Liccardi, G.; (2011) Nuclear EGFR modulation of DNA repair. Doctoral thesis , UCL (University College London).

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Abstract

Overexpression of the epidermal growth factor receptor (EGFR) is associated with resistance to chemotherapy and radiotherapy. EGFR involvement, in repair of radiation-induced DNA damage, is mediated by association with the catalytic subunit of DNA protein kinase (DNAPKcs). This study investigated the role of EGFR nuclear import, and its association with DNAPKcs, on DNA repair following treatment either with cisplatin or ionizing radiation (IR). EGFR- null murine NIH3T3 cells were transfected with wild type or with mutated EGFR (mutations found in human cancers L858R, EGFRvIII and mutations in the EGFR nuclear localization signal (NLS) sequence NLS123, LNLS123). Comet assay analysis, which measures unhooking of cisplatin crosslinks and repair of IR induced strand breaks, demonstrated that wtEGFR and EGFRvIII completely repair cisplatin and IR induced DNA damage. Immunoprecipitation studies show that repair is associated with the binding of both wtEGFR and EGFRvIII to DNAPKcs, which increases by 2- fold 18 hours following cisplatin treatment. Confocal analysis and proximity ligation assay indicated that this association takes place both in the cytoplasm and in the nucleus resulting in a significant increase of DNA-PK kinase activity. Intermediate levels of repair as shown by the L858R construct with impaired nuclear localization demonstrated that EGFR kinase activity is partially involved in repair but is not sufficient to determine EGFR nuclear expression. EGFR-NLS mutants showed impaired nuclear localization and impaired DNAPKcs association resulting in significant inhibition of DNA repair and downregulation of DNA-PK kinase activity. Our data suggest that EGFR nuclear localization is required for the modulation of cisplatin and IR induced DNA damage repair. The EGFR-DNAPKcs binding is triggered by cisplatin or IR and not by EGFR nuclear translocation per se. Understanding mechanisms regulating EGFR subcellular distribution in relation to DNA repair kinetics will be a critical determinant of improved molecular targeting and response to therapy.

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