Csig-18. hypermethylation of slit-robo pathway genes results in inactivation in glioma progression

Abstract INTRODUCTION Glioblastomas (GBM) are the most common and malignant primary brain tumors. Their rapid growth and invasion into neuronal parenchyma is devastating, with limited treatment options. Genomic alterations have been extensively studied in gliomas tumors, including aberrations of the Slit-Robo pathway genes. The Slit family of secreted proteins modulates migration of somatic cells during development and mediate their effect by binding to its receptor Roundabout (Robo). Genes in the Slit-Robo pathways have been shown to be inactivated by promoter hypermethylation in a number of human cancers. We hypothesize that Slit-Robo signaling pathways are modulated via promotor methylation, controlling GBM malignancy and regulating the invasive capacity of glioma stem cells (GSCs). METHODS We characterized expression of mRNA and protein via real-time PCR and Western blots in primary GBM tissue. We then assessed whether epigenetic alterations correlate with Slit-Robo expression by conducting in vitro demethylation assays in patient derived GSCs followed by real-time PCR. We conducted invasion assays to elucidate the role of Slit in GSC invasion. RESULTS The Cancer Genome Atlas indicates a negative correlation between Robo2 expression and glioma patient survival (17.1 months versus 37.4 months; p < 1.4E-4). We discovered differential expression of the Slit (1-3) and Robo (2, 3) genes and protein of up to 8-fold between grades 3 and 4 astrocytomas (8.34 versus 4.73 density). Treating patient derived glioma stem cells with 5-aza-2-deoxycytidine results in induction in Slit-Robo gene expression ranging from 5 – 40-fold (p < 0.01). Addition of Slit 2 and 3 in an invasion assay induced migration of GSCs in a differential and concentration dependent manner of 10 - 25% (p < 0.05). CONCLUSIONS Our results suggest that promoter methylation and gene expression of the Slit-Robo genes correlates with protein expression, tumor invasiveness, and prognosis and a potential therapeutic target.