MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth.

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding–dependent, yet RNA editing–independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.

[1]  V. Kim.  MicroRNA biogenesis: coordinated cropping and dicing , 2005, Nature Reviews Molecular Cell Biology.

[2]  M. O’Connell,et al.  The many roles of an RNA editor , 2001, Nature Reviews Genetics.

[3]  K. Nishikura,et al.  Mutagenic Analysis of Double-stranded RNA Adenosine Deaminase, a Candidate Enzyme for RNA Editing of Glutamate-gated Ion Channel Transcripts (*) , 1995, The Journal of Biological Chemistry.

[4]  N. Rajewsky,et al.  Widespread changes in protein synthesis induced by microRNAs , 2008, Nature.

[5]  Masato Nagino,et al.  let-7 regulates Dicer expression and constitutes a negative feedback loop. , 2008, Carcinogenesis.

[6]  C. Samuel,et al.  Adenosine deaminase ADAR1 increases gene expression at the translational level by decreasing protein kinase PKR-dependent eIF-2alpha phosphorylation. , 2009, Journal of molecular biology.

[7]  M. Jantsch,et al.  RNA-Regulated Interaction of Transportin-1 and Exportin-5 with the Double-Stranded RNA-Binding Domain Regulates Nucleocytoplasmic Shuttling of ADAR1 , 2009, Molecular and Cellular Biology.

[8]  C. Burge,et al.  Prediction of Mammalian MicroRNA Targets , 2003, Cell.

[9]  K. Nishikura,et al.  ADAR gene family and A-to-I RNA editing: diverse roles in posttranscriptional gene regulation. , 2005, Progress in nucleic acid research and molecular biology.

[10]  Noam Shomron,et al.  Regulation of Cancer Aggressive Features in Melanoma Cells by MicroRNAs , 2011, PloS one.

[11]  Yukio Kawahara,et al.  RNA editing of the microRNA‐151 precursor blocks cleavage by the Dicer–TRBP complex , 2007, EMBO reports.

[12]  E. Hatano,et al.  The NF90-NF45 Complex Functions as a Negative Regulator in the MicroRNA Processing Pathway , 2009, Molecular and Cellular Biology.

[13]  Yong Liu,et al.  Adenosine deaminases acting on RNA, RNA editing, and interferon action. , 2011, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[14]  Boris Zinshteyn,et al.  Adenosine‐to‐inosine RNA editing , 2009, Wiley interdisciplinary reviews. Systems biology and medicine.

[15]  A. Hamilton,et al.  Improved northern blot method for enhanced detection of small RNA , 2008, Nature Protocols.

[16]  Bjorn-Erik Wulff,et al.  Modulation of microRNA expression and function by ADARs. , 2012, Current topics in microbiology and immunology.

[17]  K. Nishikura,et al.  Editor meets silencer: crosstalk between RNA editing and RNA interference , 2006, Nature Reviews Molecular Cell Biology.

[18]  N. Amariglio,et al.  A-to-I RNA editing: a new regulatory mechanism of global gene expression. , 2007, Blood cells, molecules & diseases.

[19]  Martina Paulsen,et al.  Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene , 2003, Nature Genetics.

[20]  Molly Megraw,et al.  Frequency and fate of microRNA editing in human brain , 2008, Nucleic acids research.

[21]  Xiaoxing Luo,et al.  p150 ADAR1 isoform involved in maintenance of HeLa cell proliferation , 2006, BMC Cancer.

[22]  P. Kao,et al.  ADAR1 Interacts with NF90 through Double-Stranded RNA and Regulates NF90-Mediated Gene Expression Independently of RNA Editing , 2005, Molecular and Cellular Biology.

[23]  Shahar Alon,et al.  Systematic identification of edited microRNAs in the human brain , 2012, Genome research.

[24]  Erez Y. Levanon,et al.  Evolutionarily conserved human targets of adenosine to inosine RNA editing , 2005, Nucleic acids research.

[25]  M. O’Connell,et al.  Down-regulation of RNA Editing in Pediatric Astrocytomas , 2008, Journal of Biological Chemistry.

[26]  I. Gromova,et al.  Human BLCAP transcript: new editing events in normal and cancerous tissues , 2009, International journal of cancer.

[27]  J. Schachter,et al.  Systemic dysregulation of CEACAM1 in melanoma patients , 2010, Cancer Immunology, Immunotherapy.

[28]  B. Shalmon,et al.  Clinical Responses in a Phase II Study Using Adoptive Transfer of Short-term Cultured Tumor Infiltration Lymphocytes in Metastatic Melanoma Patients , 2010, Clinical Cancer Research.

[29]  P. Seeburg,et al.  Modulation of microRNA processing and expression through RNA editing by ADAR deaminases , 2006, Nature Structural &Molecular Biology.

[30]  M. Mathews,et al.  Proteins binding to duplexed RNA: one motif, multiple functions. , 2000, Trends in biochemical sciences.

[31]  Michael R. Green,et al.  Oncogenic BRAF Induces Senescence and Apoptosis through Pathways Mediated by the Secreted Protein IGFBP7 , 2008, Cell.

[32]  T. Cheng,et al.  ADAR1 is required for hematopoietic progenitor cell survival via RNA editing , 2009, Proceedings of the National Academy of Sciences.

[33]  M. O’Connell,et al.  Editing independent effects of ADARs on the miRNA/siRNA pathways , 2009, The EMBO journal.

[34]  A. Ferguson-Smith,et al.  Genomic imprinting at the mammalian Dlk1-Dio3 domain. , 2008, Trends in genetics : TIG.

[35]  R. Malik,et al.  RNA regulation and cancer development. , 2007, Cancer letters.

[36]  A. Hatzigeorgiou,et al.  Redirection of Silencing Targets by Adenosine-to-Inosine Editing of miRNAs , 2007, Science.

[37]  Lin He,et al.  mir-17-92, a cluster of miRNAs in the midst of the cancer network. , 2010, The international journal of biochemistry & cell biology.

[38]  M. O’Connell,et al.  Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes , 2004, Genome Biology.

[39]  Angela Gallo,et al.  A-to-I RNA editing: the "ADAR" side of human cancer. , 2012, Seminars in cell & developmental biology.

[40]  S. Orkin,et al.  Corrigendum: ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon signaling , 2009, Nature Immunology.

[41]  Gideon Rechavi,et al.  Adenosine-to-inosine RNA editing meets cancer. , 2011, Carcinogenesis.

[42]  Richard J Lamont,et al.  Overexpression of dicer as a result of reduced let‐7 MicroRNA levels contributes to increased cell proliferation of oral cancer cells , 2010, Genes, chromosomes & cancer.

[43]  T. Mikkelsen,et al.  Altered adenosine-to-inosine RNA editing in human cancer. , 2007, Genome research.

[44]  C. Samuel,et al.  Adenosine deaminases acting on RNA (ADARs) are both antiviral and proviral. , 2011, Virology.

[45]  K. Nishikura.  Functions and regulation of RNA editing by ADAR deaminases. , 2010, Annual review of biochemistry.

[46]  J. Martinez-Climent,et al.  Epigenetic regulation of miRNA genes in acute leukemia , 2012, Leukemia.

[47]  F. Major,et al.  The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data , 2008, Nature.

[48]  M. Weiss,et al.  Stress-induced Apoptosis Associated with Null Mutation of ADAR1 RNA Editing Deaminase Gene* , 2004, Journal of Biological Chemistry.

[49]  A. Nagler,et al.  A new MALDI-TOF-based assay for monitoring JAK2 V617F mutation level in patients undergoing allogeneic stem cell transplantation (allo SCT) for classic myeloproliferative disorders (MPD). , 2008, Leukemia research.

[50]  Joshua J. Forman,et al.  A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence , 2008, Proceedings of the National Academy of Sciences.

[51]  A. Rich,et al.  The Zalpha domain from human ADAR1 binds to the Z-DNA conformer of many different sequences. , 1998, Nucleic acids research.

[52]  M. Wolter,et al.  De-repression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures , 2010, Oncogene.

[53]  Albert Zlotnik,et al.  Homeostatic chemokine receptors and organ-specific metastasis , 2011, Nature Reviews Immunology.

[54]  Prachi Mishra,et al.  DNA hypermethylation as a chemotherapy target. , 2011, Cellular signalling.

[55]  S. Maas,et al.  Characterization of ADAR1-mediated modulation of gene expression. , 2008, Biochemical and biophysical research communications.

[56]  Z. Paroo,et al.  Phosphorylation of the Human MicroRNA-Generating Complex Mediates MAPK/Erk Signaling , 2009, Cell.

[57]  Ryan D. Morin,et al.  Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution , 2009, Nature.

[58]  K. Nishikura,et al.  A new function for the RNA-editing enzyme ADAR1 , 2009, Nature Immunology.

[59]  Anna M. Krichevsky,et al.  miR-21: a small multi-faceted RNA , 2008, Journal of cellular and molecular medicine.

[60]  G. Zheng,et al.  Abnormal expression of ADAR1 isoforms in Chinese pediatric acute leukemias. , 2011, Biochemical and biophysical research communications.

[61]  Zipora Y. Fligelman,et al.  Systematic identification of abundant A-to-I editing sites in the human transcriptome , 2004, Nature Biotechnology.

[62]  W. Filipowicz,et al.  The widespread regulation of microRNA biogenesis, function and decay , 2010, Nature Reviews Genetics.