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. 2026 Apr 15;16:17507. doi: 10.1038/s41598-026-48902-8

ADAM15 promotes the progression and metastasis of hepatocellular carcinoma by activating the JNK/p38 pathway

Xu Junhui 1, Su Mengyun 2,✉
PMCID: PMC13237111  PMID: 41986445

Abstract

ADAM15, as a member of the membrane-bound protease family, participates prominently in the progression and metastasis of various tumours. However, its mechanism of action in hepatocellular carcinoma remains unclear. The functional role of ADAM15 in hepatocellular carcinoma (HCC) was investigated both in vitro and in vivo. ADAM15 knockdown inhibited the proliferation, migration and invasion of HCC cells, whereas ADAM15 overexpression enhanced these malignant behaviors. The results of apoptosis assay showed that inhibition of ADAM15 expression promoted apoptosis of HCC cells, and overexpression of ADAM15 inhibited apoptosis of HCC cells. Western Blot results showed that ADAM15 knockdown inhibited EMT transition and decreased the expression of mesenchymal marker N-cadherin. Additionally, ADAM15 silencing increased the expression of the pro-apoptotic protein Bax while decreasing the anti-apoptotic protein Bcl-2. The results of subcutaneous tumor formation assay in nude mice showed that knockdown of ADAM15 expression significantly inhibited the growth of subcutaneous tumors. The results of tail vein lung metastasis assay showed that ADAM15 knockdown inhibited lung metastasis of hepatocellular carcinoma in nude mice. In the mechanistic study, overexpression of ADAM15 activated the JNK-p38MAPK pathway, thereby promoting EMT and suppressing apoptosis in HCC cells. Conversely, ADAM15 knockdown inhibited the JNK-p38 MAPK pathway, leading to enhanced apoptosis and suppressed EMT. ADAM15 is highly expressed in hepatocellular carcinoma. ADAM15 regulates the apoptosis and EMT of hepatocellular carcinoma cells by activating the JNK-p38 MAPK signaling pathway, thereby promoting the progression and metastasis of hepatocellular carcinoma.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-48902-8.

Keywords: ADAM15, HCC, JNK-p38 MAPK, Tumor progression and metastasis

Subject terms: Cancer, Cell biology, Oncology

Introduction

Liver cancer, predominantly hepatocellular carcinoma (HCC), is a highly malignant tumor1. It currently ranks as the sixth most common cancer globally and the third leading cause of cancer-related mortality worldwide2. Most liver cancer patients present at intermediate or advanced stages. Although diagnostic and therapeutic approaches have advanced significantly, the five-year survival rate remains poor, especially for patients with distant metastasis3. The traditional treatment of liver cancer is being challenged by targeted therapy and immunotherapy4,5. The current focuses on developing more effective and safer drugs targeting the immune checkpoint pathway and angiogenesis related pathways, such as vascular endothelial growth factor receptor (VEGFR), transforming growth factor β (TGF-β) signaling pathway, etc6–8. Exploring epigenetic modification and dysregulation pathways, including MAPK (Mitogen-activated Protein Kinase), PI3K(Phosphatidylinositol 3 kinase)-AKT (Protein kinase B, PKB) and Janus Kinases (JAKs)/signal transducers and activators of transcription (STATs) pathways. Some scholars have also studied the expression patterns of other target genes in liver cancer and their relationship with prognosis. At the same time, they have also explored the potential therapeutic role of microRNAs in liver cancer9,10. These investigations may help to identify safer and more effective drug combinations1,11. However, tumor cell mutations and drug resistance frequently result in poor efficacy of targeted therapy and immunotherapy. Therefore, further exploration of the molecular mechanisms underlying HCC progression and metastasis remains crucial for identifying novel therapeutic targets.

In addition to mediating cell-to-cell interactions, MAPKs also play a key role in cellular responses to various external stimuli, including growth factors, inflammatory factors, cytokines, and environmental changes12,13. The MAPK signaling cascade is one of the oldest evolved signaling pathways characterized by a series of phosphorylation events leading to specific cellular responses following activation14,15. This pathway critically contributes to cancer development, and comprises four distinct cascades, namely extracellular signal-regulated kinase 1/2 (ERK1/2), N-terminal kinase (JNK), p38 MAPK, and BMK1 cascade1. Aberrant MAPK pathway activation primarily results from gain-of-function mutations in RAS and RAF proteins, which lead to constitutive activation of the ERK1/2 pathway16. However, the precise roles of JNK and p38 isoforms in cancer promotion remain unclear, and their mechanisms in HCC require further elucidation.

The ADAM family belongs to the membrane-bound metalloproteinase family and plays a significant role in tumor progression and metastasis17,18. ADAM15, located on chromosome 1q21, uniquely contains an RGD sequence among ADAM family members, which can bind to integrin molecules and alter cell adhesion ability19,20. Studies have shown that abnormal activation and expression of ADAM15 play an important role in other types of tumors, such as in breast cancer cell lines, where ADAM15 upregulates the expression of tight junction protein Claudin-1 through the PI3K/Akt/mTOR pathway, thereby affecting the morphology and function of breast cancer cells21. Our previous work first established that ADAM15 shows elevated expression in HCC and correlates with poor prognosis in HCC22. However, the mechanism by which ADAM15 promotes the progression and metastasis of hepatocellular carcinoma remains unclear. This study has confirmed through in vitro and in vivo experiments that ADAM15 promotes the progression and metastasis of HCC by activating the JNK/p38 MAPK pathway.

Materials and methodology

All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Cell culture and transfection

Human HCC cells HLF, HepG2, HepB3, Huh7 and MHCC97h and human normal liver cells L02 were both obtained from ATCC. We selected HLF, MHCC97h and L02 for further experimental studies. All three cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM). 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (100U/ml penicillin + 0.1 mg/ml streptomycin) were added to the culture medium of all cells, and they were cultured in an incubator containing 5% CO2 and at a temperature of 37 °C. Established siRNA - ADAM15 was purchased from miaolingbio. Lnc, Wuhan, China. HLF and MHCC97h cells were both cultured in 6-well plates (5 × 105/well) with 2.4 ug siRNA using Attractence Transfection Reagent (Cat. No. 301005, QIAGEN, China). ADAM15 overexpression plasmid was purchased from Miaolingbio.Lnc, Wuhan, China. HLF, MHCC97h cells were first cultured into 6-well plates (5 × 105/well), and then the ADAM15 overexpression plasmid was transfected into the corresponding HCC cells with Attractence Transfection Reagent for subsequent experimental detection. The information of the ADAM15 knockdown plasmid (siRNA) and the overexpression plasmid can be found in Supplementary Material 1.

Western blot

Total protein was extracted from HCC cells using RIPA buffer (Beyotime, Guangzhou, China). After extraction was completed, the protein concentration was detected using the BCA kit. Proteins of the same weight were separated by 10–12% SDS-PAGE and then electrotransferred to PVDF membranes. Then, it was sealed with 5% skimmed milk, followed by incubation with different primary antibodies, then incubation with HRP-conjugated secondary antibodies, and finally development with the ECL kit (P0018FS, Beyotime, China). The primary antibodies used in this experiment were as follows: anti-E-cadherin (1:1000, Abcam, Ab40772); anti-N-cadherin (1:1000, Abcam, Ab76011); anti-Snail (1:1000, Abcam, Ab216347); anti-Bax (1:1000, Servicebio, GB11007); anti-Bcl-2 (1:800, Beyotime Biotechnology, AF0060); Anti-p38 (1:500, CST, 9212s); Anti-p-p38 (1:500, CST, 9211s); Anti-JNK (1:500, CST, 9252s); Anti-p-JNK (1:500, CST, 4668s); anti-Ki67 (1:1000, A2094, abclonal); anti-PCNA (1:1000, ab92552, Abcam); anti-Tubulin (1:1000, 10094-1-AP, Proteintech). The secondary antibodies used in this experiment are as follows: HRP-conjugted goat anti-mouse IgG (1:5000, Servicebio, GB23301). JNK inhibitor (SP600125, HY-12041, MCE, America); JNK agonist (Anisomycin, HY-18982, MCE, America). The densities of the proteins were quantified with ImageJ software.

Cell proliferation assay

Edu

Liver cancer cells were planted in 6-well plates at a certain density. Edu detection was initiated when the cell condition was good. First, an equal volume of 2× Edu working solution was added to the 6-well plate and incubate for 2 h. Then, the 1× working solution was removed from the 6-well plate, followed by addition of 1 ml of 4% paraformaldehyde to each well for fixation at room temperature for 15 min. Then, the stationary solution was removed and multiple rinses were performed. Next, 1 ml of permeable solution (PBS containing 0.3%Triton X-100) was added to each well and incubated at room temperature for 15 min. After that, the permeable solution was removed and multiple rinses were performed. Then, fluorescence detection was carried out under an inverted microscope following the steps in the Edu kit manual. Triplicate experiments were performed.

CCK8

The corresponding liver cancer cells were seeded in 96-well plates, and the cell density was maintained at 3000–5000 cells per well. Then the cells were placed in a 37 °C incubator and incubated overnight; Prepare the reagents according to the instructions of the CCK kit. After renewing the culture medium in the 96-well plate, 10 µl of CCK8 solution was added to each well, and it was incubated at 37 ° C for 1–4 h. Finally, the OD value was determined at 450 nm using a microplate reader (PerkinElmer), recorded and analyzed. All the experiments were repeated three times.

Wound healing test

The liver cancer cells (MHC97h and HLF) that were growing well in the logarithmic phase were planted in 6-well plates. After the cells adhered firmly, the corresponding plasmids or drugs were added for treatment. After about 48 h, when the cells have grown and fused to 80–90% of the area of the 6-well plate, the scratching operation was initiated. The tip of a 10–200 µl pipette tip was used to scrape and mark perpendicularly to the bottom of the 6-well plate to remove the medium. The plate was rinsed two to three times with sterile PBS, add 1 ml of fresh serum-free medium to each well, and then place the plate in a 37 °C incubator for continued culture. Photos were taken at time points such as 0 h, 24 h, and 48 h respectively, and the above experiment was repeated three times.

Cell invasion test

First, the 24-well plate was place on ice and inserted Transwell chambers into each well. 60 µl of diluted matrigel (BD Company, #356234) was then added to each upper chamber. Then, 600 µl of complete medium containing 10% FBS was added to each well of the 6-well plate, and 100 µl of serum-free medium cell suspension was added to the upper chamber, with a general density of 5 × 105 cells /ml per well. The 6-well plate was then placed in a 37 °C incubator and continue to cultivate for 24 to 48 h. The small chamber was then placed in 4% paraformaldehyde, followed by fixation at room temperature for 30 min. After that, each small chamber was stained in 0.1% crystal violet solution for 30 min; the cells and dyes inside the chamber were gently wipe off with a wet cotton swab. We observed and photographed invaded cells on the lower membrane surface under an inverted microscope, recording multiple fields of view. The above experiment was repeated three times.

Cell flow cytometry

Firstly, the HCC cells were digested with pancreatic enzymes. Then, the cells were neutralized and resuspended in DMEM complete medium containing 10% fetal bovine serum, and the cell concentration was adjusted to 5 × 106−1 × 107 cells/ml. Then the cells were collected in 15 ml EP tubes for centrifugation treatment, and the bottom cells were collected for PBS rinsing and centrifugation. After removing PBS, 195 µl of Annexin V-FITC binding solution (ThermoFisher SCIENTIFIC, No: A13199) was added to mix the cells evenly. Then, 5 µl of Annexin V-FITC reagent was added to the above mixture and gently mixed. Subsequently, 10 µl of propidium iodide staining solution (ThermoFisher SCIENTIFIC, No: A13199) was added to the above mixture and gently mixed. The above-mentioned mixed solution was incubated at room temperature in the dark for 20 min, and then placed on an ice surface for flow cytometry on the machine (FACScan flow cytometer, Becton, Dickinson and Company). The control groups included unstained cells and cells stained with only Annexin V-FITC or PI for comparative analysis. Flow cytometer laser route 488, conjugate FITC. The above experiment was repeated three times.

Subcutaneous tumor formation test in nude mice

The nude mice required for this experiment were purchased from Wuhan Shulaibao Biotechnology Co., LTD. The Ethics Committee of Renmin hospital of Wuhan University approved all procedures (Ethics Approval Number: 20250303 A). Twelve male BALB/nude mice aged 4–5 weeks were selected and randomly divided into two groups, with 6 mice in each group, namely the Lenti-shADAM15 cell group and the Lenti-shvector group. The MHCC97h cells (sh-ADAM15 stably transfected cell line and control group cell line) in the logarithmic growth phase and after treatment were selected. They were digested with trypsin and the cells were counted. The cell concentration was adjusted to 2 × 107 cells/ml. After disinfecting the right scapular region with iodophor, we injected 0.1 ml MHCC97h cell suspension (2 × 107 cells) subcutaneously into each mouse. We monitored tumor growth weekly, measuring tumor dimensions. Four weeks later, the nude mice were sacrificed for cervical dislocation after anesthesia (The anesthesia was administered by inhaling sevoflurane, which was provided by the Animal Laboratory of the First Clinical College of Wuhan University). The subcutaneous tumors were removed, their volume was measured and their weight was weighed. Volume calculation formula: V(cm3) =(a2×b)/2, where a is the short diameter of the tumor nodule and b is the long diameter of the tumor nodule; To remove the tumors of cryopreserved in liquid nitrogen, follow-up for PCR or Western blotting (WB) detection, part of the fixed in 4% paraformaldehyde, paraffin embedding slices, after line immunohistochemical staining.

Tail vein lung metastasis test of liver cancer

Six 4–6 week-old male BALB/c nude mice were acclimatized for one week before random division into two groups, with 3 mice in each group, namely the Lenti-shADAM15 cell group and the Lenti-shvector group. A 100 µl serum-free cell suspension containing 1 × 106 MHCC97h cells was slowly injected through the tail vein insulin needle. Biopsy of small animals was performed 6–8 weeks later to observe lung metastasis.

Data mining from the public databases

The expression of mRNA-seq and clinicopathological information of 371 HCC patients and 50 noncancerous patients were downloaded from the TCGA database (https://portal.gdc.cancer.gov/). Among them, only 365 tumor samples have both mRNA-seq and clinical survival data. All the raw count data were analyzed to identify differential expression genes (DEGs) in HCC samples and matched noncancerous samples by the package “limma” of R software. | log2FC | = 0 and P < 0.05 were set as the cut-off point.

The GEPIA analysis

GEPIA (Gene Expression Profiling Interactive Analysis) (http://gepia.cancer-pku.cn/) is a valuable and highly cited resource for gene expression analysis and survival analysis based on tumor and normal samples from the TCGA and the GTEx databases23. They deconvolute each sample tool in TCGA/GTEx with the bioinformatics tools CIBERSORT, EPIC and quanTIseq. This online database enables differential gene expression analysis, survival analysis, and can also conduct classification analysis based on a specific variable.

Statistical analysis

The experimental data were analyzed using GraphPad Prism 8 or Image J statistical software, presenting resultsas mean ± standard deviation. The data that conformed to the normal distribution and homogeneity of variance were compared between the two groups using the t-test, and the data that did not conform to the normal distribution were compared between the two groups using the Mann–Whitney U-sum test. p < 0.05 was considered statistically significant. between the two groups.

Results

Analysis of the expression and survival of ADAM family members

The ADAM family consists of 29 members (ADAM1-ADAM29), with only nine members (ADAM8, ADAM9, ADAM10, ADAM12, ADAM13, ADAM15, ADAM17, ADAM19 and ADAM28) demonstrating both somatic expression and metalloproteinase activity. We analyzed the expression of ADAM8, ADAM9, ADAM10, ADAM12, ADAM13, ADAM15, ADAM17, ADAM19 and ADAM28 in HCC tissues and normal tissues on the GEPIA website. The results showed that the expression of ADAM13 could not be detected nor analyzed. Among them, only ADAM15 had a significant difference in expression between HCC tissues and normal tissues (Fig. S1A–H); We further performed the survival analysis of the above-mentioned 10 members of the ADAM family in HCC. The results showed that ADAM13 could not detect the relevant information, and only the survival analyses of ADAM9, ADAM12, ADAM15, ADAM17, and ADAM19 were statistically significant (Fig. S2A–H). Importantly, only ADAM15 exhibited both significant overexpression in HCC and clinically relevant survival outcomes. Further clinical correlation analysis demonstrated that ADAM15 expression levels were elevated in HCC with TNM Ⅲ-Ⅳ (Fig. 1A), deceased HCC patients (Fig. 1B), and and patients with post-treatment recurrence (Fig. 1C). The above results further confirmed that HCC, patients with high expression of ADAM15 had a later tumor stage, were more prone to recurrence and had a shorter survival time.

Fig. 1.

Fig. 1

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The relationship between the expression level of ADAM15 and the clinical data of HCC patients. (A) The higher the TNM stage of HCC patients, the higher the expression level of ADAM15; (B) Patients with HCC who had a high expression level of ADAM15 had a shorter survival time; (C) Patients with HCC who had a high expression level of ADAM15 had a higher recurrence rate. “Deceased” indicates the death of liver cancer patients, “Living” indicates the survival of liver cancer patients, “Disease free” indicates that the patients have no recurrence of liver cancer after treatment, and “Disease recurrence” indicates that the patients have liver cancer recurrence after treatment. *P < 0.05 and **P < 0.01 indicate that there is a statistically significant difference between the two groups.

The expression of ADAM15 affects the proliferation, migration and invasion of HCC cells

Our results indicated that ADAM15 was highly expressed in MHCC97h and lowly expressed in HLF. We conducted the subsequent experiments using MHCC97h and HLF (Fig. 2A). Accordingly, siRNA-ADAM15 was transfected into MHCC97h cells, and the ADAM15 overexpression plasmid was transfected into HLF cells. CCK-8 assay results revealed that ADAM15 knockdown in MHCC97h cells significantly inhibited HCC cell proliferation, while ADAM15 overexpression in HLF cells markedly enhanced HCC proliferation (Fig. 2B and C). Similarly, we conducted the Edu cell proliferation assay. The results also indicated that knockdown of ADAM15 expression in MHCC97h cells could significantly inhibit the proliferation of HCC cells, and overexpression of ADAM15 in HLF cells could significantly promote the proliferation of HCC cells (Fig. 2D and E). Wound healing assay showed that ADAM15 knockdown in MHCC97h cells significantly impaired HCC cell migration, whereas ADAM15 overexpression in HLF cells substantially increased migratory capacity (Fig. 2F and G). The results of our cell invasion test indicated that knockdown of ADAM15 expression in MHCC97h cells could significantly inhibit the invasion of HCC cells, and overexpression of ADAM15 in HLF cells could significantly promote the invasion of HCC cells (Fig. 2J and K).

Fig. 2.

Fig. 2

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The expression and biological behavior of ADAM15 in HCC. (A) The expression level of ADAM15 in HCC cells; (B) The CCK8 test confirmed that knockdown of ADAM15 inhibited the proliferation of HCC cell (MHCC97h); (C) The CCK8 test confirmed that overexpression of ADAM15 promoted the proliferation of HCC cell (HLF); (D) The Edu test confirmed that knockdown of ADAM15 inhibited the proliferation of HCC cell (MHCC97h); (E) The Edu test confirmed that overexpression of ADAM15 promoted the proliferation of HCC cell (HLF); (F) Knockdown of ADAM15 (siADAM15) inhibited the migration of HCC cell (MHCC97h); (G) Knockdown of ADAM15 (si2ADAM15) inhibited the migration of HCC cell (MHCC97h); (H) Overexpression of ADAM15 promoted the migration of HCC cell (HLF); (I) Knockdown of ADAM15 (siADAM15) inhibited the invasion of HCC cell (MHCC97h); (J) Knockdown of ADAM15 (si2ADAM15) inhibited the invasion of HCC cell (MHCC97h); (K) Overexpression of ADAM15 promoted the invasion of HCC cell (HLF).*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicates a statistically significant difference between the two groups.

The expression of ADAM15 is associated with epithelial-mesenchymal transition in HCC cells

EMT plays a crucial role in tumor progression and metastasis. Given ADAM15’s established function in modulating cell adhesion and viability, we investigated its involvement in the EMT of HCC cells. We conducted an experiment to verify the knockdown of ADAM15 expression using two siRNA sequences.We conducted an experiment to verify the knockdown of ADAM15 expression using two siRNA sequences. The WB results indicated that knockdown of ADAM15 expression in MHCC97h cells could significantly inhibit the EMT of HCC cells, suppress the expressions of N-cadherin and Snail, and promote the expression of E-cadherin (Fig. 3A and B). Overexpression of ADAM15 in HLF cells can significantly promote the EMT of HCC cells, inhibit the expression of E-cadherin, and promote the expression of N-cadherin and Snail (Fig. 3C).

Fig. 3.

Fig. 3

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The relationship between the expression level of ADAM15 and EMT in HCC cells. (A) Knockdown of ADAM15 (siADAM15) inhibited the EMT of HCC cell (MHCC97h); (B) Knockdown of ADAM15 (si2ADAM15) inhibited the EMT of HCC cell (MHCC97h); (C) Overexpression of ADAM15 promoted the EMT of HCC cell (HLF). **P < 0.01, ****P < 0.0001 indicates a statistically significant difference between the two groups.

The expression of ADAM15 is associated with apoptosis of HCC cells

Since apoptosis critically regulates tumor progression, we examined ADAM15’s impact on HCC cell apoptosis. Flow cytometry analysis demonstrated that ADAM15 knockdown in MHCC97h cells significantly increased apoptosis (Fig. 4A), while ADAM15 overexpression in HLF cells substantially decreased apoptosis (Fig. 4B). Meanwhile, we performed Western Blot, we conducted an experiment to verify the knockdown of ADAM15 expression using two siRNA sequences. The results indicated that knockdown of ADAM15 expression in MHCC97h cells could promote the expression of apoptotic protein Bax and inhibit the expression of anti-apoptotic protein Bcl-2 (Fig. 4C and D). Overexpression of ADAM15 in HLF cells could significantly inhibit the expression of apoptotic protein Bax in HCC cells and promote the expression of the anti-apoptotic protein Bcl-2 (Fig. 4E).

Fig. 4.

Fig. 4

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The relationship between the expression level of ADAM15 and apoptosis in HCC cells. (A) Knockdown of ADAM15 promoted the apoptosis of MHCC97h cells; (B) Overexpression of ADAM15 inhibited the apoptosis of HLF cells; (C) The results of Western Blot indicated that knockdown of ADAM15 (siADAM15) promoted the apoptosis of HCC cell (MHCC97h); (D) The results of Western Blot indicated that knockdown of ADAM15 (si2ADAM15) promoted the apoptosis of HCC cell (MHCC97h); (E) Overexpression of ADAM15 inhibited the apoptosis of HCC cell (HLF). ***P < 0.001, ****P < 0.0001 indicates a statistically significant difference between the two groups.

ADAM15 affects the apoptosis and EMT of HCC cells by regulating the JNK/p38 MAPK pathway

To further explore the influence of ADAM15 on downstream signaling pathways, we conducted GSEA and KEGG analyses (www.kegg.jp/kegg/kegg1.html ). The GSEA results indicated that ADAM15 was overexpressed in HCC cells and was enriched in the MAPK signaling pathway (Fig. 5A). Meanwhile, our KEGG analysis was also enriched in the MAPK signaling pathway (Fig. 5B). The above bioinformatics analysis initially confirmed that ADAM15 could regulate the MAPK signaling pathway. Therefore, we further conducted research through in vitro experiments. We knocked down the expression of ADAM15 in MHCC97h cells and performed Western Blot detection. The results indicated that knockdown of ADAM15 (siADAM15 and si2ADAM15) expression could significantly inhibit the activity of the JNK-p38 MAPK signaling pathway, suppress the expressions of p-JNK and p-p38, simultaneously inhibit EMT in MHCC97h cells while promoting HCC cell apoptosis (Fig. 5C and D). At the same time, we knocked down ADAM15 (using siADAM15 and si2ADAM15), and then administered the JNK agonist Anisomycin. Subsequently, we conducted another Western Blot assay. The results indicated that knockdown of ADAM15 (siADAM15 and si2ADAM15) expression could significantly inhibit the activity of the JNK-p38 MAPK signaling pathway, suppress the expressions of p-JNK and p-p38, simultaneously inhibit EMT in MHCC97h cells while promoting HCC cell apoptosis, when a JNK agonist is administered, it can significantly enhance the activity of the JNK-p38 MAPK signaling pathway, promote the expressions of p-JNK and p-p38, simultaneously promote EMT while inhibiting apoptosis in MHCC97h cells (Fig. 5E and F). We further verified the effect of overexpressing ADAM15 in HLF cells on JNK-p38 MAPK. The results indicated that overexpression of ADAM15 could activate the JNK-p38 MAPK signaling pathway, promote the expression of p-JNK and p-p38, and simultaneously promote the EMT and inhibit the apoptosis in HCC cell (HLF) (Fig. 5G). We overexpressed ADAM15 in HLF cells and simultaneously administered a JNK inhibitor. The results of Western Blot indicated that overexpression of ADAM15 could activate the JNK-p38 MAPK signaling pathway, promote the expression of p-JNK and p-p38, and simultaneously promote the EMT and inhibit the apoptosis in HCC cell. Administration of JNK inhibitors can inhibit JNK-p38 MAPK signaling pathway, inhibit the expression of p-JNK and p-p38, and simultaneously inhibit the EMT and promote the apoptosis in HCC cell (HLF) (Fig. 5H). Meanwhile, we conducted the wound healing and invasion test. While knockdown the expression of ADAM15 (siADAM15 and si2ADAM15), JNK activator was administered. The results showed that knockdown the expression of ADAM15 could inhibit the migration of MHCC97h. However, after administration of JNK activator, this phenomenon could be partially reversed, and the migration ability of MHCC97h was partially restored (Fig. 5I and J). At the same time, The results showed that knockdown the expression of ADAM15 could inhibit the invasion of MHCC97h. However, after administration of JNK activator, this phenomenon could be partially reversed, and the invasion ability of MHCC97h was partially restored (Fig. 5K and H). The above results providing mechanistic evidence that ADAM15 promotes HCC progression through JNK-p38 MAPK pathway activation.

Fig. 5.

Fig. 5

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ADAM15 promoted the progression and metastasis of HCC through the JNK-p38 MAPK pathway. A The GSEA enrichment test indicated that ADAM15 was highly expressed in the MAPK signaling pathway. B The KEGG enrichment test indicated that ADAM15 was highly expressed in the MAPK signaling pathway. C Western blot showing ADAM15, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, E-cad, N-cad and Snail in MHCC97h with knockdown of ADAM15 using siADAM15. D Western blot showing ADAM15, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, E-cad, N-cad and Snail in MHCC97h with knockdown of ADAM15 using si2ADAM15. E Western blot showing ADAM15, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, E-cad, N-cad and Snail in MHCC97h treated with Anisomycin (JNK agonist) followed by transfection with siADAM15. (F) Western blot showing ADAM15, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, E-cad, N-cad and Snail in MHCC97h treated with Anisomycin (JNK agonist) followed by transfection with si2ADAM15. (G) Western blot showing ADAM15, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, E-cad, N-cad and Snail in HLF with overexpression of ADAM15. (H) Western blot suggesting ADAM15, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, E-cad, N-cad and Snail in HLF treated with SP600125 (JNK inhibitor) followed by transfection with overexpression of ADAM15. (I) Wound healing test in MHCC97h treated with Anisomycin (JNK agonist) for 0 h, 24 h, 48 h followed by transfection with siADAM15. (J) Wound healing test in MHCC97h treated with Anisomycin (JNK agonist) for 0 h, 24 h, 48 h followed by transfection with si2ADAM15. (K) Cell invasion test in MHCC97h treated with Anisomycin (JNK agonist) for 24 h followed by transfection with siADAM15. (L) Cell invasion test in MHCC97h treated with Anisomycin (JNK agonist) for 24 h followed by transfection with si2ADAM15. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 indicates that there is a statistically significant difference between the two groups.

In vivo experiments have confirmed that knockdown of ADAM15 can inhibit tumor cell proliferation, EMT, apoptosis and metastasis

Subcutaneous tumor formation assays in nude mice confirmed that ADAM15 knockdown in MHCC97h significantly inhibited tumor growth (Fig. 6A and B). Meanwhile, we performed immunohistochemical staining on subcutaneous tumor specimens to detect the expressions of proliferation indicators Ki67 and PCNA. The results indicated that knockdown of ADAM15 expression could significantly inhibit the expression of proliferation indicators Ki67 and PCNA (Fig. 6C and B). Meanwhile, we performed Western Blot tests on subcutaneous tumor specimens to examine the changes of apoptotic indicators and EMT-related indicators. The results indicated that inhibiting the expression of ADAM15 could promote the expression of apoptotic protein Bax and E-cadherin, and inhibit the expression of anti-apoptotic protein Bcl-2 and N-cadherin and Snail (Fig. 6E and F). Then we further explored the role of ADAM15 to promote the metastatic of liver cancer cell. We conducted an experiment on tail vein lung metastasis of liver cancer in nude mice. The results showed that inhibiting the expression of ADAM15 could significantly suppress the lung metastasis of liver cancer cells (Fig. 6G). The above experimental results further confirmed that inhibiting the expression of ADAM15 could suppress the progression and metastasis of liver cancer.

Fig. 6.

Fig. 6

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Knockdown of ADAM15 can inhibit tumor cell proliferation, EMT, apoptosis and metastasis in vivo experiments. (A) Knockdown of ADAM15 could inhibit the growth of HCC by the subcutaneous tumor formation experiment in nude mice; (B) The liver cancer volume in the ADAM15 knockdown group was significantly lower than that in the control group; (C) Knockdown of ADAM15 could inhibit the expression of Ki67; (D) Knockdown of ADAM15 could inhibit the expression of PCNA; (E) Knockdown of ADAM15 could inhibit the EMT of HCC; (F) Knockdown of ADAM15 could promote the apoptosis of HCC; (G) Knockdown of ADAM15 could inhibit the lung metastasis of HCC cells by tail vein tumor formation test in nude mice. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicates a statistically significant difference between the two groups.

Discussion

Liver cancer remains a highly malignant tumor24. While treatment modalities continue to diversify, the high recurrence and metastasis rates, coupled with frequent chemoresistance and targeted therapy resistance, severely compromise patient survival25. These clinical realities underscore the critical need to explore the molecular mechanisms of liver cancer progression and metastasis. Currently, some scholars are exploring how post-transcriptional regulation, epigenetic modifications and metabolic modifications in HCC affect tumor progression, providing potential theoretical basis for more precise treatment of liver cancer26–28. However, the therapeutic effects for advanced liver cancer are still unsatisfactory at present, with a high recurrence rate. Exploring new therapeutic targets is expected to provide new treatment opportunities for patients with liver cancer. Our previous work first established that ADAM15 shows elevated expression in HCC tissues, where it correlates with shortened survival and poor prognosis29. Therefore, we speculated that ADAM15 was involved in the progression and metastasis of liver cancer.

Our experimental findings demonstrated that ADAM15 overexpression promoted HCC cell proliferation, migration, and invasion, while its knockdown produced the opposite effects. Meanwhile, the results of Western Blot indicated that high expression of ADAM15 could promote the EMT of HCC cells, while knockdown of ADAM15 expression inhibited the EMT of HCC cells. Studies have shown that EMT is closely related to the progression and metastasis of tumors29. The EMT process can lead to reduce adhesion of tumor cells, increase expression of mesenchymal markers, reduce the polarity of tumor cells, increase the migration and invasion of tumor cells, and promote the progression and metastasis of tumors30,31. Similarly, our study also established ADAM15 as a regulator of apoptosis in HCC. As a programmed cell death mechanism, apoptosis normally eliminates damaged cells, but its dysregulation contributes to cancer progression, metastasis, and treatment resistance32,33. Therefore, the targeted regulation of molecules involved in apoptosis resistance is closely related to tumor treatment, which can restore the sensitivity of tumor cells to apoptosis and overcome the ineffectiveness of treatment. This study confirmed through in vitro cell experiments that the high expression of ADAM15 promoted EMT in HCC cells and inhibited apoptosis of HCC cells. The above results further confirm the potential role of ADAM15 in the progression and metastasis of liver cancer.

To further confirm the role of ADAM15 in liver cancer, we verified the role of ADAM15 in liver cancer through different animal models. Firstly, the results of the subcutaneous tumor formation test in nude mice showed that knockdown of ADAM15 expression could inhibit the growth of subcutaneous tumors. Meanwhile, the results of IHC detection on subcutaneous tumor tissues indicated that knockdown of ADAM15 inhibited the expression of proliferation markers Ki67 and PCNA. The Western Blot results showed that knockdown of ADAM15 inhibited the EMT of HCC cells and promoted the apoptosis of HCC cells. The above results were consistent with those of in vitro experiments, further confirming the role of ADAM15 in the progression and metastasis of liver cancer. Meanwhile, complementary tail vein metastasis assays showed that ADAM15 knockdown markedly reduced lung metastasis formation, collectively demonstrating ADAM15’s pro-metastatic function in HCC.

The results of in vivo and in vitro experiments have preliminarily confirmed the important role of ADAM15 in liver cancer. To further explore the specific regulatory mechanism of ADAM15 in liver cancer, we conducted GSEA and KEGG analyses through bioinformatics methods. The results all indicated that the ADAM15 high-expression group was enriched in the MAPK signaling pathway, which suggested that ADAM15 might promote the progression and metastasis of liver cancer through the MAPK pathway. The MAPK pathway is one of the most important signaling pathways for maintaining life activities in eukaryotes and changes in many diseases34. The MAPK pathway includes extracellular signal-regulated kinase 1/2 (ERK1/2), amino-terminal kinase (JNK), p38 MAPK, and the BMK1 cascade. Literature indicates that approximately half of early-stage hepatocellular carcinoma (HCC) patients exhibit abnormal activation of the MAPK signaling pathway35, and the activated MAPK signaling pathway is closely related to the poor prognosis and distant metastasis of HCC patients36. We knocked down the expression of ADAM15 in HCC cells. The results of Western Blot indicated that ADAM15 knockdown could inhibit the activation of the JNK-p38 MAPK pathway, simultaneously suppress EMT, and promote apoptosis. When the expression of ADAM15 was knockdown and JNK activator was administered simultaneously, apoptosis was inhibited as a result, promoting the EMT. Similarly, overexpression of ADAM15 could activate the JNK-p38 MAPK pathway, promote EMT, and inhibit apoptosis. When ADAM15 was overexpressed and JNK inhibitors were given simultaneously, EMT was inhibited, promoting the apoptosis. The above results confirmed that ADAM15 promoted EMT and inhibited apoptosis in liver cancer by regulating JNK-p38 MAPK, thereby promoting the progression and metastasis of liver cancer. Wound healing and invasion assays provided functional correlation, showing that while ADAM15 knockdown impaired HCC cell migration and invasion, JNK agonist co-treatment partially restored these capabilities. The results also indicated that ADAM15 promoted the progression and metastasis of liver cancer by regulating the JNK-p38MAPK pathway.

This study provides the first comprehensive mechanistic characterization of ADAM15 in the progression and metastasis of liver cancer. It was confirmed through in vivo and in vitro experiments that ADAM15 regulated EMT and apoptosis of HCC cells by regulating the JNK-p38 MAPK pathway. The discovery of the above results further enriched the relevant theories on the progression and metastasis of liver cancer and provided potential targets for the treatment of liver cancer. The molecular mechanism of ADAM15 overexpression in liver cancer deserves further investigation, as does the potential role of ADAM15 in liver cancer progression and metastasis.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (394.5KB, zip)
Supplementary Material 2 (5.3MB, rar)
Supplementary Material 3 (1.3MB, jpg)
Supplementary Material 4 (3.2MB, jpg)
Supplementary Material 5 (329KB, pdf)
Supplementary Material 6 (138.4MB, rar)
Supplementary Material 7 (47.7MB, pdf)

Acknowledgements

We would like to express our special gratitude to the Laboratory of General Surgery at Renmin hospital of Wuhan University and the Central Laboratory of the First Clinical College of Wuhan University for their assistance during our experimental period.

Author contributions

XJH, SMY collected data, designed the research; XJH, SMY conducted to review and editing; wrote the paper. All authors read and approved the final manuscript.

Funding

This study was funded by the Natural Science Foundation of Hubei Province(No. 2024AFB470), and the Funding for Scientific Research Projects from Wuhan Municipal Health Commission (No. WX23Q11), and Municipal Natural Science Foundation Exploratory Program (Morning Light Program) Key Special Project for Clinical Research in Municipal Medical Institutions (No. 2025020701020262). Funding sources for this study had no role in the study design; data collection, analyses, or interpretation; or writing of the manuscript.

Conflict of interest.

The authors state that the study was conducted without any business or financial relationship, which could be interpreted as a potential conflict of interest.

Data availability

The original contributions presented in the research are included in the article. Further inquiries can be directed to the corresponding author (mengyunsu@aliyun.com).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (394.5KB, zip)
Supplementary Material 2 (5.3MB, rar)
Supplementary Material 3 (1.3MB, jpg)
Supplementary Material 4 (3.2MB, jpg)
Supplementary Material 5 (329KB, pdf)
Supplementary Material 6 (138.4MB, rar)
Supplementary Material 7 (47.7MB, pdf)

Data Availability Statement

The original contributions presented in the research are included in the article. Further inquiries can be directed to the corresponding author (mengyunsu@aliyun.com).

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