The long-term objective of this proposal is to understand cytokine-mediated regulation of proliferation and apoptosis of malignant glioblastoma cells. Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumors, with an average survival of less than a year. GBM arises from a complex series of molecular events that include inactivation of tumor suppressor genes as well as overexpression and activation of proto-oncogenes. Consequently GBM cells become highly proliferative and resistant to apoptosis. The latent transcription factor Stat3, which is activated by IL-6-family of cytokines and other growth factors induces the expression of genes that are responsible for the suppression of apoptosis in a variety of human cancer cells. Although GBM cells secrete IL-6 and respond to it, little is known about the role of Stat3 activation in the regulation of apoptosis in GBM cells. We found that Stat3 is constitutively activated by an autocrine action of IL-6 in GBM tumor tissues and GBM cell lines. Inhibition of Stat3 activation by the Jak-specific tyrosine kinase inhibitor AG490 reduces steady state levels of anti-apoptotic proteins Bcl-2, BcI-XL and Mcl-1, and induces apoptosis in GBM cells. In contrast, AG490 does not induce apoptosis in normal human astrocytes. Interestingly, Stat3 is activated by IL-4 in GBM cells that is in part, attributable to the expression of IL-13Ra2, a decoy receptor for IL-13. IL-4 normally activates Stat6 but not Stat3 by signaling through the classical Jak-Stat pathway, and produces growth arrest in normal human astrocytes and low-grade gliomas that do not express IL-13Ra2. In consideration of these observations, we hypothesize that (i) constitutive activation of Stat3 via an autocrine action of IL-6, provides survival signal in GBM cells by inducing the expression of anti-apoptotic genes, and (ii) IL-4 induces further activation of Stat3 in these cells via an IL-13Ra2-dependent novel mechanism. To test these hypotheses we will pursue the following specific aims. 1. To define the role of activated Stat3 in the survival of GBM cells we will: (a) Express a dominant negative mutant Stat3 (DNStat3) and the suppressor of cytokine signaling (SOCS)-I in GBM cells via an ecdysone-inducible system, and determine their apoptotic response in vitro, and (b) Determine the effects of Stat3 inactivation by DNStat3, SOCS-1 and AG490 on the growth of intracranial and subcutaneous transplants of GBM cell lines in rodent brain tumor models. 2. To identify cellular and molecular mechanisms underlying the IL-4-mediated activation of Stat3 in IL-13Ra2-expressing glioma cells we will: (a) Determine if IL-13Ra2 expression level parallels the malignancy grade of glioma and is associated with aberrant Stat activation by IL-4, and (b) Define the role of IL-13Ra2 in the regulation of IL-4-dependent signal transduction in GBM cells. This investigation will define cellular and molecular mechanisms underlying Stat3-mediated survival of GBM cells, and thus significantly advance our current understanding of the molecular pathobiology of GBM, and importantly will lead to the development of novel therapies for this deadly disease.
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