N6‐methyladenosine (m6A) RNA modification in human cancer

Abstract N6‐methyladenosine (m6A) RNA modification, first discovered in 1974, is the most prevalent, abundant and penetrating messenger RNA (mRNA) modification in eukaryotes. This governs the fate of modified transcripts, regulates RNA metabolism and biological processes, and participates in pathogenesis of numerous human diseases, especially in cancer through the reciprocal regulation of m6A methyltransferases (“writers”) and demethylases (“erasers”) and the binding proteins decoding m6A methylation (“readers”). Accumulating evidence indicates a complicated regulation network of m6A modification involving multiple m6A‐associated regulatory proteins whose biological functions have been further analysed. This review aimed to summarize the current knowledge on the potential significance and molecular mechanisms of m6A RNA modification in the initiation and progression of cancer.


| INTRODUC TI ON
More than 170 kinds of chemical modifications have been found in various types of RNAs. 1 The common examples of RNA modification include N 6 -methyladenosine (m 6  (readers) to regulate the post-transcription of genes without altering base sequences. 6 The profile and characteristic of m 6 A modification remained unidentified due to technical bottleneck and less knowledge about regulators involved, until the first demethylases fat-mass and obesity-associated protein (FTO) was reported in 2011, triggering the upsurge of RNA epigenetic transcriptome researches. m 6 A methylation was discovered in purified poly(A) RNA fraction in 1974. 4 It is the most pervasive and abundant internal modification in eukaryotic cells, which has been confirmed by other researchers in various eukaryotes, from yeast, Arabidopsis and Drosophila to mammals, and even in viruses. Researchers detected an enormous number of highly conserved m 6 A sites and also determined more than 12 000 m 6 A signal peaks on 7676 mammalian genes by m 6 A sequencing. 8  especially around stop codons of the CDS and the first quarter of 3′-UTR. 9 m 6 A methylation is also found near the start codon in Arabidopsis thaliana. 10 In mammals, m 6 A modification exists widely in many tissues, with the highest level of m 6 A modification in the liver, kidney and brain, 9 showing a character of tissue universality and preference. m 6 A modification was initially thought to exist only in mRNAs primarily due to the limitations of detection techniques. Subsequent studies found that m 6 A methylation occurred in various types of RNAs, such as small ribosome RNA, transfer RNA, small nuclear RNA (snRNA), small nucleolar RNA, microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA. 11,12 The functions and roles of m 6 A methylation in different biological processes have gained renewed attention. m 6 A modification can regulate RNA metabolism (Figure 1), affect cell fate and functionally participate in a variety of pathophysiological processes, such as stem cell differentiation, cell division, immune homeostasis, mitosis, gametogenesis, sex determination and biological rhythm, and occurrence of numerous human diseases. 13,14 In this review, starting from m 6 A modification, the related regulators and research progresses on tumours were elaborated, which were expected to become novel markers for molecular diagnosis and potential therapeutic strategies for tumours.

| m 6 A-Writers
The deposition of m 6  complex. Structural studies indicated that METTL3 interacted with METTL14 to form a stable heterodimer structure in the ratio of 1:1 to contribute to recognizing target RNAs. 18 The physical relationship between METTL3 and METTL14 has a synergistic effect; METTL14 can increase the activity of METTL3 methyltransferase. 18 24,25 The knockdown of KIAA1429 results in a decrease of in the m 6 A content, which is greater than that achieved by METTL3 and METTL14 knockdown in A549 cells. 26 RBM15 and its paralog RBM15B can bind to the U-rich region and catalyse m 6 A modification in some mRNAs and lncRNA XIST. 27 In 2018, ZC3H13 was identified as a new m 6 A writer in mice and D melanogaster.

| m 6 A-Erasers
Until today, only two m 6 A demethylases, FTO and ALKB homolog 5 (ALKBH5), have been identified, both of which belong to the ALKB dioxygenase family and rely on the cofactors Fe 2+ and α-ketoglutarate to execute catalytic functions. FTO was initially identified as an obesity-related gene by genome-wide association studies. 28 FTO was the first m 6 A demethylase discovered in 2011, which confirmed that m 6 A modification was a dynamic and reversible process. 7 FTO F I G U R E 1 N6-methyladenosine (m 6 A) RNA modification in human cancer. m 6 A modification is a dynamic and reversible process. m 6 A methylation is catalysed by methyltransferase complex (writers), reversed by demethylases (erasers) and functionally facilitated by m 6 Abinding proteins (readers). m6A methylation participates in carcinogenesis and tumour progression affects mRNA stability and translation efficiency by regulating m 6 A modification. 29 In 2019, FTO was verified to demethylate the m 6 A m modification of snRNA and modulate alternative splicing of mRNAs. 30 FTO-mediated m 6 A demethylation involves two steps concomitant with two intermediates, N 6 -hydroxymethyladenosine and N 6 -formyladenosine. 31 ALKBH5 belongs to the ALKB family, but unlike other AIkBs, only ALKBH5 can demethylate m 6 A modification. 32 ALKBH5 can directly catalyse the methylation of m 6 A-modified adenosines without producing intermediates. 33 The expression patterns of FTO and ALKBH5 are different. FTO exists widely in adults and embryos, especially in the brain, while ALKBH5 is expressed in testis. Besides FTO and ALKBH5, more m 6 A demethylases need to be further discovered. inducing mRNA degradation. 38 Interestingly, YTHDF3 combined with YTHDF1 can enhance mRNA translation, while YTHDF3 combined with YTHDF2 can promote its degradation. 39,40 However, other studies found that YTHDF2 binding to the m 6 A site prevented FTO from removing m 6 A methylation in the 5′-UTR region, thereby promoting the cap-independent translation of mRNAs. 41 Besides, HNRPNPA2/B1 is involved in the transcription of miRNA precursors (pre-miRNA). 35 However, HNRPNPC can affect the secondary structures of mRNAs and lncRNAs. 6 m 6 A modification can destroy base pairing and improve the accessibility of single-stranded RNA motifs, thus being recognized by HNRPNPC and HNRPNPG. 6,42 Although HNRNPC and HNRNPG cannot directly bind to the m 6 A site, they can mediate the selective splicing of m 6 A-modified transcripts by recognizing and combining m 6 A-dependent structural switches. 6,43 Prrc2a has been recently confirmed as an m 6 A reader, and recombinant Prrc2a can stabilize m 6 A-modified transcripts required for myelin formation. 44 The newly discovered IGF2BPs are considered to belong to the m 6 A reader family. IGF2BP2 selectively binds to m 6 Amodified mRNA via K homolog and flanking domains, promoting the translation and stability of mRNA, which is quite different from that of readers with the YTH domain. 45,46

| M 6 A AND C AN CER S
The list of pathophysiology processes regulated by m 6 A modification continues to expand with increasing research and technological breakthroughs. It includes mRNA metabolism, immune modulation, biological rhythm, neural development, autophagy, R-loop regulation, embryonic and reproductive development and various diseases. 11,47,48 The m 6 A modification can be traced from systemic lupus erythematosus, 49 single nucleotide polymorphism (genetic variant), 50 type 2 diabetes, 51 inflammatory response and so on. 52 Recently, emerging evidence indicates the crucial parts of m 6 A methylation in carcinogenesis and tumour progression by regulating the expression of oncogenes and tumour suppressor genes. 1,14,53 Analogous to other epigenetic modifications, m 6 A methylation participates in cell proliferation, apoptosis, migration, angiogenesis, autophagy, chemoradiotherapy resistance, energy metabolism, immune escape, self-renewal and differentiation during the initiation and progression of cancers. 54,55 Inhibitors of m 6 A regulators have been found and identified as potential inhibitors of cancer progression, suggesting that m 6 A might be a potential target for cancer treatment. The article will review the relationships between m 6 A modification and various types of tumours were discussed further (Table 1, Figure 2).  Most noteworthy, erasers and writers provide complementary mechanisms in AML progression. FTO also acts as an oncogene in AML. 62 It is highly expressed in some subtypes of AML, such as t

| m 6 A in breast cancer (BC)
Increasing numbers of studies have reported that abnormal m 6

| m 6 A in hepatocellular carcinoma (HCC)
Most of the m 6 A-related enzymes are reported to be up-regulated in HCC, but METTL14 and YTHDF2 are still debatable. METTL3 is significantly elevated in HCC and associated with poor prognosis in patients with HCC. 79 METTL3 reduction inhibits cell migration, colony formation and proliferation in vitro and lung metastasis and tumorigenicity of HCC in vivo. 79 Mechanistically, METTL3 potentiates the degradation of SOCS2 mRNA (tumour suppressor gene) via the m 6 A-YTHDF2-dependent pathway. 79 Equally, METTL3-mediated m 6 A modification activates Wnt/β-catenin signalling by promoting CTNNB1 expression to drive the growth of hepatoblastoma. 80 Ma

| m 6 A in gastric cancer (GC)
METTL3 can serve as an oncogene-promoting GC tumorigenesis and progression. 86 miRNA-600 reverses METTL3-induced tumorigenesis and progression of lung cancer. 102 Lin et al 103 also confirmed that METTL3 promoted cell growth, proliferation and invasion in lung adenocarcinoma. Besides being an m 6 A "writer", METTL3 may serve as an m 6 A "reader" in the cytoplasm by identifying and interacting with translation initiation factor, enhancing the translation of EGFR and TAZ in lung cancer. 103 It indicates that METTL3 conveys oncogenic signals to potentiate aggressiveness in lung cancer.
FTO acts as a proto-oncogene in NSCLC. 104 The knockdown of FTO inhibits the proliferation of lung cancer cells by modulating the mRNA stability of USP7 via FTO-mediated m 6 A demethylase. 104 Furthermore, FTO rather than METTL3, METTL14 and ALKBH5 is considered as the main factor causing the dysfunction of m 6 A modification in lung squamous cell carcinoma. 105 FTO can enhance the stability of MZF1 mRNA by reversing m 6 A modification, inducing MZF1 expression and ultimately promoting the progression of lung squamous cell carcinoma. 105 All these indicate that FTO may be a potential therapeutic target in lung cancer. Intermittent hypoxia-induced ALKBH5 amplification in lung adenocarcinoma promotes cell proliferation and invasion by decreasing the m 6 A level of FOXM1 mRNA and increasing the translation of FOXM1 mRNA. 106

| m 6 A in bladder cancer and renal cell carcinoma (RCC)
Enrichment of m 6 A modification is associated with bladder cancer.
Bioinformatics analysis revealed that m 6

| m 6 A in pancreatic cancer
The expression of METTL3 increases at both protein and mRNA levels in pancreatic cancer; METTL3 knockdown inhibits cell proliferation, invasion and migration. 114 Pancreatic cancer cells with METTL3 silencing are more susceptible to irradiation, gemcitabine, 5-FU and L-OHP, but cell morphology and proliferation are not affected. 115 METTL3 and METTL14 are proposed as novel and promising targets for enhancing chemosensitivity and radiosensitivity of pancreatic cancer cells. 115 In pancreatic cancer, METTL3 correlates with carcinogenesis and may act as a potential therapeutic target. 114,115 In pancreatic ductal adenocarcinoma, increased WTAP expression is significantly related to the sex and tumour stage and appears to be an independent and valid prognostic factor. 116

| M 6 A AND REL ATED INHIB ITOR S
Considering that epigenetics is characterized by regulating transcription and post-transcriptional products, previous studies showed that m 6 A methylation has crucial role in malignant biological behaviours.
Therefore, developing specific inhibitors of m 6 A-related proteins is of great scientific significance and clinical value, thus being vital in tumour therapy. [130][131][132][133] Gemcitabine has emerged as an inducer of apoptosis in pancreatic cancer cells with low METTL3 expression. 115 CRC cells with YTHDF1 silencing are more sensitive to L-OHP and 5-FU. 98 METTL3 drives the ectopic expression of CBX8 in an m 6 Adependent manner, which obviates the sensitivity of colon cancer cells to chemotherapy of CPT-11 and L-OHP. 95 Single medication is prone to produce drug resistance in the clinical application. The aforementioned findings on m 6 A methylation and chemotherapeutic drugs provide a promising strategy for tumour therapy. 1,134 At present, the development of inhibitors based on m 6 130,133,137,138 As the first obesity-related gene identified by genome-wide association analysis analysis, 28 not only FTO is closely associated with obesity and tumour, but also its common variant rs9939609 may be associated with central nervous system diseases, such as brain volume loss and alcohol dependence. 139 Therefore, FTO inhibitors may also be developed as drugs for neurological diseases, besides their anti-cancer and weight-reducing effects in addition to anti-tumour and weight loss, may also be developed as drugs for neurological diseases. The progress on methyltransferase inhibitors is relatively slow. So far, only 3-deazaadenosine has been proven to inhibit METTL3, but it has a broad-spectrum efficacy and inhibits the activity of all m 6 A "readers". 140 Furthermore, chemical oxidation eliminates the m 6 A modification of mRNA. 141,142 Although several m 6 A demethylase agents have been reported in the literature, their specificity or efficacy cannot achieve the goal of precise and suboptimal treatment. Precise and effective m 6 A targeted drugs still need further research and development.

| CON CLUS I ON S AND PER S PEC TIVE S
Various biological processes and diseases have been revealed in detail with increasing investigations on the composition of m 6 A regulatory network and its significance in mRNA processing and metabolism. 143 As the most prevalent modifications of mRNAs, m 6 A modification, characterized by widespread existence, unique distribution and dynamic reversibility, is involved in almost the whole course of mRNA biology from production to degradation.
It participates in the regulation of biological functions of miR-NAs and lncRNAs, 12,144 which acts as an important regulator of stress response, biological rhythm, cell differentiation, immune response, virus replication and infection, adipogenesis, embryonic development, sex determination, carcinogenesis, tumour progression, and so on. 14,54 Many issues related to the function and action mechanism of m 6 A methylation remain unresolved. Novel m 6

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interests.

AUTH O R CO NTR I B UTI O N S
FCH and ZMZ wrote and discussed the manuscript. DSP designed and drafted the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.