title: Identification of RASAL1 as a major tumor suppressor gene in thyroid cancer.
abstract: BACKGROUND
  RAS-coupled MAPK and PI3K pathways play a fundamental role in thyroid tumorigenesis, and classical genetic alterations upregulating these pathways are well characterized. We hypothesized that gene abnormality of negative modulators of these signaling pathways might be an important alternative genetic background for thyroid cancer.
  
  
  METHODS
  By examining gene expression patterns of negative modulators of RAS signaling, we attempted to identify potential tumor suppressor genes. We then analyzed the methylation and mutation patterns of the identified gene in 101 thyroid tumors and tested its functions in vitro and in vivo to establish the tumor suppressor role in thyroid cancer.
  
  
  RESULTS
  Among 13 negative modulators of the RAS pathway screened, RASAL1, encoding a RAS GTPase-activating protein, was frequently hypermethylated in thyroid cancers, which was coupled to its silencing in thyroid cancer cells. We also, for the first time, identified the presence of RASAL1 mutations, with a prevalence of 4.88% (n = 2 of 41) in follicular thyroid cancer (FTC) and 16.67% (n = 5 of 30) in anaplastic thyroid cancer (ATC). RASAL1 displayed MAPK- and PI3K-suppressing and thyroid tumor-suppressing activities, which were all impaired by the mutations. Hypermethylation and mutations of RASAL1 were mutually exclusive and collectively found in zero of 20 benign thyroid tumors, 3.22% (n = 1 of 31) of papillary thyroid cancers, 31.70% (n = 13 of 41) of FTCs, and 33.33% (n = 10 of 30) of ATCs. A rate of 20.83% (n = 5 of 24) of tumors carrying RASAL1 mutation or methylation at high levels (>50%) vs 44.16% (n = 34 of 77) of tumors carrying no RASAL1 mutation or methylation at low levels (< 50%) harbored any of the classical mutations (two-sided P = .02, Fisher exact test) in RAS, BRAF, PTEN, and PIK3CA genes in the MAPK and PI3K pathways, revealing a largely mutually exclusive relationship.
  
  
  CONCLUSIONS
  We identified RASAL1 as a major tumor suppressor gene that is frequently inactivated by hypermethylation and mutations, providing a new alternative genetic background for thyroid cancer, particularly FTC and ATC.
authors.affiliation: Affiliation of authors: Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes, and Metabolism, Johns Hopkins University School of Medicine, Baltimore, MD (DL, CY, EB, AKM, MX).
authors.forename: Dingxie
authors.initials: D
authors.lastname: Liu
authors.forename: Chongfei
authors.initials: C
authors.lastname: Yang
authors.forename: Ermal
authors.initials: E
authors.lastname: Bojdani
authors.forename: Avaniyapuram Kannan
authors.initials: AK
authors.lastname: Murugan
authors.forename: Mingzhao
authors.initials: M
authors.lastname: Xing
mesh_terms: D018263:Adenocarcinoma, Follicular / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D019175:DNA Methylation; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000302:isolation & purification*; D016147:Genes, Tumor Suppressor; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D065646:Thyroid Carcinoma, Anaplastic; D013964:Thyroid Neoplasms / Q000737:chemistry* / Q000235:genetics; D020703:ras GTPase-Activating Proteins / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; C580923:RASAL1 protein, human; D020703:ras GTPase-Activating Proteins; D019869:Phosphatidylinositol 3-Kinases; D020929:Mitogen-Activated Protein Kinase Kinases