High expression of folate metabolic pathway gene MTHFD2 is related to the poor prognosis of patients and may apply as a potential new target for therapy of NSCLC

1 Introduction

Lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer, is the leading reason of malignant carcinoma associated mortality globally [1,2]. In year 2020, it was reported that 2.2 million new lung cancer cases were diagnosed and about 1.8 million were dead in the same year [3]. In China, 733.3 thousand new lung cancer cases and 610.2 death were identified in the year 2015 [4]. For US, the incidence and mortality of lung cancer were estimated as 236,740 and 130,180, respectively, in year 2022 [5]. Therefore, lung cancer is a heavy burden for the global medical system. However, the molecular mechanism of lung cancer is not completely clear yet [6].

Folic acid is an essential B vitamin for human body [7]. It is transformed into tetrahydrofolate in human body, and tetrahydrofolate can carry a carbon unit to participate in the synthesis of nucleotide and deoxynucleoside nucleic acid [8]. Folic acid plays an important role in maintaining genome stability [9,10]. The folate metabolism pathway includes methionine synthase [11], methylenetetrahydrofolate reductase, methylenetetrahydrofolate dehydrogenase 1, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and mitochondrial aminomethyltransferase 1 and 2, which play an important role in folate metabolism [12]. MTHFD2 is a nicotinamide adenine dinucleotide phosphate dependent bifunctional enzyme located in mitochondria, which is highly expressed in mitochondrial folate metabolism [13,14]. In recent years, many studies have shown that the overexpression of MTHFD2 can promote the occurrence and development of breast cancer [15], colon cancer [16], ovarian cancer [17], glioma [18], and other types of tumors. Yu and his colleges found the inhibitory effect of MTHFD2 knockdown on NSCLC may be mediated via suppressing cell cycle-related genes [19]. These findings delineate the role of MTHFD2 in the development of NSCLC and may have potential applications in the treatment of NSCLC. However, there are few reports on the expression of MTHFD2 in NSCLC and its relationship with the patient’s prognosis. The investigation of MTHFD2 in NSCLC research is supported by its roles in tumor metabolism and immune modulation, which are distinct from other metabolic enzymes and align with emerging therapeutic vulnerabilities in NSCLC. MTHFD2’s integration of metabolic reprogramming and immune evasion positions it as a priority target in NSCLC. This study proposes that MTHFD2 serves as a critical metabolic driver in NSCLC, with its overexpression in tumor tissues not only promoting tumor progression through dysregulation of folate-mediated one-carbon metabolism but also shaping the tumor immune microenvironment.

2 Data collection and analysis

3 Results

3.1 MTHFD2 expression in human solid tumors

The expression level of MTHFD2 in solid tumor tissues such as bladder cancer, esophageal cancer, head and neck squamous cell carcinoma, gastric cancer, and colorectal cancer was significantly higher than that in corresponding normal tissues (P < 0.05), Figure 1a. In NSCLC patients, the expression level of MTHFD2 in cancer tissues was significantly higher than that in adjacent tissues (P < 0.05), Figure 1b. The expression level of MTHFD2 was related to clinical stage (P < 0.05), Figure 1c. MTHFD-encoded protein was mainly expressed in the cytoplasm with positive expression of brownish yellow particle staining, Figure 1d and e (https://www.proteinatlas.org/).

Figure 1

Relative expression of MTHFD2 gene in human solid tumors and NSCLC. (a) Expression of MTHFD2 in a variety of human tumors; (b) relative expression of MTHFD2 in cancer tissues and normal bronchial epithelial tissues of NSCLC patients; (c) relationship between MTHFD2 expression level and clinical stages of NSCLC patients; (d) positive expression of MTHFD2 in the carcinoma tissues of NSCLC patients with brown-yellow staining; (e) MTHFD2 gene has negative expression in normal lung tissue; (f) expression of MTHFD2 in a variety of human cancer cell lines. *P < 0.05; **P < 0.01, ***P < 0.001.

3.2 PPI network construction of MTHFD2 and relevant genes

In the protein interaction database (STRING), the PPI network of MTHFD2 was constructed. There were 21 proteins, which interacted with MTHFD2 in the network, and the interaction relationship between proteins was 173 with the average local clustering coefficient of 0.881, Figure 2. The 21 genes interacting with MTHFD2 are listed in Table 1.

Figure 2

PPI network of MTHFD2 and relevant genes. 21 proteins interacting with MTHFD2 were constructed in the network, and the interaction relationship between proteins was 173 with the average local clustering coefficient of 0.881.The thickness of the PPI connections (edges) means the strength of data support (confidence score: 0.400). The nodes means the corresponding genes.

Table 1

Description of MTHFD2 and relevant genes

Gene name	Gene description or protein
MTHFD2	Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase
MTHFD1L	Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 like
MTHFD1	Methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1
MTHFR	Methylenetetrahydrofolate reductase
MTHFD2L	Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 like
SHMT2	Serine hydroxymethyltransferase 2
SHMT1	Serine hydroxymethyltransferase 1
GART	Phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase
ATIC	5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase
AMT	Aminomethyltransferase
GLDC	Glycine decarboxylase
ALDH18A1	Aldehyde dehydrogenase 18 family member A1
PSAT1	Phosphoserine aminotransferase 1
MTHFS	Methenyltetrahydrofolate synthetase
ALDH1L2	Aldehyde dehydrogenase 1 family member L2
ST20-MTHFS	ST20-MTHFS readthrough
FTCD	Formimidoyltransferase cyclodeaminase
PPAT	Phosphoribosyl pyrophosphate amidotransferase
MTFMT	Mitochondrial methionyl-tRNA formyltransferase
ALDH1L1	Aldehyde dehydrogenase 1 family member L1
TYMS	Thymidylate synthetase

3.4 KEGG pathway enrichment of MTHFD2 gene

The KEGG pathway of MTHFD2 and relevant genes was mainly enriched in one carbon pool by folate, metabolic pathways, antifolate resistance pathway, etc. Figure 3 and Table 4.

Figure 3

The KEGG pathway of MTHFD2 and relevant genes was mainly enriched in one carbon pool by folate, metabolic pathways and antifolate resistance pathway. The bubble (count = observed gene number) means observed genes and the negLog10_strength means the strength of data support [negLog10_strength = −log10 (p-value)].

Table 4

KEGG pathway enrichment of MTHFD2 gene

KEGG pathway description	Observed gene count	Background gene count	Strength	FDR
One carbon pool by folate	17	20	2.9	1.24 × 10−42
Metabolic pathways	18	1,447	1.06	8.75 × 10−16
Antifolate resistance	6	31	2.26	1.77 × 10−10
Glycine, serine, and threonine metabolism	5	38	2.09	6.68 × 10−8
Carbon metabolism	6	117	1.68	1.83 × 10−7
Glyoxylate and dicarboxylate metabolism	4	30	2.09	2.48 × 10−6
Biosynthesis of amino acids	4	73	1.71	6.03 × 10−5
Purine metabolism	3	127	1.34	0.0146

3.5 MTHFD2 expression and prognosis

The OS of patients with high expression of MTHFD2 gene in lung adenocarcinoma (LUAD) was significantly lower than that of patients with low expression (HR = 1.6, P = 0.0041), while the expression level of MTHFD2 was not related to the DFS of LUAD (HR = 1.4, P = 0.05), lung squamous cell carcinoma (LUSC; HR = 1.3, P = 0.16), and OS of LUSC (HR = 0.82, P = 0.15), Figure 4.

Figure 4

Survival curve of MTHFD2 expression and NSCLC patient’s prognosis. (a) DFS of LUAD between MTHFD2 high and low expression groups; (b) OS of LUAD between MTHFD2 high and low expression groups; (c) DFS of LUSC between MTHFD2 high and low expression groups; and (d) OS of LUSC between MTHFD2 high and low expression groups.

3.6 Relationship between MTHFD2 expression and lymphocyte infiltration

MTHFD2 expression level was correlated with B cells (r = −0.174, P < 0.05), CD8+ T cells (r = 0.143, P < 0.05), and CD4+ T cells (r = −0.236, P < 0.05) lymphocyte infiltration in LUAD. For LUSC, MTHFD2 expression level was correlated with CD4+ T cells (r = −0.195, P < 0.05) lymphocyte infiltration, Figure 5.

Figure 5

Scatter plot of relationship between MTHFD2 expression and lymphocyte infiltration. Correlation module draws the expression scatterplots between MTHFD2 and lymphocyte infiltration in NSCLC, together with the Spearman’s rho value and estimated statistical significance. Partial correlation conditioned on tumor purity or age are also provided. Partial correlation (partial.cor) measures the correlation between two variables while controlling for other factors. log2 TPM refers to the logarithm (base 2) of TPM, which is a normalized measure of gene expression. LUSC: Lung Squamous Cell Carcinoma; LUAD: Lung Adenocarcinoma. MTHFD2 expression level was correlated with B cells (r = −0.174, P < 0.05), CD8+ T cells (r = 0.143, P < 0.05), and CD4+ T cells (r = −0.236, P < 0.05) lymphocyte infiltration in LUAD. For LUSC, MTHFD2 expression level was correlated with CD4+ T cells (r = −0.195, P < 0.05) lymphocyte infiltration.

4 Discussion

MTHFD2 is an enzyme involved in folate metabolism and one-carbon metabolism, pathways critical for nucleotide synthesis and cellular proliferation. Its biological significance is particularly pronounced in the context of cancer, as aberrant MTHFD2 expression has been linked to various malignancies. Elevated levels of MTHFD2 are often associated with enhanced tumor growth and metastasis, suggesting its role as a facilitator of cancer cell bioenergetics and survival. This enzyme not only supports the biosynthesis of nucleotides necessary for DNA replication and repair but also influences the epigenetic landscape of cancer cells through its involvement in the generation of S-adenosylmethionine. Thus, targeting MTHFD2 presents a promising therapeutic strategy, highlighting the need for further research to fully elucidate its mechanisms and potential as a biomarker for treatment response in oncology. MTHFD2 plays a pivotal role in the mitochondrial folic acid cycle, which is crucial for cellular metabolism and proliferation. Recent studies have highlighted its significant impact on various cancer types, including colon cancer and glioma, where it influences critical processes such as cell proliferation, apoptosis, and drug resistance [19,20,21]. The enzyme’s involvement in folate metabolism not only underscores its metabolic importance but also positions it as a potential therapeutic target in cancer treatment. Understanding the mechanisms by which MTHFD2 contributes to tumor biology may lead to the development of more effective strategies for combating resistance to conventional therapies and improving patient outcomes in malignancies linked to folate metabolism dysregulation. However, the role of MTHFD2 NSCLC remains largely underexplored, as there is a paucity of research concerning its expression, biological function, and prognostic implications. Despite its significant involvement in folate metabolism and cellular processes that influence tumor progression, studies focusing specifically on MTHFD2 in the context of NSCLC have been limited. This gap in knowledge underscores the need for further investigation to elucidate the potential contributions of MTHFD2 to tumor biology and patient outcomes, which could ultimately inform therapeutic strategies and improve clinical management of NSCLC.

In the present work we performed an integrated bioinformatics analysis of expression and significance of folate metabolic pathway gene MTHFD2 in NSCLC. In this present work, it reveals a significant correlation between the expression levels of MTHFD2 and the prognosis of NSCLC patients, indicating that elevated MTHFD2 expression is associated with poorer outcomes. Interestingly, contrasting findings have emerged from research focusing on liver cancer, where high levels of MTHFD2 expression correlate with improved disease-free survival. These divergent results underscore the complexity of MTHFD2’s role in cancer biology, suggesting that its prognostic significance may vary significantly across different tumor types. The differential prognostic impact of MTHFD2 in LUAD vs squamous cell carcinoma (LUSC) may stem from intrinsic biological and metabolic disparities between these NSCLC subtypes. LUAD, often driven by mutations in EGFR or KRAS, exhibits heightened reliance on one-carbon metabolism for nucleotide synthesis and redox balance, processes where MTHFD2 is pivotal [22]. This metabolic addiction may render LUAD more vulnerable to MTHFD2 overexpression, accelerating proliferation and therapy resistance, thereby worsening survival [22]. In contrast, LUSC, typically linked to PI3K/AKT or SOX2 alterations, may depend less on folate-driven pathways, instead prioritizing glycolytic or glutaminolytic routes, diminishing MTHFD2’s prognostic relevance [23]. Additionally, the tumor microenvironment (TME) may differ between LUAD and LUSC. LUAD shows stronger correlations between MTHFD2 and immune infiltration (e.g., CD8+ T cells), suggesting its role in modulating immunosuppression. High MTHFD2 might enhance tumor immune evasion in LUAD through metabolic competition or checkpoint interactions, indirectly affecting survival. LUSC’s TME, enriched with neutrophils and distinct stromal interactions, may buffer such effects. Molecularly, LUAD’s higher anabolic demands and oncogene-metabolism crosstalk could amplify MTHFD2’s oncogenic role, while LUSC’s squamous differentiation pathways (e.g., TP63) may override folate metabolism’s influence. These subtype-specific dependencies and TME dynamics likely explain the survival association divergence [24]. Consequently, to better understand these discrepancies and validate the implications of MTHFD2 expression on patient outcomes, further investigation through large-scale clinical trials is warranted.

The growth, invasion, metastasis, and recurrence of tumors are a complex pathological process. Tumor lymphocyte infiltration may refer to the infiltrating lymphocytes in tumors and their surrounding tissues, which are the products of the specific immune response of the body to tumors and reflect the body’s anti-tumor immune response. In the correlation analysis of the present work between MTHFD2 and immune infiltrating cells, it was found that MTHFD2 was associated with a variety of immune cell infiltrates, suggesting that folic acid metabolic pathway is related to body immunity. MTHFD2 has emerged as a significant player in the field of tumor immunity due to its important role in cellular metabolism and the regulation of folate pathways. Recent studies suggest that MTHFD2 not only contributes to the metabolic reprogramming of cancer cells but also influences cancer immune evasion through PD-L1 up-regulation [25]. Elevated expression of MTHFD2 in various malignancies has been associated with immune evasion tactics employed by tumors, including the suppression of T-cell activation and the promotion of an immunosuppressive milieu [26]. Consequently, targeting MTHFD2 presents a promising therapeutic strategy, potentially enhancing anti-tumor immunity and improving patient outcomes through the restoration of effective immune responses against cancer cells. Therefore, immunotherapy for NSCLC combined with drugs targeting folic acid metabolic pathway may achieve better therapeutic effect compared to immunotherapy alone. This work also identified that the biological process of MTHFD2 and relevant genes was mainly enriched in tetrahydrofolate metabolic process, one-carbon metabolic process, and folic acid metabolic process. The KEGG pathway of MTHFD2 and relevant genes was mainly enriched in one carbon pool by folate, metabolic pathways and antifolate resistance pathway. MTHFD2 protein, also known as the four-hydrogen methylene folic acid hydrolase, is one of the mitochondrial enzymes encoded by MTHFD2 gene located in the second chromosome in human being. This enzyme was first identified in Ehrlich ascites tumor cells in 1960 by Scrimgeour and Huennekens [27]. MTHFD2 has the dual activities of methylene tetrahydrofolate dehydrogenase and cyclohydrolase, and is a key enzyme in the reciprocal conversion reaction of folic acid metabolites in vivo [28]. Therefore, MTHFD2 metabolic pathway may provide a potential cancer including NSCLC treatment target.

Tumor cells have rapid glycolysis and synthesis of amino acids, nucleotides, and lipids to support the rapid proliferation of tumor cells. In particular, the synthesis of one carbon unit carried by the tetrahydrofolate cofactor is important for cells proliferating, which are required for nucleotide synthesis and methylation reactions [29]. Previous publications have shown that MTHFD2 is expressed in fetal cells and transformed cell lines, but has low or absent expression in most adult tissues and cell types [30,31]. In recent years, MTHFD2 has been shown to be important for rapidly growing cells, such as embryonic or tumor cells, mainly by supporting the high levels of purine synthesis required [32]. Nisson and his colleges performed a study including 19 cancer types and found that MTHFD2 mRNA and protein expressions were elevated in the mitochondrial folate pathway in highly proliferative cancers [33]. Furthermore, a number of studies have shown that MTHFD2 is highly expressed in breast cancer [15], renal carcinoma [34], and is associated with poor prognosis. The increased expression of MTHFD2 has been proved to be closely related to the occurrence and development of tumors and patient’s prognosis by clinical study.

The current study presents significant insights; however, it is important to acknowledge inherent limitations. The primary findings are derived from bioinformatic analyses, which, while valuable, necessitate further validation through empirical methods. To strengthen the conclusions drawn from this research, it is essential that subsequent investigations incorporate laboratory-based cell experiments and robust clinical data. Such comprehensive validation will not only enhance the reliability of the findings but also facilitate a more thorough understanding of the underlying biological mechanisms.

5 Conclusion

In conclusion, the key gene of folate metabolism pathway MTHFD2 may play an important role in the occurrence and development of lung cancer. In addition to affecting the regulation of folate metabolic pathway, the changes in MTHFD2 in patients with lung cancer can also affect the immune microenvironment of the human body, which may involve in the occurrence and development of lung cancer by affecting the immune microenvironment and metabolic pathways.