Abstract 689: EGFR nuclear translocation modulates DNA repair following cisplatin or ionizing radiation treatment
Authors: Gianmaria Liccardi, John A. Hartley, Daniel Hochhauser
Journal: Cancer Research (2010)

Abstract Overexpression of the epidermal growth factor receptor (EGFR) is associated with resistance to chemotherapy and radiotherapy. Nuclear translocation of EGFR occurs through a tripartite nuclear localisation sequence (NLS). This process has been shown to enhance DNA repair in cells treated with ionising radiation (IR). The association of EGFR with the catalytic subunit of DNA protein kinase (DNAPKcs) contributes to this effect. The L858R mutation, found in tyrosine kinase inhibitor-sensitive non-small cell lung cancer, which shows reduced nuclear translocation, has been shown to sensitise cells to radiation. We investigated the role of EGFR nuclear import, and its association with DNAPKcs on DNA repair, following treatment with cisplatin or IR. NIH3T3 cells, which do not express EGFR, were transfected with plasmids expressing wild type EGFR (wtEGFR), L858R, EGFR variant III (EGFRvIII), wtEGFR with mutation in the NLS (NLS123) and an L858R construct with the same NLS mutation (LNLS123). Immunoprecipitation, western blot and confocal microscopy demonstrated that mutation of the NLS impairs EGFR nuclear translocation and activation following treatment with IR or cisplatin. Confocal microscopy confirmed the role of nuclear translocation in mediating association of EGFR with DNAPKcs following these treatments. Immunoprecipitation experiments showed association of EGFR with DNAPKcs only when EGFR is expressed in the nucleus. This association was shown to increase 2-fold by 18 hours following cisplatin treatment, and resulted in stimulation of DNA-PKcs activity as shown by a DNAPKcs kinase assay. The DNA interstrand crosslink (ICL) is a critical determinant of platinum cytotoxicity. Effects of nuclear translocation of EGFR on repair (unhooking) of cisplatin-induced DNA ICL and IR-induced DNA strand breaks were determined by the single cell agarose gel electrophoresis (comet assay). Unhooking of cisplatin-induced ICL was 100% at 36 hours in cells expressing wt EGFR and EGFRvIII. In contrast, in cells expressing L858R, unhooking was 46 ±3.8% as compared with peak levels of ICL. NLS123 showed 19.5 ±3.7% and LNLS123 16.5 ±5.2% unhooking at 36 hours. Additionally, following IR, repair of DNA strand breaks was 100% at 4 hours in cells expressing wtEGFR and EGFRvIII. In cells expressing the L858R, repair of DNA strand breaks was 88 ± 6.3% at four hours whereas NLS123 showed 75 ± 5.7% and LNLS123 73 ±3.9%. These results demonstrate that EGFR nuclear translocation and activation are involved in the modulation of DNA repair following treatment with cisplatin or IR. EGFR-DNAPKcs association and EGFR nuclear expression may be a critical determinant of response to chemotherapy or IR. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 689.