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DNA Damage and Repair

EGFR Mutations Compromise Hypoxia-Associated Radiation Resistance throughImpaired Replication Fork–Associated DNADamage RepairMohammad Saki1, Haruhiko Makino2, Prashanthi Javvadi3, Nozomi Tomimatsu3,Liang-Hao Ding3, Jennifer E. Clark1, Elaine Gavin1, Kenichi Takeda2, Joel Andrews1,Debabrata Saha3, Michael D. Story3, Sandeep Burma3, and Chaitanya S. Nirodi1

Abstract

EGFR signaling has been implicated in hypoxia-associatedresistance to radiation or chemotherapy. Non–small cell lungcarcinomas (NSCLC) with activating L858R or DE746-E750EGFR mutations exhibit elevated EGFR activity and down-stream signaling. Here, relative to wild-type (WT) EGFR,mutant (MT) EGFR expression significantly increases radiosen-sitivity in hypoxic cells. Gene expression profiling in humanbronchial epithelial cells (HBEC) revealed that MT-EGFRexpression elevated transcripts related to cell cycle and repli-cation in aerobic and hypoxic conditions and downregulatedRAD50, a critical component of nonhomologous end joiningand homologous recombination DNA repair pathways.NSCLCs and HBEC with MT-EGFR revealed elevated basal andhypoxia-induced g-H2AX–associated DNA lesions that were

coincident with replication protein A in the S-phase nuclei.DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalledreplication forks and decreased fork velocities in aerobic andhypoxic conditions. EGFR blockade by cetuximab significantlyincreased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality ofPARP inhibition.

Implications: This study demonstrates that within an alteredDNA damage response of hypoxic NSCLC cells, mutant EGFRexpression, or EGFR blockade by cetuximab exerts a syntheticlethality effect and significantly compromises radiation resistancein hypoxic tumor cells.Mol Cancer Res; 15(11); 1503–16.�2017 AACR.

IntroductionRelative to well-oxygenated tumors, hypoxic tumors tend to be

significantly more resistant to ionizing radiation (IR). Radiation-induced free radicals chemically react with DNA and cause DNAdamage through the formation of a DNA radical. For DNAdamage to persist, oxygen is required to oxidize the DNA radicaland extend its half-life. Hypoxic cells are more radioresistantbecause, in conditions of low oxygen, oxidation of the DNA

radical and radiation-induced DNA damage are limited (1).Oxygen enhances radiosensitivity by a radiation dose-modifyingfactor (DMF) called oxygen enhancement ratio (OER). OER isdefined as the ratio of the radiation dose under anoxic or hypoxicconditions to the dose under conditions of a specific partialpressure of oxygen to produce the same biological effect. Alter-natively, the hypoxia reduction factor (HRF) has been defined asthe ratio of radiationdose at a specific partial pressure of oxygen tothe dose under fully aerobic conditions (21% O2) for the samebiological effect (2).

Prolonged exposure to hypoxia itself can be cytotoxic, andtumor survival depends on an adaptive prosurvival responseto hypoxia that could also augment radioresistance. Activa-tion and downstream signaling of the EGFR in many cancers,including non–small cell lung cancer (NSCLC), has beenlinked to survival responses to hypoxia. These include inhibi-tion of hypoxia-induced cell death (3), stimulation of pro-proliferative pathways (4), induction of epithelial–mesenchy-mal transition (5), and establishment of a positive feedbackloop through induction of the hypoxia-inducible factor 1(HIF-1a; ref. 6).

EGFR also has roles in the repair of radiation-induced DNAdouble-strand breaks (DSB). DSB repair in tumors is catalyzed bytwo signaling pathways, nonhomologous end joining (NHEJ)and homologous recombination (HR). Several studies have dem-onstrated a role for EGFR in NHEJ, which involves radiation-induced nuclear translocation of EGFR, interactions with the

1Department of Oncologic Sciences, University of South Alabama MitchellCancer Institute, Mobile, Alabama. 2Division of Medical Oncology and MolecularRespirology, Faculty of Medicine Tottori University, Yonago, Tottori, Japan.3Department of Radiation Oncology, University of Texas Southwestern MedicalCenter, Dallas, Texas.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

M. Saki and H. Makino contributed equally to this article.

Additional data deposited: NCBI Gene Expression Omnibus (GEO) [accessionnumber GSE95564].

Corresponding Author: Chaitanya S. Nirodi, Department of Oncologic Sciences,University of South Alabama Mitchell Cancer Institute, 1660 Springhill Avenue,MCI-3029,Mobile, AL 36604, United States. Phone: 251-445-9862; Fax: 251-460-6994; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0136

�2017 American Association for Cancer Research.

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NHEJ enzyme, DNA-dependent protein kinase catalytic subunit(DNA-PKcs), andmodulation of DNA-PKcs phosphorylation (7–11). Other studies have shown that EGFR-mediated DSB repairmay also involve HR (12, 13). Recent studies have uncovered aless understood, kinase-independent role for EGFR in HR-medi-ated repair of cisplatin-induced DNA interstrand crosslinks, butnot radiation-induced DSBs (14).

We previously demonstrated thatNHEJ-mediatedDSB repair isdefective in NSCLCs harboring somatic, activating mutations inEGFR that were clinically linked to sensitivity to EGFR tyrosinekinase inhibitors (TKI; refs. 15–17). We showed that, despiteelevated EGFR tyrosine phosphorylation and downstream signal-ing, NSCLCs harboring DE746-E750, L858R, or the TKI-resistantT790M mutant (MT) EGFR are profoundly radiosensitive (18).MT-EGFR–associated radiosensitivity manifests as pronounceddelays in repair of radiation-induced DSBs, failure of EGFRnuclear import, abrogation of EGFR–DNA-PKcs interactions(8), and inhibition of DNA-PKcs phosphorylation at threonine2609 (T2609; ref. 11), a pattern that is remarkably similar to EGFRblockade by the mAb, cetuximab (19). How expression of sig-naling-hyperactive, NHEJ-defective MT-EGFR affects hypoxiaresponse is not known.

Evidence from a number of studies shows that hypoxiainduces a unique DNA damage response (DDR), which is acascade of signaling events that collectively orchestrate DNAdamage recognition, DNA repair, cell-cycle arrest, or apoptosis(20). DNA DSBs do not usually occur in hypoxic cells (21).However, in response to hypoxia, decrease in ribonucleotidereductase (RNR) activity (22) causes nucleotide insufficiency,resulting in a rapid halt of DNA synthesis and transient cell-cycle arrest through replication fork–bound replication proteinA (RPA) and activation of the ataxia telangiectasia–relatedcheckpoint kinase (ATR) Cdc25 pathway (ATR/Chk1/Cdc25pathway; ref. 23). Stalled replication forks are at a high riskfor DNA DSBs, and repair of fork-associated DSBs is essentialfor survival. During conditions of transient hypoxia (<12hours), HR but not NHEJ appears to be critical for survival(24). In contrast, during prolonged hypoxia (>12 hours), manycomponents of the HR pathway are downregulated with asignificant upregulation of several NHEJ components (25).

This study investigates the effect of activating L858R andDE746-E750 mutations in EGFR on radiation resistance in con-ditions of hypoxia. Our study has uncovered a novel relationshipbetween MT-EGFR expression and replication stress in oxygen-replete and hypoxic conditions. We demonstrate that in thecontext of an altered DDR in hypoxic cells, expression ofNHEJ-defective MT-EGFR, or an EGFR blockade by cetuximab,exerts a synthetic lethality effect and sensitizes hypoxic cells toradiation. This study has important clinical implications in thetreatment of NSCLC patients, especially those at greatest risk oftherapy failure due to tumor hypoxia.

Materials and MethodsCell culture

NSCLC cell lines with either WT-EGFR (NCI-A549), or DE746-E750-T790M (NCI-H820), DE746-E750 (NCI-HCC827) orL858R-T790M (NCI-H1975) mutated forms of EGFR, used inthis study were from the ATCC and maintained as described pre-viously (8). CDK4/hTERT immortalized human bronchial epi-thelial (HBEC) cells stably expressing the V5-tagged wild-type

EGFR (WT), L858R, and DE746-E750 forms of EGFR were main-tained as described in ref. 11. For induction of hypoxia, cells werecultured in poly-L-lysine (Sigma-Aldrich) or attachment factor(AF; Thermo Fisher Scientific) coated glass petri dishes to avoidthe problem of oxygen (O2) dissolved in plastic. Similarly, forimaging studies, cells were first seeded on glass coverslips, and thecoverslips were submerged in glass petri dishes containing medi-um. Petri dishes containing cells were incubated in anopenZiplocbag at 5%CO2, 0.1%O2, and 37�C in a ProOx 110-controlledN2/CO2 gas environment chamber, i-Glove (BioSpherix). Dissolvedoxygen in medium, measured using a ruthenium-based oxygenprobe, reached desired levels (0.1%) within 30 to 45 minutes ofincubation in hypoxia chamber. For irradiation under hypoxia,Ziploc bags were hermetically sealed while still under hypoxicconditions before being exposed to radiation in either XRAD320(dose rate 117 cGy/minute, Precision X-ray) or Cs137 irradiator(dose rate 347 cGy/minute, J.L. Shephard and associates).

All cell lineswere testedweekly formycoplasma contaminationusing theMycoAlert Kit (Lonza) and authenticated twice annuallyusing professional authentication services (Genetica-LabCorpCell line testing).

Clonogenic survival assayClonogenic survival was measured as described before (8, 11,

18). For survival response in hypoxic state, cells were exposed to0.1% O2 for 24 hours, irradiated as described above, allowed torecover for 8 hours under hypoxic conditions, and allowed toformcolonies over 7 to10days in aerobic environment (21%O2).Where inhibitors were used, cells were incubated in aerobicor hypoxic environments as above and then received vehicle,PARP1/2 inhibitor, ABT-888, (Cayman Chemical) for 2 hours, or50 mg/mL (�345 nmol/L) humanizedmAb anti-EGFR cetuximab(Imclone/Bristol-Myers Squibb/Eli Lilly) for 6 hours prior toirradiation. Plating efficiency (PE) values were used to determineeffective concentration for 50% survival (EC50) for ABT-888.Surviving fraction (SF) values were normalized to PE and plottedas a function of radiation dose. The data were fit with the linearquadratic equationusing SigmaPlot version12.5 (Systat Software;www.systatsoftware.com).

Microarray gene expression analysisHBEC cells stably expressingWT, L858R, or DE746-E750 forms

of EGFR were left under aerobic conditions (21%O2) or exposedfor 24 hours to a hypoxic environment containing 0.1% O2.Illumina Whole Genome HumanHT12 v4 Expression BeadChipwas used in this study. Total RNA was extracted, amplified,transcribed into biotin-labeled cRNA, and hybridized with strep-tavidin-Cy3 (GEHealthcare) using standard Illumina protocols asdescribed previously (26). Slides were scanned on an IlluminaBeadstation. Summarized expression values for each probesets were generated using BeadStudio 3.1 (Illumina Inc.). Thedata were background subtracted and quantile–quantile normal-ized across samples using MBCB algorithm (27). A two-sample ttest was performed between WT EGFR and MT-EGFR (L858RandDE746-E750 combined) samples or, separately, between21%O2 and 0.1% O2 samples within WT, L858R and DE746-E750cohorts. Genes with P < 0.01 and fold change greater than 2were considered as changed with statistical significance. Themicroarray data are deposited in NCBI's Gene Expression Omni-bus (GEO) (28) and are accessible through GEO accession num-ber GSE95564.

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Western blot analysisApproximately 106 NSCLC or HBEC3KT cells were exposed

to an aerobic (21% O2) or hypoxic (0.1% O2) environment for24 hours. Whole-cell lysates were prepared as described previ-ously (8, 11) Antibodies specific to RAD50 (# sc20155), RAD51(# sc-8349), MRE11 (# sc5859), b-actin (# sc-69879), SantaCruz Biotechnology, NBS1/Nibrin (# 05-616), RNR/RRM2 (#ABC106), Ku80 (# 05-393), EMD-Millipore (Upstate Biotechnol-ogy), and HIF1a (# GTX628480), GeneTex were used. Blots wereimaged using the Chemidoc MP imaging system (Bio-Rad), andband densitometric analysis was performed using the ImageLabsoftware version 4.1 (Bio-Rad). After background subtraction,density of a given band on a blot was normalized to density ofb-actin band from the same sample on that particular blot toobtain relative density.

Immunocytochemistry for gH2AX fociNSCLC cells were seeded in duplicate on glass bottom 96-well

plates and then exposed for 24 hours to aerobic or hypoxicenvironments. For reoxygenation experiments, cells werereturned to an aerobic environment and harvested at varioustime points for immune-detection of 53BP1 and gH2AX foci asdescribed previously (8, 11, 18).

For cell-cycle distribution of gH2AX foci, while still underaerobic or hypoxic states, cells were pulsed for 10 minutes with10 mmol/L of the nucleoside analogue, 5-ethynyl-20-deoxyur-idine (EdU) before being processed for immunocytochemistryusing the Alexa Fluor-488 Click-iT EdU Imaging Kit (Invitro-gen), mouse anti-gH2AX (pSer 139; # 05-636), rabbit anti-phospho histone H3 (pSer 10; # 04-817) from EMD-Millipore(Upstate Biotechnology), and rabbit anti-cyclin B1 antibody (#4138), Cell Signaling Technology. Fluorescent images wereacquired under the IN Cell Analyzer 2000 high content auto-mated imaging system (GE Healthcare) using a 40� objectiveand analyzed using the IN Cell Investigator software (GEHealthcare). gH2AX foci in EdU-positive S-phase, Cyclin B1–positive G2 phase, and histone H3–positive M-phase cells wereenumerated. The G1 subpopulation was estimated by subtract-ing the sum of S, G2, and M phase cells from the total numberof nuclei. In each phase, the fraction of nuclei harboring >5 fociwas calculated.

In a separate experiment, to evaluate extent of radiation-induced DNA damage, NSCLC cells were processed for immu-nocytochemistry at 45minutes following 0 or 1 Gy radiation and15 minutes after an EdU pulse. Images were acquired in multiplefocal z-planes using a Nikon A1rsi scanning confocal microscopewith an oil immersion 100� (NA1.49) objective. Images within aZ-stack were collapsed to generate a single composite image andtotal number of nuclei, number of gH2AX foci per nucleus, andEdUþ S-phase nuclei were enumerated using the Cell Profilerimage analysis software.

Immunocytochemistry for RPA-gH2AX foci in S-phase cellsNSCLC and HBEC3KT cells were seeded in 96-well plates and

pulse labeled as described above. To obtain clear RPA foci, cellswere subjected to in situ fractionation as described in ref. 29,labeled with Alexa 488 Click-it reagent and immunostained withrabbit anti-gH2AX and mouse anti-RPA (# MAB286, EMD Milli-pore), followed by incubation with secondary antibodies andDAPI. Fluorescent images were acquired under the IN Cell Ana-lyzer 2000 systemusing a40�objective. gH2AX foci, RPA foci and

EdU-positive (S-phase) nuclei were enumerated and number of S-phase nuclei with merged RPA and gH2AX foci was obtained.

DNA fiber analysisReplication fork dynamics were evaluated by DNA fiber assay

as described in ref. 30. Briefly, NSCLC cells were seeded in AF-coated glass 60-mmdishes and exposed for 24 hours to aerobic orhypoxic environments. Cellswere pulse labeledwith 25mmol/L 50

iodo 20deoxyuridine (IdU) for 20 minutes followed by 10-foldexcess (250 mmol/L) of 50 chloro 20 deoxy uridine (CldU) for afurther 20minutes. Cellswere detached anddiluted to adensity of7� 105 cells/mL. Nuclear suspension (2 mL) was spotted on glassslides, dried, andfixed. Samples were blockedwith 5%BSA in PBSand incubated with a 1:25 dilution of IdU-specific mouse anti-BrdUrd (BD Biosciences) and 1:400 dilution of CldU-specific ratanti-BrdUrd (Abcam). Samples were incubated with a 1:500dilution of Cy3-conjugated sheep anti-mouse and 1:400 dilutionof Alexa-488–conjugated goat anti-rat secondary antibodies andmounted. Images were acquired with the Nikon A1rsi scanningconfocal microscope. A minimum of 200 well-separated fiberswere counted per sample. The number and juxtaposition of IdU-and CldU-stained fibers were manually measured using NISElements software (v 4.0). Replication structures such as stalledforks (IdUþ/CldU�), active forks (IdUþ/CldUþ), new origins(IdU�/CldUþ), elongating forks (CldU-IdU-CldU), and termi-nating forks (IdU-CldU-IdU) were enumerated. Length of DNAsynthesized in 20-minute nucleotide pulses wasmeasured using aDNA extension factor of 2.59 kbp/mmand relative fork velocity inaerobic and hypoxic conditions was calculated as a ratio of CldU/IdU lengths. A CldU/IdU ratio approximately 1.0 indicatedunperturbed replication and CldU/IdU ratio <1 was consideredslower replication.

ResultsMutations in EGFR compromise hypoxia-associated radiationresistance

We compared radiation response in four different NSCLCcell lines following a 24-hour exposure to either an aerobic(21% oxygen) or a hypoxic (0.1% oxygen) environment. In allthreeMT-EGFRNSCLC cell lines, exposure to hypoxia reduced PEacross a wide range of oxygen concentrations, while WT-EGFRexpressing A549 cells were relatively unaffected (SupplementaryFig. S1A–S1C). When normalized for PE, hypoxic WT-EGFR–expressing A549 cells were significantly more radioresistant rel-ative to aerobic A549 cells (Fig. 1A). In contrast, hypoxia-associ-ated radioresistance inMT-EGFR–expressingNSCLCswasmarkedreduced (Fig. 1B–D).

To quantify hypoxia-associated radiation resistance, radiationDMFs, such as OER or HRF, are frequently used. Here, we use theterm hypoxia reduction factor (signaling; HRFS), which encom-passes not only physiochemicalmodification of DNAbut also thebiological or enzymatic processes that can influence survivalunder hypoxic conditions. We define HRFS as the ratio of theradiation dose at a specific oxygen concentration (0.1%) to theradiation dose under fully aerobic conditions (21% oxygen) forthe same reduction in clonogenic survival. Data in Fig. 1E revealthat HRFS across a range of SFs was consistently and significantlyhigher in WT-EGFR–expressing A549 cells (mean HRFS, 2.28)comparedwithmutant EGFR–expressing cells (meanHRFS, 1.12–1.58). Our panel consisted of NSCLCs with low (H820), medium

Hypoxia-Linked Radioresistance Is Compromised by Mutant EGFR

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(H1975), and high (HCC827) expression of MT-EGFR (Fig. 1F).Despite these wide differences in expression levels, all three celllines exhibited marked reduction in HRFS relative to wild EGFR-expressing NSCLC, A549, indicating that expression level was nota factor in HRFS reduction.

To rule out effects from genetic background, we stablyoverexpressed WT- and MT-EGFR forms in HBEC cells. Again,hypoxia alone significantly inhibited survival in HBEC cellsoverexpressing L858R or DE746-E750 EGFR but had no effecton cells overexpressing the WT-EGFR (Supplementary Fig.S1B). Cells expressing L858R or DE746-E750 EGFR-expressingcells (Fig. 2B and C) exhibited significantly reduced HRFS(means, 1.66 and 1.76) compared with WT-EGFR (mean,2.23; Fig. 2A). Figure 2E shows that WT-EGFR and MT-EGFRforms were overexpressed to similar levels in HBEC cells andwere nearly three orders of magnitude higher relative to mock-transfected controls. These levels were comparable with levelsof expression found in most MT-EGFR–expressing NSCLCs,such as H1975 and HCC827 (Fig. 1E). Again, overexpressionalone was not a factor in HRFS reduction because similarresults were obtained when L858R or DE746-E750 EGFR wereectopically expressed at lower levels in A549 NSCLCs (Sup-

plementary Fig. S5). Even though MT-EGFR expression wasonly 5.5- to 6.7-fold over endogenous WT EGFR (Supplemen-tary Fig. S5A), this level of MT-EGFR expression was sufficientto exert a strong dominant negative effect in A549 cells bysignificantly reducing PE (Supplementary Fig. S5B, left), radio-resistance (Supplementary Fig. S5B and S5C), and HRFS (Sup-plementary Fig. S5D).

Mutations in EGFR alter DDR patterns in hypoxic cellsTo examine whether mutation status of EGFR influenced hyp-

oxia-induced changes in gene expression andwhether these effectscould account for reduced HRF, we compared gene expressionpatterns associated with WT, L858R, or DE746-E750 EGFR stablyexpressed in the isogenic background of HBEC cells. In aerobicconditions, relative to WT-EGFR–expressing cells, 226 genes weredifferentially expressed 2-fold or more (P < 0.01) in MT-EGFRHBECs. In contrast, in hypoxic conditions, the number was nearly8 times larger, with 1,793 genes differentially expressed betweenthe two cohorts. Unsupervised clustering revealed significantdifferences between WT- and MT-EGFR in both aerobic andhypoxic conditions (Fig. 3A) and gene expression changes inL858R and DE746-E750–expressing cells clustered together. In

Figure 1.

Hypoxia-associated radiationresistance is compromised in NSCLCswith activating mutations in EGFR.Clonogenic survival assay in NSCLCcell lines, A549 (A), H1975 (B), H820(C), and HCC827 (D) following a24-hour exposure to 21% O2 (circles,solid line) or 0.1% O2 (squares, brokenlines). Symbols, representing meanSF normalized to PE and error barsrepresenting SD were derived from atleast three independent experiments,eachwith samples in triplicate. E,HRFSin NSCLCs. HRFS values at SF from 0.1to 0.8 are shown. Symbols (meanHRFS) and error bars (SD). Asteriskrepresents summary of an ordinaryone-way ANOVA test performedbetween A549 and MT-EGFR NSCLCswhere P < 0.001. F, Western blotanalysis (left) of whole-cell lysatesfrom indicated NSCLCs with WT(A549, H460) and MT-EGFR (H820,H1975, and HCC827). Densitometricanalysis (right) of EGFR bandintensities normalized to b-actin bandintensities in each lane. Relative toA549, levels of MT forms of EGFRwere at least 1.5 to 3 orders ofmagnitude higher.

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aerobic conditions (Fig. 3B), relative to WT-EGFR, MT-EGFRexpression was associated with upregulation of cell-cycle activa-tors, including CCNE1 and CCNE2, and downregulation of cell-cycle inhibitors, such as CDKN2A, CDKN2D, and CDKN1A. Thisis consistent with the elevated pro-proliferative signaling associ-ated with the MT-EGFR. In hypoxic conditions, a very differentpattern emerged. Although expression of NHEJ genes, PRKDC(DNA-PKcs), XRCC6 (KU70), XRCC5 (KU80), or LIG4 (ligase IV)was unchanged, the hypoxic state was associated with down-regulation of many HR DNA repair components, includingRAD50, RAD51,MRE11, andNBN (NBS1).Of these,mRNA levelsof RAD50 andMRE11 appeared significantly lower in MT-EGFR–expressing cells relative toWT-EGFR cells. Data fromWestern blot

and densitometric analysis (Fig. 3C and D) largely validatedthe microarray profile. For example, at both mRNA and proteinlevels, RAD51 levels reduced to the same extent in hypoxic WT-and MT-EGFR–expressing cells. Likewise, at both transcriptand protein levels, RAD50 remained largely unaffected by hyp-oxia in WT-EGFR cells but was significantly reduced in L858R orDE746-E750 EGFR-expressing HBEC cells. However, MRE11 andNBS1 appeared to be differentially downregulated at the mRNAlevel but not at the protein level. Remarkably similar trends wereobserved inWT- andMT-EGFR–expressing NSCLC cells. The datain Fig. 3 not only confirm hypoxia-associatedHR downregulationbut also suggest thatMT-EGFR expressionmay exert an inhibitoryeffect on additional HR components, such as RAD50.

Figure 2.

Ectopic expression of L858R andDE746-E750 EGFR significantlyreduces hypoxia-associated radiationresistance. Clonogenic survival assay inHBEC cells stably expressing WT (A),L858R (B), and DE746-E750 EGFR (C),following a 24-hour exposure to 21% O2

(circles, solid line) or 0.1% O2 (squares,broken lines). Symbols, representingmean SF normalized to PE and errorbars representing SD were derivedfrom at least three independentexperiments, each conducted withsamples in triplicate. D, HRFS values atdifferent SF. Symbols, mean HRFS;error bars, SD. Asterisk representssummary of a one-way ANOVA testperformed betweenWT- andMT-EGFRexpressing HBEC cells where P < 0.001.E, Western blot analysis: lysates fromHBEC cells that were mock transfected(M) or stably transfected with WT-EGFR (WT), L858R (LR), and DE746-E750 (DL) forms of EGFR (left).Densitometric analysis (right) of EGFRband intensities normalized to b-actinband intensities in each lane. Relativeto vector alone, levels of WT or MTforms of EGFR were at least 2.5–3orders of magnitude higher.






MT-EGFR–expressing NSCLCs and HBECs exhibit uniquepatterns of hypoxia-associated DNA lesions

We next examined the effect of hypoxia and reoxygenation ontwo well-established surrogates of DNA damage, 53BP1 andgH2AX,which localize to sites proximal toDSBs and formdistinct

foci (Fig. 4). Relative to WT-EGFR–expressing A549 cells (0.5 fociper nucleus), MT-EGFR–expressing H820, HCC827, and H1975cells harbored dramatically elevated levels of basal 53BP1 (Fig.4A) and gH2AX foci (2.5–7.5 foci per nucleus; Fig. 4B). A 24-hourexposure to 0.1% O2 resulted in a modest 5-fold increase in

Figure 3.

Microarray gene expression analysis ofmRNA isolated from HBEC cells stablyexpressing wild type (WT, black),L858R (light gray), or DE746-E750(dark gray) forms of EGFR in aerobic(21% O2) conditions or after 24-hourexposure to hypoxic (0.1% O2)environments. A, Unsupervisedclustering of genes from differentsamples. Gene expression patterns ofL858R or DE746-E750 clustered asone group (MT), which was distinctfrom WT in both aerobic and hypoxicconditions. B, Fold differences in geneexpression inMT-EGFR relative toWT-EGFR cells grown in aerobic (left) orhypoxic conditions (right). Positivevalues, fold increase; negative values,fold decrease in expression inMT-EGFR–expressing cells, relative toWT-EGFR–expressing cells. C,Western blot analysis of lysates fromHBEC cells stably transfected withWT-EGFR (WT), L858R (LR), andDE746-E750 (D) forms of EGFR (left)or indicated NSCLCs (right) isolatedfrom cells in aerobic or hypoxicconditions. Representative blots frommultiple gels from at least threeindependently performedexperiments are shown.D, Densitometry analysis: bandintensities of individual proteins in 3Cwere normalized to intensities of thecorresponding b-actin band from thesame gel as the target protein. Bars,mean fold change in hypoxicconditions relative to aerobic stateand error bars, SEM (n ¼ 3independent experiments). Asterisksummarizes an ordinary one-wayANOVA test performed betweenWT- and MT-EGFR–expressing HBECor NSCLC cells where P < 0.001.

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Figure 4.

MT-EGFR expression is associated with elevatedlevels of 53BP1 and gH2AX foci in aerobic andhypoxic state. NSCLC cells exposed to an aerobicenvironment (N) or exposed for 24 hours to 0.1%O2 (0) and then reexposed to the 21% O2 for theindicated time points.A, 53BP1 foci (A) or gH2AXfoci (B) enumerated by analysis of imagesacquired by automated fluorescencemicroscopy. Bars represent mean foci pernucleus and error bars represent SEM from twoindependent experimentswith n¼�2,500nucleiper sample in each experiment. C, HBEC cellsexpressing WT- and MT-EGFR at 24 hours afterexposure to 21% or 0.1% O2 were processed by asabove to detect nuclear gH2AX foci. Barsrepresent mean foci per nucleus and error barsare SEM from two independent experiments withn¼�1,500 nuclei per sample in each experiment.D, Cell-cycle distribution of basal gH2AXfoci in nuclei stained with cyclin B (G2), p-histoneH3 (M), Alexa-488 linked EdU (incorporated onlyin the S-phase) in aerobic (left) and hypoxic(right) conditions. Number of G1 cells wasdetermined by subtracting the sum of S, G2,M phase cells from total number of nuclei. Barsrepresent themean fraction of nuclei with >5 fociand error bars represent SEM from twoindependent experiments, eachwith n¼�5,000nuclei per sample.






gH2AX foci in A549 cells, which returned to near baseline levels, 2hours following reoxygenation. Interestingly, 53BP1 foci wereundetectable in hypoxic A549 cells, even after reexposure to 21%O2. In striking contrast, all three MT-EGFR–expressing NSCLCstested showed high basal levels of 53BP1 and gH2AX foci.Exposure to a 0.1% O2 environment resulted in 2.5- to 7-folddecrease in 53BP1. However, reexposure to an aerobic environ-ment not only led to a further increase in gH2AX foci but also adramatic resurgence of 53BP1 foci to levels that were nearly 2-foldhigher compared with aerobic conditions. Highly similar resultswere observed when WT- and MT- EGFR were ectopicallyexpressed in HBEC cells (Fig. 4C). Relative to WT-EGFR–expres-sing cells, cells expressing DE746-E750 and L858R exhibitedsignificantly higher levels of gH2AX foci at 21% O2 that furtherincreased upon exposure to a hypoxic environment.

To determine whether hypoxia-associated increase in gH2AXfoci affected all cells or only cells in specific phases of cell-cycleprogression, cell-cycle distribution of basal and hypoxia-induced gH2AX foci in WT- andMT-EGFR NSCLCs was assessed(Fig. 4D). In aerobic A549 cells, the fraction of foci-bearingcells (with >5 gH2AX) was extremely low (0.2%–0.8%) in allphases of the cell cycle. but increased approximately 23-fold (upto 14.5%) following a 24-hour exposure to 0.1% O2. Theincrease in foci-bearing fractions was observed almost exclu-sively in the S-phase subpopulation, while those in G1, G2,and M subpopulations remained essentially unchanged. Instriking contrast, foci-bearing fractions in aerobic MT-EGFR–expressing NSCLCs were significantly elevated in all phases ofthe cell cycle, although the most dramatic difference was in theS-phase subpopulation (24- to 31-fold higher relative to A549).At 24 hours of exposure to 0.1%O2, foci-bearing fractions in allthree MT-EGFR NSCLC cell lines were significantly higher (2- to3.5-fold) compared with aerobic state but, again, these increasesweremost pronounced in theG1 and S subpopulations. The datasuggest that NSCLCs with activating DE746-E750 and L858Rmutations differ significantly from WT-EGFR cells in basal andhypoxia-associated DDR.

We next used confocal microscopy to examine the extent andmagnitude of radiation-induced DNA damage in aerobic andhypoxic WT- and MT-EGFR–expressing NSCLC cells 45 minutesafter exposure to 1 Gy radiation. Representative images of con-focal scans are shown in Fig. 5A. Regardless of EGFR status, inaerobic conditions, all three NSCLCs, regardless of EGFR status,registered essentially similar levels of gH2AX foci per nucleus(39.5–42.5 � 3.5) in response to 1 Gy (Fig. 5B). Moreover, thefraction of foci-bearing nuclei (with >10 foci) in aerobic state after1 Gy was also essentially similar; 80% to 85% (�10.11) of nucleihad >10 gH2AX foci in all three cell lines. As expected, exposure to1 Gy in an oxygen-deficient environment reduced the number ofgH2AX foci but to differing extents depending on EGFR status.

Figure 5.

Elevated MT-EGFR–associated gH2AX foci. A–C, NSCLC cells exposed for 24hours to an aerobic (21%O2) or hypoxic (0.1%O2) environmentwere eithermockirradiated (0 Gy) or exposed to radiation (1 Gy), pulsed with EdU and allowed torecover in their respective environments for 45minutes before being stained forgH2AX and EdU. A, Representative images of unirradiated cells in aerobicconditions from a typical experiment showing composite images generated bycollapsing image stack of multiple confocal images acquired at �100magnification. Mean gH2AX foci per nucleus (left) and fraction of gH2AX focibearing nuclei (>10 foci, right) across the entire population (B) or the EdUþ, S-phase subpopulation (C). Bars represent mean values and error bars are SEMfrom two independent experiments, each with n ¼ �100 nuclei per sample.

NSCLCs andHBECs exposed for 24 hours to an aerobic (21%O2) or hypoxic (0.1%O2) environment stained for total nuclei (DAPI), S-phase nuclei (EdU, Alexa-647), replication protein A (Alexa-488), and gH2AX (Alexa-547). D,Representative images acquired at �40 magnification from a typicalexperiment with NSCLCs are shown. E, S-phase nuclei with gH2AX and RPA fociwere enumerated in NSCLC and HBEC cells by image analysis software. Left,NSCLCs; right, HBEC cells. Bars represent mean fraction of nuclei that are in theS-phase (EdUþ) and contain >20 RPA foci, >20 gH2AX foci. Mean of means andSEMwere derived from at least two independent experiments, eachwith�1,500nuclei (n � 1,500) per sample.

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A549 cells irradiated under hypoxic conditions showed anapproximately 40% decrease in foci relative to aerobic irradiatedcells but only 25% and 2% reductions in H1975 and HCC827,respectively (Fig. 5B). When foci-bearing EdUþ, S-phase subpop-ulation was considered, a different pattern emerged (Fig. 5C).First, in all three NSCLCs, relative to aerobic cells, the meannumber of radiation-induced gH2AX foci in EdUþ nucleiremained unchanged with hypoxia. Moreover, S-phase foci-bear-ing fractions (>10 foci) in all three cell lines was either the same(HCC827) or slightly higher (A549 and H1975) with hypoxia.The data in Figs. 4D and 5C suggested that hypoxia-associatedincreases in the S-phase gH2AX foci likely involved replicationevents in the S-phase.

To verify whether this is indeed the case, we enumeratedfractions of S-phase nuclei harboring >20 gH2AX foci that alsocontained >20 foci of RPA. In NSCLCs and HBEC cells, WT-EGFR expression was associated with a 5-fold (HBEC) to 9-fold(A549) hypoxia-associated increase in the RPAþ/H2AXþ S-phase fraction (Fig. 5D and E). In aerobic and hypoxic cellsexpressing MT-EGFRs, this fraction was significantly highercompared with WT-EGFR cells. Camptothecin, a selectiveinhibitor of topoisomerase I (Top1), which stabilizes Top1-linked single-stranded nicks, also caused a dramatic increase inthe RPAþ/H2AXþ S-phase fraction (Supplementary Fig. S3A),but this increase was evident in both WT- and MT-EGFR cells. Incontrast, the effects of hydroxyurea (Supplementary Fig. S3B),which blocks initiation and elongation phases of replication,resembled those of hypoxia, with modest HU-induced increasein RPAþ/H2AXþ fractions in WT-EGFR NSCLCs and HBECs butdramatically high basal and HU-induced fractions of theseevents in MT-EGFR cells. The data indicate that in both aerobicand hypoxic states, replicating MT-EGFR–expressing cells har-bor a significantly higher burden of gH2AX foci compared withWT-EGFR cells.

MT-EGFR expression is associated with replication stressTo conclusively determine whether MT-EGFR expression has

any effect on kinetics of replication fork progression, we examinedkinetics of fork initiation, fork extension, and fork progression inNSCLCs A549, H1975, HCC827 after a 24-hour exposure to 21%or 0.1% O2 using the DNA fiber assay (30). This assay involvessequential 20-minute pulses of two nucleotide analogues, IdUandCldU, anddetectionof their incorporation in replicatingDNAby differently labeled fluorescent antibodies. Stalled forks (redonly fibers) active forks (red–green fibers), terminating forks(red–green–red), and new origins (green only) fibers (Fig. 6Aand B) were enumerated. Ratio of CldU:IdU lengths was used todetermine relative rates of fork progression, with lower ratiosrepresenting replication stalling or slowing and higher ratiosrepresenting unperturbed replication (Fig. 6C). Data in Fig. 6Breveal that, in aerobic WT-EGFR–expressing A549 NSCLCs, asmall fraction (7.5%) of all replicons occurred as stalled replica-tion forks, while themajority of structures were active forks (70%)or successful terminating replicons (13.5%). Exposure to 0.1% for24 hours resulted in a 7-fold increase in the fraction of stalledreplication forks (52%), with a 50% reduction in the fraction ofactive forks (35%) and terminating replicons (6%). In strikingcontrast, even in the aerobic state, almost 20% to26%of repliconsin MT-EGFR–expressing H1975 and HCC827 NSCLCs appearedas stalled forks, an approximately 3 to 4 higher baseline relative toWT-EGFR–expressing A549 cells. In hypoxic conditions, this

fraction of stalled forks formed the majority of replicons(�63%) in HCC827 and equaled active forks (45%) in H1975.Active and terminating forks constituted a combined 83% of allreplicons in aerobic A549 cells, but formed a significantly lesserproportion (65%–70%) of all replicons in H1975 and HCC827.In hypoxia, there was a further reduction in the combined fractionof active and terminating forks (31% and 45% in HCC827 andH1975, respectively). The high proportion of stalled replicationforks clearly seems to have affected fork velocity. Even in aerobicconditions, relative fork velocity measured as CldU/IdU ratiowas significantly reduced in H1975 (0.65 � 0.04) and HCC827(0.66� 0.03), comparedwith A549 cells (0.89� 0.04; Fig. 5C). Inhypoxic conditions, whereas WT-EGFR NSCLC registered a ratioof 0.60� 0.035, H1975 andHCC827 exhibited ratios of 0.49 and0.39, respectively.

Many studies have shown that, during prolonged hypoxia,levels of a key enzyme in nucleotide metabolism, RNR aredrastically reduced (22) and nucleotide depletion inhibits ordelays replication initiation, leading to stalled replication forksand reduced fork velocities (31, 32). We tested whether replica-tion stress encountered in aerobic MT-EGFR NSCLCs was due toRNRdeficiency. Consistent with other reports (22, 33), prolonged24-hour exposure to 0.1% O2 almost completely abrogated RNRlevels in all cell lines, regardless of EGFR status (Fig. 6D and E).The data indicate that, despite adequate levels of RNR, replicationfork progression in MT-EGFR NSCLCs is significantly compro-mised in aerobic conditions, and RNR-depleted hypoxic condi-tions may exacerbate replication stress.

EGFR blockade by cetuximab significantly reducesradioresistance of hypoxic cells

Synthetic lethality occurs when simultaneous inactivation oftwo complementary genes or pathways causes loss of cell viabilityor reproductive capacity, whereas inactivation of only one gene/pathway does not. Synthetic lethality has been demonstrated byinhibition of the PARP, an abundant nuclear protein with animportant function in an alternate NHEJ DSB repair pathwayinvolving XRCC1 and ligase III (34).Multiple studies have shownthat inhibition of PARP-mediated alternate NHEJ has a profoundsynthetic lethality effect in the context of HR deficiency in BRCA-deficient breast cancers (35) aswell asHR-downregulated hypoxictumors (36–39). As MT-EGFR expression (8, 11, 18) or EGFRblockade by the anti-EGFR mAb, cetuximab (7, 19) also com-promises NHEJ, we reasoned that cetuximabmight have a similarsynthetic lethality effect on HR-downregulated hypoxic tumors.As a positive control for synthetic lethality, we first confirmed theeffects of the PARP inhibitor, ABT-888, on PE in NSCLCs inaerobic and hypoxic conditions (Fig. 7A). Treatment with ABT-888 had no effect on survival in aerobic A549 cells but signifi-cantly reduced of A549 cells in the hypoxic state with an effectiveconcentration (EC50) of 0.5 mmol/L. Consistent with the resultsin Fig. 1E, hypoxia alone was sufficient to reduced PE in MT-EGFR–expressingNSCLCs. Surprisingly, ABT-888hadno effect onPE of any of the MT-EGFR NSCLCs cells even in hypoxic condi-tions. ABT-888 at a concentration of 1 mmol/L had no effect onradiosensitivity of aerobic A549 NSCLC cells but significantlyincreased A549 radiosensitivity in hypoxic conditions (Fig. 7B).D37 is the radiation dose required to reduce the SF down to 37%(SF ¼ 0.37). In aerobic conditions, the difference in D37 valuesbetween untreated and ABT-888–treated A549 cells was only0.88 Gy but it was approximately 3.5-fold higher in hypoxic






Figure 6.

Replication stress in MT-EGFR–expressing cells. A, Schematicrepresentation of DNA fiber assay(top). Representative images acquiredby confocal microscopy at �100magnification (bottom) from NSCLCcells exposed to aerobic or hypoxicconditions showing of IdU-stained(red) and CldU-stained (green) fibers.Inset, replication structures identified.B, Bars, representing meanpercentage of specific replicationstructures relative to total number ofreplicons, and error bars, representingSD from two independentexperiments, each with at least n ¼200 replicons per sample are shown.C,Dot density plot showing frequencydistribution of relative fork velocityderived from CldU/IdU ratios.Horizontal bars, mean ratio. Asteriskrepresents results of a t test analysis ofbetween A549 and H1975 or A549 andHCC827 cells. D, Western blotanalysis: representative blots probedfor RNR and b-actin in lysates ofNSCLCs (top) and HBEC (bottom)cells expressingWT, L858R, or DE746-E750 forms of EGFR. E, Densitometricanalysis of EGFR band intensitiesnormalized to b-actin band intensitiesin aerobic conditions (N) and hypoxic(H) conditions. Bars, mean RNRexpression relative toA549RNR levelsfor NSCLCs, or WT-EGFR–expressingcells in HBEC. Error bars are SEM fromtwo independent experiments.

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Figure 7.

EGFR blockade sensitizes hypoxicA549 to radiation. A, Clonogenic assayin indicated NSCLCs followingtreatment with indicatedconcentrations ofABT-888 in aerobic orhypoxic conditions. B, Clonogenicsurvival assay in A549 NSCLC cells,preexposed for 24 hours to aerobic orhypoxic environment, treated for2 hours with vehicle (UNT), 1 mmol/LABT-888 (B), or 50 mg/mL cetuximab(C) and exposed to indicated doses ofradiation. Symbols represent mean SFrelative to vehicle-treated samples (A)or SF normalized to PE (B and C). Meanof means and mean SD from at leastthree independent experiments, eachwith samples in triplicate are shown.D, HRFS values determined across arange of SFs in A549 NSCLCs that wereleft untreated (square symbol), ABT-888–treated (circle), and CTX-treated(triangle). E, Dose-modifying factor forABT-888 (circles) andCTX (triangles) inaerobic (filled symbols) and hypoxicconditions (open symbols). Symbolsare mean HRFS (D) or mean DMF (E),and error bars are SEM from threeindependent experiments, each withsamples in triplicate. Asterisksummarizes a simple, paired, two-tailedt test comparing DMF trends betweenCTX and ABT treatments in aerobicconditions where P < 0.001.






conditions. EGFR blockade by cetuximab had a surprisinglysimilar effect (Fig. 7C). The difference in D37 values betweenuntreated and cetuximab-treated was 1.02 Gy in aerobic and3.05 Gy in hypoxic conditions, again 3-fold higher. Both ABT-888 and cetuximab reduced HRF to similar extents (Fig. 7D).Moreover, both ABT-888 and cetuximab were significantlymore effective in hypoxic conditions (Fig. 7E). TheDMF is definedas the ratio of the radiation dose without agent to the radiationdose with agent to decrease SF to the same extent. In aerobicconditions, comparedwithABT-888, cetuximabwas slightlymoreeffective in augmenting radiation in aerobic A549 cells (Fig. 7E).However, mean DMF for both ABT-888 and cetuximab wassignificantly higher in hypoxic conditions (2.0 and 1.78 Gy,respectively) compared with aerobic conditions (1.12 and 1.29Gy, respectively). The data indicate that, like ABT-888, EGFRblockade by cetuximab was significantly more effective in radio-sensitizing A549 cells in the hypoxic state compared with cells inthe aerobic state.

DiscussionEGFR activation and downstream signaling has well-docu-

mented roles in prosurvival mechanisms that contribute toradiation resistance not only in aerobic but also in hypoxicenvironments. This study proffers evidence that activatingDE746-E750 and L858R mutations in EGFR have the oppositeeffect on hypoxia-associated radiation resistance and survival.Compared with WT-EGFR, MT-EGFR expression in NSCLCs orHBEC significantly compromised survival (Supplementary Fig.S1A and S1B) and diminished HRF (Figs. 1 and 2). Deficiencyof the NHEJ enzyme, DNA-PKcs, similarly reduced survival inhypoxic but not aerobic conditions (Supplementary Fig. S1C).Hypoxia-induced PARP cleavage, a reliable indicator of apo-ptosis, was observed in all NSCLCs, regardless of EGFR status,but not in HBEC cells, (Supplementary Fig. S1D). Thus, thedifference in survival between WT- and MT-EGFR–expressingcells is unlikely due to apoptosis.

We used HRFS as a measure of hypoxia-associated radiationresistance from physicochemical modification of DNA as well asbiochemical signaling. Although oxygen concentration can sig-nificantly influence HRFS, variations in cellular reducing environ-ment can also impactHRFS. Reduction of theDNA radical by thiol(–SH) containing compounds restores DNA to its original(reduced) form, thereby limiting radiation-induced DNA dam-age. Thus, cells with high thiol content are generally less sensitiveto radiation, and the effect of oxygen (or hypoxia) on HRFS inthese cases is less apparent (40). Thiol content was not a factor inthe decreasedHRFS because thiol levels were similar betweenWT-andMT-EGFRNSCLCs and ectopicMT-EGFR expression inHBECcells had no impact on thiol levels (Supplementary Fig. S2).Moreover, microarray analysis of WT- and MT-EGFR–expressingHBEC cells (Fig. 3, GEO accession number GSE95564) or Onco-mine database analysis of a large dataset with 99WT- and 127MT-EGFR tumor samples from NSCLC patients (GEO accessionnumber GSE31210) showed no significant differences in antiox-idant metabolic pathway between WT- and MT-EGFR NSCLCs. Itappears unlikely that the reduced HRFS in MT-EGFR–expressingcells was due to physicochemical modulation of the DNA radicalby oxygen or thiols.

A more likely mechanism could be defective DDR. We previ-ously demonstrated that NSCLCs andHBEC expressingMT-EGFR

are defective in NHEJ-mediated repair of radiation induced DNAdamage. Chronically hypoxic tumors exhibit a markedly alteredDDR relative to aerobic tumors. A number of studies havedemonstrated that, due to a selective downregulation of HR,hypoxic cells are overreliant on NHEJ as the sole DSB repairpathway (25, 41). In agreement with these reports, our dataconfirm that key proteins in the HR DNA repair pathway, includ-ing RAD50 and RAD51, are downregulated during prolongedhypoxia. Interestingly, ectopic expression of DE746-E750 andL858R-mutant EGFR not only reduced RAD51 levels, as didWT-EGFR, but also significantly downregulated levels of RAD50,which were relatively unaffected byWT-EGFR expression. RAD51exclusively participates in HR (42). In contrast, RAD50, a crucialcomponent of the MRE11-RAD50-NBS1 (MRN) complex, isrequired for both NHEJ and HR (43). Thus, in addition to theirreported defects in NHEJ (8, 10, 11, 18), MT-EGFRs may exert anadditional level of NHEJ and HR suppression in hypoxicconditions.

In hypoxic state, replication stress appears to be independent ofEGFR mutation status and could be, in part, due to RNR down-regulation (Fig. 5D), which is known to cause nucleotide deple-tion and replication arrest (33). However, in aerobic conditions,where RNR levels were similar, MT-EGFR NSCLCs showed ele-vated replication stress compared with WT-EGFR NSCLC. Onepossibility is that MT-EGFRs are defective in NHEJ (8, 10, 11, 18)and an accumulation of unresolved DSBs in the S-phase mayarrest or slow down fork progression (Fig. 4). An alternativemechanism may involve deregulation of the S-phase checkpointcontrol, resulting in fork-associated DNA damage. A recent studyfound that, in addition to NHEJ-mediated DSB repair, DNA-PKcsfacilitates S-phase checkpoint control (44), which involves Ataxiatelangiectasia–related (ATR) protein, checkpoint kinase 1, Chk1,and Cdc25a (45). DNA-PKcs is phosphorylated at T2609 by ATRin response to replication stress (46). We previously demonstrat-ed thatMT-EGFR expression inNSCLCs andHBEC cells complete-ly abrogates DNA-PKcs T2609 phosphorylation (11). However,unlike MT-EGFR expression (Fig. 5C), DNA-PKcs deficiency hadno effect on CldU/IdU ratio in aerobic conditions (44). Thus,although S-phase checkpoint impairment may elevate DSBs, thereplication stress in aerobic MT-EGFR NSCLCs may involve addi-tional factors.

Parallels can be drawn betweenMT-EGFR andMYC oncogene–associated replication stress. MYC influences replication at mul-tiple levels, including direct interaction with prereplication com-plex proteins, regulation of replication proteins, CDC6, CDK andminichromosomemaintenance (MCM)proteins, such asMCM2-7 and 10 at origin sites (47), and upregulation of cyclin E.Microarray gene expression analysis showed no differences inMYC mRNA between WT- and MT-EGFR–expressing HBECs.However, many of MYC upregulated proteins, including CyclinE, MCM 2-7, and 10, were significantly overexpressed in MT-EGFR–expressing cells relative to WT-EGFR cells (SupplementaryFig. S4). LikeMT-EGFR–expressing cells (Fig. 4), MYC overexpres-sion also results in fork-associated DSBs (48) and slowing ofreplication due to premature origin firing and increased origindensity (47). It is therefore conceivable that overabundance ofreplication factors, together with preexisting DSBs due to NHEJdefectsmay, in part, be responsible for replication stress in aerobicMT-EGFR NSCLCs.

Our study finds interesting parallels between cetuximab-medi-ated EGFR blockade and PARP inhibition. Similar to PARP

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inhibition, expression of MT-EGFR exerted lethality, possiblythrough inhibition of NHEJ in an altered, HR-deficient DDR.First, MT-EGFR (Supplementary Fig. S1A and S1B), or DNA-PKcsdeficiency (Supplementary Fig. S1C) similarly reduced survival ofhypoxic cells but did not affect survival of aerobic cells. Second,MT-EGFR expression resulted in markedly reduced HRFS in bothNSCLCs and HBEC (Figs. 1 and 2) to the same extent as PARPinhibition (Fig. 7). Third, like PARP inhibition (Fig. 7) and (39)EGFR blockade by cetuximab was significantly more effective inincreasing radiosensitivity of A549 cells in the hypoxic state(DMF: 1.78) compared with aerobic state (DMF: 1.29).

Interestingly, PARP inhibition did not affect survival ofNHEJ-compromised MT-EGFR NSCLCs in either aerobic orhypoxic conditions (Fig. 7A), indicating that EGFR and PARPmay be epistatic, with interdependent activities and mutuallysupportive roles in NHEJ-mediated DSB repair (49). Comparedwith the transient 2-hour ABT-888 treatment in our study, a 72-hour PARP inhibition by olaparib did show MT-EGFR NSCLCsto be slightly more sensitive than WT-EGFR NSCLCs (14).Alternatively, EGFR and PARP may share a synthetic lethalinteraction. EGFR blockade by lapatinib in triple-negativebreast cancers (50) and cetuximab in head and neck cancers(13) augmented ABT-888 cytotoxicity through possible sup-pression of NHEJ, HR, or both (12).

Our data support a model to explain MT-EGFR–associatedradiosensitivity in aerobic and hypoxic conditions. In aerobicconditions, expression of NHEJ-defectiveMT-EGFR compromisesrepair of radiation-induced aswell as fork-associatedDSBs, result-ing in increased radiosensitivity. In the hypoxic state, in thecontext of an altered HR downregulated DDR, expression ofNHEJ-defective MT-EGFR or blockade of EGFR-mediated NHEJhas a catastrophic effect on DSB repair and causes syntheticlethality.

Understanding how EGFR mediates repair of fork-associatedDSBs could elucidate novel therapeutic targets. Our finding thatMT-EGFR expression or EGFR blockade has a synthetic lethaleffect in hypoxic conditions suggests that anti-EGFR therapy incombination with radiotherapy could potentially be effective in

treating not only hypoxic NSCLCs but also NSCLCs with muta-tions in HR DSB repair genes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Saki, H. Makino, J. Andrews, D. Saha, S. Burma,C.S. NirodiDevelopment of methodology: M. Saki, H. Makino, L.-H. Ding, J. Andrews,S. Burma, C.S. NirodiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Makino, N. Tomimatsu, L.-H. Ding, J.E. Clark,E. Gavin, K. Takeda, J. Andrews, M.D. Story, C.S. NirodiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M. Saki, H. Makino, L.-H. Ding, J. Andrews, D. Saha,M.D. Story, S. Burma, C.S. NirodiWriting, review, and/or revision of the manuscript: M. Saki, L.-H. Ding,J.E. Clark, J. Andrews, D. Saha, M.D. Story, C.S. NirodiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.S. NirodiStudy supervision: C.S. NirodiOther (initial experiments while in the laboratory): P. Javvadi

AcknowledgmentsThe authors gratefully acknowledge Dr. David Boothman (University of

Texas Southwestern Medical Center), Dr. Robert W. Sobol (University of SouthAlabama, Mitchell Cancer Institute), and Dr. Conchita Vens (NetherlandsCancer Institute) for their critical inputs during the course of this study.

Grant SupportThis work was supported by the NIH (R01CA129364 to C.S. Nirodi,

RO1CA149461, RO1CA197796, R21CA202403 to S. Burma, R21CA175879to D. Saha), the National Aeronautics and Space Administration(NNX16AD78G to S. Burma, NNJ05HD36G to M.D. Story), and startupfunds from the University of South Alabama Mitchell Cancer Institute.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 13, 2017; revised June 29, 2017; accepted August 3, 2017;published OnlineFirst August 11, 2017.

References1. Gray LH, Conger AD, Ebert M, Hornsey S, Scott OC. The concentration of

oxygen dissolved in tissues at the time of irradiation as a factor inradiotherapy. Br J Radiol 1953;26:638–48.

2. Carlson DJ, Keall PJ, Loo BW Jr, Chen ZJ, Brown JM. Hypofractionationresults in reduced tumor cell kill compared to conventional fractionationfor tumors with regions of hypoxia. Int J Radiat Oncol Biol Phys2011;79:1188–95.

3. Chen Y, Henson ES, XiaoW, Huang D,McMillan-Ward EM, Israels SJ, et al.Tyrosine kinase receptor EGFR regulates the switch in cancer cells betweencell survival and cell death induced by autophagy in hypoxia. Autophagy2016;12:1029–46.

4. Lee SM, Lee CT, Kim YW, Han SK, Shim YS, Yoo CG. Hypoxia confersprotection against apoptosis via PI3K/Akt and ERKpathways in lung cancercells. Cancer Lett 2006;242:231–8.

5. Misra A, Pandey C, Sze SK, Thanabalu T. Hypoxia activated EGFR signalinginduces epithelial to mesenchymal transition (EMT). PLoS One 2012;7:e49766.

6. Franovic A, Gunaratnam L, Smith K, Robert I, PattenD, Lee S. Translationalup-regulation of the EGFR by tumor hypoxia provides a nonmutationalexplanation for its overexpression in human cancer. Proc Natl Acad Sci U SA 2007;104:13092–7.

7. Dittmann K, Mayer C, Fehrenbacher B, Schaller M, Raju U, Milas L, et al.Radiation-induced epidermal growth factor receptor nuclear import is

linked to activation of DNA-dependent protein kinase. J Biol Chem2005;280:31182–9.

8. Das AK, Chen BP, Story MD, Sato M, Minna JD, Chen DJ, et al. Somaticmutations in the tyrosine kinase domain of epidermal growth factorreceptor (EGFR) abrogate EGFR-mediated radioprotection in non-smallcell lung carcinoma. Cancer Res 2007;67:5267–74.

9. Kriegs M, Kasten-Pisula U, Rieckmann T, Holst K, Saker J, Dahm-Daphi J,et al. The epidermal growth factor receptor modulates DNA double-strandbreak repair by regulating non-homologous end-joining. DNA Repair2010;9:889–97.

10. Liccardi G, Hartley JA, Hochhauser D. EGFR nuclear translocation mod-ulates DNA repair following cisplatin and ionizing radiation treatment.Cancer Res 2011;71:1103–14.

11. Javvadi P, Makino H, Das AK, Lin YF, Chen DJ, Chen BP, et al. Threonine2609 phosphorylation of the DNA-dependent protein kinase is a criticalprerequisite for epidermal growth factor receptor-mediated radiationresistance. Mol Cancer Res 2012;10:1359–68.

12. Myllynen L, Rieckmann T, Dahm-Daphi J, Kasten-Pisula U, Petersen C,Dikomey E, et al. In tumor cells regulation of DNA double strand breakrepair through EGF receptor involves both NHEJ and HR and is indepen-dent of p53 and K-Ras status. Radiother Oncol 2011;101:147–51.

13. Nowsheen S, Bonner JA, Lobuglio AF, Trummell H, Whitley AC,Dobelbower MC, et al. Cetuximab augments cytotoxicity with poly






(adp-ribose) polymerase inhibition in head and neck cancer. PLoS One2011;6:e24148.

14. Pfaffle HN, Wang M, Gheorghiu L, Ferraiolo N, Greninger P, Borgmann K,et al. EGFR-activating mutations correlate with a Fanconi anemia-likecellular phenotype that includes PARP inhibitor sensitivity. Cancer Res2013;73:6254–63.

15. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFRmutations in lung cancer: correlation with clinical response to gefitinibtherapy. Science 2004;304:1497–500.

16. Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, et al. EGFreceptor genemutations are common in lung cancers from"never smokers"and are associatedwith sensitivity of tumors to gefitinib and erlotinib. ProcNatl Acad Sci U S A 2004;101:13306–11.

17. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFRmutations in lung cancer activate anti-apoptotic pathways. Science2004;305:1163–7.

18. Das AK, SatoM, StoryMD, PeytonM,Graves R, Redpath S, et al. Non-smallcell lung cancers with kinase domain mutations in the epidermal growthfactor receptor are sensitive to ionizing radiation. Cancer Res 2006;66:9601–8.

19. Dittmann K, Mayer C, Rodemann HP. Inhibition of radiation-inducedEGFR nuclear import by C225 (cetuximab) suppresses DNA-PK activity.Radiother Oncol 2005;76:157–61.

20. Olcina M, Lecane PS, Hammond EM. Targeting hypoxic cells through theDNA damage response. Clin Cancer Res 2010;16:5624–9.

21. Hammond EM,DorieMJ, Giaccia AJ. ATR/ATM targets are phosphorylatedbyATR in response tohypoxia andATM in response to reoxygenation. J BiolChem 2003;278:12207–13.

22. Chimploy K, Tassotto ML, Mathews CK. Ribonucleotide reductase, apossible agent in deoxyribonucleotide pool asymmetries induced byhypoxia. J Biol Chem 2000;275:39267–71.

23. Hammond EM, Denko NC, Dorie MJ, Abraham RT, Giaccia AJ. Hypoxialinks ATR and p53 through replication arrest. Mol Cell Biol 2002;22:1834–43.

24. Sprong D, Janssen HL, Vens C, Begg AC. Resistance of hypoxic cells toionizing radiation is influenced by homologous recombination status. Int JRadiat Oncol Biol Phys 2006;64:562–72.

25. Chan N, Koritzinsky M, Zhao H, Bindra R, Glazer PM, Powell S, et al.Chronic hypoxia decreases synthesis of homologous recombination pro-teins to offset chemoresistance and radioresistance. Cancer Res 2008;68:605–14.

26. Ding LH, Park S, Peyton M, Girard L, Xie Y, Minna JD, et al. Distincttranscriptome profiles identified in normal human bronchial epithelialcells after exposure to gamma-rays and different elemental particles of highZ and energy. BMC Genomics 2013;14:372.

27. Ding LH, Xie Y, Park S, Xiao G, Story MD. Enhanced identification andbiological validation of differential gene expression via Illumina whole-genome expression arrays through the use of themodel-based backgroundcorrection methodology. Nucleic Acids Res 2008;36:e58.

28. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI geneexpression and hybridization array data repository. Nucleic Acids Res2002;30:207–10.

29. Mirzoeva OK, Petrini JHJ. DNA damage-dependent nuclear dynamics ofthe Mre11 complex. Mol Cell Biol 2001;21:281–8.

30. Schwab RA, Niedzwiedz W. Visualization of DNA replication in thevertebrate model system DT40 using the DNA fiber technique. J Vis Exp2011;56:e3255.

31. Greenberg GR, Hilfinger JM. Regulation of synthesis of ribonucleotidereductase and relationship to DNA replication in various systems. ProgNucleic Acid Res Mol Biol 1996;53:345–95.

32. Odsbu I, Morigen, Skarstad K. A reduction in ribonucleotide reductaseactivity slows down the chromosome replication fork but does not changeits localization. PLoS One 2009;4:e7617.

33. Graff P, Amellem O, Andersson KK, Pettersen EO. Role of ribonucleotidereductase in regulation of cell cycle progression during and after exposureto moderate hypoxia. Anticancer Res 2002;22:59–68.

34. Audebert M, Salles B, Calsou P. Involvement of poly(ADP-ribose)polymerase-1 and XRCC1/DNA ligase III in an alternative routefor DNA double-strand breaks rejoining. J Biol Chem 2004;279:55117–26.

35. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al.Targeting the DNA repair defect in BRCA mutant cells as a therapeuticstrategy. Nature 2005;434:917–21.

36. Bindra RS, Schaffer PJ, Meng A, Woo J, Maseide K, Roth ME, et al. Down-regulation of Rad51 and decreased homologous recombination in hypoxiccancer cells. Mol Cell Biol 2004;24:8504–18.

37. Chan N, Bristow RG. "Contextual" synthetic lethality and/or loss ofheterozygosity: tumor hypoxia and modification of DNA repair. ClinCancer Res 2010;16:4553–60.

38. Kumareswaran R, Ludkovski O, Meng A, Sykes J, Pintilie M, Bristow RG.Chronic hypoxia compromises repair of DNA double-strand breaks todrive genetic instability. J Cell Sci 2012;125:189–99.

39. Liu SK, Coackley C, Krause M, Jalali F, Chan N, Bristow RG. A novelpoly(ADP-ribose) polymerase inhibitor, ABT-888, radiosensitizesmalignant human cell lines under hypoxia. Radiother Oncol 2008;88:258–68.

40. Astor MB. Oxygen concentration and the OER for acutely or chronicallythiol deficient cells. Int J Radiat Oncol Biol Phys 1986;12:1131–4.

41. Bindra RS, Schaffer PJ, Meng A, Woo J, Maseide K, Roth ME, et al.Alterations in DNA repair gene expression under hypoxia: elucidating themechanisms of hypoxia-induced genetic instability. Ann N Y Acad Sci2005;1059:184–95.

42. Baumann P, West SC. Role of the human RAD51 protein in homologousrecombination and double-stranded-break repair. Trends Biochem Sci1998;23:247–51.

43. Petrini JH. The mammalian Mre11-Rad50-nbs1 protein complex: integra-tion of functions in the cellular DNA-damage response. Am J Hum Genet1999;64:1264–9.

44. Lin YF, Shih HY, Shang Z, Matsunaga S, Chen BP. DNA-PKcs is required tomaintain stability of Chk1 and Claspin for optimal replication stressresponse. Nucleic Acids Res 2014;42:4463–73.

45. Shechter D, Costanzo V, Gautier J. Regulation of DNA replication byATR: signaling in response to DNA intermediates. DNA Repair 2004;3:901–8.

46. Yajima H, Lee KJ, Chen BP. ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-inducedreplication stress. Mol Cell Biol 2006;26:7520–8.

47. Dominguez-Sola D, Ying CY, Grandori C, Ruggiero L, Chen B, Li M, et al.Non-transcriptional control of DNA replication by c-Myc. Nature2007;448:445–51.

48. Maya-MendozaA,Ostrakova J, KosarM,Hall A,DuskovaP,MistrikM, et al.Myc and Ras oncogenes engage different energymetabolism programs andevoke distinct patterns of oxidative and DNA replication stress. Mol Oncol2015;9:601–16.

49. Veuger SJ, Curtin NJ, Smith GC, Durkacz BW. Effects of novel inhibitors ofpoly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinaseon enzyme activities and DNA repair. Oncogene 2004;23:7322–9.

50. Nowsheen S, Cooper T, Stanley JA, Yang ES. Synthetic lethal interactionsbetween EGFR and PARP inhibition in human triple negative breast cancercells. PLoS One 2012;7:e46614.


Saki et al.




2017;15:1503-1516. Published OnlineFirst August 11, 2017.Mol Cancer Res Mohammad Saki, Haruhiko Makino, Prashanthi Javvadi, et al. Damage Repair

Associated DNA−Resistance through Impaired Replication Fork EGFR Mutations Compromise Hypoxia-Associated Radiation

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EGFR Mutations Compromise Hypoxia- Associated Radiation … › content › molcanres › 15 › 11 › 1503.full.pdf · DNA Damage and Repair EGFR Mutations Compromise Hypoxia-Associated