Inhibition of DNA-Dependent Protein Kinase Induces Accelerated Senescence in Irradiated Human Cancer Cells

2011;9:1696-1707. Published OnlineFirst October 18, 2011.Mol Cancer Res Arun Azad, Susan Jackson, Carleen Cullinane, et al. Senescence in Irradiated Human Cancer CellsInhibition of DNA-Dependent Protein Kinase Induces Accelerated

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DNA Damage and Cellular Stress Responses

Inhibition of DNA-Dependent Protein Kinase InducesAccelerated Senescence in Irradiated Human Cancer Cells

Arun Azad1,3, Susan Jackson1, Carleen Cullinane1,3, Anthony Natoli1, Paul M. Neilsen5, David F. Callen5,Sauveur-Michel Maira6, Wolfgang Hackl6, Grant A. McArthur1,2,3,4, and Benjamin Solomon1,2,4

AbstractDNA-dependent protein kinase (DNA-PK) plays a pivotal role in the repair ofDNAdouble-strand breaks (DSB)

and is centrally involved in regulating cellular radiosensitivity.Here, we identifyDNA-PK as a key therapeutic targetfor augmenting accelerated senescence in irradiated human cancer cells. We find that BEZ235, a novel inhibitor ofDNA-PK and phosphoinositide 3-kinase (PI3K)/mTOR, abrogates radiation-induced DSB repair resulting incellular radiosensitization and growth delay of irradiated tumor xenografts. Importantly, radiation enhancement byBEZ235 coincides with a prominent p53-dependent accelerated senescence phenotype characterized by positiveb-galactosidase staining, G2–M cell-cycle arrest, enlarged and flattened cellular morphology, and increased p21expression and senescence-associated cytokine secretion. Because this senescence response to BEZ235 is accom-panied by unrepaired DNA DSBs, we examined whether selective targeting of DNA-PK also induces acceleratedsenescence in irradiated cells. Significantly, we show that specific pharmacologic inhibition of DNA-PK, but notPI3K or mTORC1, delays DSB repair leading to accelerated senescence after radiation. We additionally show thatPRKDC knockdown using siRNA promotes a striking accelerated senescence phenotype in irradiated cellscomparable with that of BEZ235. Thus, in the context of radiation treatment, our data indicate that inhibitionof DNA-PK is sufficient for the induction of accelerated senescence. These results validate DNA-PK as animportant therapeutic target in irradiated cancer cells and establish accelerated senescence as a novel mechanism ofradiosensitization induced by DNA-PK blockade. Mol Cancer Res; 9(12); 1696–707. �2011 AACR.

Introduction

Ionizing radiation is a widely used anticancer modality.However, the high relapse rates following radiotherapyindicate the urgent requirement for novel radiosensitizingstrategies. As radiation is a potent inducer of DNA double-strand breaks (DSB; ref. 1), targeting signaling networksinvolved in DSB repair is a promising approach for enhanc-ing cellular radiosensitivity. Inmammalian cells, the primaryrepair mechanism of radiation-induced DSBs is the nonho-mologous end-joining (NHEJ) pathway (2), in whichDNA-

dependent protein kinase (DNA-PK) plays a critical role.Upon recruitment to DSB sites, the catalytic subunit ofDNA-PK (DNA-PKcs) phosphorylates key DNA repairproteins and facilitates direct ligation of broken DNA ends(3). Accordingly, DNA-PK–deficient cells have ineffectiveDSB repair and are exquisitely sensitive to DSB-inducingagents (4). Conversely, upregulation of DNA-PK promotesrepair of DSBs leading to tumor radioresistance preclinically(5) and clinically (6–8). Thus, DNA-PK is an importantmolecular target for inhibiting DSB repair and enhancingthe cytotoxicity of radiation.Another attractive target for potentiating radiation efficacy

is the phosphoinositide 3-kinase (PI3K)/mTOR signalingpathway. Aberrant upregulation of this pathway occurs inmany humanmalignancies (9) and is implicated in resistanceto radiation preclinically (10, 11) and clinically (12–14).Although radiosensitization was reported with early PI3Kinhibitors such as wortmannin and LY294002 (15), theseagents had unacceptable pharmacokinetic and toxicity pro-files (16) and have now been superseded by novel PI3Kinhibitors with superior pharmacologic properties. One ofthese is NVP-BEZ235 (Novartis Pharma AG), an orallyavailable PI3K/mTOR inhibitor (17) that is currently inphase II clinical testing.In addition to inhibiting PI3K and mTOR, it has recently

been reported that BEZ235 has potent activity againstDNA-PK (18). This suggests that treatment of irradiated

Authors' Affiliations: 1Division of Cancer Research and 2Division ofCancer Medicine, Peter MacCallum Cancer Centre, East Melbourne;Departments of 3Pathology and 4Medicine, St. Vincent's Hospital, Univer-sity of Melbourne, Parkville, Victoria; 5Cancer Therapeutics Laboratory,Discipline of Medicine, University of Adelaide and Hanson Institute, Ade-laide, South Australia, Australia; and 6Novartis Institutes for BiomedicalResearch, Oncology Disease Area, Novartis Pharma AG, Basel,Switzerland

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

Corresponding Author: Benjamin Solomon, Peter MacCallum CancerCentre, Locked Bag 1, A'Beckett Street, Melbourne 3002, Victoria, Aus-tralia. Phone: 613-9656-1697; Fax: 613-9656-1408; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-11-0312

�2011 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 9(12) December 20111696





cells with BEZ235 is likely to impair NHEJ resulting inaccumulation of unrepaired DSBs. In turn, the clonogenicpotential of irradiated tumor cells may be compromised byBEZ235 as unrepaired DSBs can trigger a range of thera-peutically desirable outcomes that include apoptosis, necro-sis, mitotic catastrophe, and terminal growth arrest (accel-erated senescence; ref. 19). Although the induction ofapoptosis underlies the antitumor efficacy of radiation incertain malignancies, solid tumors commonly exhibit resis-tance to radiation-induced apoptosis (20). Nevertheless,radiation remains an effective modality for the treatmentof many tumor types, thus emphasizing a potentially valu-able role for nonapoptotic responses to treatment. In keepingwith this, emerging evidence indicates that apoptosis is notthe sole or even predominant mechanism through whichsome DNA-damaging agents and PI3K/mTOR pathwayinhibitors increase radiosensitivity (20–23). Thus, nonapop-totic mechanisms may be a significant factor in radiosensi-tization of epithelial cancer cells by BEZ235.Herein, we examined the cellular andmolecular outcomes

of inhibiting DNA-PK in irradiated cancer cells. Afterconfirming that BEZ235 potently inhibits DNA-PK, wedetermined that treatment with BEZ235 attenuates radia-tion-induced DNA DSB repair leading to p53-dependentaccelerated senescence after radiation in vitro and in vivo.Significantly, using complementary genetic and pharmaco-logic approaches, we also found that selectively targetingDNA-PK reproduces the phenotypic changes observed withBEZ235. These findings collectively identify acceleratedsenescence as a critical determinant of outcome whenDNA-PK blockade is combined with radiation and providea sound rationale for specific therapeutic targeting of DNA-PK in irradiated tumor cells.

Materials and Methods

Cell cultureAll cell lines were obtained from the American Type

Culture Collection apart from p53-inducible H1299 cells(24). Cells were incubated at 37�C/5%CO2 in RPMI-1640(H460, PC3, SKBR3, and MCF7 cells), Dulbecco's Mod-ified Eagle's Media (DMEM)/F12 (A549 cells), a-MEM(A431 cells), or DMEM (U87-MG and H1299 cells)supplemented with 10% FBS apart from U87-MG cellswhich were supplemented with 15% FBS. SKBR3 cells werealso supplemented with insulin (0.2U/mL) andH1299 cellswere maintained in plasmocin (InvivoGen) at 1:10,000.

Inhibitors and inhibitor treatmentBEZ235, BKM120, and RAD001 were obtained from

Novartis Pharma AG. KU57788 was purchased from SYN-thesis. All compounds were dissolved in dimethyl sulfoxideexcept RAD001, which was dissolved in high-grade ethanol.

Delivery of irradiationCell lines were irradiated using a 137Cs source (Gamacell

40, Atomic Energy of Canada) at a dose rate of 0.57Gy/min.In all in vitro studies, drugs were added to cells 1 hour before

irradiation. With the exception of clonogenic survival assays(see later), drugs were maintained in growth medium untiltime of harvest.

Clonogenic survival assaysClonogenic survival assays were conducted as previously

described (25). Briefly, cells were treated with BEZ235 (50or 100 nmol/L) or vehicle control and irradiated (1–8 Gy) 1hour later. BEZ235 was removed 24 hours after irradiation,and cells were incubated in drug-free medium for 10 daysbefore formaldehyde fixation and crystal violet staining.Colonies consisting of 50 cells or more were counted in 3replicate plates. Clonogenic survival curves were fit usingmultitarget single hit (MTSH) regression to determine D0(radiation dose resulting in 37% surviving fraction).

Senescence-associated b-galactosidase assayCells were treated with drug for 48 hours and then fixed in

2% paraformaldehyde/0.2% glutaraldehyde. After additionof staining solution containing 20 mg/mL X-gal (Promega),cells were incubated for 16 to 24 hours at 37�C in a non-CO2 chamber. Images were obtained with a Leica invertedmicroscope at 200� magnification using a SPOTlightdigital camera.

Flow cytometryCell-cycle analysis was conducted using a FACSCalibur

flow cytometer (Becton Dickinson) as previously described(25). The percentage of apoptotic cells was quantified bymeasurement of sub-G1 levels using FCS Express (De NovoSoftware).

Cytokine antibody arraysAfter treatment for 96 hours, cell supernatant was col-

lected and stored at �20�C. Supernatant volumes werenormalized to cell number and then analysed using antibodyarrays (R&D Systems; #ARY005) as per the manufacturer'sinstructions.

ImmunoblottingImmunoblotting was conducted as previously described

(25).The followingantibodieswereused: phospho-AKTSer473

(#9271; 1:1,000), total AKT (#9272; 1:1,000), phospho-S6Ser235/236 (#2211; 1:2,000), total S6 (#2217; 1:2,000; CellSignaling Technology), total p53 (sc-6243; 1:500), total p21(sc-397; 1:2,000; Santa Cruz Biotechnology), and total-DNA-PK (1:1,000; gift from Dr. Kum Kum Khanna,Queensland Institute ofMedical Research,Herston, Queens-land, Australia).

ImmunofluorescenceCells were cytospun on to Superfrost plus slides (Lomb) or

grown on 4-well chamber slides (Lab-Tek), fixed in 4%paraformaldehyde for 15minutes, andblocked for 60minuteswith 10% FBS. The following primary antibodies wereincubated for 90 minutes at room temperature: gH2AXSer139

(ab22551; 1:250), phospho-DNA-PKcsThr2609 (ab18356;1:250;Abcam),phospho-ATMSer1981 (#39530;1:500;Active

DNA-PK Blockade and Radiation-Induced Senescence

www.aacrjournals.org Mol Cancer Res; 9(12) December 2011 1697





Motif),phospho-histoneH3Ser10 (06–570;1:1,000;Upstate),and b-tubulin (E7, 1:100; Developmental Studies Hybrid-oma Bank). Secondary antibodies (anti-mouse Alexa Fluor488, 1:500; anti-rabbit Alexa Fluor 568, 1:500; MolecularProbes) were incubated for 75 minutes at room temperature.Slides were mounted with Prolong antifade with 40,6-diami-dino-2-phenylindole (DAPI; Invitrogen) and images obtainedwith an Olympus BX-51 fluorescence microscope at 60�magnification using a SPOTlight digital camera.MetaMorphimage analysis software was used for quantification of fociper cell.

Terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling assayH460 and A549 cells were treated as indicated for 48

hours and then cytospun on to Superfrost plus slides. Cellswere fixed in 4% paraformaldehyde for 15 minutes, andterminal deoxynucleotidyl transferase–mediated dUTP nickend labeling (TUNEL) assay (Chemicon International) wasconducted as per the manufacturer's instructions.

Animal studiesFollowing subcutaneous injection of H460 cells into 8-

to 12-week-old athymic nude mice (Australian ResearchCouncil, Canberra, ACT, Australia), xenograft experi-ments were done as previously described (25). Briefly,once tumors had grown to approximately 100 mm3, micewere randomized into the following groups (n ¼ 10):vehicle control (NMP/PEG 300, days 1–5 and 8–12),BEZ235 (40 mg/kg/d oral, days 1–5 and 8–12), radiationwith vehicle control (2 Gy, days 2–5), or radiation withBEZ235 (administered 2 hours preradiation). Radiationwas delivered using a linear accelerator (Varian MedicalSystems) with radiation dosimetry supervised by a medicalphysicist. Experimental endpoints were tumor volume of1,000 mm3 or significant tumor-related ulceration. Allexperiments were carried out according to the guidelinesset out by the Australian National Health and MedicalResearch Council with prior Institutional Animal EthicsCommittee approval. Tumor growth delay was calculatedas previously described (25).

Immunohistochemical studiesH460 tumor xenografts were harvested 48 and 96 hours

after radiation treatment, fixed with 10% neutral-bufferedformalin, and embedded in paraffin. Staining for Ki67 wasconducted on sections (4 mm) as previously described (25).Sections were stained for gH2AX (#2577, 1:500; CellSignaling) using standard techniques in a Dako autostainerand then counterstained with hematoxylin.

Senescence-associated b-galactosidase assay in vivoH460 tumor xenografts were harvested 48 and 96 hours

after radiation treatment, frozen in Tissue-Tek optimumcutting temperature (OCT; Miles Laboratories), and storedat�80�C. Frozen sections (4 mm) were fixed and stained inaccordance with in vitro experiments and then counter-stained with nuclear fast red.

siRNAH460 cells were seeded overnight and then transfected

with siRNA targeting PRKDC or nontargeting siRNA(Dharmacon: SMARTpool catalogue no.D-005030-01 andD-001210-01-05, respectively) using Lipofectamine 2000(Invitrogen). Cells were washed twice with PBS 6 hours laterand incubated in RPMI-1640 supplemented with 10% FBSfor a total of 72 hours prior to irradiation.

Statistical analysisStatistical significance was determined using the Student t

test calculated with GraphPad Prism software (GraphPadSoftware, Inc.).

Results

BEZ235 inhibits DNA-PK and enhances G2–M growtharrest after radiationThe radiosensitizing properties of BEZ235 were tested in

H460 and A549 non–small cell lung cancer (NSCLC) cells.These cell lines have constitutive activation of the PI3K/mTOR pathway due to oncogenic KRAS mutations (26).H460 cells additionally possess activating mutations ofPIK3CA (26). Our initial experiments confirmed thatBEZ235 inhibited PI3K/mTOR signaling, as evidenced bydecreased phosphorylation of AKT and S6 (SupplementaryFig. S1). We next investigated the effects of BEZ235 on theDNA-PKcs,whichnotably has structural homologywith classIA PI3K (17). We found that 100 nmol/L BEZ235 signif-icantly decreased the number of phospho-DNA-PKcs foci percell after 4 Gy (H460, 19.6 to 8.7; A549, 16.1 to 7.3) and 10Gy radiation (H460, 31.4 to 9.9; A549, 29.1 to 9.1; Fig. 1A).Having established that BEZ235 is a potent inhibitor of

PI3K/mTOR and DNA-PK, we conducted clonogenicsurvival assays on H460 and A549 cells. Treatment withBEZ235 for 24 hours significantly increased cellular radio-sensitivity, as reflected by a dose-dependent reduction incolony formation after 10 days (Fig. 1B). The D0 (radiationdose resulting in 37% surviving fraction) decreased from 2.9to 2 and 1.4 Gy in H460 cells and from 5 to 2.2 and 1.2 Gyin A549 cells, following treatment with 50 and 100 nmol/LBEZ235, respectively. These results were normalized toaccount for the effect of BEZ235 alone, which reducedcolony numbers by approximately 15% to 30% comparedwith untreated controls.To investigate potential mechanisms of radiation

enhancement by BEZ235, fluorescence-activated cell-sort-ing (FACS) analysis was conducted on H460 and A549 cellsstained with propidium iodide. We determined that treat-ment with 100 nmol/L BEZ235 did not increase the sub-G1(apoptotic) fraction at 48 hours after radiation in either cellline (Fig. 1C). To provide an additional mechanism forquantification of apoptosis, we conducted the TUNEL assayin H460 and A549 cells. Results from these experimentsconfirmed the findings from the measurement of sub-G1fraction that BEZ235 does not enhance apoptosis in irra-diated cells (Supplementary Fig. S2). In contrast to thesefindings, we found that 100 nmol/L BEZ235 substantially

Azad et al.

Mol Cancer Res; 9(12) December 2011 Molecular Cancer Research1698





Figure 1. BEZ235 inhibits DNA-PK and enhances G2–M growth arrest after radiation. A, representative immunofluorescent images of phospho-DNA-PKcs(Thr2609) foci at 30 minutes after radiation (�60 magnification). Green, phospho-DNA-PKcs foci; blue, DAPI staining. Right, number of phospho-DNA-PKcsfoci per cell. Data are mean � SEM from 3 independent experiments. ��, P < 0.001; ��, P < 0.01. B, clonogenic survival assays of H460 and A549cells. Data points represent the mean surviving fraction � SEM of 3 independent experiments. C, cell-cycle profile of H460 and A549 cells stained withpropidium iodide after 48 hours treatment. Right, quantitation of sub-G1 fraction. Data are mean � SEM from 3 independent experiments.







increased G2–Mphase cell-cycle arrest at 48 hours following4 Gy (H460, 10%–30%; A549, 19%–30%) and 10 Gyradiation (H460, 29%–50%; A549, 35% to 47%; Fig. 1C).Consistent with our earlier observation that short-termtreatment (24 hours) with BEZ235 leads to a prolongeddecrease in postradiation clonogenic survival (Fig. 1B), thesedata suggest that BEZ235 and radiation induce irreversiblegrowth arrest in H460 and A549 cells.

BEZ235 induces accelerated senescence after radiationin vitroAs irreversible cell-cycle arrest is a feature of accelerated

senescence, H460 and A549 cells were examined for senes-

cence-associated b-galactosidase (SA-bGal) activity (Fig.2A). In both cell lines, treatment with 100 nmol/L BEZ235significantly enhanced SA-bGal staining after 4 Gy (H460,3%–32%; A549, 9%–34%) and 10 Gy radiation (H460,7%–61%; A549, 14%–44%; Fig. 2B). Untreated controlcells, in contrast, exhibited no SA-bGal staining, therebyconfirming the absence of replicative (nonaccelerated) senes-cence. Treatment with drug alone also did not result in amajor senescence response indicating that BEZ235 pro-motes accelerated senescence in combination with but notindependently of radiation.Notably, positive SA-bGal stain-ing induced by BEZ235 in irradiated cells was accompaniedby characteristic morphologic features of accelerated

Figure 2. BEZ235 induces accelerated senescence after radiation (IR) in vitro. A, representative images of SA-bGal activity after 48 hours treatment (�200magnification). B, percentage of SA-bGal positive cells. Data are mean � SEM from 3 independent experiments. ��, P < 0.001; ��, P < 0.01; �, P < 0.05. C,Western blot showing total p21 levels after 48 hours treatment in H460 and A549 cells. DMSO, dimethyl sulfoxide.

Azad et al.






senescence (flattened, enlarged, elongated, and multinucle-ated cells). Interestingly, although multinucleation is also afeature of mitotic catastrophe, M phase (phospho-histoneH3 positive) cells displaying typical changes of abnormalmitoses (multipolar spindles, multiple centrosomes, andmicrotubule misalignment; ref. 27) were infrequently seenirrespective of treatment (Supplementary Fig. S3).Senescent cells commonly secrete high levels of proin-

flammatory cytokines and growth factors, a process knownas the senescence-associated secretory phenotype (SASP;ref. 28). Using cytokine antibody arrays to analyze cellculture supernatants, we found that the combination ofBEZ235 (100 nmol/L) and 4 Gy radiation noticeablyelevated interleukin (IL) 8, macrophage migration inhibi-tory factor, (MIF) and plasminogen activator inhibitor-1(PAI-1) levels in H460 cells and MIF levels in A549 cells(Supplementary Fig. S4). Importantly, these cytokines are allrecognized as components of the SASP (29, 30). Senescentcells also typically exhibit upregulation of the cyclin-depen-dent kinase inhibitor (CDKI) p21, which is a key down-stream target of p53 (31). Using Western blot analysis, wedetermined that the addition of BEZ235 increased p21levels following 4 and 10 Gy radiation (Fig. 2C). Together,these results show that the response of irradiated H460 andA549 cells to BEZ235 is characterized by many of the keyfeatures of accelerated senescence.

BEZ235 delays repair of radiation-induced DNA DSBsin vitroSustained G2–M phase cell-cycle arrest is recognized as a

classical cellular response to DNA damage (32). As previous

reports indicate that up to 20 DNA DSBs are required forenforcing theG2–Mcheckpoint (33), we conducted stainingfor gH2AX (a marker of DNA DSBs) on H460 and A549cells. In both cell lines, the addition of 100 nmol/L BEZ235significantly increased the number of gH2AX foci per cell at24 hours following 4Gy (H460, 6.0–22.2; A549, 2.7–15.8)and 10 Gy radiation (H460, 12.7–31.3; A549, 8.0–45.3; Fig. 3). In contrast, treatment with BEZ235 did notincrease gH2AX expression at early time points after radi-ation (Supplementary Fig. S5). Indeed, the rate of appear-ance of gH2AX foci formation was delayed by BEZ235 inirradiated cells suggesting that BEZ235 causes persistence ofradiation-induced gH2AX foci by inhibiting DSB repairrather than augmenting DNA damage at earlier time points.ThemajorDNADSB repair pathways inmammalian cells

are the NHEJ and homologous recombination pathways, inwhich DNA-PK and ataxia telangiectasia mutated (ATM)play crucial roles (34). Therefore, having previously estab-lished that BEZ235 inhibits DNA-PKcs, we investigated itseffects on ATM phosphorylation in irradiated cells. Consis-tent with the findings of Maira and colleagues (17), wedetermined that treatment with BEZ235 did not inhibitradiation-induced phosphorylation of ATM at S1981 ineither H460 or A549 cells (Supplementary Fig. S6). Thus,DNA-PK, but not ATM, blockade underlies the inhibitionof DSB repair by BEZ235.

BEZ235 sustains DNA DSBs and induces acceleratedsenescence after radiation in vivoThe in vivo activity of BEZ235 and radiation was assessed

in H460 xenografts established subcutaneously in athymic

Figure 3. BEZ235 delays repair of radiation-induced DNADSBs in vitro. Representative immunofluorescence images of gH2AX (Ser139) foci in H460 (top) andA549 (bottom) cells at 24 hours after radiation (�60magnification). Green, gH2AX foci; blue, DAPI staining. Right, number of gH2AX foci per cell. Data aremean� SEM from 3 independent experiments. ��, P < 0.001; ��, P < 0.01.







nudemice. Preliminary experiments revealed that a radiationdose of 8 Gy delivered in 4 consecutive daily 2 Gy fractionselicited measurable antitumor activity without causing per-manent tumor eradication (data not shown). We also estab-lished that the maximum tolerated dose of BEZ235 was 40mg/kg administered orally once daily. Using this treatmentschedule, we subsequently found that the combination ofBEZ235 and radiation substantially increased tumor growthdelay compared with either treatment alone (BEZ235 þradiation: 9.5 days vs. radiation: 3.5 days vs. BEZ235: 3.5days; Fig. 4A).In a parallel experiment, H460 tumors were harvested

from mice 48 and 96 hours after a single 8 Gy radiationfraction and then examined for SA-bGal activity. Whilenegligible staining was shown in tumors treated withBEZ235 alone (40 mg/kg daily on days 1 and 2), therewas a striking increase in SA-bGal activity when BEZ235was combined with radiation (Fig. 4B). This effect wasmost pronounced 96 hours after radiation and coincidedwith a marked reduction in cellular proliferation (Ki67).Staining for gH2AX was also conducted on H460 xeno-grafts to assess the impact of combined treatment on the

formation of DSBs. Although treatment with BEZ235alone did not increase staining above that of untreatedcontrols, gH2AX levels at 96 hours were markedly increas-ed in irradiated tumors treated with BEZ235 (Fig. 4B).These data collectively indicate that the enhancement ofradiation efficacy by BEZ235 in H460 xenografts involvesboth the accumulation of unrepaired DNA DSBs and theinduction of accelerated senescence.

Induction of accelerated senescence by BEZ235 inirradiated cells is p53 dependentStimuli that activate the DNA damage response (DDR)

primarily drive senescence through the p53–p21 signalingpathway (35). As both H460 and A549 cells have wild-type p53 (36), we investigated whether the senescencephenotype produced by BEZ235 in irradiated cells isp53 dependent. Initially, a panel of cell lines harboringwild-type (MCF-7, U87-MG; ref. 37) and deleted/mutated p53 (HT29, SKBR3, PC3, and A431; ref. 36)was screened for SA-bGal activity. Although BEZ235strongly induced SA-bGal staining after radiation in p53wild-type MCF-7 and U87-MG cells, no staining was

Figure 4. BEZ235 inducesaccelerated senescence afterradiation (IR) in vivo. A, H460 tumorxenograft growth after treatment asindicated. Data points representmean tumor growth of each group(n ¼ 10) � SEM. Curves are shownuntil any animal in a group reachedan experimental endpoint. B,representative images of SA-bGalactivity and Ki67 and gH2AX(Ser 139) staining in H460xenografts harvested at indicatedtime points after a single 8 Gyradiation fraction(�20 magnification).

Azad et al.






observed in the cell lines with mutated or absent p53(Supplementary Fig. S7A).Given these findings, SA-bGal staining was conducted on

p53-null H1299NSCLC cells cotransfected with vectors forthe ecdysone receptor and inducible p53 (24). Addition ofthe ecdysone analogue Ponasterone A (Invitrogen) inducedp53 and p21 expression in H1299 cells (Supplementary Fig.S7B), which in turn was associated with positive SA-bGalstaining and morphologic features of senescence followingtreatment with BEZ235 and radiation (Supplementary Fig.S7C). In comparison, no SA-bGal activity was seen inH1299 cells in the absence of Ponasterone A. Together,these results confirm that accelerated senescence responsefollowing treatment with BEZ235 and radiation is depen-dent on the p53–p21 signaling axis.

Selective pharmacologic inhibition of DNA-PK leads toaccelerated senescence in irradiated cellsTo further investigate the significance of DNA-PK

inhibition in the accelerated senescence response to radi-ation, we compared the activity of a selective DNA-PKinhibitor (KU57788) with that of BEZ235 in H460 cells.Following 10 Gy radiation, we observed that KU57788did not alter sub-G1 levels (radiation: 13% vs. KU57788þ radiation: 11%) and had comparable effects to BEZ235on gH2AX expression (45 vs. 40 foci per cell), G2–Mgrowth arrest (50% vs. 55%), and SA-bGal staining (65%vs. 67%; Fig. 5A–C). Similar to BEZ235, we also estab-lished that p53 is required for KU57788 to induceaccelerated senescence in irradiated cells (Fig. 5D). Incontrast to these findings, selective PI3K (BKM120) andmTORC1 (RAD001) inhibitors used alone or in combi-nation did not reproduce the effects of BEZ235 orKU57788 on DNA DSBs, G2–M cell-cycle arrest, orSA-bGal staining. Importantly, the activity of KU57788was not related to inhibition of related kinases as itselectively inhibited DNA-PK (Supplementary Fig. S8),without influencing the activity of AKT, S6, or ATM(data not shown). Likewise, neither BKM120 norRAD001 inhibited DNA-PK (Supplementary Fig. S8) atdoses that were sufficient to suppress phosphorylation ofAKT and S6, respectively (data not shown). These datacollectively suggest that selective pharmacologic inhibitionof DNA-PK leads to p53-dependent accelerated senes-cence after radiation.

DNA-PK knockdown by siRNA induces acceleratedsenescence in irradiated cellsOn the basis of our findings withKU57788, we proceeded

to knockdown DNA-PK in H460 cells using siRNA. TotalDNA-PK expression was abolished by PRKDC siRNA inunirradiated cells, whereas a scrambled (control) siRNAsequence had no impact on DNA-PK levels (Fig. 6A).Similarly, phospho-DNA-PK expression was effectivelyreduced by PRKDC siRNA in irradiated cells (Fig. 6B, top),leading to persistence of gH2AX foci at 24 hours afterradiation (Fig. 6B, middle). Significantly, the residual DNADSBs were accompanied by a dramatic increase in SA-bGal

staining (PRKDC siRNA 67% vs. vehicle 5% and scrambledsiRNA 4%) and striking morphologic changes typical ofaccelerated senescence (Fig. 6B, bottom). These results showthat selective targeting of DNA-PK is sufficient to induceaccelerated senescence in irradiated cancer cells.

Discussion

Radiotherapy is a major treatment modality adminis-tered to approximately half of all patients with cancer (1).Unfortunately, a significant number of these patientsdevelop and ultimately die of locally recurrent disease,emphasizing the need to identify novel means of enhanc-ing radiation efficacy. In this study, we examined theradiosensitizing properties of BEZ235 in H460 and A549NSCLC cells. We found that BEZ235 potentiated theantitumor activity of radiation in vitro and in vivo,consistent with recent reports of radiation enhancementby novel PI3K/mTOR/DNA-PK inhibitors (38, 39).Significantly, BEZ235 did not increase the radiosensitivityof H460 and A549 cells by augmenting radiation-inducedapoptosis or mitotic catastrophe. Instead, irradiated cellstreated with BEZ235 displayed many of the hallmarks ofaccelerated senescence (31) including irreversible cell-cycle arrest, positive SA-bGal staining, classical morpho-logic changes, and increased expression of p21 and aSASP.Accelerated senescence is increasingly recognized as an

important and therapeutically advantageous outcome oftreatment with DNA-damaging agents (20, 40, 41).While several studies have previously shown that epithelialcancer cells readily undergo senescence after radiationtreatment in vitro (reviewed in ref. 20), a recent reportfrom Efimova and colleagues identified accelerated senes-cence in breast cancer xenografts treated with radiationand the PARP inhibitor ABT-888 (42). We similarlyfound that growth delay of irradiated H460 xenograftsby BEZ235 was associated with a robust acceleratedsenescence response. As a result, our data identify BEZ235as just the second pharmacologic agent to promote accel-erated senescence after radiation in vivo.The induction of accelerated senescence in irradiated

cells and tumors treated with BEZ235 was associated withdelayed repair of DNA DSBs. These findings mirror thoseof Efimova and colleagues and support an emergingparadigm that links cellular senescence with a prolongedDDR (33). While transient DNA damage signaling resultsin a reversible cell-cycle arrest, Rodier and colleaguesrecently showed that irreversible growth arrest and theacquisition of a SASP are dependent upon persistentactivation of the DDR (43). Our data are consistent withthis as the addition of BEZ235 to radiation resulted inSASP cytokine secretion and other features of acceleratedsenescence. In contrast, cells treated with radiation aloneexhibited neither persistent DSBs nor a prominent senes-cence phenotype. Thus, residual DNA DSBs appear to bea crucial determinant of accelerated senescence after treat-ment with BEZ235 and radiation.







Figure 5. Selective pharmacologic inhibition of DNA-PK leads to accelerated senescence after radiation (IR). A, representative immunofluorescence images ofgH2AX (Ser139) foci in H460 cells at 24 hours after 10 Gy radiation (�60 magnification). Cells were treated with vehicle, BEZ235 (250 nmol/L), KU57788(1mmol/L), BKM120 (1mmol/L) or RAD001 (10nmol/L).Green, gH2AX foci; blue, DAPI staining.Right, number of gH2AX foci per cell. Data aremean�SEM from3 independent experiments. ��, P < 0.001; ��, P < 0.01. B, cell-cycle profile of H460 cells at 48 hours after 10 Gy radiation. Right, percentage ofH460 cells arresting with 4nDNA content. Data aremean�SEM from3 independent experiments. ��,P < 0.01. C, representative images of SA-bGal activity inH460 cells at 48 hours after 10 Gy radiation (�200 magnification). Right, percentage of SA-bGal–positive cells. Data are mean � SEM from 3 independentexperiments. ��, P < 0.001; ��, P < 0.01. D, representative images of SA-bGal activity in p53-inducible H1299 cells at 48 hours after 10 Gy radiation(�200 magnification). PoA, Ponasterone A.

Azad et al.






On the basis of these findings, we postulated thatDNA-PK inhibition is a critical factor in the senescenceresponse of irradiated cells to BEZ235. This assertion issupported by the observation that a selective DNA-PKinhibitor (KU57788) exerted comparable effects toBEZ235 on DNA repair and senescence following radi-ation. To confirm that inhibition of DNA-PK is sufficientto induce accelerated senescence after radiation, we thenshowed that knockdown of PRKDC with siRNA leads toaccelerated senescence in irradiated H460 cells. Althoughprevious studies have also reported that DNA-PK inhi-bitors retard DSB repair after radiation (44–47), theprecise cellular mechanisms through which DNA-PKinhibition increases tumor radiosensitivity are currentlyunknown. Our data provide the first demonstration of amechanistic link between DNA-PK knockdown andaccelerated senescence in irradiated cells and indicate thataccelerated senescence induced by DNA-PK blockade is asignificant factor in radiosensitization of p53 wild-typetumors.The results of this study highlight the therapeutic poten-

tial of combining DNA-PK blockade with radiation. Ourfindings are complemented by earlier reports of radiosensi-tization using selective (44–47) and nonselective (15, 16, 38)DNA-PK inhibitors. Importantly, in addition to attenuatingNHEJ, pharmacologic inhibition of DNA-PK has beenshown to suppress other major DSB repair pathwaysincluding homologous recombination (48) and backup-NHEJ (B-NHEJ; ref. 49). Thus, the pronounced radio-sensitization achieved with DNA-PK inhibitors in this studyand others is likely to reflect simultaneous activity against

multiple DSB repair pathways. We speculate that concur-rently inhibiting key molecules in each of these pathwaysmay further compromise radiation-induced DSB repair, ashas been previously shown by combining DNA-PK andPARP-1 inhibitors to target NHEJ and B-NHEJ, respec-tively (50). Moreover, by prolonging the DDR, thisapproach is likely to offer additional therapeutic benefitsarising from an enhanced accelerated senescence response.In conclusion, we have shown that selective targeting of

DNA-PK induces p53-dependent accelerated senescence inirradiated human cancer cells. To the best of our knowledge,DNA-PK inhibitors such as BEZ235 are not currently beingcombined with radiation in clinical trials. Our data highlightthe potential benefits of using DNA-PK blockade to mod-ulate repair of therapeutically induced DSBs and therebypromote radiation-induced accelerated senescence. Thesefindings provide a rationale for further preclinical andclinical evaluation of DNA-PK inhibitors in combinationwith anticancer agents that induce DSBs or inhibit DSBrepair.

Disclosure of Potential Conflicts of Interest

S.-M.Maira is a stockholder and employee of Novartis Pharma AG.W.Hackl is anemployee of Novartis Pharma AG. G.A.McArthur and B. Solomon are on an advisoryboard for Novartis Pharma AG. No potential conflicts of interest were disclosed byother authors.

Acknowledgments

The authors thank Dr. Jim Hagekyriakou, Rachel Walker, and Jacqueline Noll forexpert technical assistance; and Drs. Jake Shortt, Kamil Wolyniec, and Megan Astleand Richard Young for helpful discussions.

Figure 6. DNA-PK knockdown by siRNA induces accelerated senescence after radiation. A, Western blot analysis showing total DNA-PK levels in H460 cellsafter treatment indicated. B, top, representative immunofluorescence images of phospho-DNA-PKcs (Thr2609) foci in H460 cells at 30 minutes after 10 Gyradiation (�60 magnification). Green, phospho-DNA-PKcs foci; blue, DAPI staining. Middle, representative immunofluorescence images of gH2AX (Ser139)foci in H460 cells at 24 hours after 10 Gy radiation (�60 magnification). Green, gH2AX foci; blue, DAPI staining. Bottom, representative images of SA-bGalactivity in H460 cells at 48 hours after 10 Gy radiation (�200 magnification). Left, percentage of SA-bGal–positive cells. Data are mean � SEM from 3independent experiments. ��, P < 0.001. Scr, scrambled.







Grant Support

The study was supported by National Health and Medical Research Council(NHMRC) Medical Postgraduate Scholarship (A. Azad); NHMRC, Sir EdwardWeary Dunlop Fellowship Cancer Council of Victoria (G.A. McArthur); andNHMRC, International Association for the Study of Lung Cancer Young InvestigatorAward, Victorian Cancer Agency Clinical Research Fellowship (B. Solomon).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be herebymarked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 30, 2011; revised September 22, 2011; accepted October 10, 2011;published OnlineFirst October 18, 2011.

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Inhibition of DNA-Dependent Protein Kinase Induces Accelerated Senescence in Irradiated Human Cancer Cells

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