Research ArticleExperimental Studies
Open Access

N-myristoyltransferase 1-mediated Src Myristoylation Promotes Non-receptor Tyrosine Kinase Pathways in Oral Squamous Cell Carcinoma

TAKUMA KOMIYAMA, GORO SUGIYAMA, HANAE KAMOHARA, TAKAHIRO FUJINAGA, WATARU KUMAMARU and MASAFUMI MORIYAMA
In Vivo January 2026, 40 (1) 76-88; DOI: https://doi.org/10.21873/invivo.14174

Abstract

Background Aim: N-myristoyltransferase 1 (NMT1), which myristoylates Src, is highly expressed in oral squamous cell carcinoma (OSCC). Although targeted therapies against epidermal growth factor receptor exist, their use is limited by resistance and toxicity, and NMT1-Src interactions remain unexplored. Herein, we aimed to evaluate the role of NMT1-mediated Src myristoylation in the malignant potential of OSCC.

Materials and Methods: Myristoylation and the expression of NMT1 in OSCC were assessed using click chemistry and immunocytochemistry. RNA-seq and enrichment analyses were performed to compare OSCC cells with or without NMT1-siRNA treatment. Src activity was determined by measuring Src phosphorylation via western blotting. Expression and binding of NMT1 and Src were analyzed via immunoprecipitation using specific antibodies. HSC-2, HSC-3, WK2, and WK3F derived from human OSCC in vitro were also used to confirm malignancy by siRNA of siNMT1 in OSCC cell lines.

Results: NMT1 and Src expression was detected in all OSCC cell lines. RNA-seq analysis of SAS cells transfected with NMT1-siRNA revealed decreased expression of genes related to cell adhesion and angiogenesis. WK3F cells, which exhibit high malignancy, showed markedly higher NMT1 expression than other cell lines. Immunocytochemistry showed that Src membrane localization was reduced in all of the OSCC cell lines with NMT1 knockdown. Co-immunoprecipitation analysis confirmed that NMT1 was bound to Src during myristoylation.

Conclusion: NMT1 promotes OSCC malignancy by mediating Src myristoylation.

Keywords:

Introduction

Receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), are abundantly expressed in the oral mucosal epithelium (1). Accordingly, molecular-targeted agents that inhibit ligand binding and thereby regulate EGFR activity have been developed (2). These agents play a central role in the treatment of advanced-stage oral squamous cell carcinoma (OSCC). However, clinical challenges, such as drug resistance and dose-limiting toxicities, restrict their use. Overcoming these issues to improve outcomes in patients with advanced cancers and establishing new therapeutic strategies remains an urgent task.

Recently, the involvement of non-receptor tyrosine kinases (nRTKs) has been considered as the mechanism underlying drug resistance (3). Upon activation, nRTKs bind to RTKs and function as mediators of downstream signaling. Various pathways, including the Abelson tyrosine-protein kinase, Janus kinase, Spleen Tyrosine kinase, and Src kinase families, function as secondary messengers (4). Despite various therapeutic approaches using inhibitors of these molecules, malignant cells often evade these molecules through many networks (5). Intrinsic activation mechanisms are among these strategies; however, their molecular basis remains unclear.

N-myristoyltransferase (NMT) catalyzes the attachment of myristic acid to proteins (6). In mammals, two isoforms, NMT1 and NMT2, have been identified. NMT1 knockout mice are embryonically lethal, and NMT2 cannot compensate for the loss of NMT1, suggesting that the two isoforms play distinct roles. NMT1 is a catalytic enzyme that regulates myristoylation and plays critical roles in membrane trafficking, immune responses, bone metabolisms and cancer (7, 8). NMT1 is highly expressed in OSCC (9). Thus, NMT1 has been implicated in malignant processes, such as carcinogenesis, proliferation, invasion, and metastasis (7). Although an association between increased NMT1 expression and malignant transformation has been suggested, the precise molecular mechanisms have not been elucidated. Studies on NMT1 inhibitors have suggested their therapeutic potential; however, basic data supporting their clinical application remain insufficient (10). Given NMT1’s importance in cellular homeostasis, its inhibition is predicted to cause significant adverse effects, which is a major hurdle.

Src, a product of proto-oncogenes, is an important family of nRTK that regulates diverse cellular processes, including proliferation, survival, migration, and angiogenesis (11). Elevated expression and constitutive activation of Src have been documented in many cancer cells, including OSCC cells (12). Numerous studies have reported its central role as a therapeutic target under investigation (13). The dysregulation of Src signaling has been widely implicated in tumor initiation, progression, and metastasis, underscoring its importance in oncogenesis. Src contains an N-terminal myristoylation motif (14). However, no study has investigated the interaction between NMT1 and Src. Thus, in this study, we aimed to focus on the interaction between NMT1 and Src and analyze the association between protein myristoylation and the malignant progression of OSCC.

Materials and Methods

Antibodies and reagents. NMT1 antibody and siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Click-iT Cell Reaction Buffer Kit, azidomyristic acid, Alexa-alkyne 488, Alexa Fluor 488, Alexa Fluor 532, and Lipofectamine RNAiMAX reagent were obtained from Invitrogen (Carlsbad, CA, USA). Anti-Src antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-Src antibodies (anti-phospho-Src family, Tyr416) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture and transfection. SAS (poorly differentiated, tongue carcinoma-derived), HSC-2 (well-differentiated, tongue carcinoma-derived), and HSC-3 (poorly differentiated, tongue carcinoma-derived) cell lines were purchased from the JCRB Cell Bank (Osaka, Japan). The WK2 and WK3F cell lines were established from a primary lesion of mandibular gingival squamous cell carcinoma in an adult male, according to previous reports (15). All cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Culture dishes were purchased from IWAKI (Tokyo, Japan), and chamber slides were obtained from SPL Life Sciences (Paju-si, Gyeonggi-do, Republic of Korea). The cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. After incubation, the medium was replaced with a fresh culture medium.

Click chemistry assay. Click chemistry assays were performed using the Click-iT™ Cell Reaction Buffer Kit (Invitrogen). The cells were incubated with azidomyristic acid for 12 h. Myristoylated proteins were labeled using Alexa Fluor 488, regardless of stimulation, and detected using a fluorescence microscope (Biorevo BZ-9000; Keyence, Osaka, Japan). For nuclear staining, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Immunocytochemistry. Immunocytochemical assays were performed as described previously (16). The cells were fixed with 3.7% formaldehyde and 0.2% glutaraldehyde, blocked with 5% skim milk in PBS, and incubated overnight with primary antibodies. The cells were then incubated with Alexa Fluor 488- or Alexa Fluor 532-conjugated IgG antibodies (1:500; Invitrogen) for 60 min at 37°C. Fluorescence was detected using a fluorescence microscope (BZ-X810; Keyence).

RNA interference. OSCC cell lines were transfected with 100 pM control or NMT1 siRNA using Lipofectamine RNAiMAX (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The cells were used for subsequent experiments 48-72 h after transfection. The siRNA duplexes against human NMT1 and control (scrambled) siRNAs were synthesized by Eurofins Genomics (Ebersberg, Germany).

Western blotting. Western blotting was performed according to standard protocols (16). Protein lysates from the cell homogenates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The immunoreactive bands were quantified using a computer-assisted densitometric system (Amersham Image Quant 800).

RNA-seq analysis. SAS cells were cultured in 100-mm dishes until they were semi-confluent. Total RNA was extracted from SAS cells treated with siCtrl or siNMT1 for 72 h using TRIzol Reagent (1 ml; Invitrogen). Library preparation and sequencing were performed by a commercial provider (paired-end). Raw FASTQ files were assessed with FastQC (v0.11.9), and adapters/low-quality bases were trimmed using Trimmomatic (v0.36). Trimmed reads were aligned to the human reference genome GRCh38/hg38 with HISAT2 (v2.1.0), and gene-level counts were obtained with RSEM (v1.3.0; Bowtie backend).

Unless otherwise stated, biological replication in this study consisted of one library per condition (siCtrl, siNMT1). Raw counts were normalized using TMM (trimmed mean of M-values). Dispersion was estimated in EdgeR by borrowing information across genes (common/tagwise dispersion), and a two-group design matrix (siNMT1 vs. siCtrl) was specified. Differential expression was tested in EdgeR; raw p-values were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate (FDR). Genes meeting FDR <0.05 and |log2FC| >1 were defined as differentially expressed (67 genes), while 869 genes met the unadjusted criterion of p<0.05.

Enrichment analysis. Functional enrichment of DEGs was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the DAVID functional annotation tool. Significantly enriched biological processes (BP) and KEGG pathways were determined based on adjusted p-values (Benjamini-Hochberg correction) <0.05.

Immunoprecipitation (IP). Immunoprecipitation assays were performed by incubating 2 μg of rabbit polyclonal anti-Src antibody (1:200) (Cell Signaling Technology; Cat# 2109) with cell lysates overnight at 4°C in the presence of protein G-Sepharose beads. Immunoprecipitates were analyzed via western blotting using an anti-NMT1 antibody (1:1,000) as described above. Normal rabbit IgG (MBL, Nagoya, Japan; Cat# PM035) was used as the negative control.

Transwell migration and invasion assays. Following siNMT1 treatment for 48 h, OSCC cells were harvested, counted, and used for Transwell assays. Cell migration and invasion were assessed using 24-well Transwell inserts (8-μm pore size; Corning Inc., Corning, NY, USA). Invasion assays were performed using 24-well Matrigel invasion chambers (Corning Inc., USA; 8-μm pore size). For migration assays, 1.25×104 cells in serum-free medium were seeded into the upper chamber. For invasion assays, 5.0×104 cells in serum-free medium were seeded into the upper chamber pre-coated with Matrigel. In both assays, 500 μl of complete medium was added to the lower chamber, and the plates were incubated for 36 h to allow cell migration or invasion. The cells on the lower surface of the upper membrane were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 for 3 min. Nuclei were stained with DAPI and observed under a fluorescence microscope. Four random fields per well were captured, and the number of cells was quantified using ImageJ software. All assays were conducted in triplicate.

Statistical analysis. All experiments were performed independently at least three times. Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using Python (v3.11) and the SciPy library (v1.11). Differences between the two groups were analyzed using Student’s t-test. Welch’s t-tests were used when the assumption of equal variance was not met. Differences were considered statistically significant at p<0.05.

Results

Downregulation of NMT1 expression reduced myristoylated protein levels in SAS cells. To confirm the expression and localization of NMT1 and myristoylated proteins in OSCC, immunocytochemistry and click chemistry assays were performed using SAS cells. Compared with the control, NMT1 was localized primarily in the cytoplasm, whereas myristoylated proteins were observed in the cytoplasm, with pronounced accumulation around the nucleus. Upon transfection with siNMT1, the NMT1 and myristoylated protein levels decreased (Figure 1A). Western blot analysis confirmed that siNMT1 downregulated the expression of NMT1 (Figure 1B).

Figure 1.
Figure 1.

Expression of NMT1 and myristoylated proteins and the effects of siNMT1 on their expression in SAS cells. NMT1 expression was detected by immunocytochemistry (ICC) and Western blotting (WB). (A) For ICC, cells without primary antibody incubation were used as a control (Ctrl), and nuclei were counterstained with DAPI. Merged images of NMT1 (red) and DAPI (blue) are shown. For WB, untreated cells were used as a control (Ctrl). (B) SAS cells were cultured with or without siRNA for 72 h, and cell lysates were probed with anti-NMT1 and anti-β-actin antibodies. Myristoylated proteins were detected using a click chemistry assay, with cells not treated with azide-containing myristic acid (100 μM) serving as a control (Ctrl), and nuclei were counterstained with DAPI. Merged images of Myristoylation (green) and DAPI (blue) are shown. Scale bars, 50 μm.

Alterations in the expression of tumor-related genes using RNA-seq analysis. RNA-seq analysis was performed on SAS cells following siRNA-mediated NMT1 knockdown. A total of 67 genes were significantly differentially expressed compared with the siCtrl group (FDR <0.05 and |log2FC| >1), including 21 upregulated and 46 downregulated genes. A volcano plot generated from all genes is provided (each group had n=1), and the principal component analysis (PCA) plot was included for quality-control purposes (Figure 2A, B). Gene Ontology (GO) (Figure 2C) and KEGG pathway enrichment analyses were performed (Figure 2D). GO Biological Process (GO_BP) analysis revealed that NMT1 knockdown altered the expression of genes related to actin filament organization, cell adhesion, and angiogenesis. These processes are closely associated with cytoskeletal dynamics, focal adhesion turnover, and tumor cell motility. Additionally, gene sets related to the innate immune response and antiviral defense were enriched, suggesting that NMT1 may modulate the interactions between tumor cells and the immune microenvironment. GO Cellular Component (GO_CC) analysis revealed significant alterations in genes localized not only to the cytoplasm and membrane, but also to the extracellular matrix (ECM), tight junctions, and collagen-containing ECM, indicating that NMT1 knockdown induced reorganization of the extracellular environment and cell-cell adhesion structures. KEGG pathway analysis identified enrichment in cancer-related pathways, such as PI3K-Akt, p53, JAK-STAT, and TNF signaling, as well as viral infection pathways, including Epstein-Barr virus, human papillomavirus, and COVID-19 infection. These pathways are essential for cell survival, inflammation, and immune evasion, which suggests that NMT1 promoted these processes. NMT1 knockdown downregulated genes associated with cell adhesion and angiogenesis in OSCC cells. Additionally, the genes involved in cell adhesion and angiogenesis were analyzed (Figure 2E, F). In the adhesion category, PLAU and CLDN4 were significantly downregulated (FDR <0.05). These molecules interact with cancer cell migration, invasion, adhesion, and invasiveness. In the angiogenesis category, ROBO4 (FDR <0.05) was relatively downregulated, indicating angiogenesis suppression, vascular stability, and tumor progression. These data suggest that NMT1 promotes tumor-related gene expression, particularly invasion and metastasis, in response to cell contact dysfunction.

Figure 2.
Figure 2.
Figure 2.

RNA-seq analysis and enrichment of differentially expressed genes (DEGs) in SAS cells after siCtrl or siNMT1 transfection. (A) Volcano plot of differential gene expression analyzed with EdgeR. Each point represents a gene; the x-axis shows log2 fold change (siNMT1 vs. siCtrl), and the y-axis shows −log10 (FDR). Dashed lines indicate thresholds (|log2FC|=1, FDR=0.05). Red: upregulated DEGs (FDR <0.05, log2FC >1); Blue: downregulated DEGs (FDR <0.05, log2FC <−1); Gray: not significant. (B) PCA of siCtrl and siNMT1 libraries. Because only one library per condition was available (n=1 each), PC1 explains nearly all variance, and the plot serves as a QC visualization rather than biological clustering evidence. (C) GO analysis of Biological Process (GO_BP), GO Cellular Component (GO_CC), and (D) KEGG pathway analyses were performed on DEGs. The x-axis indicates the number of genes; the y-axis lists significantly enriched terms/pathways. Adjusted p-values were used. Dot size reflects the number of genes involved, and dot color denotes statistical significance (−log10 p-value). (E) Changes in cell-adhesion-related gene expression after siCtrl or siNMT1 treatment in SAS cells. RNA-seq data are shown as log2 fold changes (siNMT1 vs. siCtrl) for adhesion-related genes. Red indicates upregulation and blue indicates downregulation. Asterisks (*) denote statistically significant genes (p<0.05, FDR<0.05). Analysis was performed with EdgeR. (F) Changes in angiogenesis-related gene expression after siCtrl or siNMT1 treatment in SAS cells. Log2 fold changes (siNMT1 vs. siCtrl) are shown for angiogenesis-related genes. Red indicates upregulation and blue indicates downregulation. Asterisks (*) denote statistically significant genes (p<0.05, FDR<0.05). Analysis was performed with EdgeR.

Comparison of NMT1 expression and myristoylated protein expression in several OSCC cell lines. Next, we investigated whether the expression of NMT1 and myristoylated proteins in OSCC was altered by the characteristics of OSCC, such as differentiation or malignancy. HaCaT cells were used as the controls. Click chemistry assays performed in several OSCC cell lines (HSC-2, HSC-3, WK2, and WK3F) revealed the perinuclear localization of myristoylated proteins compared to the control, and immunocytochemistry confirmed that the cytoplasmic expression of NMT1 was higher in all cell lines than in the control (Figure 3A). Western blot analysis was performed on these cell lines (Figure 3B). Notably, poorly differentiated HSC-3 cells exhibited higher NMT1 expression than well-differentiated HSC-2 cells. Similarly, WK3F cells, which are derived from metastatic lymph nodes and exhibit high malignancy, showed higher NMT1 levels than their paired primary tumor-derived WK2 cells. Among all cell lines examined, WK3F displayed the highest level of NMT1 expression. These data suggest that the expression of NMT1 and myristoylation was upregulated in poorly differentiated and metastatic cells.

Figure 3.
Figure 3.

Comparison of NMT1 expression and myristoylated protein expression in several OSCC cell lines. (A) NMT1 expression was detected by immunocytochemistry (ICC), and myristoylated proteins were detected using a click chemistry assay. Nuclei were counterstained with DAPI (blue) and merged images of myristoylated proteins (green) and DAPI (blue) are shown. The normal keratinocyte cell line HaCaT was used as a control for comparison with OSCC. Scale bars, 50 μm. (B) NMT1 expression was detected by Western blotting (WB). The normal keratinocyte cell line HaCaT was used as a control for comparison with OSCC. Cell lysates were probed with anti-NMT1 and anti-β-actin antibodies.

NMT1 interacts with Src via activation and localization in OSCC cell lines. We hypothesized that NMT1 regulates Src, a major kinase family involved in malignancy, as Src is a myristoylated protein. Thus, to investigate the relationship between NMT1 and Src, we examined Src expression and localization using an anti-Src antibody under NMT1 knockdown in OSCC cell lines, compared to HSC-2, HSC-3, WK2, and WK3F (Figure 4A). Immunocytochemistry showed that Src was localized in the whole cell, including the membrane, under control conditions. However, NMT1 knockdown markedly reduced membrane-localized Src, especially in HSC-3 and WK3F cells, suggesting a shift from the membrane to the cytoplasm. Western blotting was performed to investigate Src activity by measuring the phosphorylation levels (Figure 4B). The expression did not differ between HSC-2 and HSC-3 cells. However, compared with WK2 cells, its expression was decreased in WK3F cells. These results suggest that NMT1 regulated Src membrane translocation by reducing myristoylation. Finally, we investigated whether NMT1 bound to Src using immunoprecipitation. Compared to the control, NMT1 co-immunoprecipitated with Src in all OSCC cell lines, suggesting the binding of both molecules (Figure 4C). Interestingly, compared to HSC-2 and WK2 cells, the binding levels were decreased in HSC-3 and WK3F cells. These results suggest that NMT1 interacted with Src via binding, resulting in the upregulation of Src activation and localization.

Figure 4.
Figure 4.

Src expression and the effect of NMT1 knockdown on Src in OSCC cell lines. (A) Each cell line was transfected with siCtrl or siNMT1, and Src expression was detected by immunocytochemistry (ICC). Nuclei were counterstained with DAPI (blue) and merged images of Src (green) and DAPI (blue) are shown. Scale bars, 50 μm. (B) Src activity was examined by Western blotting (WB) to detect total Src and phosphorylated Src (p-Src). (C) The interaction between NMT1 and Src was assessed by immunoprecipitation (IP).

NMT1 knockdown inhibited migration and invasion in WK2 and WK3F. Transwell assays were performed using siNMT1 transfection in WK2 and WK3F cells to evaluate the functional relevance of NMT1 in OSCC progression (Figure 5A, B). NMT1 knockdown significantly reduced the migration and invasion abilities of both cell lines (p<0.01), indicating that NMT1 plays a critical role in regulating these malignant phenotypes in OSCC.

Figure 5.
Figure 5.

Effects of NMT1 knockdown on the invasive and migratory abilities of WK2 and WK3F cells. (A, B) Two OSCC cell lines were used: WK2 and WK3F. After transfection with siCtrl or siNMT1, invasion and migration abilities were evaluated using Transwell assays. For invasion assays, Matrigel-coated Transwell chambers were used, while for migration assays, uncoated Transwell inserts were employed. In each well, cells were counted in four randomly selected fields, and this procedure was repeated in three independent experiments. Data are presented as the mean ± standard deviation (SD) for each group. Statistical significance was evaluated by Welch’s t-test. For each n, mean values are shown as three individual black dots (n=3). Box plots indicate the interquartile range (IQR), with the central line representing the median and whiskers showing the minimum and maximum values. **p<0.01.

Discussion

In oral cancer, the five-year survival rate in advanced stages remains relatively low at approximately 63%, and this continues to serve as a poor prognostic factor (17). In this study, we investigated the interaction between NMT1 and Src as it is relevant to the intrinsic activity of nRTKs (18). NMT1 and Src are highly expressed in OSCC, supporting a critical molecule for malignancy and oncogenes, as reported in a previous study. Furthermore, the translocation of Src, in addition to the reduction of myristoylation in NMT1 knockdown, suggested that NMT1 promotes myristoylation, and Src, as an nRTK, is related to NMT1 in response to the activity. Although RTKs in the oral mucosal epithelium are therapeutic targets, the crucial role of nRTKs in the growth, differentiation, and survival of epithelial cells has remained unknown for a long time. Consequently, RTKs have been intensively studied as regulatory targets in carcinogenesis triggered by inflammatory stimuli, including autoimmune (19). Notably, the EGFR is abundantly expressed in the oral mucosal epithelium, and this finding has greatly contributed to the advancement of molecular-targeted therapies. It is well established that molecular drugs that inhibit EGFR activation are currently used in clinical practice as standard therapies for oral cancer (20). Moreover, the relationship between the epithelial-mesenchymal transition pathway, such as E-cadherin, and EGFR signaling has been documented in several studies (21). Drug development has also highlighted the emergence of resistance mechanisms, necessitating further elucidation of their underlying processes. The NMT1-mediated myristoylation of Src in this study provides new insights into the intrinsic mechanisms of cancer progression.

Many nRTKs reside in the cytoplasm and mediate downstream signaling. They are involved in essential cellular processes, such as proliferation, motility, adhesion, and survival (4). Representative members of this family include Src, JAK, ABL, and SYK. Aberrant expression or activation of these molecules is closely linked to pathological conditions, such as autoimmune diseases, making them important therapeutic targets in anticancer drug development (22-24). A major proto-oncogene encodes Src; however, its post-translationally modified functions remain largely undefined (25). Although this molecular association has not been previously clarified, the present study provides the first evidence supporting such an interaction. Moreover, the expression level of NMT1 was associated with the extent of myristoylation, indicating that NMT1 contributes to Src myristoylation.

A comprehensive analysis of oral cancer cell lines revealed that NMT1 was associated with cell adhesion and angiogenesis, which is consistent with the known functions of Src. Comparative studies between the oral cancer cell lines, HSC-2 and HSC-3, demonstrated higher NMT1 expression in HSC-3 cells, suggesting its involvement in malignant progression. Similarly, although no marked difference was observed in the proportion of myristoylated cells between WK2 and WK3F cells, NMT1 expression was notably higher in WK3F cells, implying that both NMT1 and myristoylation may contribute to metastatic potential. Recent studies have identified a variety of molecules implicated in the mechanisms underlying oral cancer metastasis. For example, LYVE1 on metastasis-associated pathways is reported as a marker in tongue cancer cells (26). Furthermore, NMT1 knockdown significantly reduced the invasive and migratory abilities of both WK2 and WK3F cells, indicating that NMT1 is involved in the activation of tumor functions.

Src, a proto-oncogene encoding a non-receptor tyrosine kinase, serves as a central molecule closely associated with cancer initiation and malignancy (25). The Src family comprises a major group of nRTKs activated by various catalytic enzymes, and their activity is suppressed by Csk-mediated phosphorylation. This inhibition occurs because the phosphorylated amino acid residues interacted with the SH domain, thereby maintaining an inactive conformation. Structurally, Src consists of SH domains, unique regions, and catalytic domains, and undergoes conformational changes upon activation that enable it to act on membrane-associated RTKs (27). Src also has a myristoylation motif, which is thought to allow for variation in response. In this study, we demonstrated these implications via interactions with NMT1. Myristoylation plays a role in immune response and underlies the development of cancer therapies (28). Therefore, we focused on the association with changes in the pathways resulting from immune responses. Src phosphorylation and localization were altered. Moreover, we confirmed these effects using WK2 and WK3F cells, which can be used to assess malignant potential. However, the mechanisms underlying its translocation to the plasma membrane are not fully understood. In recent years, reports have emerged describing the mechanisms of RTK activation within cells, highlighting the increasing importance of nRTKs in cancer biology (29). This represents a critical aspect related to drug resistance, and in the present study, we identified new insights into the activation mechanism of Src mediated by myristoylation, a type of post-translational modification.

Previous studies have suggested the importance of NMT1 in oral cancer. However, its precise role remains unclear. In this study, we demonstrated that NMT1 bound and interacted with Src, thereby promoting its myristoylation and altering its localization in the plasma membrane. These findings provide new insights into the role of NMT1 in myristoylation and suggest a previously unrecognized intrinsic activation mechanism of tumor cells mediated by the activation of nRTKs. The interaction between NMT1 and Src was confirmed in SAS cells and other cell lines. However, in highly malignant cells, less NMT1-Src binding was observed. This suggests that changes in Src localization and the transient nature of the NMT1-Src interaction may influence the apparent binding level in these cells. Whether the interaction between NMT1 and Src is direct or mediated by other molecules remains unclear. In the present study, Src myristoylation was not directly detected. This represents a limitation of the study and further analyses using different approaches will be necessary. Structural analyses are essential for further elucidation of the nature of this binding. Furthermore, peptides that inhibit the molecular interactions between NMT1 and Src may be novel therapeutic agents with mechanisms capable of overcoming drug resistance. Here, we present a schematic diagram illustrating the roles of NMT1 and Src in tumor activity via the myristoylation pathway, which leads to cancer activation in OSCC (Figure 6). Our findings suggest that NMT1 is involved in the activation of tumor-associated signaling pathways, including cell adhesion, invasion, metastasis, and angiogenesis, through Src myristoylation. NMT1 binds to Src, suggesting that its interaction may facilitate myristoylation. However, NMT1 is known to myristoylate a variety of substrates. Therefore, the phenotypic changes observed after NMT1 knockdown may also be influenced by substrates other than Src. In addition, the possibility that NMT2 acts in a compensatory manner cannot be excluded. Further detailed analyses will be required to clarify these points. Further in vivo studies are required to confirm the translational significance of these findings.

Figure 6.
Figure 6.

A schematic diagram illustrating the roles of NMT1 and Src in tumor activity via the myristoylation pathway. (1) NMT1 and Src are encoded by nuclear DNA, transcribed into mRNA, and translated on cytoplasmic ribosomes, where they initially localize. (2) Following translation, Src associates with NMT1 and undergoes N-myristoylation at its N-terminal glycine. (3) This modification increases membrane affinity, and in cooperation with adjacent basic residues and palmitoylation, directs Src to the plasma membrane. (4) Membrane localization via myristoylation facilitates Src activation and amplifies downstream signaling, thereby enhancing malignant traits such as cell migration, invasion, and angiogenesis.

Conclusion

We investigated the interaction between NMT1 and Src via their binding and localization. Our data suggest that intrinsic activity via NMT1-mediated Src myristoylation is the mechanism underlying tumor progression in OSCC. These findings contribute to our understanding of the importance of NMT1 and myristoylation in OSCC.

Acknowledgements

We thank Cell Innovator Inc. (Fukuoka, Japan) for performing the RNA sequencing and primary bioinformatics analysis. We appreciate the technical assistance from The Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. We would like to thank Editage (www.editage.jp) for the English language editing.

Footnotes

  • Authors’ Contributions

    Conceptualization, T. K. and G. S.; Methodology, T. K.; Software, T. K.; Validation, T. K. and H. K.; Formal Analysis, W. K.; Investigation, T. F.; Writing – Original Draft Preparation, T. K.; Writing – Review and Editing, G. S.; Visualization, T. K.; Supervision, M. M.; Project Administration, G. S.; and Funding Acquisition, G. S.

  • Data Availability

    The data generated in this study are available upon request from the corresponding author. RNA-seq data have been deposited in the European Nucleotide Archive and can be found under accession number: PRJEB101740.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Funding

    This work was supported by a Grant-in-Aid (KAKEN No. 23K09314) from the Japan Society for the Promotion of Science (to G. Sugiyama).

  • Artificial Intelligence (AI) Disclosure

    The authors acknowledge the use of the OpenAI ChatGPT in the preparation of this manuscript. Specifically, ChatGPT (GPT-4) was utilized for assistance with (1) literature search and identification of relevant studies, and (2) improving the clarity and grammar of English translations from Japanese. AI was not used for the data analysis, scientific interpretation, or generation of the original content. The authors made all intellectual decisions and critical analyses.

  • Received October 3, 2025.
  • Revision received October 21, 2025.
  • Accepted October 29, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Lemmon MA,
    2. Schlessinger J,
    3. Ferguson KM
    : The EGFR family: not so prototypical receptor tyrosine kinases. Cold Spring Harb Perspect Biol 6(4): a020768, 2014. DOI: 10.1101/cshperspect.a020768
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Belli S,
    2. Esposito D,
    3. Servetto A,
    4. Pesapane A,
    5. Formisano L,
    6. Bianco R
    : c-Src and EGFR inhibition in molecular cancer therapy: what else can we improve? Cancers (Basel) 12(6): 1489, 2020. DOI: 10.3390/cancers12061489
    OpenUrlCrossRefPubMed
  3. ↵
    1. Fuse MJ,
    2. Okada K,
    3. Oh-Hara T,
    4. Ogura H,
    5. Fujita N,
    6. Katayama R
    : Mechanisms of resistance to NTRK inhibitors and therapeutic strategies in NTRK1-rearranged cancers. Mol Cancer Ther 16(10): 2130-2143, 2017. DOI: 10.1158/1535-7163.MCT-16-0909
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Siveen KS,
    2. Prabhu KS,
    3. Achkar IW,
    4. Kuttikrishnan S,
    5. Shyam S,
    6. Khan AQ,
    7. Merhi M,
    8. Dermime S,
    9. Uddin S
    : Role of non receptor tyrosine kinases in hematological malignances and its targeting by natural products. Mol Cancer 17(1): 31, 2018. DOI: 10.1186/s12943-018-0788-y
    OpenUrlCrossRefPubMed
  5. ↵
    1. Yesilkanal AE,
    2. Johnson GL,
    3. Ramos AF,
    4. Rosner MR
    : New strategies for targeting kinase networks in cancer. J Biol Chem 297(4): 101128, 2021. DOI: 10.1016/j.jbc.2021.101128
    OpenUrlCrossRefPubMed
  6. ↵
    1. Kosciuk T,
    2. Price IR,
    3. Zhang X,
    4. Zhu C,
    5. Johnson KN,
    6. Zhang S,
    7. Halaby SL,
    8. Komaniecki GP,
    9. Yang M,
    10. DeHart CJ,
    11. Thomas PM,
    12. Kelleher NL,
    13. Fromme JC,
    14. Lin H
    : NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle. Nat Commun 11(1): 1067, 2020. DOI: 10.1038/s41467-020-14893-x
    OpenUrlCrossRefPubMed
  7. ↵
    1. Wang H,
    2. Xu X,
    3. Wang J,
    4. Qiao Y
    : The role of N-myristoyltransferase 1 in tumour development. Ann Med 55(1): 1422-1430, 2023. DOI: 10.1080/07853890.2023.2193425
    OpenUrlCrossRefPubMed
  8. ↵
    1. Kutsuna S,
    2. Sugiyama G,
    3. Komiyama T,
    4. Kamohara H,
    5. Ohyama Y,
    6. Kumamaru W,
    7. Yamada T
    : TNF-α-induced inhibition of protein myristoylation via binding between NMT1 and Sorbs2 in osteoblasts. In Vivo 38(1): 107-113, 2024. DOI: 10.21873/invivo.13416
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Shrivastav A,
    2. Sharma A,
    3. Bajaj G,
    4. Charavaryamath C,
    5. Ezzat W,
    6. Spafford P,
    7. Gore-Hickman R,
    8. Singh B,
    9. Copete M,
    10. Sharma R
    : Elevated N-myristoyltransferase activity and expression in oral squamous cell carcinoma. Oncol Rep 18(1): 93-97, 2007. DOI: 10.3892/or.18.1.93
    OpenUrlCrossRefPubMed
  10. ↵
    1. Tan XP,
    2. He Y,
    3. Yang J,
    4. Wei X,
    5. Fan YL,
    6. Zhang GG,
    7. Zhu YD,
    8. Li ZQ,
    9. Liao HX,
    10. Qin DJ,
    11. Guan XY,
    12. Li B
    : Blockade of NMT1 enzymatic activity inhibits N-myristoylation of VILIP3 protein and suppresses liver cancer progression. Signal Transduct Target Ther 8(1): 14, 2023. DOI: 10.1038/s41392-022-01248-9
    OpenUrlCrossRefPubMed
  11. ↵
    1. Pelaz SG,
    2. Tabernero A
    : Src: coordinating metabolism in cancer. Oncogene 41(45): 4917-4928, 2022. DOI: 10.1038/s41388-022-02487-4
    OpenUrlCrossRefPubMed
  12. ↵
    1. Cheng SJ,
    2. Kok SH,
    3. Lee JJ,
    4. Yen-Ping Kuo M,
    5. Cheng SL,
    6. Huang YL,
    7. Chen HM,
    8. Chang HH,
    9. Chiang CP
    : Significant association of SRC protein expression with the progression, recurrence, and prognosis of oral squamous cell carcinoma in Taiwan. Head Neck 34(9): 1340-1345, 2012. DOI: 10.1002/hed.21923
    OpenUrlCrossRefPubMed
  13. ↵
    1. Zhang S,
    2. Yu D
    : Targeting Src family kinases in anti-cancer therapies: turning promise into triumph. Trends Pharmacol Sci 33(3): 122-128, 2012. DOI: 10.1016/j.tips.2011.11.002
    OpenUrlCrossRefPubMed
  14. ↵
    1. Le Roux AL,
    2. Mohammad IL,
    3. Mateos B,
    4. Arbesú M,
    5. Gairí M,
    6. Khan FA,
    7. Teixeira JMC,
    8. Pons M
    : A myristoyl-binding site in the SH3 domain modulates c-Src membrane anchoring. iScience 12: 194-203, 2019. DOI: 10.1016/j.isci.2019.01.010
    OpenUrlCrossRefPubMed
  15. ↵
    1. Fujinaga T,
    2. Kumamaru W,
    3. Sugiura T,
    4. Kobayashi Y,
    5. Ohyama Y,
    6. Ikari T,
    7. Onimaru M,
    8. Akimoto N,
    9. Jogo R,
    10. Mori Y
    : Biological characterization and analysis of metastasis-related genes in cell lines derived from the primary lesion and lymph node metastasis of a squamous cell carcinoma arising in the mandibular gingiva. Int J Oncol 44(5): 1614-1624, 2014. DOI: 10.3892/ijo.2014.2332
    OpenUrlCrossRefPubMed
  16. ↵
    1. Suzuki A,
    2. Sugiyama G,
    3. Ohyama Y,
    4. Kumamaru W,
    5. Yamada T,
    6. Mori Y
    : Regulation of NF-kB signalling through the PR55β-RelA interaction in osteoblasts. In Vivo 34(2): 601-608, 2020. DOI: 10.21873/invivo.11813
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Jiromaru R,
    2. Yasumatsu R,
    3. Matsuo M,
    4. Hashimoto K,
    5. Kogo R,
    6. Nakagawa T
    : Clinical analysis of oral squamous cell carcinoma: a single-institution experience. Cancer Diagn Progn 4(2): 105-110, 2024. DOI: 10.21873/cdp.10294
    OpenUrlCrossRefPubMed
  18. ↵
    1. Eshaq AM,
    2. Flanagan TW,
    3. Hassan SY,
    4. Al Asheikh SA,
    5. Al-Amoudi WA,
    6. Santourlidis S,
    7. Hassan SL,
    8. Alamodi MO,
    9. Bendhack ML,
    10. Alamodi MO,
    11. Haikel Y,
    12. Megahed M,
    13. Hassan M
    : Non-receptor tyrosine kinases: their structure and mechanistic role in tumor progression and resistance. Cancers (Basel) 16(15): 2754, 2024. DOI: 10.3390/cancers16152754
    OpenUrlCrossRefPubMed
  19. ↵
    1. Solouki S,
    2. August A,
    3. Huang W
    : Non-receptor tyrosine kinase signaling in autoimmunity and therapeutic implications. Pharmacol Ther 201: 39-50, 2019. DOI: 10.1016/j.pharmthera.2019.05.008
    OpenUrlCrossRefPubMed
  20. ↵
    1. Veeraraghavan VP,
    2. Daniel S,
    3. Dasari AK,
    4. Aileni KR,
    5. Patil C,
    6. Patil SR
    : Emerging targeted therapies in oral oncology: Focus on EGFR inhibition. Oral Oncol Rep 11: 100592, 2024. DOI: 10.1016/j.oor.2024.100592
    OpenUrlCrossRef
  21. ↵
    1. Yun H,
    2. Han GH,
    3. Wee DJ,
    4. Chay DB,
    5. Chung JY,
    6. Kim JH,
    7. Cho H
    : Loss of E-cadherin activates EGFR-MEK/ERK signaling, promoting cervical cancer progression. Cancer Genomics Proteomics 22(2): 271-284, 2025. DOI: 10.21873/cgp.20501
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Shawky AM,
    2. Almalki FA,
    3. Abdalla AN,
    4. Abdelazeem AH,
    5. Gouda AM
    : A comprehensive overview of globally approved JAK inhibitors. Pharmaceutics 14(5): 1001, 2022. DOI: 10.3390/pharmaceutics14051001
    OpenUrlCrossRefPubMed
    1. Jones JK,
    2. Thompson EM
    : Allosteric inhibition of ABL kinases: therapeutic potential in cancer. Mol Cancer Ther 19(9): 1763-1769, 2020. DOI: 10.1158/1535-7163.MCT-20-0069
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Rohila D,
    2. Park IH,
    3. Pham TV,
    4. Weitz J,
    5. Hurtado de Mendoza T,
    6. Madheswaran S,
    7. Ishfaq M,
    8. Beaman C,
    9. Tapia E,
    10. Sun S,
    11. Patel J,
    12. Tamayo P,
    13. Lowy AM,
    14. Joshi S
    : Syk inhibition reprograms tumor-associated macrophages and overcomes gemcitabine-induced immunosuppression in pancreatic ductal adenocarcinoma. Cancer Res 83(16): 2675-2689, 2023. DOI: 10.1158/0008-5472.CAN-22-3645
    OpenUrlCrossRefPubMed
  25. ↵
    1. Parsons SJ,
    2. Parsons JT
    : Src family kinases, key regulators of signal transduction. Oncogene 23(48): 7906-7909, 2004. DOI: 10.1038/sj.onc.1208160
    OpenUrlCrossRefPubMed
  26. ↵
    1. Karinen S,
    2. Forero-Rodríguez J,
    3. Järvinen A,
    4. Eklund KK,
    5. Barreto G,
    6. Salem A
    : CRISPR/Cas9-mediated knockout of LYVE1 in human tongue cancer cells reveals transcriptomic changes in metastasis-associated pathways. Cancer Genomics Proteomics 22(4): 525-537, 2025. DOI: 10.21873/cgp.20519
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Reinecke J,
    2. Caplan S
    : Endocytosis and the Src family of non-receptor tyrosine kinases. Biomol Concepts 5(2): 143-155, 2014. DOI: 10.1515/bmc-2014-0003
    OpenUrlCrossRefPubMed
  28. ↵
    1. Udenwobele DI,
    2. Su RC,
    3. Good SV,
    4. Ball TB,
    5. Varma Shrivastav S,
    6. Shrivastav A
    : Myristoylation: an important protein modification in the immune response. Front Immunol 8: 751, 2017. DOI: 10.3389/fimmu.2017.00751
    OpenUrlCrossRefPubMed
  29. ↵
    1. Mehta K,
    2. Hegde M,
    3. Girisa S,
    4. Vishwa R,
    5. Alqahtani MS,
    6. Abbas M,
    7. Shakibaei M,
    8. Sethi G,
    9. Kunnumakkara AB
    : Targeting RTKs/nRTKs as promising therapeutic strategies for the treatment of triple-negative breast cancer: evidence from clinical trials. Mil Med Res 11(1): 76, 2024. DOI: 10.1186/s40779-024-00582-zw
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
N-myristoyltransferase 1-mediated Src Myristoylation Promotes Non-receptor Tyrosine Kinase Pathways in Oral Squamous Cell Carcinoma
TAKUMA KOMIYAMA, GORO SUGIYAMA, HANAE KAMOHARA, TAKAHIRO FUJINAGA, WATARU KUMAMARU, MASAFUMI MORIYAMA
In Vivo Jan 2026, 40 (1) 76-88; DOI: 10.21873/invivo.14174
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords