The role of m6A RNA methylation in human cancer

N6-methyladenosine (m6A) is identified as the most common, abundant and conserved internal transcriptional modification, especially within eukaryotic messenger RNAs (mRNAs). M6A modification is installed by the m6A methyltransferases (METTL3/14, WTAP, RBM15/15B and KIAA1429, termed as “writers”), reverted by the demethylases (FTO and ALKBH5, termed as “erasers”) and recognized by m6A binding proteins (YTHDF1/2/3, IGF2BP1 and HNRNPA2B1, termed as “readers”). Acumulating evidence shows that, m6A RNA methylation has an outsize effect on RNA production/metabolism and participates in the pathogenesis of multiple diseases including cancers. Until now, the molecular mechanisms underlying m6A RNA methylation in various tumors have not been comprehensively clarified. In this review, we mainly summarize the recent advances in biological function of m6A modifications in human cancer and discuss the potential therapeutic strategies.


Introduction
According to MODOMICS, 163 different chemical modifications in RNA have been identified in all living organisms by the end of 2017 [1]. Among these modifications, N 6 -methyladenosine (m 6 A), methylated at the N 6 position of adenosine, has been considered as the most pervasive, abundant and conserved internal transcriptional modification within eukaryotic messenger RNAs (mRNAs) [2], microRNAs (miRNAs) [3] and long non-coding RNAs (lncRNAs) [4]. RNA m 6 A is enriched near stop codon and 3′ untranslated terminal region (UTR) [5,6] and translated near 5′ UTR in a capindependent manner [7], thereby affecting RNA transcription, processing, translation and metabolism.

M 6 A modification in RNA transcript
METTL3 and FTO are implicated in regulating transcription of CCAAT-enhancer binding protein (CEBP) family. METTL3 is localized to the starting sites of CEBPZ, which is required for recruitment of METTL3 to chromatin [23]. CEBPA is identified as an exclusive transcription factor displaying a positive correlation with FTO and regulating its transcription in acute myeloid leukemia (AML) [24]. M 6 A modification in RNA processing M 6 A modifications promote the initiation of miRNA biogenesis [3] and regulate nuclear mRNA processing events [25]. METTL3 recognizes the pri-miRNAs by microprocessor protein DGCR8 and causes the elevation of mature miRNAs and concomitant reduction of unprocessed pri-miRNAs in breast cancer [3]. METTL14 interacts with DGCR8 to modulate pri-miR-126 and suppresses the metastatic potential of hepatocellular carcinoma (HCC) [26]. FTO can regulate poly(A) site and 3′ UTR length by interacting with METTL3 [25]. YTHDC1 knockout in oocytes exhibits massive defects and contributes to extensive alternative polyadenylation and 3′ UTR length alterations [27]. M 6 A modification in RNA splicing M 6 A RNA modifications that overlap in space with the splicing enhancer regions affect alternative RNA splicing by acting as key pre-mRNA splicing regulators [28]. Inhibiton of m 6 A methyltransferase impacts gene expression and alternative splicing patterns [29]. FTO regulates nuclear mRNA alternative splicing by binding with SRSF2 [25]. FTO and ALKBH5 regulate m 6 A around splice sites to control the splicing of Runtrelated transcription factor 1 (RUNX1T1) in exon [28], and removal of m 6 A by FTO reduces the recruitment of SRSF2 and prompts the skipping of exon 6, leading to a short isoform of RUNX1T1 [30]. Depletion of METTL3 is associated with RNA splicing in pancreatic cancer [31]. WTAP is enriched in some proteins involved in pre-mRNA splicing [32]. But, some studies show that, M 6 A is not enriched at the ends of alternatively spliced exons and METTL3 unaffects pre-mRNA splicing in embryonic stem cells [33].

M 6 A modification in RNA degradation
M 6 A is a determinant of cytoplasmic mRNA stability [34], and reduces mRNA stability [35]. A RNA decay monitoring system is adopted to investigate the effects of m 6 A modifications on RNA degradation [36]. Knockdown of METTL3 abolishes SOCS2 m 6 A modification and augments SOCS2 expression [37]. M 6 A-mediated  SOCS2 degradation also relies on m 6 A 'reader' YTHDFs [37], which accelerate the decay of m 6 A-modified transcripts [38] or target mRNA [39]. Knockout of m 6 A methyltransferase attenuates YTHDF2 specific binding with target mRNAs and increases their stability [40]. M 6 A RNA methylation also controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways [41] and decreases the stability of MYC/CEBPA transcripts [24].

M 6 A modification in RNA translation
M 6 A modifications occur in mRNA and noncoding RNA (ncRNAs) to regulate gene expression in its 5′ or 3′ UTR [7,42]. METTL3 enhances mRNA translation [8], while depletion of METTL3 selectively inhibits mRNAs translation in 5′UTR [43] and reduces AFF4 and MYC translation in bladder cancer [44] but increase that of zinc finger protein 750 and fibroblast growth factor 14 in nasopharyngeal carcinoma [45]. M 6 A modifications facilitate the initiated translation through interacting with the initiation factors eIF3, CBP80 and eIF4E in an RNA-independent manner [46]. Heat-shock-induced translation of heat-shock protein 70 (HSP70) alters the transcriptome-wide distribution of m 6 A [7] and affects DNA repair [47]. ABCF1-sensitive transcripts largely overlaps with METTL3-modified mRNAs and are critical for m 6 A-regulated mRNA translation [43]. In addition, FMR1 binds to hundreds of mRNAs to negatively regulate their translation [20]. YTHDF1 facilitates the translation of m 6 A-modified mRNAs in protein-synthesis and YTHDF3 acts in the initial stage of m 6 A-driven translation from circular RNAs (circRNAs) [38,48,49].

M 6 A modification in metabolic and infectious diseases
M 6 A modification is involved in metabolic abnormalities in patients with T 2 DM and obesity [56]. FTO regulates the energy homeostasis and dopaminergic pathway through FTO-dependent m 6 A demethylation [50,51], and it is ubiquitous in adipose and muscle tissues, influencing RUNX1T1 splicing in adipogenesis [28,30]. METTL3/14 reduce the abundance of Hepatitis C virus replication, but FTO promotes its production through YTHDF proteins [54]. M 6 A is also identified as a conserved modulatory symbol across Flaviviridae genomes, including dengue, Zika virus and West Nile virus [55].

M 6 A modification in infertility
Deficiency of demethylase ALKBH5 leads to the aberrant spermatogenesis and apoptosis with impaired fertility in testes and striking changes in DNA methyltransferase 1 (Dnmt1) and ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) [14]. YTHDF2 is required for maternal transcriptome during oocyte maturation [57]. YTHDC1/2 determine the germline development in mouse [58], and YTHDC1 is essential for spermatogonia in males and oocyte maturation in females [27]. M 6 A modification in nervous system development M 6 A modification regulates the pace of cerebral cortex development [59] and m 6 A-regulated histone modifications enhances self-renewal of neural stem cells by METTL3/14 [60]. M 6 A has dual effects on delaying tempo of corticogenesis by two distinct pathways: increased cell-cycle length and decreased mRNA decay [59]. M 6 A depletion decreases the decay of radial glia cells associated with stem cell maintenance, neurogenesis and differentiation [61].

M 6 A modification in inflammation and metabolism-related cancer
Cacinogenesis is characterized by stepwise accumulation of genetic/epigenetic alterations of different protooncogenes and tumor-suppressor genes following other diseases including chronic inflammation and metabolic diseases. METTL3/14 and FTO influence Hepatitis C virus replication and production, and endogenous mediators of inflammatory responses (proinflammatory cytokines, reactive oxygen, et al) can promote genetic/ epigenetic alterations [62]. FTO affects RUNX1T1 splicing in adipogenesis [28,30], and RUNX1T1 is essential for pancreas development [63]. Transcription factor forkhead box protein O1 (FOXO1) as another direct substrate of FTO, regulates gluconeogenesis in liver [64] and promotes the growth of pancreatic ductal adenocarcinoma [65].

M 6 A RNA modification in human cancer
Emerging evidence suggests that, m 6 A modification is associated with the tumor proliferation, differentiation, tumorigenesis [46], proliferation [66], invasion [46] and metastasis [26] and functions as oncogenes or antioncogenes in malignant tumors (Table 1 and Fig. 3).
Collectively, these studies corroborate the functional importance of m 6 A modifications in leukemia, such as METTL3 [23,70], METTL14 [68], FTO [24,67] and YTHDF2 [24,40] and they provide profound insights into development and maintenance of AML and selfrenewal of leukemia stem/initiation cells through the downstream MYC and Tal1 pathways.
Lung cancer M 6 A demethylase FTO is identified as a prognostic factor in lung squamous cell carcinoma (LUSC) and facilitates cell proliferation and invasion, but inhibits cell apoptosis by regulating MZF1 expression [75]. METTL3 acts as a oncogene in lung cancer by increasing EGFR and TAZ expression and promoting cell growth, survival and invasion [46]. METTL3-eIF3 caused mRNA circularization promotes the translation and oncogenesis of lung adenocarcinoma [46]. Besides, SUMOylation of METTL3 is of importance for the promotion of tumor growth at lysine residues K 177 , K 211 , K 212 and K 215 in non-small cell lung carcinoma (NSCLC) [76]. These studies provide insights into the critical roles of METTL3 and FTO in lung carcinoma.

Hepatocellular carcinoma (HCC)
METTL3 is related to a poor prognosis in HCC patients and promotes HCC cell proliferation, migration and colony formation by YTHDF2-dependent posttranscriptional silencing of SOCS2 [37]. But, METTL14 is an anti-metastatic factor and serves as a favorable factor in HCC by regulating m 6 A-dependent miRNA processing [26]. MiR-145 down-regulates YTHDF2 through targeting its mRNA 3′ UTR [77]. In conclusion, METTL3 upregulation or METTL14 downregulation predicts poor prognosis in patients with HCC and contributes to HCC progression and metastasis [26,37]. METTL3 suppresses SOCS2 expression in HCC via the miR-145/m 6 A/ YTHDF2 dependent axis [37,77]. Thus, these studies suggest a new dimension of epigenetic alteration in liver carcinogenesis.

Breast cancer and colorectal cancer (CRC)
METTL3 is associated with the expression of mammalian hepatitis B X-interacting protein (HBXIP), displaying an aggressiveness in breast cancer. HBXIP-induced METTL3 promotes the proliferation of breast cancer via inhibiting tumor suppressor let-7 g [78]. Besides, ALKBH5 decreases the levels of m 6 A in NANOG mRNA and enhances its stability, leading to an increase of NANOG mRNA and protein levels in breast cancer stem cells (BCSCs) [79]. Another m 6 A eraser 'FTO' polymorphism has no association with the risk of CRC [80], but the m 6 A 'writer' WTAP is associated with carbonic anhydrase IV (CA4), which inhibits the proliferation and induces apoptosis and cycle arrest by repressing the Wnt signaling through targeting the WTAP-WT1-TBL1 axis [81].
Brief summary of m 6 A modification-related carcinogenesis M 6 A RNA modifications regulate RNA production/metabolism and take part in the carcinogenesis. On the one hand, m 6 A-modified genes usually act a oncogenic role in cancer, leading to alterations of mRNA translation and acceleration of tumor progression, and decreasing m 6 A modification results in tumor development. On the other hand, given that SUMOylation of METTL3 represses its m 6 A methyltransferase capacity and results in tumor growth of NSCLC, modification of m 6 A methylase can determine the tumor development.   [24,68,82] and CA4 in CRC [81]. Meclofenamic acid (MA) as one of the selective FTO inhibitors is a non-steroidal anti-inflammatory drug by competing with FTO binding sites [83]. MA2, the ethyl ester derivative of MA, increases m 6 A modification, leading to the suppression of tumor progression [74,83]. The expression of ASB2 and RARA is increased in hematopoiesis and they act as key regulators of ATRAinduced differentiation of leukemia cells [84]. FTO enhances the leukemogenesis of AML by inhibition of the ASB2 and RARA expression [67]. FB23-2, as another inhibitor of m 6 A demethylase FTO suppresses AML cell proliferation and promotes the cell differentiation and apoptosis [82].
S-adenosylmethionine (SAM) serves as a cofactor substrate in METTL3/14 complex and its product Sadenosylhomocysteine (SAH) inhibits the methyltransferases by competing with adenosylmethionine [87]. 3deazaadenosine (DAA) inhibits SAH hydrolase and interrupts insertion of m 6 A into mRNA substrates [88] and its analogs suppress the replication of various viruses editing m 6 A-mRNA in cancers [89,90].
METTL14 acts an oncogenic role by regulating MYB/ MYC axis through m 6 A modification [68]. SPI1, a hematopoietic transcription factor, directly inhibits METTL14 expression in malignant hematopoietic cells [68] and may be a potential therapeutic target for AML. CA4 inhibits the tumorigenicity of CRC by suppressing the WTAP-WT1-TBL1 axis [81].
Future prospect M 6 A RNA modifications act by regulating RNA transcript, splicing, processing, translation and decay and participate in the tumorigenesis and metastasis of multiple malignancies. However, the underlying mechanisms of m 6 A modifications in cancer should be further addressed.. Besides FMR1 and LRPPRC, the function of ALKBH family in m 6 A RNA methylation is undetermined. METTL14 has different expression levels in various tumor tissues. Given a dual role of METTL14 either as a tumor suppressor [26] or an oncogene in cancer [68], its role in other cancers need be further elucidated. Though some inhibitors of m 6 A methylation have shown promising effects on cancer development [68,81], novel therapeutic strategies for m 6 A RNA methylation should be further explored in the treatment of cancer.