1 INTRODUCTION

Epigenesis refers to the heritable variation of gene functions under the condition that the DNA sequence does not change, which ultimately leads to the change in phenotype.1, 2 Epigenetics participates in and regulates multiple levels of information flow from DNA to RNA to protein.3, 4 Recent investigations have found that the epigenetic modification of RNA plays vital roles in biological processes.5 N6-methyladenosine (m6A), as one of more than 170 RNA modifications observed so far in coding and non-coding of RNAs, is the most common internal modification in mRNA,6, 7 and is almost universally presented in poly (A)+ RNA of all advanced eukaryotes.8, 9 In human cells, there are 2000 m6A peaks in beyond 7000 mRNA and 300 non-coding RNA transcripts.10 It was found that m6A is not randomly distributed on mRNA, but mainly clustered in long introns and stop codons near the 3′UTR region.11 Under similar physiological conditions, highly conserved m6A peaks have been observed in mouse and human transcriptomes, revealing significant correlation between m6A abundance and functions of specific genes.10 In mutation analysis, RRm6ACH has been defined as a consensus m6A sequence, where R = G/A (G > A) and H = U/A/C (U > A > C).12 In the early 1970s, m6A modification was first detected in mammalian cells, but due to the constraints of the research conditions and technology at that time, the functional significance of this modification was not clarified. The true biological significance of m6A was not disclosed until 2011 when it was discovered that fat mass and obesity-associated (FTO) protein reversibly inhibited the m6A level.13 Meanwhile, advancements in sequencing technology and quantitative mass spectrometry technology10, 11, 14 encouraged researchers to probe further into m6A modification. It was reported that m6A can regulate cell differentiation,15, 16 cell circadian rhythm,17 cell cycle,18, 19 and cell stress response20, 21 by participating in various aspects of RNA physiological processes, such as mRNA maturation,22-24 transport from nucleus to cytoplasm,25, 26 translation efficiency21, 27, 28 and stability29, 30 (Figure 1).

RNA m6A modification is dynamically and reversibly co-regulated by RNA m6A modification machineries EEE. RNA m6A modification machineries EEE are consisted of Editors, Erasers, and Effectors. M6A is responsible for cell differentiation, cell circadian rhythm, cell cycle, and cell stress response by affecting various aspects of RNA metabolism, such as mRNA maturation, nucleus transport, RNA splicing, stability, and protein translation efficiency

RNA m6A modification is co-mediated by the modification machineries EEE (Editor, Eraser, and Effector).31 The modification Editor—multicomponent methyltransferase complex (MTC), positioned in the nuclear spot area, catalyzes the dislocation of methyl group to the certain adenine of the target RNA from S-adenosylmethionine (SAM) and methylates the hydrogen atom on the adenine atom N6,32, 33 by recruiting two predominant units, that is, methyltransferase-like enzyme 3 (METTL3) and methyltransferase-like enzyme 14 (METTL14), and with other auxiliary managing distinct units, such as Wilm's tumor-1-associated protein (WTAP), vir-like m6A methyltransferase-associated (VIRMA), KIAA1429, Hakai, RNA-binding motif protein 15 (RBM15), and methyltransferase-like enzyme 16 (METTL16). The modification Erasers—FTO and alkB homolog 5 (ALKBH5), are both Fe (II)/α-ketoglutarate-dependent alkB dioxygenase family members, which perform as m6A erasers and catalyze the reversible oxidative demethylation of the m6A methyl group.34, 35 The messages embedded in the methyl group are decoded by a certain type of protein component called m6A Effectors, including YTH domain family proteins (YTHDC1/2 and YTHDF1/2/3), insulin-like growth factor 2 mRNA-binding protein (IGF2BP1/2/3), and heterogeneous ribonucleoprotein (hnRNPC and hnRNPG), which bind m6A to modulate the nuclear transportation, degradation, and steadiness of mRNAs, thereby determining the fate of the modified target RNA.21, 36, 37

Methyltransferase and its components can regulate cell cycle, cell growth, cell differentiation, cell apoptosis, and other cellular biological processes. The correlation between methyltransferase and tumors has become a new research focus. It has been found that m6A modification and methyltransferase are related to tumor augmentation, differentiation, tumor formation, infiltration, and metastasis. Recently, increasing proof has attested to the involvement of METTL14, an indispensable constituent of MTC, in cancer. Here, we review the role methyltransferase METTL14 in tumor occurrence and expansion.

2 STRUCTURE AND FUNCTIONS OF METTL14 IN MTC

In the last decade of the 20th century, Tuck38 isolated two methylase constituents of 200 kDa (MT-A) and 800 kDa (MT-B), from the nuclear extract of Hela cells, and identified one key methylase subunit 70 kDa, named METTL3 or MT-A70, from MT-A. This investigation marks a considerable breakthrough in the research of m6A methyltransferase. The METTL3 protein, which has 580 amino acids, is comprised of a zinc finger domain (ZFD) and a methyltransferase domain (MTD). The ZFD contains two tandem CCCH-type zinc fingers (ZnF1 and ZnF2) connected by an anti-parallel β-sheet (Figure 2A,B), which is responsible for target recognition, specifically for binding to single-stranded RNAs containing 5′-GGACU-3′ consensus sequence.39 Phylogenetic analysis revealed that METTL14, a homologue of METTL3 in the human genome, shares 43% homologous sequence with METTL3.40 The METTL14 gene is 30.06 kb in size, contains 11 exons, and is positioned on chromosome 4q26. This gene is expressed in many tissues at different developmental stages. As relevant research deepens, more knowledge about the METTL14 gene, including its expression products, structure, and functions, is revealed. The METTL14 protein has 456 amino acids and is predominantly composed of a MTD, while without a ZFD as the METTL3 protein does (Figure 2C,D). METTL14 and METTL3 form a compact and stable asymmetric heterodimer with a 1:1 stoichiometric ratio, and there are extensive hydrogen bond interactions between the two.41 X-ray crystallography was adopted to inspect the structure of the MTD of the METTL3-14 heterodimer. The MTD of METTL14 encompasses residues 110–404, while that of METTL13 consists of residues 358–580. The crystal structure of the heterodimer revealed dense presence of SAM in the catalytic cavity of METTL3; the MTD of METTL14, though similar to that of METTL3, has a closed catalytic cavity with no SAM binding sites, which indicates that METTL3 is the only catalytic subunit in the MTC, and METTL14 in the complex does not serve a role to catalyze methyl group transfer.40 The overall structure of MTD3 resembles that of Class I DNA N6-adenine MTD. Nevertheless, neither MTD3 nor MTD14 possesses a target recognition domain (TRD) similar to DNA m6A, which appears as a substrate-binding platform. Analysis of the surface electrostatic potential of the METTL3/METTL14 complex demonstrated that there is a groove with positive charges between METTL3 and METTL14. Ten positively charged residues, including R245, R249, R254, R255, K297, and R298 from METTL14, and R465, R471, H474, and H478 from METTL3, together shapes this groove (Figure 2E). In the case that the above residues from METTL14 in the groove are mutated, the activity of groove-bound RNA decreases and the activity of methyltransferase drops,42 indicating that the groove formed by the positively charged residues of METTL3 and METTL14 may affect the binding of the complex to the RNA substrate. A recent study revealed that the C-terminus of METTL14 was induced arginine methylation by binding to protein arginine methyltransferase 1 (PRMT1), which promotes the interaction of RNA substrates to METTL14, and enhances the RNA methylation catalytic capacity of the MTC and interaction with RNA polymerase II (RNAPII).43 Importantly, the level of m6A modification is dependent on arginine methylation of METTL14. Analysis of transcriptome m6A modification levels revealed that nearly 2000 m6A sites, which depend on arginine methylation of METTL14, distribute over 1000 genes involved in cellular physiological processes including DNA repair. Specifically, the m6A modification of DNA double-stranded cross-linking repair-related genes relies on arginine methylation of METTL14, which improves the translation efficiency of these genes. In addition, the N-terminus of METTL14 can discern and directly interact with histone 3 trimethylated at Lys 36 (H3K36me3). The combination promotes recruitment of the MTC to RNAP II nearby, revealing that the MTC exerts cotranscriptional selective deposition by moving to the site of immature RNA.44 Liu et al.41 reported that the catalytic activity of the METTL3-METTL14 complex has considerably stronger catalytic activity than the METTL3 protein isolated in vitro. The above results indicate that though METTL3 plays a catalytic role in the MTC, its catalytic capability relies on METTL14. Though METTL14 itself has no catalytic effect, it acts as a premier RNA-binding platform, which promotes the recognition to RNA substrates, activates, and escalates the methyltransferase activity of METTL3, and significantly improves the methylation efficiency of the MTC. The role of METTL14 in the METTL3-METTL14 MTC is much like that of Dnmt3L in the DNA methyltransferase complex Dnmt3a-Dnmt3L: Dnmt3L activates and enhances the methylation activity of the DNA methyltransferase complex by binding to Dnmt3a, but Dnmt3L itself has no methylation activity.45 Therefore, in general, METTL14 is similar to Dnmt3L in that they both contain MTD without catalytic activity, but activate and strengthen the methylation of the chaperone.

Structure and functional subunit of METTL14. (A) Schematic domain structure of METTL3. (B) Structure of the zinc finger domain (ZFD) (PDB ID: 5YZ9) and methyltransferase domain (MT-A70) of METTL3 (PDB ID: 5L6D). (C) Schematic domain structure of METTL14. (D) Structure of the methyltransferase domain of METTL14 (PDB ID: 5IL0). (E) The RNA-binding groove with positive charges between METTL3 and METTL14. All structure figures were prepared using PyMOL

3 MODULATION OF METTL14 EXPRESSION

The abnormal expression of METTL14 in cancers and other diseases is triggered by multiple mechanisms. It was reported that METTL14 overexpression in acute myeloid leukemia is negatively regulated by SPI1 and mediates downstream targets, MYB and MYC, to accelerate acute myeloid leukemia (AML) oncogenesis.46 In breast cancer, aurora kinase A (AURKA) positively regulates METTL14 protein expression by inhibiting its ubiquitylation-mediated degradation to elevate DROSHA m6A content to improve DROSHA mRNA stability in breast cancer stem-like cells.47 METTL14 expression is also regulated by transcription factors. In pancreatic cancer, transcription factor P65 positively regulates METTL14 expression by interconnecting with the promoter region of METTL14. Upregulated METTL14 hampers the attenuation of cytidine deaminase (CDA) transcript, enhances its stability, and induces chemotherapy resistance of pancreatic cancer cells.48 Non-coding RNAs (ncRNAs) also play a role in regulation of METTL14 expression. In breast cancer, the stability and expression of METTL14 are positively regulated by LNC942, which elevates the m6A content in downstream targets CXCR4 and CYP1B1, stabilizes protein expression and translation, and further promotes tumorigenesis.49 The expression of METTL14 is regulated by miR-103-3p to inhibit osteogenesis, and m6A modification executed by METTL14 in turn promotes osteogenesis by inhibiting the treatment of miR-103-3p by DiGeorge critical region 8 (DGCR8), which reveals the key role of miR-103-3p - METTL14 - m6A modification axis in osteoblast activity.50 In vascular endothelial cells, the expression level of METTL14, modulated by miR-4729, is reduced, resulting in declination in m6A modification level of TIE1 mRNA 3′UTR specific site, TIE1 mRNA stability reduction, and angiogenesis inhibition.51 In renal cell carcinoma, METTL14 is regulated by the competitive binding effect of circRNAs and miRNAs, and affects the expression of downstream molecule PTEN and changes in related AKT/PKB signals.52 In skin tumors, METTL14 acts as the target of NBR1-dependent selective autophagy, and its expression level is down-regulated. METTL14 depletion reduces the translation efficiency of damaged DNA binding protein 2 (DDB2), causes global genome repair (GGR) repair defect, and provokes UVB-induced skin tumorigenesis.53 In Epstein-Barr virus (EBV)-related cancers, METTL14 expression and stability is upregulated by Epstein-Barr nuclear antigen 3C (EBNA3C) through interaction at the specific amino domain of EBNA3C.54

4 METTL14 AND CANCER

In recent decades, increasing investigations have shown that METTL14, as an m6A methyltransferase, is involved in tumorigenesis and development, whether as an oncogene or an anti-oncogene, as shown in Table 1.

TABLE 1. METTL14 acts as anti-oncogene and oncogene in human cancers Role Cancer type Regulator Targets Molecular mechanism Signaling pathway/Axis Ref. Anti-oncogene Bladder cancer Notch1 mRNA stabilization METTL14 - m6A modification - Notch1 axis 55 Colorectal cancer SOX4 mRNA degradation METTL14 - YTHDF2 - SOX4 axis; SOX4-mediated EMT process and PI3K/AKT signaling pathway partly 56 DGCR8 miR-375 Modulate pri-miR-375 process in an m6A-dependent manner METTL14 - miR-375 - YAP1 axis and METTL14 - miR-375 - SP1 axis 57 lncRNA XIST LncRNA degradation METTL14 - YTHDF2 - lncRNA XIST axis 58 MeCP2 KLF4 mRNA stabilization MeCP2 - METTL14 - KLF4 axis 75 Endometrial cancer PHLPP2; mTORC2 Promote translation by YTHDF1; mRNA degradation by YTHDF2 METTL14- PHLPP2/mTORC2-AKT pathway 60 Gastric cancer Wnt and PI3K signals Regulate Wnt and PI3K/AKT/mTOR pathway Wnt and PI3K/AKT/mTOR signaling pathways 61, 79 LINC01320 LncRNA stabilization METTL14 - LINC01320 - miR-495-5p - RAB19 axis 62 PTEN mRNA stabilization METTL14 - m6A modification - PTEN axis 80 Glioblastoma ADAM19 mRNA m6A modification METTL14 - m6A modification - ADAM19 axis 63 Hepatocellular carcinoma DGCR8 miR-126 Modulate pri-miR-126 process in an m6A-dependent manner DGCR8 - METTL14 – miR-126 axis 64 USP48 mRNA stabilization METTL14 - USP48 - SIRT6 axis 65 EGFR Stimulate PI3K/AKT signals EGFR/PI3K/AKT signaling pathway 66 Renal cell carcinoma P2RX6 pre-mRNA splicing ATP-P2RX6-Ca2+-p-ERK1/2 -MMP9 signals 68 circRNAs PTEN mRNA stabilization circRNAs - miRNAs - METTL14 - PTEN axis 52 Papillary thyroid carcinoma OIP5-AS1 Regulate expression EGFR, MEK/ERK signaling pathway 86 Osteosarcoma Caspase-3 Activate caspase-3 Apoptosis 87 Skin tumor NBR1-dependent autophagy DDB2 Translation efficiency NBR1-dependent autophagy - METTL14 – DDB2 axis 53 Oncogene Acute myeloid leukemia SPI1 MYB; MYC Accelerate hematopoietic stem cells proliferation and reduce monocyte differentiation SPI1 - METTL14 - MYB/MYC axis 46 MALAT1 PML-PARα mRNA exportation lncRNA - fusion gene - METTL14 loop 92 Breast cancer LNC942 CXCR4; CYP1B1 mRNA stabilization and translation LNC942 - METTL14 - CXCR4/CYP1B1 axis 49 AURKA DROSHA mRNA stabilization AURKA - METTL14 - DROSHA axis 47 Pancreatic cancer PERP mRNA turnover METTL14 - m6A modification - PERP axis 95 P65 CDA mRNA stabilization P65 - METTL14 - CDA axis 48 Prostate cancer CLK1-SRSF5 axis Alternative splicing CLK1 - SRSF5 - METTL14exon10 skipping axis 97 Head and neck squamous cell carcinoma LNCAROD LncRNA stabilization METTL14 – LNCAROD -YBX1/HSPA1A axis 99 EBV-related tumors EBNA3C Maintain METTL14 protein stability 54 5 METTL14 AS AN ANTI-ONCOGENE

In most cases, METTL14 is identified as a tumor suppressor gene, which inhibits oncogenesis and progression of various cancers, such as bladder cancer,55 colorectal cancer,56-58 endometrial cancer,59, 60 gastric cancer,61, 62 glioblastoma,63 hepatocellular carcinoma,64-66 and renal cell carcinoma,52, 67, 68 by reducing m6A modification on key transcripts.

5.1 Bladder cancer

Bladder cancer is a widespread malignant cancer worldwide.69 Bladder cancer stem cells (CSCs), a subgroup of bladder cancer cells also known as tumor-initiating cells (TICs), can self-renew and differentiate into large cell populations, and are rich in tumorigenic properties.70 Gu et al.55 found that METTL14 expression is reduced in bladder cancer and bladder TICs, and it is the main regulator for the decline of m6A content in bladder cancer and bladder TICs. METTL14 reduction promotes cell propagation, aggression, and self-renewal of bladder TICs. Studies have shown that neurogenic locus notch homolog protein 1 (Notch1) plays a part in accelerating bladder cancer and bladder TICs. The m6A modification of Notch1 is reduced after overexpression of METTL14, which in turn depresses the stability of Notch1 mRNA and inhibits protein expression. The above results indicate that METTL14 may target Notch1 to inhibit progression of bladder cancer and spread of bladder TICs, which reveals that the METTL14-m6A-Notch1 axis has the potential to serve as an underlying target for the treatment of bladder cancer.

5.2 Colorectal cancer

Colorectal cancer (CRC), the third most prevalent digestive tract malignancy worldwide, has seen an increasing incidence rate of CRC in the younger population.71, 72 The expression of METTL14 is remarkably depressed in CRC, and a reduction in METTL14 is associated with poor overall survival (OS). Cox regression analysis has revealed that METTL14 is an independent prognostic molecule for CRC, and it is positively associated with the level of immune infiltration.73 It was proved that METTL14 impedes the metastasis of CRC cells and exerts a tumor suppressor effect. Studies have found that SRY-related high-mobility-group box 4 (SOX4) is controlled by m6A modification mediated by METTL14, and METTL14 represses the progression of CRC via the SOX4-mediated EMT process and PI3K/AKT signals.56 It was found in another study that METTL14, as the upstream target of miR-375, suppresses CRC cells propagation via the miR-375—Yes-associated protein 1 (YAP1) axis, and depresses migration and aggression of CRC cells via the miR-375 - SP1 axis.57 Yang et al.,58 using RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation (Me-RIP), found that the oncogene lncRNA XIST is the downstream molecule of METTL14, which down-regulates lncRNA XIST in an m6A-dependent process and inhibits tumor-driving effects such as the growth and metastasis of CRC. In CRC that is resistant to immunotherapy, mismatched repair proficiency or low microsatellite instability (pMMR-MSI-L) tumors account for about 85% of all patients. Studies have proved that reducing m6A modification by knocking down METTL14 escalates the response of pMMR-MSI-L CRC to anti-PD-1 treatment.74 METTL14 and methyl CpG binding protein 2 (MeCP2) jointly regulate the m6A modification of the specific site of the tumor suppressor Kruppel-like factor 4 (KLF4) mRNA through the protein interaction between the two proteins. The M6A reader IGF2BP2 regulates the stability of KLF4 mRNA and the expression of KLF4 protein by reading unique methylation modification information, and ultimately affects the progression and metastasis of CRC.75

5.3 Endometrial cancer

Endometrial cancer (EC) is a common gynecologic malignancy, originating from endometrium that grows out of control.76, 77 Ma et al.59 described METTL14 as a probable indicator for the diagnosis and prognosis of EC. Liu et al.60 sequenced clinical specimens of EC and found that METTL14 has a hot spot R298P mutation, which induces about 70% of EC to reduce m6A methylation compared with matched normal endometrium, contributing to the proliferation of EC cells and tumorigenicity. Analysis of m6A-seq characteristics of EC samples from patients and cell lines shows that the decline in m6A mRNA methylation motivated by the METTL14 mutation may cause a decreased level of the negative effector PHLPP2 of the AKT signals and an increase in the level of the positive effector mTORC2, resulting in increased AKT activity and enhanced cell proliferation. METTL14 was found to be an important governor of the AKT signals and cell propagation in EC.

5.4 Gastric cancer

Gastric cancer (GC), also known as stomach cancer, is a leading digestive system cancer worldwide and still contributes to human death in less developed countries.78 METTL14 is lowly expressed in GC and is a key regulatory factor that decreases m6A levels in GC.61 METTL14 overexpression curbs the propagation of GC cells by inhibiting Wnt and PI3K/AKT/mTOR signals, and suppresses aggression of gastric cancer cells by obstructing the EMT process, while an increase in m6A caused by FTO knockdown reverses the above changes.61, 79 Hu et al.62 demonstrated that METTL14 methylates LINC01320 to upregulate LINC01320 expression, accelerating aggression of GC cells through the miR-495-5p-RAB19 axis. Yao et al.80 verified that METTL14 enhances phosphatase and tensin homologue (PTEN) m6A modification by interconnecting the m6A characteristic sequence GGACT with PTEN mRNA, improving RNA stability of PTEN and playing a role in cancer inhibition in stomach adenocarcinoma (STAD).

5.5 Glioblastoma

Glioblastoma (GBM), occurring predominantly among adults, is the most common and aggressive type of glioma.81 METTL14 deficiency considerably accelerates the propagation, self-renewal, and oncogenesis of glioblastoma stem cells (GSCs). After METTL14 knockdown GSCs transplanted into immunodeficiency non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice, it was found that METTL14 depletion results in significant tumor progression initiated by GSCs in the cerebra of the mice. The results of m6A sequencing showed that METTL14 knockdown leads to overexpression of the oncogene ADAM19 mRNA in GSCs.63 It implies that METTL14 and m6A modification may have the potential to be a brand-new molecular target for GBM treatment.

5.6 Hepatocellular carcinoma

Hepatocellular carcinoma (HCC), accounting for more than 80% of primary liver carcinoma, is the most common malignant carcinoma of liver cells and is an urgent health threat worldwide.82, 83 In hepatocellular carcinoma (HCC), particularly in metastatic HCC, m6A modification is reduced, METTL14 is identified as the core molecule regulating abnormal m6A modification of HCC. In addition, down-regulation of METTL14 is a detrimental prognostic factor for recurrence-free survival of HCC and is appreciably related to tumor aggression. Furthermore, miR-126, as an anti-oncogene, is the target of METTL14 in the process of HCC metastasis. METTL14 interacts with DGCR8 and regulates the pri-miR-126 process to reduce miR-126 expression, thereby promoting HCC metastasis, which accounts for an important function of METTL14 and m6A in HCC metastasis.64 Du et al.65 found that ubiquitin-specific peptidase 48 (USP48) bound SIRT6 through deubiquitination at specific si