Defining a role for Sonic hedgehog pathway activation in desmoplastic medulloblastoma by identifying GLI1 target genes

4568 The oncogenic transcription factor GLI1 mediates Sonic hedgehog (Shh) signaling and transforms cells in culture. As expected, GLI1 is expressed in the subset of medulloblastomas, usually of desmoplastic histology, that contains Patched-1 mutations and demonstrates up-regulation of the downstream genes in the Shh signal transduction pathway. Desmoplastic histology has recently been found to be associated with a favorable prognosis. To identify roles that Shh pathway activation may play in this subset of medulloblastomas, we identified GLI1 target genes in transformed rat kidney epithelial cells (RK3E) and then compared these targets genes with the previously published gene expression profile that defines the subset of medulloblastomas defined by up-regulation of Shh pathway genes (Thompson et al., J Clin Oncol, 24, 1924-31, 2006) in order to identify a subset of GLI1 targets specifically expressed in medulloblastomas. Since studies of RK3E cells have the advantage over studies of human tumor tissue of allowing identification of specific direct GLI1 target genes following experimental manipulation of GLI1 expression, we used oligonucleotide microarrays, containing over 31,000 DNA sequences to provide a set of downstream target genes in RK3E cells transformed by GLI1 (RK3E + GLI1). We identified 1823 genes whose expression was altered more than 2 fold in 2 independent RK3E + GLI1 cell lines; 572 genes were up-regulated and 1251 down-regulated. Of these, we identified 30 whose expression was similarly and uniquely altered in medulloblastomas showing up-regulation of Shh pathway genes. Of interest, up-regulation of p53 was observed in the RK3E + GLI1 cells and the medulloblastomas showing up-regulation of Shh pathway genes. We identified potential GLI1 binding elements in either the promoter region or regulatory introns in 13 of these genes, suggesting that these may represent immediate targets (Bcl2, PRPSAP1, MGMT, GLI1, CSCC4, MYCL1, SLC19A1, NTRK2, PRKCH, PLTP, PRNP, SLIT2, and CCPG1). Using gel mobility shifts we confirmed that GLI1 binds the regulatory regions of Bcl2 and PRPSAP1, and using cotransfection assays showed that it activates transcription through the PRPSAP1 promoter. Roles previously defined for some of the 30 targets include regulation of cell growth and proliferation, cell-cell interactions, signal transduction, cell metabolism, and apoptosis. Collectively, review of the literature reveals that the targets may affect cell behavior by converging on the MYC and JUN oncogenic pathways and on the Bcl2 and p53 cell survival pathways.