Multiple functions of m⁶A RNA methylation in cancer

Abstract

First identified in 1974, m⁶A RNA methylation, which serves as a predominant internal modification of RNA in higher eukaryotes, has gained prodigious interest in recent years. Modifications of m⁶A are dynamic and reversible in mammalian cells, which have been proposed as another layer of epigenetic regulation similar to DNA and histone modifications. m⁶A RNA methylation is involved in all stages in the life cycle of RNA, ranging from RNA processing, through nuclear export, translation modulation to RNA degradation, which suggests its potential of influencing a plurality of aspects of RNA metabolism. All of the recent studies have pointed to a complicated regulation network of m⁶A modification in different tissues, cell lines, and space–time models. m⁶A methylation has been found to have an impact on tumor initiation and progression through various mechanisms. Furthermore, m⁶A RNA methylation has provided new opportunities for early stage diagnosis and treatment of cancers.

Herein, we review the chemical basis of m⁶A RNA methylation, its multiple functions and potential significance in cancer.

Background

Termed as “epitranscriptome,” RNA harbors the potential of being dynamically and reversibly regulated by the addition and removal of distinct chemical moieties, which extends the RNA repertoire and alters its chemistry in various ways [1].

Among more than 100 types of post-transcriptional modifications identified in RNAs so far [2–4], N⁶-methyladenosine (m⁶A) RNA methylation is one of the most prevalent modifications, accounting for about 50% of total methylated ribonucleotides and 0.1–0.4% of all adenosines in total cellular RNAs [5, 6]. Most mammalian m⁶A sites are found within the consensus sequence Rm⁶ACH (R = G or A, H = A, C, or U), which is consistent with the enriched binding motifs observed in studies of methyltransferase-like 14 (METTL14), methyltransferase-like 3 (METTL3), and Wilms tumor 1-associated protein (WTAP) (GGAC, GGAC, and GACU respectively) [7]. There have been plenty of researches concerning DNA and histone methylation. DNA methylation is an important biochemical process which comprises the addition of a methyl group to the carbon 5 of the pyrimidine ring of cytosine or the nitrogen 6 of the purine ring of adenine. DNA methylation is brought about by a group of enzymes known as the DNA methyltransferases (DNMT) and can be de novo (when CpG dinucleotides on both DNA strands are unmethylated) or maintenance (when CpG dinucleotides on one strand are methylated). The enzymes that actively demethylate DNA include 5-methylcytosine glycosylase and MBD2b. Accumulating evidence has shown that DNA methylation plays an important role in all aspects of cellular processes including tumorigenesis [8].

Histone methylation in localized promoter regions is one kind of histone codes for chromatin packing and transcription. Generally, methylation of H3K4, H3K36, and H3K79 is linked to gene expression activation, whereas H3K9me2, H3K9me3, H3K27me3, and H4K20 are associated with gene repression [9–12]. Modifications of m⁶A RNA methylation are dynamic and reversible in mammalian cells, which have been proposed as another layer of epigenetic regulation similar to DNA and histone methylation. The biological function of m⁶A RNA methylation is highly variable depending on context and little is known about the underlying mechanisms; however, emerging evidence has suggested that m⁶A modification plays a pivotal role in pre-mRNA splicing, 3′-end processing, nuclear export, translation regulation, mRNA decay, and miRNA processing [13]. Growing appreciation of the biological significance of m⁶A RNA methylation has implied its important and diverse biological functions in mammals including tissue development, circadian rhythm, DNA damage response, sex determination, and tumorigenesis [14].

In this review, we will discuss how m⁶A RNA methylation participates in tumorigenesis, invasion, metastasis, and drug resistance and how to use m⁶A modification as new diagnostic biomarkers and therapeutic targets.

Chemical basis of m⁶A RNA methylation

The m⁶A methylase complex is consisted of at least five “writer” proteins where METTL3 acts as the catalytic core. METTL14 serves as structural support for METTL3, while WTAP stabilizes the core complex. RNA-binding motif protein 15 (RBM15) helps to recruit the complex to its target sites. The molecular function of KIAA1429 is still elusive [15]. On the other hand, “erasers” consisting of fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5) act as demethylases to reverse m⁶A modification [16, 17]. The functional interplay among the m⁶A methyltransferases and demethylases probably determines the dynamic regulation of m⁶A modification.

YT521-B homology (YTH) domain-containing proteins including YTHDF1-3, YTHDC1, and YTHDC2 have recently been identified as “readers” of m⁶A marks on mRNA. They display a 10- to 50-fold higher affinity for m⁶A-methylated mRNA than for unmethylated mRNA [18–22]. When perturbed in different cellular contexts, individual YTH domain family proteins interact with distinct subsets of m⁶A sites and produce different effects on gene expression. For example, YTHDF2, the first identified m⁶A reader, increases turnover of m⁶A-modified mRNA by promoting co-localization with decay factors [23, 24]. Conversely, YTHDF1 can be bound to m⁶A sites near the stop codon, which then interacts with the translation initiation factor eIF3, causing a stimulatory effect on translation in mammals [25]. Another mechanism for the regulation of translation by m⁶A RNA methylation comes into play during stress responses where preferential m⁶A methylation of the 5′-UTR of stress response genes leads to cap-independent translation, which involves nuclear relocalization of YTHDF2 to prevent m⁶A removal by FTO [26, 27]. YTHDF3 has been shown to interact cooperatively with YTHDF2 to accelerate mRNA decay while it also promotes translation of methylated RNAs in cooperation with YTHDF1 [28, 29]. YTHDC1 recruits the splicing factor Serine and arginine-rich splicing factor (SRSF) 3, promotes exon inclusion, and restricts binding of the exon-skipping factor SRSF10 [30].

As to the processing of pri-miRNAs, hnRNPA2B1 has the capability of recruiting DGCR8 to RNA by targeting m⁶A sites, indicating its important role in promoting pri-miRNA processing [31, 32].

A special combination of direct and indirect mediation has also been observed and studied. m⁶A RNA methylation has been shown to modify RNA structure, as a direct effect, in a way that improves accessibility for RNA-binding proteins such as hnRNPC, which in turn mediates an indirect effect. This phenomenon is termed as “m⁶A-switch” [33].

To sum up, the emerging new data and discoveries have revealed a complex picture concerning epitranscriptome (Fig. 1).

Fig. 1.

Chemical basis and diverse molecular functions of m⁶A RNA methylation. m⁶A RNA methylation is modulated by its “writers,” “erasers,” and “readers.” Writers refer to the m⁶A methylase complex which is consisted of METTL3, METTL14, WTAP, RBM15, and KIAA1429. Erasers are m⁶A demethylases including FTO and ALKBH5. Readers are proteins that bind to m⁶A modifications and exert various functions, some of whom identified so far are YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, and hnRNPA2B1. m⁶A RNA methylation is known to be involved in all stages in the life cycle of RNA including pre-mRNA splicing, pri-miRNA processing, through nuclear export, RNA translation modulation and RNA degradation

Methods of detecting m⁶A RNA methylation

The research related with m⁶A modification has been reinvigorated due to the advent of potent analytical methods, which harness the effects of m⁶A RNA methylation on RNA structure and metabolism, in combination with novel, high-throughput methods.

Several methods, including dot-blot and high-performance liquid chromatography coupled to triple–quadrupole mass spectrometry (LC–MS/MS) can be utilized to yield important quantitative information about existence and abundance of m⁶A modification, while they are not suitable for widespread identification and localization of modified sites [34].

Thus, an emerging method termed m⁶A-seq or MeRIP-seq has received considerable attention recently due to its accuracy and reproducibility. This novel method is based on the high specificity of one antibody raised against m⁶A in combination of high-throughput sequencing, making the mapping of m⁶A in the mammalian transcriptome possible [23, 35].

However, the immunoprecipitation-based approach m⁶A-seq localizes m⁶A residues to 100- to 200-nt-long regions of transcripts and is incapable of single-nucleotide-resolution detection of methylation sites. Contrary to other base modifications, such as 5-methylcytosine (5^mC), which can make use of chemical conversion of cytosine as a mapping approach at single-nucleotide resolution in RNA [36], m⁶A RNA methylation has no such chemical conversion. Additionally, m⁶A does not introduce errors during reverse transcription that would allow direct mapping of its position [37]. A novel method called m⁶A individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) has marked a major step forward in the field, which augments m⁶A-seq with UV-induced crosslinking of the antibody to the immunoprecipitated RNA fragments [38]. Reverse transcription of crosslinked RNA then results in a highly specific pattern of mutations or truncations in the cDNA, and these specific mutational signatures are crucial for mapping m⁶A residues throughout the transcriptome at single-nucleotide resolution.

Moreover, it is currently possible to directly test the influence of altering any modification site in many organisms with the use of CRISPR-based genome engineering. CRISPR multiplexing strategies could potentially permit interrogation of many sites in parallel and hasten functional discoveries and would be valuable for the research of m⁶A RNA methylation as a complementary approach [39].

Multiple functions of m⁶A RNA methylation in cancer

Currently, m⁶A RNA methylation has been found to have diverse biological regulatory functions in cancer initiation and progression and dysregulated expression of m⁶A RNA methylation is closely associated with various kinds of cancers. Herein, we mainly discuss four aspects of these functions exerted by m⁶A RNA methylation in cancer (Table 1, Fig. 2).

Table 1.

Multiple functions exerted by m⁶A RNA methylation in various cancers

Molecule	Role in cancer	Cancer	Biological function	Mechanism	Refs
METTL3	Oncogene	AML	Promote tumorigenesis	Promote MYC et al. translation	[43]
	Oncogene	BC	Promote tumor growth	Promote HBXIP translation	[68]
	Oncogene	HCC	Promote tumor growth	Promote SOCS2 degradation	[70]
	Suppressor gene	GBM	Suppress tumorigenesis	Downregulate ADAM19	[58]
METTL14	Oncogene	AML	Promote tumorigenesis	Stabilize MYC and MYB	[44]
	Suppressor gene	HCC	Suppress metastasis	Promote miR126 processing	[71]
	Suppressor gene	GBM	Suppress tumorigenesis	Downregulate ADAM19	[58]
NSun2	Oncogene	CRC	Promote migration	Suppress miR125b processing	[73]
FTO	Oncogene	AML	Promote tumorigenesis	Destabilize ASB2 and RARA	[52]
	Oncogene	AML	Promote tumorigenesis	Stabilize MYC and CEBPA	[47]
ALKBH5	Oncogene	GBM	Promote tumorigenesis	Stabilize FOXM1 mRNA	[61]
	Oncogene	BC	Promote tumorigenesis	Stabilize NANOG and KLF4	[57]
YTHDF2	Oncogene	PC	Promote tumor growth	Activate Akt/GSK3b/CyclinD1	[75]
	Suppressor gene		Suppress metastasis	Destabilize YAP mRNAs	[75]

GBM glioblastoma, CRC colorectal carcinoma, PC pancreatic cancer

Fig. 2.

Multiple functions of m⁶A RNA methylation in cancer. m⁶A RNA methylation exerts multiple functions in cancer initiation and progression. The circle in the middle represents the reversible process of m6A RNA methylation, which plays a pivotal role in modulating CSC pluripotency, cancer proliferation, cancer metastasis, and tumor immunity. Distinct mechanisms through which m⁶A RNA methylation exerts these four aspects of functions in various types of cancers are shown in a graphical form in this picture

m⁶A RNA methylation influences cancer stem cell pluripotency and cell differentiation

In recent years, a new dimension of intratumor heterogeneity and a hitherto-unappreciated subclass of neoplastic cells within tumors, termed cancer stem cells (CSCs), have aroused interests of researchers [40]. CSCs, typically rare within tumors, may prove to regenerate all facets of a tumor as a result of their stem cell-like capacity to self-renew, survive, and become dormant in protective microenvironments, representing a reservoir of self-sustaining cells that give rise to many types of cancers [41, 42].

Hematopoietic stem cells (HSCs) have well-defined developmental trajectories, and it is plausible to monitor and quantify their differentiation, providing an ideal model system in which to explore differentiation states. Abnormal or blocked differentiation is a common feature of myeloid hematological malignancies.

According to the recent research conducted by Ly P Vu and colleagues, leukemia cells show an elevated abundance of METTL3 as compared to normal hematopoietic cells. Utilizing miCLIP coupled with ribosome profiling, it is elucidated that METTL3 augments m⁶A levels of its target genes including myelocytomatosis (MYC), B cell lymphoma 2 (BCL2), and phosphatase and tensin homolog (PTEN) genes in the human acute myeloid leukemia (AML) MOLM-13 cell line, thereby promoting the translation of these mRNAs. These data suggest that AML cells regulate their translational state through m⁶A RNA methylation of specific transcripts to retain pluripotency properties and inhibit cell differentiation. Depletion of METTL3 in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo [43].

Consistent with METTL3, METTL14 is also highly expressed in AML cells carrying t(11q23), t(15;17), or t(8;21) and is downregulated during myeloid differentiation. Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets (e.g., myelocytomatosis (MYB) and MYC) through m⁶A modification, while the protein itself is negatively regulated by spleen focus-forming virus proviral integration oncogene (SPI1), collectively forming a SPI1/METTL14/MYB, MYC, et al. signaling axis in myelopoiesis and leukemogenesis. This highlights the critical roles of METTL14 and m⁶A modification in normal and malignant hematopoiesis [44].

RBM15, another component of the m⁶A writer complex, is linked to myeloid leukemia as well where AML initiation is mediated by a chromosomal translocation t (1;22) of RBM15 (also called OTT1) with the myelin and lymphocyte (MAL) gene [45]. Bansal and colleagues also suggested a role for m⁶A RNA methylation in myeloid leukemia that WTAP expression was elevated in cells derived from 32% of patients with AML and knockdown of WTAP resulted in reduced proliferation, increased differentiation, and increased apoptosis in a leukemia cell line [46].

However, upregulated m⁶A RNA methylation expression can result in anti-proliferation effects in some circumstances. Su and colleagues have elaborated that R-2-hydroxyglutarate (R-2HG) exhibits broad and variable anti-proliferation effects in leukemia and glioma since it increases global m⁶A RNA modification in the sensitive cells via suppressing FTO. The R-2HG/FTO/m⁶A axis regulates MYC and CCAAT/enhancer-binding protein alpha (CEBPA) gene expression and downstream pathways [47].

On the other hand, decreased m⁶A RNA methylation levels may exert oncogenic functions in some certain types of AML. For example, FTO, an m⁶A demethylase, is highly expressed in AML with t(11q23)/MLL rearrangements, t(15;17)/PML-RARA, FLT3-ITD, and/or NPM1 mutations and functions as an oncogene that promotes leukemic oncogene-mediated cell transformation and inhibits all-trans-retinoic acid (ATRA)-mediated leukemia cell differentiation. The oncogenic role of FTO is exerted through regulating expression of its target genes such as a suppressor of cytokine signaling box-2 (ASB2) and the retinoic acid receptor alpha (RARA) by reducing m⁶A RNA methylation levels in these mRNA transcripts. RNA stability assays have proposed that FTO-induced repression of ASB2 and RARA expression is at least in part due to the decreased stability of ASB2 and RARA mRNA transcripts upon FTO-mediated decrease of m⁶A RNA methylation levels. However, the reader(s) that targets m⁶A modification and impairs mRNA stability remains to be identified, which indicates an additional reading process controlling the stability of FTO target transcripts. Studies have [48–51] demonstrated the anti-leukemic effects of ASB2 and RARA, suggesting that the FTO/ASB2 or RARA axis likely plays a critical role in the pathogenesis of AML [52].

In breast cancer, Zhang and colleagues proposed that the exposure of breast cancer cells to hypoxia could stimulate hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of ALKBH5, which induced m⁶A demethylation and stabilization of NANOG mRNA. Intratumoral hypoxia is a critical feature of the tumor microenvironment caused by dysregulated cell proliferation in combination with abnormal blood vessel formation and function, which drives cancer progression [53–55]. NANOG, as a pluripotency factor, is required for primary tumor formation and metastasis since they play a pivotal role in the maintenance and specification of cancer stem cells. This explains the correlation between decreased m⁶A RNA methylation level and promotion of breast cancer stem cell (BCSC) phenotype [56]. The researchers have also reported that the exposure of breast cancer cells to hypoxia induces zinc finger protein 217 (ZNF217)-dependent inhibition of m⁶A methylation of mRNAs encoding NANOG and Kruppel-like factor 4 (KLF4), which is another pluripotency factor that mediates BCSC speciation [57].

When it comes to glioblastoma (GBM), a study carried out by Cui et al. demonstrates that reduced mRNA m⁶A level is critical for maintaining glioblastoma stem-like cell (GSC) growth, self-renewal, and tumor development as downregulation of METTL3 or METTL14 expression reduces mRNA m⁶A levels of theirs target gene A disintegrin and metallopeptidase domain 19 (ADAM19) and promotes ADAM19 expression. ADAM19 is a metalloproteinase disintegrin gene that exhibits elevated expression in glioblastoma cells and promotes glioblastoma cell growth and invasiveness [58–60].

Similarly, Zhang and colleagues have identified that m⁶A demethylase ALKBH5 is highly expressed in GSCs and demethylates forkhead box protein M1 (FOXM1) nascent transcripts [61]. The nuclear RNA-binding protein HuR, which reportedly regulates both pre-mRNA splicing and expression [62, 63], has been shown to bind with RNAs with no m⁶A modification and exert stabilizing effects on its bound RNAs [24]. Recruiting HuR to the unmethylated 3′UTR makes FOXM1 nascent transcripts more stable and upregulates its expression. Accumulating evidence has shown that the transcription factor FOXM1 functions as a key cell-cycle molecule required for G1/S and G2/M transition and M-phase progression [64] and is overexpressed in GBM, playing a pivotal role in regulating GSC proliferation, self-renewal, and tumorigenicity [65–67]. Taken together, lower m⁶A level mediated by ALKBH5 in GBM helps to promote tumor progression.

m⁶A RNA methylation involves in cancer cell proliferation

m6A RNA methylation has been found involved in cancer cell proliferation in many kinds of cancers. There remains a coherent natural link between the oncoprotein hepatitis B virus X-interacting protein (HBXIP) and METTL3 in the development of breast cancer (BC). Mechanistically, HBXIP upregulates METTL3 in breast cancer cells by inhibiting miRNA let-7g, which downregulates the expression of METTL3 by targeting its 3′UTR. METTL3 then promotes the expression of HBXIP gene through m⁶A modification, forming a positive feedback loop of HBXIP/let-7g/METTL3/HBXIP, which leads to accelerated cell proliferation in breast cancer [68].

In human hepatocellular carcinoma (HCC), METTL3 has been elucidated to be frequently upregulated and contributes to HCC progression through a distinct mechanism. SOCS2, one of the members of suppressor of cytokine signaling (SOCS) family, functions as a tumor suppressor gene by negatively regulating the JAK/STAT pathway [69]. METTL3 substantially augments SOCS2 mRNA m⁶A modification and downregulates SOCS2 mRNA expression by degrading SOCS2 mRNA transcripts through m⁶A “reader” protein YTHDF2-dependent degradation pathway, suggesting a new dimension of epigenetic alteration in liver carcinogenesis [70].

m⁶A RNA methylation promotes cancer cell migration and tumor metastasis

The topic that m⁶A RNA methylation can act on cancer cell migration and tumor metastasis has now blossomed into a full-fledged field of research.

In HCC, especially in metastatic HCC, a decreased tendency of m⁶A modifications is observed and METTL14 is addressed to be the main factor involved in aberrant m⁶A modification [71]. It has been demonstrated by Alarcon and colleagues that m⁶A modification can mark pri-miRNAs for processing by recognizing DiGeorge critical region 8 (DGCR8) in a manner dependent on METTL3/m⁶A, highlighting the important role of m⁶A modification in RNA processing, including mRNAs and pri-miRNAs [32]. Similarly, METTL14 manipulates pri-miRNA processing by regulating the recognition and binding of DGCR8 to pri-miRNAs in an m⁶A-dependent manner and thus METTL14 depletion in HCC results in pri-miR126 processing arrest and reduces the expression of mature miR126 who has been identified as a metastasis suppressor, leading to advanced metastasis capability.

Besides the genetic background of cancer cells, alteration in microenvironment has emerged as a vital layer of regulating cancer metastasis. In colorectal carcinoma (CRC), serine proteases are important component in microenvironment and can selectively activate protease-activated receptor 2 (PAR2) through proteolysis of the receptor [72]. The research conducted by Yang and colleagues has unraveled that PAR2 activation decreases the level of miR-125b through NOP2/Sun RNA methyltransferase family, member 2 (NSun2)-mediated pre-miR-125b2 methylation in CRC. NSun2 is discovered to methylate precursor of miR-125b, interferes with its processing, and reduces the level of mature miR-125b [73]. The downregulation of miR-125b augments the expression of its target gene GRB2-associated-binding protein 2 (Gab2), thereby dramatically promoting cancer cell migration, which provides a novel epigenetic mechanism by which m⁶A modification on miRNAs promotes cancer metastasis [74].

In human pancreatic cancer (PC), the research conducted by Chen et al. showed that YTHDF2 was upregulated at both mRNA and protein levels and orchestrated proliferation and epithelial–mesenchymal transition (EMT) dichotomy [75]. Two of the main characteristics of tumor growth are uncontrolled proliferation and abnormal cell migration [76, 77]. However, cells are usually not supposed to respond to gene alterations by proliferating or migrating both at the same time, which is called migration–proliferation dichotomy [78]. YTHDF2 functions as the main regulator in this phenomenon who can promote the ability of proliferation via Akt/GSK3b/CyclinD1 pathway, while it can suppress the migration, invasion, and adhesion ability by inhibiting EMT probably via downregulation of yes-associated protein (YAP) gene. There exist two m⁶A RNA methylation sites in YAP mRNA transcript with one site in coding DNA sequences (CDS) and the other site in exon [35]. Therefore, it seems reasonable to come to the hypothesis that YTHDF2 might bind to m⁶A sites of YAP mRNA to decrease the stability of mRNA, while the direct link between YTHDF2 and YAP remains to be clarified.

m⁶A RNA methylation may contribute to tumor immunity

As elucidated by Li et al., m⁶A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathway. This prompts the very first attempt at studying m⁶A RNA methylation as key regulators of T cell differentiation, suggesting their involvement in tumor–immune system communication and importance in tumor growth and spread [79].

In other circumstances, toll-like receptors (TLRs) establish the first line of defense against besieging pathogens as the most conserved molecules of the innate immune system, which recognize pathogen-associated molecular patterns to facilitate an immune response. RNAs with m⁶A modification are found incapable of activating TLR3, and those with 5^mC and/or m⁶A do not activate TLR7 or TLR8, leading to non-recognition of pathogens carrying these RNA modifications by TLR receptors (e.g., a viral nucleic acid). The methylation in m⁶A interferes with Watson–Crick base pairing; thus, its presence destabilizes RNA duplexes, which may explain why RNAs containing m⁶A modification are incapable of stimulating TLR3 [80]. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. These undetected viral components may then stimulate a pathway involved in cancer development [81–83].

The future of m⁶A RNA methylation

m⁶A RNA methylation as diagnostic and therapeutic targets

Huang et al. have demonstrated a significant increase of m⁶A RNA methylation in circulating tumor cells (CTCs) compared with whole blood cells for the first time, constituting the first step for the investigation of RNA methylation in CTCs from lung cancer patients. This research outcome may facilitate uncovering the metastasis mechanism of cancers in the future. It is worth noting that early detection and characterization of upregulated m⁶A expression in CTCs can make contributions to monitoring and preventing the development of overt metastatic diseases [84].

Moreover, m⁶A RNA methylation represents brand new therapeutic targets. All the subtypes of AMLs with high levels of endogenous FTO expression, such as those carrying t(11q23), t(15;17), NPM1 mutations, and/or FLT3-ITD, are more sensitive to ATRA treatment than the other AML subtypes [52]. It is plausible to hypothesize that the proliferation of these subtypes of AML cells relies more on the FTO signaling, and thus they are more responsive to ATRA treatment, as ATRA can release the expression/function of ASB2 and RARA, two negative targets of FTO, and thereby trigger cell differentiation.

Meclofenamic acid (MA) is a US Food and Drug Administration (FDA)-approved non-steroidal anti-inflammatory drug primarily known for its inhibition of prostaglandins synthesis as well as for its more specific inhibition of cyclooxygenase enzymes and lipoxygenases [85]. Intriguingly, recently identified FTO inhibitors are also derived from dual cyclooxygenase/lipoxygenase inhibitors [86]. Mechanistic studies conducted by Huang et al. indicated that MA specifically competed with FTO binding for the m⁶A-containing nucleic acid and treatment of HeLa cells with the ethyl ester form of MA (MA2) led to elevated levels of m⁶A modification in mRNA [87]. The identification of MA as a highly selective inhibitor of FTO will certainly shed light on more research into specific inhibitors existing within our resources of known drugs. Studies of MA2 have presented possibilities for therapeutic targets in cancers. For example, as elucidated by Cui and colleagues, MA2 suppresses glioblastoma progression and prolongs lifespan of GSC-grafted animals, which suggests that m⁶A methylation could be a promising target for anti-glioblastoma therapy [58].

The cellular components of m⁶A regulatory complex themselves can also function as novel options for cancer treatment. For example, researchers have endeavored to design a peptide mimicking the critical METTL14 domain and investigate whether it has a potential therapeutic effect in HCC [71].

However, the correlation between m⁶A RNA methylation and clinical features including predict treatment-free survival (TFS) and overall survival (OS) in cancer still warrants further research.

Challenges of future research on m⁶A RNA methylation

As described above, the rapid development of m⁶A RNA methylation research has contributed to revealing underlying mechanisms of cancer initiation and progression. However, there remain a large number of challenges.

The first issue we should pay attention to is that whether the multiple functions proposed in cancer is actually dependent on m⁶A modification. Cellular components of m⁶A methylation complex are well established to be involved in cancer carcinogenesis and may function as tumor promoters or suppressors [56, 88–93]. It is identified by Lin and colleagues that METTL3 boosts translation of certain mRNAs including epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ, thus promoting growth, survival, and invasion of human lung cancer cells. METTL3 activates mRNA translation through an interaction with translation initiation machinery rather than invoking m⁶A reader proteins downstream of nuclear METTL3, since both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA [56].

Additionally, the elevated level of the S-adenosyl methionine (SAM) donor of the methyl group in the m⁶A methylation process has been shown to suppress cell growth in cancer [94–98]. However, a direct causative link between m⁶A RNA methylation caused by SAM and its cancer growth-inhibitory effect remains to be established.

Secondly, we need to identify through which mechanism m⁶A RNA methylation is introduced. Despite the strong consensus, only a small fraction of RACH sites is detectably methylated in vivo, arguing that the sequence motif is not sufficient to determine the distribution of m⁶A [39]. It has been elucidated by Zhou and colleagues that miRNAs regulate m⁶A modification via a sequence pairing mechanism, through which miRNA sequences alter m⁶A modification levels via modulating the binding of METTL3 to mRNAs containing miRNA targeting sites. These results reveal how miRNAs involves in regulating the formation of m⁶A and partially explain the site selection mechanism of m⁶A [99]. Similarly, as reported by Zhang et al., the interaction between ALKBH5 and FOXM1 nascent transcripts is facilitated by a nuclear antisense lncRNA FOXM1-AS [61]. More than 70% of mammalian transcriptomes have antisense transcription [100]. It is worthwhile to determine whether there are other antisense lncRNAs that give rise to the specificity of m⁶A methylation so that the current view of methylation regulation can be completed.

Another mechanism by which the emplacement of m⁶A is regulated refers to that METTL3 can associate with chromatin and localize to the transcriptional start sites of active genes. Thereby, METTL3 induces m⁶A modification within the coding region of the associated mRNA transcript and enhances its translation by relieving ribosome stalling. One example is that METTL3 modulates nn n zSP1, an oncogene in several cancers which regulates c-MYC expression in this way [101], identifying a new paradigm for selecting RNAs to be modified, namely the stable recruitment of the RNA-modifying enzyme to specific genomic loci [102].

Iron metabolism also participates in m⁶A recruitment modulation. ALKBH5 belongs to the AlkB family of non-heme Fe(II)/a-ketoglutarate-dependent dioxygenases, whose activity is iron dependent [17]. Gene expression profiling carried out by Schonberg et al. revealed ferritin-dependent regulation of FOXM1 signaling. Given that Zhang et al. have proposed the interaction between ALKBH5 and FOXM1 nascent transcripts, it is plausible to indicate that ALKBH5 activity can be modulated by iron metabolism, consequently affecting FOXM1 expression [61, 66].

The third issue is that although there exist emerging links between m⁶A RNA methylation and transcript-specific fates in different cell types, it remains to be determined whether m⁶A is differentially recognized by distinct YTH domain proteins or whether m⁶A RNA methylation is used in a cell type-specific manner to either direct translational enhancement or mRNA decay.

Interestingly, METTL14 promotes stability of MYC mRNA through affecting m⁶A abundance mainly on the 3′-terminal exon, while FTO promotes stability of MYC mRNA through inhibition of YTHDF2-mediated RNA decay which can be attributed to decreased m⁶A abundance on the 5′-terminal and internal exons of MYC mRNA [52]. This phenomenon indicates that m⁶A modifications on different regions of the same mRNA transcript (e.g., MYC) may result in distinct fates likely due to recognition by different readers. It also leads to very open and constructive discussion that YTH domain family proteins may depend on sequence context to control their selectivity towards m⁶A sites and may be dynamically regulated.

However, much remains to be learned before we can understand the dynamic regulation of the co-transcriptional installation of m⁶A RNA methylation and its impacts on downstream mRNA processing events and cancer progression.

Conclusion

As the most prevalent modifications of mRNA, m⁶A RNA methylation has a deep root in modulating gene expression and is implicated in affecting many cellular processes and diseases, including cancers. Over the past few years, empowered by the availability of highly specific antibodies and the accessibility of high-throughput sequencing technologies, it has become feasible to identify the chemical basis and multiple functions of m⁶A RNA methylation. There remain many other kinds of RNA post-transcriptional modifications which may share similar properties with m⁶A RNA methylation, shedding light on the advancement of epitranscriptome.

Abbreviations

5^mC

5-Methylcytosine

ADAM19

A disintegrin and metallopeptidase domain 19

ALKBH5

alkB homolog 5

AML

Acute myeloid leukemia

ASB2

A suppressor of cytokine signaling box-2

ATRA

All-trans-retinoic acid

BC

Breast cancer

BCL2

B cell lymphoma 2

BCSC

Breast cancer stem cell

CDS

Coding DNA sequences

CEBPA

CCAAT/enhancer-binding protein alpha

CRC

Colorectal carcinoma

CSCs

Cancer stem cells

CTCs

Circulating tumor cells

DC

Dendritic cells

DGCR8

DiGeorge Critical Region 8

DNMT

DNA methyltransferases

EGFR

Epidermal growth factor receptor

EMT

Epithelial–mesenchymal transition

FDA

Food and Drug Administration

FOXM1

Forkhead box protein M1

FTO

Fat mass and obesity-associated protein

Gab2

GRB2-associated-binding protein 2

GBM

Glioblastoma

GSCs

Glioblastoma stem-like cells

HBXIP

Hepatitis B virus X-interacting protein

HCC

Hepatocellular carcinoma

HIF

Hypoxia-inducible factor

HSCs

Hematopoietic stem cells

KLF4

Kruppel-like factor 4

LC–MS/MS

Liquid chromatography coupled to triple–quadrupole mass spectrometry

m⁶A

N⁶-Methyladenosine

MA

Meclofenamic acid

MA2

The ethyl ester form of MA

MAL

Myelin and lymphocyte

METTL

Methyltransferase-like

miCLIP

m6A individual-nucleotide-resolution crosslinking and immunoprecipitation

MYB

Myeloblastosis

MYC

Myelocytomatosis

NSun2

NOP2/Sun RNA methyltransferase family, member 2

OS

Overall survival

PAR2

Protease-activated receptor 2

PC

Pancreatic cancer

PTEN

Phosphatase and tensin homolog

R-2HG

R-2-hydroxyglutarate

RARA

Retinoic acid receptor alpha

RBM15

RNA-binding motif protein 15

SAM

S-Adenosyl methionine

SOCS

Suppressor of cytokine signaling

SPI1

Spleen focus-forming virus proviral integration oncogene

SRSF

Serine and arginine-rich splicing factor

TFS

Treatment-free survival

TLRs

toll-like receptors

WTAP

Wilms tumor 1-associated protein

YAP

yes-associated protein

YTH

YT521-B homology

ZNF217

Zinc finger protein 217

Authors’ contributions

YP, PM, and YL drafted the manuscript. YL and WL discussed and revised the manuscript. YS, WL, and PM designed the research and drafted the manuscript. All authors read and approved final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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