TRIM39 aggravates hepatocellular carcinoma growth through targeting the p62-KEAP1-NRF2 axis

Abstract

The tripartite motif (TRIM) protein family plays important roles in the initiation and progression of various tumors through different mechanisms. However, the biological functions of TRIM39 and its underlying mechanism in hepatocellular carcinoma (HCC) remain largely unknown. In this study, we found that the expression of TRIM39 was increased in HCC tissues compared to adjacent normal tissues and negatively correlated with the prognosis of HCC patients. Functional studies demonstrated that TRIM39 promoted the proliferation of HCC cells in vitro and in vivo. Mechanistically, TRIM39 interacted with p62 and facilitated its ubiquitination, leading to a decrease of p62 protein stability. The downregulation of p62 released KEAP1, which subsequently inhibited NRF2-HO-1 pathway. Moreover, overexpression p62 counteracted the pro-tumor phenotypes induced by TRIM39 in HCC. In conclusion, our study elucidates the oncogenic roles and the relevant mechanisms of TRIM39 in HCC, highlights the TRIM39-p62-Keap1-NRF2 axis in HCC progression. These findings may provide potential therapeutic targets for HCC treatment.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-32976-x.

Introduction

Hepatocellular carcinoma (HCC) represents a major global health challenge with high incidence and mortality rates¹. Due to the limitation of current therapeutic options, it is of great significance to elucidate the mechanisms driving HCC tumorigenesis and progression, and develop novel therapeutic targets². Tripartite motif (TRIM) family comprises a large group of proteins with E3 ubiquitin ligase activity, and defined by three conserved structural domains: a RING finger domain at the N-terminal, a B-box domain and a coiled-coil domain (CCD)³. The TRIM family is implicated in various biological processes, such as autophagy, immune regulation, and tumor progression^4–7. However, the functional roles and underlying mechanisms of TRIM39 in cancer remain largely unexplored, especially in HCC. A study reported that TRIM39 acted as an oncogene in colorectal cancer (CRC) by suppressing autophagic degradation of p53⁸. TRIM39 also modulate the cell cycle and apoptosis through decreasing the stability of p53 and its downstream target p21⁹.

p62 is a selective autophagy receptor that facilitates the autophagosome-lysosomal degradation of intracellular substrates by interacting with autophagy related protein LC3¹⁰. Beyond its role in selective autophagy, p62 also serves as a signaling scaffold in various pathologic and physiologic processes, such as inflammation, ferroptosis and oxidative stress response^11–14. Notably, the functional roles of p62 in HCC appear to be contradictory. Several studies showed that p62 was overexpressed and had oncogenic roles in HCC progression by regulating autophagy, NF-κB or mTOR signal pathways¹⁵. However, p62 deficiency was found to enhance liver tumorigenesis induced by diethylnitrosamine and high-fat diet¹⁶. Another study reported that TRAF2 promoted the growth of HCC cells by reducing p62 protein levels, the down-regulation of p62 then activated mTORC1 signal by enhancing the lysosome location of mTORC1¹⁷. These studies suggest that whether p62 promotes or suppresses HCC development is context-dependent. To date, the underlying molecular mechanism is still unclear.

Here, we found that TRIM39 was upregulated in HCC tumor tissues and associated with poor clinical outcomes in HCC patients. In vitro and in vivo results demonstrated that TRIM39 promoted the growth of HCC cells. Mechanistically, p62 was identified as a novel TRIM39-interacting protein, and this interaction led to a decrease in p62 protein levels. The downregulation of p62 results in the release of KEAP1, which subsequently inhibits NRF2-HO-1 pathway. Furthermore, TRIM39 promoted HCC progression in a p62-dependent manner. This study may provide new approaches and targets for HCC treatment.

Methods

Antibodies and reagents

All antibodies and chemical reagents utilized in this study are cataloged in Supplementary Table S1.

Cell culture and transfection

HEK293T cells and the panel of human hepatocellular carcinoma (HCC) cell lines (HepG2, Huh7, QGY-7703, MHCC97, HepG3B, SMMC-7721, HCCLM3) were sourced from Procell Life Science & Technology Co., Ltd. and the American Type Culture Collection (ATCC). Cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified 5% CO₂ incubator, following standard cell culture protocols. Plasmid’s transfections were performed using Lipofectamine 3000 reagent (Invitrogen)¹⁸. Cells were typically harvested for analysis 48 h post-transfection.

Plasmid construction

Human TRIM39 was amplified from QGY-7703 cell cDNA library and subsequently cloned into the pLenti-CMV-puro and pFLAG-CMV vectors. Similarly, human p62 was amplified from cDNA library of HEK293T cells and inserted into the pcDNA3.1/Myc-His and pLenti-neo vectors. For knockdown experiments, short hairpin RNA (shRNA) sequences targeting human TRIM39 and non-specific control shRNA were designed, synthesized, and cloned into the pLKO.1-puro lentiviral vector. The integrity of all constructed plasmids was verified by Sanger sequencing. The oligonucleotide primers used for cloning are listed in Supplementary Table S2.

Lentiviral production and transduction

Lentiviral particles were produced and used for transduction as previously described⁸. In brief, HEK293T packaging cells were co-transfected with the lentiviral transfer vector (e.g., pLKO.1-shRNA, pLenti-CMV-puro) along with the psPAX2 (packaging) and pMD2.G (envelope) plasmids. The viral supernatant was collected 48 h post-transfection, filtered, and HCC cells were infected with lentiviral particles and subsequently selected by puromycin (Selleck) or neomycin (Beyotime) for 1 weeks. The knockdown and overexpression efficiency were confirmed by western blotting. The shRNA target sequences are provided in Supplementary Table S3.

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from cells using Trizol Reagent (Takara). First-strand cDNA was synthesized from RNA using the HiScript II 1 st Strand cDNA Synthesis Kit (Vazyme). qRT-PCR was subsequently performed using the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme) on a real-time PCR system. The relative mRNA expression levels of target genes were normalized to GAPDH and calculated using the 2^−ΔΔCt method¹⁹. All primer sequences are listed in Supplementary Table S4.

Western blot analysis

Cells were lysed by RIPA lysis buffer (Beyotime) containing a cocktail of protease and phosphatase inhibitors (MCE) on ice. Lysates were clarified by centrifugation at 12,000 × g for 15 min at 4 °C. Protein concentrations of the supernatants were determined using a BCA protein assay kit (Thermo Scientific). Equal amounts of protein (30 µg per lane) were separated by SDS-PAGE (Epizyme) and transferred onto nitrocellulose membranes (Pall). After blocking with 5% (w/v) non-fat milk for 1 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4 °C. Following incubation with appropriate HRP-conjugated secondary antibodies, the signals were visualized using a chemiluminescence detection system (Odyssey), as described in the standard protocol²⁰.

Co-immunoprecipitation (Co-IP)

HCC cells transfected with the indicated plasmids were collected and lysed by Western & IP lysis buffer (Beyotime) containing protease inhibitors. Cell lysates were incubated with 2 µg of the specified primary antibody or control IgG overnight at 4 °C with gentle rotation. Protein A/G agarose beads (Santa Cruz Biotechnology) were then added, and the incubation continued for an additional 2 h. The bead-bound immunocomplexes were washed three times with lysis buffer, eluted in 2× SDS loading buffer by boiling at 98 °C for 10 min, and analyzed by western blotting, following established methodologies.

Liquid chromatography-mass spectrometry (LC-MS) analysis

FLAG-TRIM39 stably overexpressing HCC cells were lysed, and the lysates were subjected to immunoprecipitation with anti-FLAG antibody as described in the Co-IP section. After extensive washing, the bound proteins were eluted and submitted for analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify TRIM39-interacting partners.

In vitro ubiquitination assay

The assay was performed as previously described with minor modifications²¹. Briefly, Purified recombinant His-p62 and GST-TRIM39 proteins were incubated in a mixture of ubiquitin ligation reaction containing 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 4 mM ATP, 1 µg ubiquitin (Yeasen, 20431ES08), 300 ng E1-UBA1 (Targetmol, TMPY-02840), and 500 ng E2-UBE2L3 (Targetmol, TMPY-02842) at 30 °C for 3 h. The reaction was terminated by boiling for 10 min in 2× SDS sample buffer, and the proteins were resolved by SDS-PAGE followed by western blotting with the indicated antibodies.

Immunofluorescence (IF) staining

For immunofluorescent staining, cells grown on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 10% FBS. The cells were then incubated with primary antibodies overnight at 4 °C, followed by appropriate fluorescently labeled secondary antibodies for 1 h at room temperature in the dark. Nuclei were counterstained with Hoechst 33,342 for 5 min. Fluorescent images (n = 3 field per experimental group)were captured using a laser-scanning confocal microscope (Zeiss)²².

EdU proliferation assay

BeyoClick™ EdU-594 (Beyotime) was used to assess the proliferation ability of HCC cells according to the manufacturer’s instructions. Briefly, cells were incubated with EdU solution for 2 h, fixed, and permeabilized. The Click reaction was performed to label the incorporated EdU, and nuclei were stained with Hoechst. The percentage of EdU-positive cells (n = 3 per experimental group) was quantified from images acquired with a fluorescence microscope (Olympus).

Colony formation assay

Cells were seeded at a low density (500 cells per well) in 12-well plates and cultured for 10–14 days to allow colony formation (n = 3 per experimental group). The resulting colonies were fixed with 4% PFA and stained with 0.1% (w/v) crystal violet. The number of colonies was quantified using image analysis software.

Animal studies

All animal procedures were reviewed and approved by the Animal Ethics Committee of the first affiliated hospital of Henan University of Science and Technology (2024-03-K0107). Mice were housed under specific pathogen-free (SPF) conditions. For the subcutaneous xenograft model, 8-week-old male Balb/c nude mice were randomly assigned to experimental groups (n = 5 mice per experimental group). Mice were anesthetized via inhalation of isoflurane, and then inoculated subcutaneously in the flank with the indicated HCC cells (1 × 10⁶ cells/100 uL PBS). Tumor dimensions were measured with calipers every 2 days, and the volume was calculated using the formula: Volume = (Length × Width²)/2. 21 days post-inoculation, mice were humanely euthanized. The euthanasia method was performed in accordance with the AVMA Guidelines. Briefly, mice were placed in a transparent induction chamber and exposed to a controlled flow of 100% carbon dioxide (CO₂) at a displacement rate of 20% of the chamber volume per minute. Tumors were then excised, weighed, and photographed. For survival analysis, the tumor-bearing mice were removed from the study (terminated) when the tumor volume reached to 1.5 cm³.

Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded (FFPE) sections from human HCC specimens or mouse xenograft tumors were deparaffinized and rehydrated. Antigen retrieval was performed, and endogenous peroxidase activity was quenched. Sections were then blocked and incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies (Dako). Staining was developed using 3,3’-diaminobenzidine (DAB, Dako), and nuclei were lightly counterstained with hematoxylin. Staining intensity and extent were evaluated by two independent pathologists, following established scoring guidelines³⁰. The use of human HCC tissue samples was approved by Human Ethics Committee of the first affiliated hospital of Henan University of Science and Technology. The written informed consents were obtained from all patients.

Statistical analysis

Quantitative data are presented as the mean ± standard error of the mean (SEM). Statistical comparisons between two groups were performed using an unpaired, two-tailed Student’s t-test. Survival curves were generated using the Kaplan-Meier method, and differences were assessed with the log-rank (Mantel-Cox) test. p values < 0.05 was considered statistically significant. All analyses were conducted using GraphPad Prism software (GraphPad Software, Inc.).

Results

TRIM39 expression is elevated in HCC and correlated with poor clinical prognosis

We initially assessed the expression of TRIM39 in human HCC tissues by using The Cancer Genome Atlas (TCGA) and GSE14520 database. TRIM39 was found to be upregulated in HCC tumor tissues compared to normal tissues (Fig. 1A-B). We further evaluated the prognostic effects of TRIM39 in clinical HCC specimens. Consistently, the increased expression of TRIM39 in HCC tissues was confirmed by RT-PCR, western blot and immunohistochemistry (IHC) (Fig. 1C-H). Kaplan-Meier survival analysis revealed that HCC patients with high TRIM39 levels usually had poorer survival rates (Fig. 1I-J). These results indicate that TRIM39 expression in HCC is associated with a worse clinical prognosis.

Fig. 1.

TRIM39 is upregulated and correlated with unfavorable prognosis in HCC. (A) Analysis of TRIM39 mRNA expression in primary HCC tumor (n = 453) and adjacent normal tissues (n = 41) in TCGA database. ****, P < 0.0001. (B) Analysis of TRIM39 mRNA expression in primary HCC tumor (n = 252) and adjacent normal tissues (n = 239) in GSE14520 database. ****, P < 0.0001. (C) Analysis of TRIM39 mRNA expression in primary tumor (n = 52) and adjacent normal tissues (n = 50). **, P < 0.01. (D) Analysis of TRIM39 mRNA expression in 50 paired of tumor and adjacent normal tissues. **, P < 0.01. (E) Western blot analysis of TRIM39 expression in 18 paired HCC tissues and peritumoral tissues. (F) Quantification of TRIM39 expression in tissues from (E). ****, P < 0.0001. (G) Representative images of TRIM39 immunohistochemistry staining in tumor (n = 22) and adjacent normal tissues (n = 22) in HCC tissue microarray. Scale bars, 200 μm for 5 x magnification. (H) Quantification of mean density of TRIM39 staining in tissue microarray from (G). **, P < 0.01. (I-J) Kaplan-Meier survival analysis of overall and disease-free survival based on TRIM39 expression in the HCC tissues from (https://kmplot.com/).

TRIM39 overexpression enhances the proliferation abilities of HCC cells in vitro and in vivo

We tested TRIM39 expression in several HCC cell lines and observed that TRIM39 was expressed at relatively low levels in HepG2, Huh7 and HepG3B cells, whereas it was highly expressed in QYG-7703, MHCC97, SMMC7721 and HCCLM3 cells (Fig. 2A-B). To investigate the functional roles of TRIM39, we conducted gain-of-function studies by stably overexpressing TRIM39 in HepG2 and Huh7 cell lines (Fig. 2C-D). The colony formation, CCK8 and EdU assays revealed that TRIM39-overexpressing HCC cells exhibited higher proliferation rates compared to control cells (Fig. 2E-J). Additionally, we assessed the impact of TRIM39 on tumor growth in vivo by establishing xenograft models. Overexpression of TRIM39 significantly enhanced tumor formation in nude mice, with increased tumor volume and weight observed in TRIM39 overexpression group compared to the control group (Fig. 2K-M). Immunohistochemical staining showed elevated levels of the proliferation marker Ki67 and reduced expression of the apoptosis-related marker Caspase-3 in the TRIM39 overexpression group (Fig. 2N-O). These findings demonstrate that TRIM39 promotes HCC tumor growth in vitro and in vivo.

Fig. 2.

TRIM39 overexpression promotes HCC progression. (A) Analysis of TRIM39 mRNA expression in indicated HCC cell lines. (B) Analysis of TRIM39 protein levels in indicated HCC cell lines. (C) qPCR analysis of TRIM39 expression in TRIM39 stably overexpression HepG2 and Huh7 cells. (D) Western blot analysis of TRIM39 expression in TRIM39 stably overexpression HepG2 and Huh7 cells. (E-F) The colony formation assay of TRIM39 stably overexpression HepG2 and Huh7 cells. **, P < 0.01. (G-H) The CCK8 assay of TRIM39 stably overexpression HepG2 and Huh7 cells. **, P < 0.01. (I-J) The EdU proliferation assay of TRIM39 stably overexpression HepG2 and Huh7 cells. The average number of EdU positive cells per field were calculated. n = 3 samples per group, four fields per sample. *, P < 0.05; **, P < 0.01. (K-M) TRIM39 stably overexpression or control HepG2 cells were subcutaneously injected into nude mice. Representative tumor sizes (K), tumor growth curves (L), and tumor weight (M) are shown. n = 5 per group; Scale bar, 50 mm. **, P < 0.01. (N-O) The representative immunohistochemical staining and quantification of Ki67 and Caspase-3 in xenograft tumor from TRIM39 stably overexpression group and control group. Scale bar, 100 μm. **, P < 0.01.

TRIM39 knockdown inhibited HCC tumor growth in vitro and in vivo

To further confirmed the roles of TRIM39 in HCC progression, we knockdown TRIM39 in MHCC97 and HCCLM3 cell lines, which expressed TRIM39 at relatively high levels (Fig. 3A-B). In contrast to the results observed in TRIM39 overexpression cells, TRIM39 knockdown significantly impaired the proliferation ability of HCC cells in vitro (Fig. 3C-G). In vivo, TRIM39 knockdown led to a reduced tumor growth rate and decreased terminal tumor weight (Fig. 3H-J). Immunohistochemical analysis showed that the expression of proliferation marker Ki67 was decreased, while the apoptosis related marker Caspase-3 was upregulated in TRIM39 knockdown groups (Fig. 3K-M). Together with the results in Fig. 2, we conclude that TRIM39 acts as an oncogene in HCC progression.

Fig. 3.

TRIM39 knockdown suppresses HCC progression. (A) qPCR analysis of TRIM39 expression in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. (B) Western blot analysis of TRIM39 expression in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. (C-D) The colony formation assay of TRIM39 stably knockdown MHCC97 and HCCLM3 cells. **, P < 0.01, ***, P < 0.001. (E) The CCK8 assay of TRIM39 stably knockdown MHCC97 and HCCLM3 cells. **, P < 0.01, ***, P < 0.001. (F-G) The EdU assay of TRIM39 stably knockdown MHCC97 and HCCLM3 cells. The average number of cells per field were calculated. n = 3 samples per group, four fields per sample. **, P < 0.01; ***, P < 0.001. (H-J) TRIM39 stably knockdown or shControl (shNC) MHCC97 cells were subcutaneously injected into nude mice. Representative tumor sizes (H), tumor growth curves (I), and tumor weight (J) are shown. n = 5 per group; Scale bar, 50 mm. **, P < 0.01; ***, P < 0.001. (K-M) The representative immunohistochemical staining and quantification of Ki67 and Caspase-3 in xenograft tumor from TRIM39 stably knockdown or control groups. Scale bar, 100 μm. *, P < 0.05; **, P < 0.01.

TRIM39 interacts with p62 and decreases its protein stabilization

To elucidate the molecular mechanism underlying the oncogenic effect of TRIM39 in HCC, we conducted an immunoprecipitation-coupled mass spectrometry screen in HCC cells and identified p62 as a potential TRIM39-interacting protein (Fig. 4A). The interaction between TRIM39 and p62 was confirmed by reciprocal co-immunoprecipitation (Co-IP) experiments (Fig. 4B). Immunofluorescent staining also showed that TRIM39 co-localized with p62 in HCC cells (Fig. 4C-D). To identify the interaction domains of TRIM39 and p62, a series of truncation were generated based on their putative functional domains (Fig. S2A). Co-IP experiments revealed that TRIM39_1 − 70 and the p62_{340 − 440} was essential for their interaction (Fig. S2B-C). Next, we determined whether the expression of TRIM39 or p62 was affected by their interactions. TRIM39 negatively regulated the protein levels of p62, while its mRNA level remained unchanged, indicating that TRIM39 regulates the stability of p62 protein through post-translational mechanisms (Fig. 4E-I). The cycloheximide chase assays showed that TRIM39 accelerated p62 protein degradation, which could be reversed by the proteasome inhibitor MG132 (Fig. 4J-L). Given that TRIM39 is an E3 ubiquitin ligase, we assessed the impact of TRIM39 on the ubiquitination status of p62 and found that TRIM39 overexpression significantly enhanced p62 ubiquitination, whereas TRIM39 deficiency had the opposite effect (Fig. 4M-N). In vitro ubiquitination assay confirmed that TRIM39 directly ubiquitinated p62 (Fig. 4O). K48 and K63 are common ubiquitin chain-types. Using ubiquitin mutants in which the indicated lysine is retained and all other lysines are replaced with arginine, we found that TRIM39 promoted K48-linked ubiquitination of p62 (Fig. 4P). To identify the specific lysine (K) residues to which ubiquitin was covalently attached, we mutated the conserved lysine residues (K420, K435) in p62_{340 − 440} to arginine (R) to generate the corresponding p62 mutants. The degradation of p62 induced by TRIM39 overexpression was completely blocked when we mutated 420 K to R, indicating that K420 was responsible for TRIM39-mediated p62 ubiquitination (Fig. 4Q). These results indicate that TRIM39 interacts with p62 and destabilizes it by promoting its ubiquitination.

Fig. 4.

p62 is a binding partner of TRIM39. (A) Cell lysates from HepG2 cells transfected with empty vector or Flag-TRIM39 were immunoprecipitated with anti-Flag antibody. p62 was identified via mass spectrometry. (B) HepG2 cells were co-transfected with Flag-TRIM39 and Myc-p62 for 48 h. Total cell lysates were immunoprecipitated with anti-Flag or anti-Myc antibodies. Myc-p62 and Flag-TRIM39 were detected by western blot. (C-D) Immunofluorescence assay of Flag-TRIM39 and Myc-p62 in HepG2 cells. Representative confocal microscopy images were shown. Scale bars, 5 μm. The colocalization of TRIM39 and p62 in HepG2 cells was analyzed. (E) The analysis of p62 mRNA levels in TRIM39 stably overexpression or knockdown HCC cells. (F-G) The analysis of p62 protein levels in TRIM39 stably overexpression HepG2 and Huh7 cells. *, P < 0.05. (H-I) The analysis of p62 protein levels in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (J) HepG2 cells transfected with Flag-TRIM39 were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. (K) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. (L) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) and MG132 (10µM) for indicated time and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. (M) TRIM39 stably overexpression and control cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. (N) TRIM39 stably knockdown and shNC cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. (O) TRIM39 facilitated p62 Ub conjugates by in vitro ubiquitination assay. (P) TRIM39 stably overexpression cells were co-transfected with indicated HA-Ub (K-R) mutants for 48 h. Total lysates were immunoprecipitated with anti-p62 antibody. The immunoprecipitation complex was analyzed by western blot with anti-HA and anti-p62 antibodies. (Q) TRIM39 stably overexpression cells were transfected with Flag-p62 and its mutants (K420R, K435R). The expression of Flag-p62 was analyzed by western blot.

TRIM39 facilitates the proliferation of HCC cells dependent on p62

To determine whether p62 is involved in TRIM39-mediated HCC progression, the functional rescue experiments were performed in TRIM39 stably overexpression HCC cells. Overexpression of p62 significantly abrogated the enhanced cell proliferation abilities induced by TRIM39 in vitro (Fig. 5A-H). Furthermore, p62 overexpression reversed the increased tumor volumes and weights observed in the TRIM39-overexpressing subcutaneous tumor model (Fig. 5I-K). The upregulation of Ki67 and downregulation of Caspase-3 in TRIM39 overexpression group were also abolished upon p62 overexpression (Fig. 5L-M). These data demonstrate that TRIM39 promotes HCC progression in a p62 dependent manner.

Fig. 5.

TRIM39 facilitates the proliferation of HCC cells in a p62-dependent manner. (A-B) The colony formation assay of TRIM39 stably overexpression HepG2 and Huh7 cells with or without p62 overexpression. ***, P < 0.001. (C-D) The CCK8 assay of TRIM39 stably overexpression HepG2 and Huh7 cells with or without p62. **, P < 0.01. (E-H) The EdU proliferation assay of TRIM39 stably overexpression HepG2 and Huh7 cells with or without p62 overexpression. The average number of cells per field were calculated. n = 3 samples per group, four fields per sample. **, P < 0.01; ***, P < 0.001. (I-K) TRIM39 stably overexpression HepG2 cells with or without p62 overexpression and control cells were subcutaneously injected into nude mice. Representative tumor sizes (I), tumor growth curves (J), and tumor weight (K) are shown. n = 5 per group; Scale bar, 50 mm. ***, P < 0.001. (L-M) The representative immunohistochemical staining and quantification of Ki67 and Caspase-3 in xenograft tumor from control group or TRIM39 stably overexpression groups with or without p62 overexpression. Scale bar, 100 μm. *, P < 0.05, **, P < 0.01.

TRIM39 inhibits the Keap1/Nrf2/HO-1 pathway

NRF2 is a transcription factor that activates genes involved in cellular defense against oxidative stress, such as HMOX-1 (HO-1), NQO1 and G6PD. p62 can recruit and sequester Keap1, an adaptor of the cullin-3 E3 ubiquitin ligase complex, which targets NRF2 for proteasomal degradation. We examined the effects of TRIM39 on the regulation of Keap-NRF2 axis by western blot and immunofluorescence. TRIM39 overexpression significantly reduced the levels of Keap1, while increasing the levels of NRF2 and its target gene HMOX-1 (HO-1) (Fig. 6A-B and E-J). In contrast, TRIM39 knockdown resulted in elevated Keap1 levels and decreased NRF2 and HO-1 levels (Fig. 6C-D and K-P). More importantly, we conducted colony formation, CCK8, and EdU assays and found that overexpression of p62 substantially abrogated the enhanced proliferation abilities observed in TRIM39-overexpressing CRC cells (Fig. S1A-D). Together, these findings suggest that TRIM39 negatively regulates the Keap1/Nrf2/HO-1 pathway.

Fig. 6.

TRIM39 inhibits the Nrf2/HO-1 signaling pathway. (A-B) Western blot analysis of KEAP1, NRF2 and HO-1 in TRIM39 stably overexpression HepG2 and Huh7 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C-D) Western blot analysis of KEAP1, NRF2 and HO-1 in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (E-F) Representative images and quantification of KEAP1 staining in HepG2 cells with TRIM39 stably overexpression. *, P < 0.05. (G-H) Representative images and quantification of NRF2 staining in HepG2 cells with TRIM39 stably overexpression. **, P < 0.01. (O-P) Representative images and quantification of HO-1 staining in HepG2 cells with TRIM39 stably overexpression. *, P < 0.05. (K-L) Representative images and quantification of KEAP1 staining in MHCC97 cells with TRIM39 stably knockdown. **, P < 0.01. (M-N) Representative images and quantification of NRF2 staining in MHCC97 cells with TRIM39 stably knockdown. *, P < 0.05; **, P < 0.01. (I-J) Representative images and quantification of HO-1 staining in MHCC97 cells with TRIM39 stably knockdown. **, P < 0.01; ****, P < 0.0001.

TRIM39 inhibits the activation of the KEAP1/NRF2/HO-1 pathways via p62

To investigate whether TRIM39 mediated inhibition of Keap1/Nrf2/HO-1 is p62 dependent, we overexpressed of p62 in TRIM39 stably overexpression cells. Western blot and immunofluorescence results showed that p62 overexpression significantly reversed the downregulation of NRF2 and HO-1 induced by TRIM39 (Fig. 7A-H). We further explored the clinical relevance between TRIM39 and p62 in CRC. The expression of TRIM39 was negatively correlated with p62 in the CRC specimens (Fig. 7I-J). These findings confirmed that TRIM39 negatively regulates Nrf2 signaling by binding to p62.

Fig. 7.

TRIM39 inhibits the activation of the Keap1/Nrf2/HO-1 pathways depending on p62. (A-B) Western blot analysis of Keap1/Nrf2/HO-1 in TRIM39 stably overexpression HepG2 cells with or without p62 overexpression. *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001. (C-D) Quantification of mean immunofluorescence density of KEAP1 staining in TRIM39 stably overexpression HepG cells with or without p62 overexpression. **, P < 0.01, ***, P < 0.001. (E-F) Quantification of mean immunofluorescence density of NRF2 staining in TRIM39 stably overexpression HepG cells with or without p62 overexpression. **, P < 0.01, ***, P < 0.001. (G-H) Quantification of mean immunofluorescence density of HO-1 staining in TRIM39 stably overexpression HepG cells with or without p62 overexpression. *, P < 0.05, ***, P < 0.001. (I) Representative immunohistochemistry staining of TRIM39 and p62 in CRC tissue from the CRC cohort collected by us. (J) The pearson correlation between TM9SF1 and Vimentin expression in (I).

Discussion

Unlike other well-studied members of TRIM family in many types of cancer, the functional roles and mechanism of TRIM39 in carcinogenesis remain largely unclear. Research on TRIM39 in tumors has primarily focused on its regulation of p53. TRIM39 loss enhanced etoposide-induced apoptosis in p53-positive cells by stabilizing p53²³. It directly binding and ubiquitylating p53, leading to p53 degradation. Another study reported that TRIM39 knockdown inhibited CRC progression through promoting autophagic degradation of p53⁸. To date, the cellular functions of TRIM39 and the underlying mechanisms remain unexplored in HCC. In this study, we showed that the TRIM39 was upregulated in HCC and associated with poor survival of HCC patients. TRIM39 was able to promote the proliferation of HCC cells in vitro and in vivo. Therefore, TRIM39 acts as an oncogene in HCC progression. Mechanistically, we identified that the p62-Keap1-NRF2 axis was responsible for the oncogenic roles of TRIM39 in HCC progression.

The well-known function of p62 is to deliver polyubiquitinated proteins and organelles for autophagosome-lysosomal degradation²⁴. p62 also serves as a signaling scaffold to participate in the regulation of multiple biological processes, including metabolism, inflammation, and programmed cell death^25–27. p62 has a close relationship with HCC and many studies have explored its role in the development of HCC. p62-positive cytoplasmic inclusion bodies are commonly observed in the livers of HCC patients²⁸. Liver-specific deletion of Atg7 or Atg5 results in the accumulation of p62 and the spontaneous development of liver adenoma²⁹. Several studies showed that p62 was highly expressed in HCC samples and prompted tumor progression. However, a study showed that p62 acted as a tumor suppressor in mouse models with persistent mTOR activation and defective autophagy¹⁶. p62 knockdown partially abolished the growth suppression induced TRAF2 depletion¹⁷. These studies suggest that whether p62 promotes or inhibits HCC development is context-dependent. In our study, we found that TRIM39 interacted with p62 and destabilized it by promoting its ubiquitination. Overexpression of p62 significantly abrogated the enhanced cell proliferation abilities induced by TRIM39 in vitro and in vivo. Thus, p62 may function as a tumor suppressor in HCC with high TRIM39 expression.

p62 can interacted with Keap1, an adaptor of the cullin-3 E3-ubiquitin ligase complex for NRF2, thereby inhibiting the proteasomal degradation of NRF2³⁰. This leads to NRF2 translocating to the nucleus, where it activates antioxidant target genes. We identified that TRIM39 inhibits the activation of the KEAP1/NRF2/HO-1 pathways via p62 in HCC cells. Similar to p62, NRF2 plays a paradoxical role in cancer. On one hand, NRF2 is essential to prevent tumorigenesis by maintaining redox homeostasis through activating antioxidant target genes. On the other hand, several studies have demonstrated that NRF2 activation accelerates tumor progression. For example, NRF2 mediates metabolic reprogramming in cancer cells by increasing glucose uptake and activating the pentose phosphate pathway³¹. NRF2 also targets the genes that control proliferation and angiogenesis to promote tumor progression^32,33. Future experiments should be performed to examine the role and mechanisms of NRF2 in TRIM39 induced tumorigenesis in HCC.

In conclusion, we demonstrate for the first time that TRIM39 promotes tumor proliferation by targeting the p62-KEAP1-NRF2 axis pathway. TRIM39 interacts with p62 and decreases its protein stabilization. TRIM39 promotes the proliferation of HCC cells and inhibits the activation of the KEAP1/NRF2/HO-1 pathways dependent on p62. Our findings underscore the functional roles of TRIM39 in HCC, contribute to the understanding of p62-KEAP1/NRF2/HO-1 pathways regulation in HCC progression. The work may provide new therapeutic targets for HCC therapy.

Author contribution

Junjun Sun: review & editing. Tianbao Yang: original draft, Validation, Methodology, Data curation. Zhigang Chen: Writing – review & editing, Project administration, Data curation, Conceptualization. Fang Yi & Cheng Yang: Visualization. Yanhui Yang: Investigation. Zhijie Chu & Longhui Ruan: Data curation. Weifeng Liu: Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Junjun Sun: review & editing. Tianbao Yang: original draft, Validation, Methodology, Data curation. Zhigang Chen: Writing – review & editing, Project administration, Data curation, Conceptualization. Fang Yi & Cheng Yang: Visualization. Yanhui Yang: Investigation. Zhijie Chu & Longhui Ruan: Data curation. Weifeng Liu: Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition.

Funding

This study was supported by the Henan Medical Science and Technology Research and Development Program Project (No. LHGJ20220688).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The study was conducted in accordance with the Declaration of the ARRIVE guidelines (https://arriveguidelines.org), and approved by the clinical research ethics committee of the first affiliated hospital of Henan University of Science and Technology (2024-03-K0107).

Statement

All methods in the methods section were performed in accordance with the relevant guidelines and regulations.