synergistic effect of olaparib with combination of ... · to calculate the combined drug effect...

Cell Death and Survival

Synergistic Effect of Olaparib with Combination of Cisplatinon PTEN-Deficient Lung Cancer Cells

Daisuke Minami1, Nagio Takigawa2, Hiromasa Takeda1, Minoru Takata3, Nobuaki Ochi1, Eiki Ichihara1,Akiko Hisamoto1, Katsuyuki Hotta1, Mitsune Tanimoto1, and Katsuyuki Kiura1

AbstractPARP enzyme plays a key role in the cellular machinery responsible for DNA damage repair. PTEN is a tumor-

suppressor gene deactivating PI3KdownstreamofEGFR signaling.Wehypothesize thatPTEN-deficient lung cancercells suppressed DNA damage signaling and that the absence of PTEN can sensitize these cells to a concurrenttreatment of aDNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olapariband cisplatin on PTEN-deficient lung tumors, two EGFR-mutant (deletion in exon19) non–small cell lung cancer(NSCLC) cell lines, PC-9 (PTENwild-type) andH1650 (PTEN loss), were used.We transfected intact PTEN geneinto H1650 cells (H1650PTENþ) and knocked down PTEN expression in the PC-9 cells (PC-9PTEN�) using shorthairpin RNA (shRNA). Combination of cisplatin with olaparib showed a synergistic effect in vitro according tothe combination index in H1650 cells. Restoration of PTEN in the H1650 cells decreased sensitivity to thecombination. Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin. We also examined theeffectiveness of cisplatin and olaparib in a xenograft model using H1650 and PC-9PTEN� cells. The combination ofcisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograftmodel using H1650PTENþ and PC-9 cells. Mechanistic investigations revealed that PTEN deficiency causedreductions in nuclear RAD51 and RPA focus formation and phosphorylated Chk1 and Mre11. Thus, geneticinactivation of PTEN led to the suppression of DNA repair. Mol Cancer Res; 11(2); 140–8. �2012 AACR.

IntroductionPARP inhibitor is one of the most promising new ther-

apeutic approaches to cancers, either as a single agent or incombination with other DNA-damaging agents includingradiation therapy (1).WhenPARP is inhibited, single-strandbreaks (SSB) degenerate to more lethal double-strand breaks(DSB) that require repair by homologous recombination(HR). Therefore, cells that are deficient in HR are highlysusceptible to PARP inhibitors (2–4), and this finding hasbeen clinically validated (5–7).As many cancer chemotherapeutic drugs and radiation

therapy cause DNA damage, tumor cells defective in DNArepair pathways are predicted to be sensitive to their effects(8). Indeed, cell lines deficient in BRCA1 and BRCA2 have

been shown to be sensitive to the DNA crosslinking agentscisplatin and mitomycin C (9, 10), the topoisomeraseinhibitor etoposide (11), and oxidative DNA damage (12).PARP1 has been suggested to be involved in base excision

repair and SSB repair (13). Moreover, PARP-1 was reportedto bind to DNA damages induced by platinum compounds,suggesting a direct role of PARP-1 in the repair of suchdamages (14, 15). The exquisite sensitivity of these cells tothe PARP inhibitor olaparib (AZD2281), alone or in com-bination with cisplatin, provides strong support for olaparibas a novel targeted therapeutic against BRCA-deficientcancers (16). Olaparib alone and in combination withcarboplatin greatly inhibit growth in BRCA2-mutated ovar-ian serous carcinoma (17). The exquisite sensitivity ofBRCA1- or BRCA2-mutant cells to PARP inhibitors formsthe rationale behind clinical trials that are now assessing thepotential of these agents (18). The preliminary results fromthese clinical trials are promising, with favorable toxicity andsustained tumor responses to the drug (3).Mutations in the phosphatase and tensin homolog

(PTEN) gene and loss of PTEN expression have both beenassociated with a wide range of human tumors (19). Approx-imately, 2% to 9% of non–small cell lung cancer (NSCLC)tumors are considered to have PTEN loss. PTEN loss andEGF receptor (EGFR) mutation co-occurred in 1 of 24EGFR-mutant patients with lung adenocarcinoma (20, 21),and a recurrent gross mutation of the PTEN gene is iden-tified in lung cancer with deficient DNA DSB repair. In

Authors' Affiliations: 1Department of Hematology, Oncology, and Respi-ratory Medicine, Okayama University Graduate School of Medicine, Den-tistry and Pharmaceutical Sciences; 2Department of General Internal Med-icine, Kawasaki Medical School, Okayama; and 3Radiation Biology Center,Kyoto University, Kyoto, Japan

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

Corresponding Author: Nagio Takigawa, Department of General InternalMedicine 4, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku,Okayama 700-8505, Japan. Phone: 81-86-225-2111; Fax: 81-86-232-8343; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-12-0401

�2012 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 11(2) February 2013140

on April 22, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst December 13, 2012; DOI: 10.1158/1541-7786.MCR-12-0401

http://mcr.aacrjournals.org/

other studies, 24% of early NSCLC samples lacked PTENexpression, which correlated with PTEN promoter methyl-ation (22) and PTEN protein expression was reduced or lostin 74% of lung tumors, and was associated with low oraberrant TP53 staining (23). In a later study, PTEN hasnovel nuclear functions, including transcriptional regulationof the RAD51 gene, whose product is essential for HR repairof DNA breaks (24, 25). McEllin and colleagues reportedthat loss of PTEN in astrocytes resulted in increased sensi-tivity toN-methyl-N0-nitro-N-nitrosoguanidine, a function-al analogue of temozolomide, and PARP inhibitor, due toinefficient repair (26).We hypothesized that PTEN-deficient lung cancer cells

suppressedDNAdamage signaling and investigated whetherthe absence of PTEN could sensitize these cells to a con-current treatment of cisplatin and olaparib.

Materials and MethodsCell linesCells were cultured at 37�Cwith 5%CO2 in RPMI 1640

supplemented with 10% heat-inactivated FBS. H1650 is alung adenocarcinoma cell line with co-occurrence of anEGFR mutation (in-frame deletion in exon 19) and homo-zygous deletion of PTEN. PC-9 is a lung adenocarcinomacell line having the same in-frame deletion mutation ofEGFR with wild-type PTEN. PTEN transfected intoH1650 cells (H1650PTENþ) and PTEN expression knockeddown in the PC-9 cells (PC-9PTEN�) using shRNA, wereestablished in our laboratory (27). H1299 and A549 areNSCLC lines with wild-type EGFR and wild-type PTEN.PC-3 is a prostate cancer cell line with PTEN loss (Supple-mentary Fig. S1). PC-9 was obtained from Immuno-Bio-logical Laboratories. H1650, H1299, A549, and PC-3 werepurchased from the American Type Culture Collection. TheMre11 expression vector was kindly provided byDrs. KenshiKomatsu and Junya Kobayashi (Kyoto University, Kyoto,Japan). The expression vector was transfected toH1650 cellsusing Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol.

Sensitivity testAntiproliferative activity was determined by a modified 3-

(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bro-mide (MTT) assay in terms of 50% inhibitory concentration(IC50) values. Briefly, the cells were plated on 96-well platesat a density of 1,000 to 3000 cells per well, and continuouslyexposed to each drug for 144 hours. Each assay was con-ducted in triplicate or quadruplicate, and IC50 values wereexpressed as mean � SD.

Design for drug combinationThe constant-ratio design for the combination assay is

highly recommended as it allows the most efficient dataanalysis (28). The multiple drug effect analysis of Chouand Talaly, based on the median-effect principle, was usedto calculate the combined drug effect (29). H1650,H1650PTENþ, PC-9, PC-9PTEN�, H1299, A549, andPC-3 cells were seeded in triplicate in 96-well plates and

were treated with cisplatin and olaparib at the indicateddoses. After simultaneous exposure of the cells to 2 drugsfor 144 hours, growth inhibition was determined using anMTT assay and the multiple drug effect analysis (Sup-plementary Method). Computer programs based on themedian-effect plot parameters and combination index(CI) equation have been used for data analysis in thepresent study (30). Experiments were repeated intriplicate.

Immunoblotting analysisCells were exposed to cisplatin (10 mmol/L) or/and

olaparib (20 mmol/L) for 6 hours. The cells at the pointof 6 hours after irradiation at a dose of 10 Gy using aHitachi MBR-1520-R irradiator (150 kV; 20 mA; filter:0.5-mm aluminum and 0.1-mm copper) were also used.They were lysed in radioimmunoprecipitation assay buffer[1% Triton X-100, 0.1% SDS, 50 mmol/L Tris-HCl (pH7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/LEGTA, 10 mmol/L b-glycerolphosphate, 10 mmol/LNaF, 1 mmol/L sodium orthovanadate containing prote-ase inhibitor tablets (Roche Applied Sciences GmbH)].Proteins were separated by electrophoresis on polyacryl-amide gels, transferred onto nitrocellulose membranes,and probed with specific antibodies followed by detectionwith enhanced chemiluminescence plus (GE HealthcareBiosciences).

Reagents and antibodiesOlaparib and cisplatin were kindly provided by AstraZe-

neca and Nippon Kayaku Kogyo Co. Ltd., respectively.Rabbit antisera against Akt, phosphorylated (p)Akt (Ser473;D9E), PTEN, pChk1 (Ser345),Mre11 (31H4), and b-actinwere purchased from Cell Signaling Technology. Rabbitantiserum against RPA70 was purchased from Abcam.Mouse antisera against Chk1 (G-4) was purchased fromSanta Cruz Biotechnology.

ImmunohistochemistryFormalin-fixed paraffin-embedded tissue blocks from the

samples were cut to a thickness of 5 mm, placed on glassslides, then deparaffinized in xylene and graded alcohol for10 minutes. The antigen was incubated in 10 mmol/Lsodium citrate buffer, pH 6.0, for 10 minutes in a 95�Cwater bath. The sections were then blocked for endogenousperoxidase with 0.3% hydrogen peroxide in methanol. Theslides were rinsed with TBS containing 0.1% Tween 20 andthe sections were blocked with goat normal serum for 60minutes. The sections were incubated with 1:200 dilution ofcleaved caspase-3 (Asp175; 5A1E; Cell Signaling) antibodyovernight at 4�C. The sections were amplified using bioti-nylated anti-rabbit antibodies and avidin–biotinylatedhorseradish peroxidase conjugate for 10 minutes (LSAB 2Kit, Dako Cytomation) then reacted with 3,30-diaminoben-zidine. Finally, the sections were counterstained with hema-toxylin. Cleaved caspase-3 expression was scored as positiveif more than 10% of the tumor cells exhibited cytoplasmicstaining (31).

Olaparib with Cisplatin on PTEN-Deficient Lung Cancer

www.aacrjournals.org Mol Cancer Res; 11(2) February 2013 141




Immunofluorescence stainingWe fixed cells 8 hours after exposure to cisplatin (10

mmol/L), or/and olaparib (20 mmol/L), or treatment with10 Gy. Cells were cultured on glass coverslips, fixed with4% formaldehyde, and permeabilized in PBS-0.25% Tri-ton X-100. For DNA damage and repair analyses, cellswere stained with 1:50 dilution of rabbit polyclonal anti-RAD51 (Santa Cruz Biotechnology) and 1:500 dilution ofmouse monoclonal anti-gH2AX (Millipore) for 2 hours atroom temperature. Cells were washed with PBS andincubated for 30 minutes at room temperature with eitherAlexa Fluor 488 or Alexa Fluor 555 (Invitrogen) second-ary antibody for RAD51 or gH2AX, respectively. Nucleiwere visualized by staining with 40, 6-diamidino-2-phe-nylindole (DAPI). For quantification of RAD51 andgH2AX foci, at least 100 cells from each group werevisually scored. Cells showing more than 5 foci werecounted as positive for g-H2AX or RAD51.These slideswere examined under a fluorescence microscope (BIOR-EVO BZ-9000; Keyence).

Xenograft modelFemale athymic mice at 7 weeks of age were purchased

from Japan Charles River Co. All mice were provided withsterilized food and water and housed in a barrier facilityunder a 12-hour light/dark cycle. Cells (1 � 106) wereinjected bilaterally subcutaneously into the backs of 7-week-old female athymic mice. At 10 days after injection, micewere randomly assigned to 4 groups (5 mice/group) thatreceived either vehicle, 5mg/kg/week of cisplatin, 50mg/kg/

day of olaparib, and 5 mg/kg/week of cisplatin plus 50mg/kg/day of olaparib. Vehicle and olaparib were adminis-tered once a day, 5 times a week by intraperitoneal injection,and cisplatin was administered once a day, once a week byintraperitoneal injection. Tumor volume (width2 � length/2) and body weight were determined periodically. Tumorvolume was expressed as mean � SD. After the completionof the treatment, all mice per group were sacrificed and thetumor specimens were obtained for analysis. All experimentsinvolving animals were conducted under the auspices ofthe Institutional Animal Care and Research AdvisoryCommittee at the Department of Animal Resources, Oka-yama University Advanced Science Research (Okayama,Japan).

ResultsOlaparib synergizes cisplatin in PTEN-deficient lungcancer cellsSynergy between PARP inhibitors and platinum drugs

was expected (14, 15) in triple-negative breast cancer andBRCA2 ovarian cancer cells (8, 17). We expected that thecombination of cisplatin with olaparib showed a syner-gistic effect in PTEN-deficient lung cancer cell lines. Cellswere treated either with 10 mmol/L cisplatin and 10 to 50mmol/L olaparib or 20 mmol/L cisplatin and 20 to 100mmol/L olaparib. Combination of cisplatin with olaparibshowed a synergistic effect according to the combinationindex in the H1650 cells (Fig. 1A, 1B; Table 1). Com-bination indices were 0.23, 0.20, 0.57, and 0.29 whenconcentration ratios of cisplatin and olaparib were

Figure 1. Combination index andsurviving fraction of H1650 andH1650PTENþ cells treated withcisplatin in combination witholaparib simultaneously for 144hours. Synergistic effects in H1650cells are shown, designating to bemolar ratios of 1:2 (A) and 1:5 (B).Antagonistic effects inH1650PTENþ

cells are shown, designating to bemolar ratios of 1:2 (C) and 1:5 (D).

Minami et al.

Mol Cancer Res; 11(2) February 2013 Molecular Cancer Research142




designed to be molar ratios of 1:1, 1:2, 1:3, and 1:5,respectively. Restoration of PTEN in the H1650 cellsdecreased sensitivity to olaparib and cisplatin (CI>1; Fig. 1C, 1D). Ablation of PTEN in PC-9 cellsincreased sensitivity to olaparib and cisplatin (CI <1),and PC-9 cells decreased sensitivity to olaparib and cis-platin (CI >1). Our results showed that PTEN-deficientlung cancer cell lines, H1650 and PC-9PTEN�, exhibitedsynergism for all combinations of olaparib doses, whereasH1650PTENþ and PC-9 cells exhibited antagonistic effectsfor most dose combinations. A synergistic effect was alsoshown in the PC-3 cells, whereas antagonistic effects atmost dose combinations were shown in H1299 and A549cells (Supplementary Table S1).Sensitivity of cisplatin or olaparib monotherapy is shown

in Table 2. IC50 values of olaparib in H1650 andH1650PTENþ cells were 15.47 � 6.8 mmol/L and 50.83� 7.7 mmol/L, respectively. PTEN-restored H1650 thatbecame resistant to olaparib (P < 0.05). On the other hand,

the IC50 values of olaparib in PC-9 and PC-9PTEN� cellswere 5.88 � 1.4 mmol/L and 6.52 � 6.7 mmol/L, respec-tively. PC-9PTEN� cells did not confer sensitization toolaparib alone. IC50 values of cisplatin or olaparib alone inPC-3, H1299, and A549 cells are shown in SupplementaryTable S2.

PTEN inactivation suppresses DNA damage signalingOncogenic activation of Akt frequently results from loss of

PTEN expression or function leads to suppression of DNAdamage signaling (32). In this study, immunoblotting assayrevealed that H1650 and PC-9PTEN� cells exhibited muchhigher levels of pAkt than H1650PTENþ and PC-9 cells,respectively (Fig. 2A). In addition, pChk1 was not over-expressed in both H1650 and PC-9PTEN� cells despite drugtreatment. pChk1 was expressed after irradiation; however,the increase was less in PTEN-deficient lung cancer cellscompared with their counterparts (Fig. 2B). Meanwhile,PTEN-deficient lung cancer cells exhibited lower levels ofMre11 compared with their counterparts (Fig. 2C). Toaddress how lower expression levels of Mre11 affects thesynergism between cisplatin and olaparib, Mre11 expression

Table 1. Combination index

Concentration ratio (molar) ofcisplatin to olaparib

Cell line 1:1 1:2 1:3 1:5

H1650 0.23 0.20 0.57 0.29H1650PTENþ 1.76 1.59 1.77 1.07PC-9 4.38 5.16 10.8 0.55PC-9PTEN� 0.43 0.40 0.66 0.78

NOTE: Combination index according to various concentra-tion ratios of cisplatin and olaparib in each cell line isdescribed. CI < 1, CI ¼ 1, and CI > 1 indicate synergism,additive effect, and antagonism, respectively.

Table 2. Drug sensitivity

Cell line Cisplatin (mmol/L) Olaparib (mmol/L)

H1650 2.12 � 0.72 15.47 � 6.8H1650PTENþ 1.65 � 0.97 50.83 � 7.7PC-9 0.21 � 0.019 5.88 � 1.4PC-9PTEN� 0.42 � 0.10 6.52 � 6.7

NOTE: Values are presented asmean�SDof 50% inhibitoryconcentration (IC50) of the drug. H1650PTENþ: PTEN-restored H1650; PC-9PTEN�: PTEN-ablated PC-9;aP < 0.05.

]a

Figure 2. PTEN inactivation andDNAdamage signaling. A, PTEN lossactivated Akt and suppressedpChk1. pChk1 was notoverexpressed in both H1650 andPC-9PTEN� cells (PTEN-deficientlung cancer cells) despite drugtreatment. B, pChk1 was expressedafter irradiation; however, theincrease was less in PTEN-deficientlung cancer cells comparedwith theircounterparts. C,PTEN-deficient lungcancer cells exhibited lower levels ofMre11 compared with theircounterparts.






vector was transfected into H1650 cells, and a stable trans-formant in which Mre11 was overexpressed was obtained(designated H1650Mre11þ cells; Supplementary Fig. S2). Inthis cell line, combination indices were 0.76, 0.91, 0.97, and0.77 when concentration ratios of cisplatin and olaparibwere designed to be molar ratios of 1:1, 1:2, 1:3, and 1:5,respectively in H1650Mre11þ (Supplementary Table S3).Although these combination index values were somewhatelevated compared with those observed in original H1650cells, these results indicated that lower levels of Mre11 alonecould not be the sole reason for this synergism.On the other hand, PTEN has other nuclear functions,

including transcriptional regulation of the RAD51 gene,whose product is essential for HR repair of DNA breaks(24, 25). Replication protein A (RPA) is displaced fromsingle-stranded DNA by RAD51 to initiate HR (32). In thisstudy, we investigated whether the formation of RAD51 andRPA foci was reduced in PTEN-deficient lung cancer celllines. Subcellular localization of RAD51, g-H2AX, and RPAis shown in Fig. 3A and in Supplementary Fig. S3A. PTENdeficiency resulted in significant reduction in RAD51 andRPA focus formation after drug exposure or g-irradiation

compared with H1650PTENþ cells (P < 0.05); althoughg-H2AX was similarly increased in both cells (Fig. 3B andSupplementary Fig. S3B).

Effectiveness of the cisplatin with olaparib in a xenograftmodelWe examined xenograft tumors to determine the effec-

tiveness of the cisplatin with olaparib in PTEN-deficientlung cancer cells in vivo. H1650 andH1650PTENþ xenografttumors grew at almost same rate. The immunostaining ofcleaved caspase-3 is shown in Fig. 4A. The combination ofcisplatin and olaparib induced significant higher positivecells than other groups in H1650 xenografts (P < 0.05). Thepositive cells were 43� 3% for cisplatin plus olaparib, 10.6� 1.1% for cisplatin alone, 16 � 3.6% for olaparib alone,and 3� 2% for vehicle. However, the combination did notdisplay synergistic effect in H1650PTENþ xenografts. Thepositive cells were 10� 2.6% for cisplatin plus olaparib, 17.3� 2.0% for cisplatin alone, 6.6 � 0.5% for olaparib alone,and 4.6 � 0.5% for vehicle (Fig. 4A).Treatment with cisplatin plus olaparib significantly sup-

pressed growth of the H1650 tumors compared with

Figure 3. PTEN inactivation and expression of RAD51 and g-H2AX. A, subcellular localization of RAD51 and g-H2AX in H1650 or H1650PTENþ cells afterexposure of olaparib (20mmol/L) or irradiation (10Gy). B,PTEN deficiency resulted in significant reduction inRAD51 focus formation after exposure of cisplatin(10mmol/L) and/or olaparib (20mmol/L), or irradiation (10Gy) comparedwithH1650PTENþcells (P<0.05); although g-H2AXwas similarly increased inboth cells.

Minami et al.





cisplatin alone, olaparib alone, and the untreated controls(Fig. 4B). The tumor sizes (mm3) at day 22 were 124.8 �33.1, 351.8 � 150.9, 413.1 � 66.0, and 847.8 � 98.5,respectively. H1650PTENþ xenograft tumors did not showsignificant response to the combination of cisplatin andolaparib compared with cisplatin alone, olaparib alone, andthe untreated controls. The tumor sizes (mm3) at day 22were 467.8 � 103.0, 373.9 � 113, 524.5 � 145.7, and801.4 � 113.2, respectively.Next, we examined whether the combination of cisplatin

with olaparib in PC-9 and PC-9PTEN� xenografts waseffective or not. Cleaved caspase-3 expressions in PC-

9PTEN� xenografts treated with the combination displayedsignificantly higher number of positive cells than those ofother groups (44.3� 4.0% for the combination, 7.3� 3.2%for cisplatin, 15.3 � 1.5% for olaparib, 1.0 � 1.7% forvehicle). In PC-9 xenografts, there were no differencesamong 4 groups: 10.6 � 2.0% for the combination, 7.3� 1.5% for cisplatin, 8.3� 1.5% for olaparib, 4.0 � 1.7%for vehicle (Fig. 4A).Western blotting also indicated that thecombination of cisplatin with olaparib seemed to inducemore cleaved caspase-3 expressions than other treatments inH1650 and PC-9PTEN� xenografts, but not in PC-9 andH1650PTENþ xenografts (Supplementary Fig. S4).

Figure 4. A, cleaved caspase-3staining (�400) of tumor sectionsfrom each treatment group in H1650,H1650PTENþ, PC-9, and PC-9PTEN�

cells, included as graphicalrepresentation. B, growth ofxenograft tumors. Growth curves ofH1650, H1650PTENþ, PC-9, andPC-9PTEN� cells xenograft tumors inanimals receiving the indicated drugs(50 mg/kg/d of olaparib, 5 mg/kg/wkof cisplatin, and both agents) orvehicle intraperitoneally werecompared using Student t test.Bars, SD.






In the PC-9PTEN� xenograft model, cisplatin plus ola-parib inhibited tumor growth than other treatment (152.6�8.06 mm3 for the combination, 336.6 � 45.7 mm3 forcisplatin, 411.2 � 67.2 mm3 for olaparib, 774.3 � 95.8mm3 for vehicle). PC-9 xenograft tumors did not showsignificant response to the combination compared withcisplatin alone, olaparib alone, and the untreated controls(279.1 � 69.5 mm3, 254.2 � 42.2 mm3, 296.0 � 57.5mm3, and 642.3� 133.2 mm3, respectively; Fig. 4B).In toxicity evaluation, all the treatment animals did not

show substantial loss of body weight (>10%) and theaddition of olaparib to cisplatin did not significantly increaseweight loss compared with cisplatin single agent treatment(data not shown).

DiscussionWe here showed that PTEN-deficient lung cancer cell

lines suppressed DNA damage signaling, and were sensitiveto the combination of olaparib with cisplatin. Synergybetween PARP inhibitor and platinum drugs was expectedin triple-negative breast cancer and BRCA2 ovarian cancercells (8, 17). In other study, addition of PARP inhibitor afteralkylating agent, demethyl sulfate, treatment increased SSBlevels indicating ongoing repair even at this late time-point(33). Recently, evidence suggested that PTEN was impor-tant for the maintenance of genome stability (24, 25). TheHR impairment caused by PTEN deficiency sensitizedtumor cells to potent inhibitors of the DNA repair enzyme,both in vitro and in vivo (34). Our results were in agreementwith their studies.We showed that xenograft tumors bearingPTEN-deficient lung cancer cells were sensitive to thecombination of cisplatin with olaparib, although this effectwas not observed in a xenograft model using PTEN wild-type cells (Fig. 4B). However, it is possible that the resultsmay be cell specific or have a different effect of PTEN loss onHR capacity because PTEN-deficient prostate cancer cellshad only mild PARP inhibitor and DNA damaging agent'ssensitivity (35).Drug interaction between cisplatin and olaparib in PTEN-

deficient lung cancer cells has not been elucidated. Ourinvestigation revealed that PTEN deficiency caused a reduc-tion of pChk1 (Fig. 2A) and decreased drug or radiation-induced nuclear RAD51 and RPA focus formation (Fig. 3and Supplementary Fig. S3). Oncogenic activation of Aktfrequently resulted from loss of PTEN expression or func-tion (36). How PTEN loss affects DNA damage signalingshould be clarified. H1650 and PC-9PTEN� cells exhibitedmuch higher levels of pAkt than H1650PTENþ and PC-9cells, respectively. Activation of Chk1 after irradiation wasattenuated in PTEN-deficient cells (Fig. 2B). Chk1 has acritical role in maintaining genomic stability by delaying S-and G2 phase progression of cells containing DNA damageto allow time for repair before mitosis and, the DSBs thatarise when Chk1 is inhibited are apparently related to aspecific S-phase role whereby Chk1 suppresses aberrantinitiation of DNA replication that wound generate DNAlesions (37). Chk1 is reportedly required for HR repair andChk1-depleted cells failed to form RAD51 nuclear foci after

exposure to hydroxyurea (38). McEllin and colleaguesshowed a significant decrease in mRNA expression onRAD51B, C, and D, and reduced HR-mediated repairin PTEN-null astrocytes (26). Meanwhile, Xu and collea-gues observed PTEN knockdown in HCT116 cells atten-uated Mre11, which was a key gene in HR repair of DSBs(32). In addition, Fraser and colleagues reported PARPinhibitor sensitivity associated with a defect in Mre11expression (35). Our observation that PTEN-deficientlung cancer cells exhibited lower levels of Mre11 com-pared with their counterparts was in agreement with theirstudies. To investigate the effect of Mre11 itself on thesynergy, we examined the combination effect of olaparibwith cisplatin using H1650Mre11þ cells. Unexpectedly,restored levels of Mre11 did not suppress the phenotypesobserved in PTEN-deficient cells in this study (Table 1and Supplementary Table S3). Further investigationsshould be required to clarify whether and to what extentthe molecular events including Chk1, Mre11, and RAD51are responsible for the synergic effect. Interestingly, Shenand colleagues recently showed that PTEN is importantfor maintaining basal levels of transcription of the RAD51gene in mouse embryonic fibroblasts (24). Although therewere no significant differences of RAD51 and RPA levelsby Western blotting (Supplementary Fig. S5), the forma-tion of RAD51 and RPA foci was reduced in PTEN-deficient lung cancer cells (Fig. 3 and Supplementary Fig.S3). As RPA binds to single-stranded DNA, the RPA focuscould be a marker for end resection at the double-strandedDNA ends (32). Thus, inactivation of PTEN might leadto suppression of DNA damage signaling, leading to thelower levels of end resection and, hence, less RPA focusformation. As shown above, reduced levels of Mre11 alonecould not provide a sufficient explanation for this, thoughMre11 is involved in the molecular mechanisms of the endresection (39).A number of clinical trials to treat triple-negative breast

cancer, metastatic melanoma, malignant glioma, advancedcolorectal cancer, ovarian cancer, and lung cancer are nowunderway to test the efficacy of PARP inhibitors or PARPinhibitors in combination with DNA-damaging agents (40).Later, phase II studies using olaparib established proof-of-concept of selectively killing of HR-deficient breast cancerand ovarian cancer cells with BRCA1 or BRCA2 mutations,resulting in a substantial clinical benefit with minimaltoxicity (6, 7, 41). DNA repair biomarkers from multipleDNA repair pathways on treatment response and cancersurvival offers opportunity to evaluate patient tumor samplesand determine their status of DNA repair pathways beforeand during therapy for individual patients. In recent years,our understanding of how to treat NSCLCs has undergone aparadigm shift by the identification of EGFR mutations(42, 43) and EML4–ALK translocation (44). In BRCA1-deficient lung cancer, PARP inhibition induced BAX/BAK-independent synthetic lethality (45). Knowledge of thestatus of multiple DNA repair profiling of patients and maydiscriminate patients with likelihood to respond to PARPinhibitors.

Minami et al.





We hypothesized that tumor cell with HR deficiency(such as PTEN-mutated cancer cells) were hypersensitiveto PARP inhibitors in combination with cisplatin, resultingin killing of tumor cells based on the synthetic lethalityprinciple. A major important solution to these barriers is tobuild biomarker testing into patient tumor identification,and to use the biomarker panels during treatment. Thecombination of cisplatin with olaparib in PTEN-deficientlung tumors might be further pursued in clinical trials.

Disclosure of Potential Conflicts of InterestN.Takigawa has a honoraria from speakers' bureau fromAstraZeneca. K. Kiura has

a honoraria from speakers' bureau from AstraZeneca. No potential conflicts of interestwere disclosed by other authors.

Authors' ContributionsConception and design: D. Minami, N. Takigawa, E. Ichihara, K. KiuraDevelopment of methodology: D. Minami, N. Takigawa, N. Ochi, E. Ichihara, K.KiuraAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): D. Minami, N. Takigawa, K. Kiura

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): D. Minami, N. Takigawa, H. Takeda, M. Takata, E. Ichihara, K.KiuraWriting, review, and/or revision of the manuscript: D. Minami, N. Takigawa, A.Hisamoto, K. Hotta, K. KiuraAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): N. Takigawa, E. Ichihara, K. KiuraStudy supervision: N. Takigawa, M. Tanimoto, K. Kiura

AcknowledgmentsThe authors thank Drs. Takashi Ninomiya, Toshi Murakami, and Noboru Asada

for expert technical support, Drs. Kenshi Komatsu and Junya Kobayashi, Departmentof Genome Repair Dynamics, Radiation Biology Center, Kyoto University (Kyoto,Japan) for providing the Mre11 expression vector and AstraZeneca for providingolaparib.

Grant SupportThis study was supported by the Ministry of Education, Culture, Sports, Science,

and Technology, Japan grants 21590995 (to N. Takigawa) and 23390221 (to K.Kiura).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received June 29, 2012; revised October 26, 2012; accepted November 16, 2012;published OnlineFirst December 13, 2012.

References1. Jin-xue He, Chun-hao Yang, Ze-hong Miao. Poly(ADP-ribose) poly-

merase inhibitors as promising cancer therapeutics. Acta Pharmaco-logica Sinica 2010;31:1172–80.

2. BryantHE, Schultz N, ThomasHD, Parker KM, Flower D, Lopez E, et al.Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 2005;434:913–7.

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

4. McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S, et al.Deficiency in the repair of DNAdamage by homologous recombinationand sensitivity to poly(ADP-ribose) polymerase inhibition. Cancer Res2006;66:8109–15.

5. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, et al.Inhibition of poly (ADP-ribose) polymerase in tumors from BRCAmutation carriers. N Engl J Med 2009;361:123–34.

6. Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN,et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patientswith BRCA1 or BRCA2 mutations and advanced breast cancer: aproof-of-concept trial. Lancet 2010;376:235–44.

7. Audeh MW, Carmichael J, Penson RT, Friedlander M, Powell B, Bell-McGuinn KM, et al. Oral poly(ADP-ribose) polymerase inhibitor ola-parib in patients with BRCA1 or BRCA2 mutations and recurrentovarian cancer: a proof-of-concept trial. Lancet 2010;376:245–51.

8. Hastak K, Alli E, Ford JM. Synergistic chemosensitivity of triple-negative breast cancer cell lines to poly (ADP-ribose) polymeraseinhibition, gemcitabine, and cisplatin. Cancer Res 2010;70:7970–80.

9. Moynahan ME, Cui TY, Jasin M. Homology-directed DNA repair,mitomycin-C resistance, and chromosome stability is restored withcorrection of a Brca1 mutation. Cancer Res 2001;61:4842–50.

10. Bhattacharyya A, Ear US, Koller BH, Weichselbaum RR, Bishop DK.The breast cancer susceptibility gene BRCA1 is required for subnu-clear assembly of Rad51 and survival following treatmentwith theDNAcross-linking agent cisplatin. J Biol Chem 2000;275:23899–903.

11. Treszezamsky AD, Kachnic LA, Feng Z, Zhang J, Tokadjian C, PowellSN, et al. BRCA1- and BRCA2-deficient cells are sensitive to etopo-side-induced DNA double-strand breaks via topoisomerase II. CancerRes 2007;67:7078–81.

12. Alli E, Sharma VB, Sunderesakumar P, Ford JM. Defective repair ofoxidative DNA damage in triple-negative breast cancer confers sen-sitivity to inhibition of poly(ADP-ribose) polymerase. Cancer Res2009;69:3589–96.

13. Helleday T. The underlying mechanism for the PARP and BRCAsynthetic lethality: clearing up the misunderstandings. Mol Oncol2011;5:387–93.

14. Guggenheim ER, Ondrus AE, Movassaghi M, Lippard SJ. Poly (ADP-ribose) polymerase-1 activity facilitates the dissociation of nuclearproteins from platinum-modified DNA. Bioorg Med Chem 2008;16:10121–8.

15. ZhuG, Chang P, Lippard SJ. Recognition of platinum-DNA damage bypoly (ADP-ribose) polymerase-1. Biochemistry 2010;49:6177–83.

16. Evers B, Drost R, Schut E, de Bruin M, van der Burg E, Derksen PW,et al. Selective inhibition of BRCA2-deficient mammary tumor cellgrowth by AZD2281 and cisplatin. Clin Cancer Res 2008;14:3916–25.

17. Kortmann U, McAlpine JN, Xue H, Guan J, Ha G, Tully S, et al. Tumorgrowth inhibition by olaparib in BRCA2 germline-mutated patient-derived ovarian cancer tissue xenografts. Clin Cancer Res 2011;17:783–91.

18. Ashworth A. A synthetic lethal therapeutic approach: poly (ADP) ribosepolymerase inhibitors for the treatment of cancers deficient in DNAdouble-strand break repair. J Clin Oncol 2008;26:3785–90.

19. Salmena L, Carracedo A, Pandolfi PP. Tenets of PTEN tumor sup-pression. Cell 2008;133:403–14.

20. Jin G, Kim MJ, Jeon HS, Choi JE, Kim DS, Lee EB, et al. PTENmutations and relationship to EGFR, ERBB2, KRAS, and TP53 muta-tions in non-small cell lung cancers. Lung Cancer 2010;69:279–83.

21. Sos ML, Koker M, Weir BA, Heynck S, Rabinovsky R, Zander T, et al.PTEN loss contributes to erlotinib resistance in EGFR-mutant lungcancer by activation of Akt and EGFR. Cancer Res 2009;69:3256–61.

22. Soria JC, LeeHY, Lee JI,Wang L, Issa JP,KempBL, et al. Lackof PTENexpression in non-small cell lung cancer could be related to promotermethylation. Clin Cancer Res 2002;8:1178–84.

23. MarsitCJ, ZhengS,AldapeK,HindsPW,NelsonHH,WienckeJK, et al.PTEN expression in non-small cell lung cancer: evaluating its relationto tumor characteristics, allelic loss, and epigenetic alteration. HumPathol 2005;36:768–76.

24. Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP, et al.Essential role for nuclear PTEN in maintaining chromosomal integrity.Cell 2007;128:157–70.

25. Yin Y, Shen WH. PTEN: a new guardian of the genome. Oncogene2008;27:5443–53.

26. McEllin B, CamachoCV,Mukherjee B, HahmB, Tomimatsu N, BachooRM, et al. PTEN Loss compromises homologous recombinationrepair in astrocytes: implications for glioblastoma therapy with






temozolomide or poly(ADP-ribose) polymerase inhibitors. Cancer Res2010;70:5457–64.

27. Takeda H, Ohashi K, Kataoka I, Ichihara E, Takigawa N, Tanimoto M,et al. Efficacy of vandetanib on EGFR mutant lung cancer cells withPTEN loss [abstract]. In: Proceedings of the 101st Annual Meeting ofAmerican Association for Cancer Research; 2010 Apr 20; WashingtonD.C. Philadelphia (PA): AACR; 2010. Abstract nr. 3620.

28. Chang TT, Chou TC. Rational approach to the clinical protocol designfor drug combinations: a review. Acta Paediatr Taiwan 2000;41:294–302.

29. Chou TC, Talaly P. A simple generalized equation for the analysis ofmultiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem1977;252:6438–42.

30. Aoe K, Kiura K, Ueoka H, Tabata M, Matsumura T, Chikamori M, et al.Effect of docetaxel with cisplatin or vinorelbine on lung cancer celllines. Anti Cancer Res 1999;19:291–9.

31. Takata S, Takigawa N, Segawa Y, Kubo T, Ohashi K, Kozuki T, et al.STAT3 expression in activating EGFR-driven adenocarcinoma of thelung. Lung Cancer 2012;75:24–9.

32. Xu N, Hegarat N, Black EJ, Scott MT, Hochegger H, Gillespie DA, et al.Akt/PKB suppresses DNA damage processing and checkpoint acti-vation in late G2. J Cell Biol 2010;190:297–305.

33. Str€om CE, Johansson F, Uhl�en M, Szigyarto CA, Erixon K, Helleday T,et al. Poly (ADP-ribose) polymerase (PARP) is not involved in baseexcision repair but PARP inhibition traps a single-strand intermediate.Nucleic Acids Res 2011;39:3166–75.

34. Mendes-Pereira AM,Martin SA, BroughR,McCarthy A, Taylor JR, KimJS, et al. Synthetic lethal targeting of PTEN mutant cells with PARPinhibitors. EMBO Mol Med 2009;1:315–22.

35. FraserM,ZhaoH, LuotoKR, LundinC,CoackleyC,ChanN, et al. PTENdeletion in prostate cancer cells does not associatewith loss of RAD51function: Implications for radiotherapy and chemotherapy. Clin CancerRes 2012;18:1015–27.

36. Chalhoub N, Baker SJ. PTEN and the PI3-kinase pathway in cancer.Annu Rev Pathol 2009;4:127–50.

37. Syljua�sen RG, Sørensen CS, Hansen LT, Fugger K, Lundin C, Johans-

son F, et al. Inhibition of human Chk1 causes increased initiation ofDNA replication, phosphorylation of ATR targets, and DNA breakage.Mol Cell Biol 2005;25:3553–62.

38. Sørensen CS, Hansen LT, Dziegielewski J, Syljua�sen RG, Lundin C,

Bartek J, et al. The cell-cycle checkpoint kinase Chk1 is required formammalian homologous recombination repair. Nat Cell Biol 2005;7:195–201.

39. Hashimoto Y, Ray Chaudhuri A, LopesM, Costanzo V. Rad51 protectsnascent DNA fromMre11-dependent degradation and promotes con-tinuous DNA synthesis. Nat Struct Mol Biol 2010;17:1305–11.

40. Wang X, Weaver DT. The ups and downs of DNA repair biomarkers forPARP inhibitor therapies. Am J Cancer Res 2011;1:301–27.

41. Fong PC, Yap TA, Boss DS, Carden CP, Mergui-Roelvink M, GourleyC, et al. Poly(ADP)-ribose polymerase inhibition: frequent durableresponses in BRCA carrier ovarian cancer correlating with platinum-free interval. J Clin Oncol 2010;28:2512–9.

42. Paez JG, J€anne 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.

43. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA,Brannigan BW, et al. Activating mutations in the epidermal growthfactor receptor underlying responsiveness of non-small-cell lung can-cer to gefitinib. N Engl J Med 2004;350:2129–39.

44. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S,et al. Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561–6.

45. Paul I, Savage KI, Blayney JK, Lamers E, Gately K, Kerr K, et al. PARPinhibition induces BAX/BAK-independent synthetic lethality ofBRCA1-deficient non-small cell lung cancer. J Pathol 2011;224:564–74.

Minami et al.





2013;11:140-148. Published OnlineFirst December 13, 2012.Mol Cancer Res Daisuke Minami, Nagio Takigawa, Hiromasa Takeda, et al.

-Deficient Lung Cancer CellsPTENSynergistic Effect of Olaparib with Combination of Cisplatin on

Updated version

10.1158/1541-7786.MCR-12-0401doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2012/12/13/1541-7786.MCR-12-0401.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/11/2/140.full#ref-list-1

This article cites 44 articles, 19 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/11/2/140.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/11/2/140To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-12-0401

http://mcr.aacrjournals.org/content/suppl/2012/12/13/1541-7786.MCR-12-0401.DC1

http://mcr.aacrjournals.org/content/11/2/140.full#ref-list-1

http://mcr.aacrjournals.org/content/11/2/140.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/11/2/140


synergistic effect of olaparib with combination of ... · to calculate the combined drug effect...

Documents