ErbB2 expression through heterodimerization with erbB1 is necessary for ionizing radiation- but not EGF-induced activation of Akt survival pathway

Purpose: ErbB1-dependent Akt phosphorylation improves post-irradiation cellular survival. In the present study, we investigated the contribution of erbB2 as a heterodimerization partner of erbB1 in activation of Akt survival signaling after irradiation or EGF treatment. Materials and methods: Pattern of receptor dimerization and protein phosphorylation were investigated by Western and immunoblotting as well as immunoprecipitation techniques. Residual DNA double-strand breaks (DNA-DSB) and clonogenic activity were analyzed by gamma H2AX and standard clonogenic assay. To knocked erbB2 expression siRNA was used. Results: In lung carcinoma cell lines A549 and H661, the erbB1-tyrosine kinase (TK) inhibitor erlotinib blocked EGF as well as ionizing radiation (IR)-induced Akt and DNA-PKcs phosphorylation. Targeting Akt and erbB1 induced cellular radiation sensitivity while, the erbB2-TK inhibitor AG825 neither affected phosphorylation of Akt and DNA-PKcs nor induced radiosensitization. ErbB2-siRNA and the anti-erbB2 antibody trastuzumab blocked IR-induced, but not EGF-stimulated Akt phosphorylation and impaired the repair of DNA-DSB. Likewise, IR but not EGF enhanced erbB1/erbB2 heterodimerization and resulted in the release of phosphorylated erbB2 cleavage products p135 and p95. Trastuzumab prevented radiation-induced formation of an active erbB1/erbB2 heterodimer and increased cellular radiation sensitivity. ErbB1- but not erbB2-TK inhibition stabilized erbB2 (p185) through preventing its cleavage. Conclusions: The data indicates that ErbB2 through heterodimerization with erbB1 is necessary for the activation of Akt signaling following irradiation but not following EGF treatment. (C) 2010 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 97 (2010) 338-345