Roles of METTL3 in cancer mechanisms and thera.pdf

REVIEWOpen AccessRoles of METTL3 in cancer:mechanismsand therapeutic targetingChengwu Zeng1,2,Wanxu Huang1,3,Yangqiu Li2*and Hengyou Weng1,4*AbstractN6-methyladenosine(m6A)is the most abundant mRNA modification and is catalyzed by the methyltransferasecomplex,in which methyltransferase-like 3(METTL3)is the sole catalytic subunit.Accumulating evidence in recentyears reveals that METTL3 plays key roles in a variety of cancer types,either dependent or independent on its m6ARNA methyltransferase activity.While the roles of m6A modifications in cancer have been extensively reviewedelsewhere,the critical functions of METTL3 in various types of cancer,as well as the potential targeting of METTL3as cancer treatment,have not yet been highlighted.Here we summarize our current understanding both on theoncogenic and tumor-suppressive functions of METTL3,as well as the underlying molecular mechanisms.The well-documented protein structure of the METTL3/METTL14 heterodimer provides the basis for potential therapeutictargeting,which is also discussed in this review.Keywords:RNA modification,METTL3,m6A,Cancer,Non-coding RNA,Drug discoveryIntroductionThere are more than 170 modifications in RNA,amongwhich N6-methyladenosine(m6A)is the most prevalentinternal modification in messenger RNA(mRNA)14.Over 7000 human transcripts harbor at least one m6A site,which is found within the consensus motif RRACH(where R=A/G,H=A/C/U),and most of the m6A sitesare enriched in the coding sequence(CDS)and the 3 un-translated region(3UTR)of mRNA,especially aroundthe stop codons 5,6.Although m6A was discoveredmore than 40 years ago 7,it failed to spark enthusiasmin this field until the identification of FTO as an m6Ademethylase in 2011 8,which reveals that m6A can bedynamically regulated and might play vital roles in devel-opment and diseases.Since then,FTO and ALKBH5,bothbelonging to the AlkB family of Fe(II)/a-ketoglutarate(a-KG)-dependent dioxygenases 9,were classified as m6A“eraser”proteins that remove m6A modifications fromRNA(Fig.1a and Table 1).In contrast to being removedby“eraser”proteins,m6A can be recognized by a set ofRNA-binding proteins called m6A“reader”proteins thatcan specifically recognize and bind to m6A-modified tran-scripts.The list of m6A“reader”proteins is increasing(Table 1),including the YTH domain family proteins(YTHDC1/2,YTHDF1/2/3)28,36,37,4143,theinsulin-like growth factor 2 mRNA-binding proteins(IGF2BP1/2/3)38,and the heterogeneous nuclear ribo-nucleoproteins(hnRNPC,hnRNPG),which were shownto mediate the regulation of RNA stability,translation effi-ciency,RNA splicing,and RNA exporting 4450.The enzymes catalyzing the formation of m6A are re-ferred to as m6A“writer”proteins,first being purified asa protein complex in 1994 by Bokar et al.51,and werefurther characterized in recent years as a multicompo-nent m6A methyltransferase complex(MTC)comprisedof a METTL3-METTL14 heterodimer core and otherbinding partners(Fig.1a),such as WTAP,ZC3H13,The Author(s).2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use,sharing,adaptation,distribution and reproduction in any medium or format,as long as you giveappropriate credit to the original author(s)and the source,provide a link to the Creative Commons licence,and indicate ifchanges were made.The images or other third party material in this article are included in the articles Creative Commonslicence,unless indicated otherwise in a credit line to the material.If material is not included in the articles Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use,you will need to obtainpermission directly from the copyright holder.To view a copy of this licence,visit http:/creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver(http:/creativecommons.org/publicdomain/zero/1.0/)applies to thedata made available in this article,unless otherwise stated in a credit line to the data.*Correspondence:;weng_hengyougrmh-Chengwu Zeng and Wanxu Huang contributed equally to this work.2Institute of Hematology,School of Medicine,Key Laboratory forRegenerative Medicine of Ministry of Education,Jinan University,Guangzhou510632,China1Bioland Laboratory(Guangzhou Regenerative Medicine and HealthGuangdong Laboratory),Guangzhou 510005,ChinaFull list of author information is available at the end of the articleZeng et al.Journal of Hematology&Oncology (2020)13:117 https:/doi.org/10.1186/s13045-020-00951-wVIRMA,and RBM15/15B 1014.In addition to theMTC,other m6A writers have also been identified inrecentyears,includingMETTL16,METTL5,andZCCHC4(Fig.1a and Table 1),which are responsiblefor the deposition of m6A into structured RNAs,suchas U6 snRNA,28S rRNA,and 18S rRNA,and insome cases,the introns of mRNA 1522.The MTCcore component METTL3-METTL14 heterodimer cat-alyzes most of m6A methylations in mRNA,withMETTL3 being the only catalytic subunit that uses S-adenosylmethionine(SAM)as the methyl donor 5254.The full-length METTL3 has 580 amino acids and iscomprised of a zinc finger domain(ZFD)and a methyl-transferase domain(Fig.1b),both of which are needed forthe enzymatic activity.Huang et al.characterized the ZFDsolution structure using nuclear magnetic resonance(NMR),showing that the domain contains two tandemCCCH-type zinc fingers(ZnF1 and ZnF2)connected byan anti-parallel-sheet(Fig.1c),which is responsible fortarget recognition,specifically for binding to single-stranded RNAs containing 5-GGACU-3 consensus se-quence 55.The structure of the methyltransferase do-main of METTL3,named MT-A70,has been determinedusing X-ray crystallography in a complex with the corre-sponding domain of METTL14 by three independentgroups 5254.It is demonstrated that METTL14 onlyplays a structural role for RNA-binding and stabilizationof the complex,while METTL3 is the catalytically activesubunit,with a co-factor binding pocket for SAM or S-adenosylhomocysteine(SAH)52,53.The crystal struc-ture reveals that the MT-A70 domain of METTL3 isformed by a Rossman fold comprising a central,curved,eight-stranded-sheet flanked by four-helixes,as wellas an interface loop to interact with METTL14 and twogate loopsthat have importantroles inadenosineFig.1 m6A RNA methylation and the structure of METTL3,the catalytic subunit of the m6A methyltransferase complex.a The writers and erasers of thedynamic m6A modification.b Schematic domain structure of METTL3.c Structure of the zinc finger domain(ZFD)of METTL3(PDB ID:5yz9).d Structure of themethyltransferase domain(MT-A70)of METTL3(PDB ID:5l6d)Zeng et al.Journal of Hematology&Oncology (2020)13:117 Page 2 of 15recognition(Fig.1d).The conserved DPPW motif(resi-dues 395399)of the enzyme is located in the gate loop 1,which undergoes a significant conformational change to-gether with gate loop 2 upon SAM/SAH binding,resultingin the closure of the co-factor binding pocket 52.Regulation of METTL3 expression and m6AdepositionThe expression of METTL3 is dysregulated in cancer viadifferent mechanisms(Fig.2a).It was demonstrated inpancreatic cancer that cigarette smoke condensate induceshypomethylation of METTL3 promoter and subsequentlythe recruitment of transcription factor NFIC to induceMETTL3 overexpression 56.An intestinal microbial me-tabolite,butyrate,was suggested to downregulate the ex-pression of METTL3 and inhibit the development ofcolorectal cancer;however,the detailed mechanism is un-clear 57.Wang et al.showed in gastric cancer that P300mediateshistoneH3acetylationatlysine27(H3K27ac)and promotes METTL3 transcription 58.It was also reported that microRNA miR-24-2 mightpromote METTL3 transcription;however,the detailedmechanism remains elusive 59.Several other micro-RNAs,including miR-186,miR-4429,miR-600,andlet-7g 6063,were proposed to suppress METTL3by targeting METTL3 mRNA.In addition,SUMOyla-tion of METTL3 protein was reported to repress itsmethyltransferase activity without affecting the proteinstability or localization,although the mechanism re-mains unclear 64.The methyltransferase activity of METTL3 is guided indiverse manners(Fig.2be).It is suggested that m6Adeposition could be governed in cis via the sequencecode and structure at the modified site 65;however,the mechanism underlying was unknown.Recently,itwas shown that histone H3 trimethylation at lysine 36(H3K36me3)could guide m6A RNA modification co-transcriptionally through direct interaction with METTL14 and the subsequent recruitment of the MTC com-plex,allowing for the selective deposition of m6A inCDS and 3UTR where H3K36me3 is often found 66.Increasing efforts have also been paid to trans regula-tors;among which,zinc finger protein 217(ZFP217)was reported to sequester METTL3 and counteractsm6A deposition on stemness associated transcripts 67,while the TGF signaling factor SMAD2/3 could recruitMETTL3/14 complex to a subset of transcripts in-volved in early cell fate decisions 68.Another tran-scriptional factor,CAATT-box-binding protein CEBPZ,was demonstrated to directly recruit METTL3 to chro-matin68.Alarge-scalecomputationalscreeningaimed at identifying RNA-binding proteins as cell-specific trans regulators of m6A,and validated experi-mentally that TRA2A and CAPRIN1 could interactwith METTL3 69.Fish et al.found that the RNA-binding protein TARBP2 recruits METTL3 and de-posits m6A on the introns of the target mRNA,regulat-ing RNA splicing and stability 70.Two long non-coding RNAs(ARHGAP5-AS1 and LINC00470)havealso been revealed to guide METTL3 to specific targets71,72,among which,the natural anti-sense transcriptARHGAP5-AS1 mediates METTL3 to deposit m6Amarks on ARHGAP5 mRNA,promoting the mRNAstabilization and inducing chemoresistance 71.Table 1 m6A modification-related factorsCategories FactorsFunctionRef.WriterMETTL3/METTL14/WTAP/VIRMA/ZC3H13/RBM15The m6A methyltransferase complex for the majority of m6A deposition1014METTL16Responsible for m6A modification of U6 snRNA,lncRNAs,and introns of pre-mRNAs1517METTL5Responsible for m6A modification of 18S rRNA18,19ZCCHC4Responsible for m6A modification of 28S rRNA2022EraserFTODemethylates m6A,also has activity towards m6Amand m1A2325ALKBH5Mainly demethylates m6A9,26,27ReaderYTHDC1Alternative splicing and RNA export28,29YTHDC2mRNA degradation and translation initiation30,31YTHDF1Promotes translation32,33YTHDF2Promotes RNA decay34,35YTHDF3Promotes mRNAs translation and degradation36,37IGF2BP1/2/3Promotes mRNA stability and translation38hnRNPC/hnRNPGRegulates mRNA structure and alternative splicing39,40Zeng et al.Journal of Hematology&Oncology (2020)13:117 Page 3 of 15METTL3 functions as an m6A methyltransferase incancerAccumulating evidence in recent years has demonstratedthat METTL3 plays critical roles in cancer as an m6Amethyltransferase,either as an oncogene or a tumorsuppressor,as summarized in Table 2.METTL3 as an oncogeneIn most cases,METTL3 was reported as an oncogene topromote the initiation and development of a variety ofcancers,including hematopoietic malignancies and solidtumors,through depositing m6A modification on criticaltranscripts(Fig.3).Acute myeloid leukemiaAcute myeloid leukemia(AML)is one of the most com-mon hematopoietic malignancies with diverse geneticand molecular abnormalities,in which the hematopoieticstem and progenitor cells(HSPCs)retain the self-renewal capacity,while the myeloid differentiation cap-acity is hampered 121123.Vu et al.found that METTL3 was more abundant in AML cells than in normalHSPCs,and when overexpressed,the wild-type but notthe catalytically inactive form of METTL3 could inhibitthe differentiation of HSPCs.In AML cells,depletion ofMETTL3 induced cell differentiation and apoptosis anddelayed leukemia progression.The authors further dem-onstrated that METTL3 mediated m6A modification onFig.2 Regulation on the expression of METTL3 and its function on m6A deposition.a Multi-level regulation of METTL3.b Sequester or recruitment ofMETTL3 by RNA binding proteins(RBP).c Histone H3K36me3 directed deposition of m6A via recruiting of METTL14 and METTL3.d Recruitment ofMETTL3 by transcriptional factors(TF).e METTL3 guided by long non-coding RNAs(lncRNA).M3,METTL3;M14,METTL14Zeng et al.Journal of Hematology&Oncology (2020)13:117 Page 4 of 15Table 2 Roles of METTL3 as an m6A methyltransferase in human cancersRoleCancer typeRegulatorTargetsMolecular mechanismCellular functionRef.OncogeneAcute myeloid leukemiaMYC,BCL2,PTENPromote translationDifferentiation,apoptosis73CEBPZSP1,SP2Promote translationCell cycle regulation,differentiation74Breast cancerlet-7gHBXIPPromote translation(?)Cell proliferation63BCL2Promote translationProliferation,apoptosis75Liver cancerSOCS2RNA decay by YTHDF2Proliferation,migration76RDM1Proliferation77LINC00958RNA stabilizationLipogenesis,proliferation,migration,invasion78SUMO1SnailRNA stabilizationMetastasis79Snailpromote translation by YTHDF1EMT80miR24-2miR-6097,Pim1Tumor growth59CTNNB1RNA stabilizationTumor growth81miR-186Wnt/-cateninProliferation,migration,invasion60GlioblastomaSOX2RNA stabilization by ELAVL1Dedifferentiation82SRSFsNonsense-mediated mRNA decay(NMD)by YTHDC1Tumor growth andprogression83Bladder cancermiR-221/222miRNA maturationProliferation84AFF4,RELA,IKBKB,MYCProliferation,apoptosis85ITGA6Promote translation by YTHDF1/3Adhesion,migration,invasion86chemicalcarcinogenesisCPCPPromote translationMalignant transformation87SETD7,KLF4RNA decay by YTHDF2Proliferation,metastasis88Gastric cancermiR-4429SEC62RNA stabilization by IGF2BP1Proliferation,apoptosis62AKT signalingpathwayProliferation,migration,invasion89CBP/P300-mediatedH3K27acHDGFRNA stabilization by IGF2BP3Tumor angiogenesis andglycolysis58ZMYM1RNA stabilization by ELAVL1EMT90LncRNA ARHGAP5-AS1ARHGAP5 mRNARNA stabilizationChemoresistance71MYC targetgenesProliferation,migration,invasion91LINC00470PTENRNA decay by YTHDF2Proliferation,migration,invasion72Prostate cancerGLI1Proliferation,migration,apoptosis,92MYCProliferation,migration,invasion.93ITGB1RNA stabilizationCell adhesion94Lung cancermiR-600-catenin,TAZ,EGFR,DNMT3AProliferation,metastasis,apoptosis61miR-25-3pmiRNA maturationBrain metastasis95TGF-JUNBRNA stabilizationEMT96YAP,MALAT1promote translation by YTHDF1/3;RNA stabilization by YTHDF3Drug resistance andmetastasis97Zeng et al.Journal of Hematology&Oncology (2020)13:117 Page 5 of 15MYC,BCL2,and PTEN mRNAs and promoted theirtranslation 73.By performing two independent CRISPR screens,Bar-bieri et al.showed that METTL3 was necessary forAML cell survival and further revealed that the CAATT-box-binding protein CEBPZ was required for recruit-ment of METTL3 to chromatin.Promoter boundMETTL3 introduces m6A modification within the cod-ing region of SP1 and SP2 transcripts and enhancestheir translation,thus promoting cell proliferation andAML maintenance 74.Liver cancerLiver cancer is the fourth most common cause ofcancer-related death worldwide,among which,hepato-cellular carcinoma(HCC)accounts for the majorit