DNPEP promotes the growth, metastasis, and cisplatin resistance of tongue squamous cell carcinoma through RACK1/ERK signaling pathway

Introduction

Head and neck malignancies represent the sixth most common type of cancer worldwide, with tongue squamous cell carcinoma (TSCC) being a significant subtype (1,2). The management of TSCC has evolved from exclusive surgical intervention to a multimodal approach incorporating surgery, radiotherapy, and chemotherapy. However, the prognosis for advanced TSCC remains poor, with a reported 5-year survival rate below 50% (3,4). Increasing evidence indicates that chemotherapy resistance, particularly resistance to cisplatin, is a major contributor to treatment failure and poor outcomes in TSCC. Despite ongoing research, the mechanisms underlying cisplatin resistance in TSCC remain incompletely understood. This study aims to elucidate the molecular basis of cisplatin resistance in TSCC and identify potential therapeutic targets to overcome this challenge (5-8).

To investigate the mechanisms of cisplatin resistance, next-generation sequencing was performed on a cisplatin-resistant TSCC cell line (HSC3/CDDP) and its parental TSCC cell line (HSC3) to identify differentially expressed messenger RNAs (mRNAs). Aspartyl aminopeptidase (DNPEP) was found to be significantly upregulated in HSC3/CDDP, prompting further investigation.

DNPEP is a zinc-dependent metalloenzyme of approximately 55 kDa, classified within the M18 aminopeptidase family (9). In vertebrates, DNPEP is the sole encoded M18 family peptidase and is broadly expressed across human tissues and bodily fluids, underscoring its essential biological functions. Emerging evidence suggests that DNPEP is implicated in multiple tumor types, where it is frequently overexpressed and may serve as a potential biomarker for cancer diagnosis and prognosis (10-12). However, the role of DNPEP in TSCC and its involvement in cisplatin resistance have not been previously characterized.

The present study demonstrated that DNPEP expression was significantly elevated in TSCC tissues compared to adjacent non-tumorous tissues. Furthermore, DNPEP expression correlated with increased tumor malignancy and poorer prognosis. Functional studies in vitro and in vivo revealed that DNPEP promoted TSCC proliferation, epithelial-mesenchymal transition (EMT), tumor growth, and cisplatin resistance. Mass spectrometry and co-immunoprecipitation analyses further indicated that DNPEP interacts with RACK1, leading to the activation of the ERK signaling pathway. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2697/rc).

Methods

Cells and human tissues

The TSCC cell lines CAL27 and HSC3 were purchased from the Institute of Cell Research, Chinese Academy of Sciences. Cisplatin resistant cell lines were generated using a low-dose shock method. Resistance was confirmed when the half-maximal inhibitory concentration (IC₅₀) of the resistant cell lines increased to at least 2.5 times that of the parental sensitive cells. TSCC cell lines were maintained in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin, and 100 mg/mL streptomycin. Cells were cultured at 37 ℃ in a humidified incubator with 5% CO₂. Forty-eight pairs of TSCC tumor tissues and corresponding adjacent non-tumorous tissues were collected from patients undergoing surgical resection at The First Affiliated Hospital of Nanchang University between November 2023 and December 2024. Histopathological examination confirmed the diagnosis of TSCC, and no patients received preoperative treatment. All methods in this study were carried out in accordance with relevant guidelines and regulations (CDYFY IACUC-202311QR045). Written informed consent was obtained from all participants, and the study was approved by the Human Ethics Committee of The First Affiliated Hospital of Nanchang University (No). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Next-generation RNA sequencing analysis

RNA sequencing was conducted by Oebiotech (Shanghai, China). Total RNA was isolated using the mirVana^TM microRNA (miRNA) Isolation Kit. Sequencing libraries were prepared with the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina platform (HiSeq^TM 2500 or HiSeq X Ten). Differential expression analysis was performed using the DESeq R package, with normalization and statistical testing carried out via the estimateSizeFactors and nbinomTest functions. Genes with a P<0.05 and a fold change >2 or <0.5 were considered significantly differentially expressed.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted from TSCC tissues or cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was then reversely transcribed into cDNA using PrimeScript RT kit (Takara, Shiga, Japan). qRT-PCR was performed using the SYBR Premix Ex Taq^TM II (DimerEraser) kit (Takara) following the manufacturer’s instructions. Relative mRNA expression levels were calculated using the ΔΔCt method.

RNA interference and overexpression

Small interfering RNAs (siRNAs) against human DNPEP and RACK1 were obtained from RiboBio (Guangzhou, China). The pcDNA3.1-DNPEP and pcDNA3.1-RACK1 plasmids for overexpression were likewise provided by RiboBio. Cells were transfected using Lipofectamine^TM 2000 (Invitrogen, USA) following the manufacturer’s instructions. In addition, lentiviral constructs encoding short hairpin RNA (shRNA) targeting DNPEP or DNPEP overexpression were purchased from RiboBio.

Western blot analysis

Total cellular proteins were obtained from tissue samples and cell lines following lysis with Beyotime buffer (Shanghai, China), and protein levels were subsequently measured using a bicinchoninic acid (BCA) assay kit (Bio-Rad Laboratories Inc., California, USA). The protein samples were denatured by heating to 100 ℃ for 5 min and then incubated at 30 ℃ prior to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. After electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and subsequently blocked with 5% nonfat milk for 60 min. The membranes were thereafter incubated overnight at 4 ℃ with the following primary antibodies: E-cadherin (1:2,000, Rabbit polyclonal, Proteintech, Rosemont, IL, USA), N-cadherin (1:2,000, Rabbit polyclonal), Vimentin (1:2,000, Rabbit polyclonal, Proteintech), Bax (1:2,000, Rabbit polyclonal, Proteintech), Bcl-2 (1:1,000, Rabbit polyclonal, Proteintech), PCNA (1:2,000, Mouse monoclonal, Cell Signaling Technology, Danvers, MA, USA), and ERK/p-ERK (1:1,000, Rabbit polyclonal, Proteintech). After incubation, the membranes were thrice washed and then incubated with goat anti-mouse or goat anti-rabbit secondary antibodies (1:5,000, Proteintech) for 1 h at room temperature. The membranes were washed several times before imaging with X-ray film, and the protein signals were detected using an enhanced chemiluminescence (ECL) detection system. Finally, band intensities were quantified using ImageJ software. LM22B-10, a selective ERK pathway activator, was purchased from Beijing Solarbio Science & Technology Co., Ltd., and used to activate the ERK pathway in cells. The treatment was applied for 24 hours as described in the “Results” section.

Immunohistochemistry

Tissue sections were subjected to antigen retrieval in 0.01 M citrate buffer (pH 6.0; Wellbio, China). The retrieval solution was heated to boiling for 20 min and then cooled to room temperature. Sections were rinsed with phosphate-buffered saline (PBS) (pH 7.2–7.6) and incubated with appropriately diluted primary antibodies at 4 ℃ overnight. After washing with PBS, 50–100 µL anti-rabbit IgG HRP polymer (Thermo Fisher Scientific, Waltham, MA, China) was applied. Color development was achieved by adding 50–100 µL DAB working solution (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). The tissue was counterstained with hematoxylin and subsequently sealed with neutral glue (Sigma-Aldrich, St. Louis, MO, USA). The tissue sections were examined under a microscope (BX 43, Olympus, Tokyo, Japan). The antibodies used for immunohistochemical (IHC) staining included Ki-67 (1:200, ServiceBio, Wuhan, China), Vimentin (1:200, Proteintech), Bax (1:2,000, Proteintech), Bcl-2 (1:1,000, Proteintech), E-cadherin (1:200, Proteintech), and N-cadherin (1:200, Proteintech). The expression levels of Ki-67 were graded based on the percentage of stained cells. For Bax, Bcl-2, Vimentin, E-cadherin, and N-cadherin, expression levels were evaluated using the histochemical score (H-score), which was calculated as follows:

H-score=SPI×(I+1)[1]

where I represents the intensity fraction, and SPI represents the percentage of cells stained.

In vitro experiment

After transfection, drug-resistant and parental TSCC cells were plated in 96-well plates at a density of 5×10³ cells per well. Following a 24 h attachment period, the cells were exposed to culture medium supplemented with graded concentrations of cisplatin (CDDP). After 24 h of drug treatment, cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) in accordance with the manufacturer’s instructions. Absorbance at 450 nm was measured, and the 50% growth inhibition (IC₅₀) value of CDDP was calculated using a dose-response curve.

In vivo experiment

CAL27 cells (1.0×10⁶ cells in 100 µL) stably transfected with lentiviral vector (LV) shRNA DNPEP, LV or DNPEP, or LV control were subcutaneously injected into 4-week-old female nude mice. Tumor size was measured weekly, and tumor volume was calculated using the following formula: volume = 0.5 × length × width². Mice were sacrificed 4 weeks post-implantation. The mice were euthanized by intraperitoneal injection of 150 mg/kg sodium pentobarbital and cervical dislocation after anesthesia. Tumors were excised, weighed, and photographed. IHC staining was performed to assess all tumor grafts. All in vivo experiments were performed at the Animal Experiment Center of The First Affiliated Hospital of Nanchang University. All animal procedures were approved by the Medical Ethics Committee of The First Affiliated Hospital of Nanchang University ([2024]CDYFYYLK[07-008]) and were performed in accordance with the Guidelines for the Care and Use of Animals for Scientific Research.

Co-immunoprecipitation (Co-IP) assay

CAL27/CDDP cells (2×10⁷) were lysed in IP lysis buffer (Beyotime) supplemented with protease inhibitors (Yeasen, China) on ice for 30 min. After centrifugation at 12,000 ×g for 15 min, the cleared supernatants were incubated with 2.5 µg of anti-immunoglobulin G (anti-IgG) or anti-DNPEP antibody at 4 ℃ overnight. Protein A/G magnetic beads (20 µL; MedChemExpress, USA) were then added and rotated at 4 ℃ for 6 h. Beads were collected using a magnetic rack and washed three times with PBS. Subsequently, the beads were resuspended in 60 µL lysis buffer and heated at 100 ℃ for 10 min. The eluted immunoprecipitates were subjected to Western blot analysis.

Cell proliferation

The cell proliferation rate was assessed using the CCK-8 and 5-ethynyl-2’-deoxyuridine (EdU) assays. For CCK-8 analysis, cells were plated in 96-well plates at 1×10³ cells per well. After adding CCK-8 reagent, optical density at 450 nm was recorded at 24 h intervals for up to 72 h. Cell proliferation was also evaluated using an EdU incorporation assay with the BeyoClick^TM EdU kit (China). Cells were incubated with EdU working solution for 2 h, followed by fixing and treating with a click reaction solution at room temperature in the dark for 30 min. Hoechst solution was applied for 10 min before imaging using a fluorescence microscope. Cell counting was performed using the “Cell Counter” plugin in ImageJ. The counting process was done manually, where relevant cells were identified and counted in each image.

Wound healing assay

Cell migratory capacity was evaluated using a wound-healing assay. After transfection, cells were plated in 6-well dishes at 1×10⁵ cells per well and maintained at 37 ℃ in high-glucose DMEM lacking FBS until they reached ~80% confluence. A 100 µL pipette tip was utilized to create a scratch, and cells were incubated at 37 ℃. Migration into the scratch was observed at 8 and 24 h.

Transwell invasion assay

Cell invasion was assessed using 8 µm chamber inserts coated with Matrigel in 24-well plates. Serum-free medium containing 3×10⁴ cells was added to the upper chamber, while medium with 20% FBS was added to the lower chamber. After 48 h, invaded cells were fixed and stained with 0.1% crystal violet for 10 min.

Statistical analysis

Biological replicates: for all in vitro experiments, we performed at least three independent biological replicates for each condition. Technical replicates: for key assays [e.g., CCK-8, EdU, Western blotting (WB), qRT-PCR], each experiment was conducted with at least two technical replicates to ensure consistency and accuracy of the measurements. Data are expressed as the mean ± standard deviation (SD). Differences in DNPEP expression between TSCC and matched adjacent tissues were analyzed using a paired Student’s t-test. Comparisons between two independent groups were performed with an unpaired t-test, whereas multiple-group analyses were conducted using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc correction. Categorical variables were analyzed using the Chi-squared test, Fisher’s exact test, or Pearson’s Chi-squared test. Kaplan-Meier analysis with the log-rank test was used for survival analysis. Statistical significance was defined as P<0.05.

Results

DNPEP was differentially expressed between cisplatin-sensitive and resistant TSCC cells

To identify transcripts associated with CDDP resistance in TSCC cells, next-generation sequencing was performed to analyze the mRNA expression levels of HSC3/CDDP and its parental HSC3 cells. Scatter and volcano plots illustrated the differentially expressed mRNAs between these two cell lines (Figure 1A). A total of 734 upregulated and 842 downregulated mRNAs were identified in HSC3/CDDP cells (fold change >2.0 or <0.5; P<0.05). Hierarchical clustering analysis further highlighted these differentially expressed mRNAs (Figure 1B). To validate the sequencing results, five upregulated and five downregulated mRNAs were randomly selected for qRT-PCR (Figure S1). The results of qRT-PCR confirmed the reliability of the sequencing data and revealed significant dysregulation of a cluster of mRNAs in HSC3/CDDP cells. Among them, DNPEP was selected for further analysis. DNPEP expression level was significantly higher in CAL27/CDDP and HSC3/CDDP cells compared with their respective parental lines, involving CAL27 and HSC3 cell lines (Figure 1C,1D).

Figure 1 Expression profile of mRNA in TSCC cells. (A) An MA diagram illustrates the differential mRNA expression between HSC3 and HSC3/CDDP cells. Volcano plots were generated based on fold change values and P values, the red and blue dots indicated statistically significant and differentially expressed mRNAs. (B) A clustered heatmap displays differentially expressed mRNAs with fold changes greater than 2.0. Red represents high expression levels, while blue denotes low expression levels. (C,D) The drug sensitivity of CAL27, HSC3, CAL27/CDDP, and HSC3/CDDP cells to CDDP was assessed, along with the mRNA and protein expression levels of DNPEP in these cells. **, P<0.01; ***, P<0.001. FC, fold change; mRNA, messenger RNA; TSCC, tongue squamous cell carcinoma.

The expression level of DNPEP in TSCC tissues determines TSCC patients’ prognosis

To investigate the potential involvement of DNPEP in malignancies, we obtained patient datasets from The Cancer Genome Atlas (TCGA) via the Genomic Data Commons portal (https://portal.gdc.cancer.gov/). DNPEP showed elevated expression in 13 tumor types, including head and neck squamous cell carcinoma (HNSCC) (Figure 2A). Consistently, a focused analysis of the HNSCC cohort indicated that DNPEP expression was significantly higher in tumor tissues than in matched adjacent non-tumor samples (Figure 2B). Kaplan-Meier survival analysis was conducted in patients with HNSCC stratified by DNPEP expression. The results showed that individuals with high DNPEP expression exhibited a better overall survival compared with those in the low-expression group (Figure 2C). These results indicated a promising diagnostic potential of DNPEP for HNSCC [area under the curve (AUC) =0.767] (Figure 2D). According to the correlation analysis, DNPEP showed a positive correlation with tumor proliferation-related genes (Figure 2E). Immunohistochemistry was used to detect DNPEP expression levels in cancer and adjacent tissues of 48 patients with TSCC. Taken together, DNPEP expression level in cancer tissues was significantly higher than that in adjacent tissues (Figure 2F).

Figure 2 Bioinformatic analysis of DNPEP expression level in TCGA-HNSC dataset. (A) DNPEP was highly expressed in 13 cancers, including HNSCC. (B) The expression level of DNPEP in HNSC tissues was significantly higher than in adjacent normal tissues. (C) Kaplan-Meier analysis revealed that patients with high DNPEP expression levels had a higher survival rate than those with low DNPEP expression, based on the optimal cut-off value. (D) ROC curve analysis demonstrated that DNPEP exhibited strong diagnostic potential for HNSCC. (E) DNPEP expression correlated positively with tumor proliferation-related genes, including MKI67, PCNA, and HDAC1. (F) DNPEP expression level in TSCC tumor tissues was significantly higher than in adjacent normal tissues (stained). *, P<0.05; **, P<0.01; ***, P<0.001. AUC, area under the curve; CI, confidence interval; FPR, false positive rate; HNSCC, head and neck squamous cell carcinoma; HR, hazard ratio; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TPR, true positive rate; TSCC, tongue squamous cell carcinoma.

In vitro study on the ability of DNPEP to regulate the proliferation, migration, invasion, and apoptosis of TSCC cells

To explore the role of DNPEP in TSCC, siRNA containing DNPEP was transfected into CAL27 and HSC3 cells, and pcDNA3.1 DNPEP plasmid was transfected into CAL27/CDDP and HSC3/CDDP cells to downregulate DNPEP expression level. The efficiency of DNPEP knockdown and overexpression was confirmed by qRT-PCR (Figure 3A). Silencing DNPEP markedly suppressed the viability of CAL27/CDDP and HSC3/CDDP cells (Figure 3B). Consistent with this, EdU incorporation assays showed a significant decrease in proliferative capacity following DNPEP depletion in both CAL27/CDDP and HSC3/CDDP cells (Figure 3C). In wound-healing assays, DNPEP knockdown significantly impaired cell migration (Figure 3D). Moreover, Transwell analyses demonstrated that depletion of DNPEP substantially reduced the invasive potential of CAL27/CDDP and HSC3/CDDP cells (Figure 3E). The results of flow cytometry revealed that knockdown of DNPEP significantly promoted the apoptotic ability of CAL27/CDDP and HSC3/CDDP cells (Figure 3F). The results of WB demonstrated that after DNPEP knockdown, the expression levels of PCNA, N-cadherin, vimentin, and Bcl-2 in CAL27/CDDP and HSC3/CDDP cells decreased, while the expression levels of Bax and E-cadherin increased (Figure 3G).

Figure 3 The relationship between the low expression of DNPEP and the tumoral behavior of TSCC cells. (A) qRT-PCR confirmed successful transfection. (B,C) EdU (200× magnification) and CCK-8 assays showed that DNPEP knockdown significantly reduced the proliferation of CAL27/CDDP and HSC3/CDDP cells. (D) Wound healing assays revealed that DNPEP knockdown inhibited cell migration (magnification ×100). (E) Transwell assays indicated that DNPEP knockdown suppressed cell invasion (stained; magnification ×100). (F) Flow cytometry demonstrated that DNPEP knockdown enhanced apoptosis in CAL27/CDDP and HSC3/CDDP cells. (G) Western blot analysis showed that DNPEP knockdown increased the expression of PCNA, N-cadherin, vimentin, and Bcl-2, while decreasing Bax and E-cadherin levels in CAL27/CDDP and HSC3/CDDP cells. -, **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; qRT-PCR, quantitative reverse transcription polymerase chain reaction; si-DNPEP, small interfering RNA targeting DNPEP; si-NC, small interfering RNA targeting negative control; TSCC, tongue squamous cell carcinoma.

PcDNA-3.1-DNPEP plasmid was transfected into CAL27 and HSC3 cells to upregulate DNPEP expression level. The efficiency of DNPEP overexpression was confirmed by qRT-PCR (Figure 4A). DNPEP overexpression significantly enhanced the viability of CAL27 and HSC3 cells (Figure 4B). The results of EDU assay further demonstrated that DNPEP overexpression markedly promoted the proliferation of these cells (Figure 4C). Additionally, wound healing assay revealed a significant increase in the migration capacity of CAL27 and HSC3 cells following DNPEP overexpression (Figure 4D). Transwell assay indicated that overexpression of DNPEP significantly increased the invasive ability of CAL27 and HSC3 cells (Figure 4E). The results of flow cytometry demonstrated that overexpression of DNPEP significantly inhibited the apoptotic ability of CAL27 and HSC3 cells (Figure 4F). The results of WB revealed that after DNPEP overexpression, the expression levels of PCNA, N-cadherin, vimentin, and Bcl-2 in CAL27 and HSC3 cells increased, while the expression levels of Bax and E-cadherin decreased (Figure 4G).

Figure 4 The relationship between the high expression of DNPEP and the development of TSCC cells. (A) qRT-PCR confirmed successful transfection. (B,C) EdU (200× magnification) and CCK-8 assays indicated that DNPEP overexpression significantly promoted the proliferation of CAL27 and HSC3 cells. (D) Wound healing assay indicated enhanced migration ability following DNPEP overexpression (magnification). (E) Transwell assay demonstrated increased invasion capacity of DNPEP-overexpressing cells (stained; magnification). (F) Flow cytometry analysis revealed that DNPEP overexpression suppressed apoptosis of CAL27 and HSC3 cells. (G) Western blotting results showed that DNPEP overexpression upregulated the expression levels of PCNA, N-cadherin, vimentin, and Bcl-2, while downregulating the expression levels of Bax and E-cadherin. *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; p-DNPEP, plasmid-DNPEP; p-NC, plasmid-negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TSCC, tongue squamous cell carcinoma.

Effect of DNPEP knockdown or overexpression on tumor growth in vivo

To evaluate the effect of DNPEP on TSCC cells in vivo, CAL27/CDDP and CAL27 cells stably transfected with LV-shRNA-DNPEP, LV-ov-DNPEP, or a control vector were subcutaneously injected into nude mice (n=20). Tumor size was measured every five days using a micrometer. Three weeks after inoculation of tumor cells, all nude mice were sacrificed, the tumors were removed, and the tumor weight was quantified (Figure 5A,5B).

Figure 5 Effect of DNPEP knockdown or overexpression on tumor growth in vivo. (A) Effect of overexpression of DNPEP in CAL27 cells on tumor size, growth rate, and quality. (B) Effect of overexpression of DNPEP in CAL27/CDDP cells on tumor size, growth rate, and quality. (C) The changes in the expression levels of PCNA, vimentin, Bcl-2, Bax, E-cadherin, and N-cadherin after overexpression of DNPEP were detected by immunohistochemistry. (D) The changes in the expression levels of PCNA, vimentin, Bcl-2, Bax, E-cadherin, and N-cadherin after DNPEP knockdown were detected by immunohistochemistry. (C,D) 400× magnification. **, P<0.01. LV, lentiviral vector; LV-OV-DNPEP, lentiviral vector overexpressing DNPEP; LV-sh-DNPEP, lentiviral vector expressing short hairpin RNA targeting DNPEP.

In order to further study the relationship between DNPEP and PCNA, vimentin, Bcl-2, Bax, E-cadherin, and N-cadherin, IHC staining was performed to determine the expression levels of PCNA, vimentin, Bcl-2, Bax, E-cadherin, and N-cadherin in tumor tissues with high and low DNPEP expression. The results indicated that in DNPEP-overexpressing tissues, the expression levels of PCNA, N-cadherin, vimentin, and Bcl-2 were upregulated, whereas the expression levels of Bax and E-cadherin were downregulated (Figure 5C). Conversely, in DNPEP-knockdown tissues, the expression levels of PCNA, N-cadherin, vimentin, and Bcl-2 were reduced, while the expression levels of Bax and E-cadherin were elevated (Figure 5D).

DNPEP upregulated RACK1 protein level and activated ERK signaling pathway to regulate the occurrence and development of TSCC cells

In order to investigate the underlying molecular mechanism of DNPEP in promoting TSCC cell proliferation, migration, and invasion, mass spectrometry-based proteomic analysis was performed as an exploratory approach to screen for candidate proteins potentially associated with DNPEP overexpression. Among the identified candidates, RACK1 was selected for further validation (Figure 6A). The results of co-IP and molecular docking indicated that there was a strong binding between DNPEP and RACK1 (Figure 6B,6C). In order to study the effect of DNPEP on RACK1, DNPEP expression level was modulated in TSCC cells, and the mRNA and protein levels of RACK1 were analyzed. The results indicated that silencing or overexpressing DNPEP had no significant impact on RACK1 mRNA level (Figure 6D). However, knockdown of DNPEP led to a decrease in RACK1 protein level in CAL27 and HSC3 cells, whereas DNPEP overexpression resulted in the increased RACK1 protein level in these cell lines (Figure 6E). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DNPEP plays a regulatory role in the ERK pathway (Figure 6F). Additionally, data from TCGA database demonstrated a positive correlation between DNPEP, RACK1, and ERK (Figure 6G). WB was performed to assess the regulatory effect of the DNPEP/RACK1/ERK axis in TSCC. The results showed that RACK1 knockdown significantly inhibited ERK phosphorylation (Figure 6H). Overexpression of DNPEP enhanced ERK phosphorylation, while RACK1 knockdown suppressed it. Furthermore, simultaneous DNPEP overexpression and RACK1 knockdown mitigated the increase in ERK phosphorylation induced by DNPEP overexpression (Figure 6I). Meanwhile, RACK1 knockdown reduced the expression levels of Bcl-2, PCNA, vimentin, and N-cadherin while increasing the expression levels of Bax and E-cadherin. Notably, treatment with LM22B-10, an ERK pathway activator, reversed the protein expression changes caused by RACK1 knockdown (Figure 6J).

Figure 6 DNPEP interacts with RACK1 and activates ERK signaling pathway to regulate the occurrence and development of TSCC. (A) Mass spectrometry analysis identified differentially expressed proteins, following DNPEP overexpression. (B) Co-IP confirmed the interaction between DNPEP and RACK1. (C) AutoDock molecular docking predicted a strong binding affinity between DNPEP and RACK1. (D) qRT-PCR analysis indicated that DNPEP knockdown or overexpression had no significant effect on RACK1 mRNA level. (E) Western blot analysis demonstrated that DNPEP knockdown reduced RACK1 protein level, whereas DNPEP overexpression increased RACK1 expression. (F,G) KEGG enrichment analysis and TCGA database analysis revealed a significant positive correlation between DNPEP, RACK1, and the ERK pathway. (H) Western blotting results showed that RACK1 knockdown decreased ERK phosphorylation. (I) Western blot analysis further demonstrated that DNPEP overexpression increased ERK phosphorylation, whereas simultaneous RACK1 knockdown attenuated this effect. (J) The addition of LM22B-10 following RACK1 knockdown restored the protein level changes induced by RACK1 depletion. ns, not significant; **, P<0.01; ***, P<0.001. IB, immunoblotting; IP, immunoprecipitation; KEGG, Kyoto Encyclopedia of Genes and Genomes; LV-OE-DNPEP, mRNA, messenger RNA; p-DNPEP, plasmid-DNPEP; p-NC, plasmid-negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; si-DNPEP, small interfering RNA targeting DNPEP; si-NC, small interfering RNA targeting negative control; si-RACK1, small interfering RNA targeting RACK1; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TSCC, tongue squamous cell carcinoma.

Discussion

Studies have found that the gene spectrum of drug resistance plays a crucial regulatory role in tumorigenesis and the progression of various cancer types. Cisplatin is a first-line chemotherapy drug for TSCC, while its resistance mainly results in poor treatment efficacy and prognosis. DNPEP, a metalloenzyme belonging to the M18 aminopeptidase family, has been shown to regulate the development of breast cancer (13). Recent research demonstrated that DNPEP also influences the progression of TSCC and cisplatin resistance.

Molecular docking and co-IP experiments revealed that DNPEP binds to RACK1, enhancing its stability. As a key protein kinase activator, RACK1 plays a role in regulating EMT, thereby promoting tumor cell proliferation and invasion, ultimately leading to metastasis (14). The ERK pathway regulates processes, such as cell cycle progression, differentiation, protein synthesis, metabolism, survival, migration, invasion, and senescence. Activation of ERK kinase can even confer tumor-like characteristics to normal cells. Prior research and STRING database analyses indicated a significant link between RACK1 and the ERK signaling pathway, and RACK1 could promote tumor progression through ERK phosphorylation (15,16).

Findings of this study demonstrate that DNPEP enhances cisplatin resistance in TSCC by promoting proliferation, migration, and invasion while inhibiting apoptosis. Recent studies suggest that acquired drug resistance in tumor cells is often associated with EMT-like changes, as EMT enhances cancer cell metastasis, indirectly diminishing chemotherapy efficacy. Additionally, both processes may share common signaling molecules (17). For instance, research has identified EMT regulators such as Snail, Twist, and α-catenin as mediators of chemoresistance in various tumors. The current study further establishes a positive correlation between DNPEP expression, TSCC proliferation, and EMT, highlighting DNPEP’s role in cisplatin resistance in TSCC.

Conclusions

In conclusion, our study demonstrates that DNPEP functions as a key regulator of malignant progression and cisplatin resistance in TSCC. Mechanistically, DNPEP overexpression promotes tumor cell proliferation, EMT, and survival, at least in part through stabilizing RACK1 and activating the ERK signaling pathway, thereby attenuating cisplatin-induced apoptosis.

Notably, emerging evidence has implicated DNPEP in the progression and aggressiveness of several other cancer types, suggesting that DNPEP-mediated signaling may represent a shared mechanism underlying tumor progression and therapy resistance across malignancies. Our findings extend these observations to TSCC and highlight a previously underappreciated role of DNPEP in chemoresistance within head and neck cancers.

From a translational perspective, the DNPEP/RACK1/ERK axis may represent a promising therapeutic target for overcoming cisplatin resistance in TSCC. Targeting this pathway, either alone or in combination with conventional chemotherapy, may offer a potential strategy to improve treatment efficacy and patient outcomes. Further studies are warranted to explore DNPEP-targeted interventions and their clinical applicability.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2697/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2697/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2697/prf

Funding: This research was supported by the National Natural Science Foundation of China (grant Nos. 82260194 and 82403716), Jiangxi Natural Science Foundation (grant Nos. 20232BAB216073 and 20242BAB25514), and Key Projects of Jiangxi Administration of Traditional Chinese Medicine (grant No. GZY-KJS-2023-028).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2697/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Human Ethics Committee of The First Affiliated Hospital of Nanchang University (No.) and adhered to the principles outlined in the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all participants. All animal experiments were approved by the Medical Ethics Committee of The First Affiliated Hospital of Nanchang University ([2024]CDYFYYLK[07-008]) and conducted according to the Guidelines for the Care and Use of Animals for Scientific Research.

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

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