RAS mediated proliferation of human astrocytomas

Introduction: Malignant astrocytomas are the most common adult human primary brain tumor. They overexpress PDGF and EGF receptors, activation of which contribute to tumor growth. Oncogenic Ras mutations although present in a large number of human cancers are not prevalent in astrocytomas. This study examined whether the Ras pathway is activated by these growth factor receptors and are they important to their proliferation. Methods: Four established human malignant astrocytoma cell lines were examined and thirteen glioblastoma (GBM) specimens for activation level of Ras. Results: EGF or PDGF stimulation for four human malignant astrocytoma cell lines, resulted in their cognate receptors to complex with Shc and GRB2, both of which are involved in activation of Ras. Levels of Ras-GTP were increased in the astrocytoma cell lines and similar to oncogenic Ras transformed control fibroblasts by 32P Ras loading experiments. The Asn17 Ras dominant-negative mutant was transfected into the astrocytoma cells to decrease Ras-GTP levels, hence determining if this signal transduction pathway contributed to their proliferation. Pooled growth and colony formation assay showed decreased proliferation in astrocytoma cell lines. Stable Asn-17 transfected astrocytoma clones also had decreased growth. Phosphorylation of MAP Kinase, a downstream target of activated Ras, was reduced in the Asn-17 transfected astrocytoma cells, correlating with their decreased proliferation. We have recently verified that the levels of activated Ras-GTP in GBM specimens was 53.5% of total Ras, compared to less than 5% in normal brain, using a enzyme based assay. Discussion: These results support the hypothesis that the proliferative signal of human malignant astrocytoma cells, which are known to overexpress receptor tyrosin, kinases, utilizes Ras mediated signal transduction. Pharmacological (i.e., farnesyl-transferase inhibitors) and genetic inhibitors of the Ras pathway may therefore be of therapeutic value in these tumors.