MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Interference with MTHFD2 induces ferroptosis in ovarian cancer cells through ERK signaling to suppress tumor malignant progression.

Ovarian cancer (OC) is a deadliest gynecological cancer with the highest mortality rate. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a crucial tumor-promoting factor, is over-expressed in several malignancies including OC. The present study aimed to explore the role and mechanisms of MTHFD2 in OC malignant progression. Thus, cell proliferation, cycling, apoptosis, migration, and invasion were evaluated by CCK-8 assay, EdU assay, flow cytometry, wound healing, transwell assay and western blotting. Additionally, glycolysis was assessed by measuring the level of glucose and lactate production, as well as the expressions of GLUT1, HK2 and PKM2. Then the expression of ferroptosis-related proteins and ERK signaling was detected using western blotting. Ferroptosis was detected through the measurement of iron level, GSH, MDA and ROS activities. The results revealed that MTHFD2 was highly expressed in OC cells. Besides, interference with MTHFD2 induced ferroptosis, promoted ROS accumulation, destroyed mitochondrial function, reduced ATP content and inhibited glycolysis in OC cells. Subsequently, we further found that interference with MTHFD2 affected mitochondrial function and glycolysis in OC cells through ERK signaling. Moreover, interference with MTHFD2 affected ferroptosis to inhibit the malignant progression of OC cells. Collectively, our present study disclosed that interference with MTHFD2 induced ferroptosis in OC to inhibit tumor malignant progression through regulating ERK signaling.

Metabolic subtypes and immune landscapes in esophageal squamous cell carcinoma: prognostic implications and potential for personalized therapies.

BACKGROUND: This study aimed to identify metabolic subtypes in ESCA, explore their relationship with immune landscapes, and establish a metabolic index for accurate prognosis assessment. METHODS: Clinical, SNP, and RNA-seq data were collected from 80 ESCA patients from the TCGA database and RNA-seq data from the GSE19417 dataset. Metabolic genes associated with overall survival (OS) and progression-free survival (PFS) were selected, and k-means clustering was performed. Immune-related pathways, immune infiltration, and response to immunotherapy were predicted using bioinformatic algorithms. Weighted gene co-expression network analysis (WGCNA) was conducted to identify metabolic genes associated with co-expression modules. Lastly, cell culture and functional analysis were performed using patient tissue samples and ESCA cell lines to verify the identified genes and their roles. RESULTS: Molecular subtypes were identified based on the expression profiles of metabolic genes, and univariate survival analysis revealed 163 metabolic genes associated with ESCA prognosis. Consensus clustering analysis classified ESCA samples into three distinct subtypes, with MC1 showing the poorest prognosis and MC3 having the best prognosis. The subtypes also exhibited significant differences in immune cell infiltration, with MC3 showing the highest scores. Additionally, the MC3 subtype demonstrated the poorest response to immunotherapy, while the MC1 subtype was the most sensitive. WGCNA analysis identified gene modules associated with the metabolic index, with SLC5A1, NT5DC4, and MTHFD2 emerging as prognostic markers. Gene and protein expression analysis validated the upregulation of MTHFD2 in ESCA. MTHFD2 promotes the progression of ESCA and may be a potential therapeutic target for ESCA. CONCLUSION: The established metabolic index and identified metabolic genes offer potential for prognostic assessment and personalized therapeutic interventions for ESCA, underscoring the importance of targeting metabolism-immune interactions in ESCA. MTHFD2 promotes the progression of ESCA and may be a potential therapeutic target for ESCA.

p53 deficiency induces MTHFD2 transcription to promote cell proliferation and restrain DNA damage.

Cancer cells acquire metabolic reprogramming to satisfy their high biogenetic demands, but little is known about how metabolic remodeling enables cancer cells to survive stress associated with genomic instability. Here, we show that the mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) is transcriptionally suppressed by p53, and its up-regulation by p53 inactivation leads to increased folate metabolism, de novo purine synthesis, and tumor growth in vivo and in vitro. Moreover, MTHFD2 unexpectedly promotes nonhomologous end joining in response to DNA damage by forming a complex with PARP3 to enhance its ribosylation, and the introduction of a PARP3-binding but enzymatically inactive MTHFD2 mutant (e.g., D155A) sufficiently prevents DNA damage. Notably, MTHFD2 depletion strongly restrains p53-deficient cell proliferation and sensitizes cells to chemotherapeutic agents, indicating a potential role for MTHFD2 depletion in the treatment of p53-deficient tumors.

MTHFD2 promotes PD-L1 expression via activation of the JAK/STAT signalling pathway in bladder cancer.

Although combination chemotherapy is widely used for bladder cancer (BC) treatment, the recurrence and progression rates remain high. Therefore, novel therapeutic targets are required. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) contributes to tumourigenesis and immune evasion in several cancers; however, its biological function in BC remains unknown. This study aimed to investigate the expression, prognostic value and protumoural function of MTHFD2 in BC and elucidate the mechanism of programmed death-ligand 1 (PD-L1) upregulation by MTHFD2. An analysis using publicly available databases revealed that a high MTHFD2 expression was correlated with clinical features and a poor prognosis in BC. Furthermore, MTHFD2 promoted the growth, migration, invasion and tumourigenicity and decreased the apoptosis of BC cells in vivo and in vitro. The results obtained from databases showed that MTHFD2 expression was correlated with immune infiltration levels, PD-L1 expression, and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. The expression of MTHFD2, PD-L1 and JAK/STAT signalling pathway-related proteins increased after interferon gamma treatment and decreased after MTHFD2 knockdown. Moreover, addition of a JAK/STAT pathway activator partially reduced the effect of MTHFD2 knockdown on BC cells. Collectively, our findings suggest that MTHFD2 promotes the expression of PD-L1 through the JAK/STAT signalling pathway in BC.

MTHFD2 suppresses glioblastoma progression via the inhibition of ERK1/2 phosphorylation.

Glioblastoma (GBM) is a WHO grade 4 tumor and is the most malignant form of glioma. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme involved in folate metabolism, has been reported to be highly expressed in several human tumors. However, little is known about the role of MTHFD2 in GBM. In this study, we aimed to explore the biological functions of MTHFD2 in GBM and identify the associated mechanisms. We performed experiments such as immunohistochemistry, Western blot, and transwell assays and found that MTHFD2 expression was lower in high-grade glioma than in low-grade glioma. Furthermore, a high expression of MTHFD2 was associated with a favorable prognosis, and MTHFD2 levels showed good prognostic accuracy for glioma patients. The overexpression of MTHFD2 could inhibit the migration, invasion, and proliferation of GBM cells, whereas its knockdown induced the opposite effect. Mechanistically, our findings revealed that MTHFD2 suppressed GBM progression independent of its enzymatic activity, likely by inducing cytoskeletal remodeling through the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, thereby influencing GBM malignance. Collectively, these findings uncover a potential tumor-suppressor role of MTHFD2 in GBM cells. MTHFD2 may act as a promising diagnostic and therapeutic target for GBM treatment.

Mitochondrial one-carbon metabolic enzyme MTHFD2 facilitates mammary gland development during pregnancy.

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis.

Mitochondrial folate pathway regulates myofibroblast differentiation and silica-induced pulmonary fibrosis.

BACKGROUND: Silica-induced pulmonary fibrosis (silicosis) is a diffuse interstitial fibrotic disease characterized by the massive deposition of extracellular matrix in lung tissue. Fibroblast to myofibroblast differentiation is crucial for the disease progression. Inhibiting myofibroblast differentiation may be an effective way for pulmonary fibrosis treatment. METHODS: The experiments were conducted in TGF-ß treated human lung fibroblasts to induce myofibroblast differentiation in vitro and silica treated mice to induce pulmonary fibrosis in vivo. RESULTS: By quantitative mass spectrometry, we revealed that proteins involved in mitochondrial folate metabolism were specifically upregulated during myofibroblast differentiation following TGF-ß stimulation. The expression level of proteins in mitochondrial folate pathway, MTHFD2 and SLC25A32, negatively regulated myofibroblast differentiation. Moreover, plasma folate concentration was significantly reduced in patients and mice with silicosis. Folate supplementation elevated the expression of MTHFD2 and SLC25A32, alleviated oxidative stress and effectively suppressed myofibroblast differentiation and silica-induced pulmonary fibrosis in mice. CONCLUSION: Our study suggests that mitochondrial folate pathway regulates myofibroblast differentiation and could serve as a potential target for ameliorating silica-induced pulmonary fibrosis.

SNHG3/hsa-miR-455-5p Axis-mediated High Expression of MTHFD2 Correlates with Tumor Immune Infiltration and Endometrial Carcinoma Progression.

Purpose: Endometrial carcinoma (EC) is one of the three most common female genital tract cancers, and it contributes to the leading deaths of gynecologic cancer. MTHFD2 was reported up-regulated and associated with poor prognosis in many malignancies. However, its biological functions and mechanisms in EC are unclear. The present study aimed to identify the biological functions and potential molecular mechanisms of MTHFD2 in EC. Methods: The gene expression and information of patients used in this study were derived from TCGA, GEO and HPA databases. KM survival analysis was used to explore the clinical outcomes of EC patients and correlation analysis was applied to find the correction between MTHFD2 expression level and immune infiltration in EC. We used GO and GSEA analysis to explore the biological functions and mechanisms of MTHFD2. The CCK8 assay, the colony formation assay and the transwell migration assay were conducted to validate the function of MTHFD2 in EC cells. We applied STRING to find the protein that interacted with MTHFD2. Finally, ENCORI was used to explore the potential upstream regulation of MTHFD2 in EC and it was validated in EC cells. Results: In the present study, we found that MTHFD2 was up-regulated in EC and its high expression level was associated with patients' poor prognosis and adverse clinical parameters. MTHFD2 level was shown to be correlated with immune infiltration. Knockdown of MTHFD2 inhibited the malignant phenotype of HEC-1A and Ishikawa cells, including proliferation, colony formation and migration. Furthermore, we found the SNHG3/hsa-miR-455-5p axis as the potential upstream of MTHFD2. Conclusion: SNHG3/hsa-miR-455-5p axis-mediated high expression of MTHFD2, and the MTHFD2 expression level was associated with tumor immune infiltration and endometrial carcinoma progression. Knockdown of MTHFD2 significantly inhibited the malignant phenotype of EC cells. MTHFD2 may be a valuable predictive biomarker, and targeting MTHFD2 may be an effective way to improve the therapeutic effect in EC.

Knockdown of MTHFD2 inhibits proliferation and migration of nasopharyngeal carcinoma cells through the ERK signaling pathway.

PURPOSE: Folate-mediated one-carbon metabolism (FOCM) plays a vital role in supporting cancer cells hyperproliferation. Malignant cells, including nasopharyngeal carcinoma (NPC) cells, are characterized by rapid proliferation and thus need large numbers of nucleotides and nutrients generated from FOCM. However, the mechanism and key genes involved in FOCM playing a vital role in NPC progression are still unclear. This study aimed to find out the key gene, and its functions in NPC and explore the potential mechanism. METHODS: Bioinformatics analysis based on TCGA and GSEA database were performed to screen the key FOCM related gene in HNSCC. The effects of MTHFD2 on cell proliferation, apoptosis and migration were conducted through MTHFD2 knockdown cell lines in vitro experiments. Cell proliferation was explored by CCK8 assay and colony formation assay. Cell apoptosis was tested through flow cytometry. Transwell migration assay was performed to study the cell migration. The potential pathway was explored by RNA-seq and the ERK inhibitor SCH772984 and the ERK activator tBHQ were applied to verify the effect of MTHFD2 in NPC via the ERK pathway. Finally, xenograft tumor model was used to explore the tumorigenicity of NPC cells in vivo and IHC was performed to study the expression of related proteins. RESULTS: MTHFD2 was highly expressed in NPC and associated with a poor prognosis. MTHFD2 knockdown inhibited the proliferation, migration and induced apoptosis of NPC cells in vitro. In consistent with cellular results, knockdown of MTHFD2 suppressed the tumorigenicity of NPC cells in vivo. MAPK pathway was enriched among DEGs between MTHFD2 knockdown cells and control cells. And the level of p-ERK1/2 and p-p38 MAPK was decreased in MTHFD2 knockdown cells and xenograft tumors of MTHFD2 knockdown cells. Furthermore, the application of the selective ERK inhibitor SCH772984 and the ERK activator tBHQ confirmed that MTHFD2-knockdown inhibited the proliferation and migration of NPC cells via the ERK signaling pathway. CONCLUSION: MTHFD2 was up-regulated in NPC tissues and its high expression was linked to a poor prognosis. Knockdown of MTHFD2 inhibited proliferation and migration of NPC cells through the ERK signaling pathway, which may provide new clues and targets for the treatment of NPC.

MTHFD2 is a potential oncogene for its strong association with poor prognosis and high level of immune infiltrates in urothelial carcinomas of bladder.

BACKGROUND: The bifunctional methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) has been reported to play an oncogenic role in various types of cancers. However, the function of MTHFD2 in urothelial carcinomas of bladder (UCB) and its association with tumor immune infiltration remains unknown. We aim to examine the suitability of MTHFD2 to be a novel biomarker of bladder cancer and whether MTHFD2 is linked to immune infiltration. METHODS: RNA sequencing data and clinical information (bladder cancer samples: normal samples = 414: 19) were downloaded from The Cancer Genome Atlas official website. Western blot analysis was performed to detect MTHFD2 expression in human bladder cancer (BLCA) cells and normal urothelial cell line SV-HUC-1. Associations between MTHFD2 expression and clinicopathological features were analyzed using Mann Whitney U test or Kruskal-Wallis H test. The "survival" and "survminer" packages were utilized to plot Kaplan-Meier survival curves. Moreover, the gene set enrichment analysis (GSEA) was conducted using a clusterProfiler package. The correlation of MTHFD2 expression with immune infiltration level was estimated using the single sample GSEA (ssGSEA) algorithm. Furthermore, associations between MTHFD2 and immune checkpoint genes were evaluated using the correlation analysis. RESULTS: Transcriptome analysis manifested that MTHFD2 was highly expressed in UCB tissues than normal bladder tissues, which was further confirmed by western blot analysis in human BLCA cells and SV-HUC-1 cells. Moreover, MTHFD2 high expression was significantly associated with the advanced disease progression. Also, the high expression of MTHFD2 was correlated with poor prognosis, and MTHFD2 was considered as an independent prognostic factor for disease specific survival. Furthermore, a number of cancer-related pathways were enriched in MTHFD2 high group, including NF-κB activation, JAK/STAT, and cancer immunotherapy by PD1 blockade. Several immune checkpoint molecules were also strongly associated with MTHFD2 expression, including PDCD1, CD274, CTLA4, CD276, LAG3, HAVCR2, and TIGIT. CONCLUSIONS: MTHFD2 expression was remarkably elevated in UCB, suggesting that MTHFD2 could be a promising biomarker for BLCA as well as novel target for anti-cancer immunotherapy since its close association with immune infiltration.

MTHFD2 promotes tumorigenesis and metastasis in lung adenocarcinoma by regulating AKT/GSK-3ß/ß-catenin signalling.

Recent studies have demonstrated that one-carbon metabolism plays a significant role in cancer development. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme of one-carbon metabolism, has been reported to be dysregulated in many cancers. However, the specific role and mechanism of MTHFD2 in lung adenocarcinoma (LUAD) still remains unclear. In this study, we evaluated the clinicopathological and prognostic values of MTHFD2 in LUAD patients. We conducted a series of functional experiments in vivo and in vitro to explore novel mechanism of MTHFD2 in LUAD. The results showed that MTHFD2 was significantly up-regulated in LUAD tissues and predicted poor prognosis of LUAD patients. Knockdown of MTHFD2 dramatically inhibited cell proliferation and migration by blocking the cell cycle and inducing the epithelial-mesenchymal transition (EMT). In addition, MTHFD2 knockdown suppressed LUAD growth and metastasis in cell-derived xenografts. Mechanically, we found that MTHFD2 promoted LUAD cell growth and metastasis via AKT/GSK-3ß/ß-catenin signalling. Finally, we identified miR-30a-3p as a novel regulator of MTHFD2 in LUAD. Collectively, MTHFD2 plays an oncogenic role in LUAD progression and is a promising target for LUAD diagnosis and therapy.

Non-metabolic function of MTHFD2 activates CDK2 in bladder cancer.

Bladder cancer is a common tumor with a high recurrence rate and high fatality rate, and its mechanism of occurrence and development remains unclear. Many proteins and metabolites reprogram at different stages of tumor development to support tumor cell growth. The moonlighting effect happens when a protein performs multiple functions simultaneously in a cell. In this study, we identified a metabolic protein, MTHFD2, which participates in the cell cycle by binding to CDK2 in bladder cancer. MTHFD2 has been shown to affect bladder cancer cell growth, which is independent of its metabolic function. We found that MTHFD2 was involved in cell cycle regulation and could encourage cell cycle progression by activating CDK2 and sequentially affecting E2F1 activation. In addition, moonlighting MTHFD2 might be regulated by the dynamics of the mitochondria. In conclusion, MTHFD2 localizes in the nucleus to perform a distinct function of catalyzing metabolic reactions. Moreover, the nuclear MTHFD2 activates CDK2 and promotes bladder cancer cell growth by modulating the cell cycle.

A novel oral inhibitor for one-carbon metabolism and checkpoint kinase 1 inhibitor as a rational combination treatment for breast cancer.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

Detection and characterisation of novel alternative splicing variants of the mitochondrial folate enzyme MTHFD2.

Through the process of alternative splicing, proteins with distinct biological functions and localisations are generated from a single gene. The mitochondrial folate metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has been receiving attention in recent years as one of the most frequently upregulated metabolic enzymes across multiple tumour types. We hypothesized that alternative splicing of MTHFD2 could be a mechanism that generates novel isoforms of this enzyme, with potentially distinct and important biological functions. Multiple alternatively spliced MTHFD2 transcripts were first characterized in the UCSC and Ensemble genome browser. Subsequently, investigating the transcriptomic data for the Genotype-Tissue Expression (GTeX) project it was found that beyond the canonical MTHFD2 transcript, alternative transcripts lacking the second exon of MTHFD2 are also common. The presence of MTHFD2 transcripts lacking the second exon was confirmed by RT-PCR in normal and cancer cells. Translation of MTHFD2 transcripts lacking this second exon are predicted to generate a truncated protein lacking the first 102 N-terminal amino acids of the full-length protein, including the mitochondrial transport sequence. Hence, the truncated MTHFD2 protein could be an isoform with distinct localisation and functions. However, we were not able to confirm the generation of a stable truncated MTHFD2 protein in eukaryotic cells. This study characterizes for the first time alternative spliced transcripts of the enzyme MTHFD2, although further work is required to investigate their biological significance.

Suppression of MTHFD2 in MCF-7 Breast Cancer Cells Increases Glycolysis, Dependency on Exogenous Glycine, and Sensitivity to Folate Depletion.

Methylenetetrahydrofolate dehydrogenase (NAD(P)+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) is a mitochondrial enzyme involved in folate metabolism. A number of recent studies have highlighted this enzyme as being highly expressed in many solid tumors, including breast cancer, and to be correlated with poor survival. However, the metabolic functions of MTHFD2 in cancer cells have not been well-defined. To investigate the function of MTHFD2 in breast cancer cells, we generated and characterized MCF-7 cells with stable suppression of MTHFD2 expression using a combination of cellular assays and metabolic profiling. Loss of MTHFD2 caused MCF7 cells to become glycine auxotrophs, that is, reliant on exogenous glycine, and more sensitive to exogenous folate depletion. Another prominent metabolic alteration observed as a consequence of MTHFD2 suppression was a more glycolytic phenotype, consistent with widespread modifications of cellular metabolism. Collectively, these data suggest that targeting MTHFD2 activity is likely to influence multiple metabolic pathways in breast cancer and could be combined with a range of antimetabolite therapies.

The one-carbon metabolic enzyme MTHFD2 promotes resection and homologous recombination after ionizing radiation.

The one-carbon metabolism enzyme bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is among the most overexpressed proteins across tumors and is widely recognized as a promising anticancer target. While MTHFD2 is mainly described as a mitochondrial protein, a new nuclear function is emerging. Here, we observe that nuclear MTHFD2 protein levels and association with chromatin increase following ionizing radiation (IR) in an ataxia telangiectasia mutated (ATM)- and DNA-dependent protein kinase (DNA-PK)-dependent manner. Furthermore, repair of IR-induced DNA double-strand breaks (DSBs) is delayed upon MTHFD2 knockdown, suggesting a role for MTHFD2 in DSB repair. In support of this, we observe impaired recruitment of replication protein A (RPA), reduced resection, decreased IR-induced DNA repair protein RAD51 homolog 1 (RAD51) levels and impaired homologous recombination (HR) activity in MTHFD2-depleted cells following IR. In conclusion, we identify a key role for MTHFD2 in HR repair and describe an interdependency between MTHFD2 and HR proficiency that could potentially be exploited for cancer therapy.

Radiological and Molecular Analysis of Radioiodinated Anastrozole and Epirubicin as Innovative Radiopharmaceuticals Targeting Methylenetetrahydrofolate Dehydrogenase 2 in Solid Tumors.

In the dynamic field of radiopharmaceuticals, innovating targeted agents for cancer diagnosis and therapy is crucial. Our study enriches this evolving landscape by evaluating the potential of radioiodinated anastrozole ([125I]anastrozole) and radioiodinated epirubicin ([125I]epirubicin) as targeting agents against MTHFD2-driven tumors. MTHFD2, which is pivotal in one-carbon metabolism, is notably upregulated in various cancers, presenting a novel target for radiopharmaceutical application. Through molecular docking and 200 ns molecular dynamics (MD) simulations, we assess the binding efficiency and stability of [125I]anastrozole and [125I]epirubicin with MTHFD2. Molecular docking illustrates that [125I]epirubicin has a superior binding free energy (∆Gbind) of -41.25 kJ/mol compared to -39.07 kJ/mol for [125I]anastrozole and -38.53 kJ/mol for the control ligand, suggesting that it has a higher affinity for MTHFD2. MD simulations reinforce this, showing stable binding, as evidenced by root mean square deviation (RMSD) values within a narrow range, underscoring the structural integrity of the enzyme-ligand complexes. The root mean square fluctuation (RMSF) analysis indicates consistent dynamic behavior of the MTHFD2 complex upon binding with [125I]anastrozole and [125I]epirubicin akin to the control. The radius of gyration (RG) measurements of 16.90 Å for MTHFD2-[125I]anastrozole and 16.84 Å for MTHFD2-[125I]epirubicin confirm minimal structural disruption upon binding. The hydrogen bond analysis reveals averages of two and three stable hydrogen bonds for [125I]anastrozole and [125I]epirubicin complexes, respectively, highlighting crucial stabilizing interactions. The MM-PBSA calculations further endorse the thermodynamic favorability of these interactions, with binding free energies of -48.49 ± 0.11 kJ/mol for [125I]anastrozole and -43.8 kJ/mol for MTHFD2-. The significant contribution of Van der Waals and electrostatic interactions to the binding affinities of [125I]anastrozole and [125I]epirubicin, respectively, underscores their potential efficacy for targeted tumor imaging and therapy. These computational findings lay the groundwork for the future experimental validation of [125I]anastrozole and [125I]epirubicin as MTHFD2 inhibitors, heralding a notable advancement in precision oncology tools. The data necessitate subsequent in vitro and in vivo assays to corroborate these results.

Downregulation of MTHFD2 Inhibits Proliferation and Enhances Chemosensitivity in Hepatocellular Carcinoma via PI3K/AKT Pathway.

BACKGROUND: Despite the substantial impact of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) on cancer progression, its significance in the regulation of hepatocellular carcinoma (HCC) cell proliferation and chemosensitivity remains poorly defined. METHODS: We evaluated MTHFD2 expression in a total of 95 HCC tissues by immunohistochemistry and analyzed the association of MTHFD2 with clinicopathologic features. qRT-PCR and Western blotting were conducted to verify MTHFD2 expression levels. Bioinformatics analysis such as gene set enrichment analysis (GSEA) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were used to predict the signaling pathways involved in MTHFD2. In addition, to investigate the anti-tumor effects of MTHFD2 knockdown, Cell Counting Kit-8 (CCK-8) and EdU assays were used. RESULTS: We found that MTHFD2 was frequently upregulated in HCC, and the combination of increased expression of MTHFD2 and Ki67 was associated with poor HCC prognosis. MTHFD2 knockdown significantly inhibited HCC cell proliferation and effectively sensitized HCC cells to sorafenib and lenvatinib. PI3K/AKT pathway was involved in MTHFD2-mediated modulation of proliferation and chemosensitivity. CONCLUSIONS: These findings indicate that MTHFD2 plays an important role in proliferation and chemosensitivity of HCC, indicating that it may serve as a novel pharmacological target for improving HCC therapy.