A RAF-INDEPENDENT EPIDERMAL GROWTH-FACTOR RECEPTOR AUTOCRINE LOOP IS NECESSARY FOR RAS TRANSFORMATION OF RAT INTESTINAL EPITHELIAL-CELLS

We recently have shown that activated Ras, but not Raf, causes transfo rmation of intestinal (RIE-1, IEC-6) epithelial cells, whereas both ac tivated Ras and Raf transform NIH 3T3 fibroblasts (Oldham, S. M., Clar k, G. J., Gangarosa, L. M., Coffey, R. J., and Her, C. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6924-6928). The observations that condi tioned medium from Ras-, but not Raf-, transfected RIE-1 cells, as wel l as exogenous transforming growth factor alpha (TGF alpha), promoted morphological transformation of parental RIE-1 cells prompted us to id entify epidermal growth factor (EGF) receptor (EGFR) ligands produced by Ras-transformed RIE-1 cells responsible for this autocrine effect. Since studies in fibroblasts have shown that v-Src is transforming, we also determined if v-Src could transform RIE-1 cells. H- or K-Ras-tra nsformed cells secreted significant amounts of TGF alpha protein, and mRNA transcripts for TGF alpha, amphiregulin (AR), and heparin-binding EGF-like growth factor (HB-EGF) were induced. Like Ras, v-Src caused morphological and growth transformation of parental RIE-1 cells. Howev er, TGF alpha protein was not secreted by RIE-1 cells stably expressin g v-Src or activated Raf, and only minor increases in EGFR ligand mRNA expression were detected in these cells. A selective EGFR tyrosine ki nase inhibitor PD153035 attenuated the Ras-, but not Src-, transformed phenotype. Taken together, these observations provide a mechanistic a nd biochemical basis for the ability of activated Ras, but not activat ed Raf, to cause transformation of RIE-1 cells. Finally, we suggest th at an EGFR dependent mechanism is necessary for Ras, but not Src, tran sformation of these intestinal epithelial cells.