egfr nuclear translocation modulates dna repair following...

Therapeutics, Targets, and Chemical Biology

EGFR Nuclear Translocation Modulates DNA Repairfollowing Cisplatin and Ionizing Radiation Treatment

Gianmaria Liccardi, John A. Hartley, and Daniel Hochhauser

AbstractEpidermal 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 catalyticsubunit of DNA protein kinase (DNA-PKcs). We investigated the role of EGFR nuclear import and its associationwithDNA-PKcs onDNA repair after exposure to cisplatin or ionizing radiation (IR). Themodel systemwas based onEGFR-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-linksand repair of IR-induced strand breaks. In contrast, cells expressing L858R or NLS mutations showed reducedunhooking of interstrand cross-links and repair of strand breaks. Immunoprecipitation showed wtEGFR andEGFRvIII binding to DNA-PKcs, increasing 2-fold 18 hours after cisplatin therapy. Confocal microscopy andproximity ligation assay showed that this interaction in the cytoplasm and nucleus was associated with increasedDNA protein kinase complex (DNA-PK) activity. Cells expressing the EGFR L858Rmutation, which has constitutivekinase activity, exhibited reduced DNA repair without nuclear localization. EGFR-NLS mutants showed impairednuclear localization and DNA-PKcs associationwith 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 showthat EGFR subcellular distribution canmodulateDNA repair kinetics,with implications for design of EGFR-targetedcombinational therapies. Cancer Res; 71(3); 1103–14. �2011 AACR.

Introduction

The epidermal growth factor receptor (EGFR) promotes theactivation of survival signaling pathways including RAS/MAPK (mitogen activated protein kinase), PI3K (phosphoi-nositide 3-kinase)/AKT, and JAK (Janus activated kinase)/STAT (1, 2). Increased EGFR activation and overexpressionis strongly associated with tumorigenesis and cancer progres-sion (3). EGFR is an important target for cancer therapiesincluding antibodies disrupting ligand–receptor interactionssuch as cetuximab (4, 5) and small molecules inhibiting EGFRkinase activity including gefitinib (6, 7) and erlotinib (8). Therehas been extensive investigation on the mechanisms by whichEGFR inhibition modulates the activity of chemotherapy and

radiation (3). Combinations of the monoclonal antibody(mAb) cetuximab with cisplatin or radiation have been usefulclinically in the treatment of head and neck and colon cancer(9, 10). In contrast, despite effects in vitro (11), only smallbenefits have been obtained combining small molecules inhi-biting EGFRwith conventional treatment. Several studies haveshown the association of EGFR with the catalytic subunit ofDNA-dependent protein kinase (DNA-PKcs), a central com-ponent of the nonhomologous end-joining (NHEJ) pathwayinvolved in the repair of DNA strand breaks (12, 13).

Recently, it has been reported that a fraction of intracellularEGFR is located within the nucleus where it may activatetranscription of genes, including cyclin D1, iNOS, c-myb, andCOX-2, that are associated with cell proliferation and the nitricoxide pathway (14–17). Evidence for the expression andactivity of nuclear EGFR has been found using a variety oftechniques including fractional immunoblotting, confocalmicroscopy, electron microscopy, reporter assays, and chro-matin immunoprecipitation. Nuclear EGFR has been shown tocorrelate with worse prognosis in a variety of malignanciesincluding breast, head and neck, and ovarian cancer (16, 18).The intracellular localization of EGFR may therefore haveprofound effects on response both to chemotherapy and tonovel therapies inhibiting the EGFR pathway.

EGFR cellular distribution is dictated by several regulatorymotifs within the juxtamembrane (JX) domain (19). Two

Authors' Affiliation: Cancer Research UK Drug-DNA InteractionsResearch Group, UCL Cancer Institute, University College London, Lon-don, United Kingdom

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Daniel Hochhauser, UCL Cancer Institute, PaulO’Gorman Building, University College London, 72 Huntley Street, LondonWC1E 6BT, United Kingdom. Phone: 44-20-7679-6006; Fax: 44-20-7679-6925; E-mail: d.hochhauser@ucl.ac.uk

doi: 10.1158/0008-5472.CAN-10-2384

�2011 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1103

Published OnlineFirst January 25, 2011; DOI: 10.1158/0008-5472.CAN-10-2384

basolateral signals control EGFR re-sorting to the transmem-brane (20), whereas the lysosomal signal (accessible followingEGFR activation) determines EGFR degradation (21). Thenuclear localization signal (NLS) sequence, comprising 13amino acids 645 to 657 (RRRHIVRKLLRR; ref. 22), has a dualrole. It allows nuclear translocation via sequence recognitionand binding to importin b (23) and mediates EGFR allostericconformational change and dimer stabilization, which areindispensable for the receptor activation (24, 25).

Studies on the somatically acquired, constitutively activeEGFR mutant L858R, found in certain non–small cell lungcancers, have shown impaired nuclear localization and DNA-PKcs binding (26). This suggests that EGFR activation andnuclear translocation are related and that nuclear localizationmay modulate DNA repair.

Nuclear translocation of EGFR following ionizing radia-tion (IR) has been shown to result in increased repair of DNAstrand breaks (12). The effects of nuclear translocation onrepair of chemotherapy-induced DNA damage are less clear.We therefore investigated the significance of nuclear loca-lization for the repair of cisplatin- and IR-induced DNAdamage, using EGFR constructs with mutations in theNLS and mutations found in human cancers (EGFRvIII,L858R). Cells expressing EGFR with mutations that impairnuclear transport showed reduced repair of DNA strandbreaks following IR and reduced unhooking of interstrandcross-links following treatment with cisplatin, as comparedwith cells expressing wild-type (wt) EGFR. Immunoprecipi-tation experiments confirmed association of EGFR withDNA-PKcs following treatment with cisplatin or IR. Confocalmicroscopy demonstrated that cells with mutations in theNLS failed to translocate to the nucleus following IR andcisplatin treatment. These findings confirm the importanceof nuclear translocation of EGFR in mediating effects onDNA repair and emphasize the significance of subcellularEGFR expression in determining responses to therapy.

Materials and Methods

MaterialsCisplatin (DBL 1 mg/mL) was obtained from Mayne

Pharma PLC. Epidermal growth factor (EGF) was obtainedfrom Sigma-Aldrich.

Irradiation conditionCells were plated at a concentration of 1 � 105/mL. Follow-

ing 48 hours transfection, cells were serum starved for 24hours and irradiated with a dose of 4 Gy with the A.G.O. HS321kV X-ray system.

Cell lines and culture conditionsNIH3T3 mouse fibroblast cell lines (obtained from CR-UK

London Research Institute) were grown in Dulbecco's minimalessential medium (Autogen Bioclear). Transfected NIH3T3cells were grown in the same medium containing G418-selective agent (Sigma-Aldrich) at a concentration of 1 mg/mL. All cells were supplemented with 10% fetal calf serum and1% glutamine and incubated at 37�C in 5% CO2.

Plasmids and site-directed mutagenesisThe plasmid DNA used was the pUSEamp vector (Upstate

Cell Signaling Solutions).The wtEGFR and the L858R constructs were kindly pro-

vided by Dr. Daphne Bell and Matthew Meyerson from theMGH Cancer Centre, Harvard Medical School, Boston, MA.

The NLS mutant constructed used the wtEGFR and theL858R plasmids as a template. The 2 designed and SDS-purified mutagenic primers (forward: 50-CCTCTTCATGG-CAGCGGCCCACATCGTTGCGAAGGCCACGCTGGCGGCGC-TGCTGCAGG-30; and reverse: 50-CCTGCAGCAGCGCCGCCA-GCGTGGCCTTCGCAACGATGTGGGCCGCTGCCATGAAGA-GG-30) were utilized according to the site-directed mutagen-esis XL kit protocol (Stratagene) to change the EGFR NLSsequence 645-RRRHIVRKRTLRR-657 into 645-AAAHIVAKA-TLAA-657.

EGFRvIII was kindly provided by Prof. William Gullick fromthe Department of Biosciences University of Kent, Canterbury,UK.

EGFR M1, M12, KMT, and DNLS encoding point mutationsof the EGFR NLS sequence (M1: AAAHIVRKRTLRR; M12:AAAHIVAAATLRR), the deletion of the NLS sequence (DNLS),and a mutation within the kinase domain (KMT: K821A) werekindly obtained from Prof. M.C. Hung (MD Anderson CancerCenter, Houston, TX).

Plasmid transfectionCells were plated at 1� 105/mL, and following 2 hours, cells

were transfected according to the GeneJuice transfectionreagent protocol (Novagen EMD Bioscience). Cells were thentreated 48 hours following transfection, or serum starved for24 hours and then treated.

Alkaline single-cell gel electrophoresis (comet) assayMeasurement of DNA interstrand cross-links was done as

previously described (27).Repair of DNA strand break in cells irradiated with a dose of

15 Gy was measured using the comet assay as previouslydescribed (28).

Data were presented as a percentage of tail moment, that is,as a percentage of the amount of strand breaks resultingimmediately following IR treatment.

Statistical analysisThe 2-way ANOVA, Bonferroni's posttests, and Student's t

test were used for calculating the significance of the differ-ences in repair and DNA-PK activity. All the cell lines wereconsidered individually and compared with the wtEGFR-expressing cell line. Statistical values of P < 0.01 were con-sidered significant.

ImmunoprecipitationStably expressing NIH3T3 cell lines were plated at 2 � 105/

mL and left overnight before treatment. Cells were thenincubated in growing medium or treated with 50 mmol/Lcisplatin for 1 hour in serum-free media and then left indrug-free medium for 18 hours, or serum starved for 24 to 36hours and treated with 100 ng/mL EGF or treated with 4-Gy IR

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Cancer Res; 71(3) February 1, 2011 Cancer Research1104

and incubated 20 minutes in serum-free media. Approxi-mately 5 � 106 cells were lysed on ice in 500 mL of CelLyticMCell lysis reagent (Sigma) supplemented with protease andphosphatase inhibitor (Roche) and Benzonase (Merck)according to manufacturer's protocol. A 1.5 mg sample ofprotein was incubated with 2 mg of anti-EGFR antibody (cloneR19/48; Invitrogen) and left rotating at 4�C for 2.5 hours.Immunoprecipitation was done as previously described (29).

Western blottingWestern blotting was done as previously described (27).

Proteins were probed using anti-EGFR (Cell Signaling; 1:1,000),anti-DNA-PKcs (AbCam; 1:400), anti-PY20 (Santa Cruz;1:1,000). Finally, the primary antibody was probed with horse-radish peroxidase–conjugated polyclonal antibodies for che-miluminescence detection with the ECL System (AmershamBiosciences).

Immunofluorescence stainingA total of 2 �104 cells were plated on 13-mm glass cover

slips (VWR). Cells were then treated with cisplatin and IR asdetailed earlier and stained as previously described (26).Respective primary antibodies were added as follows: anti-rabbit EGFR (1:50, clone 15F8; Cell Signaling) and anti-mouseDNA-PKcs (1:50; AbCam). Then, secondary fluorescent con-jugated antibodies were added as follows: 1:100 Alexa Fluor647 goat anti-rabbit and 1:100 Alexa Fluor 488 goat anti-mouse(Molecular Probes and Invitrogen Life Technologies). Cellswere visualized by confocal microscopy (objective: �40; LeicaTCS SP2). Nuclear slice images were acquired by sequentialscanning by using the LAS AF Lite program.

DNA-PK functional assayDNA-PK activity was detected using the Promega Signa-

TECT DNA-PK assay system, according to the manufacturer'sprotocol. The enzymatic activity of DNA-PK was analyzed byscintillation counting and expressed as a percentage change ofcontrol DNA-PK activity as measured in untreated cells.

Proximity ligation assayProximity ligation was done according to the man-

ufacturer's protocol, using the Duolink Detection Kit (Cam-bridge BioScience Ltd.). NIH3T3 cells were grown on 13-mmglass cover slips (VWR) and treated with cisplatin or IR asdetailed earlier. Immunofluorescence staining protocol wascarried out until the primary antibody incubation. Cy3 signalamplification was utilized for the assay. Cells were examinedwith a confocal microscope (objective: �40; Leica TCS SP2).

Results

EGFR nuclear translocation modulates DNA repairThe synergistic effects of both cisplatin and IR with EGFR

inhibition have been well described (12, 23). However, the roleand consequences of EGFR nuclear translocation and kinaseactivation in the repair of drug-induced DNA interstrandcross-links, or radiation-induced DNA strand breaks, havenot been fully examined. To probe the role of EGFR in

modulating DNA repair, the EGFR-negative cell line NIH3T3was transfected with each of 10 plasmids [wtEGFR, NLS123,L858R, LNLS123, EGFRvIII, M1, M12, KMT, DNLS, and vectorcontrol (VC)], and therapy-induced DNA damage and itsrepair were assessed. The resulting transfectants expresseither wtEGFR or mutations within the NLS sequence(NLS123, LNLS123, M1, M12, and DNLS), kinase domain(L858R, KMT), or extracellular domain (EGFRvIII) of EGFR(Fig. 1A). Their expression was confirmed over a period of 72hours following transfection (Supplementary Data S1). Trans-fected cells were incubated with 50 mmol/L cisplatin for 1 houror treated with 15 Gy IR. Formation and repair of cisplatin-induced interstrand cross-links, critical cytotoxic lesions pro-duced following drug treatment, were measured over a 48-hour period by a modification of the comet assay as previouslydescribed (ref. 27; Fig. 1B). Repair of IR-induced strand breakswas measured over a 4-hour period (Fig. 1C).

There was no alteration in the formation of the peak of DNAinterstrand cross-links by cisplatin in cells expressing any ofthe constructs (Supplementary Fig. S2 and SupplementaryTable S1). However, cells expressing mutations of the NLSsequence (NLS123, LNLS123) and of the kinase domain(L858R, KMT, DNLS) clearly showed reduction in repair(unhooking) of interstrand cross-links (Fig. 1B). Unhookingof cisplatin-induced interstrand cross-links was greater than95% by 36 hours and complete by 48 hours in cells expressingwtEGFR and EGFRvIII. In contrast, cells expressing mutationsNLS123 and LNLS123 showed only 26% � 4.07% and 19.3% �4.8% unhooking at 48 hours, respectively. Intermediate levelsof unhooking were observed for the L858R (46.57% � 2.13%),KMT (54.95% � 2,56%), and DNLS (57.21% � 9.72%) mutants,whereas M1- and M12-expressing cell lines showed 84.09% �4.87% and 97.41% � 2.73% unhooking of interstrand cross-links, respectively, at 48 hours following cisplatin treatment.Statistical analysis showed significance (P < 0.01) at the 48-hour time points and/or at earlier time points among thedifferent mutants (Table 2A Supplementary Data).

The effect of the EGFR constructs on repair of DNA strandbreaks induced by IR was also investigated. Repair of IR-induced DNA strand breaks is shown as the percentage of theIR-induced tail moment calculated from the comet assay data(Fig. 1C). Decrease of tail moment was 100% in both wtEGFR-and EGFRvIII-expressing cell lines at 4 hours following treat-ment, indicating complete repair of strand breaks. Significantdifferences (P < 0.001) in repair kinetics between wtEGFR andthe mutant EGFR-expressing cell lines were found at 30minutes following IR (Table 2B Supplementary Data). At 4hours, cells expressing NLS123 and LNLS123 showed signifi-cant delay in repair of strand breaks with 22.48% � 3.72% and24.94 � 1.45% unrepaired strand breaks, respectively. Inter-mediate levels of repair were observed for cells expressingL858R (12.33 � 1.00%), KMT (18.86 � 3.45%), DNLS (17.38 �5.06%), M1 (8.51 � 1.12%), and M12 (9.28 � 2.26%) plasmids.

wtEGFR and EGFRVIII associate with DNA-PKcsfollowing treatment with IR or cisplatin

Previous studies have shown association of EGFR and DNA-PKcs following IR treatment (12, 30, 31). However, this

EGFR Nuclear Localization and DNA Repair

www.aacrjournals.org Cancer Res; 71(3) February 1, 2011 1105

association and its significance have not been describedfollowing cisplatin treatment. NIH3T3 cells were transfectedwith wtEGFR and treated with 50 mmol/L cisplatin for 1 hour.Cells were then collected at various time points up to 24 hoursfollowing treatment, protein extracts were prepared, immu-noprecipitated using an anti-DNA-PKcs mAb, and blottedwith an anti-EGFR antibody (Fig. 2A). There was a time-dependent association of EGFR and DNA-PKcs, resulting

in a 2.7-fold increase at 18 hours following the cisplatintreatment.

To determine the levels of EGFR–DNA-PKcs associationand whether it is induced by cisplatin or IR, we compared thelevels of this association following cisplatin, IR, and EGFtreatment. Experiments were carried out using extracts fromcells expressing wtEGFR, NLS123, L858R, LNLS123, and EGFR-vIII (Fig. 2B), immunoprecipitated using an anti-EGFR

0 1 2 3 4

12 24 36 48

Time (h)

wtEGFR

NLS123

LNLS123

EGFRvIII

wtEGFR

NLS123

LNLS123

EGFRvIII

constitutive activeconstitutive active

Figure 1. Effects of EGFRmodulation in repair of interstrandcross-links and DNA strandbreaks. A, graphic representationof the EGFR constructs employedin the study. B and C,measurement of drug-inducedDNA interstrand cross-links orstrand breaks in NIH3T3 cellstransfected with wtEGFR,NLS123, L858R, LNLS123,EGFRvIII, M1, M12, KMT, DNLS,and VC were treated with (B) 50mmol/L cisplatin alone and (C) 15-Gy IR. Interstrand cross-linkformation is represented as apercentage decrease of the peakof cross-link, and strand breaksare shown as a percentage of tailmoment.

Liccardi et al.

antibody, and blotted using an anti-DNA-PKcs antibody.Western blot analysis showed that in cells expressing wtEGFRand EGFRvIII, there is association with DNA-PKcs followingtreatment with IR or cisplatin. However, cells expressingNLS123, L858R, and LNLS123 showed no interaction betweenEGFR and DNA-PKcs. The immunoprecipitated samples werealso blotted with a pan-phosphotyrosine antibody to deter-mine the activity of EGFR. Cells expressing wtEGFR showedmaximal activation of the receptor following EGF treatment.Intermediate levels of activation were detected following IR orcisplatin treatment. L858R-, LNLS123-, and EGFRvIII-expres-sing cells showed a constitutive activation of the receptor,whereas NLS123 showed no receptor activation. Therefore, theEGFR-DNA-PKcs binding is triggered by cisplatin or IR treat-ment and not by the EGFR nuclear translocation per se.

DNA-PKcs and EGFR localize in the same cellularcompartments following IR or cisplatin treatment

Having established an association between EGFR and DNA-PKcs following cisplatin or IR treatment, we investigated theircellular localization by confocal microscopy following IR andcisplatin treatment. Cell transiently transfected with wtEGFRand EGFRvIII showed clear EGFR nuclear expression follow-ing cisplatin (Fig. 3) or IR treatment (Fig. 4). In contrast, cellstransiently transfected with NLS123, L858R, LNLS123, KMT,and DNLS showed a lack of EGFR nuclear accumulation. M1-and M12-transfected cells showed only reduced EGFR nuclearexpression following IR or cisplatin treatment. Next, stablytransfected cells were utilized to investigate this pattern. Cellsexpressing wtEGFR and EGFRvIII showed nuclear expressionof both EGFR and DNA-PKcs following cisplatin (Fig. 5) or IR

Figure 2. A, wtEGFR transfectedNI3T3 cells were treated with 50mmol/L cisplatin for 1 hour inserum-free media. Cells were thenlysed 1, 9, 12, 15, 18, and 24 hoursfollowing treatment. A total of 750mg of protein lysate wereimmunoprecipitated using anti-DNA-PKcs and blotted with anti-EGFR and anti-DNA-PKcs. EGFRpull-down was quantified by 2-dimensional densitometricanalysis and shown as a bindingfold compared with the untreatedcontrol. Mouse unrelated antibody(M IGg) was used as negativecontrol. B, stable NIH3T3 cellsexpressing wtEGFR, NLS123,L858R, LNLS123, EGFRvIII, andVC were treated with 50 mmol/Lcisplatin or 4-Gy IR or treated with100 ng/mL EGF as described inthe Materials and Methodssection. A total of 1.5 mg ofprotein lysate was thenimmunoprecipitated using anti-EGFR mAb and blotted with anti-DNA-PKcs, anti-PY20, and anti-EGFR. IP, immunoprecipitation.

- 1 9 12 15 18 24 TIme (h)

50 μmol/L cisplatin

IP: DNA-PKcs

IP: EGFR

FCSX-ray

Cisplatin

IP: EGFR

FCSX-ray

Cisplatin

WB:DNA-PKcs

pTYr (py20)

WB:DNA-PKcs

pTYr (py20)

DNA-PKcs

1 1.16 1.5 1.6 1.6 2.7 2.8

wtEGFR VC NLS123

EGFRvIII L858R LNLS123

treatment (Fig. 6). In contrast, L858R-expressing cells showedimpaired EGFR nuclear localization following cisplatin or IRtreatment. Expression of EGFR and DNA-PKcs was exclusivelycytosolic in NLS123- and LNLS123-expressing cell lines follow-ing cisplatin (Fig. 5) or IR treatment (Fig. 6). Therefore, theNLS123 mutation inhibits EGFR nuclear localization and alsoindirectly inhibits DNA-PKcs subcellular localization follow-ing IR or cisplatin treatment. Nuclear translocation of EGFRwas verified via cellular fractionation following cisplatin andIR treatment (data not shown).

EGFR and DNA-PKcs association following cisplatin orIR treatment

Previous studies have shown binding between EGFR andDNA-PKcs in the nucleus following EGFR nuclear transloca-tion (23, 32). To investigate the colocalization of EGFR andDNA-PKcs following treatment with cisplatin or IR, we con-ducted a Duolink proximity assay. This assay allows visualiza-tion of the interaction between 2 proteins in fixed cells. Eachinteraction is represented via a single red fluorescent dot(Fig. 7A). In cells expressing vector and NLS123 constructs, nointeraction was detectable. In contrast, cells expressingwtEGFR and EGFRvIII showed subcellular interaction

between EGFR and DNA-PKcs following IR or cisplatintreatment.

EGFR modulation of DNA-PK activityWe previously showed that the association of EGFR and

DNA-PKcs resulted in stimulation of DNA-PKcs activity (31).The experiments detailed earlier showed that nuclear trans-location of EGFR is required for the association with DNA-PKcs. To investigate whether this association resulted in analteration in enzyme activity, we investigated the effects ofexpression of different EGFR constructs on DNA-PK kinaseactivity. As compared with individual untreated control,cells expressing wtEGFR showed a 27.5% � 7.22% increasein DNA-PK activity following IR treatment (4 Gy) and a37.52% � 4.01% increase following 50 mmol/L cisplatintreatment for 1 hour (Fig. 7B). In cells expressing EGFRvIII,there was a 32.42% � 16.58% increase in DNA-PK activityfollowing IR treatment and a 26.6% � 8.49% increase follow-ing cisplatin treatment. In contrast, no significant change inDNA-PK activity compared with controls was found inL858R- and LNLS123-expressing cells following IR (�5.29%� 8.27% and 2.72% � 7.06%) or cisplatin treatment (9.04%� 3.46% and 2.25% � 6.07%). Results are shown in

Nuclei

wtEGFR

NLS123

LNLS123

EGFRvIII

DNA-PKcs EGFR Merge Nuclei DNA-PKcs EGFR MergeUntreated Cisplatin 50 μmol/L (18 h)

Figure 3. EGFR and DNA-PKcscellular localization followingcisplatin treatment. NIH3T3transiently transfected withwtEGFR, NLS123, L858R,LNLS123, EGFRvIII, M1, M12,KMT, DNLS, and VC were treatedwith 50 mmol/L cisplatin for 1 hourin serum-freemedia and then fixedwith 4% paraformaldehyde (PFA)18 hours following treatment.Cells were stained with goat anti-rabbit Alexa Fluor 647 (EGFR),goat anti-mouse Alexa Fluor488 (DNA-PKcs), and DAPI(40,6-diamidino-2-phenylindole;nucleus).

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Table 2C Supplementary Data. Only cells expressing NLS123showed a clear decrease in DNA-PK kinase activity com-pared with control following IR (�36.95% � 8.08%) orcisplatin treatment (�43.30% � 5.82%). Therefore, nuclearlocalization is required for EGFR-induced stimulation ofDNA-PK activity. EGF treatment did not induce a significantchange in DNA-PK kinase activity (P > 0.05) in NLS123-,LNLS123-, and EGFRvIII-expressing cell lines; only L858Rshowed borderline significant differences (P < 0.05) com-pared with the wtEGFR-expressing cells.

Discussion

Inhibitors of EGFR play a major role in cancer therapeutics.However, the activity of these agents as monotherapies is low,and it is important to investigate regimens with optimalcombinations by using chemotherapy and radiation (3). Thereis evidence of the effects of EGFR inhibition on DNA repairfollowing irradiation. In this study, we show the importance ofnuclear EGFR in modulating the repair of DNA damagefollowing cisplatin chemotherapy or radiation.

Nuclei

wtEGFR

NLS123

LNLS123

EGFRvIII

DNA-PKcs EGFR Merge Nuclei DNA-PKcs EGFR Merge

Serum starved 4 Gy 20 min

Figure 4. EGFR and DNA-PKcs cellular localization following IR treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII,M1, M12, KMT, DNLS, and VC were serum starved for 24 hours, treated with 4-Gy IR, and then fixed with 4% PFA 20 minutes following treatment.Cells were stained with goat anti-rabbit Alexa Fluor 647 (EGFR), goat anti-mouse Alexa Fluor 488 (DNA-PKcs), and DAPI (nucleus).

Expression of EGFR in the nucleus is well established, butthe implications on the effects of therapy are not clear.According to recent reports, EGFR nuclear translocationrequires receptor dimerization and activation, as, followinginternalization, mature and active EGFR may become a poorsubstrate for lysosomal degradation (33, 34). This allows eitherindirect sorting of the receptor through the Golgi or directsorting through the endoplasmic reticulum. Subsequent asso-ciation with Sec61 results in retrotranslocation to the cytosolwhere EGFR is stabilized following association with HSP70(33). Binding of importin b, mediated through the NLSsequence, translocates the receptor to the nucleus (22, 33).Inactive receptors (or those without an active conformation)are usually sent back to the plasma membrane via therecognition of a basolateral signal in the JX domain (19).

The interaction of EGFR with DNA-PKcs has been shown inseveral studies (12). The DNA-PK complex plays a key role inNHEJ, the major method of repair of DNA strand breaksfollowing IR treatment. Interaction of EGFR with DNA-PKcshas been shown to contribute to the repair of DNA strand

breaks. Inhibition of EGFR, by cetuximab or gefitinib, inhibitsrepair of IR-induced DNA strand breaks and impairs EGFR–DNA-PKcs interaction (4, 11).

It is well known that interstrand cross-links contributesignificantly to cisplatin cytotoxicity and that unhooking ofinterstrand cross-links may be used to determine clinicalsensitivity (35). In previous studies, we showed that theunhooking of cisplatin DNA interstrand cross-links was inhib-ited by gefitinib and that this is mediated through the DNA-PKpathway (31). This pathway has been shown to have relevancein the repair of cisplatin-induced DNA damage (36).

In this study, we show that EGFR nuclear expression intransfected EGFR-null cells modulates repair of DNA damagethrough the DNA-PK pathway. Cells expressing EGFR con-structs with mutated NLS sequence are inhibited in theirability to unhook DNA interstrand cross-links (35). Abrogationof nuclear expression of EGFR results in significant delay inrepair of interstrand cross-link in these cells. This correlateswith reduced association of EGFR with DNA-PKcs. Cellsexpressing constructs that do not translocate to the nucleus

Nuclei

wtEGFR

NLS123

LNLS123

EGFRvIII

Untreated Cisplatin 50 μmol/L (18 h)

Figure 5. EGFR and DNA-PKcs cellular localization following cisplatin treatment. NIH3T3 cells stably expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII,and VC were treated with 50 mmol/L cisplatin for 1 hour in serum-free media and then fixed with 4% PFA 18 hours following treatment. Cells werestained with goat anti-rabbit Alexa Fluor 647 (EGFR), goat anti-mouse Alexa Fluor 488 (DNA-PKcs), and DAPI (nucleus).

Liccardi et al.

showed increased sensitivity to cisplatin (SupplementaryFig. S3). Previous studies have shown that EGFR nucleartranslocation correlated with repair of IR-induced strandbreaks.It has been shown previously that the association between

DNA-PKcs and EGFR peaks at 20 minutes following IR treat-ment (37), but association following cisplatin treatment hasnot been described. Here, we show that binding peaks at 18hours following cisplatin treatment. The different timingfollowing cisplatin and IR treatment likely reflects differenttypes of DNA lesions and repair mechanisms. There wasreduced repair of DNA IR-induced strand breaks in cellsexpressing EGFR constructs that do not translocate to thenucleus. Repair of DNA strand breaks following IR treatmenthas been shown to be modulated by EGFR (37) and the lessmarked effects of impaired EGFR nuclear translocation ascompared with the repair of cisplatin-induced interstrandcross-links may be secondary to the activation of otherDNA repair pathways (12).

In this study, the nuclear translocation of EGFR was shownby confocal microscopy. Interestingly, these experiments sug-gest that there is colocalization of EGFR and DNA-PKcs bothwithin the nucleus and the cytoplasm. Although the primarylocation of DNA-PKcs is in the nucleus in the formation ofcomplexes on damaged DNA, cytoplasmic expression of DNA-PKcs has been shown in several studies (38–40). The results ofthe Proximity ligation assay show physical proximity of DNA-PKcs and EGFR following DNA damage by cisplatin or IRtreatment in cells with intact nuclear localization. This wasnot shown in cells expressing EGFR constructs deficient innuclear localization.

There is contradictory evidence regarding nuclear expres-sion of EGFRvIII. Although EGFRvIII and STAT3 colocaliza-tion within the nucleus was shown in some studies (17, 41),other studies have reported a lack of nuclear expression inglioma models (42). Cells expressing EGFRvIII show elevatedactivation of DNA-PKcs and enhancement of DNA strandbreaks repair. In this study, we show that EGFRvIII and

Nuclei

wtEGFR

NLS123

LNLS123

EGFRvIII

Serum starved 4 Gy 20 min

Figure 6. EGFR and DNA-PKcs cellular localization following IR treatment. NIH3T3 cells stably expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII,and VC were serum starved for 24 hours, treated with 4-Gy IR and then fixed with 4% PFA 20 minutes following treatment. Cells were stained withgoat anti-rabbit Alexa Fluor 647 (EGFR), goat anti-mouse Alexa Fluor 488 (DNA-PKcs), and DAPI (nucleus).

wtEGFR undergo nuclear translocation and binding withDNA-PKcs following cisplatin or IR treatment.

Although cells expressing kinase-dead EGFR showed a lackof nuclear translocation, expression of the L858R mutant alsoresulted in impaired nuclear expression despite constitutivekinase activity. This resulted in a reduction in DNA strandbreaks repair that is consistent with the observation that non–small cell lung cancer lines expressing L858R show increased

sensitivity to IR (43) and reduced nuclear expression (26).Moreover, the difference in repair between cells expressingL858R (kinase active but with impaired nuclear localization)and M1or M12 (kinase active and expressed in the nucleus)suggests that it is the impaired nuclear EGFR accumulation(which is a consequence of the lack of allosteric activation)and not kinase activity per se that determines the reducedDNA repair in these models.

Serumstarved

Cisplatin 50 μmol/L(18 h)

Untreated(growing cells)

wtEGFR

NLS123

EGFRvIII

wtEGFR

NLS123

LNLS123

EGFRvIII

IR Cisplatin EGF

4 Gy20 min

Figure 7. A, EGFR–DNA-PKcscomplex cellular localization.Stable NIH3T3 cells expressingwtEGFR, NLS123, EGFRvIII, andVC were treated with 50 mmol/Lcisplatin for 1 hour in serum-freemedia and then fixed with 4% PFA18 hours following treatment or4-Gy IR and then fixed with 4%PFA 20 minutes followingirradiation. Cells were thenimmunoblocked with anti-rabbitEGFR and anti-mouse DNA-PKcs.Interacting complexes were thenvisualized via the Duolinkproximity assay. Each red spotrepresents a single interaction. B,EGFR modulation of DNA-PKkinase activity. Stable NIH3T3cells expressing wtEGFR,NLS123, L858R, LNLS123, andEGFRvIII were treated with 50mmol/L cisplatin for 1 hour or 4-GyIR or 100 ng/mL EGF in serum-freemedia. Eighteen hours followingthe treatment with cisplatin, 20minutes following the treatmentwith IR, and at 1 hour followingEGF incubation, samples wereprepared for the DNA-PK kinaseassay. The graph shows thepercentage change in DNA-PKactivity following each treatmentcompared with untreated control.Stars, statistical significance.

Liccardi et al.

The impaired kinase activity shown by the NLS123 mutantsupports the previously described allosteric mode of activa-tion of EGFR and the importance played by the third cluster ofarginines (646-RR-647) within the NLS sequence in adoptingan a-helical conformation that is indispensable for EGFRactivation (25, 34). The impairment of other EGFR functions,as a result of mutations in the NLS sequence, has not beenexcluded. Interestingly, the LNLS123 mutant shows kinaseactivation despite bearing the same NLS mutation that ren-ders the NLS123 kinase dead. This suggests that the L858Rmutation, which has been shown to thermodynamically sta-bilize EGFR (44, 45), allows receptor activation that does notrequire the allosteric conformational change. Further workwill be required to investigate whether the L858R inhibition ofnuclear translocation is due to a structurally hidden NLSsequence.EGFR inhibition by gefitinib has been shown to suppress

DNA repair following treatment with radiation and cisplatin(31). Similarly, in this study, the kinase-dead mutant KMT(mutation K721A) shows no nuclear localization, suggestingthat the targeting of the EGFR kinase domain interferes withnuclear translocation and consequently with repair. Maximaleffect of gefitinib on the inhibition of interstrand cross-linkswas observed only in EGFR constructs translocating to thenucleus (Supplementary Fig. S4). A recent study showed thatcisplatin resistance and DNA repair were dependent onnuclear translocation (23). Here, we have shown that a varietyof EGFR mutants deficient in nuclear expression showimpaired repair and that nuclear accumulation is a majordeterminant of repair of cisplatin-induced interstrand cross-links. In addition, we recently showed that the expression ofHER2 modulated repair of cisplatin-induced interstrandcross-links and that this also requires nuclear expression(27). Other factors, including ubiquitination of EGFR inducedby cisplatin in head and neck cancer cells, may contribute tothe interaction between cisplatin and the EGFR pathway intherapy (46).There has been extensive study on the interaction of the

EGFR and DNAPK pathways. Following the initial observationthat cetuximab treatment inhibits EGFR–DNA-PKcs interac-tion (47), the role of this interaction in the modulation of DNA

repair has been confirmed. Cells expressing specific EGFRmutations found in human cancer such as the L858R in non–small cell lung cancer have been found to show sensitivity toIR. This includes delayed DNA repair kinetics, defective IR-induced arrest in DNA synthesis, and increased apoptosis (26).Here, we show that inhibition of EGFR nuclear translocationalters DNA-PKcs cellular distribution.

Stimulation of DNA-PK kinase activity was associated withnuclear expression and binding. There are likely other factorsapart from EGFR–DNA-PKcs binding that may influence theeffect of EGFR on DNA repair. EGF induces nuclear transloca-tion of EGFR, but this is not associated with EGFR–DNA-PKcsinteraction. Receptor kinase activation may influence, indir-ectly, DNA-PK possibly by the activation of the AKT pathway,which has been shown to be a kinase of DNA-PKcs (48, 49).

These results suggest that nuclear expression of EGFR playsa significant role in response to cisplatin and radiation. Theintracellular localization of EGFR may play a critical part inresponse to therapies combining inhibitors of the EGFR path-way with chemotherapy or radiation. Understanding of themechanisms by which nuclear expression modulates thera-peutic effects of these modalities will optimize design ofclinical studies in the future.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Prof. William Gullick and Prof. M.C. Hung for providing reagents.

Grant Support

G. Liccardi was supported by CRUK for the studentship (C2259/A7475). Thiswork was also funded by CRUK through a program grant to J. A. Hartley and D.Hochhauser (C2259/A9994).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 30, 2010; revised October 5, 2010; accepted December 1, 2010;published OnlineFirst January 25, 2011.

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2011;71:1103-1114. Published OnlineFirst January 25, 2011.Cancer Res Gianmaria Liccardi, John A. Hartley and Daniel Hochhauser following Cisplatin and Ionizing Radiation TreatmentEGFR Nuclear Translocation Modulates DNA Repair

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