SPP1 promotes lymph node metastasis and malignant progression of head and neck squamous cell carcinoma by activating FAK-AKT signaling pathway

Abstract

Background

Lymph node metastasis is a critical determinant of poor prognosis in head and neck squamous cell carcinoma (HNSCC). Secreted phosphoprotein 1 (SPP1) has been implicated in the progression of multiple malignancies; however, its clinical relevance and potential molecular mechanisms in HNSCC remain not fully understood. This study aims to comprehensively characterize the expression profile, biological functions, and potential molecular mechanisms of SPP1 during HNSCC progression.

Methods

Public datasets were analyzed to assess SPP1 expression and its association with clinicopathological features and patient outcomes. Forty surgical specimens of head and neck squamous cell carcinoma (including adjacent normal tissues, non-metastatic tumors and lymph node metastatic tumors) were collected and verified by immunohistochemistry, quantitative PCR and Western blotting. Then, cellular functional assays including CCK-8, EdU synthesis, wound healing, and Transwell assays were performed to evaluate the effects of SPP1 knockdown and overexpression on the proliferation and invasion of head and neck squamous cell carcinoma cells. Transcriptome sequencing was performed in SPP1 knockdown cells and control cells (n = 3 in each group), followed by differential expression analysis, enrichment analysis, and gene set enrichment analysis to explore potential mechanisms.

Results

The results showed that SPP1 expression was significantly increased in HNSCC tissues compared with adjacent normal tissues and was associated with lymph node metastasis, advanced tumor stage, and poor prognosis. Experimental validation in clinical samples confirmed that SPP1 expression was increased in tumors, especially in cases with lymph node metastasis. The results of the cell function experiments showed that the knockout of the SPP1 gene significantly inhibited the proliferation and migration abilities of HNSCC, while when the SPP1 gene was overexpressed, these functions of the cells were enhanced. Transcriptomic profiling identified 1,853 differentially expressed genes following SPP1 knockdown, with enrichment in pathways related to extracellular matrix organization, focal adhesion, and oncogenic signaling. Gene set enrichment analysis further supported the involvement of extracellular matrix remodeling and migration-related biological processes.

Conclusions

SPP1 promotes the malignant progression of HNSCC and is closely associated with lymph node metastasis. Its tumor-promoting effects may be mediated, at least in part, through regulation of extracellular matrix- and adhesion-related signaling pathways. These findings highlight SPP1 as a potential biomarker and therapeutic target in HNSCC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12957-026-04377-5.

Introduction

Cancer progression is a dynamic and multifactorial process involving genetic alterations, tumor microenvironment remodeling, and continuous evolution of therapeutic strategies. Despite substantial advances in cancer treatment, metastasis remains the leading cause of cancer-related mortality [1]. Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies worldwide and is associated with high morbidity and mortality [2]. Despite the advancements in surgery, radiotherapy, chemotherapy and immunotherapy, the overall survival rate of patients with HNSCC has not significantly improved over the past few decades [3, 4]. This is largely attributed to the high incidence of regional lymph node metastasis and locoregional recurrence, both of which are strongly associated with poor survival outcomes [5, 6].

Lymph node metastasis is an important prognostic factor in head and neck squamous cell carcinoma, which is closely related to the advanced stage of the tumor, treatment resistance and poor clinical prognosis [2, 7]. Tumor invasion and metastasis are complex biological processes, in which there is a dynamic interaction between tumor cells and tumor microenvironment [8]. These processes include extracellular matrix (ECM) remodeling, immune regulation, angiogenesis, and activation of multiple signaling pathways [9–11]. Notably, signaling pathways related to cell adhesion and migration, such as focal adhesion kinase (FAK) and protein kinase B (AKT) pathways, which have been widely implicated in tumor progression and signaling regulation [12–14]. Therefore, it is important to investigate the key molecules that promote lymphatic metastasis and their drivers to improve risk assessment as well as to develop new treatment strategies.

Secreted phosphoprotein 1 (SPP1), encoded by the SPP1 gene, is also known as osteopontin (OPN). It is a highly phosphorylated glycoprotein that participates in various biological processes, including inflammation, cell survival, and wound healing [15, 16]. Increasing evidence has shown that SPP1 is abnormally overexpressed in a variety of malignant tumors and plays a key role in the development, invasion and metastasis of tumors [17, 18]. SPP1 is widely distributed in a variety of tissues and is also produced in large amounts by cancer cells. Previous studies have shown that elevated SPP1 expression levels are associated with poor prognosis in a variety of cancers [19–21]. In addition, emerging evidence suggests that SPP1 can promote tumor invasion and metastasis by interacting with integrins and activating downstream signaling pathways, including FAK-AKT pathway, to regulate cell adhesion and migration [22].

In HNSCC, many existing studies have shown that SPP1 expression is significantly higher in tumor tissues than in normal mucosal tissues and may be associated with tumor progression as well as patient survival [23]. However, although SPP1 has been reported to be upregulated in HNSCC and associated with tumor progression, its specific role in lymph node metastasis and the underlying molecular mechanisms remain insufficiently understood. In particular, whether SPP1 contributes to HNSCC progression through defined signaling pathways has not been fully elucidated. Therefore, the present study aimed to systematically investigate the clinical relevance and biological functions of SPP1 in HNSCC, with a particular focus on lymph node metastasis. Furthermore, we sought to explore the potential molecular mechanisms underlying its effects, especially its involvement in adhesion-related signaling pathways such as the FAK–AKT axis.

Materials and methods

TCGA data mining and bioinformatics analysis

RNA-seq (FPKM) expression data and corresponding clinical information for TCGA-HNSC (tumor :502 and normal samples :44) were downloaded from The Cancer Genome Atlas (https://portal.gdc.cancer.gov/). Clinical variables included age, sex, survival status, and AJCC T and N stage. Ethical approval was not required. SPP1 expression was compared between clinical subgroups (N0 vs. N+, T1–2 vs. T3–4). Differentially expressed genes (DEGs) between tumor and normal samples were identified using |log2 fold change| > 1 and FDR < 0.05, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Survival analyses were performed to evaluate the prognostic value of SPP1. Although TCGA provides a valuable resource for large-scale transcriptomic analysis, it is important to note that bulk RNA-seq data may be affected by tumor heterogeneity and technical biases. Therefore, the findings derived from TCGA analyses should be interpreted with caution and further validated through experimental approaches.

HNSCC clinical specimens

The 40 pairs of HNSCC tissue samples and their adjacent normal tissue samples in this study were collected from the First Affiliated Hospital of Chongqing Medical University between 2023 and 2025. All the samples met the condition of not having received chemotherapy or radiotherapy before the surgery. The collected tissues were stored at − 80 ° C for use in subsequent experiments. This research has been approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. All participants signed written informed consent forms.

Cell culture

The human pharyngeal squamous cell carcinoma cell line FaDu was purchased from Cellverse Co., Ltd. (Shanghai), and the tongue squamous cell carcinoma cell line SCC − 15 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai). Cells were cultured in DMEM (Procell, China) medium supplemented with 10% FBS and 1% penicillin-streptomycin solution. The cells were cultured in an incubator at 37 °C with 5% CO₂, and the culture medium was replaced every 1 to 2 days.

Immunohistochemistry (IHC)

Immunohistochemical (IHC) staining was performed on 40 HNSCC tissue samples, including 22 node-positive (LNM+) samples and 18 node-negative (LNM-) samples. The collected surgical tissues were fixed with 4% paraformaldehyde (PFA), embedded in paraffin and then sectioned. Paraffin sections were oven dried for 3 to 6 h, then subjected to successively different concentrations of alcohol and immersed in xylene solution for 20 min. EDTA (pH 9.0) was heated to approximately 100 °C, sectioned for 15 min and allowed to cool naturally at room temperature. This was followed by a 15-minute immersion with catalase inhibitors. Then, the slices were treated with goat serum (Biosharp) at room temperature for 15 to 30 min. Additional primary antibodies (1:300 dilution; Proteintech, China) were added and placed in a wet box at 4 °C overnight. Hrp-labeled goat secondary antibody conjugated to rabbit anti-protein was dripped after washing with PBS for 10 min and placed in the incubator to rest for 30 min. Then, DAB staining and hematoxylin counterstaining were performed. After dehydration treatment, slides were immersed in neutral resin and three randomly selected areas from each section were observed using an Olympus microscope (200x, 400x magnification). The immunohistochemical score was calculated as the positive intensity multiplied by the percentage of positive cells. Negative: 0, weak positive: 1, positive: 2, strong positive: 3; In the percentage of positive cells, < 25% : 1, 26–50% : 2, 51–75% : 3, > 75% : 4. The immunohistochemical score was calculated as the positive intensity multiplied by the percentage of positive cells. Patients were divided into SPP1-high and SPP1-low groups based on the median H-score as the cutoff value.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from tissue samples using the SteadyPure RNA Extraction Kit (AG Biology, China) according to the manufacturer’s protocol. RNA concentration and purity were determined by spectrophotometry, where samples with A260/A280 ratio between 1.8 and 2.1 were considered eligible for subsequent experiments. Complementary DNA (cDNA) was synthesized with ABScript Neo RT Master Mix and qPCR was performed with gDNA Remover (ABclonal, China). The resulting cDNA was diluted fivefold with nuclease-free water before quantitative analysis. Quantitative real-time PCR was performed using 2× Universal SYBR Green Fast qPCR Mix (ABclonal, China) on a real-time PCR system (Bio-Rad CFX96). Polymerase chain reaction (PCR) cycles were performed after the addition of primers (Sangon Biotech, China): A 30-s denaturation treatment at 95 °C was followed by 40 5-s denaturation treatment cycles each and a final 30-s annealing/extension treatment at 60 °C. GAPDH was used as the internal reference gene. The relative expression level of mRNA was calculated using the 2^−ΔΔCt method. Each sample was analyzed in triplicate.

Protein extraction and Western blot analysis

It was extracted at a ratio of 100:1:2 using RIPA buffer (Beyotime, China) supplemented with PMSF (Biosharp, China) as well as phosphatase inhibitor mixture (Beyotimel, China) to ensure that the phosphorylated state of the protein was maintained. After dissociation on ice for 30 min, the samples were centrifuged at 12,000×g for 15 min at 4 °C, and then the supernatants were collected. Protein concentrations were determined using a BCA kit (Beyotime, China). The total amount of protein was set to 30 µg and electrophoresis was performed at 80 V followed by electroconversion at 300 mA. Membranes were blocked with 5% nonfat milk for 2 h and then incubated with primary antibodies (1:2000) overnight at 4 ° C. After washing with TBST, secondary antibodies (1:500) were incubated for 1 h and kept at room temperature. Protein bands were visualized using ECL reagent (SparkJade, China) with a Bio-Rad imaging system. GAPDH was used as the internal control. The developed bands were quantified using ImageJ. All experiments were performed in triplicate.

Lentiviral transduction and establishment of stable cell lines

FaDu and SCC-15 cell lines were seeded into 6-well plates at a density of 5 × 10⁴ cells per well. When cell confluence reached approximately 50%, cells were infected with lentivirus (Tsingke Biotechnology Co., Ltd., China) at a multiplicity of infection (MOI) of 10. After 8 h of incubation, the medium was replaced with complete culture medium. Transduction efficiency was evaluated under a fluorescence microscope. Stable cell lines were selected using puromycin (5 µg/mL), with medium refreshed every two days. After 72 h of selection, surviving cells were considered stably transduced. The efficiency of transduction was further validated by Western blot and quantitative real-time PCR.

Cell counting kit 8

A cell counting kit (CCK-8; Beyotime, China) was used. Lentivirus-transduced FaDu and SCC-15 cells were seeded into 96-well plates at a density of 1,000 cells per well. Cell viability was measured at 0, 24, 48, 72, 96, and 120 h. At each time point, The plates were incubated at 37 ° C for 1 h after the addition of reagents, and subsequently the absorbance at 450 nm was recorded using a microplate reader. Blank wells containing medium only were used for background subtraction. Each experiment was repeated three times.

EdU assay

An EdU assay kit (Beyotime, China) was used. Using 96-well plates, each well was seeded with 2000 cells. After 24 h, EdU solution was added to continue incubation in the incubator for 2 h. Then follow the instructions in the kit. Finally, observe and count using a fluorescence microscope. Each experiment was repeated three times.

Wound healing assay

Cells were cultured using 6-well culture plates until the density reached more than 90%. A sterile 200 µl pipetter tip was used to make a scratch, and then the exfoliated cells were washed with PBS. Cells were then cultured in serum-free medium during the migration assay. Wound images were captured at fixed positions under a microscope at 0, 24, and 36 h for the knockdown group, and at 0, 12, and 24 h for the overexpression group. The remaining wound area at each time point was quantified using ImageJ, and the wound closure rate was calculated. Each experiment was repeated three times.

Transwell migration assay

Cell migration was assessed using Transwell inserts (BIOFIL Tissue Culture Plate Insert). The cells (5 × 10⁴ cells/well) were seeded in the upper chamber and cultured with serum-free medium, and the lower chamber was added with medium containing 20% FBS. After 36 h, the cells were washed with PBS, and the migrated cells were fixed with 4% paraformaldehyde (Servicebio, China) and stained with crystal violet (Beyotime, China). The inserts were gently washed with PBS to remove excess dye.

Five areas per well were selected, photographed under a microscope, and quantified by ImageJ. Each experiment was repeated three times.

Transwell invasion assay

Cell invasion was assessed using Transwell inserts (BIOFIL Tissue Culture Plate Insert) pre-coated with Matrigel (Corning, USA). Briefly, Matrigel was applied to the upper chamber and allowed to solidify at 37 °C. Cells (5 × 10⁴ cells/well) were seeded into the upper chamber in serum-free medium, while the lower chamber contained medium with 20% FBS.After 36 h of incubation, non-invading cells were removed from the upper surface. Invaded cells on the lower surface were fixed, stained with crystal violet, and quantified under a microscope using ImageJ. Each experiment was performed in triplicate.

Transcriptome sequencing analysis

Transcriptome sequencing was performed on three SPP1-knockdown FaDu samples and three control FaDu samples. Differential expression analysis and functional enrichment analysis were conducted using the BMKCloud online platform (www.biocloud.net).

Statistical analysis

R software (version 4.2.0) and GraphPad Prism (version 9.0) were used. The results were expressed as mean ± standard deviation (SD). Comparisons between two groups were performed using Student’s t test or the Mann–Whitney U test, as appropriate. Comparisons among multiple groups were conducted using one-way ANOVA. For TCGA data, the Wilcoxon rank-sum test or Kruskal–Wallis test was applied. Kaplan-Meier method and log-rank test were used for survival analysis, and Cox regression model was used for univariate and multivariate analysis. Two-sided P values of less than 0.05 were considered to indicate statistical significance.

Results

SPP1 is significantly upregulated in HNSCC and predicts poor prognosis

SPP1 expression in HNSCC was first analyzed using the TCGA-HNSC dataset. Differential expression analysis showed that the expression of SPP1 in tumor tissues was higher than that in normal tissues, as illustrated by the volcano plot (Fig. 1A), heatmap (Fig. 1B), and boxplot analysis (Wilcoxon test, p = 1.1 × 10⁻¹³; Fig. 1C). Kaplan-Meier survival analysis showed that the overall survival of patients with high SPP1 expression was significantly lower than that of patients with low SPP1 expression (log-rank p = 0.027; Fig. 1D). Multivariate Cox regression analysis further identified SPP1 expression as an independent prognostic factor after adjustment for clinicopathological variables (Fig. 1E). In addition, SPP1 expression was significantly higher in patients with lymph node metastasis (N⁺) compared with those without metastasis (N0) (p = 0.0092; Fig. 1F), and was also elevated in advanced T stage tumors (T3–T4) compared with early-stage tumors (T1–T2) (p = 0.00094; Fig. 1G). These results indicate that increased SPP1 expression is associated with tumor progression and metastatic potential in HNSCC.

Fig. 1.

Bioinformatic evidence supporting the clinical significance of SPP1 in head and neck squamous cell carcinoma (HNSCC). A Volcano plot showing differentially expressed genes between tumor and normal tissues in the TCGA-HNSCC cohort. Red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots indicate genes without significant differential expression. B Heatmap of the top 50 differentially expressed genes between tumor and normal samples, with SPP1 highlighted. C Boxplots showed that the expression of SPP1 in tumor tissues was significantly higher than that in normal tissues in TCGA-HNSCC dataset (Wilcoxon test, p = 1.1 × 10^-13). D Kaplan-Meier overall survival analysis was compared between patients with high and low SPP1 expression (top 25% vs. bottom 25%) in the TCGA-HNSCC cohort. (p = 0.027, log-rank test). E The prognostic value of SPP1 expression after adjusting for clinicopathological factors was demonstrated. F Comparison of SPP1 expression between lymph node-negative (N0) and lymph node-positive (N+) patients in TCGA-HNSCC, showing significantly higher SPP1 levels in the N+ group. G Comparison of SPP1 expression in early (T1-T2) and advanced (T3-T4) tumors showed that SPP1 expression was increased in advanced tumors.

SPP1 is upregulated in clinical HNSCC tissues and correlates with lymph node metastasis

Immunohistochemical (IHC) staining showed that SPP1 protein expression was significantly higher in HNSCC tissues than in adjacent normal tissues, with the strongest staining observed in lymph node metastatic tumors (Fig. 2A). H-score analysis further confirmed that SPP1 expression was significantly increased in the lymph node metastatic group compared with the non-metastatic group and normal tissues (Fig. 2B). Western blot analysis of representative clinical samples demonstrated a stepwise increase in SPP1 protein levels from normal tissues to non-metastatic tumors and metastatic tumors, which was supported by densitometric quantification (Fig. 2C). Consistently, quantitative real-time PCR analysis showed that SPP1 mRNA expression was significantly elevated in HNSCC tissues, particularly in tumors with lymph node metastasis (Fig. 2D). In addition, clinicopathological analysis of 40 HNSCC patients revealed that high SPP1 expression was significantly associated with poor differentiation (P < 0.001) and lymph node metastasis (P = 0.027), but not with gender, age, T stage, tumor site, or smoking history (Table 1).

Fig. 2.

SPP1 expression is increased in metastatic HNSCC tissues. A Representative immunohistochemical (IHC) staining images of SPP1 in normal tissues (NT), non-metastatic HNSCC [LNM(−)], and lymph node metastatic HNSCC [LNM(+)]. Upper panels show low-magnification images, and lower panels show corresponding high-magnification views. Scale bars: 50 μm (upper) and 25 μm (lower). B Quantification of SPP1 expression by IHC H-score in NT, LNM(−), and LNM(+) tissues. C Representative western blot images showing SPP1 protein expression in NT, LNM(−), and LNM(+) tissues, with GAPDH as a loading control, and corresponding densitometric analysis. D qRT-PCR analysis of SPP1 mRNA expression. Data are presented on a log10 scale as relative expression (2^−ΔΔCt). Data are shown as individual data points. Kruskal-Wallis test and Dunn’s multiple comparison test were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Table 1.

Association between SPP1 expression and clinicopathological characteristics in patients with HNSCC

Clinicopathological characteristics	Total (n = 40)	Expression of SPP1		P value
		Negative(n = 9)	Positive(n = 31)
Gender
Male	37 (92.5)	8 (88.9)	29 (93.5)	0.545
Female	3 (7.5)	1 (11.1)	2 (6.5)
Age (Years)
≤ 60	12 (30.0)	2 (22.2)	10 (32.3)	0.697
> 60	28 (70.0)	7 (77.8)	21 (67.7)
Differentiation
Poorly	23 (57.5)	0 (0.0)	23 (74.2)	<0.001
Moderately /Well	17 (42.5)	9 (100.0)	8 (25.8)
T stage
Ⅰ+Ⅱ	13 (32.5)	3 (33.3)	10 (32.3)	0.690
Ⅲ+Ⅳ	27 (67.5)	6 (66.7)	21 (67.7)
N stage
N0	22 (55.0)	8 (88.9)	14 (45.2)	0.027
N+	18 (45.0)	1 (11.1)	17 (54.8)
Tumor site
Larynx	27 (67.5)	6 (66.7)	21 (67.7)	0.690
Hypopharynx	13 (32.5)	3 (33.3)	10 (32.3)
Smoking history
Yes	35 (87.5)	6 (66.7)	29 (93.5)	0.065
No	5 (12.5)	3 (33.3)	2 (6.5)

Values are presented as n (%). P values were calculated using the chi-square test or Fisher’s exact test, as appropriate

Establishment and validation of SPP1 knockdown and overexpression in HNSCC cell lines

Stable SPP1 knockdown and overexpression models were generated in FaDu and SCC-15 cells. Western blot analysis demonstrated that SPP1 protein levels were markedly reduced in FaDu and SCC-15 cells transduced with two independent shRNAs targeting SPP1 compared with shCtrl cells (Fig. 3A, C).which was further supported by densitometric analysis. Consistently, qRT-PCR showed significant downregulation of SPP1 mRNA following SPP1 silencing (Fig. 3B, D). Conversely, SPP1 overexpression led to a pronounced increase in SPP1 protein abundance in FaDu and SCC-15 cells (Fig. 3E, G), accompanied by elevated SPP1 mRNA expression (Fig. 3F, H). Together, these results confirm successful establishment of SPP1 knockdown and overexpression cell models for subsequent functional assays.

Fig. 3.

Validation of SPP1 knockdown and overexpression efficiency in HNSCC cell lines. A, B The knockdown efficiency of SPP1 in FaDu cells was confirmed by Western blotting and quantitative real-time PCR (qRT-PCR). C, D The knockdown efficiency of SPP1 in SCC-15 cells was further validated by WB and qRT-PCR. E, F The protein and mRNA levels of SPP1 were successfully overexpressed in the cells. G, H SPP1 overexpression in SCC-15 cells was similarly confirmed using WB and qRT-PCR. GAPDH was used as an internal control

SPP1 promotes the migratory and invasive abilities of HNSCC cells in vitro

Wound healing assays showed that SPP1 knockdown reduced the migratory capacity of both FaDu and SCC-15 cells compared with control cells (Fig. 4A). In contrast, SPP1 overexpression markedly enhanced wound closure in both cell lines (Fig. 4B). Consistent with these findings, Transwell migration assays demonstrated that silencing of SPP1 significantly decreased the number of migrated cells, whereas enforced SPP1 expression increased cell migration in both FaDu and SCC-15 cells compared with their respective controls (Fig. 4C, D). Furthermore, Matrigel invasion assays revealed that SPP1 knockdown significantly impaired the invasive ability of HNSCC cells, while SPP1 overexpression markedly enhanced cell invasion (Fig. 4E). Taken together, these results suggest that SPP1 promotes both the migratory and invasive capacities of HNSCC cells in vitro.

Fig. 4.

SPP1 promotes migration and invasion of HNSCC cells in vitro. A Knockdown of SPP1 significantly inhibited the migration ability of FaDu and SCC-15 cells. Compared with the control cells, the difference was significant. B On the contrary, overexpression of SPP1 significantly enhanced the migration ability of FaDu and SCC-15 cells, and the related images and quantitative analysis were also shown. C Knockdown of SPP1 significantly reduced the number of cell migration. D Overexpression of SPP1 significantly enhanced cell migration ability. E Transwell invasion assays using Matrigel-coated inserts demonstrated that SPP1 knockdown significantly decreased, whereas SPP1 overexpression increased, the invasive capacity of HNSCC cells. Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by post hoc multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

SPP1 promotes proliferation of HNSCC cells in vitro

The effects of SPP1 on cell proliferation were evaluated through EdU assay and CCK-8 assay. EdU assays showed that SPP1 knockdown significantly decreased the proportion of EdU-positive cells in both FaDu and SCC-15 cells compared with shCtrl, whereas SPP1 overexpression markedly increased EdU incorporation (Fig. 5A). Similarly, CCK-8 assays demonstrated that silencing SPP1 suppressed cell growth over time, while SPP1 overexpression promoted proliferation in both cell lines (Fig. 5B). These findings suggest that SPP1 enhances the proliferative capacity of HNSCC cells in vitro.

Fig. 5.

SPP1 promotes proliferation of HNSCC cells in vitro. A EdU incorporation assays showing that SPP1 knockdown (shSPP1#1 and shSPP1#2) reduced, whereas SPP1 overexpression (SPP1 OE) increased, the proliferative capacity of FaDu and SCC-15 cells compared with their respective controls (shCtrl or Vector). Representative images and quantification of EdU-positive cells are shown. B CCK-8 assays demonstrating that SPP1 knockdown inhibited, whereas SPP1 overexpression enhanced, proliferation of FaDu and SCC-15 cells at the indicated time points. Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by post hoc multiple comparisons test

Transcriptomic profiling identifies ECM remodeling and migration-related pathways following SPP1 knockdown

To further explore the molecular mechanisms involved in SPP1, transcriptome sequencing was performed in shSPP1 and shCtrl FaDu cells (n = 3 per group). PCA revealed distinct differences between the two groups, indicating that the two groups had distinct transcriptomic outcomes after SPP1 knockdown (Fig. 6A). The heat map further confirmed the consistency of the expression pattern within each group, while also clarifying the differences between the different groups (Fig. 6C). Differential expression analysis revealed that the expression of 1853 genes was significantly changed by SPP1 knockdown, including 654 up-regulated genes and 1199 down-regulated genes (Fig. 6B), suggesting that SPP1 regulates a wide range of transcriptional programs in HNSCC cells. The results showed that these differentially expressed genes were closely related to the pathways involved in tumor progression and interaction between cells and microenvironment. KEGG pathway analysis showed that there were significant enrichment in focal adhesion, extracellular matrix-receptor interaction, PI3K-Akt signaling pathway and cancer-related pathways (Fig. 6D). Consistent with these results, Gene ontology (GO) bioprocess analysis revealed significant enrichment of components related to the regulation of cell migration, intracellular signaling, regulation of angiogenesis, and regulation of cell death (Fig. 6E). Gene sets associated with collagen trimer organization were significantly enriched in shCtrl cells, whereas pathways related to intrinsic apoptotic signaling and guanyl-nucleotide exchange factor activity were suppressed following SPP1 knockdown (Fig. 6F). Together, these transcriptomic changes support a role for SPP1 in regulating extracellular matrix remodeling, cell migration, and survival-related signaling, which is consistent with the phenotypic effects observed in functional assays. Among these, several genes related to extracellular matrix organization and focal adhesion, such as COL、FN1、MMP and ITGA、ITGB、VCL, were significantly altered following SPP1 knockdown. The top differentially expressed genes are listed in Supplementary Table S1.

Fig. 6.

Transcriptomic profiling reveals ECM- and migration-related pathway alterations following SPP1 knockdown. A PCA of the RNA sequencing data showed a clear distinction between shSPP1 samples and shCtrl samples (three samples in each group). B The volcano plot shows the profile of differentially expressed genes between shSPP1 cells and shCtrl cells. Red dots represent up-regulated genes, blue dots represent down-regulated genes, and grey dots represent genes with no significant difference (threshold: |log2FC| ≥ 1, FDR < 0.05). C Heat maps of differentially expressed genes showed significantly different expression patterns between the shSPP1 and shCtrl groups. D The KEGG results focus on the pathways related to cancer and the microenvironment. E Gene ontology (GO) biological process enrichment analysis of differentially expressed genes. F Representative gene set enrichment analysis (GSEA) plots showed that collagen trimer, endogenous apoptosis signaling pathway, and guanyl exchange factor activity gene sets were significantly enriched in shSPP1 cells compared with shCtrl cells.

SPP1 activates the FAK–AKT signaling pathway in HNSCC cells

To further investigate the molecular mechanisms underlying the pro-tumorigenic effects of SPP1, we examined the activation status of the FAK–AKT signaling pathway by Western blot analysis. The results showed that SPP1 knockdown markedly reduced the phosphorylation levels of FAK (p-FAK) and AKT (p-AKT) in FaDu cells, while the total protein levels of FAK and AKT remained largely unchanged (Fig. 7A, B). Conversely, overexpression of SPP1 in SCC-15 cells significantly increased the phosphorylation of FAK and AKT, without affecting the total levels of these proteins (Fig. 7C, D). Quantitative analysis further confirmed that the ratios of p-FAK/FAK and p-AKT/AKT were significantly decreased in SPP1 knockdown cells and increased in SPP1-overexpressing cells.

Fig. 7.

SPP1 activates the FAK–AKT signaling pathway in HNSCC cells. A Western blot analysis of p-FAK, FAK, p-AKT, and AKT expression in SPP1 knockdown cells. B Quantification of p-FAK/FAK and p-AKT/AKT ratios in knockdown cells. C Western blot analysis in SPP1 overexpression cells. D Quantification of p-FAK/FAK and p-AKT/AKT ratios in overexpression cells. Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using Student’s t-test or one-way ANOVA as appropriate

These findings provide direct experimental evidence that SPP1 promotes activation of the FAK–AKT signaling pathway in HNSCC cells.

Discussion

HNSCC remains a highly challenging malignancy. Despite advances in surgery, radiotherapy, and chemotherapy, the overall survival rate of patients remains unsatisfactory [3, 24]. High rates of regional lymph node metastasis and locoregional recurrence are the main causes of treatment failure and poor prognosis in HNSCC, as lymph node metastasis is an established independent prognostic factor and is closely associated with reduced survival [25]. Therefore, identifying key molecules involved in lymphatic dissemination and malignant progression is of considerable clinical importance. In this study, we systematically investigated the clinical relevance and biological function of SPP1 in HNSCC. Our analyses based on public datasets and clinical specimens demonstrated that SPP1 expression is significantly upregulated in HNSCC with lymph node metastasis and is closely associated with lymph node involvement and poor clinical outcomes. These findings suggest that SPP1 may serve as a potential biomarker for aggressive disease and metastatic risk in HNSCC.

SPP1, also known as osteopontin, is a multifunctional secreted glycoprotein involved in various biological processes, including inflammation [26], extracellular matrix remodeling, cell adhesion, and immune regulation [27]. Accumulating evidence indicates that SPP1 acts as a key regulator in the progression of multiple malignancies. Elevated SPP1 expression has been reported to promote tumor growth and metastasis in breast cancer [28], hepatocellular carcinoma [29], colorectal cancer [30], lung cancer [31], and glioma, and is frequently associated with poor patient prognosis [32]. Mechanistically, SPP1 can interact with integrins and CD44 receptors, thereby regulating the interaction between cells and the extracellular matrix, and activating downstream signaling pathways involved in cytoskeleton remodeling, migration, and survival [33–35]. Although there are research reports indicating that compared with normal mucosa, the expression of SPP1 is increased in HNSCC tissues [23], its specific role in lymph node metastasis and malignant progression in this tumor type has not been fully elucidated. Our findings extend these observations by demonstrating a stepwise increase in SPP1 expression from normal tissues to non-metastatic tumors and lymph node metastatic tumors. Functional assays further demonstrated that SPP1 contributes to malignant phenotypes in HNSCC cells.

Using both knockdown and overexpression approaches in two independent cell lines, SPP1 was found to enhance cell proliferation, migration, and invasion. These results are consistent with previous studies in other tumor types, where SPP1 has been shown to promote tumor cell motility, invasion, and growth [4], and further support a role for SPP1 in the malignant progression of HNSCC.

To further explore the molecular basis of these phenotypic changes, we performed transcriptome analysis following SPP1 knockdown. Differential expression and enrichment analyses indicated that SPP1-associated genes were primarily enriched in pathways related to extracellular matrix organization, focal adhesion, and oncogenic signaling. These pathways are known to play central roles in tumor cell–microenvironment interactions and metastatic dissemination [36, 37]. In particular, signaling pathways associated with cell–matrix interactions, including the PI3K–AKT pathway, are widely recognized to regulate cancer cell survival, migration, and invasion [37, 38]. Previous studies have shown that SPP1 can regulate integrin-mediated signaling in multiple tumor types [39], Consistent with these observations, our results suggest that SPP1 may promote HNSCC progression by modulating extracellular matrix remodeling and adhesion-related signaling networks. In addition, recent studies have highlighted the importance of tumor microenvironment remodeling and metabolic reprogramming in cancer progression [40]. Increasing evidence suggests that metabolic–microenvironmental interactions contribute to tumor invasion and metastatic dissemination by reshaping extracellular matrix dynamics and associated signaling networks [40, 41]. Furthermore, inflammation-related biomarkers have been increasingly recognized as important indicators of tumor progression and prognosis, reflecting the complex interplay between tumor cells and the immune microenvironment [41]. Meanwhile, pan-cancer multidimensional molecular analyses have provided new insights into the coordinated regulation of oncogenic signaling pathways across different tumor types [42]. Taken together, these findings support the concept that tumor progression is driven by integrated signaling networks rather than individual pathways. In this context, our results indicating that SPP1 promotes HNSCC progression through activation of the FAK–AKT signaling axis are consistent with this broader biological framework [43].)

It should be noted that this study has some limitations. Firstly, although transcriptome analysis has provided us with crucial information about the potential signaling pathways related to SPP1, we have not directly investigated the key downstream mechanisms at the protein level. Additional mechanistic experiments, For instance, the verification of pathway activation and the functional recovery experiments, etc., all require additional confirmation in order to clarify the specific molecular mechanisms. Second, the number of clinical samples analyzed in this study was relatively small. Larger-scale multi-center research cohorts are needed to further confirm the clinical application value of SPP1 as a prognostic biomarker. Third, this study is mainly based on in vitro experiments, and lacks in vivo validation. In particular, appropriate in vivo metastasis models are required to further evaluate the role of SPP1 in lymph node metastasis and to better assess the translational relevance of our findings.

In conclusion, our study demonstrates that SPP1 is significantly upregulated in HNSCC with lymph node metastasis and is associated with aggressive clinicopathological features. Functional experiments indicate that SPP1 promotes malignant phenotypes, including proliferation, migration, and invasion, in HNSCC cells. These findings suggest that SPP1 may serve as a potential biomarker and therapeutic target in HNSCC, and provide a basis for future studies aimed at elucidating its precise molecular mechanisms.

Conclusion

This study shows that SPP1 is significantly upregulated in HNSCC and is associated with lymph node metastasis and unfavorable clinical outcomes. Functional analyses indicate that SPP1 promotes malignant phenotypes of HNSCC cells, including proliferation, migration, and invasion in vitro. Transcriptomic analysis further suggests that SPP1 is involved in extracellular matrix remodeling and adhesion-related signaling pathways, including the FAK–AKT axis. Taken together, these findings suggest that SPP1 may serve as a potential biomarker and therapeutic target in HNSCC. However, further studies are required to clarify its precise molecular mechanisms and to validate its clinical applicability.

Abbreviations

HNSCC

Head and neck squamous cell carcinoma

SPP1

Secreted phosphoprotein 1

OPN

Osteopontin

LNM

Lymph node

DEGs

Differentially expressed genes

ECM

Extracellular matrix

Authors’ contributions

Jingyan Li: conceived and designed the study, collected the clinical samples, performed data analysis, and drafted the manuscript; Quan Zeng and Zhihai Wang: contributed to study conception, data analysis, and interpretation of the results; Min Pan and Mengna Wang drafted the manuscript; Tianhao Wu and Youlan Zhang collected the clinical samples. All authors read and approved the final manuscript.

Funding

This study was supported by the Chongqing Natural Science Foundation (Grant No. CSTB2025NSCQ-GPX1140).

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Publicly available datasets analyzed in this study are available in The Cancer Genome Atlas (TCGA) database ( https://portal.gdc.cancer.gov/ ).

Declarations

Ethics approval and consent to participate

This study was approved by the ethics committee of Chongqing Medical University and was conducted in accordance with the Declaration of Helsinki.

Consent for publication

All participants signed written informed consent for anonymous data publication.

Competing interests

The authors declare no competing interests.