title: Epigenetic silencing of Id4 identifies a glioblastoma subgroup with a better prognosis as a consequence of an inhibition of angiogenesis.
abstract: BACKGROUND
  Inhibitors of DNA binding/differentiation (Id1 to Id4) are a family of helix-loop-helix transcription factors, which are highly expressed during embryogenesis and at lower levels in mature tissues. Id4 plays an important role in neuronal stem cell differentiation, and its deregulation has been implicated in glial neoplasia.
  
  
  METHODS
  The methylation status of Id4 was analyzed by methylation-specific polymerase chain reaction (PCR) in 62 glioblastoma (GBM) cases and in 20 normal brain tissues. Methylation status of Id4 was confirmed by sequencing after subcloning and messenger RNA (mRNA) and protein expression. We also evaluated the mRNA expression of MGP (matrix GLA protein), TGF-β1 (transforming growth factor beta 1), and VEGF (vascular endothelial growth factor) by real-time PCR analysis. Clinical and histological assessment of tumor angiogenesis was performed by evaluating the relative enhancing tumor ratio on magnetic resonance imaging and microvessel density on von Willebrand factor-stained sections, respectively.
  
  
  RESULTS
  The promoter of Id4 was methylated in 23 of 62 (37%) GBMs. In methylated GBMs, Id4 mRNA was significantly reduced, compared with unmethylated GBMs (P = .0002). A significant reduction of protein expression was detected in all hypermethylated cases. GBMs with methylated Id4 showed a significant reduction of MGP, TGF-β1, and VEGF mRNA expression and had significantly lower relative enhancing tumor ratio (P = .0108) and microvessel density (P = .0241) values with respect to unmethylated GBMs. Finally, Id4 methylation was significantly associated with a favorable clinical outcome (P = .0006).
  
  
  CONCLUSIONS
  These data suggest that methylation of Id4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment throughout MGP-mediated neoangiogenesis.
authors.affiliation: Institute of Anatomic Pathology, Università Cattolica del Sacro Cuore, Rome, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Martini
authors.forename: Tonia
authors.initials: T
authors.lastname: Cenci
authors.forename: Giorgio Quintino
authors.initials: GQ
authors.lastname: D'Alessandris
authors.forename: Valeriana
authors.initials: V
authors.lastname: Cesarini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Cocomazzi
authors.forename: Lucia
authors.initials: L
authors.lastname: Ricci-Vitiani
authors.forename: Ruggero
authors.initials: R
authors.lastname: De Maria
authors.forename: Roberto
authors.initials: R
authors.lastname: Pallini
authors.forename: Luigi Maria
authors.initials: LM
authors.lastname: Larocca
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001932:Brain Neoplasms / Q000098:blood supply / Q000235:genetics*; D019175:DNA Methylation; D005909:Glioblastoma / Q000098:blood supply / Q000235:genetics*; D006801:Humans; D051796:Inhibitor of Differentiation Proteins / Q000235:genetics*; D008875:Middle Aged; D009389:Neovascularization, Pathologic; D011379:Prognosis; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D055815:Young Adult
chemical_list: C490128:ID4 protein, human; D051796:Inhibitor of Differentiation Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A