The goal of this proposal is to characterize the effects of Epstein Barr Virus (EBV) on the Writ/Wingless signaling pathway which is a critical regulator of cell growth and differentiation during development and is frequently affected during cancer development. In this application, we will determine if this pathway is activated infected cell lines and in EBV-associated cancers, identify the viral proteins that affect regulation of this pathway, determine the mechanisms that underlie this activation, and identify the cellular targets that are affected by this activation. Specifically we will: 1) Characterize beta-catenin levels, localization, and activity in EBV positive and negative cell lines and in EBV-associated and AIDS-related cancers using cell fractionation and immunoblotting, electrophoretic mobility shift assays (EMSA) on beta-catenin responsive elements, reporter assays, and immunohistochemistry to identify specific phosphorylated protein components of the pathway and determine intracellular localization of beta-catenin. 2) Identify the effects of LMP 1 on expression and localization of components of writ/wingless using protein and gene array analyses. 3) Determine the effects of LMP2 on Akt activation, inactivation of glycogen synthase kinase beta, B-catenin localization, and wnt-regulated gene expression. Compare and contrast LMP2 with mutated forms of LMP2 and with constitutively activated forms of PI3-kinase and Akt. Determine the contribution of activation of this pathway on LMP2-mediated effects on cellular growth using fibroblast and epithelial cell transformation assays and perform genomic expression array analysis on cell lines expressing LMP2, mutant forms of LMP2, or activated components of wnt signaling to determine effects on gene expression. 4) Analyze LEF/TCF regulated gene expression in cell lines expressing EBV latent proteins. Identify components of the transcription complex and determine how EBV proteins affect this complex. The proposed studies represent the first studies on a completely new pathway likely affected in EBV-associated cancers. These studies will determine the contribution of this pathway in the development of EBV-associated cancers and identify the specific effects of EBV proteins on the regulation of wnt/wingless signaling. A detailed understanding of the activation of this pathway may provide new opportunities for treatment of EBV-associated cancers and also of similar cancers considered EBV-negative, that develop in the immunosuppressed.
