VCP interaction with HMGB1 promotes hepatocellular carcinoma progression by activating the PI3K/AKT/mTOR pathway

The RNA sequencing data of the HCC cohort, including 371 HCC and 50 adjacent non-tumorous samples, was downloaded from The Cancer Genome Atlas Program (TCGA) database (https://​portal.​gdc.​cancer.​gov/​). The HCC datasets, including GSE121248 (70 HCC and 37 normal samples), GSE136247 (39 HCC and 30 normal samples), GSE41804 (20 HCC and 20 normal samples), and GSE14520 (247 HCC and 241 normal samples) were screened from the Gene Expression Omnibus (GEO) database (https://​www.​ncbi.​nlm.​nih.​gov/​geo/​). Concerning the HCC cohort from TCGA database, the GEPIA tool (http://​gepia.​cancer-pku.​cn/​) was operated to analyze the correlation of VCP expression and survival probability following the criteria of the best cut-off algorithm based on VCP transcriptional level in patients. Meanwhile, the UALCAN tool (http://​ualcan.​path.​uab.​edu/​) was performed to obtain all candidate genes co-expressed with VCP according to Pearson’s correlation coefficient.

Gene set enrichment analysis (GSEA) is a computational algorithm utilized to assess whether a previously defined gene set indicates a statistical significance and concurrent difference between two biological phenotypes [23]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets were retrieved from the Molecular Signatures Database (MSigDB, version 7.5.1). HCC patients from TCGA database were divided into the high and low expressed groups according to the median transcriptional value of VCP and HMGB1, respectively. The normalized enrichment score (NES) and false discovery rate (FDR) were determined by GSEA software (version 4.1.0). FDR value < 0.05 was thought of as statistical significance.

Gene ontology (GO) analysis, including biological process (BP), cell components (CC), and molecular function (MF), and pathway enrichment including KEGG and Reactome panels, were performed using the Metascape online tool (https://​metascape.​org/​). The protein–protein interaction (PPI) network was constructed in the STRING database (https://​string-db.​org/​) and visualized via Cytoscape software (version 3.8.2). The hub genes were identified using the Cytohubba plug-in.

Human HCC cell lines of Huh7 and MHCC-LM3 cells, as well as 293T cells, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The immortalized hepatocyte cell line L02 was obtained from the China Center for Type Culture Collection (CCTCC, China). Huh7, MHCC-LM3, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), and L02 cells were maintained in RPMI medium (Gibco). All were supplemented with 10% fetal bovine serum (FBS, Gibco, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml), and cultured in a humidified incubator with 5% CO₂ at 37 °C.

The stable cell line was constructed with lentivirus transduction using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Huh7 cells were transfected by PLKO.1-puro-VCPshRNA (VCP-sh1 and VCP-sh2) or PLKO.1-puro-nonspecific shRNA (as control), MHCC-LM3 cells were treated with PLV-EGFP-puro-Myc-VCP or PLV-EGFP-puro plasmid (as control). Then, HCC cells with successful lentivirus transfection were screened by puromycin for 10 to 14 days. Small interfering RNA (siRNA) and various plasmids were transfected into cells using Lipofectamine 2000. HMGB1-siRNA target sequences (5′ to 3′) #1: CCCGTTATGAAAGAGAAATTT. #2: GGAGGAAGATGAAGAAGATTT. VCP-siRNA target sequences (5′ to 3′) #1: GAATAGAGTTGTTCGGAAT. #2: GGCCAAAGCCATTGCTAAT.

Whole-cell lysates were collected using the protein lysis buffer containing proteinase and phosphatase inhibitors. The BCA assay was operated to measure the protein concentration. The protein was separated by 12% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gel was then transferred to polyvinylidene difluoride (PVDF) membranes (0.25 μm, Millipore, Billerica, MA, USA), and blocked with 5% fat-free dry milk resolved in TBST buffer. The membranes were then washed three to five times (5 min/time) and incubated with the indicated primary antibodies including VCP (2648, CST, USA), HMGB1 (ab18256, Abcam, USA), E-cadherin (20874-1-AP, Proteintech, China), β-catenin (51067-2-AP, Proteintech, China), Snail (sc-271977, Santa Cruz, USA), Twist2 (66544-1-Ig, Proteintech, China), PI3K (4249, CST, USA), AKT (10176-2-AP, Proteintech, China), pAKT (66005-1-Ig, CST, USA), mTOR (20657-1-AP, Proteintech, China), pmTOR (5536, CST, USA), ub (sc-8017, Santa Cruz, USA), GAPDH (60004-1-Ig, Proteintech, China), Myc-tag (60003-2-Ig, Proteintech, China), His-tag (12698, CST, USA), and Flag-tag (66008-3-Ig, Proteintech, China). The bound antibodies were examined using enhanced chemiluminescent reagents (34577, Thermo Fisher, USA) after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (FSM0075 and FSM0056, Fushen, China). The relative protein quantification was analyzed using ImageJ software.

Total RNA was extracted using TRIzol reagent (15596018, Invitrogen, USA) following the manufacturer’s instructions. Reverse transcription (PrimeScript RT reagent Kit, RR047A, Takara, Japan) and SYBR (1725124, Bio-Rad, USA) green-based real-time PCR were performed. GAPDH expression was regarded as endogenous control, and the value of 2^−ΔΔCT was adopted to determine the relative gene transcriptional expression. The qPCR primers included VCP forward: 5′-CTGGAGCCGATTCAAAAGGTG-3′, reverse: 5′-ACACTGTGTCACCTCGGAAC-3′. HMGB1 forward: 5′-GCGAAGAAACTGGGAGAGATGTG-3′, and reverse: 5′-GCATCAGGCTTTCCTTTAGCTCG-3′.

Forty-four HCC samples and adjacent non-tumorous tissues from HCC patients who underwent surgical resection were collected at Xiangya Hospital, Central South University (CSU), Changsha, China, between March 2017 and March 2020. Written informed consent was obtained from all patients. This project was approved by the Medical Ethics Committee of the Xiangya Hospital, CSU. Immunohistochemistry was performed as described previously [24]. The VCP expressed level was evaluated using Image J software, and semi-quantitatively scored as highly positive (4), moderately positive (3), low positive (2), and negative/undetectable (1) staining.

The LC-ESL-LTQ-Orbitrap-MS method was used to identify VCP-interacting proteins as described in a previous study [25]. Briefly, the protein bands were cut from the gel and transferred to 100 mM NH₄HCO₃ with 50% acetonitrile for excising and destaining. Subsequently, the proteins were reduced, alkylated, and dried in a vacuum centrifuge. The gel pieces harboring proteins were incubated in digestion solution at 37 °C for 18–24 h. The tryptic peptide mixture was purified with a ZipTipC18 microcolumn (ZTC18S096, Millipore, Germany) and subjected to separation on a Pep Map C18 trap column (75 μm, 15 cm) with column flow rates of 200 nL/min. MS/MS analysis of the seven strongest ions in the LTQ was conducted using MS and MS spectra. Next, Xcalibur and Proteome Discoverer software were used to analyze the MS data.

Cells were cultured in twelve-well plates with coverage of glass coverslips up to 30–40% confluency. The cells were washed and fixed with 4% paraformaldehyde before blocking with 5% BSA containing 0.1% Triton-100. Then, the cells were incubated with mouse HMGB1(SAB1403925, Sigma, USA) and rabbit VCP (2648, CST, USA) primary antibodies at 4 °C overnight. Next, the cells were stained with Alexa Fluor 488-conjugated anti-mouse IgG (A-21202, Thermo Fisher, USA) or Alexa Fluor 594-conjugated anti-rabbit IgG (A-11012, Thermo Fisher, USA) for 1 h at room temperature. After intermediate washes, fluorescent signals were detected under a confocal microscope (Olympus, Tokyo, Japan).

GST Sefinose Resin (C600031, Sangon Biotech, China) was used to purify the GST-tagged VCP protein complex. The purified proteins were examined using Coomassie blue staining. The lysate from MHCC-LM3 cells was incubated with GST-tagged proteins at 4 °C overnight with gentle rotation. The precipitated pellets were washed to elute resins and bound proteins were analyzed by SDS-PAGE.

For Cell Counting Kit-8 (C0038, Beyotime, China), cells were seeded at a density of 1000 cells/well into 96-well plates and cultured for 24 h, 10 μL CCK8 was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using a PerkinElmer spectrophotometer. For the EdU Cell Proliferation Kit (C0078S, Beyotime, China), appropriate cells were seeded into 24-well plates and incubated for 24–36 h. EdU (20 μM) was added to each well and incubated for 2 h. Then, the cells were fixed with 4% paraformaldehyde and incubated with phosphate-buffered saline (PBS) containing 0.3% Triton X-100. Next, the cells were incubated with 0.5 mL Click Additive solution for 30 min followed by the nuclei stained with DAPI (1:1000) for 10 min in the dark. The fluorescence was examined under a fluorescence microscope.

For the wound-healing assay, 1 × 10 ⁶ cells were seeded into six-well plates. When the cells reached 90% confluence, the cell layer was scratched with a 10 μL sterile plastic tip and cultured for 72 h in serum-free medium. Images were taken under a microscope at the indicated time point to evaluate the healing rate of gap closure.

For the transwell system, 5 × 10⁴ cells were plated in the upper compartment of a transwell chamber (8 μm size, 3422, Corning, USA) in a serum-free medium. The upper chamber was coated with 10% Matrigel (356234, Corning, USA) when for invasion assay. The lower chamber was filled with fresh medium containing 10% FBS. After incubation of 24–48 h, the cells on the lower membrane were fixed with 4% paraformaldehyde and stained with crystal violet (C0121, Beyotime, China). Matrigel-invading or migratory cells were counted under a microscope.

Male BALB/c nude mice aged 4–5 weeks were purchased from HFK Bioscience (Beijing, China) and randomized into various groups, with 6–7 mice in each group. One million MHCC-LM3 cells with stable overexpressing VCP or 2.5 million Huh7 cells with stable silencing VCP (suspended in 0.1 mL PBS) were implanted subcutaneously under the right armpit of nude mice. Body weight and tumor size were measured every three days. The mice were sacrificed three weeks later or when they are moribund. Tumor volume (mm³) was calculated using the longest diameter × (shortest diameter)² × 0.5. This experiment was performed in compliance with the requirements of the Department of Laboratory Animals, Xiangya Hospital, CSU, China.

For quantitative variables, a student’s t-test with one-tailed was used to compare the difference between two groups. When more than two groups are included in the experiments, the one-way ANOVA with a Brown-Forsythe test was accepted for multiple comparisons. The chi-square test was conducted for the qualitative variables. Kaplan–Meier method with a Log-rank test was operated to compare the survival distributions. All analyses were performed in GraphPad Prism software (version 8.0) and the results are presented as the mean ± SD or mean ± SEM. Differences were considered statistical significance at a P value < 0.05. Experimental data were obtained from three independent experiments unless otherwise presented. All *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns: no significance.

The expression of VCP was first evaluated using public cancer databases. Data analysis using TCGA database showed that VCP is elevated in several tumor types, such as breast cancer, colorectal cancer, and HCC (Additional file 1: Fig. S1A). Data analysis using the GEO database showed that VCP expression was significantly increased in tumor samples compared to the corresponding normal tissue samples (Fig. 1A–D). Further analysis of the difference in VCP expression between tumor and adjacent normal tissues was conducted in the HCC cohort from TCGA, and consistent results were obtained (Fig. 1E, F; Additional file 1: Fig. S1B). In addition, a poorer prognosis, including shorter overall survival (OS) and disease-free survival (DFS), was observed in HCC patients with a higher VCP expression versus those with lower expression levels (Fig. 1G, H). The correlation between VCP expression and clinicopathological factors in HCC patients was further analyzed in GSE14520 dataset. The results indicated that the up-regulated VCP expression was significantly associated with the advanced tumor-node-metastasis (TNM) stage (P-value = 0.03) (Additional file 2: Table S1).

Furthermore, 44 pairs of HCC and adjacent normal liver samples were collected from Xiangya Hospital. The results of IHC staining indicated the markedly increased VCP expression in tumor tissues compared to the adjacent normal tissues (Fig. 1I). Consistently, the VCP protein expression level in HCC cells was higher than that in immortalized normal liver cells (Fig. 1J). Additionally, we used subcutaneous xenograft mice models to determine whether VCP facilitates HCC growth in vivo. The results revealed that tumor growth rate was significantly accelerated in the group overexpressing VCP. Reversely, the growth ability was inhibited in the group of VCP depletion (Fig. 1K–P). Overall, these data suggest that the overexpression of VCP may serve as a poor prognostic factor in HCC and promotes tumor progression.

We next examined the biological functions of VCP in HCC. Specific siRNAs targeting VCP were transfected into Huh7 cells and MHCC-LM3 cells were treated with exogenous VCP (Fig. 2A). The CCK8 assay targeting the cytoplasm and the EdU assay targeting the nucleus were conducted to detect cell proliferative ability. The results found that knockdown of VCP markedly inhibited the growth ability of HCC cells, as indicated by both assays (Fig. 2B–E). Flow cytometry was performed to identify the potential mechanism by which VCP promotes cell proliferation in HCC. The results indicated that the inhibition of VCP induced an obvious increase in apoptotic levels in Huh7 cells. Conversely, overexpression of VCP significantly decreased the fraction of apoptotic cells in MHCC-LM3 cells (Fig. 2F, G).

The correlation analysis of clinicopathologic traits and VCP expression showed that the elevated expression of VCP is associated with poor prognosis in HCC patients. The influence of VCP on the migratory and invasive abilities of HCC cells was further assessed. The wound-healing assay revealed that Huh7 cells transfected with VCP siRNA exhibited lower migratory ability than cells transfected with non-specific siRNA. A similar result was observed in MHCC-LM3 cells in which overexpressing VCP accelerated the migratory speed (Fig. 3A, B). Consistently, the transwell assay also revealed that VCP depletion decreased the ratio of migration and invasion in Huh7 cells, whereas overexpressing VCP enhanced this process in MHCC-LM3 cells (Fig. 3C, D). These data indicated that VCP may correlate with tumor metastasis. The markers related to epithelial-mesenchymal transition (EMT) were further examined by western blot. It was noticed that VCP overexpression promoted the expressed level of β-catenin, Snail1, and Twist2, but the phenomenon was attenuated by VCP knockdown in HCC cells. In contrast, the expression level of E-cadherin was downregulated when overexpressing VCP (Fig. 3E–G).

To identify possible tumor-related pathways involved in the process of VCP facilitating the migration and invasion in HCC cells, GSEA analysis was performed in the HCC cohort from TCGA based on the transcriptional expression of VCP. The median value was regarded as the cut-off to classify patients into high- and low-VCP expression groups. The results indicated the up-regulation of mTOR signaling pathway in the group with high VCP expression, which is known to associate with tumor progression (Fig. 3H) [2, 26]. Several critical biomarkers within the mTOR pathway were determined by western blot in HCC cells with silencing or exogenously overexpressing VCP. The results demonstrated that the expression of PI3K was upregulated by increasing VCP expression, and the phosphorylated levels of AKT and mTOR were also elevated. These effects were attenuated when VCP expression was inhibited (Fig. 3I–K).

It was reported that VCP belongs to the AAA-ATPase family, which contains two critical ATPase domains, namely the D1 and D2 areas, empowering its ability to bind to a variety of substrates. Sequentially, it exhibits diverse functions in multiple biological processes, such as autophagy and proteasome-dependent protein degradation [8]. In the current study, aiming to clarify the potential regulatory mechanism of VCP promoting HCC cell migration and invasion, the CoIP/MS technology was adopted to identify VCP-interacting proteins in MHCC-LM3 cells, and a total of 330 proteins were identified (Additional file 3: Table S2). The overall profile of the biological functions was analyzed for the candidate proteins via GO and pathway enrichment. The results indicated that VCP is involved in several biological processes, such as protein binding and various diseases, including HCC (Additional file 4: Figure S2).

Meanwhile, a total of 6768 potential VCP co-expressed genes were retrieved in the HCC cohort from TCGA database according to Pearson's correlation coefficient. Finally, 79 VCP-interacting proteins were obtained after overlapping the protein candidates identified in CoIP/MS and TCGA (Fig. 4A). GO analysis showed that the proteins exhibited significant roles in diverse BPs, CCs, and MFs, including protein complex, cell-to-cell adhesion, ATP binding, and ubiquitin-specific protease binding (Fig. 4B–D; Additional file 5:Table S3). The KEGG and Reactome pathway enrichment indicated that diversified signal panels were enriched, such as protein processing in the endoplasmic reticulum and protein ubiquitination (Fig. 4E, F). The PPI network of VCP and its interacting proteins was constructed in the STRING database, and the hub genes that have the highest possibility of binding with VCP were further identified (Fig. 4G). HMGB1 was part of the protein complexes after VCP immunoprecipitation in MHCC-LM3 cells as well as in the hub gene network (Figs. 4G, 5C). Then, HMGB1 was selected for further experimental investigation. The MS/MS spectrum of HMGB1 is shown, and the identified amino acid sequence that interacted with VCP is labeled in red color (Fig. 5A, B).

We next sought to validate the interaction between VCP and HMGB1 in HCC cells. First, the GST-tagged VCP fusion protein was successfully purified using GST agarose beads (Fig. 5D, E). The GST pull-down experiment was conducted by incubating GST-tagged VCP with whole lysates from MHCC-LM3 cells. HMGB1 was detected in the GST-tagged VCP pulled down samples but not in the GST-tagged control group (Fig. 5F). Consistently, the CoIP assay demonstrated that endogenous VCP and HMGB1 could be precipitated with each other in HCC cells as well (Fig. 5G, H). Moreover, the confocal assay was performed to show the co-localization of VCP and HMGB1 in HCC cells in vivo (Fig. 5I, J).

Next, we aimed to elucidate the crucial protein domains that are responsible for the interaction between VCP and HMGB1. Based on their protein structures [5, 8], truncated mutation plasmids of VCP (wild type (WT), ΔN, ΔD1, ΔD2, and ΔC tail) and HMGB1 (WT, ΔA box, ΔB box, and ΔC tail) were constructed as indicated (Fig. 6A, C). Then, the series of VCP or HMGB1 mutant plasmids were transfected into 293T cells and CoIP was performed separately. The result revealed that the deletion of the D1 domain in VCP and A box in HMGB1 significantly disrupted the interaction (Fig. 6B, D). The influence of the D1 domain on VCP in promoting HCC progression was also investigated. To this end, exogenous VCP-ΔD1 was transfected into MHCC-LM3 cells, and the transfection efficacy was determined (Fig. 6E). The CCK8 assay showed that the cell proliferative ability was not increased in cells treated only with VCP-ΔD1, but it was enhanced significantly within the existing full-length VCP (Fig. 6F). Similarly, the D1 mutant did not promote cell migration and invasion (Fig. 6G, H). These data support that the D1 region plays a critical role in VCP exhibiting pro-HCC activities.

At first, the HMGB1 expression in HCC cells and immortalized hepatocytes was examined by western blot, showing the elevated expression of HMGB1 in tumor cells (Additional file 6: Fig. S3). Moreover, the results found that the increased VCP expression significantly up-regulated the protein level of HMGB1 instead of the transcriptional expression (Additional file 7: Fig. S4). Then, the impact of VCP on the half-life of HMGB1 was investigated. Under normal circumstances, HMGB1 is rapidly degraded within 24 h. In contrast, the half-life of HMGB1 protein was dramatically prolonged in cells with VCP overexpression, and this phenomenon was reversed in cells with VCP knockdown (Fig. 7A–C).

Next, GSEA in the HCC cohort from TCGA database showed that the process of proteasome and ubiquitin-mediated proteolysis was enriched in the group with high VCP expression. Simultaneously, the up-regulated signal of ubiquitin-mediated proteolysis was also detected in the group with elevated HMGB1 expression (Fig. 7D–F). The overall ubiquitination level of HCC cells was examined after incubation with MG132 (an inhibitor of proteasome) or other indicated treatment. The results demonstrated a distinct increase in cellular ubiquitylated proteins in cells with VCP inhibition. The corresponding decreased ubiquitylated level in cells overexpressing VCP was observed (Fig. 7G). Concurrently, the ubiquitylated level of HMGB1 in HCC cells displayed the same tendency following different VCP expression levels (Fig. 7H). Furthermore, we detected the overall expression of HMGB1 in HCC cells after treating with MG132 (20 μM, 24 h), it was noticed that the protein expressed level of HMGB1 was enhanced in cells overexpressing VCP, and largely reduced when silencing VCP expression (Fig. 7I, J). These findings show that HMGB1 is a substrate of VCP-dependent proteasomal degradation.

To elucidate the role of HMGB1 in oncogenic activities mediated by VCP in HCC progression. Firstly, the efficacy of siRNA targeting HMGB1 was verified in HCC cells, among which HMGB1-si-1 was used for the following experiments, referred to here as HMGB1-siRNA (Additional file 8: Fig. S5). In vitro data showed that the enhanced invasive ability of HCC cells induced by VCP was dramatically counteracted by HMGB1 inhibition. In contrast, when overexpressing HMGB1 in HCC cells with VCP silencing, cell invasion was rescued (Fig. 8A–F). Simultaneously, the strengthened EMT mediated by elevated VCP expression was impeded by HMGB1 knockdown (Additional file 9: Fig. S6). The findings indicate that HMGB1 is critical for the biological function of VCP in promoting HCC. Furthermore, the impact of HMGB1 on VCP in activating the mTOR signaling was evaluated. Functionally, the increased levels of phosphorylated AKT, mTOR, and PI3K by VCP overexpression were significantly attenuated by knockdown of HMGB1. In contrast, the exogenous expression of HMGB1 enhanced the phosphorylated levels of biomarkers within the mTOR pathway in cells with VCP knockdown (Fig. 8G, H). GSEA in the HCC cohort from TCGA database also displayed the enrichment of the mTOR pathway in the high-VCP expression group (Fig. 8I). These results reveal the essential function of HMGB1 for VCP promoting the PI3K/AKT/mTOR pathway activation in HCC.