Radioresistance of K-Ras mutated human tumor cells is mediated through EGFR-dependent activation of PI3K-AKT pathway

Background and purpose: In the context of EGFR-targeting strategies we investigated autocrine/paracrine factors leading to in vitro radioresistance of K-Ras mutated tumor cells through activation of EGFR mediated signal transduction. Patients and methods: Ras mutated (Ras(mt)) and normal Ras (Ras(wt)) presenting human tumor cell lines were used to analyze the potential of conditioned media (CM) of both cell types to mediate radioresistance and to activate EGFR-signaling cascades. Therefore, clonogenic assays as well as SDS-PAGE combined with immunoblotting was performed. Additionally, Ras-mutated cells were transfected with K-Ras-siRNA to investigate, how downregulation of mutated K-Ras affects secretion of EGFR-ligands, stimulation of EGFR-signating and modulation of radiation response. Results: TGF alpha, Amphiregulin (ARG) and CM from Ras(mt) cells (Ras(mt)-CM) resulted in an increased clonogenic survival of irradiated Ras(wt) cells. Both, EGFR ligands as well as Ras(mt)-CM led to a strong phosphorylation of EGFR and activation of downstream pathways, i.e. PI3K-AKT. However, neutralization of TGF alpha or ARG in Ras(mt)-CM led to a marked reduction of P-AKT. Furthermore, Ras(mt)-CM from K-Ras-siRNA transfected Ras(mt)-cells markedly inhibited phosphorylation of AKT in Ras(wt) cells and enhanced radiation sensitivity of A549 cells transfected with the siRNA. Conclusion: The data suggest that constitutively upregulated autocrine/paracrine secretion of EGF receptor ligands, especially ARG from K-Ras mutated cells, mediates radioresistance in Ras(mt)-cells through stimulation of EGFR-PI3K-AKT pathway. (C) 2005 Elsevier Ireland Ltd.