BackgroundThe Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors.MethodsWe silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples.ResultsSilencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas.ConclusionsOur results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

Abstract. We investigated a role for Hedgehog signalling in glioblastoma, neuroblastoma and medulloblastoma by studying the transcription of PTCH, SMO, GLI1 and GLI3 in a total of 25 cell lines by standard RT-PCR and qRT-PCR, before and after 5-aza-2'-deoxycytidine and trichostatin A (TSA) treatments. Also 25 glioblastoma samples were tested by qRT-PCR. We also performed real-time methylated specific PCR (qMSP) of the SMO promoter region in DNA from 80 tumor samples (40 glioblastomas and 40 neuroblastomas) and from the 25 cell lines. We detected SMO promoter methylation in more than half of the cell lines and tumor samples. PTCH expression in cell lines was lower than in normal controls, just the opposite to GLI1. SMO and GLI3 expression were high and fully correlated in glioblastoma and medulloblastoma, although partially in neuroblastoma. Our results support the existence of Hedgehog signalling in glioblastoma and medulloblastoma, and to a lesser extent, in neuroblastoma.

A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.