Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression

Collaboration among writers-readers-erasers of m6A regulates the stability of tumor-specific genes. The importance of RNA methylation in biological processes is an emerging focus of investigation. We report that altering m6A levels by silencing either N6-adenosine methyltransferase METTL14 (methyltransferase-like 14) or demethylase ALKBH5 (ALKB homolog 5) inhibits cancer growth and invasion. METTL14/ALKBH5 mediate their protumorigenic function by regulating m6A levels of key epithelial-mesenchymal transition and angiogenesis-associated transcripts, including transforming growth factor–β signaling pathway genes. Using MeRIP-seq (methylated RNA immunoprecipitation sequencing) analysis and functional studies, we find that these target genes are particularly sensitive to changes in m6A modifications, as altered m6A status leads to aberrant expression of these genes, resulting in inappropriate cell cycle progression and evasion of apoptosis. Our results reveal that METTL14 and ALKBH5 determine the m6A status of target genes by controlling each other’s expression and by inhibiting m6A reader YTHDF3 (YTH N6-methyladenosine RNA binding protein 3), which blocks RNA demethylase activity. Furthermore, we show that ALKBH5/METTL14 constitute a positive feedback loop with RNA stability factor HuR to regulate the stability of target transcripts. We discover that hypoxia alters the level/activity of writers, erasers, and readers, leading to decreased m6A and consequently increased expression of target transcripts in cancer cells. This study unveils a previously undefined role for m6A in cancer and shows that the collaboration among writers-erasers-readers sets up the m6A threshold to ensure the stability of progrowth/proliferation-specific genes, and protumorigenic stimulus, such as hypoxia, perturbs that m6A threshold, leading to uncontrolled expression/activity of those genes, resulting in tumor growth, angiogenesis, and progression.

[1]  Minoru Yoshida,et al.  RNA-Methylation-Dependent RNA Processing Controls the Speed of the Circadian Clock , 2013, Cell.

[2]  Zhike Lu,et al.  m6A RNA Methylation Regulates the Self-Renewal and Tumorigenesis of Glioblastoma Stem Cells , 2017, Cell reports.

[3]  E. Buratti,et al.  TDP‐43: new aspects of autoregulation mechanisms in RNA binding proteins and their connection with human disease , 2011, The FEBS journal.

[4]  J. Steitz,et al.  Identification of HuR as a protein implicated in AUUUA‐mediated mRNA decay , 1997, The EMBO journal.

[5]  J. Malter,et al.  Regulation of AU-Rich Element RNA Binding Proteins by Phosphorylation and the Prolyl Isomerase Pin1 , 2015, Biomolecules.

[6]  J. Arbiser,et al.  Inhibition of FoxM1-Mediated DNA Repair by Imipramine Blue Suppresses Breast Cancer Growth and Metastasis , 2016, Clinical Cancer Research.

[7]  Chuan He,et al.  RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation , 2015, Genes & development.

[8]  R. Gregory,et al.  The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. , 2016, Molecular cell.

[9]  J. Massagué,et al.  How cells read TGF-β signals , 2000, Nature Reviews Molecular Cell Biology.

[10]  Shu-Bing Qian,et al.  Dynamic m6A mRNA methylation directs translational control of heat shock response , 2015, Nature.

[11]  Olivier Elemento,et al.  5′ UTR m6A Promotes Cap-Independent Translation , 2015, Cell.

[12]  C. Heldin,et al.  Transforming growth factor-beta-induced mobilization of actin cytoskeleton requires signaling by small GTPases Cdc42 and RhoA. , 2002, Molecular biology of the cell.

[13]  H. Huber,et al.  A crucial function of PDGF in TGF-β-mediated cancer progression of hepatocytes , 2006, Oncogene.

[14]  Chuanzhao Zhang,et al.  Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells , 2016, Oncotarget.

[15]  Gideon Rechavi,et al.  Gene expression regulation mediated through reversible m6A RNA methylation , 2014, Nature Reviews Genetics.

[16]  David Padua,et al.  Roles of TGFβ in metastasis , 2009, Cell Research.

[17]  Qi Zhou,et al.  m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. , 2015, Cell stem cell.

[18]  Chuanzhao Zhang,et al.  Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA , 2016, Proceedings of the National Academy of Sciences.

[19]  Yael Mandel-Gutfreund,et al.  RBPmap: a web server for mapping binding sites of RNA-binding proteins , 2014, Nucleic Acids Res..

[20]  Samie R. Jaffrey,et al.  Emerging links between m6A and misregulated mRNA methylation in cancer , 2017, Genome Medicine.

[21]  Xiaodong Cui,et al.  Exome-based analysis for RNA epigenome sequencing data , 2013, Bioinform..

[22]  Chengqi Yi,et al.  N6-Methyladenosine in Nuclear RNA is a Major Substrate of the Obesity-Associated FTO , 2011, Nature chemical biology.

[23]  Chuan He,et al.  Reading RNA methylation codes through methyl-specific binding proteins , 2014, RNA biology.

[24]  Samir Adhikari,et al.  Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase , 2014, Cell Research.

[25]  Mircea Ivan,et al.  Post-Transcriptional Control of the Hypoxic Response by RNA-Binding Proteins and MicroRNAs , 2011, Front. Mol. Neurosci..

[26]  Yi Xing,et al.  m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. , 2014, Cell stem cell.

[27]  Jian Sun,et al.  A HuR/TGF-β1 feedback circuit regulates airway remodeling in airway smooth muscle cells , 2016, Respiratory Research.

[28]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[29]  S. Kwon,et al.  Expression of connective tissue growth factor in pancreatic cancer cell lines. , 2007, International journal of oncology.

[30]  L. Nabors,et al.  HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs. , 2001, Cancer research.

[31]  Joan Massagu,et al.  How cells read TGF-|[beta]| signals , 2000 .

[32]  S. Rabbani,et al.  Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion , 2013, Breast Cancer Research.

[33]  Miao Yu,et al.  A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation , 2013, Nature chemical biology.

[34]  W. Zwart,et al.  Supplementary Materials and Methods , 2013 .

[35]  Jie Jin,et al.  FTO Plays an Oncogenic Role in Acute Myeloid Leukemia as a N6-Methyladenosine RNA Demethylase. , 2017, Cancer cell.

[36]  Samie R. Jaffrey,et al.  The dynamic epitranscriptome: N6-methyladenosine and gene expression control , 2014, Nature Reviews Molecular Cell Biology.

[37]  C. Heldin,et al.  Transforming growth factor-β employs HMGA2 to elicit epithelial–mesenchymal transition , 2006, The Journal of cell biology.

[38]  Yufei Huang,et al.  A protocol for RNA methylation differential analysis with MeRIP-Seq data and exomePeak R/Bioconductor package. , 2014, Methods.

[39]  M. Gorospe,et al.  Role of RNA binding protein HuR in ductal carcinoma in situ of the breast , 2011, The Journal of pathology.

[40]  Yuval Kluger,et al.  m6A mRNA methylation controls T cell homeostasis by targeting IL-7/STAT5/SOCS pathway , 2017, Nature.

[41]  J. Massagué,et al.  TGFβ in Cancer , 2008, Cell.

[42]  Yufei Huang,et al.  Guitar: An R/Bioconductor Package for Gene Annotation Guided Transcriptomic Analysis of RNA-Related Genomic Features , 2016, BioMed research international.

[43]  R. Coutts,et al.  Current Evidence and Future Perspectives on HuR and Breast Cancer Development, Prognosis, and Treatment , 2016, Neoplasia.

[44]  Olivier Elemento,et al.  Reversible methylation of m6Am in the 5′ cap controls mRNA stability , 2016, Nature.

[45]  Yang Wang,et al.  N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells , 2014, Nature Cell Biology.

[46]  Feng Liu,et al.  METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6‐methyladenosine‐dependent primary MicroRNA processing , 2017, Hepatology.