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The m6A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-κB/MYC signaling network

Abstract

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in many bioprocesses. However, its functions in bladder cancer (BCa) remain elusive. Here, we discovered that methyltransferase-like 3 (METTL3), a major RNA N6-adenosine methyltransferase, was significantly up-regulated in human BCa. Knockdown of METTL3 drastically reduced BCa cell proliferation, invasion, and survival in vitro and tumorigenicity in vivo. On the other hand, overexpression of METTL3 significantly promoted BCa cell growth and invasion. Through transcriptome sequencing, m6A sequencing and m6A methylated RNA immuno-precipitation quantitative reverse-transcription polymerase chain reaction, we revealed the profile of METTL3-mediated m6A modification in BCa cells for the first time. AF4/FMR2 family member 4 (AFF4), two key regulators of NF-κB pathway (IKBKB and RELA) and MYC were further identified as direct targets of METTL3-mediated m6A modification. In addition, we showed that besides NF-κB, AFF4 binds to the promoter of MYC and promotes its expression, implying a novel multilevel regulatory network downstream of METTL3. Our results uncovered an AFF4/NF-κB/MYC signaling network operated by METTL3-mediated m6A modification and provided insight into the mechanisms of BCa progression.

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Data availability

The authors declare that all relevant data are available within the article and its Supplementary information files or from the corresponding author upon reasonable request. The m6A-sequencing and RNA-sequencing datasets have been submitted to the NCBI database under the accession number PRJNA498900, SAMN10337857 and SAMN10337858.

References

  1. 1.

    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA. 2018;68:7–30.

    PubMed  Google Scholar 

  2. 2.

    Felsenstein KM, Theodorescu D. Precision medicine for urothelial bladder cancer: update on tumour genomics and immunotherapy. Nat Rev Urol. 2018;15:92–111.

    CAS  Article  Google Scholar 

  3. 3.

    Ribas A, Tumeh PC. The future of cancer therapy: selecting patients likely to respond to PD1/L1 blockade. Clinical cancer research: an official journal of the American Association for. Cancer Res. 2014;20:4982–4.

    CAS  Google Scholar 

  4. 4.

    Oing C, Rink M, Oechsle K, Seidel C, von Amsberg G, Bokemeyer C. Second line chemotherapy for advanced and metastatic urothelial carcinoma: vinflunine and beyond-a comprehensive review of the current literature. J Urol. 2016;195:254–63.

    CAS  Article  Google Scholar 

  5. 5.

    van Kessel KE, Zuiverloon TC, Alberts AR, Boormans JL, Zwarthoff EC. Targeted therapies in bladder cancer: an overview of in vivo research. Nat Rev Urol. 2015;12:681–94.

    Article  Google Scholar 

  6. 6.

    Desrosiers R, Friderici K, Rottman F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci USA. 1974;71:3971–5.

    CAS  Article  Google Scholar 

  7. 7.

    Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485:201–6.

    CAS  Article  Google Scholar 

  8. 8.

    Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, Elemento O. et al. 5′ UTR m(6)A promotes cap-independent translation. Cell. 2015;163:999–1010.

    CAS  Article  Google Scholar 

  9. 9.

    Bokar JA, Rath-Shambaugh ME, Ludwiczak R, Narayan P, Rottman F. Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex. J Biol Chem. 1994;269:17697–704.

    CAS  PubMed  Google Scholar 

  10. 10.

    Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10:93–95.

    CAS  Article  Google Scholar 

  11. 11.

    Ping XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ. et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24:177–89.

    CAS  Article  Google Scholar 

  12. 12.

    Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC. N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol. 2014;16:191–8.

    CAS  Article  Google Scholar 

  13. 13.

    Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7:885–7.

    CAS  Article  Google Scholar 

  14. 14.

    Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013;49:18–29.

    CAS  Article  Google Scholar 

  15. 15.

    Batista PJ, Molinie B, Wang J, Qu K, Zhang J, Li L. et al. m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell. 2014;15:707–19.

    CAS  Article  Google Scholar 

  16. 16.

    Meyer KD, Jaffrey SR. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol. 2014;15:313–26.

    CAS  Article  Google Scholar 

  17. 17.

    Alarcon CR, Lee H, Goodarzi H, Halberg N, Tavazoie SF. N6-methyladenosine marks primary microRNAs for processing. Nature. 2015;519:482–5.

    CAS  Article  Google Scholar 

  18. 18.

    Niu Y, Zhao X, Wu YS, Li MM, Wang XJ, Yang YG. N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function. Genom Proteom Bioinform. 2013;11:8–17.

    CAS  Article  Google Scholar 

  19. 19.

    Wu Y, Xie L, Wang M, Xiong Q, Guo Y, Liang Y. et al. Mettl3-mediated m(6)A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis. Nat Commun. 2018;9:4772

    Article  Google Scholar 

  20. 20.

    Wu Y, Zhou C, Yuan Q. Role of DNA and RNA N6-adenine methylation in regulating stem cell fate. Curr Stem Cell Res Ther. 2018;13:31–38.

    CAS  PubMed  Google Scholar 

  21. 21.

    Merkurjev D, Hong WT, Iida K, Oomoto I, Goldie BJ, Yamaguti H. et al. Synaptic N(6)-methyladenosine (m(6)A) epitranscriptome reveals functional partitioning of localized transcripts. Nat Neurosci. 2018;21:1004–14.

    CAS  Article  Google Scholar 

  22. 22.

    Weng YL, Wang X, An R, Cassin J, Vissers C, Liu Y. et al. Epitranscriptomic m(6)A regulation of axon regeneration in the adult mammalian nervous system. Neuron. 2018;97:313–25.e316.

    CAS  Article  Google Scholar 

  23. 23.

    Shi H, Zhang X, Weng YL, Lu Z, Liu Y, Lu Z. et al. m(6)A facilitates hippocampus-dependent learning and memory through YTHDF1. Nature. 2018;563:249–53.

    CAS  Article  Google Scholar 

  24. 24.

    Tong J, Cao G, Zhang T, Sefik E, Amezcua Vesely MC, Broughton JP. et al. m(6)A mRNA methylation sustains Treg suppressive functions. Cell Res. 2018;28:253–6.

    Article  Google Scholar 

  25. 25.

    Zheng Q, Hou J, Zhou Y, Li Z, Cao X. The RNA helicase DDX46 inhibits innate immunity by entrapping m(6)A-demethylated antiviral transcripts in the nucleus. Nat Immunol. 2017;18:1094–103.

    CAS  Article  Google Scholar 

  26. 26.

    Lin S, Choe J, Du P, Triboulet R, Gregory RI. The m(6)A methyltransferase METTL3 promotes translation in human cancer cells. Mol Cell. 2016;62:335–45.

    CAS  Article  Google Scholar 

  27. 27.

    Chen M, Wei L, Law CT, Tsang FH, Shen J, Cheng CL. et al. RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2. Hepatology. 2018;67:2254–70.

    CAS  Article  Google Scholar 

  28. 28.

    Cai X, Wang X, Cao C, Gao Y, Zhang S, Yang Z. et al. HBXIP-elevated methyltransferase METTL3 promotes the progression of breast cancer via inhibiting tumor suppressor let-7g. Cancer Lett. 2018;415:11–19.

    CAS  Article  Google Scholar 

  29. 29.

    Barbieri I, Tzelepis K, Pandolfini L, Shi J, Millan-Zambrano G, Robson SC. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature. 2017;552:126–31.

    CAS  Article  Google Scholar 

  30. 30.

    Taketo K, Konno M, Asai A, Koseki J, Toratani M, Satoh T. et al. The epitranscriptome m6A writer METTL3 promotes chemo- and radioresistance in pancreatic cancer cells. Int J Oncol. 2018;52:621–9.

    PubMed  Google Scholar 

  31. 31.

    Cao G, Li HB, Yin Z, Flavell RA. Recent advances in dynamic m6A RNA modification. Open Biol. 2016;6:160003.

    Article  Google Scholar 

  32. 32.

    Zhang C, Samanta D, Lu H, Bullen JW, Zhang H, Chen I. et al. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m(6)A-demethylation of NANOG mRNA. Proc Natl Acad Sci USA. 2016;113:E2047–2056.

    CAS  Article  Google Scholar 

  33. 33.

    Cui Q, Shi H, Ye P, Li L, Qu Q, Sun G. et al. m(6)A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells. Cell Rep. 2017;18:2622–34.

    CAS  Article  Google Scholar 

  34. 34.

    Zhu F, Qian W, Zhang H, Liang Y, Wu M, Zhang Y. et al. SOX2 is a marker for stem-like tumor cells in bladder cancer. Stem Cell Rep. 2017;9:429–37.

    CAS  Article  Google Scholar 

  35. 35.

    Mukherjee N, Houston TJ, Cardenas E, Ghosh R. To be an ally or an adversary in bladder cancer: the NF-kappaB story has not unfolded. Carcinogenesis. 2015;36:299–306.

    CAS  Article  Google Scholar 

  36. 36.

    Jeong KC, Kim KT, Seo HH, Shin SP, Ahn KO, Ji MJ. et al. Intravesical instillation of c-MYC inhibitor KSI-3716 suppresses orthotopic bladder tumor growth. J Urol. 2014;191:510–8.

    CAS  Article  Google Scholar 

  37. 37.

    Mahe M, Dufour F, Neyret-Kahn H, Moreno-Vega A, Beraud C, Shi M. et al. An FGFR3/MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers. EMBO Mol Med. 2018; 10:pii: e8163.

    Article  Google Scholar 

  38. 38.

    Zhu J, Li Y, Chen C, Ma J, Sun W, Tian Z. et al. NF-kappaB p65 overexpression promotes bladder cancer cell migration via FBW7-mediated degradation of RhoGDIalpha protein. Neoplasia. 2017;19:672–83.

    CAS  Article  Google Scholar 

  39. 39.

    Zheng J, Kong C, Yang X, Cui X, Lin X, Zhang Z. Protein kinase C-alpha (PKCalpha) modulates cell apoptosis by stimulating nuclear translocation of NF-kappa-B p65 in urothelial cell carcinoma of the bladder. BMC Cancer. 2017;17:432.

    Article  Google Scholar 

  40. 40.

    Luo Z, Lin C, Guest E, Garrett AS, Mohaghegh N, Swanson S. et al. The super elongation complex family of RNA polymerase II elongation factors: gene target specificity and transcriptional output. Mol Cell Biol. 2012;32:2608–17.

    CAS  Article  Google Scholar 

  41. 41.

    Deng X, Su R, Weng H, Huang H, Li Z, Chen J. RNA N(6)-methyladenosine modification in cancers: current status and perspectives. Cell Res. 2018;28:507–17.

    CAS  Article  Google Scholar 

  42. 42.

    Ma JZ, Yang F, Zhou CC, Liu F, Yuan JH, Wang F. et al. METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6)-methyladenosine-dependent primary MicroRNA processing. Hepatology. 2017;65:529–43.

    CAS  Article  Google Scholar 

  43. 43.

    Vu LP, Pickering BF, Cheng Y, Zaccara S, Nguyen D, Minuesa G. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med. 2017;23:1369–76.

    CAS  Article  Google Scholar 

  44. 44.

    Weng H, Huang H, Wu H, Qin X, Zhao BS, Dong L. et al. METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m(6)A modification. Cell Stem Cell. 2018;22:191–205. e199.

    CAS  Article  Google Scholar 

  45. 45.

    Su R, Dong L, Li C, Nachtergaele S, Wunderlich M, Qing Y. et al. R-2HG exhibits anti-tumor activity by targeting FTO/m(6)A/MYC/CEBPA signaling. Cell. 2018;172:90–105. e123.

    CAS  Article  Google Scholar 

  46. 46.

    Doyle GA, Betz NA, Leeds PF, Fleisig AJ, Prokipcak RD, Ross J. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins. Nucleic Acids Res. 1998;26:5036–44.

    CAS  Article  Google Scholar 

  47. 47.

    Huang H, Weng H, Sun W, Qin X, Shi H, Wu H. et al. Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol. 2018;20:285–95.

    CAS  Article  Google Scholar 

  48. 48.

    Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H. et al. N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell. 2015;161:1388–99.

    CAS  Article  Google Scholar 

  49. 49.

    Seo HK, Ahn KO, Jung NR, Shin JS, Park WS, Lee KH. et al. Antitumor activity of the c-Myc inhibitor KSI-3716 in gemcitabine-resistant bladder cancer. Oncotarget. 2014;5:326–37.

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Stine ZE, Walton ZE, Altman BJ, Hsieh AL, Dang CV. MYC, metabolism, and cancer. Cancer Discov. 2015;5:1024–39.

    CAS  Article  Google Scholar 

  51. 51.

    Gabay M, Li Y, Felsher DW. MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harbor Perspect Med. 2014; 4:pii: a014241.

    Article  Google Scholar 

  52. 52.

    Watters AD, Latif Z, Forsyth A, Dunn I, Underwood MA, Grigor KM. et al. Genetic aberrations of c-myc and CCND1 in the development of invasive bladder cancer. Br J Cancer. 2002;87:654–8.

    CAS  Article  Google Scholar 

  53. 53.

    Tabach Y, Kogan-Sakin I, Buganim Y, Solomon H, Goldfinger N, Hovland R. et al. Amplification of the 20q chromosomal arm occurs early in tumorigenic transformation and may initiate cancer. PLoS One. 2011;6:e14632

    CAS  Article  Google Scholar 

  54. 54.

    Shiina H, Igawa M, Shigeno K, Terashima M, Deguchi M, Yamanaka M. et al. Beta-catenin mutations correlate with over expression of C-myc and cyclin D1 Genes in bladder cancer. J Urol. 2002;168:2220–6.

    CAS  Article  Google Scholar 

  55. 55.

    Li Y, Liu H, Lai C, Du X, Su Z, Gao S. The Lin28/let-7a/c-Myc pathway plays a role in non-muscle invasive bladder cancer. Cell Tissue Res. 2013;354:533–41.

    CAS  Article  Google Scholar 

  56. 56.

    Li Y, Xu Z, Wang K, Wang N, Zhu M. Network analysis of microRNAs, genes and their regulation in human bladder cancer. Biomed Rep. 2013;1:918–24.

    CAS  Article  Google Scholar 

  57. 57.

    Duyao MP, Buckler AJ, Sonenshein GE. Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter. Proc Natl Acad Sci USA. 1990;87:4727–31.

    CAS  Article  Google Scholar 

  58. 58.

    Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L. et al. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell. 2010;37:429–37.

    CAS  Article  Google Scholar 

  59. 59.

    Deng P, Wang J, Zhang X, Wu X, Ji N, Li J. et al. AFF4 promotes tumorigenesis and tumor-initiation capacity of head and neck squamous cell carcinoma cells by regulating SOX2. Carcinogenesis. 2018;39:937–47.

    CAS  Article  Google Scholar 

  60. 60.

    Kamat AM, Tharakan ST, Sung B, Aggarwal BB. Curcumin potentiates the antitumor effects of Bacillus Calmette-Guerin against bladder cancer through the downregulation of NF-kappaB and upregulation of TRAIL receptors. Cancer Res. 2009;69:8958–66.

    CAS  Article  Google Scholar 

  61. 61.

    Visvanathan A, Patil V, Arora A, Hegde AS, Arivazhagan A, Santosh V. et al. Essential role of METTL3-mediated m(6)A modification in glioma stem-like cells maintenance and radioresistance. Oncogene. 2018;37:522–33.

    CAS  Article  Google Scholar 

  62. 62.

    Choe J, Lin S, Zhang W, Liu Q, Wang L, Ramirez-Moya J. et al. mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis. Nature. 2018;561:556–60.

    CAS  Article  Google Scholar 

  63. 63.

    Li Y, Deng H, Lv L, Zhang C, Qian L, Xiao J. et al. The miR-193a-3p-regulated ING5 gene activates the DNA damage response pathway and inhibits multi-chemoresistance in bladder cancer. Oncotarget. 2015;6:10195–206.

    PubMed  PubMed Central  Google Scholar 

  64. 64.

    Liang Y, Zhu F, Zhang H, Chen D, Zhang X, Gao Q. et al. Conditional ablation of TGF-beta signaling inhibits tumor progression and invasion in an induced mouse bladder cancer model. Sci Rep. 2016;6:29479

    CAS  Article  Google Scholar 

  65. 65.

    Dominissini D, Moshitch-Moshkovitz S, Salmon-Divon M, Amariglio N, Rechavi G. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. Nat Protoc. 2013;8:176–89.

    CAS  Article  Google Scholar 

  66. 66.

    El‐Sheikh A, Fan R, Birks D, Donson A, Foreman NK, Vibhakar R. Inhibition of Aurora Kinase A enhances chemosensitivity of medulloblastoma cell lines. Pediatr Blood Cancer. 2010;55:35–41.

    PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81872313 and 81672776 to YL, 81802391 to QG, 31501838 to XH Z), and Anhui Provincial Natural Science Foundation (1808085QH266 to QG)

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Cheng, M., Sheng, L., Gao, Q. et al. The m6A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-κB/MYC signaling network. Oncogene 38, 3667–3680 (2019). https://doi.org/10.1038/s41388-019-0683-z

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