ATXN2-mediated translation of TNFR1 promotes esophageal squamous cell carcinoma via m6A-dependent manner
Authors: Rui Li, Lingxing Zeng, Hongzhe Zhao, Junge Deng, Ling Pan, Shaoping Zhang, Guandi Wu, Ying Ye, Jialiang Zhang, Jiachun Su, Yanfen Zheng, Shuang Deng, Ruihong Bai, Lisha Zhuang, Mei Li, Zhixiang Zuo, Dongxin Lin, Jian Zheng, Xudong Huang
Journal: Molecular Therapy (2022)

N6-methyladenosine (m6A) is the most prevalent RNA modification, and the effect of its dysregulation on esophageal squamous cell carcinoma (ESCC) development remains unclear. Here, by performing transcriptome-wide m6A sequencing in 16 ESCC tissue samples, we identified the key roles of m6A in TNFRSF1A (also known as TNFR1)-mediated MAPK and NF-κB activation in ESCC. Mechanistically, a functional protein involved in m6A methylation, ATXN2, is identified that augments the translation of TNFRSF1A by binding to m6A-modified TNFRSF1A mRNA. Upregulation of the TNFRSF1A protein level, a vital upstream switch for TNFRSF1A-mediated signaling events, activates the NF-κB and MAPK pathways and thus promotes ESCC development. Furthermore, TNFRSF1A m6A modifications and protein levels are upregulated in ESCC, and high levels of TNFRSF1A m6A and protein are correlated with poor ESCC patient survival. These results collectively indicate that the m6A-TNFRSF1A axis is critical for ESCC development and thus may serve as a potential druggable target. N6-methyladenosine (m6A) is the most prevalent RNA modification, and the effect of its dysregulation on esophageal squamous cell carcinoma (ESCC) development remains unclear. Here, by performing transcriptome-wide m6A sequencing in 16 ESCC tissue samples, we identified the key roles of m6A in TNFRSF1A (also known as TNFR1)-mediated MAPK and NF-κB activation in ESCC. Mechanistically, a functional protein involved in m6A methylation, ATXN2, is identified that augments the translation of TNFRSF1A by binding to m6A-modified TNFRSF1A mRNA. Upregulation of the TNFRSF1A protein level, a vital upstream switch for TNFRSF1A-mediated signaling events, activates the NF-κB and MAPK pathways and thus promotes ESCC development. Furthermore, TNFRSF1A m6A modifications and protein levels are upregulated in ESCC, and high levels of TNFRSF1A m6A and protein are correlated with poor ESCC patient survival. These results collectively indicate that the m6A-TNFRSF1A axis is critical for ESCC development and thus may serve as a potential druggable target. IntroductionEsophageal cancer is the sixth leading cause of cancer-related death worldwide,1Chen W. Zheng R. Baade P.D. Zhang S. Zeng H. Bray F. Jemal A. Yu X.Q. He J. Cancer statistics in China, 2015.CA Cancer J. Clin. 2016; 66: 115-132Google Scholar and the incidence and histological type of esophageal cancer varies with geographic location. Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype, with poor prognosis and an extremely high prevalence in China.2Lin Y. Totsuka Y. Shan B. Wang C. Wei W. Qiao Y. Kikuchi S. Inoue M. Tanaka H. He Y. Esophageal cancer in high-risk areas of China: research progress and challenges.Ann. Epidemiol. 2017; 27: 215-221Google Scholar Comprehensive analysis of exome sequencing in tumor-normal paired ESCC tissues identified 22 significantly mutated driver genes (SMGs) (including TP53, NOTCH1, NFE2L2, KMT2D, CDKN2A, ZNF750, PIK3CA, RB1, FAT1, EP300, FBXW7, TGFBR2, AJUBA, CREBBP, FAT2, NOTCH3, PTCH1, KDM6A, FAM135B, TET2, PTEN, and ADAM29), along with other somatic genomic alterations contribute to the development of ESCC,3Lin D.C. Wang M.R. Koeffler H.P. Genomic and epigenomic aberrations in esophageal squamous cell carcinoma and implications for patients.Gastroenterology. 2018; 154: 374-389Google Scholar but even so, there is still a lack of effective biomarkers for early detection, and the 5-year survival rate of ESCC patients is < 20%.4Adams J.M. Cory S. Modified nucleosides and bizarre 5'-termini in mouse myeloma mRNA.Nature. 1975; 255: 28-33Google Scholar Discovering the molecular mechanisms underlying ESCC development is necessary for developing effective biomarkers and targets for early diagnosis and clinical treatment.N6-methyladenosine (m6A) is the most abundant internal reversible chemical modification in eukaryotic mRNA, and "writer" and "eraser" proteins play key roles in the deposition and removal of m6A methylation.5Zaccara S. Ries R.J. Jaffrey S.R. Reading, writing and erasing mRNA methylation.Nat. Rev. Mol. Cell Biol. 2019; 20: 608-624Google Scholar RNA m6A modification can be recognized by a "reader" that influences multiple aspects of RNA fate, such as pre-mRNA processing,6Yue Y. Liu J. Cui X. Cao J. Luo G. Zhang Z. Cheng T. Gao M. Shu X. Ma H. et al.VIRMA mediates preferential m6A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation.Cell Discov. 2018; 4: 10Google Scholar, 7Xiao W. Adhikari S. Dahal U. Chen Y.S. Hao Y.J. Sun B.F. Sun H.Y. Li A. Ping X.L. Lai W.Y. et al.Nuclear m6A reader YTHDC1 regulates mRNA splicing.Mol. Cell. 2016; 61: 507-519Google Scholar, 8Liu N. Dai Q. Zheng G. He C. Parisien M. Pan T. N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.Nature. 2015; 518: 560-564Google Scholar, 9Alarcón C.R. Goodarzi H. Lee H. Liu X. Tavazoie S. Tavazoie S.F. HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events.Cell. 2015; 162: 1299-1308Google Scholar translation,10Shi H. Wang X. Lu Z. Zhao B.S. Ma H. Hsu P.J. Liu C. He C. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA.Cell Res. 2017; 27: 315-328Google Scholar and stability.11Wang X. Lu Z. Gomez A. Hon G.C. Yue Y. Han D. Fu Y. Parisien M. Dai Q. Jia G. et al.N6-methyladenosine-dependent regulation of messenger RNA stability.Nature. 2014; 505: 117-120Google Scholar Emerging evidence has uncovered that m6A modification exerts either oncogenic or tumor-suppressive effects in different conditions.12Ma J.Z. Yang F. Zhou C.C. Liu F. Yuan J.H. Wang F. Wang T.T. Xu Q.G. Zhou W.P. Sun S.H. METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6-methyladenosine-dependent primary microRNA processing.Hepatology. 2017; 65: 529-543Google Scholar,13Weng H. Huang H. Wu H. Qin X. Zhao B.S. Dong L. Shi H. Skibbe J. Shen C. Hu C. et al.METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m6A modification.Cell Stem Cell. 2018; 22: 191-205Google Scholar Nevertheless, whether and how aberrant m6A abundance can translate to a pro-tumorigenic signal in ESCC is still not understood.Tumor necrosis factor (TNF), an inflammatory cytokine, plays an important role in the pathogenesis of many chronic inflammatory diseases and cancer by binding two cell-membrane receptors (TNFR1 and TNFR2).14Liu Z.G. Molecular mechanism of TNF signaling and beyond.Cell Res. 2005; 15: 24-27Google Scholar It is well known that TNFR1 (also known as TNFRSF1A) is ubiquitously expressed and is the primary receptor mediating a majority of the biological effects of TNF.15Smith C.A. Farrah T. Goodwin R.G. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death.Cell. 1994; 76: 959-962Google Scholar A previous multiple sclerosis genome-wide association study (GWAS) reported that rs1800693 polymorphism, located in the intron of TNFR1, is the most significant signal for multiple sclerosis susceptibility. The risk G allele resulted in the production of a novel, soluble form of TNFR1 that can block TNF. This result indicates that the multiple sclerosis-associated TNFR1 variant simulates the effect of TNF-blocking drugs.16Gregory A.P. Dendrou C.A. Attfield K.E. Haghikia A. Xifara D.K. Butter F. Poschmann G. Kaur G. Lambert L. Leach O.A. et al.TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis.Nature. 2012; 488: 508-511Google Scholar TNFR1-mediated signaling has recently been shown to enhance tumor formation during liver,17Knight B. Yeoh G.C. Husk K.L. Ly T. Abraham L.J. Yu C. Rhim J.A. Fausto N. Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice.J. Exp. Med. 2000; 192: 1809-1818Google Scholar skin,18Arnott C.H. Scott K.A. Moore R.J. Robinson S.C. Thompson R.G. Balkwill F.R. Expression of both TNF-α receptor subtypes is essential for optimal skin tumour development.Oncogene. 2004; 23: 1902-1910Google Scholar and gastric19Oshima H. Ishikawa T. Yoshida G.J. Naoi K. Maeda Y. Naka K. Ju X. Yamada Y. Minamoto T. Mukaida N. et al.TNF-α/TNFR1 signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor cells.Oncogene. 2014; 33: 3820-3829Google Scholar carcinogenesis and promote the metastasis of cancer cells.20Popivanova B.K. Kitamura K. Wu Y. Kondo T. Kagaya T. Kaneko S. Oshima M. Fujii C. Mukaida N. Blocking TNF-α in mice reduces colorectal carcinogenesis associated with chronic colitis.J. Clin. Invest. 2008; 118: 560-570Google Scholar Despite that the mechanism of TNFR1-mediated signaling events during tumor promotion has been partially delineated, little is known about ESCC development, especially TNFR1-involved epigenetic regulation.Here, we revealed an aberrant increase in m6A modification of mRNAs involved in TNFR1-mediated signaling pathways in ESCC tissues. TNFR1 RNA, an upstream switch for TNFR1-mediated signaling events, is aberrantly m6A modified due to the elevated expression of METTL3. An excessive m6A level of TNFR1 promotes protein translation through ATXN2-dependent regulation and evokes mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) activation, which may be an important molecular mechanism for ESCC development and progression.ResultsTNFR1-involved signaling pathways exhibited elevated m6A modification levels in ESCCTo describe the global landscape of m6A modification in ESCC, we performed m6A sequencing (m6A-seq) of total RNAs of 16 tumor tissues and paired adjacent normal tissues from 8 ESCC patients. We detected 6,877, 4,524, and 4,245 m6A peaks, representing 6,643, 4,386, and 4,113 transcripts in all of the samples, ESCC tumors, and adjacent normal tissues, respectively (Figures 1A , S1A, and S1B). These m6A modifications occurred mainly in mRNA and were predominantly enriched in coding sequence (CDS) and stop codons, consistent with previous studies21Dominissini D. Moshitch-Moshkovitz S. Schwartz S. Salmon-Divon M. Ungar L. Osenberg S. Cesarkas K. Jacob-Hirsch J. Amariglio N. Kupiec M. et al.Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.Nature. 2012; 485: 201-206Google Scholar (Figures 1B, S1A, and S1B). In addition, the canonical motif of GGm6ACU was also enriched in these detected peaks (Figures S1A and S1B). Among the 6,877 m6A peaks, 6,546 (95.19%) were recorded in the RMBase: http://mirlab.sysu.edu.cn/rmbase/,22Xuan J.J. Sun W.J. Lin P.H. Zhou K.R. Liu S. Zheng L.L. Qu L.H. Yang J.H. RMBase v2.0: deciphering the map of RNA modifications from epitranscriptome sequencing data.Nucleic Acids Res. 2018; 46: 327-334Google Scholar indicating that the m6A-seq data are reliable (Figure S1C). Interestingly, we found that m6A mRNA methylation was increased globally in ESCC tissues compared with adjacent normal tissues (Figure 1C). Among the dysregulated m6A peaks, the number of peaks exhibiting increased m6A levels in at least 4 paired tumor-normal samples was 1,392 (92.49%), but only 113 peaks (7.51%) showed decreased m6A levels in at least 4 paired tumor-normal samples (Figure 1D).The enrichment theory was adapted to transcripts exhibiting increased m6A levels in at least 4 paired tumor-normal samples, and the results indicated that the transcripts participating in the TNFR1 pathway and its mediated NF-κB and MAPK signaling were perturbed by increased m6A modification (Figure 1E). The binding of TNF to TNFR1 triggers a series of intracellular events that ultimately result in the activation of NF-κB and MAPK,23Hehlgans T. Pfeffer K. The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: players, rules and the games.Immunology. 2005; 115: 1-20Google Scholar,24Baud V. Karin M. Signal transduction by tumor necrosis factor and its relatives.Trends Cell Biol. 2001; 11: 372-377Google Scholar which promotes tumor formation in some types of cancers. Therefore, we hypothesized that increased m6A methylation may promote ESCC development mainly through initiating TNFR1-mediated MAPK and NF-κB activation. Interestingly, most transcripts involved in TNFR1-mediated signaling events showed elevated m6A modification in tumor tissues versus adjacent normal tissues (Figures 1F−1H and S1D; Table S1). These results suggested that an aberrant increase in global m6A modification in ESCC may play an oncogenic role via canonical oncogenic pathways, especially the TNFR1-mediated MAPK and NF-κB pathways.Elevated m6A modification of TNFR1 RNA augments its protein level in ESCCAs mentioned above, the transmembrane receptor TNFR1 is of critical importance and acts as a trigger for TNFR1-mediated oncogenic pathways. Transcripts encoding TNFR1 showed increased m6A modification levels in ESCC tumors based on m6A-seq (Figure 2A ). Then, we detected the m6A levels of the TNFR1 transcript in 215 pairs of ESCCs and adjacent normal esophageal samples (SYSUCC cohort; Table S2) by using methylated RNA immunoprecipitation sequencing (MeRIP)-coupled quantitative real-time-PCR (qRT-PCR) and found that its m6A modification was elevated in ESCC tumors (Figure 2B). Next, we attempted to determine which component in m6A methyltransferase complex participated in m6A modification on TNFR1 transcripts. We found that the mRNA levels of METTL14, WTAP, and VIRMA were not associated with TNFR1 m6A levels in ESCC tumor tissues (N = 215, SYSUCC cohort; Figures S2A−S2C). However, the m6A levels of the TNFR1 transcript were positively correlated with METTL3 mRNA levels in ESCC tumors (N = 215, SYSUCC cohort; Figure 2C), indicating that METTL3 is the main methyltransferase that catalyzes the m6A modification of the TNFR1 transcript. Meanwhile, we analyzed the expression of METTL3 from three ESCC datasets. METTL3 mRNA expression was significantly upregulated in ESCC tissues compared with adjacent normal tissues in an independent cohort (GSE53625)25Li J. Chen Z. Tian L. Zhou C. He M.Y. Gao Y. Wang S. Zhou F. Shi S. Feng X. et al.LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma.Gut. 2014; 63: 1700-1710Google Scholar (p < 0.0001, two-sided paired Wilcoxon signed-rank test; Figure S2D). These results were confirmed in our cohort consisting of 215 pairs of ESCC and adjacent normal esophageal samples (SYSUCC cohort; Table S2) by using qRT-PCR (p = 0.0010, two-sided paired Wilcoxon signed-rank test; Figure S2E). In addition, immunohistochemistry (IHC) analysis with another 58 paired normal esophageal and ESCC samples showed similar results (SYSUCC cohort; Figures S2F and S2G; Table S3). Kaplan-Meier estimation showed that ESCC patients with high METTL3 mRNA levels (greater than or equal to median) had shorter survival times than ESCC patients with low METTL3 mRNA levels (less than median) (Figure S2H). We then explored the role of METTL3 by disrupting its expression in ESCC cell lines. The overexpression of METTL3 significantly increased the m6A levels of the TNFR1 transcript in two ESCC cell lines, while silencing METTL3 had the opposite effect (Figure 2D). Furthermore, in ESCC cells with METTL3 silencing, the overexpression of wild-type METTL3 (M3-WT), but not the enzyme-inactivated mutant METTL3 (M3-MUT, amino acids [aa]395-398, DPPW-APPA26Lin S. Choe J. Du P. Triboulet R. Gregory R.I. The m6A methyltransferase METTL3 promotes translation in human cancer cells.Mol. Cell. 2016; 62: 335-345Google Scholar), restored the m6A levels in the TNFR1 transcript (Figure 2E), suggesting that m6A modification of the TNFR1 transcript is dependent on the methyltransferase activity of METTL3. We next attempted to determine the function of m6A modification on the TNFR1 transcript. We found that the m6A levels of TNFR1 were not associated with its mRNA abundance (N = 215, SYSUCC cohort; Figure 2F). Moreover, we found that forced changes in METTL3 levels in KYSE30 and EC109 cells did not significantly alter TNFR1 mRNA levels (Figure 2G). However, we observed that upregulation of METTL3 expression significantly increased TNFR1 protein levels and decreased TNFR1 protein levels when METTL3 was silenced (Figure 2H). These results suggested that elevated m6A modification of the TNFR1 transcript may affect its protein translation in ESCC.Figure 2Elevated m6A modification of TNFR1 RNA augments its protein level in ESCCShow full caption(A) The average read density from m6A-seq analysis of 8 tumor-normal pairs showing the m6A peak identified in the TNFR1 transcript. (B) TNFR1 m6A levels were significantly higher in ESCC tumors than in paired normal tissues (N = 215). Wilcoxon rank-sum tests (two-sided) were used. (C) Spearman's correlation analysis between TNFR1 m6A levels and METTL3 mRNA levels in ESCC (N = 215). (D) Effects of METTL3 overexpression or knockdown on TNFR1 m6A levels in KYSE30 and EC109 cell lines. (E) Effects of wild-type (M3-WT) or catalytic mutant METTL3 (M3-MUT, aa395-398, DPPW→APPA) overexpression on TNFR1 m6A levels in cells with METTL3 knockdown (shM3). (F) Spearman's correlation analysis between m6A levels and mRNA levels of TNFR1 in ESCC (N = 215). (G) Effects of METTL3 overexpression or knockdown on TNFR1 mRNA levels in KYSE30 and EC109 cell lines. (H) Effects of METTL3 overexpression or knockdown on TNFR1 protein levels in KYSE30 and EC109 cell lines. (I) The average reads density in the m6A peak region of the TNFR1 transcript based on m6A-seq. The sequences around the exact m6A site are highlighted, and the purple arrow indicates the exact m6A site according to 2 public miCLIP-seq datasets (GEO: GSM4084010 and GSM1828594). (J) The TNFR1 m6A levels were measured by the SELECT method in METTL3 overexpression or knockdown cell lines. (K) The schematic represents the domain organization of the dCas13b-ALKBH5 expression cassette (upper panel) and the positions of the m6A site within TNFR1 RNA and regions targeted by 2 gRNAs (lower panel). (L) m6A-TNFR1 RNA levels in ESCC cells treated with doxycycline (DOX)-inducible dCas13b-ALKBH5 plasmid and non-targeting (NT)-gRNA (control) or gRNAs with DOX pretreatment. (M and N) The TNFR1 m6A and protein levels were measured by using the dm6ACRISPR system. Data are the means ± SEMs from at least 3 independent experiments in (D), (E), (G), (J), (L), and (M). Two-sided Student's t tests were used in , (D), (E), (G), (J), (L), and (M) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and p > 0.05, not significant). β-Actin served as loading control in (H) and (N).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next ascertained an identical m6A modification location at the TNFR1 peak region by using the m6A individual-nucleotide resolution cross-linking and immunoprecipitation sequencing datasets (miCLIP-seq) derived from Gene Expression Omnibus (GEO) database: http://www.ncbi.nlm.nih.gov/geo/ (Figure 2I). Furthermore, the m6A site in the 3′ UTR of TNFR1 was verified by a single-base elongation- and ligation-based quantitative PCR amplification method (called SELECT)27Xiao Y. Wang Y. Tang Q. Wei L. Zhang X. Jia G. An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N6-methyladenosine modification.Angew. Chem. Int. Ed. Engl. 2018; 57: 15995-16000Google Scholar in KYSE30 and EC109 cells (Figure 2J). The recently discovered CRISPR-Cas13b-based tool could demethylate targeted m6A-modified mRNA,28Li J. Chen Z. Chen F. Xie G. Ling Y. Peng Y. Lin Y. Luo N. Chiang C.M. Wang H. Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.Nucleic Acids Res. 2020; 48: 5684-5694Google Scholar and we applied the dm6ACRISPR system to TNFR1. The mRNA of TNFR1 was targeted by two guide RNAs (gRNAs) at distinct positions, and the expression of the two gRNAs combined with dCas13b-ALKBH5 significantly decreased the m6A levels of the targeted site (Figures 2K, 2L, and S2I). We examined the mRNA and protein levels of TNFR1 and found that the dm6ACRISPR system significantly reduced the protein level of TNFR1 (Figures 2M and 2N). Overall, excessive m6A modification of TNFR1 RNA augments its protein level in ESCC.ATXN2 promotes TNFR1 protein translation in an m6A-dependent mannerTo gain further insights into the mechanism for how m6A methylation affects the function of TNFR1, we examined common m6A recognition proteins, that is, "reader" proteins, which may be involved in m6A-mediated TNFR1 dysregulation. However, RNA immunoprecipitation (RIP) assays with a series of reader proteins, including YTHDF1-3, YTHDC1-2, and IGF2BP1-3 showed that these common m6A recognition proteins could not interact with TNFR1 transcript (Figure S3A), indicating that there may be other functional proteins involved in this process. We then performed mass spectrometry analysis of proteins generated by RNA pulldown using 50-bp unmethylated or m6A-methylated TNFR1 probes and identified four proteins that potentially interact with m6A-methylated TNFR1 (Figure 3A ; Table S4). Western blotting and RIP-coupled qRT-PCR analysis indicated that only ATXN2 bound to m6A-methylated TNFR1 (Figures 3B and 3C). RNA electrophoretic mobility shift assays (REMSA) verified that ATXN2 preferentially bound to m6A-methylated TNFR1 but not unmethylated TNFR1 (Figure 3D). We further examined the mRNA and protein levels of ATXN2 in our ESCC cohort. qRT-PCR and IHC analysis of ATXN2 both revealed that the levels were significantly higher in tumors than in adjacent normal tissue (Figures S3B−S3D), suggesting that ATXN2 may play a role in ESCC development. Analysis of public ATXN2 PAR-CLIP (photoactivatable ribonucleoside-enhanced CLIP) data (POSTAR2 database: http://lulab.life.tsinghua.edu.cn/POSTAR/) from HEK293T cells showed that the ATXN2 binding region covered the m6A site of TNFR1 (Figure 3E). Meanwhile, integration analysis revealed that there is a high degree of co-occupancy between the binding region of ATXN2 and m6A residues (Figure 3F). Furthermore, METTL3 overexpression significantly increased the interaction of ATXN2 with TNFR1 mRNA while it was reduced in METTL3 depletion (Figure 3G). As expected, in METTL3-silenced ESCC cells, overexpression of WT METTL3 but not mutant METTL3 restored the interaction of ATXN2 with TNFR1 mRNA (Figure 3H). We also used the dm6ACRISPR system to remove the m6A modification of TNFR1. CLIP coupled with qRT-PCR assays showed that the interaction between ATXN2 and TNFR1 was significantly decreased after removing the m6A modification (Figure 3I). These results suggested that m6A modification is necessary for the interaction between ATXN2 and TNFR1 RNA.Figure 3ATXN2 recognizes the TNFR1 m6A site and promotes its translationShow full caption(A) Scatterplot of proteins interacting with 50-bp TNFR1 probes with or without m6A modification in KYSE30 cells. The filled red dots indicate potential TNFR1[m6A] binding proteins that have higher affinity for TNFR1[m6A] probes. (B) RNA pull-down coupled with immunoblot analysis shows a specific interaction between ATXN2 and m6A-modified TNFR1. (C) Association of ATXN2 with TNFR1 determined by RNA immunoprecipitation (RIP) assays. IgG served as the negative control. (D) Electrophoretic mobility shift assays of recombinant ATXN2 with unmethylated or methylated TNFR1 probes. The probes were maintained constantly, while a gradient of 0–10 μM recombinant ATXN2 was added to the reactions. (E) Published ATXN2 PAR-CLIP-seq data in HEK293T cells (DRS012391) showed an approximately identical binding region in the TNFR1 transcript with m6A sites. The green bar represents the ATXN2 binding region detected by ATXN2 PAR-CLIP, the sequence of the ATXN2-binding region is highlighted, and the purple arrow indicates the exact m6A site according to 2 public miCLIP-seq datasets. (F) Shown are the intensity of ATXN2 binding centered at m6A residues and the intensity of the m6A CLIP signal centered at ATXN2 binding sites. (G) The levels of ATXN2 bound to TNFR1 RNA determined by CLIP-quantitative PCR in ESCC cells with METTL3 knockdown or overexpression. (H) Effects of WT (M3-WT) or catalytic mutant METTL3 (M3-MUT, aa395–398, DPPW→APPA) overexpression on the association of ATXN2 and TNFR1 in cells with METTL3 knockdown (shM3) determined by ATXN2 CLIP-quantitative PCR. (I) Association of ATXN2 with TNFR1 determined by CLIP-quantitative PCR assays in ESCC cells co-transfected with dCas13b-ALKBH5 and NT-gRNA or gRNAs. (J and K) Effects of ATXN2 knockdown on the mRNA and protein levels of TNFR1 in KYSE30 and EC109 cell lines. (L and M) Polysome fraction analysis in cells with the indicated treatments. The TNFR1 mRNA level in each gradient fraction was measured by quantitative PCR and plotted as a percentage. Data are the means ± SEMs from at least 3 independent experiments in (G)–(J), (L), and (M). Two-sided Student's t tests were used in (G)–(J), (L), and (M) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and p > 0.05, not significant). β-Actin served as loading control in (B) and (K).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we wanted to characterize the molecular mechanism underlying interaction between ATXN2 and m6A-modified TNFR1 transcript. We found that ATXN2 silencing in ESCC cells markedly suppressed the protein level of TNFR1 without affecting its mRNA levels (Figures 3J and 3K). Further treatment of ESCC cells silencing METTL3 or ATXN2 with the protein synthesis inhibitor cycloheximide resulted in no difference in half-life for TNFR1 compared with control cells (Figure S3E). We then hypothesized that the potential m6A mediator ATXN2 promotes the translation of TNFR1 in an m6A-dependent manner. The polysome profile showed that ATXN2 knockdown decreased the association of the TNFR1 transcript with actively transcribing ribosomes to a similar extent as METTL3 depletion (Figures 3L, S3F, and S3G). In addition, in METTL3-overexpressing cells, ATXN2 knockdown reversed the increased proportion of TNFR1 mRNA distribution in the polysome fraction (Figures 3M and S3H). These results indicated that the m6A mediator ATXN2 promotes the translation of TNFR1 in an m6A-dependent manner.m6A methylation regulates the activation of MAPK and NF-κB signaling pathways via TNFR1Accumulating evidence has indicated that dysregulation of TNFR1 expression contributes to tumorigenesis via its downstream MAPK and NF-κB pathways.29Ting A.T. Bertrand M.J.M. More to life than NF-κB in TNFR1 signaling.Trends Immunol. 2016; 37: 535-545Google Scholar,30Egusquiaguirre S.P. Yeh J.E. Walker S.R. Liu S. Frank D.A. The STAT3 target gene TNFRSF1A modulates the NF-κB pathway in breast cancer cells.Neoplasia. 2018; 20: 489-498Google Scholar We examined the mRNA level of TNFR1 in our ESCC cohort by using qRT-PCR and found that there was no significant difference in TNFR1 mRNA levels between ESCC tumor and adjacent normal tissues (Figure 4A ). We further performed western blotting to measure the protein level of TNFR1 in 10 paired randomly selected ESCC and adjacent normal tissues. These results revealed that the TNFR1 protein level was increased in ESCC tissues compared with adjacent normal tissues (Figure 4B). IHC analysis with another 58 paired normal esophageal and ESCC samples showed consistent results with western blotting analysis (N = 58, SYSUCC cohort; Figures 4C and 4D). We also found that TNFR1 overexpression activated its mediated MAPK and NF-κB pathways in ESCC cells, as indicated by the enhanced phosphorylation of ERK, p38, and p65 without an alteration in their total protein levels, while TNFR1 knockdown exhibited the opposite results (Figure 4E). We next explored whether m6A modification was involved in the activation of TNFR1-mediated signaling pathways. After removing the m6A modification of TNFR1, we found that the phosphorylation of ERK, p38, and p65 protein was significantly decreased compared to that in the control cells, along with TNFR1 protein level reduction (Figure 4F). Furthermore, we investigated the effects of TNF treatment on MAPK and NF-κB activation in ESCC cells by using the dm6ACRISPR system. The results indicated that MAPK and NF-κB signaling were attenuated and persisted for fewer periods of time in ESCC cells when TNFR1 m6A modifications were removed (Figure 4G). These results demonstrated that the upregulation of TNFR1 protein levels caused by elevated m6A modification activates TNFR1-mediated MAPK and NF-κB signaling pathways in ESCC.Figure 4Effects of m6A methylation on TNFR1-mediated MAPK and NF-κB signaling pathway activationShow full caption(A) mRNA levels of TNFR1 in ESCC samples compared with paired normal tissues (SYSUCC cohort, N = 215). (B) Immunoblot analysis of the protein levels of TNFR1 in ESCC tissues and corresponding adjacent normal tissues (N = 10). β-Actin was used as a loading control. T, tumor tissues; N, adjacent normal tissues. (C and D) Representative IHC images of TNFR1 in ESCC tumors and paired adjacent normal tissues (C) and quantification of IHC staining (N = 58) (D). Scale bars, 500 and 100 μm. (E and F) Immunoblot assays showed alterations in p-ERK, p-p38, and p-p65 in ESCC cells when TNFR1 was overexpressed or knocked down (E) or when m6A modification was removed by the dm6ACRISPR system (F). (G) Immunoblot assays showed the time course of ERK, p38, and p65 phosphorylation after TNF-α stimulation in ESCC cells treated with the dm6ACRISPR system. p values were calculated by two-sided paired Wilcoxon signed-rank test in (A) and (D) (