Investigating the oncogenic potential of MTHFD2 in oral squamous cell carcinoma

Abstract

Purpose

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial bifunctional enzyme, is reportedly upregulated in several malignancies, demonstrating cancer-associated dysregulation. However, its functional contribution to oral squamous cell carcinoma (OSCC) progression remains unexplored. Therefore, this study aimed to investigate the role of MTHFD2 in OSCC development.

Methods

MTHFD2 expression was assessed in OSCC biospecimens using integrated bioinformatics, immunohistochemistry, and immunoblotting techniques. Survival analysis was conducted utilizing the Kaplan–Meier methodology. The MTHFD2-associated mechanisms involved in OSCC were explored via enrichment analysis. Genetic suppression of MTHFD2 was employed to evaluate the functional consequences on OSCC cell proliferation (Cell Counting Kit-8 assay), apoptosis (flow cytometry), migration (wound healing assay), and invasion (Matrigel transmigration).

Results

MTHFD2 expression was considerably elevated in OSCC lesions and cellular models. MTHFD2 overexpression in OSCC tissues correlated with advanced Tumor, Node, and Metastasis stage (P = 0.002), regional lymph node metastasis (P = 0.043), and elevated Ki67 expression (P < 0.001). Furthermore, increased MTHFD2 levels were associated with poor overall survival outcomes in patients with OSCC (P < 0.05). Pathway enrichment analysis revealed that MTHFD2 upregulation was closely linked to the dysregulation of key neoplastic processes. Stable MTHFD2 depletion attenuated cell proliferation in vitro and diminished xenograft tumorigenicity in vivo (P < 0.05). Additionally, MTHFD2 ablation induced OSCC cell apoptosis and reduced cell migration and invasion.

Conclusion

This study indicates that MTHFD2 is critical for OSCC progression and may serve as a potential therapeutic target, though further validation is needed to confirm its independent prognostic value and clinical applicability.

Introduction

Oral squamous cell carcinoma (OSCC) ranks among the most prevalent malignant tumors in the head and neck region (Bray et al. 2018). Currently, surgery, radiotherapy, chemotherapy, and biotherapy are the primary treatment modalities for OSCC. Despite significant advancements in early diagnosis and therapeutic strategies, the 5-year survival rate for patients with OSCC remains approximately 50%. This is largely attributed to an inadequate understanding of the mechanisms underlying OSCC development (Du et al. 2020) .Therefore, identifying innovative molecular targets for OSCC therapeutics is imperative.

Tumor cells frequently rewire their metabolic signaling pathways to facilitate rapid proliferation (Hanahan 2022; Pavlova et al. 2022). One-carbon metabolism, a crucial aspect of metabolic reprogramming in cancer, plays a pivotal role in nucleotide synthesis, the methionine cycle, methylation reactions, adenosine triphosphate production, and redox homeostasis (Ducker and Rabinowitz 2017; Zhao et al. 2021). Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is an essential enzyme for one-carbon folate metabolism in the mitochondria (Nilsson et al. 2014). Growing evidence indicates that MTHFD2 is markedly upregulated in various cancers, exhibiting minimal expression in numerous healthy adult tissues (Shang et al. 2021; Shi et al. 2023). Considering these characteristics, MTHFD2 serves as a potential diagnostic marker and a candidate anticancer target. Elevated MTHFD2 expression has been reported to predict an unfavorable prognosis and may promote cancer cell proliferation and metastasis – findings that are further supported by our current observations in OSCC (Lin et al. 2018; Liu et al. 2016; Shi et al. 2021). Moreover, the suppression of MTHFD2 expression has been associated with reduced tumor burden and prolonged survival (Ju et al. 2019; Zhang et al. 2023; Zhou et al. 2023). These findings position MTHFD2 as a promising target for cancer therapeutic strategies. However, the relationship between MTHFD2 expression and OSCC development remains unclear.

We previously detected a pronounced upregulation of MTHFD2 mRNA expression in OSCC specimens compared with that in adjacent normal tissues within The Cancer Genome Atlas (TCGA) patient cohort. Furthermore, receiver operating characteristic curve analysis demonstrated that MTHFD2 expression exhibited robust predictive capability in distinguishing OSCC tissues from adjacent normal tissues (Wang et al. 2022). Therefore, this study aimed to investigate the role of MTHFD2 in OSCC development. MTHFD2 was significantly upregulated at the protein level in clinical OSCC tissues and cell lines. MTHFD2 overexpression in OSCC tissues was closely associated with advanced tumor, node, and metastasis (TNM) stage, regional lymph node involvement, and poor survival outcomes. Additionally, MTHFD2 knockdown notably hindered OSCC cell proliferation in vitro and tumor-forming capacity in vivo, induced apoptosis, and reduced migration and invasion. Our study potentially provides a novel prognostic marker and therapeutic target for OSCC.

Materials and methods

Clinical specimens

The study included 45 patients diagnosed with primary OSCC tumors at the Stomatological Hospital, Southern Medical University, from January 2021 to December 2021. A total of 45 OSCC tissues and 12 adjacent normal tissues were excised following surgical resection. Two pathologists independently reviewed each tissue sample. All enrolled participants completed standardized consent documentation with notarized signatures. The research protocol was approved by the Ethics Committee of the Stomatological Hospital, Southern Medical University (No. AFSQ-0105).

Immunohistochemical staining

Formalin-fixed, paraffin-embedded specimens of OSCC and adjacent normal tissues were sectioned to a thickness of 4 μm and processed for immunostaining. Following dewaxing and rehydration, the tissue sections underwent antigen retrieval through heat induction in citrate buffer (pH 6.0). Subsequently, the tissue specimens were incubated overnight at 4 °C with primary antibodies targeting MTHFD2 (1:200, NBP1-33200, Novus) and Ki67 (1:100, AF0198, Affinity Biosciences). Thereafter, the slides were incubated for 1 h with secondary antibodies conjugated to horseradish peroxidase (HRP; 1:10,000, GK600705A, GeneTech). Finally, immunoreactivity was detected using 3,3’‑diaminobenzidine (ZLI-9018, OriGene) for chromogenic development, after which the sections were counterstained with hematoxylin to enhance tissue structural visualization. A light microscope (Olympus, Japan) was employed to examine the stained sections at 200× magnification.

Target protein immunolabeling quantification was performed independently by two senior pathologists, following double-blinded protocols wherein clinical case details were systematically masked throughout the assessment. Cellular chromogenic deposition intensity was graded using a four-point semiquantitative scale under optical microscopy: 0 (absent staining), 1 (weak, pale yellow), 2 (moderate, yellow brown), and 3 (strong, brown). The percentage of cells displaying positive staining was classified based on the following scale: 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The final score for each specimen was calculated by multiplying the staining intensity by the proportion of positive cells. Samples were classified as exhibiting high expression if their total score was ≥ 6, whereas low expression was assigned to those with a total score of < 6.(Nilsson et al. 2014).

Cell culture

Human OSCC cell lines (HSC3, HSC6, SCC15, CAL27, and CAL33) and normal oral keratinocytes (NOK) were utilized in this study. HSC3, HSC6, CAL33, and NOK cell lines were generously provided by J. Silvio Gutkind (NIH, Bethesda, MD, USA). SCC15 and CAL27 cells were procured from the American Type Culture Collection (Rockville, MD, USA). The HSC3, HSC6, CAL27, and CAL33 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). SCC15 cells were cultured in DMEM-F12 (Gibco, USA) with the addition of 10% FBS. All cells were maintained at 37 °C in a humidified incubator with 5% CO₂. NOK cells were cultured in serum-free keratinocyte growth medium (ScienCell, SC-2611-b), supplemented with bovine pituitary extract (25 µg/mL; Gibco, USA), epidermal growth factor (1 ng/mL; Gibco, USA), and a 1% antibiotic-antimycotic solution (penicillin/streptomycin; Gibco, USA).

Cell transfection

Lentivirus-based empty vector (5′-TTCTCCGAACGTGTCACGT-3′) and MTHFD2 gene-specific short hairpin RNAs (shRNAs; #1: 5′-CGAGAAGTGCTGAAGTCTAAA-3′, #2: 5′-GCAGTTGAAGAAACATACAAT-3′, and #3: 5′-GCTGGGTATATCACTCCAGTT-3′) were procured from GeneChem Co., Ltd. (Shanghai, China). Subsequently, stable CAL27 cell lines with MTHFD2 knockdown were established via puromycin-based screening. CAL27 cells were transduced with viral particles following the manufacturer’s guidelines. Puromycin (2 µg/mL; Sigma, USA) was administered for 3 weeks to select and validate polyclonal populations exhibiting stable MTHFD2 knockdown. Transfection efficiency was determined using a Western blot assay.

Western blot

Cellular proteins were extracted by homogenizing samples in radioimmunoprecipitation assay lysis buffer (Cowin Biotech, Taizhou, China). Protein content was quantified using a bicinchoninic acid assay kit (Sigma–Aldrich, Santa Clara, CA, USA). Equalized amounts of protein lysates underwent fractionation via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electrotransfer to polyvinylidene difluoride membranes (Millipore, USA). After blocking with 5% non-fat dried milk for 1 h, the membranes were probed with primary antibodies at 4 °C for 16 h, followed by HRP-conjugated secondary antibodies (1 h). Signal acquisition was conducted using an enhanced chemiluminescence detection system (Millipore, USA). The following primary antibodies were employed: MTHFD2 (NBP1-33200; Novus, USA) and GAPDH glyceraldehyde-3-phosphate dehydrogenase (Proteintech, Wuhan, China).

Cell viability assay

Cells transfected with shMTHFD2 or the empty vector shRNA (shNC) were plated in 96-well plates (4 × 10³ cells/well). Cell viability was assessed at 12, 24, 36, 48, and 72 h post-transfection using a Cell Counting Kit-8 (CCK-8; GLPBIO, USA) in accordance with the manufacturer’s protocol. Following a 1-h incubation at 37 °C, absorbance was measured using a microplate reader (iMark™, Bio-Rad) at a wavelength of 450 nm.

Cell apoptosis assay

Apoptosis in OSCC cells (shMTHFD2/shNC-transfected) was evaluated using an annexin V-fluorescein isothiocyanate/propidium iodide (PI) dual-staining kit (KGA108; KeyGEN, China). Briefly, collected cells were resuspended in binding buffer and incubated with the fluorochrome for 15 min at room temperature, protected from light, prior to flow cytometric analysis. The cells were reconstituted in binding buffer suspension, followed by the quantification of apoptotic cell proportions via flow cytometry using a BD FACSCalibur platform.

Wound healing and invasion assays

Cells were seeded in 6-well plates until they reached full confluence. A mechanical wound was created in the monolayer using a sterile tip, followed by two gentle washes to remove detached cells. The plates were subsequently maintained for 24 h in a low-serum medium (1% FBS). Wound closure was assessed by analyzing the gap width at three randomly selected fields per well, and images were recorded using a Zeiss imaging system (Germany).

Cell invasion capacity was evaluated using Matrigel-coated Transwell inserts (Corning Inc.). Briefly, serum-starved cells were plated in the upper compartment, while the lower chamber contained medium supplemented with 10% FBS as a chemoattractant. After a 24-h incubation period, migratory cells adhering to the lower membrane surface were fixed, stained with crystal violet (0.1%), and microscopically quantified at 50× magnification (Carl Zeiss, Germany).

Animal experiment

All animal experimental procedures complied with the ethical guidelines established by the Animal Care Committee of Sun Yat-sen University. Female BALB/c nude mice (4 weeks old) were acquired from the Guangdong Laboratory Animal Center (Guangdong, China) and acclimatized in sterile isolators prior to randomization into control (shNC) and experimental (shMTHFD2) groups (n = 5 per group). Subcutaneous xenografts were generated by injecting 3 × 10⁶ transfected CAL27 cells per mouse. Tumor growth was assessed every 3 days using calipers, with volume calculated as follows: length × width²/2. Following a 30-day observation period, tumors were harvested post-euthanasia for mass recording and fixation in 4% formaldehyde.

Bioinformatics analysis

We downloaded and organized RNA sequencing data from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov) for the TCGA-head and neck squamous cell carcinoma (HNSCC) cohort. The OSCC samples were derived from HNSCC data. To delineate the prognostic impact of MTHFD2, we stratified TCGA-OSCC cases by median expression thresholds and analyzed overall survival (OS) between high- and low-expression cohorts. Subsequently, the influence of MTHFD2 expression on OS was assessed through subgroup analyses based on age, race, tumor stage, cervical lymph node metastasis, alcohol consumption, and smoking status using the Kaplan-Meier plotter.

To investigate MTHFD2-associated functions in OSCC, Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analyses were performed on differentially expressed genes (DEGs) utilizing the R package GOplot (version 1.0.2).

Statistical analysis

All data were analyzed using GraphPad Prism (version 8.0). In intergroup comparisons, Student’s t-test and one-way analysis of variance were employed for two-group and multi-group analyses, respectively. Statistical significance was set at P < 0.05.

Results

Elevated expression of MTHFD2 in OSCC

We examined MTHFD2 protein expression levels in clinical specimens and OSCC cell lines using immunohistochemistry and Western blot. MTHFD2 was detected in the nucleus and cytoplasm, predominantly exhibiting cytoplasmic expression. Notably, MTHFD2 protein expression was significantly elevated in OSCC samples compared with that in adjacent normal tissues (Fig. 1A, C). Furthermore, MTHFD2 expression levels demonstrated significant associations with key clinicopathological parameters in patients with OSCC, including primary tumor stage (P < 0.001), regional lymph node metastasis (P = 0.043), and TNM stage (P = 0.002, Table 1). Consistent with the tissue findings, MTHFD2 protein expression was persistently elevated in a panel of five OSCC cell lines (HSC3, SCC15, CAL33, HSC6, and CAL27) relative to that in NOK cells (Fig. 1E). Among these cell lines, CAL27 exhibited the most pronounced upregulation; consequently, CAL27 cells were selected for subsequent experiments.

Fig. 1.

Expression pattern of MTHFD2 in OSCC. A, C MTHFD2 immunohistochemical localization and quantitative assessment in OSCC and adjacent normal tissues. B, D Comparative Ki67 immunohistochemistry and quantification in OSCC and normal tissues. E Western blot analysis of MTHFD2 protein levels in NOK and OSCC cell lines. ***P < 0.001. Scale bar, 50 μm. NOK, normal oral keratinocytes

Table 1.

Association of MTHFD2 levels with clinicodemographic parameters in patients with OSCC

Characteristics	Cases	MTHFD2 staining score		P value
		Low	High
Age
≥ 50	28	4	24	0.028
< 50	17	3	14
Sex
Male	30	4	26	0.898
Female	15	3	12
T-primary tumor
T1- T2	25	6	19	< 0.001
T3 -T4	20	1	14
N-regional lymph node				0.043
Non-metastasis	26	7	19
Metastasis	19	0	19
Histological grade
Well differentiated	30	6	24	0.070
Moderately + poorly differentiated	15	1	14
TNM stage
I - II	18	6	12	0.002
III - IV	27	1	26
Ki67
High	37	21	16	0.047
Low	8	1	7

T, tumor; N, node

Immunohistochemical staining results revealed that Ki67 is primarily localized in the nucleus, with its expression significantly elevated in OSCC specimens compared with that in adjacent normal tissues (P < 0.001, Fig. 1B, D). Furthermore, elevated MTHFD2 levels positively correlated with Ki67 expression in OSCC tissues (P = 0.047, Table 1). Therefore, the data obtained indicate that MTHFD2 is overexpressed in OSCC tissues and may play a role in disease progression.

Prognostic significance of MTHFD2 in patients with OSCC

Our previous research revealed that upregulated MTHFD2 expression significantly correlated with diminished OS in the TCGA-OSCC cohort, suggesting that MTHFD2 potentially serves as a prognostic biomarker for adverse outcomes in OSCC. In this study, we conducted subgroup analyses to further substantiate this association. Significantly reduced OS was consistently observed in high MTHFD2 expressers across multiple clinical subsets, including pathologic T3–4, pathologic N1–3, M0, individuals aged > 60 years, smokers, and white ethnic subgroups (all P < 0.05, Fig. 2). Collectively, MTHFD2 upregulation is indicative of poor survival in patients with OSCC, suggesting its functional involvement in OSCC progression.

Fig. 2.

MTHFD2 expression stratification predicts survival outcomes in patients with OSCC. Overall survival curves of MTHFD2 for OSCC are presented across diverse subgroups, including age, race, tumor stage, cervical lymph node metastasis, alcohol consumption, and smoking status

Functional enrichment analysis of MTHFD2 in OSCC

To delineate the regulatory networks involving MTHFD2 in OSCC pathogenesis, we utilized the LinkedOmics platform to identify DEGs correlated with MTHFD2. The subsequent biological characterization of candidate genes was performed using GO terms and KEGG pathway enrichment analyses. GO analysis revealed a strong association of MTHFD2 with oncogenic biological processes, including cell differentiation, keratinization, keratinocyte differentiation, muscle contraction, intermediate filament-based processes, and intermediate filament cytoskeleton organization. Significantly enriched cellular components comprised the collagen-containing extracellular matrix, keratin filament, ion channel complex, Golgi lumen, and myosin complex. Molecular function analysis indicated that these proteins are associated with receptor–ligand activity, G protein-coupled receptor binding, serine hydrolase activity, peptidase regulator activity, endopeptidase regulator activity, and cytoskeletal structural constituents (Fig. 3A–C). The KEGG enrichment analysis revealed that high MTHFD2 expression was significantly associated with the one-carbon metabolism pathway, neuroactive ligand–receptor interaction, histidine metabolism, and endopeptidase regulator activity (Fig. 3D).

Fig. 3.

Functional enrichment analysis of the DEGs associated with MTHFD2 in OSCC. A–C GO enrichment analysis of DEGs associated with MTHFD2. D KEGG pathway enrichment mapping for MTHFD2-correlated transcriptional signatures in OSCC. BP, biological process; CC, cellular component; MF, molecular function

MTHFD2 depletion suppresses OSCC cell growth in vitro and in vivo

To ascertain whether elevated MTHFD2 levels are functionally implicated in OSCC progression, we knocked down MTHFD2 in CAL27 cells using stable shRNA. Efficient MTHFD2 protein knockdown was attained with shMTHFD2-#1 and shMTHFD2-#3 in CAL27 cells (Fig. 4A). Consequently, shMTHFD2-#1 and shMTHFD2-#3 were selected for follow-up experiments. The CCK-8 assay demonstrated that MTHFD2 knockdown inhibited OSCC cell viability throughout the 12–72 h period post-transfection, relative to the empty vector group (Fig. 4B). This effect was most pronounced at 72 h (P < 0.01).

Fig. 4.

MTHFD2 depletion attenuated malignant growth in cellular systems and inhibited xenograft development in OSCC animal models. A Immunoblot confirmation of MTHFD2 knockdown efficiency in CAL27 cells. B CCK-8 proliferation kinetics were assessed in MTHFD2-depleted OSCC lineages compared to control shRNA at 0–72 h post-transfection. C–F Representative images of OSCC orthotopic xenograft and corresponding tumor tissue pathology assessment (n = 5). D–E Comparative quantification of tumor volume and mass in the shMTHFD2 group versus the shNC group. *P < 0.05 and **P < 0.01

To validate the tumorigenic role of MTHFD2 in OSCC pathogenesis in vivo, we developed subcutaneous xenografts in BALB/c nude mice. CAL27 cells, with shMTHFD2 or shNC, were subcutaneously injected into the nude mice. Consistent with our in vitro findings, MTHFD2 deficiency significantly hindered tumor growth compared with that in the shNC group, yielding substantially lower endpoint tumor burdens as quantified by both mass and volume (P < 0.05, Fig. 4C–F). Collectively, these findings demonstrate that MTHFD2 depletion reduces proliferative capacity in vitro and tumorigenic potential in vivo, thereby identifying MTHFD2 as an oncogenic driver in OSCC.

MTHFD2 depletion induces OSCC cell apoptosis

The assessment of MTHFD2 depletion-induced apoptosis was conducted using Annexin V/PI staining. We observed a significantly elevated apoptotic index in shMTHFD2-#1-transduced CAL27 cells (20.93 ± 2.07%, P = 0.0005; Fig. 5A, B) compared with that in the shNC group (7.94 ± 1.44%). Flow cytometry revealed that MTHFD2 depletion significantly increased OSCC cell apoptosis.

Fig. 5.

Functional consequences of MTHFD2 knockdown across apoptotic, migratory, and invasive phenotypes in OSCC. A, B Apoptotic incidence was assessed using Annexin V/PI dual-labeling cytometry in OSCC cells with stable MTHFD2 knockdown compared to control shRNA. C, D Cell invasion capacity under MTHFD2 knockdown was evaluated using a Transwell assay, with representative fields and cell penetration indices quantified. E–F Cell migratory capacity under MTHFD2 knockdown was assessed using scratch-wound closure kinetics, along with representative images and quantification of migrated OSCC cells. **P < 0.01, ***P < 0.001

MTHFD2 depletion suppresses OSCC cell migration and invasion

Our clinicopathological analyses indicated a positive correlation between MTHFD2 expression and advanced TNM staging in OSCC cohorts (Table 1). Given the vital role of invasion in OSCC pathogenesis, we further investigated the functional role of MTHFD2 in modulating cellular motility. Scratch-wound closure assays and Matrigel invasion chambers demonstrated significantly diminished migratory and invasive phenotypes in MTHFD2-depleted OSCC cells compared with those in the controls (P < 0.001, P < 0.01, respectively; Fig. 5C–F). These findings establish MTHFD2 as a critical regulator of OSCC cell invasion dynamics.

Discussion

OSCC is the predominant oral cavity malignancy, characterized by unfavorable clinical outcomes. The limited availability of molecularly targeted interventions contributes to persistently low survival rates. Increasing evidence indicates that mitochondrial MTHFD2 is a critical regulator of one-carbon metabolism, exhibiting oncogenic upregulation in multiple cancer types. Selectively targeting MTHFD2 likely presents a superior therapeutic opportunity. Our investigation identified significant MTHFD2 overexpression in OSCC specimens, correlating with reduced OS. MTHFD2 depletion hampered OSCC cell growth in vitro and in vivo. Furthermore, MTHFD2 depletion induced apoptosis in OSCC cells and diminished their migratory and invasive capacities. Collectively, our results establish MTHFD2 as a promising clinically relevant target for precision oncology strategies in oral cancer, warranting further validation in larger cohorts.

While MTHFD2 upregulation has been reported in multiple cancers with prognostic implications (He et al. 2020; Ju et al. 2019; Zhang et al. 2023), its expression patterns in OSCC have remained incompletely characterized. We substantiate elevated MTHFD2 protein levels in both clinical OSCC specimens and cellular models. Transcriptional validation using TCGA datasets further revealed concomitant mRNA upregulation in neoplastic tissue compared with that in adjacent normal tissue. This work provides a novel demonstration of concurrent transcriptional and translational MTHFD2 dysregulation in OSCC pathogenesis.

The relationships between MTHFD2 expression and clinicopathological determinants were next evaluated in patients with OSCC. Our findings indicate that elevated MTHFD2 expression is significantly associated with advanced TNM stages (T3–4) and regional lymph node metastasis. Furthermore, MTHFD2 upregulation significantly correlates with high Ki67 expression in OSCC. Ki67 is a well-known, widely used marker of tumor proliferation (Menon et al. 2019). Accordingly, we hypothesized that MTHFD2 promotes tumor cell proliferation. Subsequent functional analyses demonstrated that MTHFD2 depletion attenuates neoplastic expansion and enhances apoptotic pathways in OSCC cells. Concordantly, the genetic suppression of MTHFD2 expression mitigated xenograft tumorigenicity in vivo. These observations align with the established oncogenic functions of MTHFD2 in diverse malignancies, where its overexpression accelerates proliferative programs. Consistent with findings in non-small cell lung cancer (NSCLC) and colorectal cancer (Wei et al. 2019; Yu et al. 2020), our data suggest that MTHFD2 may serve as a master regulator of tumor growth through proliferation enhancement.

Further clinicopathological assessments confirmed that MTHFD2 expression levels positively correlate with disease progression and regional lymph node metastasis in OSCC cohorts. Complementary functional studies validated that MTHFD2 depletion reduces motility phenotypes and matrix-infiltrative capacity in OSCC models. Given the established role of invasion in OSCC metastatic dissemination (Routray et al. 2021), these findings carry significant therapeutic implications. This prometastatic function appears to be conserved across various malignancies. For instance, MTHFD2 depletion impairs migratory and invasive capabilities in ovarian carcinoma (Li et al. 2021), while its overexpression enhances vimentin-mediated migratory programs in renal cell carcinoma (Lin et al. 2018). Likewise, MTHFD2 inhibition disrupts ILK-mediated metastatic signaling in NSCLC (Zhou et al. 2023). Taken together, these data indicate that upregulated MTHFD2 expression facilitates OSCC progression by endowing cancer cells with a more aggressive malignant phenotype.

The mechanistic basis of MTHFD2-driven oncogenesis remains inadequately characterized. Yu et al. demonstrated that MTHFD2 depletion disrupts proliferative capacity by modulating cell cycle regulators (CCNA2, MCM7, and SKP2) in NSCLC (Yu et al. 2020). Concurrently, Li et al. established that the MTHFD2-dependent activation of signal transducer and activator of transcription 3 orchestrates epithelial–mesenchymal transition programming and invasive phenotypes in ovarian carcinoma (Li et al. 2021). Moreover, breast cancer studies implicate MTHFD2 in promoting tumorigenesis through AKT pathway activation (Huang et al. 2021). Importantly, MTHFD2 regulates serine-glycine metabolism and mitochondrial one-carbon metabolism in response to oxidative stress, with its suppression compromising cellular proliferation and invasion by disrupting the nicotinamide adenine dinucleotide phosphate/redox axis (Hitzel et al. 2018; Ju et al. 2019). Our pathway enrichment analysis in OSCC revealed that MTHFD2 overexpression correlates with the dysregulation of core neoplastic processes, particularly the one-carbon metabolism pathway, such as neuroactive ligand–receptor interaction, histidine metabolism, and endopeptidase regulator activity. The dysregulation of endopeptidase regulator activity—particularly that of asparagine endopeptidase and prolyl endopeptidase—is often characterized by the overexpression of these enzymes. Additionally, it is frequently observed across diverse cancer types and is linked to unfavorable prognosis and enhanced proliferative advantage (Zhu et al. 2016). These enzymes facilitate metastatic dissemination through extracellular matrix remodeling (Perez et al. 2020; Qi et al. 2017). Furthermore, dysregulated histidine metabolism drives oncogenesis by modulating tumor cell metabolic processes (Liu et al. 2025). Collectively, these pathways constitute metabolic circuits through which OSCC reprograms fundamental processes within the tumor to facilitate cell proliferation. While warranting experimental validation, this study delineates novel MTHFD2-associated networks in oral carcinogenesis. The foregoing research suggests that MTHFD2 exerts oncogenic effects on tumor progression across various cancers by modulating multiple cancer-related signaling pathways.

There were some potential limitations to this study. Firstly, functional experiments were mainly conducted using the single cell line (CAL27). Notably, the functional phenotypes observed in CAL27-including reduced proliferation, enhanced apoptosis, and impaired migration and invasion ability are consistent and statistically significant, providing strong preliminary evidence for MTHFD2’s oncogenic role in OSCC. Future studies are need to validate the observed phenotypes in additional OSCC cell lines (e.g., SCC15, HSC3) to confirm the universality of MTHFD2’s oncogenic role. Secondly, the size of the clinical cohort is relatively small; therefore, the results of subgroup stratification should be interpreted with considerable caution. Larger, well-powered cohorts are warranted to validate these exploratory findings and further confirm the independent prognostic value of MTHFD2. Finally, the lack of pharmacologic inhibition studies represents a key limitation of this work, and future investigations will focus on evaluating MTHFD2-specific inhibitors in preclinical models.

Overall, MTHFD2 exhibits considerable overexpression in OSCC clinical specimens and cellular models, demonstrating robust prognostic stratification value. Elevated MTHFD2 levels correlate with advanced primary tumors, progressive TNM staging, regional lymphatic dissemination, and increased Ki67 expression in OSCC tissues. MTHFD2 knockdown dramatically suppresses cell growth in vitro and tumorigenicity in vivo. Furthermore, MTHFD2 depletion enhances OSCC cell apoptosis and reduces cell migration and invasion. These findings enhance our understanding of the functional role of MTHFD2 in OSCC and highlight its potential as a potential therapeutic target for this malignancy. Further validation using pharmacological inhibitors is warranted to confirm its clinical applicability. Additionally, future studies are needed to elucidate the specific molecular mechanisms underlying MTHFD2-mediated regulation of OSCC tumorigenesis and progression.

Author contributions

JW: Conceptualization, Methodology, Validation, Writing review \& editing, Supervision, Project administration, Funding acquisition. JH: Methodology, Validation, Resources, Investigation, Data curation, Formal Analysis, Visualization, Writing-original draft preparation, Funding Acquisition. WW: Methodology, Validation, Resources, Investigation, Data curation, Formal Analysis, Visualization, Writing-original draft preparation. SL: Software, Validation, Resources, Visualization. All authors reviewed the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant no. 81800969), Medical Science and Technology Foundation of Guangdong Province (Grant no. B2020109), and Stomatological Hospital, Southern Medical University Research Incubation Foundation (Grant no. PY2019034).

Data availability

The data presented in this study are available on reasonable request from the corresponding author.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Stomatological Hospital, Southern Medical University (approval number AFSQ-0105). All animal experimental procedures complied with the ethical guidelines established by Sun Yat-sen University’s animal care committee.