Importance of EGFR/ERCC1 interaction following radiation-induced DNA damage.
OBJECTIVE
The epidermal growth factor receptor (EGFR) plays an important role in cellular response to chemotherapy and radiotherapy through modulation of DNA repair. EGFR activates DNA-dependent protein kinase (DNA-PK) stimulating repair of DNA strand breaks (SB) and interstrand crosslinks (ICL). We investigated the role of EGFR in repair of ionizing radiation (IR)-induced SB independently of DNA-PK.


METHODS
The EGFR interactome was investigated via mass spectrometry. IR-induced EGFR-ERCC1 binding was validated biochemically and via proximity ligation assay in different cell lines including the M059K and M059J glioma cell lines, proficient and deficient for the expression of DNAPKcs, respectively. EGFR-ERCC1 functional significance following IR-induced SB was investigated in knockdown experiments with the Comet and γH2AX foci assays. The effect of this interaction was tested with EGFR-ERCC1 knockdown in combination with gefitinib and NU7026 using the MTT and apoptosis assays.


RESULTS
This study demonstrates that EGFR inhibition further impairs IR-induced DNA repair in cells lacking expression of DNAPKcs or in combination with the DNAPK inhibitor NU7026. Our data suggest a role for EGFR in DNA repair independent of DNAPKcs but dependent on ERCC1. Alkaline comet and γH2AX foci assays in cells depleted of EGFR, ERCC1, or EGFR-ERCC1 expression demonstrated involvement of this interaction in DNA repair. Cellular survival and apoptosis data correlate with levels of residual DNA damage underlying the importance of this complex following SB.


CONCLUSIONS
These data emphasize the importance of understanding the various mechanisms by which EGFR modulates DNA repair to optimize targeted therapy for patients with cancer.
Authors' Affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, United Kingdom.
Gianmaria
G
Liccardi
Authors' Affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, United Kingdom.
John A
JA
Hartley
Authors' Affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, United Kingdom d.hochhauser@ucl.ac.uk.
Daniel
D
Hochhauser
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011839:Radiation, Ionizing
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D066246:ErbB Receptors; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C071447:ERCC1 protein, human; D004720:Endonucleases