RESUMO
AIMS: Proteins of the Polycomb repressive complex 2 (PRC2) are epigenetic gene silencers and are involved in tumour development. Their oncogenic function might be associated with their role in stem cell maintenance. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is a key member of PRC2 function: we have investigated its expression and function in gliomas. METHODS: EZH2 expression was studied in grade II-IV gliomas and in glioma stem-like cells (GSC) by quantitative PCR and immunohistochemistry. Effects of EZH2 down-regulation were analysed by treating GSC with the histone deacetylase (HDAC) inhibitor suberoylanide hydroxamic acid (SAHA) and by shRNA. RESULTS: DNA microarray analysis showed that EZH2 is highly expressed in murine and human GSC. Real-time PCR on gliomas of different grade (n = 66) indicated that EZH2 is more expressed in glioblastoma multiforme (GBM) than in low-grade gliomas (P = 0.0013). This was confirmed by immunohistochemistry on an independent set of 106 gliomas. Treatment with SAHA caused significant up-regulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was associated with a significant decrease of glioma proliferation. CONCLUSION: The data suggest that EZH2 plays a role in glioma progression and encourage the therapeutic targeting of these malignancies by HDAC inhibitors.
Assuntos
Neoplasias Encefálicas/genética , Proteínas de Ligação a DNA/genética , Glioma/genética , Fatores de Transcrição/genética , Animais , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Separação Celular , Imunoprecipitação da Cromatina , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Citometria de Fluxo , Glioma/metabolismo , Glioma/patologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Imuno-Histoquímica , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacos , VorinostatRESUMO
Over the last decade, the knowledge on the molecular genetic background of gliomas has dramatically increased. This information provides the basis for the molecular target therapies and molecular tests serve to complement the subjective nature of histopathologic criteria and add useful data regarding response to treatments and prognosis. In particular, the use of loss of heterozygosity (LOH) and methylation specific polymerase chain reaction (PCR) (MSP) based testing of gliomas is already in place and used clinically in several centers. This paper provides a brief overview of these molecular genetic aberrations and discusses the clinical utility, as well as the advantages and disadvantages of such approach. Newly developed molecular techniques, such as LOH testing, fluorescence in situ hybridization (FISH), DNA sequencing and MSP, are currently being employed in assessment of gliomas in some laboratories. However, the clinical use of some markers and the context in which the information obtained should be used are still not entirely understood. Therefore, this paper will focus on validation and implementation of molecular testing in gliomas, with emphasis on LOH on chromosomes 1p, 19q, 17p and 10q and O(6)-methylguanine-DNA methyltransferase (MGMT) methylation status.