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

• Published in: Clinical cancer research 2006-07, Vol.12 (13), p.4119-4126
• Main Authors: Toulany, Mahmoud, Kasten-Pisula, Ulla, Brammer, Ingo, Wang, Shaomeng, Chen, Jianyong, Dittmann, Klaus, Baumann, Michael, Dikomey, Ekkehard, Rodemann, H Peter
• Format: Article
• Language: English
• Subjects: Carcinoma - drug therapy; Carcinoma - genetics; Carcinoma - radiotherapy; Cell Line, Tumor; Chromones - pharmacology; DNA repair; DNA Repair - drug effects; Dose-Response Relationship, Drug; EGFR; Ellipticines - pharmacology; Enzyme Inhibitors - pharmacology; Genes, ras - genetics; H2AX; Humans; Morpholines - pharmacology; Mutation; Organic Chemicals - pharmacology; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; PI3K-AKT; Proto-Oncogene Proteins c-akt - antagonists & inhibitors; Radiation Tolerance - drug effects; radiosensitization; Receptor, Epidermal Growth Factor - antagonists & inhibitors; Signal Transduction - drug effects; Structure-Activity Relationship
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.CCR-05-2454

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 (Ser 139 ), ATM (Ser 1981 ), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr 2609 ) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of γH2AX 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 Thr 2609 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.