Oncogenic Ras blocks anoikis by activation of a novel effector pathway independent of phosphatidylinositol 3-kinase

Activated Ras, but not Rat, causes transformation of RIE-1 rat intestinal e pithelial cells, demonstrating the importance of Raf-independent effector s ignaling in mediating Ras transformation. To further assess the contributio n of Raf-dependent and Raf-independent function in oncogenic Ras transforma tion, we evaluated the mechanism by which oneogenic Ras blocks suspension-i nduced apoptosis, or anoikis, of RIE-1 cells. We determined that oncogenic versions of H-, K-, and N-Ras, as well as the Ras-related proteins TC21 and R-Ras, protected RIE-1 cells from anoikis. Surprisingly, our analyses of R as effector domain mutants or constitutively activated effectors indicated that activation of Raf-1, phosphatidylinositol 3-kinase (PI3K), or RalGDS a lone is not sufficient to promote Ras inhibition of anoikis. Treatment of R as-transformed cells with the U0126 MEK inhibitor caused partial reversion to an anoikis-sensitive state, indicating that extracellular signal-regulat ed kinase activation contributes to inhibition of anoikis. Unexpectedly, on cogenic Ras failed to activate Akt, and treatment of Ras-transformed RIE-1 cells with the LY294002 P13K inhibitor did not affect anoikis resistance or growth in soft agar. Thus, while important for Ras transformation of fibro blasts. P13K may not be involved in Ras transformation of RIE-I cells. Fina lly, inhibition of epidermal growth factor receptor kinase activity did not overcome Ras inhibition of anoikis, indicating that this autocrine loop es sential for transformation is not involved in anoikis protection. We conclu de that a P13K- and RalGEF-independent Ras effector(s) likely cooperates wi th Raf to confer anoikis resistance upon RIE-1 cells, thus underscoring the complex nature by which Ras transforms cells.