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Blockage of Epidermal Growth Factor Receptor-Phosphatidylinositol 3-Kinase-AKT Signaling Increases Radiosensitivity of K-RAS Mutated Human Tumor Cells In vitro by Affecting DNA Repair

Authors' Affiliations: ¹ Divison of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany; ² Department of Radiotherapy and Radiooncology, Laboratory of Experimental Radiation Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; ³ Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan; and ⁴ Department of Radiation Oncology, University of Dresden, Dresden, Germany

Requests for reprints: H. Peter Rodemann, Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, Eberhard-Karls University Tuebingen, Roentgenweg 11, 72076 Tuebingen, Germany. Phone: 49-0-7071-29-85962; Fax: 49-0-7071-29-5900; E-mail: hans-peter.rodemann{at}uni-tuebingen.de.

Purpose: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA double-strand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS_mt) cells.

Experimental Design: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS_mt (A549) and K-RAS_wt (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser¹³⁹), ATM (Ser¹⁹⁸¹), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr²⁶⁰⁹) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of H2AX foci and frequency of micronuclei.

Results: BIBX reduced clonogenic survival of K-RAS_mt-A549 cells, but not of K-RAS_wt-FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS_mt-A549 and MDA-MB-231 cells, but not in K-RAS_wt-FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr²⁶⁰⁹ was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS_mt nor in K-RAS_wt cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation.

Conclusion: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.

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