Differential epigenetic regulation of sonic hedgehog GLI1 on downstream target genes in medulloblastoma and astrocytomas

Abstract

Sonic hedgehog (Shh) signaling in astrocytic tumorigenesis has not been as thoroughly investigated as in medulloblastoma. We have attempted to understand the regulatory status of GLI1, the immediate activator of Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We attempted to correlate the expression pattern of GLI1 and its regulated genes in 14 cell lines (8 of astrocytoma and 6 of medulloblastoma) and 41 primary samples (27 astrocytomas and 14 medulloblastomas). We also determined the promoter methylation of Cyclin D2 and PTCH1 in the 14 cell lines and in a total of 58 primary samples (44 astrocytomas and 14 medulloblastomas). The dependence of the GLI1 modulated genes on expression of GLI1 was confirmed by knockdown experiments with the help of GLI1-siRNA. We also found that Shh-GLI1 signaling shows different patterns of regulation of downstream target genes in medulloblastomas and astrocytomas: Cyclin D2 and Plakoglobin were upregulated in medulloblastoma but downregulated in astrocytoma; however, PAX6 and NKX2.2 showed over all a low expression pattern of expression in both tumors. However, in several cell lines (A172, SW1783, T98G, CCF-STTG-1 and GOS-3), exposure to both 5-Aza-2'deoxycytidine and Trichostatin A increased the expression of the GLI1 downstream target gene Cyclin D2 (p=0.0014). These results were verified by promoter methylation studies in the 14 cell lines and the 58 primary samples. Our promoter methylation studies revealed a significant number of astrocytoma cell lines (63%) and primary astrocytomas (32%) showing that methylation of Cyclin D2 related to its own level of expression; something that did not occur in medulloblastoma. Compared to Cyclin D2, PTCH1 promoter methylation did not influence on PTCH1 expression in the cell lines and tumor samples. Our results demonstrate that Shh-GLI1 signaling acts differentially for the regulation of target genes in medulloblastoma and astrocytoma. Secondly, PTCH1 and Cyclin D2 are under epigenetic regulation besides the transcriptional regulation exerted by GLI1. This finding may open new ways for treating patients by demethylating agents.