title: BRAF activation induces transformation and then senescence in human neural stem cells: a pilocytic astrocytoma model.
abstract: OBJECTIVE
  BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain.
  
  
  METHODS
  The constitutively active BRAF(V600E) allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16(INK4a) levels in pilocytic astrocytoma.
  
  
  RESULTS
  BRAF(V600E) expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF(V600E)-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16(INK4a) whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16(INK4a) immunoreactivity in the majority of cases, but patients with tumors negative for p16(INK4a) had significantly shorter overall survival.
  
  
  CONCLUSIONS
  BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16(INK4a) expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.
authors.affiliation: Division of Pediatric Oncology and Departments of Pathology and Pediatrics and Adolescent Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. eraabe2@jhmi.edu
authors.forename: Eric H
authors.initials: EH
authors.lastname: Raabe
authors.forename: Kah Suan
authors.initials: KS
authors.lastname: Lim
authors.forename: Julia M
authors.initials: JM
authors.lastname: Kim
authors.forename: Alan
authors.initials: A
authors.lastname: Meeker
authors.forename: Xing-Gang
authors.initials: XG
authors.lastname: Mao
authors.forename: Guido
authors.initials: G
authors.lastname: Nikkhah
authors.forename: Jarek
authors.initials: J
authors.lastname: Maciaczyk
authors.forename: Ulf
authors.initials: U
authors.lastname: Kahlert
authors.forename: Deepali
authors.initials: D
authors.lastname: Jain
authors.forename: Eli
authors.initials: E
authors.lastname: Bar
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Cohen
authors.forename: Charles G
authors.initials: CG
authors.lastname: Eberhart
mesh_terms: D001254:Astrocytoma / Q000235:genetics / Q000473:pathology*; D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D016922:Cellular Senescence; D004789:Enzyme Activation; D019942:Genes, p16; D006801:Humans; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D058953:Neural Stem Cells / Q000201:enzymology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases; C021502:acid beta-galactosidase; D001616:beta-Galactosidase