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


METHODS
Ras mutated (Rasmt) and normal Ras (Raswt) 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
TGFalpha, Amphiregulin (ARG) and CM from Rasmt cells (Rasmt-CM) resulted in an increased clonogenic survival of irradiated Raswt cells. Both, EGFR ligands as well as Rasmt-CM led to a strong phosphorylation of EGFR and activation of downstream pathways, i.e. PI3K-AKT. However, neutralization of TGFalpha or ARG in Rasmt-CM led to a marked reduction of P-AKT. Furthermore, Rasmt-CM from K-Ras-siRNA transfected Rasmt-cells markedly inhibited phosphorylation of AKT in Raswt cells and enhanced radiation sensitivity of A549 cells transfected with the siRNA.


CONCLUSIONS
The data suggest that constitutively upregulated autocrine/paracrine secretion of EGF receptor ligands, especially ARG from K-Ras mutated cells, mediates radioresistance in Rasmt-cells through stimulation of EGFR-PI3K-AKT pathway.
Toulany
Mahmoud
M
Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Germany.
Dittmann
Klaus
K
Krüger
Maren
M
Baumann
Michael
M
Rodemann
H Peter
HP
D066258:Amphiregulin; D045744:Cell Line, Tumor / Q000528:radiation effects*; D017077:Culture Media, Conditioned; D066255:EGF Family of Proteins; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D011905:Genes, ras; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011836:Radiation Tolerance; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
C587848:AREG protein, human; D066258:Amphiregulin; D017077:Culture Media, Conditioned; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors