Abstract B33: Promoter hypermethylation in pediatric malignant astrocytoma: Identification of candidate genes and therapeutic possibilities

Abstract

Abstract Brain tumors account for the most frequent type of solid tumors and the main cause of cancer deaths in children. Recent investigations have suggested that many tumors are initiated not only by genomic abnormalities, but also caused by epigenetic changes. Unlike DNA sequence changes, epigenetic abnormalities are reversible and could be targeted therapeutically. At present, little is known about DNA methylation (a key epigenetic mechanism that controls the regulation of gene expression) in pediatric malignant astrocytoma WHO grade IV. In this study, we have used a combination of three different microarray strategies to develop an integrated approach to identify methylated genes in short-term cell cultures derived from pediatric malignant astrocytoma. In parallel, we evaluated reversal of gene silencing as a therapeutic strategy using clinically relevant doses of 5-aza-2′-deoxycytidine (5-Aza-dC) on these tumor cells in an attempt to reactivate these genes and prevent tumor cell growth Global methylation and expression analysis was performed in 23 tumor samples comprising 18 short-term cultures and 5 biopsies using the Illumina-Infinium 450K methylation array and Affymetrix U133A Expression Array, respectively. In addition, Affymetrix U133 plus 2.0 arrays were used to determine re-expression in 1 short-term tumor culture following pharmacological unmasking with the demethylating agent 5-Aza-dC. The results of these 3 analyses were used to identify frequently silenced genes which were shortlisted based on specific criteria: β score ≥ 0.75 for methylated and β score ≤ 0.25 for un-methylated genes (methylation array), genes down-regulated ≥3 fold and present in NHA (Expression array U133A) and genes absent in untreated tumor culture and re-expressed in 5-Aza-dC treated sample. Shortlisted genes met these criteria in more than 50% of tumor samples and also had CpG Island around the promoter region. The methylation and down-regulation of candidate genes were validated using CoBRA and RT-PCR respectively. Low-dose long term epigenetic therapy was performed by treating the tumor samples for 3 days with biweekly re-treatment and assessing the anti-tumor effect using a colony formation assay after 21 days and long term growth curve analysis for 10 weeks. Efficacy of low-dose 5-Aza-dC treatment on re-expression of methylated genes was also examined using RT-PCR. Genome-wide array analysis identified 25 genes which were silenced in □ 50% of samples. Validation experiments showed there was good correlation between methylation and under expression. Re-expression of the silenced genes after treatment with the 5-Aza-dC was also confirmed in 3 cultures for 5 known methylated genes. The most frequently epigenetically silenced genes were CXCL14, PRR5L, PTPRE, PION and SH2D4A. These genes have critical roles in carcinogenesis such as regulation of cell proliferation, migration, apoptosis, angiogenesis and metastasis. We also confirmed the effect of epigenetic therapy using low-dose 5-Aza-dC on inhibition of tumor colony formation by more than 90% compared to untreated samples and reduction of growth rate by 26 population doublings after 10 weeks. RT-PCR indicated low-dose 5-Aza-dC causes re-expression of methylated genes after 3 days treatment. Frequent silencing of these candidate genes has not been previously associated with development of pediatric astrocytoma. Our research revealed that promoter hypermethylation was reversible by treatment with 5-Aza-dC for all these genes. Our investigations also demonstrated the use of low-doses of 5-Aza-dC to reverse the gene silencing as a potential treatment of brain tumors. This work contributes to a more detailed understanding of the effect of epigenetic silencing on pediatric brain tumors. Citation Format: Hoda Kardooni, Nicola Potter, John Darling, Mark Morris, Tracy Warr. Promoter hypermethylation in pediatric malignant astrocytoma: Identification of candidate genes and therapeutic possibilities. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B33.