Epigenetic genome-wide analysis identifies BEX1 as a candidate tumour suppressor gene in paediatric intracranial ependymoma

• Published in: Cancer letters Vol. 346; no. 1; pp. 34 - 44
• Main Authors: Karakoula, Katherine, Jacques, Thomas S, Phipps, Kim P, Harkness, William, Thompson, Dominic, Harding, Brian N, Darling, John L, Warr, Tracy J
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 28.04.2014; Elsevier Limited
• Subjects: 5-Aza-dC; Angiogenesis; Apoptosis; BEX1; Brain cancer; Brain Neoplasms - genetics; Cell cycle; Cell growth; Cells, Cultured; Child; Deoxyribonucleic acid; DNA; DNA Methylation; Ependymoma - genetics; Epigenesis, Genetic; Epigenetics; Gene expression; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genome-Wide Association Study; Genomes; Hematology, Oncology and Palliative Medicine; Humans; Insects; Kinases; Medical prognosis; Nerve Tissue Proteins - genetics; Oligonucleotide Array Sequence Analysis; Paediatric ependymoma; Pathogenesis; Pharmacological unmasking; Polymerase chain reaction; Protein folding; Real-Time Polymerase Chain Reaction; Rodents; Signal transduction; Stress response; Systems development; Transfection; Tumors; DNA methylation; 5-Aza-dC; BEX1; Paediatric ependymoma; Pharmacological unmasking
• ISSN: 0304-3835; 1872-7980
• DOI: 10.1016/j.canlet.2013.12.005

Abstract Promoter hypermethylation and transcriptional silencing is a common epigenetic mechanism of gene inactivation in cancer. To identify targets of epigenetic silencing in paediatric intracranial ependymoma, we used a pharmacological unmasking approach through treatment of 3 ependymoma short-term cell cultures with the demethylating agent 5-Aza-2′-deoxycytidine followed by global expression microarray analysis. We identified 55 candidate epigenetically silenced genes, which are involved in the regulation of apoptosis, Wnt signalling , p 53 and cell differentiation. The methylation status of 26 of these genes was further determined by combined bisulfite restriction analysis (COBRA) and genomic sequencing in a cohort of 40 ependymoma samples. The most frequently methylated genes were BEX1 (27/40 cases), BAI2 (20/40), CCND2 (18/40), and CDKN2A (14/40). A high correlation between promoter hypermethylation and decreased gene expression levels was established by real-time quantitative PCR, suggesting the involvement of these genes in ependymoma tumourigenesis. Furthermore, ectopic expression of brain-expressed X-linked 1 ( BEX1 ) in paediatric ependymoma short-term cell cultures significantly suppressed cell proliferation and colony formation. These data suggest that promoter hypermethylation contributes to silencing of target genes in paediatric intracranial ependymoma. Epigenetic inactivation of BEX1 supports its role as a candidate tumour suppressor gene in intracranial ependymoma, and a potential target for novel therapies for ependymoma in children.