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Background
Recurrence and metastasis represent the primary causes of therapeutic failure in head and neck squamous cell carcinoma (HNSCC). However, the underlying molecular mechanisms driving these processes remain incompletely elucidated.
Methods
WNK1 (with-no-lysine kinase 1) expression in both normal and HNSCC tissues was analyzed using the TCGA, TIMER2.0 and Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets, and further validated in HNSCC cell lines by Western blot. Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and orthotopic xenograft assays were conducted to assess the biological role of WNK1 in HNSCC proliferation and metastasis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) -based quantitative proteomics and post-translational modification profiling were performed to explore the underlying molecular mechanisms.
Results
WNK1 overexpression was significantly elevated in HNSCC tissues and cell lines and was correlated with reduced overall survival. WNK1 knockdown suppressed tumor growth and metastasis both in vivo and in vitro. Proteomic and phosphoproteomic profiling of WNK1-driven alterations identified critical signaling pathways closely associated with tumor malignancy. Specifically, WNK1 promotes NF-κB activation through RELA (p65) phosphorylation and nuclear accumulation, resulting in the upregulation of pro-tumorigenic effectors.
Conclusions
Elevated WNK1 drives invasive progression and distant metastasis in HNSCC through NF-κB-dependent transcriptional reprogramming, highlighting its potential as a novel therapeutic target for HNSCC.
Introduction
Head and neck squamous cell carcinoma (HNSCC), a malignant tumor originating in the squamous epithelium of the mucous membranes of the oral cavity, nasal cavity, paranasal sinuses, and throat, is the seventh most common human cancer globally, characterized by marked heterogeneity and high mortality [1, 2]. Although advances in disease management (e.g., surgery, chemoradiotherapy, and targeted therapies) have improved clinical outcomes over the past decades, the 5-year survival rate of HNSCC remains below 60% even after multimodal therapy [3, 4]. Notably, over 50% of patients with locally advanced HNSCC develop recurrence or regional lymph node metastases within 3 years of treatment [5]. While immunotherapy prolongs both overall survival (OS) and progression-free survival (PFS), it remains associated with a low objective response rate in HNSCC [6, 7]. There is a critical need to elucidate the molecular drivers of malignant progression and identify novel therapeutic targets.
Emerging studies have revealed that nuclear factor kappa B (NF-κB) is a key driver of tumor invasion, proliferation, and metastasis in human cancers, including HNSCC [8, 9–10]. The activation of NF-κB-dependent transcriptional programs requires its phosphorylation and subsequent nuclear translocation, which enables the regulation of downstream oncogenic targets [11, 12]. As reported in The Cancer Genome Atlas (TCGA), HPV-negative HNSCC demonstrated significantly elevated NF-κB pathway activity compared to HPV-positive tumors. This difference was particularly pronounced in cases with PIK3CA mutations or deletions of TRAF3 or CASP8. These findings indicate that specific genomic alterations in HNSCC may contribute to the activation of the NF-κB pathway [13, 14]. Although HPV-positive patients have lower NF-κB activity, studies suggest a transient E6/ E7-mediated NF-κB activation during viral integration during HPV infection [15]. Compared to HPV-positive HNSCC, ferroptosis-inducing stressors stress upregulates PD-L1 via the NF-κB signaling pathway and shapes an immunosuppressive microenvironment in HPV-negative HNSCC [16]. Although the pro-tumorigenic functions of the NF-κB signaling pathway highlight its therapeutic potential, systemic inhibition of this pathway directly poses substantial clinical challenges due to off-target cytotoxicity and immune dysregulation [17].
As pivotal regulators of intracellular signaling cascades, protein kinases are central to tumorigenesis and malignant progression by mediating post-translational modifications (phosphorylation) that dynamically regulate protein activity and function [18, 19]. Owing to the functional versatility of protein kinases and the reversibility of their regulation, they have emerged as promising therapeutic targets in targeted cancer therapies, exemplified by tyrosine kinase inhibitors (TKIs) [20]. Following the landmark 2001 approval of imatinib by the US Food and Drug Administration (FDA) for chronic myeloid leukemia (CML), over 50 TKIs targeting receptor tyrosine kinases (e.g., EGFR, ALK, ROS1, HER2, VEGFR, FGFR, PDGFR) and non-receptor kinases (e.g., KIT, RET, MET, MEK) have been clinically approved, demonstrating durable efficacy and favorable safety profiles across solid tumors and hematologic malignancies [21, 22]. Therefore, developing selective inhibitors against understudied kinase family members represents a promising therapeutic strategy that can achieve sustained tumor suppression and reduce toxic side effects. However, the correlation between HNSCC, as well as their potential involvement in activating the NF-κB pathway, remains to be elucidated.
In our previous study, we identified a 13-gene prognostic signature capable of effectively predicting the risk of metastasis in nasopharyngeal carcinoma (NPC) patients. Among these 13 signature genes, WNK1 (with-no-lysine kinase 1) was the most significantly differentially expressed gene when comparing patients with or without distant metastasis. WNK1, a member of the WNK family of serine/threonine protein kinases, plays a key role in blood pressure regulation and renal homeostasis [23]. Furthermore, WNK1 has been implicated in the progression of various cancers linked to poor prognosis [24, 25]. However, the precise role of WNK1 in HNSCC has not yet been completely clarified. In this study, we established that WNK1 is upregulated in HNSCC and drives tumor progression by promoting malignant proliferation and metastasis. Elevated WNK1 expression enhances NF-κB signaling through promoting phosphorylation of RELA (p65) and facilitating its nuclear translocation, thereby activating oncogenic transcription programs. Collectively, our findings position WNK1 as both a diagnostic/prognostic biomarker and a novel therapeutic target for HNSCC.
Results
WNK1 is upregulated and associated with metastasis in HNSCC
Our previously published study identified a 13-genes prognostic signature that stratifies NPC patients into high- and low-risk groups. This signature demonstrated significant discriminative capacity for distant metastasis-free survival (DMFS), disease-free survival (DFS), and overall survival (OS) in multicenter validation cohorts [26]. Among these 13 signature genes, WNK1 as the most differentially expressed gene between patients with and without distant metastasis, suggesting its critical role in stratifying high- and low-risk groups in the prognostic model (Supplementary Table S1). To identify the role of WNK1 in cancer progression, we initially utilized the TIMER2.0 database to analyze mRNA expression profile across various cancer types (pan-cancer). Comprehensive pan-cancer analysis demonstrated significantly elevated WNK1 expression in tumor versus matched normal tissues across multiple malignancies, including cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSCC), liver hepatocellular carcinoma (LIHC), and pheochromocytoma & paraganglioma (PCPG). Subsequently, a paired-sample differential expression analysis using TCGA data showed that WNK1 was significantly upregulated in tumor samples. (Fig. 1A, B). We also examined WNK1 protein expression using datasets from the Human Protein Atlas (HPA) and samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). A strong positive staining indicated that WNK1 was significantly upregulated in HNSCC tumor tissues (Fig. 1C, D). Subsequently, western blot analysis with anti-WNK1 antibody validated significant overexpression of WNK1 protein in 5 HNSCC cell lines (HSC2, HSC3, CAL27, CAL33, SCC1) compared to normal oral keratinocytes (NOK) (Fig. 1E). This protein-level confirmation aligns with our bioinformatics results.
In recent years, the application of single-cell RNA sequencing (scRNA-seq) technology has improved the accuracy of tumor research [27]. To explore the contribution of WNK1 to HNSCC metastasis, we analyzed an scRNA-seq dataset (GSE234933) comprising clinically annotated specimens from primary tumors, locoregional recurrences, and distant metastases. Comparative analysis revealed that WNK1 expression was significantly elevated in lung metastatic lesions compared to primary tumors (Fig. 1F). Moreover, we evaluated the prognostic value of WNK1 in HNSCC using Kaplan-Meier analysis. Patients with higher WNK1 expression exhibited poorer overall survival (OS) compared to those with lower expression (Fig. 1G). Collectively, these findings confirm that WNK1 is upregulated in HNSCC and is positively associated with metastasis.
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Fig. 1
WNK1 expression is significantly elevated in HNSCC and is linked to poor survival. A Analysis of the TIMER2.0 database reveals WNK1 expression across multiple cancer types (pan-cancer). B The mRNA levels of WNK1 were analyzed across multiple cancer types (pan-cancer) using The Cancer Genome Atlas (TCGA) dataset for paired samples. C. Characteristic images of WNK1 protein expression in normal oral tissue and HNSCC tissues from Human Protein Atlas (HPA). D The protein levels of WNK1 in HNSCC were analyzed using samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). E Western blotting was used to detect the protein expression levels of WNK1 in HNSCC cell lines. F Comparative analysis of WNK1 expression in lung metastatic lesions compared with primary tumors reveals significant differences (GSE234933). G The overall survival of HNSCC patients with high (n = 49) or low (n = 48) expression of WNK1 was assessed (GSE41613). Mean ± SD; NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; Student’s t test
WNK1 promotes HNSCC cell proliferation, migration and invasion in vitro
Given the elevated expression of WNK1 in HNSCC, we hypothesized that WNK1 drives oncogenesis through specific signaling cascades. To investigate this, we performed comprehensive transcriptomic profiling of TCGA-HNSCC cohorts. Compared to the WNK1-low group, the WNK1-high group exhibited significant enrichment in hallmark cancer pathways (e.g., proliferation, migration) and demonstrated activation of tumorigenic processes (Fig. 2A, B). These findings align with prior studies implicating WNK1 in malignant progression across solid tumors [28, 29].
To elucidate the role of WNK1 in HNSCC cells, we transduced CAL27 and HSC3 cell lines, both of which exhibit high endogenous WNK1 expression, with an shRNA targeting WNK1 to generate stably transduced cell lines. Western blot analysis confirmed efficient WNK1 knockdown in transfected cells. Functional validation through CCK-8 assays showed markedly reduced proliferation rates in shWNK1 cells (Fig. 2C). Consistently, colony formation assays revealed a great reduction in clonogenic capacity of CAL27 and HSC3 cells following WNK1 silencing (Fig. 2D). Subsequently, Transwell assays were performed to evaluate the role of WNK1 in HNSCC cell invasion and migration. The results showed that WNK1 downregulation significantly inhibited migration and invasion in CAL27 and HSC3 cells. Conversely, the negative control exhibited markedly higher invasive cell counts (Fig. 2E). Collectively, these findings confirm that WNK1 knockdown suppresses HNSCC proliferation, migration, and invasion in vitro, suggesting its potential as a therapeutic target for head and neck cancer.
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Fig. 2
WNK1 promotes HNSCC cell proliferation, migration and invasion. A Gene Ontology (GO) enrichment for the differentially expressed genes (DEGs) between high and low WNK1. B Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) stratified by WNK1 expression levels. C The Cell Counting Kit-8 (CCK-8) assay was used to assess the proliferative capacity of cells. D Representative and quantified results of colony formation assays in WNK1-knockdown or control HNSCC cells. E Representative and quantified results of the migration and invasion assays in WNK1-knockdown or control HNSC cells. Scale bar,200 μm. Mean ± SD; NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; one-way ANOVA
WNK1-driven variation of proteome and phosphoproteome in HNSCC
The WNK family is a class of serine/threonine protein kinases that could regulate the biological behavior of cells through phosphorylation [30, 31–32]. To explore the molecular mechanisms by which WNK1 promotes HNSCC progression, we performed proteomic and phosphoproteomic sequencing in CAL27 cells transfected with shWNK1 (n = 3) or non-targeting control shRNA (n = 3). As shown in Fig. 3A, Principal component analysis (PCA) demonstrated the separation of the shWNK1 CAL27 cells from the shNC group in the proteomic profile. Proteomic profiling of WNK1-knockdown CAL27 cells identified 198 significantly upregulated and 66 downregulated proteins (|fold change (FC)| >1.5 and p < 0.05). In addition, differential expression analysis of phosphoproteomics identified 312 up-regulated phosphorylated sites on 225 proteins and 188 down-regulated phosphorylated sites on 154 proteins (Fig. 3B–F).
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Fig. 3
Proteome and phosphoproteome profiling in WNK1-knockdown HNSCC cells. A Principal component analysis (PCA) of proteomics. B Principal component analysis (PCA) of phosphoproteomics. C, D Volcano plot and Heatmap of differently expressed proteins in WNK1-knockdown cells. E, F Volcano plot and Heatmap displaying differential proteins and phosphorylated sites. G Differentially expressed proteins were categorized into four groups based on fold change. H, I Biological functions and KEGG enrichment results of proteins exhibiting various fold change. J Differentially expressed phosphorylated sites were categorized into four groups based on fold change. K, L Biological functions and KEGG enrichment results of sites displaying different fold change. Mean ± SD; NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; Student’s t test
To delineate the functional implications of WNK1-driven molecular alterations in HNSCC, we stratified differentially expressed proteins and phosphorylated sites into four quantiles (Q1-Q4) based on the magnitude of fold changes (Fig. 3G and J). Subsequently, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for each group (Fig. 3I–L). Functional annotation revealed that WNK1-regulated proteins orchestrate multiple oncogenic processes beyond cell adhesion molecules, including protein localization, mRNA catabolic process, and signal transduction regulation. Our enrichment analysis also highlighted functions and pathways associated with tumors, consistent with Fig. 2A. Integrative analysis of transcriptomic, proteomic, and phosphoproteomic datasets coupled with functional validations establishes WNK1 as a central signaling orchestrator in HNSCC pathogenesis.
WNK1 activates the NF-κB signaling pathway to promote malignant progression in HNSCC
To further investigate the mechanisms by which WNK1 regulates HNSCC malignant progression, we focused on 66 proteins showing significant downregulation following WNK1 knockdown. Subsequent GO enrichment analysis via Metascape (https://metascape.org/gp/index.html#/main/step1) revealed predominant involvement in TNF-alpha/NF-kappa B signaling complex 6 (Fig. 4A) [33]. Nuclear factor kappa-B (NF-κB) is a nuclear transcription factor that plays a key role in promoting inflammation, which can contribute to cancer development in chronic inflammatory environments. The NF-κB signaling pathway has been extensively characterized as a pivotal driver of tumorigenesis in HNSCC, mediating key oncogenic processes such as inflammation, apoptosis evasion, and metastatic dissemination [34]. The correlation between WNK1 and NF-κB was further confirmed in HNSCC patients through analysis of bulk RNA sequencing data obtained from the TCGA database, as shown in Fig. 4B. Phosphorylation of RELA (p65) and its subsequent nuclear translocation, which represent the critical steps in canonical NF-κB signaling activation, serve as key mediators facilitating transcriptional activation of target genes. To investigate this mechanism, we next examined WNK1 involvement in this signaling cascade in HNSCC. Western blot analysis revealed that WNK1 knockdown induced a significant reduction in total p65 protein expression with a concomitant decrease in phosphorylation levels (Fig. 4C). Furthermore, immunofluorescence analysis demonstrated a significant reduction in nuclear translocation of the NF-κB subunit p65 following WNK1 knockdown (Fig. 4D). To directly explore the functional dependency of WNK1-driven oncogenic phenotypes on NF-κB, we performed critical rescue experiments using an NF-κB pathway inhibitor(NFκBi: IKK-IN-1). The NF-κB inhibitor exhibited an anti-tumor effect similar to that induced by WNK1 knockdown in HNSCC cells; however, combining the treatments failed to significantly enhance the overall anti-tumor efficacy (Fig. 4E). Taken together, these findings demonstrate that WNK1 drives oncogenic progression through post-translational modifications (PTMs)-mediated activation of the NF-κB pathway; however, the exact phosphorylation sites responsible for this regulatory mechanism require further experimental validation.
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Fig. 4
Alterations in the NF-κB pathway in WNK1-knockdown HNSCC cells. A Gene Ontology (GO) Biological Processes significantly enriched through Metascape analysis. B The Gene Set Enrichment Analysis (GSEA) results highlight the significant association between high WNK1 expression and the HALLMARK_TNFA_SIGNALING_VIA_NFKB pathway. C Western blotting was used to detect indicated proteins in WNK1-knockdown HNSCC cells. D Fluorescence microscopy analysis of WNK1-knockdown or control HNSCC cells. Scale bar, 10 μm. E Representative and quantified results of the migration and invasion assays in HNSCC cells with or without NFκBi (IKK-IN-1; 0.2 µg/ml). Scale bar, 200 μm. Mean ± SD; NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; one-way ANOVA
WNK1 knockdown inhibits tumor growth and metastatic dissemination of HNSCC cells in vivo
To investigate the in vivo oncogenic function of WNK1, we established orthotopic xenograft models through submucosal tongue implantation of lentivirus-mediated WNK1-knockdown CAL27 cells and isogenic control counterparts in nude mice. The xenografts were harvested for tumor growth calculation and lymph nodes were dissected and collected for local metastasis evaluation after the mice were sacrificed (Fig. 5A). As shown by Hematoxylin and Eosin (H&E) staining, mice bearing WNK1-knockdown CAL27 tumors exhibited smaller tumor sizes compared to those bearing control CAL27 cells (Fig. 5B, C).
To histologically identify metastatic epithelial tumor cells within cervical lymph nodes, we conducted pan-cytokeratin (Pan-CK) immunohistochemical staining. WNK1 knockdown significantly reduced the metastatic burden in cervical lymph nodes, with a 42% decrease in Pan-CK-positive foci compared to controls (Fig. 5D). These results suggest that WNK1 acts as an oncogene to promote HNSCC tumor growth and metastasis in vivo, suggesting great therapeutic value of inhibiting tumor metastasis This functional dependency identifies WNK1 as a druggable target with first-in-class potential for metastasis-interception therapy in HNSCC.
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Fig. 5
WNK1 promotes HNSCC tumor growth and metastasis in vivo. A WNK1-knockdown CAL27 cells were orthotopically injected into the tongue submucosal layer to establish a cervical lymph node metastasis mouse model. B Representative images of xenograft tumors formed by WNK1-knockdown or control HNSCC cells. C Representative hematoxylin and eosin (H&E) staining images of HNSCC orthotopic xenograft model (Left panel). Scale bar, 1 mm. Quantification of tumor volume of HNSCC orthotopic xenograft (Right panel). **P<0.01 by Student’s t test. D Representative pan-cytokeratin immunohistochemical (IHC) staining of lymph node metastatic carcinoma (LNM) from WNK1-knockdown and control groups (Left panel). Scale bar, 100 μm. Quantification of metastatic burden in cervical lymph nodes (Right panel). *P<0.05 by Chi-square test
Discussion
Accumulating evidence indicates that protein kinases play essential roles in tumorigenesis and malignant progression across various human cancers [35, 36]. However, the specific roles and underlying mechanisms of protein kinases in HNSCC remain largely unexplored. Our integrated multi-omics analysis and functional validation establish WNK1 – a serine/threonine kinase aberrantly overexpressed in HNSCC – as both a prognostic biomarker and a mechanistic driver of tumor proliferation and metastatic dissemination. Mechanistically, WNK1 promotes the phosphorylation and nuclear translocation of p65, thereby triggering the transcription of multiple genes involved in cell fate determination (Fig. 6).
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Fig. 6
Schematic model of the role of WNK1 in regulating NF-κB signaling pathway in HNSCC cells. HNSCC cells upregulate WNK1 to enhances NF-κB signaling through phosphorylating p65 protein and facilitating p65 nuclear translocation to activate oncogenic transcription program, which ultimately induced HNSCC cell proliferation, migration and invasion
Globally, over 780,000 new cases of head and neck cancer are diagnosed annually, resulting in nearly 400,000 deaths. More than 90% of these cancers are squamous cell carcinoma (SCC), with 60% presenting as locally advanced head and neck squamous cell carcinoma with lymph node metastasis [37]. Deciphering the intricate tumor biology of HNSCC, particularly the mechanisms regulating metastasis, has significant therapeutic potential. In our prior investigation, we conducted microarray-based transcriptomic profiling of 24 paired locoregionally advanced nasopharyngeal carcinoma (NPC) specimens – stratified by distant metastasis status – from patients treated with radical chemotherapy. The mRNA expression levels of NPC patients were analyzed before treatment to identify a set of molecular signatures composed of 13 genes. These signatures were validated and used to stratify patients into high-risk and low-risk groups, providing improved differentiation of distant metastasis-free survival, disease-free survival, and overall survival. Based on these data, we constructed a novel prognostic model by integrating gene expression profiles, N stage, sex, C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels. This model increased the prediction accuracy of distant metastasis in NPC from 57% to 75% [26]. Among the 13 signature genes, WNK1 exhibited the most significant differential expression in patients with distant metastasis, highlighting its critical role in the prognostic model for stratifying high-risk and low-risk groups. Elucidation of the functional mechanisms underlying WNK1-driven tumor progression will pave the way for therapeutic targeting of this pathway, thereby enhancing patient outcomes. In this study, we expanded the scope of our investigation to characterize the transcriptomic signatures of WNK1 in HNSCC, revealing that both mRNA and protein levels of WNK1 are consistently elevated in tumor tissues compared to normal counterparts. Notably, WNK1 acts as an oncogene by driving HNSCC cell growth and invasion in both in vitro and in vivo models. These results align with previous findings that WNK1 is significantly upregulated in multiple cancers, including breast cancer, colorectal cancer, renal-cell carcinoma and multiple myeloma [24, 25, 38, 39]. Beyond its role in regulating migratory functions in both cancer cells and innate immune cells, Azucena Esparís-Ogando et al. demonstrated that WNK1 impairs the therapeutic efficacy of trametinib in ovarian cancer models [40, 41]. These findings suggest that WNK1 may modulate chemoradiotherapy resistance and sensitivity, highlighting a critical area for further investigation.
WNK463 is a WNK kinase inhibitor with oral bioavailability that specifically targets the catalytic domain of WNK, ensuring strong binding affinity and selectivity for the kinase [42]. Previous research has demonstrated that inhibiting WNK1 expression in zebrafish reduced the development of tumor-induced ectopic blood vessels and hindered tumor progression. Additionally, employing chemical inhibitors to target WNK1 or its downstream kinases, OSR1 (oxidative stress responsive kinase 1) and SPAK (Ste20-related proline alanine-rich kinase), resulted in a reduction of ectopic vessel formation and suppressed the proliferation of transplanted hepatoma cells [43]. These findings highlight the potential value of WNK463 in preclinical models and emphasize the therapeutic importance of molecules downstream of WNK1 [44]. For example, OSR1 plays a crucial role in various oncogenic signaling pathways; however, its pathological function in HNSCC has yet to be fully understood [45, 46]. Subsequent research could further elaborate and refine the logical framework. Overall, the inhibition of WNK1 in HNSCC may represent a promising therapeutic strategy.
Mounting evidence underscores that NF-κB serves as a central regulator of genes involved in immunity, inflammation, and cell survival [47]. Pathological dysregulation of NF-κB signaling has been implicated in inflammatory and autoimmune diseases, viral infections, and multiple cancer types, including HNSCC. Aberrant activation of NF-κB promotes cancer stem cell recruitment to drive invasion and metastasis, enhances ferroptosis resistance, and compromises immunotherapy efficacy through heightened secretion of immunosuppressive factors [48]. However, the mechanisms that activate NF-κB in HNSCC pathogenesis have yet to be fully elucidated. Using proteomics and phosphoproteomics based on mass spectrometry techniques, which provided critical datasets and comprehensive insights into tumor biology and molecular regulation, we identified WNK1 as a protein kinase critical for NF-κB activation in HNSCC cells. Of note, four ribosomal proteins—RPL4, RPL6, RPL8, and RPS11—were specifically downregulated in WNK1-low cells and associated with the TNF-α/NF-κB signaling complex 6 (Supplementary Table S2). These ribosomal proteins are recruited to the 3’-UTR of NF-κB1 mRNA and critically regulate its translation [49, 50]. We also demonstrated that WNK1 enhanced p65 phosphorylation and nuclear localization in HNSCC cells, which is aligned with the increased nuclear p65 associated with the acquisition of progressive cancer. In addition, our proteomic and phosphoproteomic profiling further identified key candidate effectors and oncogenic pathway - including mitogen-activated protein kinase (MAPK) signaling pathway, homologous recombination - that are mechanistically linked to therapy resistance [51, 52–53]. Notably, these understudied datasets offer a foundational framework for elucidating WNK1 gene regulation and HNSCC pathogenesis. We also acknowledge that the mechanisms by which WNK1 regulates NF-κB signaling and its downstream oncogenic programs merit further investigation. Furthermore, the clinical value of WNK1 should be further validated in large-scale multicenter studies before clinical application.
Conclusion
In summary, our study demonstrates that upregulated WNK1 drives invasive proliferation and metastasis in HNSCC. Inhibition of WNK1 expression significantly suppresses tumor growth and cervical lymph node metastases by reducing phosphorylated p65 levels, thereby blocking its nuclear accumulation. Therefore, the WNK1—NF-κB axis represents a novel therapeutic strategy for HNSCC.
Materials and methods
Cell culture
The HNSCC cell lines (HSC2, HSC3, CAL27, CAL33 and SCC1) and normal oral keratinocytes (NOK) were obtained from Guangdong Provincial Key Laboratory of Stomatology. These cell lines were cultured in Dulbecco’s modified Eagle medium (Gibco) with 10% fetal bovine serum (FBS, Excell, China). All cells were cultured in a standard humidified incubator with 5% CO2 at 37 °C. Cell lines tumor location correspondence: HSC2: from a patient-derived primary buccal mucosal tumor; HSC3: from a patient-derived metastatic lymph node tumor in a 63-year-old male with primary lingual squamous carcinoma; CAL27: from a mid-tongue lesion in a 56-year-old Caucasian male; CAL33: from lesion fragments of a moderately differentiated primary tumor in a 69-year-old male; SCC1: from a tumor at the floor of the mouth in a male patient.
Plasmids and lentiviruses
DNA sequences encoding WNK1-targeting shRNAs (shWNK1-1: 5′-GCGTAGTTTCAAGTATCACAA − 3′; shWNK1-2: 5′-GCAGGAGTGTCTAGTTATATT-3′) were cloned into the PLKO.1-puro lentiviral vector (RRID: Addgene_8453). The recombinant plasmids were co-transfected into HEK293FT cells with psPAX2 (RRID: Addgene_12260) and pMD2.G (RRID: Addgene_12259) packaging plasmids using Lipofectamine 3000. Lentiviral particles were harvested at 48 h post-transfection, filtered through 0.45 μm PVDF membranes, and used to transduce HNSCC cell.
Western blot
Total protein lysates were separated by 10% SDS-PAGE (Epizyme, China) and electrophoretically transferred onto 0.45 μm PVDF membranes (Millipore, USA).The membranes were blocked with 5% (w/v) BSA in TBST for 1 h and incubated with primary antibodies overnight at 4 °C. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, the target bands were visualized using ECL™ Western Blotting Substrate (Thermo Fisher). The antibodies used are listed in Supplementary Table S3.
Cell counting kit-8 (CCK-8) & colony formation assays
Cell viability was assessed using the CCK-8 assay (Dojindo, Japan). Briefly, 1 × 10^3 cells were seeded per well in 96-well plates. Following incubation for the specified times, the cells were exposed to a mixture of 10 µl CCK-8 and 100 µl serum-free DMEM for an additional 2 h prior to measurement. The OD value was measured at 450 nm using a spectrophotometric plate reader (SpectraMax i3X). For colony formation assays, 1,000 cells per well were plated in 6-well plates. Following the designated incubation period, colonies were fixed with methanol, stained with 0.5% crystal violet, and manually quantified under an inverted microscope.
Transwell migration and invasion assays
Transwell chambers with 8-µm pore membranes (Corning) – Matrigel-coated for invasion assays or uncoated for migration assays – were employed to evaluate cellular migratory and invasive potential. Briefly, 1 × 10⁵ CAL27/HSC3 cells in serum-free medium were plated in the upper chambers, whereas the lower chambers contained medium with 10% FBS. Following 24-hour incubation, the cells that had migrated or invaded were fixed with methanol, stained with 0.5% crystal violet, and then counted using an inverted microscope.
Immunofluorescence assay
Cells were collected and fixed with methanol. After permeabilization with PBS containing 0.5% Triton X-100, cells were incubated with primary antibodies at 4 °C overnight. Subsequently, cells were stained with species-matched secondary antibodies corresponding to the host species of the primary antibodies. Nuclei were counterstained with DAPI. Slides were analyzed using confocal laser scanning microscopes (LSM 980, ZEISS). The antibodies used are listed in Supplementary Table S3.
Immunochemistry (IHC) staining
Tissue sections were subjected to deparaffinization and rehydration, followed by endogenous peroxidase blocking with 3% H₂O₂, nonspecific antigen blocking with 10% normal goat serum, overnight incubation with primary antibodies at 4 °C, and subsequent incubation with horseradish peroxidase HRP-conjugated species-specific secondary antibodies (rabbit/mouse). Chromogenic detection was performed using diaminobenzidine (DAB), with haematoxylin as a nuclear counterstain. The antibodies used are listed in Supplementary Table S3.
Liquid chromatography‑mass spectrometry (LC–MS) analysis
Proteomic and phosphoproteomic data were obtained by liquid chromatography-mass spectrometry. Peptide samples were reconstituted in mobile phase A (0.1% formic acid, 2% acetonitrile in water) and loaded onto an Easy-nLC 1000 nanoflow LC system (Thermo Fisher Scientific). The binary mobile phase system contained: Mobile phase A: 0.1% (v/v) formic acid in water/acetonitrile (98:2, v/v); A nonlinear gradient was delivered at 450 nL/min as follows:0–70 min: 9% → 25% B;70–82 min: 25% → 35% B; 82–86 min: 35% → 90% B; 86–90 min: isocratic elution at 90% B. Eluted peptides were ionized via a captive spray ion source (1.5 kV ion spray voltage) and analyzed on a timsTOF Pro mass spectrometer (Bruker Daltonics) operated in data-dependent acquisition (DDA) mode with 10 PASEF MS/MS scans per cycle.
Animal studies
A total of 12 female BALB/c nude mice (5–6 weeks old, 16–20 g) were purchased from Zhuhai BesTest Bio-Tech Co., Ltd. (Guangdong, China). All animals were housed in a specific pathogen-free (SPF) facility at the Laboratory Animal Center of Nanfang Hospital, where they were provided with a continuous supply of sterile air maintained at a temperature of 25 °C. HNSCC orthotopic models were established by intralingual injection of 2.5 × 10⁵ WNK1-knockdown CAL27 cells or control cells in 25µL DMEM-10% (v/v) Matrigel solution. The xenografts were harvested for tumor volume (V) calculation with the following formula: V = Length × width2/2, and lymph nodes were dissected and collected for local metastasis evaluation after the mice were sacrificed. All animal procedures were conducted in accordance with the guidelines approved by the Animal Ethics Committee of Nanfang Hospital (application No. IACUC-LAC-20240516-001).
Statistics and reproducibility
All statistical analyses were performed using the SPSS version 21.0 statistical software, R (version 4.2.2) and Graph-Pad Prism version 9.0 software. Data were presented as the mean ± SD. Two-tailed Student’s t test and analysis of variance (ANOVA) were performed for comparing mean values, and the difference was considered statistically significant with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001.
Acknowledgements
None.
Author contributions
Ru-zhen Chen: Conceptualization, Data curation, Methodology, Project administration, Writing—original draft, Writing – review & editing. Rui Li: Methodology, Software, Writing—original draft, Writing—review & editing. Bing-lin Cheng: Methodology, Software, Writing—review & editing. Si-bei Lu: Investigation, Methodology. Wen Hu: Investigation. Feng Lin: Investigation. Xin Wen: Formal analysis. Yue Chen: Funding acquisition. Ying-lei Wang: Investigation. Mo-ting Zhang: Methodology, Software. Xiao-hong Hong: Funding acquisition, Supervision, Writing—original draft, Writing—review & editing. Xin-ran Tang: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.
Funding
This study was supported by the National Natural Science Foundation of China (Grant No. 82172673, 82203822, 82403798), Funding by Science and Technology Projects in Guangzhou (Grant No.2024A04J5202, 2024A04J5234), the Natural Science Foundation of Guangdong Province (Grant No. 2023A1515012396, 2021A1515111001) and the Youths Development Scheme of Nanfang Hospital, Southern Medical University (2021J004).
Data availability
The public high-throughput sequencing data used in this study are available in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/ ) under accession codes GSE234933, GSE41613. Others public data used in this study are available in The Cancer Genome Atlas (TCGA; https://tcga-data.nci.nih.gov/ ), Human Protein Atlas (HPA: https://www.proteinatlas.org/ ) and Clinical Proteomic Tumor Analysis Consortium (CPTAC; https://gdc.cancer.gov/about-gdc/contributed-genomic-data-cancer-research/clinical-proteomic-tumor-analysis-consortium-cptac ). Finally, our mass spectrometry (MS) proteomic data were deposited in the ProteomeXchange Alliance ( https://proteomecentral.proteomexchange.org ) using the iProX partner repository with the dataset identifier IPX0013316001.
Declarations
Ethics approval and consent to participate
As these databases of GEO, TCGA, HPA and CPTAC are open and accessible, approval from the local ethics committee was not required. Furthermore, our experiment does not involve human subjects and thus is Not applicable. All mice experiments conducted in this study were reviewed and approved by the Animal Ethics Committee of Nanfang Hospital (Approval No. IACUC-LAC-20240516-001). All procedures were performed in full compliance with the committee’s relevant guidelines and regulations.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Abbreviations
Head and neck squamous cell carcinoma
With-no-lysine kinase 1
Nuclear factor kappa B
Tyrosine kinase inhibitors
The Cancer Genome Atlas
Human Protein Atlas
Clinical Proteomic Tumor Analysis Consortium
Gene Ontology
Kyoto Encyclopedia of Genes and Genomes
Pan-cytokeratin
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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