TGF-BETA REGULATION OF INTESTINAL EPITHELIAL CELLS

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Colorectal neoplasia is the second leading cause of cancer death in the United States. Identification of specific molecular "lesions" in colorectal tumors cell has elevated the prospects for design of more specific treatments. The interface between two specific molecular lesions are proposed for study herein. The general goal is to identify mechanisms by which Ras, a mutationally activated gene in about 50 percent of colorectal cancers, decreases the activity of a potent tumor suppressor systems, the transforming growth factor beta (TGFbeta) ligand/receptor axis. The end result is unregulated growth due to TGFbeta resistance. The rate intestinal epithelial cell line, RIE-1 and a battery of transfected RIE-1 clones and human colon carcinoma cells will be used. Six Specific Aims are proposed: 1) a step-wise analysis of the impact of Ras over expression of TGF beta signaling in the Smad pathway from the cells membrane to the nucleus will be performed; 2) related observations on TGFbeta signaling will be made in human colon carcinoma cells and correlated with Ras activity; 3) and 4). The effects of two Ras- stimulated growth factor pathways, the epidermal growth factor pathway and TGFbeta ligand production on TGFbeta resistance will be studied; 5) the reversibility of Ras transformation and reversion of the neoplastic phenotype will be tested by transfection of a functional TGFbetaRII gene into RIE-Ras cells and 6) preliminary studies described herein support Ras-mediated TGFbeta resistance reduction of TGFbetaRII occurs by a Raf- independent pathway leading us to further study of the conventional Raf/MAPKK/MAPK signaling pathway, the phosphatidylinositol 3 kinase pathway and the Rho pathway. The techniques employed herein are generally standard techniques such as cellular transfection, growth assays, Northern analysis, and Western analysis. The proposal is unique in its collection and use of a large variety of transfected cell lines expressing key, relevant components of the Ras effector system. The long term goal of this project is to identify signaling pathways eligible for novel strategies for therapeutic intervention in colorectal cancer and to further understanding of the interaction between Ras and TGFbeta signaling.