerous tissues (N). (D) Representative IHC images showing TRIM37 level in HCC tissues (T) and corresponding neighboring non-cancerous tissues (N) (magnification ×). (E) Quantification of TRIM37 IHC staining scores in paired HCC (T) and neighboring healthy tissues (N). (F) WB analysis of TRIM37 protein level in HCC cell lines (HCCLM3, Huh7, HepG2, and Hep3B) compared to healthy liver epithelial cells (LO2). Each experiment was repeated at least three independent biological replicates. Data are reported as mean ± SD (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. HCC, hepatocellular carcinoma; HR, hazard ratio; IHC, immunohistochemistry; LIHC, mRNA, messenger RNA; N, normal; qRT-PCR, quantitative real‑time polymerase chain reaction; SD, standard deviation; T, tumor; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; WB, Western blot.

TRIM37 promotes the proliferative capacity of HCC cells

To examine the TRIM37 function in HCC progression, we utilized shRNA to suppress TRIM37 in HCCLM3 cells and transfected Huh7 cells with a TRIM37 overexpression plasmid. WB confirmed successful TRIM37 knockdown and overexpression at the protein level (Figure 2A,2B). Next, the TRIM37 effects on HCC cell growth were estimated via CCK-8 and colony formation assessments. Notably, CCK-8 assay outcomes illustrated that TRIM37 suppression decreased cell viability in HCCLM3 cells, while TRIM37 upregulation improved cell viability in Huh7 cells (Figure 2C). Consistent pattern was observed in the colony formation assay: TRIM37 knockdown caused a significant impairment of the colony-forming capability of HCCLM3 cells, whereas TRIM37 overexpression promoted colony formation in Huh7 cells (Figure 2D,2E). Collectively, these outcomes indicate that TRIM37 enhances the proliferative capacity of HCC cells.

Figure 2 TRIM37 enhances the proliferative potential of HCC cells. (A,B) WB was conducted to evaluate TRIM37 protein expression in cells transfected with TRIM37-specific shRNA or TRIM37 upregulation vectors. (C-E) The proliferative capacity of HCCLM3 and Huh7 cells was estimated using CCK-8 and colony formation assessments. HCCLM3 cell transfection was conducted with TRIM37 shRNA, and Huh7 cells with TRIM37 overexpression vectors. (D,E) stained. Each experiment was repeated at least three independent biological replicates. Data are reported as mean ± SD (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; HCC, hepatocellular carcinoma; OD, optical density; SD, standard deviation; shRNA, short hairpin RNA; WB, Western blot.

TRIM37 triggers cell invasion and EMT in HCC

To elucidate the TRIM37 function in HCC progression, we performed Transwell invasion assessment to observe the impacts of TRIM37 overexpression and knockdown. The outcomes illustrated that TRIM37 overexpression in Huh7 cells significantly enhanced invasive capacity, whereas TRIM37 knockdown in HCCLM3 cells markedly reduced invasion (Figure 3A,3B).

Figure 3 TRIM37 promotes invasion and EMT in HCC cells. (A,B) Transwell invasion assessments were conducted to evaluate the invasive capabilities of HCCLM3 and Huh7 cells transfected with TRIM37-specific shRNA or overexpression vectors (magnification ×; stained). (C,D) WB was used to examine the expression of EMT markers’ expression in each transfected group. Each experiment was repeated at least three independent biological replicates. Data are reported as mean ± SD (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; SD, standard deviation; shRNA, short hairpin RNA; WB, Western blot.

Research has established EMT as a key process in the distant metastasis of malignant tumors. We hypothesized that TRIM37 facilitates HCC invasion by modulating the EMT process. To test this, WB analysis was conducted to evaluate the EMT-associated markers’ expression (Vimentin, E- and N-cadherin). Notably, TRIM37 knockdown in HCCLM3 cells caused N-cadherin and Vimentin suppression, along with E-cadherin overexpression compared to control cells (Figure 3C). Conversely, TRIM37 overexpression in Huh7 cells caused overexpressed N-cadherin and Vimentin and suppressed E-cadherin levels (Figure 3D). These findings indicate that TRIM37 regulates HCC cell invasion by modulating the EMT process.

TRIM37 facilitates HCC progression via the AKT/GSK-3β/β-Catenin pathway

The AKT/GSK-3β/β-Catenin pathway is critically involved in the HCC progression. To examine the mechanism by which TRIM37 regulates HCC progression via this pathway, we first examined alterations in the pathway activity following TRIM37 knockdown or overexpression. WB analysis illustrated that TRIM37 knockdown in HCCLM3 cells caused reduced phosphorylated AKT (p-AKT), phosphorylated GSK-3β (p-GSK-3β), and β-Catenin levels, while total AKT and GSK-3β levels were still unchanged (Figure 4A). Conversely, TRIM37 overexpression in Huh7 cells overexpressed p-GSK-3β, p-AKT, and β-Catenin, with the total AKT and GSK-3β concentrations remaining unchanged (Figure 4B).

Figure 4 The PI3/AKT inhibitor LY294002 impairs TRIM37-mediated functions in Huh7 cells. (A,B) WB analysis of p-AKT, AKT, β-Catenin, p-GSK-3β, and GSK-3β expression in HCCLM3 and Huh7 cells transfected with TRIM37-specific shRNA or overexpression vectors. (C) WB analysis of the same proteins in TRIM37-overexpressing Huh7 cells exposed to LY294002. (D) CCK-8, (E) colony formation (stained), and (F) Transwell invasion assessments were utilized to estimate the functional effects of LY294002 treatment on TRIM37-overexpressing Huh7 cells (magnification ×; stained). Each experiment was repeated at least three independent biological replicates. Data are reported as mean ± SD (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; SD, standard deviation; shRNA, short hairpin RNA; WB, Western blot.

To further validate this mechanism, rescue experiments were conducted using the PI3K inhibitor LY294002 in TRIM37-overexpressing Huh7 cells. We found that LY294002 treatment significantly suppressed p-GSK-3β, p-AKT, and β-Catenin compared to the TRIM37 + DMSO control group (Figure 4C).

Additionally, CCK-8, colony formation, and Transwell assessments illustrated that LY294002 treatment significantly lessened the proliferative, cologenic, and invasive abilities of TRIM37-overexpressing Huh7 cells (Figure 4D-4F). These outcomes indicate that TRIM37 may trigger HCC progression by stimulating the AKT/GSK-3β/β-Catenin pathway.

TRIM37 interacts with PTEN to promote its ubiquitination

In order to facilitate substrate-specific protein degradation by means of the ubiquitin-proteasome system, TRIM37 possesses E3 ubiquitin ligase activity, which is conferred by its RING domain. The publicly accessible HitPredict database (http://www.hitpredict.org/) was subsequently analyzed to examine the molecular mechanism by which TRIM37 controls the AKT/GSK-3β/β-Catenin pathway. The results showed that TRIM37 interacts primarily with PTEN.

WB was then used to determine whether TRIM37 influences PTEN expression. The outcomes illustrated that TRIM37 overexpression significantly decreased PTEN protein concentrations, while its knockdown led to higher PTEN levels than in the control groups (Figure 5A,5B). However, qRT-PCR analysis illustrated that TRIM37 knockdown did not change PTEN mRNA levels (Figure 5C), indicating that TRIM37 regulates PTEN at the post-transcriptional level.

Figure 5 TRIM37 interacts with PTEN and promotes its ubiquitination. (A,B) WB analysis of PTEN protein level in HCCLM3 and Huh7 cells with TRIM37-specific shRNA or overexpression vectors. (C) qRT-PCR analysis of PTEN mRNA levels in HCCLM3 cells with TRIM37-specific shRNA. (D) Co-IP assays were conducted to evaluate the TRIM37 and PTEN interaction. (E) Ubiquitination assays were used to detect polyubiquitinated PTEN in HCCLM3 cells transfected with TRIM37-specific shRNA. Each experiment was repeated at least three independent biological replicates. Data are reported as mean ± SD (n=3). ns, not significant; *, P<0.05; **, P<0.01. Co-IP, co-immunoprecipitation; mRNA, messenger RNA; qRT-PCR, quantitative real‑time polymerase chain reaction; SD, standard deviation; shRNA, short hairpin RNA; WB, Western blot.

Co-IP assays showed that TRIM37 co-precipitated with PTEN, further confirming its physical interaction with PTEN in HCC cells (Figure 5D). Moreover, compared to the control group, ubiquitination assays revealed that TRIM37 knockdown significantly decreased PTEN polyubiquitination (Figure 5E). These findings illustrated that TRIM37 promotes PTEN degradation by facilitating its ubiquitination.

TRIM37 facilitates HCC progression by inhibiting PTEN

Given that TRIM37 promotes tumor cell progression and interacts with PTEN, we further investigated whether PTEN mediates TRIM37-driven HCC progression. Notably, transfection of PTEN-overexpressing plasmids into TRIM37-overexpressing Huh7 cells restored PTEN expression.

Cells in the TRIM37 + PTEN group showed significantly reduced cell viability, colony formation, and invasive capacity (Figure 6A-6C) compared to the TRIM37 group. Furthermore, PTEN overexpression reversed the EMT phenotype and suppressed p-AKT, p-GSK-3β, β-Catenin, N-cadherin, and Vimentin, while increasing E-cadherin levels (Figure 6D,6E). These results suggest that TRIM37 facilitates HCC proliferation, invasion, and EMT, at least in part through PTEN inhibition.

Figure 6 TRIM37 facilitates HCC progression by inhibiting PTEN expression. (A) CCK-8 assessment was conducted to estimate the TRIM37-overexpressing Huh7 cell growth with or without PTEN upregulation. (B) Colony formation (stained), and (C) Transwell invasion assessments were carried out to estimate the proliferative and invasive capabilities of TRIM37-upregulating Huh7 cells with or without PTEN upregulation (magnification ×; stained). (D) WB analysis of p-AKT, AKT, β-Catenin, p-GSK-3β, and GSK-3β expression in TRIM37-overexpressing Huh7 cells with or without PTEN overexpression. (E) WB analysis of EMT markers (Vimentin, E- and N-cadherin) in the same groups. Each experiment was repeated at least three independent biological replicates. Data are reported as mean ± SD (n=3). **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; SD, standard deviation; WB, Western blot.

Discussion

HCC is one of the most predominant malignant neoplasms and is linked to increased death rates (12). Despite advancements in diagnostic techniques and developing the targeted therapies, the majority of patients experience suboptimal prognostic outcomes. Therefore, there is an urgent requirement to determine potential molecular biomarkers and therapeutic targets to enhance patient results. Herein, we illustrated that TRIM37 is increased in HCC and induces tumor progression by triggering PTEN degradation via ubiquitination, thereby stimulating the AKT/GSK-3β/β-Catenin pathway.

Emerging research has illustrated that TRIM37 functions as an oncogene and may represent a potential therapeutic target for various malignancies. Notably, TRIM37 is overexpressed in numerous tumor types, such as lung, pancreatic, gastric, and ovarian cancers (13). Additionally, it has been recognized as a negative prognostic factor. Consistently, our initial analyses illustrated that TRIM37 showed a significant overexpression in HCC tissues and cell lines. Bioinformatics analysis further established higher TRIM37 mRNA levels in tumor tissues than in healthy tissues, with increased levels linked to reduced OS.

We conducted various functional assays to elucidate the TRIM37 function in the HCC cell biology. The outcomes revealed that TRIM37 overexpression enhanced cell viability, colony formation, and invasion, whereas its knockdown reversed these effects. These results illustrate that TRIM37 may act as a potential molecular prognostic biomarker and therapeutic target in HCC.

EMT is a well-established driver of cancer progression and metastasis. Our study demonstrated that TRIM37 upregulation caused an overexpression of mesenchymal markers (N-cadherin and Vimentin), while it caused suppression of epithelial marker (E-cadherin). Conversely, TRIM37 knockdown reversed this expression pattern. These outcomes are in line with earlier investigations reporting that TRIM37 triggers tumor cell migration and metastasis by controlling EMT in HCC (14). Collectively, our results indicate that TRIM37 promotes HCC cell proliferation, invasion, and EMT, thereby supporting its role as a prognostic marker and therapeutic target in HCC.

PTEN is a widely expressed tumor inhibitor and a vital negative modulator of the PI3K/AKT/mTOR pathway. Therefore, it controls essential cellular functions, including survival, growth, and energy metabolism (15,16). Notably, PTEN harbors point mutations across various tumor types, with particularly high frequencies in glioblastoma and lung, endometrial, prostate, and liver cancers (17).

Previous studies have demonstrated that ubiquitination of PTEN by E3 ubiquitin ligases regulates its catalytic activity, protein stability, and subcellular localization. Importantly, PTEN stability is tightly controlled through ubiquitin-mediated proteasomal degradation. Several E3 ligases, including NEDD4 (neuronal precursor cell-expressed developmentally suppressed 4, also called NEDD4-1), WW domain-containing protein 2 (WWP2), and TRIM37, have been shown to specifically ubiquitinate PTEN, thereby promoting its degradation. For instance, Wang et al. illustrated that the E3 ubiquitin ligase RBCK1 interacts with PTEN, thereby facilitating its K48-linked ubiquitination and degradation in ovarian cancer (18).

Based on these findings, we hypothesized that TRIM37 promotes HCC progression by promoting PTEN degradation via ubiquitination. In this study, Co-IP and ubiquitination assays revealed that TRIM37 physically interacts with PTEN, thereby triggering its ubiquitination-mediated degradation. Moreover, previous investigations illustrated that PTEN inhibits gastric cancer progression by controlling the AKT/GSK-3β/β-Catenin pathway (19). Consistent with this, this investigation revealed that PTEN overexpression suppresses the AKT/GSK-3β/β-Catenin pathway and attenuates cancer progression in HCC.

The AKT/GSK-3β/β-Catenin pathway has an essential function in numerous pathophysiological functions, such as cell fate determination, motility, differentiation, angiogenesis, tumor growth, metastasis, and anti-tumor immunity (20). Aberrant stimulation of the PI3K/AKT pathway is often distinguished in numerous human malignancies and is associated with enhanced tumor malignancy and reduced OS rates (21). Notably, GSK-3β regulates β-Catenin degradation through phosphorylation, and its activity is blocked by AKT-mediated phosphorylation (22). To further elucidate the mechanism by which TRIM37 modulates this pathway in HCC, we examined the TRIM37 effects on the AKT/GSK-3β/β-Catenin axis in Huh7 cells. Previous investigations have shown that TRIM37 promotes chemoresistance and stemness in pancreatic malignant cells by mediating PTEN ubiquitination and stimulating the AKT/GSK-3β/β-Catenin pathway (23). In agreement with these findings, our outcomes demonstrate that either PTEN overexpression or pharmacological inhibition of PI3K/AKT signaling using LY294002 reverses the impacts of TRIM37-overexpression, including the stimulation of the AKT/GSK-3β/β-Catenin pathway and the enhancement of growth, invasion, and EMT in HCC. Our findings suggest that TRIM37 triggers HCC cell growth, invasion, and EMT by facilitating PTEN degradation through ubiquitination and stimulating the AKT/GSK-3β/β-Catenin pathway. This study has several limitations. First, our current data support that TRIM37 regulates PTEN ubiquitination under knockdown conditions, the complementary evidence from TRIM37 overexpression assays remains to be verified in future study. Second, we did not perform cycloheximide chase assays with or without MG-132 to directly determine the effect of TRIM37 on PTEN stability and half-life. Finally, although the LO2 cell line is commonly utilized as a normal hepatic epithelial model, it should be noted that immortalized cell lines may exhibit certain genetic and phenotypic differences from primary normal liver cells.

Conclusions

This investigation demonstrates that TRIM37 is upregulated in HCC and triggers tumor cell growth, invasion, and EMT. Furthermore, TRIM37 interacts with PTEN, facilitating its ubiquitination-mediated degradation, resulting in the stimulation of the AKT/GSK-3β/β-Catenin pathway. Collectively, our outcomes elucidate a key molecular mechanism underlying HCC development, highlight TRIM37 as a potential biomarker, and offer new therapeutic insights for improving the treatment and prognosis of patients with HCC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0447/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0447/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0447/prf

Funding: This work was supported by the Medical Science and Technology Project of Zhejiang Province (No. 2025KY1586) and the Jiaxing Key Discipiline of Medcine-Surgery Med (Hepatobiliary Pancreatology) (No. 2023-ZC-005).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0447/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. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Hospital of Jiaxing (China) (No. 2024-KY-514). Informed consent was obtained from all participants in this study.

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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