Many recent studies have focused on the role of folic acid, especially its relationship with the risk of cancer. These studies discuss the relationship between folic acid and various solid tumors (1,2). The incidence and mortality of cancer in patients receiving folic acid and VB12 were explored in two double-blind, randomized, controlled clinical trials in Norway. Surprisingly, folate therapy was associated with increased cancer incidence, cancer mortality, and all-cause mortality (3). Low levels of folic acid can lead to DNA replication, methylation and repair dysfunction. Moreover, researchers assessed the relationship between folate intake and risks of head and neck cancer (HNC) using prospective data from prostate, lung, colorectal cancer, and ovarian cancer. One particular study included 186 cases with a confirmed incidence of HNC. Using a Cox proportional hazard model, the authors found that higher dietary folate intake and enhanced folate intake were dose-related to HNC risk reduction (4).
Folic acid is an essential nutrient with a biological function that provides methylation materials for intracellular DNA methylation and thymidine synthesis. Folate metabolism disorders may disrupt methylation and affect DNA synthesis and repair, which can lead to cancer (5). The human gene for methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) encodes monofunctional C1-tetrahydrofolate synthase, which is a mitochondrial protein with monofunctional 10-formyl-tetrahydrofolate synthetase activity (6,7). As a component of folate-mediated one-carbon metabolism, human mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase is tightly associated with the matrix side of the mitochondrial inner membrane and catalyzes the conversion of 10-formyl-tetrahydrofolate to formate in both embryonic and adult mammalian mitochondria. Then, formate is transported from the mitochondria to the cytoplasm, where it is involved in the cytoplasmic branch of folate-dependent one-carbon metabolism, and this makes it indispensable in cytoplasmic processes such as purine biosynthesis, thymidylate biosynthesis, and the methyl cycle (8).
MTHFD1L is critical for cell growth, which is associated with multiple malignant tumors (9-11). Li et al. investigated the roles of MTHFD1L silencing in cell proliferation and apoptosis in tongue squamous cell carcinoma (TSCC) but its exact mechanism remains unclear (9). Carcinogenesis is a multifactorial phenomenon involving numerous genetic and epigenetic events and can alter the function of tumor suppressor genes, oncogenes, and other related molecules (12).Concurrently, OSCC is a highly complex multistep process involving the progressive acquisition of genetic and epigenetic alterations and dynamic genomic changes. Current methods of molecular testing remain deficient in their effect on treatment selection for oral cancer (13). Therefore, the identification of suitable biomarkers for early diagnosis and treatment is urgently required in OSCC. We present the following article in accordance with the MDAR checklist (available at http://dx.doi.org/10.21037/tcr-20-2798).
Data from head and neck squamous cell carcinoma (HNSCC) patients from TCGA (TCGA public data; November 2, 2017, http://tcga-data.nci.nih.gov/) were analyzed. In total, 528 samples were available in the TCGA database, including 520 RNAseqV2 samples and 40 pairs of samples with pathological data and HNSCC tumor tissue with corresponding adjacent normal tissues. This expression profile was based on RNAseqV2 data from 40 paired samples. Gene expression data were normalized to RNA normalization using the Trimmed Mean of M-values (TMM) method. To prevent incorrect sample-grouping errors, a quality control procedure was performed based on of the biological coefficient of variation (BCV).
Immunohistochemical (IHC) staining and survival analysis
Tumor and matched para-tumor tissues were acquired from 96 OSCC patients who were histologically diagnosed at Beijing Hospital. The tissue samples were immediately frozen in liquid nitrogen after removal and stored at −80 °C until ready for use. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by ethics board of Beijing Hospital (BJ200768X2). Written informed consent form was obtained from all patients. Surgical specimens of OSCC were obtained from June 2007 to July 2016, and 96 patients were involved in a follow-up cohort study until August 2017.
The tissue sections were deparaffinized and blocked in 0.03% hydrogen peroxidase. After heating in a microwave for 10 min and cooling to room temperature, the sections were incubated with blocking serum (10% Tris-buffered saline) for 30 min and incubated with rabbit anti-human MTHFD1L antibody (1:200, SIGMA, Cat. No. HPA029040, San Francisco, USA) at 4 °C overnight. IHC staining was blindly evaluated by two pathologists.
Cell lines and cell culture
OSCC cell lines CAL-27 and SCC-25 and the human embryonic kidney cell line 293 were purchased from the Cell Resource Centre, Shanghai Institute for Biological Sciences, Chinese Academy of Science. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, NY, USA) that was supplemented with 10% fetal calf serum (Cat. # A11-102, Ausbian) at 37 °C with 5% CO2 in a humidified atmosphere. The cell lines were tested and authenticated on Oct 20th, 2017, via short tandem repeat (STR) analysis.
MTHFD1L short hairpin RNA (shRNA) design and lentivirus construction
To repress MTHFD1L expression, a shRNA fragment specifically targeting MTHFD1L was designed (GeneChem, Shanghai, China). The lentiviral vectors expressing MTHFD1L shRNA were constructed, and the knockdown efficiency was confirmed Briefly, shRNA with a target sequence of 5'-ATTCCAGTTCCTGTATGAT-3' targeting MTHFD1L was designed, and a scrambled shRNA with sequence 5'-TTCTCCGAACGTGTCACGT-3' was used as the negative control (NC). Related stem-loop DNA oligonucleotides were synthesized, annealed, and inserted into the GV115 PSCSI-GFP lentiviral vector at AgeI/EcoRI sites. The final lentiviral plasmid constructs were co-transfected into 293 cells to generate lentiviruses. For the lentivirus infection, cells were seeded into six-well plates and cultured until the cell monolayers were 70% confluent. The lentiviruses were then added to the cells at a multiplicity of infection (MOI) of 20. After 72 h, the cells were examined using a fluorescence microscope and harvested for subsequent experiments. The knockdown efficiency was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot.
RNA extraction and RT-qPCR analysis
The total cellular RNA was isolated from cultured cells using TRIzol reagent (Cat. #3101-100, Shanghai Pufei Biotechnology Co. Ltd., Nanjing, China). RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and reverse transcription of 2 μg of RNA and using the Moloney Murine Leukemia Virus (M-MLV) reverse transcription kit in accordance with the manufacturer’s instructions. qPCR was conducted using the two-step method and following the manufacturer’s procedure. The procedure utilized the following amplification: hold at 95 °C for 30 seconds for pre-denaturation; 40 cycles of 5 s at 95 °C for denaturation and 30 s at 60 °C for elongation. Primers were purchased from Ribobio (Guangzhou, China). The primers for RT-qPCR included a MTHFD1L forward primer (5'-TCACTCACATTTGCCTCCCTC-3') and a reverse primer (5'-CTGAAGGGCAAGGCCATGTA-3') as well as GAPDH forward primer (5'-TGACTTCAACAGCGACACCCA-3') and a reverse primer (5'-CACCCTGTTGCTGTAGCCAAA-3'). GAPDH was used as an internal control. The 2−ΔΔCt method was used to quantitatively analyze the relative changes in gene expression.
Western blot assay
For the whole-cell protein extraction, cells were harvested and lysed on ice for 15 min in a lysis buffer containing 100 mM Tris-HCl, 2% mercaptoethanol, 20% glycerin, and 4% sodium dodecyl sulfate (SDS). The protein concentration in the clarified lysates was determined using a BCA protein assay kit (Ding Guo Biotechnology, Beijing, China) using the manufacturer’s instructions. Equal amounts of proteins were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked for 1 h at room temperature with 5% fat-free milk in Tris-buffered saline and Tween-20 (TBST) buffer. Then, membranes were incubated with primary antibodies, including anti-MTHFD1L (1:200, Sigma-Aldrich Cat# HPA029040, RRID:AB_2672879), anti-TP53 (1:300, Atlas Antibodies Cat# HPA051244, RRID:AB_2681403), anti-JUN (1:200, Atlas Antibodies Cat# HPA059474, RRID:AB_2684030), anti-GADD45A (1:200, Atlas Antibodies Cat#HPA053420, RRID:AB_2682144), anti-FAS (1:300, Atlas Antibodies Cat#HPA027444, RRID:AB_10601378), anti-MYC (1:100, Santa Cruz Biotechnology Cat#sc-789, RRID:AB_631274), or anti-GAPDH (1:2,000, Atlas Antibodies Cat#HPA040067, RRID:AB_10965903), overnight at 4 °C. After incubation, membranes were washed in TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000) for 1.5 h at room temperature. Then, membranes were washed and developed in TBST buffer. The immunoreactive bands were visualized using a chemiluminescent PierceTM ECL Western Blotting Substrate Kit (Thermo Fisher Scientific) with subsequent exposure to X-ray film (Cat. #038401501, Carestream).
Cell proliferation assays
After infection with lentiviruses expressing sh-MTHFD1L or scrambled shRNA, OSCC cells were cultured for 48 h and harvested during the logarithmic phase. Next, cell suspensions were generated and seeded in triplicate in 96-well plates at a density of 1,000 cells per well. After culturing at 37 °C with 5% CO2 for an additional 24 h, images of the cell were captured daily for 5 d using a Celigo image cytometer system (Nexcelom; Lawrence, MA, USA). Cell numbers per well were also quantified using the Celigo system, and cell growth curves were plotted for each condition.
The cell viability was assessed using the MTT (Genview, Beijing, China) assay. Briefly, 1,500 shRNA-treated or untreated cells per well were seeded in 96-well plates. After 24 h at 37 °C, MTT (5 mg/mL) was added to each well before the terminal point of cell culture, and cells were incubated for an additional 4 h. Then, the medium was removed and formazan crystals were dissolved in 100 μL dimethyl sulfoxide (DMSO). The cell viability was determined by measuring the absorbance of each well at 490/570 nm and using a multi-well plate reader (Cat. #M2009PR; Tecan Infinite).
Colony formation assay
After infection with lentivirus expressing sh-MTHFD1L or scrambled shRNA, OSCC cells were enumerated and plated at a density of 800 cells per well into six-well plates in triplicate. After incubation in a 5% CO2 incubator at 37 °C for 14 d, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent, Shanghai, China) for 30–60 min. Then, colonies were stained with Giemsa (Shanghai DingGuo Biotech, Shanghai, China) for 20 min, and images of cell plates were captured using a digital camera and colonies were enumerated for each treatment group.
Cell apoptosis analysis
An annexin-V-APC apoptosis detection kit (eBioscience, Cat. No. 88-8007, San Diego, CA, USA) was used to detect apoptosis using the manufacturer’s instructions. Prepared cells were harvested, washed with PBS, and suspended at a final density of 1×106–1×107 cells/mL. Then, 10 μL annexin-V-APC was mixed with 200 μL of the cell suspension and incubated at room temperature in the dark for 10–15 min. Cells were analyzed using a Guava easyCyte HT flow cytometer (Millipore, Darmstadt, Germany), and data were analyzed using Guava InCyte software.
In vivo xenograft tumor assay
Experiments were performed under a project license (LA2019103) granted by ethics board of Peking University, in compliance with institutional guidelines for the care and use of animals. Four-week-old female BALB/c nude mice were obtained from the Shanghai Laboratory Animal Centre. CAL-27 cells were stably transduced with lentivirus vectors harboring the NC (scrambled shRNA) or MTHFD1L-shRNA, which were suspended in PBS (Invitrogen, Waltham, MA, USA). The cells (5×106) were subcutaneously injected into the right axilla. Tumor sizes were initially assessed using a caliper, and the body weight was measured 8 d post-grafting. The tumor volume was calculated using the formula V = π/(6× length × width × width). At 27 d, mice were euthanized and the tumors were excised.
DNA microarrays and ingenuity pathway analysis (IPA)
Total RNA from OSCC cells infected with lentivirus expressing either scrambled shRNA (n=3) or MTHFD1L-shRNA (n=3) was extracted using TRIzol reagent. The RNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and Agilent 2100 bioanalyzer. The Affymetrix GeneChip PrimeView Human Gene Expression Array was used for microarray processing to determine gene expression profiling in accordance with the manufacturer’s instructions. Significantly and differentially expressed genes in OSCC were selected based on the following criteria: false discovery rate (FDR) <0.05 and FCAbsolute >1.5 based on volcano plots. Pathway enrichment analysis was performed for all significantly and differentially expressed genes using IPA.
Data are presented as the mean ± standard deviation (SD) deviation and three independent experiments were performed. All statistical analyses were conducted using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). The differences between two groups were evaluated using a Student’s t-test or one-way analysis of variance. All statistical analyses were two-sided and P values <0.05 were considered statistically significant unless stated otherwise.
Identification of MTHFD1L as a potential gene related to OSCC
We compared the MTHFD1L level in 40 HNSCC tumor specimens registered in the TCGA database with those in the corresponding paired non-tumor adjacent tissues from the same individuals. The TMTHFD1L level was significantly higher in HNSCC than in the adjacent tissue (P<0.05; Figure 1A,B). We obtained information regarding adjacent tissue and tumor tissue to evaluate MTHFD1L expression using IHC staining in oral specimens in accordance with TCGA. The expression level of MTHFD1L in OSCC tissues from 96 patients was also significantly higher than that in normal tissues (Figure 1C). The correlations between MTHFD1L and clinicopathological variables of OSCC are summarized in Table 1. MTHFD1L levels correlated with T classification (P=0.002) and local recurrence (P<0.001) in patients with OSCC. No significant difference was observed between the MTHFD1L expression level and any other clinicopathological factor.
Furthermore, a Kaplan-Meier analysis was performed to determine whether MTHFD1L expression was clinically and significantly associated with overall survival in HNSCC patients. To review the TCGA dataset, the mRNA level of MTHFD1L in HNSCC tissues was used as the cut-off point to categorize all cases into MTHFD1L-high (n=116) and -low (n=383) groups. An IPA analysis revealed that the overall survival rates were higher in MTHFD1L-low groups than in MTHFD1L-high groups (Figure 1D).
Confirmation of the lentivirus transfection by Western blot and qRT-PCR
A lentivirus-based shRNA strategy was used to knockdown MTHFD1L in human OSCC cell lines. CAL-27 and SCC-25 cells were infected with lentivirus vectors expressing MTHFD1L-shRNA (the knockdown) or scrambled-shRNA (the control). Western blot and qRT-PCR were performed to determine the MTHFD1L expression at both mRNA and protein levels. MTHFD1L mRNA levels were reduced by 95.1% in CAL-27 cells and 80.6% for SCC-25 cells compared to the corresponding controls (Figure 2A). Accordingly, protein levels were distinctly reduced in the sh-MTHFD1L group compared to the sh-Ctrl group (Figure 2B).
MTHFD1L silencing reduces proliferation and clonogenicity of human OSCC cells
We investigated the knockdown effect of MTHFD1L on the cell proliferation of CAL-27 and SCC-25 cells. First, a Celigo cell counting assay was performed to monitor cell proliferation daily for five consecutive days. The number of cells in the sh-Ctrl group temporally increased, whereas only a slight increase in cell number was observed in the sh-MTHFD1L group. A significant difference in cell numbers between the sh-Ctrl and sh-MTHFD1L groups was observed at 5 d post-infection (P<0.01). An obvious impairment of cell proliferation was observed at 2 d post-infection in sh-MTHFD1L groups (P<0.05; Figure 3A). The MTT assay was performed to evaluate the cell proliferation of both cells. Cell proliferation was also significantly inhibited at 5 d post-infection for the sh-MTHFD1L group compared to the sh-Ctrl group (P<0.01; Figure 3B). Moreover, colony formation analysis results showed that cells treated with sh-MTHFD1L had less clonogenic ability than cells treated with sh-Ctrl (Figure 3C).
MTHFD1L silencing induces apoptosis of human OSCC cells
Compared to the sh-Ctrl group, the MTHFD1L knockdown group significantly increased the rate of cell apoptosis in CAL-27 and SCC-25 cells (P<0.01; Figure 4). Therefore, MTHFD1L may play an important anti-apoptotic role in OSCC cells.
Silencing of MTHFD1L inhibits tumorigenicity in vivo
Furthermore, to determine whether MTHFD1L promoted OSCC cell tumorigenesis, a xenograft model with stably transfected CAL-27 cells was used in BALB/c nude mice. At 27 d post-grafting, the volumes of MTHFD1L-deficient xenograft tumors were significantly less than those of the NC group (Figure 5A,B). After sacrifice, the average tumor weight of the NC group was significantly heavier than that in the MTHFD1L knockdown group (Figure 5C). Together, these results indicate that MTHFD1L knockdown effectively reduced the growth of OSCC cells in vivo.
Correlation between MTHFD1L expression and the p53 signaling pathway activation level
In total, 673 genes were differentially expressed in OSCC cells expressing sh-MTHFD1L compared to sh-Ctrl cells. Of the differentially regulated genes, 259 were upregulated and 414 were downregulated (Figure 6A). Following pathway enrichment analysis, some fundamental biological signaling pathways, such as the p53 and IL-8 signaling pathway, were potentially activated (Z-score ≥2).
To further investigate the related regulatory network of the MTHFD1L-induced malignancy, we analyzed the knowledge-based interactome and constructed a network map of MTHFD1L using IPA. Several genes reportedly involved in tumorigenesis were differentially regulated by MTHFD1L such as c-MYC, TP53, GADD45A, the Fas cell surface death receptor (FAS), and JUN (Figure 6B). These genes were further confirmed via Western blot analysis, which showed that c-MYC and p53-associated proteins TP53, GADD45A, FAS and JUN levels showed a certain degree of difference in MTHFD1L-silenced cells compared with those in control cells (Figure 6C). Therefore, these results indicate that MTHFD1L might act as a key regulator of OSCC progression via the c-MYC gene and the p53-dependent pathway.
A possible relationship between MTHFD1L expression and clinicopathological indices in HNSCC was determined through exploration of the TCGA database. Furthermore, MTHFD1L expression in tumor tissue and normal tissues was reviewed using data from the Human Protein Atlas (HPA). IHC revealed that MTHFD1L expression is higher in tumor tissue than in normal tissues. More importantly, a Kaplan-Meier analysis was also performed to determine whether MTHFD1L expression was clinically and significantly associated with overall survival in HNSCC patients. The results indicate that the MTHFD1L gene was highly expressed and related to survival.
Cells that preferentially utilize glucose and glutamine to supply and maintain cell mitosis were grown (14). It is clear that metabolites of the folic acid cycle, such as methionine, antioxidant NADPH and nucleotides, are important for cell growth (15,16). Interestingly, the key enzymes in the folate cycle of MTHFD1L not only play an important role in supporting cancer growth but also increase the susceptibility of targeted therapy (10). However, little is known about the specific function of this gene, especially in OSCC. Based on the biosignal analysis, a differential expression of MTHFD1L in OSCC and paracancerous tissues was found. Furthermore, we analyzed the correlation between MTHFD1L expression and survival in OSCC patients and found a powerful connection. Therefore, it is hypothesized that MTHFD1L could play a critical role in the progression of this disease.
We verified the effect of MTHFD1L silencing on cell proliferation, apoptosis and tumorigenesis in OSCC. We found that the value-added rate decreased by 40% and the apoptotic rate decreased by 20% compared with the control group. This finding is also consistent with a previous discovery, which suggests that this gene plays an important regulatory role in the biological behavior of OSCC. In a recent study, these effects were attributed to NADPH reduction and reactive oxygen species (ROS) accumulation. MTHFD1L contributes to the production of NADPH, which is sufficient in combating oxidative stress in OSCC. In addition, it was determined that the expression of MTHFD1L was significantly affected by the mTORC1-4EBP1-eIF4E axis (9). However, the mechanism of shRNA that inhibited MTHFD1L gene expression in the cell growth of OSCC is unclear.
In humans, abnormal patterns of protein-protein interactions cause or indicate certain diseased states (17). MTHFD1L directly interacts with the c-MYC proto-oncogene and basic helix-loop-helix (bHLH) transcription factor (18). c-MYC is located on chromosome 8q24 and belongs to the MYC oncogene family. MYC family proteins regulate various biological responses, including cell proliferation, apoptosis, differentiation and senescence. For example, c-MYC promotes cell proliferation by indirectly activating cyclin E/CDK2 through direct transcriptional activation or the downstream target iron transferrin receptor 1 (TFRC1) (19-21). Furthermore, c-MYC inactivation restored RAS/BRAF-induced senescence, which indicates that continuous MYC signaling is necessary for RAS/BRAF-induced tumorigenesis and tumor progression (21). When combined with the present experimental data, this may indicate that the suppression of MTHFD1L affects c-MYC expression and thereby inhibits tumor cell proliferation and induces apoptosis.
Multiple studies have reported that the regulation of folic acid and its metabolites on cell proliferation and apoptosis are closely related to the p53 pathway in cancer (22,23). Abnormal activation of the p53 pathway may also be involved in the development of OSCC (24). In our study and according to IPA, the p53 pathway might contribute to the abnormal expression of MTHFD1L in OSCC. Therefore, we hypothesized that MTHFD1L may act on OSCC via the p53 pathway. Surprisingly, our data showed that the downregulation of MTHFD1L regulated the activation levels of the p53 signaling pathway. In the molecular regulatory network constructed by IPA analysis, some molecules have changed and several proteins closely related to the p53 pathway have also changed including TP53, GADD45A, FAS and JUN. MTHFD1L directly interacts with the MAGE family member D1 (MAGED) (25,26). MAGED enhances the phosphorylation of the JUN activator protein 1 (AP-1) transcription factor subunit and activates JUN (27). Similar to p53, JUN is also a very unstable protein that is restricted by various ligases and its control of cell cycle progression is dependent on p53 (28). Thus, MTHFD1L knockdown may also affect JUN expression through MAGED and induce cell apoptosis of the tumor. Similarly, proteomic research was used to analyze blood and subcellular fractions from OSCC and the corresponding control samples and found that the c-MYC and p53-related pathways may be involved in the occurrence of OSCC (29).
For the first time, this study revealed a novel MTHFD1L/c-MYC/p53 signaling pathway regulatory network in OSCC. While current data are limited and unable to support an in-depth hypothesis, they provide a direction for future research. Future studies on the role of MTHFD1L in OSCC pathogenesis will facilitate the development of tumor-specific therapeutics that target folate metabolism.
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at http://dx.doi.org/10.21037/tcr-20-2798
Data Sharing Statement: Available at http://dx.doiorg/10.21037/tcr-20-2798
Peer Review File: Available at http://dx.doi.org/10.21037/tcr-20-2798
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr-20-2798). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by ethics board of Beijing Hospital (BJ200768X2). Written informed consent form was obtained from all patients. Experiments were performed under a project license (LA2019103) granted by ethics board of Peking University, in compliance with institutional guidelines for the care and use of animals.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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