MTHFD2 is a folic acid metabolism enzyme which could mediate mitochondrial one-carbon production[6]. The generation of one carbon unit in folic acid metabolism is a key step for the nucleotide and DNA synthesis [24]. The alteration in the metabolic process of one carbon unit will exert an effect on cellular nucleic acid replication. Cumulative evidences has proved that MTHFD2 functioned in tumor cell growth, the development of drug resistance and tumor stem cell-like characteristics by affecting folate metabolism, one carbon unit synthesis and intracellular redox balance [25]. It is reported that MTHFD2 could facilitate the methylation level of hypoxia-inducible factor (HIF)-2a mRNA and elevate its translational level, which eventually leaded to the activation of glycolysis and increase of MTHFD2 expression [16]. The positive feedback cycle could enhance metabolic reprogramming and tumor development.
The reactive oxygen species (ROS) have a cytotoxic effect on tumor cells, and therefore, the increase level of ROS in tumor cells was considered as a protective factor for cancer patients [26]. As an NADP + dependent enzyme, MTHFD2 can regulate intracellular level of NADPH/NADH and affect intracellular redox balance [27]. By downregulating the expression of MTHFD2, the intracellular level of NAPDH and glutathione could be reduced while the intracellular oxidation level could be elevated, contributing to the elimination of tumor cells. The latest study found that MTHFD2 could modulate the stabilization of ubiquinol-cytochrome C reductase core protein 2 (UQCRC2), which belongs to the Complex III family. Moreover, MTHFD2 exhaustion could result in mitochondrial functional disturbance by restraining the activity of Complex III via regulating UQCRC2 expression [28]. However, the function of MTHFD2 in modulating the proliferation of bladder cancer cells through altering the level of ROS and its potential mechanisms is still required to be investigated.
In the present study, we performed a bioinformatics analysis of RNA sequencing data retrieved from the TCGA and GEO databases. Firstly, we found that MTHFD2 was generally overexpressed (Fig. 1), and higher MTHFD2 expression was related with worse overall survival in in pan-cancers (Fig. 2–3). Especially, we focused on the expression level of MTHFD2 in bladder cancer and its effect on patient’s prognosis. The findings demonstrated that MTHFD2 expression was elevated in bladder cancer (Fig. 3A-3B) and overexpression of MTHFD2 is associated with unfavorable clinical pathological features, shorter survival time as well as poorer prognosis (Fig. 3C-3H). In addition, we further explored the function of MTHFD2 by enrichment analysis in bladder cancer. GSEA enrichment test manifested that the overexpression of MTHFD2 was related with biological processes including cell cycle, DNA replication and repair, and the activation of signaling pathway including MYC, MAPK, Wnt, NF-κB and ATM (Fig. 6). GO and KEGG analysis revealed that MTHFD2 might be involved in the process of tumor initiation, which was consistent with the previous results (Fig. 7). The study implied that MTHFD2 might be a possible prognostic biomarker and promising targets for the treatment of bladder cancer.
It is confirmed that ncRNA modulated the gene expression through the ceRNA mechanism, which plays an important role in tumor initiation [29]. To explore miRNA that possibly regulate MTHFD2 expression, we used Starbase databases and Tarbase databases to predict miRNA that might potentially bind to MTHFD2. We found that there were 30 miRNAs that simultaneously exist in two databases, and were predicted to be implicated in the modulation of MTHFD2 expression level (Fig. 8A). According to the mechanism of miRNA regulating the targeted genes, the predicted miRNA should be down-regulated and negatively correlated with MTHFD2 expression in bladder cancer and miR-383-5p met the two required conditions. Previous studies also showed that miR-383-5p suppressed the proliferation and induced the apoptosis in gastric cancer cells [30]. Hence, miR-383-5p was assumed to be the most potential upstream miRNA of MTHFD2.
Circular RNA (circRNA) is usually considered to be the upstream molecule of miRNA and plays a vital role in regulating miRNA. The data from Starbase database predicted there were the 12 circRNAs that were likely to bind to miRNAs (Fig. 9A). The potential circRNAs involved in miR-383-5p/MTHFD2 axis should be up-regulated and associated with unfavorable prognosis in bladder cancer following the ceRNA hypothesis [31]. The results indicated that, among the most potential up-regulated circRNAs, hsa_circ_0046140 and hsa_circ_0006769, were likely to be implicated in miR-383-5p/MTHFD2 axis (Table 2). In other words, hsa_circ_0046140 and hsa_circ_0006769/miR-383-5p/MTHFD2 axis were defined as the possible regulatory pathways in bladder cancer, which is required to be further experimentally verified.
The TME is mainly composed of tumor cells, stromal cells and numerous extracellular components (such as cytokines, growth factors, hormones, etc.) [32, 33]. It is acknowledged that the immune cell infiltration in TME could inhibit proliferation of tumor cells [34]. For example, cytotoxic CD8 + T cells exert an inhibitory effect on tumor survival by recognizing tumor cell surface antigens, releasing cytotoxins or activating death ligand [35]. We found that the expression of MTHFD2 was positively correlated with the tumor infiltrating immune cells in bladder cancer, by analyzing the immune cell infiltration landscape (Fig. 10A-B).
Immunotherapy, as a supplement to the classical anticancer therapy, has been applied in clinical practice in recent years. It could induce a potent antitumor response through activating the immune system and reverse the immune tolerance, which greatly prolonged the survival of tumor patients and provided a new approach for cancer therapy [36]. Immune checkpoint inhibitors, such as PD-1/PD-L1 and CTLA-4 were widely used in tumor immunotherapy as crucial therapeutic targets. The overexpression of immune checkpoints is an additional factor that determines the efficacy of immunotherapy [37]. We then evaluated the association between MTHFD2 and immune checkpoints and the findings manifested a positive correlation between MTHFD2 expression and PD-1/PD-L1 and CTLA-4 in bladder cancer. (Fig. 11), suggesting that targeting MTHFD2 may help improve the efficacy of immunotherapy for bladder cancer.
However, some recent evidences have found that the immune cell infiltration in TME could prevent tumor cells from being killed. Tumor-associated macrophages (TAMs) have been verified to enhance the growth, angiogenesis and invasion of tumor cells by secreting growth factors, cytokines and proteases, and develop resistance to treatment [38]. TAMs also induce chronic inflammation by releasing various cytokines such as tumor necrosis factor, IL-6, IL-12 and transforming growth factor-β, leading to immune-associated tumor cytotoxicity suppression events [39]. Therefore, the role of activation and infiltration of immune cells in the TME in tumorigenesis, progression and therapeutic effect is still required to be further elucidated.