Gliomas are the most common primary malignant tumour in the central nervous system, with characteristics of high malignancy and poor prognosis. Their incidence accounts for 80% of all brain tumours. Despite the use of a variety of high-intensity treatment regimens, such as surgery combined with chemoradiotherapy, the median survival time of patients with GBM is still only 12–15 months, and only 3% -5% of patients have a survival time longer than 3 years [
1‐
3]. Therefore, exploring the biological origin and occurrence of gliomas and finding potential diagnostic and therapeutic targets have been the focus of research in the field of molecular biology.
Li et al. recently reported that m
6A methylation is reduced in glioma tissues, and that ectopically increasing m
6A levels by METTL3 overexpression in one glioma cell line could impair its proliferation and migratory ability, while increasing apoptosis [
77]. But they did not dig into the mechanism through which this epitranscriptomic modification may affect glioblastoma growth.
Cui’s group addressed the above point and described the involvement of m
6A RNA methylation and of m
6A-related proteins in glioblastoma in 2017 [
78]. The model they chose were glioblastoma stem cells (GSCs), considered the initiating cells of glioblastoma, usually enriched in restricted niches and deemed responsible not only for glioblastoma onset but also for its resistance to therapy and eventual recurrence [
79].
But on the other hand, Liu et al. found that WTAP expression predicts poor prognosis in malignant glioma patients [
80]. As WTAP is a crucial interactor of the methyltransferase complex, so this works suggested that m
6A modification related enzymes and m
6A methylation processes may play an oncogenic role in glioma [
81]. Visvanathan et al. published the first mechanistic work linking m
6A modification and oncogenesis in glioblastoma. They studied the levels of m
6A RNA methylation in three GSC lines and showed that they were reduced uponin vitro differentiation. Moreover, they also found that METTL3 mRNA was clearly more abundant in GSCs compared to counterparts [
82,
83].
To sum up, the expression of m6A in glioma is different. This indicates that m6A modification may not only promote cancer but also inhibit it during the occurrence and development of glioma. So there’s been a lot of interests and researches from biomedical scientists.
Writers and gliomas
GSCs are a group of cells with the ability to promote tumour growth and invasion and have strong resistance to both radiotherapy and chemotherapy. Therefore, the presence of GSCs indicates a poor prognosis for patients with GBM [
84]. One study showed that in GSCs, the expression levels of METTL3 increased, and the expression levels of METTL14 and ALKBH5 decreased, while FTO did not show significant changes. By installing m6A on the SOX2 3’-untranslated region (3’-UTR), METTL3 mediates GSC maintenance and dedifferentiation by regulating the stability of SOX2 mRNA. The complete structure of METTL3 and human antigen R (HuR) is critical for maintaining this process. In addition, METTL3 knockdown inhibited GSC growth and neurosphere formation and reduced the expression levels of stem cell-specific markers, stage-specific embryonic antigen-1 (SSEA1), and glioma reprogramming factors (including POU class 3 homeobox 2 (POU3F2), oligodendrocyte transcription factor 2 (OLIG2), spalt like transcription factor 2 (SALL2) and SOX2). Of which, SOX2 has a high affinity for METTL3 [
82].
The deep sequencing of m6A and mRNA showed that the knockdown of METTL3 and/or METTL14 led to the upregulation of oncogenes and genes coding downstream proteins, including ADAM metallopeptidase domain 19 (ADAM19), EPH receptor A3 (EPHA3), Kruppel-like factor 4 (KLF4) and tumour-inhibiting factors, resulting in the inhibition of GSC growth and self-renewal [
85]. METTL3 overexpression or treatment with the FTO inhibitor MA2, the ethyl ester form of meclofenamic acid (MA), can cause an increase in m6A levels in GBM cells [
78]. However, another study reported the opposite effect of METTL3 in GBM; this effect was related to a decrease in m6A levels during differentiation. Silencing METTL3 expression in GBM can significantly inhibit tumour growth and prolong mouse survival time, which is consistent with clinical observations that an increase in METTL3 expression is consistent with the poor survival of patients with GBM. Further studies on the mechanisms of action have shown that METTL3 is involved in the RNA processing and carcinogenic pathways of GSCs and has a variety of complexities. METTL3 plays a major role in m6A modification in GSCs and participates in the expression and alternative splicing of GSC-specific genes. In addition, METTL3 reduced A-to-I RNA editing by downregulating ADAR and ADAMRB1 and increased the editing abundance of C-U RNA by upregulating apolipoprotein B mRNA editing enzyme catalytic subunit 1 (APOBEC1) and APOBEC3A [
59].
METTL3 expression is upregulated in GSCs and weakens during differentiation. SOX2 was identified as an important target of METTL3-mediated m6A, whereas METTL3 promoted the recruitment of HuR to m6A-modified SOX2 mRNA and enhanced SOX2 stability [
85]. In addition, after the downregulation of METTL3 expression, GSCs showed strong radiosensitivity and a weak DNA repair capacity [
82]. Therefore, the above studies also revealed that METTL3-mediated m6A modification was important in GSC maintenance and radiotherapy resistance. As a zinc finger protein, zinc finger CCCH-type containing 13 (ZC3H13), is also an important regulator in the m6A-METTL-associated complex (MACOM) and can anchor WTAP, virilizer and Hakai in the nucleus [
78]. A recent study showed that the ZC3H13 mutation and retinoblastoma 1 (RB1) mutation could replicate human GBM in a mouse model. In addition, the ZC3H13 mutation also changed the gene expression profile of the RB1 mutant to enhance the resistance of GBM tumours to TMZ [
86].
In addition, WTAP is overexpressed in GBM, and WTAP enhances the proliferation, migration, invasion, and tumourigenicity of GBM cells in xenografts by mediating the phosphorylation of epidermal growth factor receptor (EGFR) and protein kinase B (AKT). In addition, WTAP also regulates the expression of certain genes associated with cancer cell movement, such as chemokine ligand 2 (CCL2), CCL3, matrix metalloproteinase 3 (MMP3), lysyl oxidase like 1 (LOXL1), hyaluronic acid synthase 1 (HAS1) and thrombospondin 1 (THBS1) [
81]. High WTAP expression is an independent prognostic factor that is positively correlated with age and World Health Organization (WHO) classification and indicates poor overall survival in GBM patients [
80]. Cell-based experiments have shown that WTAP plays an important role in the miR-29a/Quaking isoform 6 (QKI-6) axis-mediated inhibition of cell proliferation, migration, and invasion as well as a downstream target for promoting GSC apoptosis [
87].
In addition to the direct impact on pluripotent genes, MeRIP-seq analyses based on m6A-Seq techniques showed that m6A-modification peaks tend to be enriched in metabolic pathway-related transcripts [
88]. METTL3 can cause changes through the downregulation of adenosine deaminase 1 (ADAR1) and apolipoprotein B mRNA expression, e.g., a reduction in editing events such as adenosine to inosine (A to I) and cytidine to uridine (C to U) (such as APOBEC3A) in GSCs [
89]. In addition, gene ontology analysis indicated that the direct target of METTL3 seems to be enriched in some major oncogenic pathways, including the Notch signalling pathway, vascular endothelial growth factor (VEGF) signalling pathway, angiogenesis, glycolysis and the Hedgehog signalling pathway; the indirect target is enriched in the RAS pathway, mitogen-activated protein kinase (MAPK) pathway, G-protein coupled receptor (GPCR) pathway, cadherin signalling pathway and cell cycle [
89]. In addition, in GSCs, METTL3-mediated m6A modification can also affect expression levels of serine and arginine rich splicing factors (SRSF) by upregulating BCL-X or nuclear receptor corepressor 2 and can prevent YTHDC1-dependent nonsense-mediated mRNA decay (NMD) [
88]. Compared with protein-coding genes, METTL3-mediated m6A-tagged lncRNAs are also highly expressed in GSCs. Furthermore, the m6A marker in the 3’-UTR appears to block the binding process of microRNA-related genes in GSCs [
89].
In summary, m6A writers are critical for the occurrence and development of GBM, and most are upregulated in GBM and show carcinogenic effects by regulating specific signalling pathways, especially helping to maintain cell stemness. However, some opposite findings indicate that the expression of some writers in GBM is downregulated and that some writers may have anticancer properties. Therefore, the existence of this conflict provides more research possibilities on the role of m6A modification-related methylation in the biological pathogenesis of gliomas.
Erasers and gliomas
Similar to writers, m6A erasers also play vital roles in GBM. The latest research shows that ALKBH5 is elevated in GSCs, enhancing cell self-renewal, proliferation and tumourigenicity [
78]. In terms of a mechanism, ALKBH5 demethylates m6A-modified bases and enhances the expression level of the key target gene forkhead box protein M1 (FOXM1) in GBM patients by reducing the abundance of m6A in the target mRNA transcript (especially in the 3’-UTR) [
90].
As an important functional target of ALKBH5, FOXM1 overexpression can reverse the function of ALKBH5 or inhibit FOXM1 long noncoding RNA antisense (FOXM1-AS) and restore GSC tumour growth. FOXM1-AS is a lncRNA on human chromosome 12 that is opposite to and partially overlaps with FOXM1. FOXM1-AS can promote the interaction between ALKBH5 and FOXM1 nascent transcripts, thereby promoting the recruitment of HuR. In general, under the combined action of FOXM1-AS, ALKBH5 enhances the self-renewal and proliferation of GSCs by regulating the expression of FOXM1 and promoting the occurrence and development of GBM [
90]. On the other hand, ALKBH5 knockdown inhibits the proliferation of GSCs, while wild-type ALKBH5 rescues the proliferation of GSCs. After ALKBH5 knockdown, the m6A level in nascent FOXM1 transcripts is elevated, and the binding of FOXM1 pre-mRNA to HuR is reduced; therefore, the recruitment of HuR to m6A-modified RNA is crucial for stabilizing FOXM1 mRNA [
90].
Su’s study showed that the inhibition of FTO hindered the self-renewal ability and carcinogenicity of GBM stem cells
in vitro and in mouse models. FTO plays carcinogenic roles through maintaining the stability of gliomas, especially the stability of oncogene homologues (c-Myc) and CCAAT-enhancer-binding protein-α (CEBPA) transcripts in IDH1/2 mutant gliomas. In addition, the inhibitory effect of MA2 on FTO significantly increases the tumourigenicity of GSC-transplanted mice [
75,
78]. The above evidence also potentially reveals that FTO may be a promising target for the drug treatment of GBM.