title: Identification of tumour-specific epigenetic events in medulloblastoma development by hypermethylation profiling.
abstract: Medulloblastoma arises in the cerebellum and is the most common malignant brain tumour of childhood, however its molecular basis is not well understood. To assess the role of aberrant epigenetic events in medulloblastoma and identify critical genes in its development, we profiled the promoter methylation status of 11 candidate tumour-suppressor genes (TSGs; p14(ARF), p15(INK4b), p16(INK4a), CASP8, HIC1, EDNRB, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in medulloblastoma cell lines, primary tumours and the normal cerebellum. Gene-specific TSG methylation was a significant feature of both medulloblastomas and the cerebellum. Extensive hypermethylation of RASSF1A was detected frequently in medulloblastomas but not in the normal cerebellum (41/44 primary tumours versus 0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a subset of primary tumours (17/44 and 14/39) occurred against a consistent background of partial methylation in the normal cerebellum. These data therefore indicate that extensive methylation of RASSF1A, HIC1 and CASP8 are tumour-specific events in medulloblastoma. Moreover, methylation of these genes in medulloblastoma cell lines was associated with their epigenetic transcriptional silencing and methylation-dependent re-expression following treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. The remaining genes studied showed either low frequency methylation (p14(ARF), p16(INK4a), RIZ1; <7% of cases), no evidence of methylation (p15(INK4b), TIMP3, TP73, TSLC1), or comparable patterns of methylation in the normal cerebellum (EDNRB), suggesting that their hypermethylation does not play a major role in medulloblastoma. Our data demonstrate that tumour-specific hypermethylation affects only a subset of genes, and does not support the existence of a concordant methylation phenotype in this disease. We conclude that epigenetic TSG inactivation is a significant feature of medulloblastoma, and identify RASSF1A, HIC1 and CASP8 as potentially critical genes in its pathogenesis. Furthermore, methylation observed in the normal cerebellum emphasises the requirement for appropriate control tissues when assessing the tumour-specificity of TSG hypermethylation.
authors.affiliation: Northern Institute for Cancer Research, The Medical School, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, UK.
authors.forename: Janet C
authors.initials: JC
authors.lastname: Lindsey
authors.forename: Meryl E
authors.initials: ME
authors.lastname: Lusher
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Anderton
authors.forename: Simon
authors.initials: S
authors.lastname: Bailey
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Gilbertson
authors.forename: Andrew D J
authors.initials: AD
authors.lastname: Pearson
authors.forename: David W
authors.initials: DW
authors.lastname: Ellison
authors.forename: Steven C
authors.initials: SC
authors.lastname: Clifford
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000964:Antimetabolites, Antineoplastic; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002277:Carcinoma / Q000235:genetics; D018287:Carcinoma, Large Cell / Q000235:genetics; D016022:Case-Control Studies; D053181:Caspase 8; D020169:Caspases / Q000235:genetics; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins / Q000235:genetics; D002531:Cerebellum; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000235:genetics; D000077209:Decitabine; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D007136:Immunoglobulins; D007223:Infant; D007231:Infant, Newborn; D051741:Kruppel-Like Transcription Factors; D008527:Medulloblastoma / Q000235:genetics*; D008565:Membrane Proteins; D008875:Middle Aged; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D000072160:Tumor Protein p73; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D000964:Antimetabolites, Antineoplastic; C403514:CADM1 protein, human; C493966:CDKN2B protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; C096870:Hic1 protein, mouse; D007136:Immunoglobulins; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011506:Proteins; C411762:RASSF1 protein, human; C000606460:TP73 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D014157:Transcription Factors; C000606368:Trp73 protein, mouse; D000072160:Tumor Protein p73; D025803:Tumor Suppressor Protein p14ARF; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D015254:DNA Modification Methylases; D011495:Histone-Lysine N-Methyltransferase; C093563:PRDM2 protein, human; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases; D001374:Azacitidine