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

Abstract

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-signaling and modulation of radiation response. Results TGFα, 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 TGFα 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.