The Mitogen-activated Protein Kinase (MAPK)/Extracellular Signal-regulated Kinase (ERK) 1/2 Kinase Inhibitor, AZD6244 (ARRY-142886) Enhances Radiosensitivity by Interfering with Radiation-induced TGF-alpha Signaling

Abstract

The MAPK/ERK pathway is important for cell proliferation, survival, and differentiation and is activated as a prosurvival pathway after irradiation (IR). AZD6244 is a potent, selective inhibitor of MEK 1/2 kinases. We previously reported that exposure to AZD6244 prior to IR enhanced radiosensitivity of cancer cell lines and xenografts, however the degree of sensitization varied among cell lines. We studied the role of soluble TGF-alpha in the degree of sensitization to IR with AZD6244. U251 (glioma) and A549 (non-small cell lung cancer) cell lines were used for all studies. Levels of soluble TGF-alpha in media from cells treated with AZD6244 +/- IR or vehicle +/- IR were assayed by ELISA. The importance of TGF-alpha on the radiation response was evaluated by treating cells with a TGF-alpha neutralizing antibody in clonogenic assays. Additional clonogenic assays were performed to determine the effects of AZD6244, recombinant human TGF-alpha, or the combination of AZD6244 and TGF-alpha on the in vitro radiosensitivity of the U251 and A549 cell lines. Phosphorylated Erk1/2 was evaluated after different treatments by immunoblotting. Mitotic catastrophe was measured using nuclear fragmentation. Basal and IR-induced TGF-alpha secretion was undetectable in U251 cells (not-sensitized with AZD6244). In A549 cells (sensitized), secretion of soluble TGF- alpha increased more than 2 fold at 24 h after IR compared to non-irradiated cells. Pretreatment of cell lines with AZD6244 reduced TGF-alpha production. The use of a neutralizing antibody to TGF-alpha reduced survival in A549 cells after IR. Conversely, the addition of TGF-alpha to AZD6244 treated A549 cells decreased the radiosensitizing effects of AZD6244. The addition of TGF-alpha to A549 cells treated with AZD6244 and IR resulted in similar levels of ERK phosphorylation and mitotic catastrophe as seen in cells treated with IR alone, in contrast to the elevated levels seen in cells treated with AZD6244 and IR. These results indicate that TGF-alpha is a survival factor in A549 cells after IR. AZD6244 enhanced radiosensitivity in vitro through an inhibition of TGF-alpha secretion leading to an increase in mitotic catastrophe.