Insights into roles of METTL14 in tumors

Abstract N6‐Methyladenosine (m6A) is considered the most common and endogenous modification of eukaryotic RNAs. Highly conserved in many species, m6A regulates RNA metabolism, cell differentiation, cell circadian rhythm, and cell cycle; it also responds to endogenous and exogenous stimuli and is associated with the development of tumors. The m6A methyltransferase complex (MTC) regulates the m6A modification of transcripts and involves two components, methyltransferase‐like enzyme 3 (METTL3) and methyltransferase‐like enzyme 14 (METTL14), and other auxiliary regulatory distinct components. Though with no catalytic effect, METTL14 serves as an RNA‐binding scaffold in MTC, promotes RNA substrate recognition, activates, and escalates the catalytic capability of METTL3, thus accounting for a pivotal member of the complex. It was reported that METTL14 regulates tumor proliferation, metastasis, and self‐renewal, and plays a part in tumorigenesis, tumor progression, and other processes. The present work is a review of the role of METTL14 both as a tumor suppressor and a tumor promoter in the oncogenesis and progression of various tumors, as well as the potential molecular mechanisms.


| INTRODUC TI ON
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 N6methyladenosine (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 technology 10,11,14 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 response 20,21 by participating in various aspects of RNA physiological processes, such as mRNA maturation, [22][23][24] transport from nucleus to cytoplasm, 25,26 translation efficiency 21,27,28 and stability 29,30 (Figure 1). 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 Sadenosylmethionine (SAM) and methylates the hydrogen atom on the adenine atom N6, 32 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.

| S TRUC TURE AND FUN C TI ON S OF ME T TL14 IN MTC
In the last decade of the 20th century, Tuck 38 isolated two methylase constituents of 200 kDa (MT-A) and 800 kDa (MT-B), from F I G U R E 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 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 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 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.

| MODUL ATION OF ME T TL14 E XPRE SS I ON
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,

| ME T TL1AND C AN CER
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.

| 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.

| 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

| Endometrial cancer
Endometrial cancer (EC) is a common gynecologic malignancy, originating from endometrium that grows out of control. 76 53 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.

| 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

| 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 the level of METTL14 in RCC presents significantly negative correlation to the expression of four miRNAs, miR-130a-3p, miR-106b-5p, miR-130b-3p, and miR-301a-3p, which regulate the METTL14 expression by interacting with a series of 24 circRNAs, such as circ-0023414 and circ-0031772.

| Other cancer
In papillary thyroid carcinoma (PTC), OIP5-AS1 is a downstream target of METTL14. METTL14 upregulation hinders the expression of OIP5-AS1, regulates EGFR, MEK/ERK signals, and depresses PTC cell proliferation and aggression. 86 METTL14 exerts a tumor suppressor effect by activating caspase-3 to constrain the propagation, migration, and aggression of osteosarcoma cells, and is considered a potential target for osteosarcoma treatment. 87

| ME T TL14 A S AN ON COG ENE
Although METTL14 has a tumor suppressor effect in most cancer types, it has also been found to act as a contributing factor to tumorigenesis and development. to the m6A modified transcript, hence increasing DROSHA mRNA stability and ultimately enhancing BCSC phenotype. 47

| Pancreatic cancer
Wang et al. 95 found increased m6A modification in more than half of pancreatic cancer tissues, and identified METTL14 as the major molecule that regulates m6A methylation in pancreatic cancer. is considered to be an innovative molecular target for treatment of EBV-related tumors.

| CON CLUS I ON S AND PER S PEC TIVE S
In conclusion, m6A affects various aspects of RNA physiological processes, such as mRNA processing, translation efficiency, and transcription stability, and contributes to developmental regulation, cell cycle, and other cellular physiological processes of tumors and other diseases. METTL14, as an m6A methyltransferase, regulates m6A contents. Existing works have confirmed that METTL14 plays a pivotal role in many aspects of tumor progression, including cell accumulation, aggression, apoptosis, and self-renewal, as either an oncogenic and anti-oncogenic factor. However, the role of METTL14 in tumors and its potential molecular mechanism is far from being fully clarified. It should be noted that, however, the in tumors such as AML, breast cancer, and pancreatic cancer. In tumors such as HCC and gastric cancer, however, these two proteins work in opposite ways. The causes for the opposing roles of these two constituents of MTC in the same tumor may be tumor heterogeneity, their different protein structures, and, in some cases, different study models. The discrepancy remains to be investigated further.
Since METTL14 has extensive impacts on tumor progression and plays different roles in various tumors, it is expected to become a novel molecule for tumor diagnosis and treatment, but specific METTL14-targeted therapy strategies are yet to be explored.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
XL conducted research and drafted the manuscript. YPD, ZHH, HLQ, and JWC provided assistance in the process of revised drafting manuscript and figure and tables construction. YZ contributed to conceptual framework, supervised the study, and revised the manuscript. All authors read the final manuscript and approved.

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.