Glioblastoma multiforme (GBM) are highly vascularized tumors of the brain. One of the most frequently observed genetic alterations in GBM is the rearrangement of the epidermal growth factor receptor (EGFR). This mutation EGFRvIII, the result of exons 2-8, results in a receptor that no longer binds ligand and is constitutively activated. The expression of this receptor results in the activation of a number of signaling pathways and confers an increase in the proliferative capacity and tumorigenicity of cells expressing the receptor. Signal transducers and activators of transcription (Stat) proteins are a family of latent transcription factors normally activated by numerous cytokines and growth factors. One member of the family, Stat3, has been implicated in aberrant cell proliferation and constitutively activated Stat3cx has been seen in several types of neoplastic cells and solid tumors. In experiments designed to explore Stat3 signaling in human brain tumors, we have found that Stat3a is constitutively activated in low- and high-grade glioma (compared to normal brain tissue). In other preliminary experiments, we have demonstrated a direct interaction between Stat3a and EGFRvIII in extracts from cells that express both proteins. The expression of EGFRvIII leads to the activation of Stat3a with a concomitant increase in Stat3a-mediated transcription and this activation required serine phosphorylation on serine residue 727 indicating a convergence of more than one signaling event in Stat3a activation by EG FRvIII. In experiments described here, we propose to determine the role of Stat3a in the growth properties imparted by EGFRvIII expression both in vitro and in vivo using EGFRvIII derived mutants that cannot activate Stat3a and by the use of dominant-negative Stat3a molecules to directly block Stat3cx mediated signaling. The in vitro studies will be performed using cultured glioma cells that express EGFRvIII while the In vivo experiments will be performed using a novel intracranial induction system developed in our laboratory.
