EGFR Nuclear Translocation Modulates DNA Repair following Cisplatin and Ionizing Radiation Treatment

Abstract

Epidermal growth factor receptor (EGFR) overexpression is associated with resistance to chemotherapy and radiotherapy. It modulates DNA repair after radiation-induced damage through association with the catalytic subunit of DNA protein kinase (DNA-PKcs). We investigated the role of EGFR nuclear import and its association with DNA-PKcs on DNA repair after exposure to cisplatin or ionizing radiation (IR). The model system was based on EGFR-null murine NIH3T3 fibroblasts in which EGFR expression was restored with isoforms that were wild-type (wt), derived from human cancers (L858R, EGFRvIII), or mutated in the nuclear localization signal (NLS) sequence. In cells expressing wtEGFR or EGFRvIII, there was complete unhooking of cisplatin-induced interstrand cross-links and repair of IR-induced strand breaks. In contrast, cells expressing L858R or NLS mutations showed reduced unhooking of interstrand cross-links and repair of strand breaks. Immunoprecipitation showed wtEGFR and EGFRvIII binding to DNA-PKcs, increasing 2-fold 18 hours after cisplatin therapy. Confocal microscopy and proximity ligation assay showed that this interaction in the cytoplasm and nucleus was associated with increased DNA protein kinase complex (DNA-PK) activity. Cells expressing the EGFR L858R mutation, which has constitutive kinase activity, exhibited reduced DNA repair without nuclear localization. EGFR-NLS mutants showed impaired nuclear localization and DNA-PKcs association with reduced DNA repair and DNA-PK kinase activity. In summary, EGFR nuclear localization was required for modulation of cisplatin and IR-induced repair of DNA damage. EGFR-DNA-PKcs binding was induced by cisplatin or IR but not by EGFR nuclear translocation per se. Our findings show that EGFR subcellular distribution can modulate DNA repair kinetics, with implications for design of EGFR-targeted combinational therapies.

RON Nuclear Translocation under Hypoxia Potentiates Chemoresistance to DNA Double-Strand Break–Inducing Anticancer Drugs

“…In contrast, experiments using functional deletion mutants of RON in J82-stable cell lines provided additional support of the survival advantage of nuclear RON for cancer cells under hypoxia. Because nuclear EGFR-induced activation of NHEJ repair leads to cisplatin resistance (38,40), our data suggest that DSB-inducing anticancer drugs are not recommended for treating patients with RON-overexpressing cancer.…”

“…EGFR is the most well-known example of the nuclear import of RTK. Nuclear EGFR stimulates tumorigenesis, partly because it activates NHEJ repair by interacting with Ku70 and DNA-PKcs in response to ionizing radiation and cisplatin treatments (37)(38)(39)(40). We recently reported (41,42) that nuclear RON provided a survival advantage for cancer cells in response to serum starvation and hypoxia (41,42).…”

Role of epidermal growth factor receptor in DNA damage repair

“…Radicals generated by ionizing radiation are also involved in the induction of the transport process [2,[5][6][7][8]. A model of integral trafficking from the endoplasmic reticulum (ER) to the nuclear envelope (NE) transport (INTERNET) provides another scenario for the nuclear translocation of EGFR, that is, integral inner nuclear membrane (INM) proteins initially inserted into the ER membrane are targeted to the INM of the NE through the outer nuclear membrane (ONM) and nuclear pore complex (NPC).…”

“…DNA-PKcs and Ku70/ Ku80 are considered predominantly nuclear proteins, but there are reports that DNA-PK subunits are independently localized in lipid rafts, where they may interact with membrane proteins also localized in lipid rafts. Ionizing radiation can induce translocation of DNA-PKcs from the nucleus to the cytosol [8]. In the human bronchial carcinoma cell line A549, the human squamous carcinoma cell line FaDu and the transformed human fibroblast cell line HH4dd, ionizing radiation results in a clear translocation of the perinuclear EGFR to the nucleus along with the nuclear import of Ku subunits 80 and 70 and phosphatase PP1 [5,7].…”

“…DNA-PKcs plays a critical role in EGFR-mediated response to radiation, because in a DNA-PKcs-deficient genetic background, the radioprotective effect of EGFR is lost. Thus, interactions with DNA-PKcs constitute a critical component of EGFRmediated radioprotection [8,15].…”