3、Nat. Cell Biol. 2018-m6a基因甲基化调控Akt活性促进子宫内膜癌的增殖和致瘤性(1).pdf

Articleshttps:/doi.org/10.1038/s41556-018-0174-41Department of Chemistry and Institute for Biophysical Dynamics,The University of Chicago,Chicago,IL,USA.2Howard Hughes Medical Institute,Chicago,IL,USA.3Department of Obstetrics and Gynecology/Section of Gynecologic Oncology,The University of Chicago,Chicago,IL,USA.4Center for Gene Diagnosis,Zhongnan Hospital of Wuhan University,Wuhan,China.5College of Chemistry,Sichuan University,Chengdu,China.6Committee on Cancer Biology and Medical Scientist Training Program,The University of Chicago,Chicago,IL,USA.7Department of Pathology,Zhongnan Hospital of Wuhan University,Wuhan,China.8Hubei Key Laboratory of Tumor Biological Behaviors&Hubei Cancer Clinical Study Center,Zhongnan Hospital of Wuhan University,Wuhan,China.9Department of Obstetrics and Gynecology,Zhongnan Hospital of Wuhan University,Wuhan,China.10MOE Key Laboratory of Macromolecular Synthesis and Functionalization,Department of Polymer Science and Engineering,Zhejiang University,Hangzhou,China.11Department of Biochemistry and Molecular Biology,The University of Chicago,Chicago,IL,USA.12These authors contributed equally:Jun Liu,Mark A.Eckert,Bryan T.Harada,Song-Mei Liu.*e-mail:elengyeluchicago.edu;chuanheuchicago.eduN6-methyladenosine(m6A)is the most prevalent messenger RNA modification in humans1,2.This modification is revers-ible3,and its biological effects are mostly mediated through writer,eraser and reader proteins1,2.A writer complex,consisting of a core METTL3METTL14 m6A methyltransferase along with regulatory subunits48,catalyses the m6A methylation of mRNA.At least two eraser enzymes,FTO and ALKBH5,mediate the reversal of this methylation3,9.m6A methylated transcripts are recognized by reader proteins that regulate pre-mRNA processing1014,trans-lation1519 and degradation10,19,20.m6A-dependent mRNA regulation is essential in mammals21,and defects in m6A methylation affect diverse biological processes1,2.In particular,m6A mRNA meth-ylation regulates the self-renewal and differentiation of stem cells by affecting mRNA turnover during cell differentiation and plays critical roles in transcriptome switching during embryonic develop-ment8,2123.Consistent with these roles,m6A mRNA methylation is emerging as a pathway affecting cancer initiation and progression in a variety of cancers2435.m6A mRNA methylation affects the growth and proliferation of stem cells and cancer cells8,21,22,2635.However,how m6A methylation affects cell growth and which underlying pathways and mechanisms mediate these changes are still not fully elucidated.Herein,we study this question in endometrial cancer,where sequencing studies have identified frequent mutation of the m6A methyltransferase subunit METTL14(ref.36).We found that about 70%of endometrial tumours exhibit reduced m6A methylation when compared with matched,normal endometrium.These reductions in m6A meth-ylation were probably caused by either mutation of METTL14 or reduced expression of the METTL3 methyltransferase.Reducing m6A mRNA levels in endometrial cancer cells through either METTL14 mutation or METTL3 downregulation could enhance cell proliferation and tumorigenicity in vitro and in vivo.m6A-seq characterization of endometrial cancer patient tumours and cell lines revealed that reduced m6A mRNA methylation could pro-mote cell proliferation by altering the expression of key enzymes that affect the AKT signalling pathway.Inhibition of AKT activation reversed the increased proliferation caused by reduced m6A meth-ylation.Together,these results characterize a somatic mutation of the m6A methylation machinery as an important factor promoting cancer progression,reveal that reduced m6A mRNA methylation is most likely an oncogenic mechanism underlying a large portion of endometrial cancers and identify m6A methylation as a regulator of the AKT pathway and cell growth.ResultsLoss-of-function METTL14 mutations in endometrial can-cer.Sequencing studies have found that the METTL14 subunit of the core m6A methyltransferase complex is frequently mutated m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancerJun Liu1,2,12,Mark A.Eckert 3,12,Bryan T.Harada 1,2,12,Song-Mei Liu4,12,Zhike Lu1,2,Kangkang Yu1,2,5,Samantha M.Tienda3,Agnieszka Chryplewicz3,Allen C.Zhu1,2,6,Ying Yang4,Jing-Tao Huang4,Shao-Min Chen4,Zhi-Gao Xu7,Xiao-Hua Leng8,Xue-Chen Yu9,Jie Cao10,Zezhou Zhang10,Jianzhao Liu 10,Ernst Lengyel 3*and Chuan He 1,2,11*N6-methyladenosine(m6A)messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and pro-liferation in development and cancer.To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity,we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltrans-ferase complex(METTL14).We found that about 70%of endometrial tumours exhibit reductions in m6A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3,another component of the methyltransferase complex.These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells,likely through activation of the AKT pathway.Reductions in m6A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2.Together,these results reveal reduced m6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signalling.NATuRE CELL BioLoGY|VOL 20|SEPTEMBER 2018|10741083| CELL BiOLOgyin endometrial tumours36,but the relevance of these mutations and of m6A mRNA methylation to the disease has not yet been established.The predominant mutation occurs at position 298 of METTL14,is more prevalent than other mutations in endometrial tumours and occurs in about 1.5%of endometrial cancer patients36.Crystal structures of the METTL3METTL14 complex reveal that the Arg 298 residue lies in the putative RNA-binding groove at the interface between the two subunits3739.Consistent with previous observations38,we found that the R298P hotspot mutation signifi-cantly reduced the RNA methylation activity of the writer complex in vitro(Fig.1a).Whereas overexpression of wild-type METTL14 promoted m6A methylation of cellular poly(A)RNAs in HEC-1-A endometrial cancer cells,the mutant METTL14 appeared inac-tive upon overexpression(Fig.1b).While overexpression of wild-type METTL14 decreased cell proliferation,overexpression of the mutant had no noticeable effect on cell proliferation(Fig.1c),sug-gesting that the METTL14 mutation is probably a loss-of-function allele that shows no evidence of further dominant negative effects on m6A methylation or cell proliferation.To examine the consequence of the mutation in tumour tis-sue,we identified three endometrial tumour samples bearing the METTL14(R298P)mutation and purified mRNA from these tumours as well as from adjacent benign endometrial tissues(Methods).Compared with mRNA from the wild-type adjacent Percentage m6A/Ain mRNA0.00.10.2d3-m6A/GabP=1 108Controlwt METTL14mu METTL140.00.20.40.6Percentage m6A/Ain mRNAPercentage m6A/Ain mRNAP=0.0057 P=0.0105wt METTL14mu METTL140204060801.01.52.0Controlwt METTL14mu METTL14ProliferationTime(h)cTumourTumour adjacentd0.00.20.4Patient 2Patient 3Patient 1eP=6 1011TumourTumour adjacent0.00.20.4g0.00.10.20.30.40.50.00.51.01.52.0Relative METTL3mRNA levelPercentage m6A/Ar=0.62P=3 105hNormal endometriumEndometrial cancer(G2)0.00.51.0P=0.0005Proportion of samples0123NormalTumour50 mfP=0.0044P=0.22P=1 104P=5 105P=0.0045P=0.33METTL3METTL14FTOALKBH5YTHDF1YTHDF20.00.51.01.52.02.53.03.5Expression in tumour/tumour adjacentFig.1|The METTL14(R298P)mutation and reduced METTL3 expression contribute to decreased m6A mRNA methylation in endometrial cancer patients a,The methyltransferase activity of the METTL3METTL14 complex containing either the METTL14(R298P)mutant(mu)or wild-type(wt)METTL14 was determined by measuring the d3-m6A/G ratio by liquid chromatographytandem mass spectrometry(LC-MS/MS)after incubation of the methyltransferase complex with RNA probe.We independently purified two batches of protein and carried out two independent trials per protein preparation for a total of n=4 independent trials.b,LC-MS/MS quantification of the m6A/A ratio in poly(A)RNA isolated from HEC-1-A cells overexpressing wild-type METTL14,mutant METTL14 or empty vector control.n=3 biological replicates.c,Cell proliferation of HEC-1-A cells was measured by MTS assay after transfection with the indicated reagents.n=3 biological replicates.For ac,error bars indicate mean s.e.m.d,LC-MS/MS quantification of the m6A/A ratio in poly(A)RNA isolated from three endometrial tumours with a METTL14(R298P)mutation and adjacent normal endometrium.The bar shows the mean from n=3 technical replicates per patient.e,Box plot of the relative m6A levels in poly(A)RNA isolated from endometrial tumour tissues versus tumour-adjacent tissues,n=38 tumournormal pairs.f,Box plot of the expression levels of METTL3,METTL14,FTO,ALKBH5,YTHDF1 and YTHDF2 in tumour tissues relative to tumour-adjacent tissues,n=22 tumournormal pairs for METTL14 and FTO,and n=38 tumournormal pairs for the others.For ac,e and f,the P-values were determined by two-tailed t-test.See methods for box-plot characteristics.g,Scatter plot showing the correlation of m6A methylation level with the expression of METTL3.The linear best fit line is shown in red.The Pearson correlation coefficient(r)and P-value(P)from a two-tailed t-test of r=0 are shown,n=38 tumournormal pairs.h,Left:IHC staining of endometrial tissue microarray cores for METTL3.Right:Quantification of IHC staining in normal endometrium(n=10 cores)and epithelial endometrial tumours(n=30 cores).Staining was assessed using automated software51 and scored on a scale of 0(no staining)to 3(high staining).Scale bars,50 m.P-value determined by 2-test.NATuRE CELL BioLoGY|VOL 20|SEPTEMBER 2018|10741083| CELL BiOLOgyb020406080048WTMETTL14+/wt METTL14mu METTL14Cell proliferationTime(h)i0200400No.of coloniesshControlshMETTL3.15shMETTL3.18mu METTL14mu METTL14wt METTL14WTMETTL14+/METTL14+/wt METTL14WTP=0.0003P=0.00030.00.20.4Relative migrationdistancejshControlshMETTL3.15shMETTL3.18P=0.0080P=0.0208g040801234shControlshMETTL3.15shMETTL3.18Cell proliferationTime(h)hshControlshMETTL3.15shMETTL3.180.01.01.5No.of cells(105)0.5P=0.0001P=7 1050.00.20.4Percentagem6A/A in mRNAfshControlshMETTL3.15shMETTL3.18mu METTL14wt METTL14WTMETTL14+/P=0.0298P=0.0083k0.00.51.0Total tumourweight(g)1.5P=0.0004WTMETTL14+/04080No.of peritonealtumoursP=5 105WTMETTL14+/lP=2 105shControlshMETTL30.000.050.10Total tumourweight(g)0.15P=7 107shControlshMETTL302040No.of peritonealtumours60wt METTL14mu METTL140.00.20.4Total tumour weight(g)P=0.0356wt METTL14mu METTL1404080No.of peritonealtumoursP=4 105ma0.00.20.4Percentagem6A/A in mRNAwt METTL14mu METTL14WTMETTL14+/P=0.0054P=0.016000.20.4Relative migrationdistanceP=0.0003P=0.0005ec0.01.02.0No.of cells(105)P=0.0025P=9 105d0100200Colonies300P=0.0002P=0.0007Fig.2|Reduced m6A methylation increases cell proliferation,anchorage-independent growth,migration and in vivo tumour growth.a,LC-MS/MS quantification of the m6A/A ratio in poly(A)RNA from the indicated HEC-1-A cell lines.b,Cell proliferation measured by MTS assay of wild-type HEC-1-A cells(WT),METTL14+/knockout cells,and knockout cells rescued by stable transfection of wild-type METTL14(wt)or METTL14(R298P)(mu).Cell numbers were normalized to the MTS signal about 5h after cell seeding.ce,Anchorage-independent cell growth(c),colony formation(d)and cell migration in a wound-healing experiment(e)were assessed for wild-type HEC-1-A cells,METTL14+/knockout cells,and knockout cells rescued with wild-type or mutant METL14.f,LC-MS/MS quantification of the m6A/A ratio in poly(A)RNA from the indicated HEC-1-A cell lines.(g)Cell proliferation measured by MTS assay of HEC-1-A cells stably expressing control shRNA(shControl)versus shRNA targeting METTL3(shMETTL3.15,shMETTL3.18).Cell numbers were normalized to the MTS signal about 5h after cell seeding.hj,Anchorage-independent cell growth(h),colony formation(i)and cell migration in a wound-healing assay(j)were assessed for HEC-1-A cells stably expressing control shRNA or shRNA targeting METTL3.For aj,n=3 biological replicates.Error bars indicate mean s.e.m.P-values determined by two-tailed t-test.km,Wild-type HEC-1-A cells and METTL14+/knockout cells(k),knockout cells rescued with wild-type or mutant METTL14(l)and HEC-1-A cells with shRNA knockdown of METTL3 or control shRNA(m)were injected into mice.The total tumour weight(left)and the total number of tumours(right)were recorded after 23 weeks.For k,n=8 and for l and m n=10 mice per group.Error bars indicate mean s.e.m.P-values determined by two-tailed t-test.NATuRE CELL BioLoGY|VOL 20|SEPTEMBER 2018|10741083| CELL BiOLOgynormal tissues,mRNA from the three mutant tumours had reduced overall m6A methylation(P=0.04,paired two-tailed t-test),sug-gesting that the METTL14(R298P)mutation inhibits m6A mRNA methylation in tumours(Fig.1d).Endometrial cancer is associated with low levels of m6A mRNA methylation.Intriguingly,about 70%of all tumours we examined(including a majority of tumours with wild-type METTL14)exhib-ited reduced total m6A mRNA methylation when compared with adjacent,normal endometrial tissues(Fig.1e).Thus,we hypoth-esized that endometrial cancer could be more broadly associated with the altered expression of factors that regulate m6A mRNA methylation.To test this hypothesis,we evaluated the expression of m6A writers,erasers and readers in tumour and adjacent normal endometrial tissues by quantitative PCR with reverse transcription(RT-qPCR)(Fig.1f and Supplementary Fig.1a).We found that a majority of endometrial cancers exhibited significantly reduced expression of the METTL3 m6A methyltransferase when compared with adjacent normal tissues.Decreased METTL3 expression cor-relates with reduced m6A methylation in these tumour tissues(Fig.1g).Immunohistochemistry of a tissue microarray with both nor-mal endometrium and epithelial endometrial cancer specimens revealed a significant decrease in METTL3 expression in tumour tis-sue at the protein level(Fig.1h).METTL14 mutation and decreased METTL3 expression appear to be mutually exclusive,as all three of the tumours with the METTL14 mutation had normal expression of METTL3 relative to adjacent normal tissues.Analysis of the TCGA endometrial cancer dataset did not reveal any significant correla-tion between the mutation status of frequently mutated genes in endometrial cancer and low METTL3 expression(Supplementary Fig.1b).Taken together,these results suggest that a large proportion of human endometrial tumours are characterized by reduced m6A mRNA methylation,through either METTL14 loss-of-function mutation or decreased METTL3 expression.Reduced m6A met