Although aberrant Ras function is involved in a wide range of human malignancies, the mechanism of cellular transformation by Ras is still unclear. Recent work has elucidated many details of the Ras signal transduction pathway in which signals from the cell surface are received by membrane-associated tyrosine kinases and transmitted through Ras to a cytoplasmic cascade of serine/threonine kinases. Although this pathway leads to the nucleus to cause alterations in gene expression, little is currently known about the final steps that regulate gene transcription. The overall goal of the proposed research is to determine how oncogenic Ras regulates the expression of key cellular genes, and the consequences of this altered gene expression on neoplastic transformation. An important clue to identifying the critical gene targets of Ras transformation has been provided by the identification of oncogene responsive promoter elements (OREs). Whereas the best characterized OREs consist of two closely linked binding sites for transcription factors from the ETS, AP-1 (Jun, Fos) or NF-kB families, two adjacent Ets- binding sites can also serve as an ORE. In each case, both sites are require for ORE function. Although not all such """"""""ORE"""""""" sequences will define genes import for Ras transformation, the ability of Ets, Jun and R)s dominant negative proteins to block Ras transformation provides strong and compelling evidence that the critical genes deregulated by oncogenic Ras will contain these elements. Thus, it is vital to determine what constitutes an ORE, whether different Ras-mediated pathways activate different ORE-containing genes, how oncogenes alter the activity of transcription factors that bind to OREs, and whether the transforming and transactivation activities of Ras are directly related. The three specific aims of this proposal are: (l) Define the correlation between transactivation of ORE-containing genes and cellular transformation by oncogenic Ras. (2) Determine whether different Ras- mediated pathways trigger distinct transactivation events that account for their diverse physiological consequences. (3) Analyze the role of ETS domain proteins in Ras-mediated transformation and transactivation. To achieve, these goals, cotransfection analyses in rodent fibroblasts or human tumor cells will be used to study the transcriptional activation of reporter genes containing four different classes of OREs: two adjacent Ets, AP-1, or NF-kB sites, or single adjacent Ets and AP-1 binding sites. Activation of these reporter genes by Ras mutants with a range of transformation activities, as well as a variety of non-nuclear oncogenes, will be analyzed. Finally, the effect of Ras on the DNA binding of ETS family proteins in OREs will be characterized, along with the molecular basis for the requirement of OREs to possess two adjacent binding sites. Altogether, these studies will further define what constitutes an ORE, help determine the factors that contribute to ORE activation, lead to a better understanding of the biological role and molecular mechanisms of the transcriptional activation that occurs in Ras-mediated transformation. Finally, this analysis may identify new targets downstream of Ras for therapeutic intervention.
