PLAU accelerates extracellular matrix remodeling through partial epithelial-mesenchymal transition in head and neck squamous cell carcinoma

Introduction

Head and neck squamous cell carcinoma (HNSCC) constitutes a significant global health challenge and accounts for approximately 4.5% of all malignancies, with an estimated 890,000 new cases and 450,000 related deaths annually (1-3). HNSCC can include malignancies of different anatomical subsites, including the oral cavity, nasopharynx, oropharynx, and hypopharynx (4). The primary risk factors for HNSCC include smoking, alcohol consumption, and infection with human papilloma virus (5). Although comprehensive therapies including surgery, chemotherapy, radiation therapy, and antitumor immunotherapy are widely used, the 5-year survival rate of patients with HNSCC is only 40–50% (6,7). Metastasis to regional lymph nodes or distant organs is the most significant contributor to the decreased survival time among these patients (4).

Metastasis is a complicated process that includes cancer cell dissemination, invasion, migration, intravasation, extravasation, and colonization (8). The key driver of cancer metastasis is epithelial-mesenchymal transition (EMT). EMT is a dynamic, multistep process in which epithelial cells lose polarity and cell-cell adhesion while gaining migratory and invasive abilities (9). Partial EMT (p-EMT) occurs during disease progression, wherein cells concurrently exhibit both epithelial and mesenchymal traits, including the coexpression of epithelial and mesenchymal proteins (9-11). Integrin subunit alpha 5 (ITGA5) and podoplanin (PDPN) are considered to be p-EMT markers in HNSCC and may be involved in cancer cell metastasis (12,13). One study investigated the transcriptional profiles of 6000 cells from 18 patients with primary HNSCC tumors and matched lymph nodes (13). The results suggested that a subset of cells also express a p-EMT program that include extracellular matrix (ECM) proteins but not classical EMT transcription factors. p-EMT cells are generally located at the anterior margin of the primary tumors, close to cancer-associated fibroblasts (CAFs). Therefore, the p-EMT program may be an independent predictor of HNSCC.

Despite the research into p-EMT intensifying in recent years, the exact nature of its molecular mechanisms remain unclear. We identified 12 differentially expressed genes (DEGs) from HNSCC datasets in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases and selected PLAU for further investigation. PLAU is one component of the plasminogen activator system, which is involved in a wide array of physiological and pathological conditions, figuring prominently in basement membrane and ECM remodeling (14). Numerous studies have shown that PLAU plays an important role in the migration of head and neck cancer. Therefore, we conducted a series of experiments to determine the correlation between PLAU and p-EMT and its role in HNCSS metastasis. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0478/rc).

Methods

Data collection and normalization

Gene expression profiles were obtained from the GEO (https://www.ncbi.nlm.nih.gov/geo/) (15). Dataset GSE51985 includes 20 samples from 10 patients; dataset GSE58911, 30 samples from 15 patients; and dataset GSE83519, 44 samples from 22 patients. The DEGs between HNSCC and adjacent normal tissue were screened with GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) (16). Three volcano plots were generated using GEO2R to identify significant DEGs in HNSCC, with cutoff criteria set at |log2 fold change (FC)| >1 and an adjusted P value <0.01. A diagram visualizing the intersection of DEGs between datasets was constructed with an online Venn diagram tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) (17).

Functional and pathway enrichment analysis of DEGs

To analyze the function of PLAU, Gene Ontology (GO) biological process terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis were applied via the Database for Annotation, Visualization and Integrated Discovery and the clusterProfiler package in R v. 4.0.4 software (https://www.r-project.org; The R Project for Statistical Computing, Vienna, Austria) (18). The enriched GO terms and KEGG pathways with a P value <0.05 were considered significantly enriched biological processes or signaling pathways.

HNSCC fresh tissue specimen acquisition and tissue fixing

Thirty-six pairs fresh HNSCC tissues were obtained from patients with primary HNSCC who underwent surgery at the Department of Otolaryngology of The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, from October 2020 to August 2021. Tissues (including dysplasia) confirmed to be free of cancer according to the examination of intraoperative frozen sections were selected as the normal control (NC) for HNSCC. Tumor tissues and adjacent normal tissue were immediately stored in 4% paraformaldehyde for 6 hours and then embedded in paraffin. Thirty-nine pairs of sections were obtained from the Pathology Department of The Second Hospital of Hebei Medical University. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of The Second Hospital of Hebei Medical University (No. 2020-R561). Informed consent was provided by all patients before surgery.

Target gene prediction

Gene expression profiles and clinicopathological characteristics of patients with HNSCC were obtained from the TCGA. The online database, Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html), was used to further confirm the significantly correlated p-EMT genes in HNSCC (19). The coexpression of DEGs and p-EMT markers were determined through the calculation of Pearson correlation coefficients (PCCs), with PCCs >0.4 indicating a p-EMT-related gene (P<0.01).

Survival analysis

The survival package in R was used to conduct survival analysis of the overlapping DEGs obtained from the bioinformatics analysis. The Kaplan-Meier method was used for the survival analysis of PLAU expression in HNSCC tissue microarrays (the HNSCC tissue microarrays are available in the table available at https://cdn.amegroups.cn/static/public/tcr-2026-0478-1.xls), with the median PLAU expression level serving as the cutoff value. Survival distributions were compared with the log-rank test, with statistical significance set at P<0.05.

Cell culture and transfection

The HNSCC cell lines HEp-2 and FaDu were purchased from the China Center for Type Culture Collection. HEp-2 was cultured in Dulbecco’s Modified Eagle Medium (DMEM)/high glucose (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% streptomycin-penicillin (Solarbio, Wuhan, China). FaDu was cultured in the same medium with 15% FBS. Both cell lines were cultured in a six-well plate with a density of 2×10⁵ cells per well. After 24 hours, 100 pmol of small interfering RNA (siRNA) was mixed with LipoMAX transfection reagent (ABclonal, Wuhan, China) at a ratio of 1:1 in volume, incubated for 10 minutes, diluted with FBS-free medium, and then transfected into the HNSCC cell lines. After 4–6 hours, the half-volume solution were removed and replaced with fresh medium containing 10% FBS. After 48 hours, cells were harvested for protein extraction. si-PLAU (5’-TCACCACCATCGAGAACCA-3’) was synthesized by RiboBio (Guangzhou, China).

Immunohistochemistry and scoring

Unstained serial sections were acquired from paraffin blocks of 30 paired patients with HNSCC. Immunohistochemical (IHC) staining on HNSCC tissue microarrays was conducted with antibodies against PLAU (1:700; cat. no. 17968-1-AP; Proteintech, Rosemont, IL, USA), ITGA5 (1:4000; cat. no. 10569-1-AP; Proteintech), or PDPN (1:750; cat. no. 11629-1-AP; Proteintech). Phosphate-buffered saline (PBS) was used as a negative control. The IHC reagent was obtained from ZSGB-BIO (Beijing, China). All slides were scanned with a ScanScope (Zeiss, Wetzlar, Germany). The histoscore quantification for each slide was conducted via an analysis of the percentage of dyed area with the quantification software Image-Pro Plus (https://imagej.net; Media Cybernetics, Rockville, MD, USA) (20).

Transwell cell invasion and migration assay

After transfection for 12 hours, HEp-2 cells and FaDu cells were starved in serum-free culture medium for 12 hours. Cells were centrifuged, and the culture medium was discarded after digestion with trypsin-EDTA solution (0.25%, without phenol red) (Solarbio), washed twice with PBS and recultured in serum-free medium. Transwell chambers (8.0-µm pores; Corning Inc., Corning, NY, USA) coated with 50 µL of Matrigel (5× dilution; 50 µL/well; Solarbio) were used for cell invasion assays, and migration assays were performed with Matrigel-free Transwell chambers in 24-well plates. The cell suspension (200 µL containing 4.0×10⁴ cells or 100 µL with 2.0×10⁴ cells) was placed in the upper chamber, and 600 µL DMEM containing 30% FBS was placed in the lower chamber. Care was taken to avoid bubbles between the lower culture medium and the chamber. After 36-hour incubation, the Transwell chambers were removed and washed twice with PBS. The cells on the upper surface of the membrane were removed with a cotton swab, and the cells underneath the membrane were fixed with 4% paraformaldehyde for 20 minutes. Cells were stained with 0.1% crystal violet for 15 minutes. Attached cells were washed twice with PBS. Stained cells were observed with a microscope (Zeiss).

Wound healing assay

Wound healing assays were performed to examine the effect of si-PLAU on cell migration. The cells (5×10⁵) were seeded in six-well plates containing DMEM medium with FBS and incubated at 37 ℃ in an atmosphere of 5% CO₂ for 24 hours. HEp-2 and FaDu cells were transfected when the cell density reached 60–70%.

When the cell density reached 90%, a 200-µL pipette tip was used to scratch the cell culture dish while the complete medium was changed to 1% FBS medium. Cell migration into the wound area was quantified at 0, 24, and 48 hours with an inverted microscope (Zeiss), with results expressed as the percentage of wound closure.

Cell Counting Kit-8 (CCK-8) cell proliferation assay

First, HEp-2 and FaDu cells were transfected with the siRNA and si-PLAU. Subsequently, the cells were seeded into 96-well plates at a density of 5,000 cells/well with a 12-hour transfection and incubated for 0, 12, 24, and 48 hours at 37 ℃ in 5% CO₂. At each time point, CCK-8 medium (10 µL) was added to each well, and plates were incubated for another 1 hour. Absorbance at 450 nm was measured with a microplate reader.

Western blotting

Total protein was extracted from HEp-2 and FaDu cells transfected with si-PLAU via radioimmunoprecipitation buffer (Reportbio, Shijiazhuang, China). Protein concentration was measured with a bicinchoninic acid protein concentration assay kit (Solarbio). Protein samples (30 µg/lane) were separated on 10% polyacrylamide gels (Beijing Biotides, Beijing, China) via electrophoresis and transferred onto polyvinylidene fluoride membranes. Subsequently, 5% skim milk was used to block membranes with tris-buffered saline with Tween (TBST) for 2 hours at room temperature with shaking at 80 rpm. Membranes were then incubated with antibodies against PLAU (1:700; cat. no. 17968-1-AP; Proteintech), ITGA5 (1:4,000; cat. no. 10569-1-AP; Proteintech), or PDPN (1:750; cat. no. 11629-1-AP; Proteintech) overnight at 4 ℃. After three 15-minute washes, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (Bioworld Technology, Bloomington, MN, USA) diluted to 1:10,000 in TBST for 1 hour at room temperature. Protein bands were visualized with enhanced chemiluminescence (Advansta, San Jose, CA, USA) after three 15-minute washes with TBST. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference. Experiments were completed with three technical replicates.

Statistical analysis

All experiments performed at least three times, and data are presented as the mean ± standard deviation. SPSS 21.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (Dotmatics, Boston, MA, USA) were used for statistical analyses. Multiple-group comparisons were conducted via the Student t-test or one-way analysis of variance. The prognostic value of PLAU for the overall survival of patients with HNSCC was evaluated via univariate and multivariate Cox regression analyses. Survival probabilities were estimated via the Kaplan-Meier method and were compared with the log-rank test, with P<0.05 indicating statistical significance.

Results

Identification of DEGs in HNSCC

Three gene expression microarray datasets for HNSCC, namely GSE51985, GSE58911, and GSE83519, were obtained from GEO. Volcano plots showing the DEGs between the three datasets were downloaded from GEO (Figure 1A-1C). The data were screened with |logFC| >1 and adjusted P<0.01 serving as the cutoff criteria. Venn diagram indicated that the number of genes related to HNSCC in the GSE51985, GSE58911, and GSE83519 datasets were 1,253, 459, and 1,649, respectively (Figure 1D). Twelve genes were differentially expressed in all three datasets.

Figure 1 Bioinformatics analysis for PLAU screening and target gene prediction. (A-C) Volcano plots of DEGs for HNSCC based on the GEO. Black dots represent genes with no significant differential expression (Padj ≥ 0.05). (D) Venn diagram of DEGs from the GEO (12 genes were selected from the intersection of the three GEO databases related to HNSCC, including GSE51985, GSE58911, and GSE83519). (E) Overall survival analysis assay for PLAU based on the GEPIA. (F,G) Coexpression relationship between PLAU and p-EMT markers (ITGA5 and PDPN) based on GEPIA. DEGs, differentially expressed genes; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; HNSCC, head and neck squamous cell carcinoma; HR, hazard ratio; TPM, transcripts per million.

Survival analysis of PLAU expression in HNSCC and the relationship between PLAU and p-EMT markers

Among the 12 DEGs found to be consistently associated with HNSCC across datasets (Figure 1D), GEPIA was applied to determine which genes had the strongest association with the overall survival rate. Patients with lower PLAU expression had a longer overall survival, suggesting that PLAU is involved in HNSCC (Figure 1E). The relationship between PLAU and ITGA5 and PDPN was analyzed via GEPIA. The expression of PLAU was positively correlated with the expression of p-EMT markers (Figure 1F-1G).

GO analysis and signaling pathway enrichment of PLAU-related messenger RNAs in HNSCC

GO term and signaling pathway enrichment analyses of PLAU-related messenger RNAs (mRNAs) were performed via the clusterProfiler package in R. Thirty GO terms and four pathways were identified as enriched (Figure 2A-2D). Focal adhesion and cell-substrate junction were the most significantly enriched metastasis-related GO terms (Figure 2A), and the most significantly enriched pathways were complement and coagulation cascades and proteoglycans in cancer.

Figure 2 Enrichment analyses of PLAU-associated transcripts and histopathological evaluation. (A,B) GO enrichment analysis of PLAU-correlated mRNAs. The vertical axis indicates significantly enriched GO categories, while the horizontal axis indicates the count of enriched transcripts and the corresponding enrichment ratio, respectively. (C,D) KEGG pathway analysis of PLAU-correlated mRNAs. The vertical axis indicates the significantly enriched pathways. (C) The number of enriched transcripts. (D) The enrichment ratio. (E) Representative HE staining of HNSCC specimens and matched peritumoral tissues. Bubble color intensity and diameter correspond to statistical significance and transcript abundance, respectively. A significance threshold of P<0.05 was applied for all enrichment analyses. BP, biological process; CC, cellular component; GO, Gene Ontology; HE, hematoxylin and eosin; HNSCC, head and neck squamous cell carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; NC, normal control.

Expression of PLAU, ITGA5, and PDPN in HNSCC tissue and adjacent normal tissue

The expression of PLAU, ITGA5, and PDPN was detected in 30 paired HNSCC tissues and adjacent normal tissues. HNSCC tissue slices were confirmed with hematoxylin and eosin (HE) staining (Figures 2E,3A-3I). The results showed that expression of these genes in the HNSCC tissue group was higher compared to that of the adjacent normal tissues.

Figure 3 The expression of PLAU, ITGA5, and PDPN in human HNSCC tissue. (A-F) IHC of PLAU, ITGA5, and PDPN in HNSCC and adjacent normal tissues (upper: magnification 100×; lower: magnification 200×). (G-I) Histoscore of PLAU, ITGA5, and PDPN in tumor tissues and adjacent tissue. Statistical comparisons were made via the Student t-test, with significance defined as P<0.05. **, P<0.01. HNSCC, head and neck squamous cell carcinoma; IHC, immunohistochemical; NC, normal control.

Relationship between PLAU expression and clinical characteristics in patients with HNSCC

The clinical information of 499 patients with HNSCC was obtained from TCGA, and the relationship between PLAU and clinical characteristics was analyzed via the chi-squared test. The results (Table 1) indicated that PLAU expression was significantly associated with metastatic lymph node (χ²=6.788; P=0.03). Univariate analysis based on information from 499 patients with HNSCC indicated that survival was significantly associated with PLAU expression [hazard ratio (HR) =1.002, 95% CI: 1.001–1.004; P=0.003], stage (HR =1.726, 95% CI 1.092–2.729; P=0.02), and TNM stage (T: HR =1.403, 95% CI: 1.020–1.930, P=0.04; N: HR=76.994, 95% CI: 6.981–849.133; P<0.001; M: HR =1.944, 95% CI: 1.379–2.741; P<0.001). Multivariate Cox regression analysis revealed that the independent prognostic factors of survival were overall stage (HR =0.515, 95% CI: 0.326–0.814; P=0.004), T stage (HR =1.982, 95% CI: 1.430–2.747; P<0.001), and N stage (HR =2.400, 95% CI: 1.683–3.424; P<0.001) (Table 2).

Knockdown of PLAU inhibited HNSCC cell proliferation, migration, invasion, and p-EMT

Based on the effectiveness of siRNAs as confirmed by Western blotting, we selected siRNA and si-PLAU for subsequent experiments. CCK-8 assays showed that the HNSCC cell growth rate significantly declined at 48 hours in the PLAU-knockdown group. In the wound healing assays, the healing rate at 48 hours was significantly reduced in si-PLAU HEp-2 (P<0.01; Figure 4A) cells. In the cell invasion assay, the number of HEp-2 cells that migrated through the filter coated with Matrigel was significantly lower in the si-PLAU than in the NC and siRNA groups (P<0.01; Figure 4B). The results were the same for the cell migration assay, when the experiment was conducted without Matrigel (Figure 4C). Knockdown of PLAU inhibited the proliferation of in HEp-2 (P<0.01; Figure 4D) cells. To investigate whether PLAU drives p-EMT in HNSCC metastasis, we performed Western blotting in HEp-2 and FaDu cells following PLAU knockdown. Knockdown of PLAU was found to promote the expression of ITGA5 and inhibit the expression of PDPN in HEp-2 (P<0.05; Figure 4E,4F) cells compared to the NC and siRNA conditions. In line with the results obtained from HEp-2 cells, PLAU knockdown in Fadu cells led to a significant decrease in migration, invasion, scratch wound healing, and proliferation abilities compared with both the NC control and siRNA groups (P<0.01; Figure 5A-5D). Western blot analysis demonstrated that PLAU knockdown in FaDu cells produced similar alterations in the expression of p-EMT markers ITGA5 and PDPN as observed in HEP-2 cells, with statistically significant differences (P<0.05; Figure 5E,5F). These results suggest that knockdown of PLAU inhibits HNSCC cell migration, invasion and p-EMT.

Figure 4 Effect of PLAU silencing on HEp-2 cell proliferation, invasiveness, and metastatic potential. (A) Wound healing assay assessing the migratory capacity of HEp-2 cells following PLAU knockdown. Representative images were acquired at 0, 24, and 48 hours postwounding (magnification 10×) for the si-PLAU, NC, and siRNA negative control groups. The bar graph quantifies the mean wound closure rate across three independent replicates per group. (B,C) Transwell invasion and migration assays evaluating the effect of PLAU depletion on cellular invasiveness and motility. Representative micrographs were captured at 48 hours (magnification 10×) for both Matrigel-coated and uncoated filters. The bar graphs depict the average cell counts traversing each filter type. All experiments were conducted in triplicate. The crystal violet staining method was used to visualize and quantify the invasion and migration of cells. (D) Quantitative analysis demonstrating a significant reduction in cell number in the si-PLAU group compared with both the NC and siRNA groups. (E) Western blot analysis evaluating the effect of PLAU silencing in HEp-2. PLAU silencing markedly suppressed the expression of the p-EMT marker PDPN, whereas ITGA5 expression was elevated. (F) The protein expression levels of p-EMT-related markers, including ITGA5 and podoplanin, were examined in HEP-2 cells from three treatment groups: NC control, siRNA transfection, and si-PLAu transfection. Asterisks denote statistically significant differences determined according to the Student t-test (P<0.05). Data are expressed as the mean ± SEM from three biological replicates. *, P<0.05; **, P<0.01. NC, normal control; OD, optical density; SEM, standard error of mean.

Figure 5 Effect of PLAU silencing on FaDu cell proliferation, invasiveness, and metastatic behavior. (A) Wound healing assay evaluating the effect of PLAU knockdown on FaDu cell migration. Representative images were acquired at 0, 24, and 48 hours postwounding (magnification 10×) for the si-PLAU, NC, and siRNA negative control groups. The bar graph quantifies the mean wound closure rates across three experiments. (B,C) Transwell assays assessing the effects of PLAU knockdown on invasion and migration. Micrographs were captured at 48 hours (magnification 10×) for Matrigel-coated and uncoated filters. Bar graphs show the average count of cells traversing each filter, with three replicates per group. The crystal violet staining method was used to visualize and quantify the invasion and migration of cells. (D) Quantitative analysis demonstrating significantly reduced cell numbers in the si-PLAU group as compared with the NC and siRNA groups. (E) Western blot analysis revealing marked PDPN suppression but elevated ITGA5 protein levels following PLAU knockdown in FaDu cells. (F) The protein expression levels of p-EMT-related markers, including ITGA5 and podoplanin, were examined in FaDu cells from three treatment groups: NC control, siRNA transfection, and si-PLAu transfection. Asterisks denote statistically significant differences according to the two-sample t-test (P<0.05). Data are the mean ± SEM from three biological replicates. *, P<0.05; **, P<0.01. NC, normal control; OD, optical density; SEM, standard error of mean.

Discussion

In recent years, bioinformatics analysis of high-throughput tumor-related datasets has been widely applied to clarify the genetic changes related to malignancy. According to numerous studies using single-cell sequencing, the p-EMT process is highly implicated in the invasiveness of HNSCC (13). The ITGA5 and PDPN genes were identified as key genes in the p-EMT process. In our study, bioinformatics analysis revealed a strong association between PLAU expression and p-EMT marker levels and demonstrated its prognostic significance in patients with HNSCC. Moreover, according to data from TCGA, PLAU is strongly associated with lymph node metastasis (Table 1).

GO and KEGG function enrichment analyses of PLAU-related mRNAs were conducted. The results suggested that PLAU expression in HNSCC was associated with several biological processes, such as cell-substrate junction, focal adhesion, extracellular matrix, and integrin binding. The majority of enriched pathways and biological processes identified have established roles in modulating cancer metastasis.

We further aimed to determine the function and clinical significance of PLAU’s role in p-EMT among patients with HNSCC. PLAU encodes urokinase plasminogen activator (uPA) which contributes to transforming inactive plasminogen into active plasminogen. High levels of PLAU gene expression have been detected in various tumor types, are correlated to overall survival rate, and may therefore be used as an independent predictor of prognosis. Moreover, elevated levels of PLAU in cancer tissue compared to normal tissue have been shown to be a risk factor contributing to lymphatic metastasis in patients with tongue cancer (21). ITGA5 and PDPN are p-EMT markers. Integrins are heterodimeric transmembrane glycoproteins consisting of an α and β subunit. ITGA5 is a transmembrane receptor that promotes cell-ECM adhesion. Ligand binding to the integrin can activate a series of signal transduction pathways that regulate processes including cell cycle and cytoskeleton organization (22). PDPN is a type 1 transmembrane sialomucin-like glycoprotein that belongs to the podoplanin family (23,24) and plays a crucial role in tumor migration and invasion, thus contributing to a poor prognosis. For instance, a high expression of PDPN in esophageal squamous carcinoma, especially in cells at the invasive edge, was reported to be correlated with poor prognosis (25,26). However, the function of PLAU and the mechanisms by which it contributes to p-EMT in HNSCC remain unclear.

In our study, we further elucidated the functions of PLAU in controlling p-EMT by knocking down PLAU with siRNA in the HEp-2 and FaDu cell lines. As anticipated, the PLAU-knockdown group exhibited reduced cell proliferation, migration, and invasion. Surprisingly, PLAU knockdown also led to the upregulation of ITGA5, which differs from the coexpression relationship identified in GEPIA. ITGA5 can bind to the β1 (TGF-β1) subunit of integrin to form the integrin α5β1 heterodimer, which can recognize specific fibronectins (27). ITGA5 has been confirmed to function in oral squamous cell carcinoma (OSCC) via the PI3K/AKT pathway (28), and it has been demonstrated that PLAU can increase cell proliferation and migration depending on levels of uPAR and integrin α5β1 (14,29,30). Combination of uPA and uPAR on the cell surface initiates a protease cascade, which includes the formation of plasmin and an interaction between uPAR and the α5β1 and αvβ5 integrins (31). Activation of uPA with uPAR at cellular plasmin leads to the release of cytokines and pro-matrix metalloproteinases (MMPs) as well as the breakdown of ECM components at the leading edges, providing a path for cells to migrate to distant sites (32). Therefore, knockdown of PLAU can reduce interaction with the uPA receptor, thereby suppressing the protein cascade reaction and reducing the consumption of ITGA5. This can explain the inconsistent results between GEPIA and our study.

Additionally, it has been demonstrated that the activity of MMPs may degrade the tumor-surrounding ECM and thus promote PDPN-mediated invasion (32-34). Moreover, PDPN has been shown to be involved in the collective migration of MCF7 cells without inducing EMT (35) and that CAFs may mediate PDPN expression (36). Overexpression of PDPN in OSCC cells enhances fibroblast invasiveness through MMP2, MMP14, and αv/β6 integrin (ITGA5/ITGB6)-mediated signaling. Additionally, CAF can trigger PDPN upregulation via TGF-β1/Smad-mediated signaling. Therefore, PLAU is likely to trigger p-EMT by inducing the release of MMPs and thereby reshaping the ECM degradation process.

PLAU expression can also activate the signaling pathways downstream of Akt and Rac1, which can promote cell invasion under hypoxic conditions (37). Hence, in line with our findings from the KEGG pathway analysis, PI3K/Akt may be a potential regulation pathway. Moreover, a study based on the TCGA-HNSC cohort and neck node status found that a higher expression of PLAU in patients with HNSCC was significantly associated with positivity for human papillomavirus and that PLAU may be an independent predictor of prognosis in patients with HNSCC (38).

Conclusions

The upregulation of PLAU in patients with HNSCC was associated with a poor prognosis. Knocking down PLAU expression significantly inhibited HNSCC cell proliferation, cell invasion, and metastasis. Most importantly, PLAU expression levels influenced the expression of p-EMT-related markers, including ITGA5 and PDPN. The results indicate that PlAU can serve as a valuable biomarker for HNSCC and can alter the p-EMT process via ECM remodeling. However, the changes in the tumor microenvironment related to how PLAU affects p-EMT and the molecular signaling pathways involved have not been clarified. In the cell models used in this study, PLAU expression was positively correlated with p-EMT, and this effect may be primarily mediated through uPAR-dependent mechanisms. However, the independent contribution of uPA-HSPG interactions cannot be excluded at this stage and remains to be further validated in future studies. In subsequent work, we hope to apply in vivo experiments to verify the effect of PLAU on the tumor microenvironment and identify the signaling pathways contributing to p-EMT.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0478/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0478/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0478/prf

Funding: This study was supported by the Hebei Natural Science Foundation (No. H2020206475).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0478/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Committee of The Second Hospital of Hebei Medical University (No. 2020-R561). Informed consent was provided by all patients before surgery.

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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