title: GAS5 long non-coding RNA in malignant pleural mesothelioma.
abstract: BACKGROUND
  Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA "sponge". Our aim was to investigate the possible role of the GAS5 in the growth of MPM.
  
  
  METHODS
  Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry.
  
  
  RESULTS
  GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression.
  
  
  CONCLUSIONS
  The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.
authors.forename: Arun
authors.initials: A
authors.lastname: Renganathan
authors.forename: Jelena
authors.initials: J
authors.lastname: Kresoja-Rakic
authors.forename: Nohemy
authors.initials: N
authors.lastname: Echeverry
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ziltener
authors.forename: Bart
authors.initials: B
authors.lastname: Vrugt
authors.forename: Isabelle
authors.initials: I
authors.lastname: Opitz
authors.forename: Rolf A
authors.initials: RA
authors.lastname: Stahel
authors.affiliation: Laboratory of Molecular Oncology, Clinic of Oncology, University Hospital Zürich, Zürich, Switzerland. emanuela.felley-bosco@usz.ch.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Felley-Bosco
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008654:Mesothelioma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000086002:Mesothelioma, Malignant; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D062085:RNA, Long Noncoding / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000098853:Podoplanin
chemical_list: D053823:Hedgehog Proteins; D019394:Ki-67 Antigen; D008156:Luciferases; D008562:Membrane Glycoproteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases; C561567:GAS5 long non-coding RNA, human; C117468:PDPN protein, human; D000098853:Podoplanin; C546842:MTOR protein, human