Aberrant Ras function contributes to the development of a wide spectrum of human cancers. Mutated Ras proteins cause deregulated signaling to the nucleus and stimulation of cell proliferation. Although many of the details of Ras signaling have now been discovered (e.g. the Raf/MEK/ERK cascade), our understanding of the genes whose expression are deregulated by Ras remain poorly understood. Some of the most downstream characterized targets of oncogenic Ras are transcription factors, including the Ets family, whose altered activities can mediate widespread changes in gene expression. Ras signaling increases the transcriptional activity of several members of the Ets family, including Ets2. The ability of two distinct types of dominant inhibitory Ets2 constructs to reverse Ras-meditated fibroblast transformation by yet uncharacterized mechanisms, has indicated that Ets proteins are essential mediators of Ras transformation. This stable reversal of the Ras transformed phenotype through altered Ets activity occurs without inhibition of normal cell growth. Likely through a distinct mechanism, high level stimulation of Ets-dependent transcription can also specifically reverse Ras transformation. These findings with broad acting inhibitors or activators of Ets function, suggest that the Ets family has multiple roles in signaling to cellular transformation. There is recent evidence that Ets signaling is also important in human tumor cells, and could additionally regulate features such as invasiveness and apoptosis. Because reversal of transformation by dominant acting Ets constructs likely acts through steps well downstream of Ras, analysis of the mechanisms of this reversion provides a framework to both characterize the functions of Ets family proteins in transformation, and to identify downstream targets essential in Ras- mediated cellular transformation. Therefore, we propose to apply such analysis to characterize the mechanisms by which Ets proteins mediate Ras transformation, including DNA binding, transcriptional activation and repression, or protein-protein interactions. In addition, we will determine whether Ets proteins function similarly in mediating the transformed phenotype of Ras-associated human tumor cells. Finally, downstream targets of Ets involved in cellular transformation will be identified and characterized by two distinct approaches. Together, these studies will provide insight into the role of Ets factors in cellular transformation, further elucidate the pathways leading to tumorigenicity, and may lead to the identification of novel and more specific downstream targets for intervention in human cancers.
