Abstract B240: Combined gefitinib and API-2 induced interaction of DNA-PK with EGFR and Akt, leading to impaired DNA-PK-mediated DNA repair and increased autophagy.

Abstract

Abstract Resistance to epidermal growth factor receptor (EGFR) inhibitors and pro-apoptotic agents frequently occurs in malignant glioma due to excessive stimulation of growth factor receptors (eg. EGFR) and up-regulation of PI3K/Akt pathway. We aimed to overcome resistance of glioblastoma cells to gefitinib (an EGFR inhibitor) with API-2 (a selective Akt inhibitor) by increasing autophagy cell death. We investigated whether combined gefitinib and API-2 induced interaction of DNA-PK with EGFR and Akt, leading to impaired DNA-protein kinase (DNA-PK)-mediated DNA repair and autophagy. Methods: U251MG, MO59J (DNA-PKcs-deficient) and MO59K (DNA-PKcs-proficient) cells treated with gefitinib, API-2 or both for 72 hours were analyzed by cell viability (MTT assay) and autophagy (FACS of acridine orange staining and LC3 expression) assays. U251MG cells treated with gefitinib and API-2 were assayed for nuclear expression of phosphorylated and native DNA-PKcs, Akt and EGFR, as well as DNA repair activity (immunostaining of γ-H2AX, a marker of DNA damage). Interaction of DNA-PK and EGFR/Akt in gefitinib and API-2-treated cells were analyzed by proximity ligation assay. Findings: Combined gefitinib and API-2 significantly reduced viability of U251MG and MO59J cells (but not MO59K cells), compared with gefitinib alone. Single treatments of gefitinib and API-2 increased acridine orange staining and LC3-II expression in U251MG cells, with further increase by combined gefitinib and API-2. Increased acridine orange staining by combined gefitinib and API-2 was greater in MO59J than MO59K cells. In U251MG cells, combined gefitinib and API-2 significantly inhibited nuclear phosphorylation of DNA-PKcs (S2056), EGFR (T1173) and Akt (S473), compared with single therapies. Gefitinib and API-2 independently impaired DNA repair (time-dependent increase of γ-H2AX immunostaining) of U251MG cells, with further inhibition by combined gefitinib and API-2. There was an increased binding of DNA-PKcs with EGFR and Akt following combined gefitinib and API-2 treatment (compared to single agents) in U251MG cells. Conclusion: Our findings reveal that API-2 enhances chemosensitivity of gefitinib by inducing interaction of DNA-PKcs with EGFR and Akt, leading to impaired DNA-PK-mediated DNA repair and induction of autophagic cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B240.