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

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

Molecular Cancer Cell Death and Survival Research

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

Abstract PARP enzyme plays a key role in the cellular machinery responsible for DNA damage repair. PTEN is a tumor- suppressor gene deactivating PI3K downstream of EGFR signaling. We hypothesize that PTEN-deficient lung cancer cells suppressed DNA damage signaling and that the absence of PTEN can sensitize these cells to a concurrent treatment of a DNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olaparib and cisplatin on PTEN-deficient lung tumors, two EGFR-mutant (deletion in exon19) non–small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild-type) and H1650 (PTEN loss), were used. We transfected intact PTEN gene þ into H1650 cells (H1650PTEN ) and knocked down PTEN expression in the PC-9 cells (PC-9PTEN ) using short hairpin RNA (shRNA). Combination of cisplatin with olaparib showed a synergistic effect in vitro according to the combination index in H1650 cells. Restoration of PTEN in the H1650 cells decreased sensitivity to the combination. Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin. We also examined the effectiveness of cisplatin and olaparib in a xenograft model using H1650 and PC-9PTEN cells. The combination of cisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograft þ model using H1650PTEN and PC-9 cells. Mechanistic investigations revealed that PTEN deficiency caused reductions in nuclear RAD51 and RPA focus formation and phosphorylated Chk1 and Mre11. Thus, genetic inactivation of PTEN led to the suppression of DNA repair. Mol Cancer Res; 11(2); 140–8. 2012 AACR.

Introduction been shown to be sensitive to the DNA crosslinking agents PARP inhibitor is one of the most promising new ther- cisplatin and mitomycin C (9, 10), the topoisomerase apeutic approaches to cancers, either as a single agent or in inhibitor etoposide (11), and oxidative DNA damage (12). combination with other DNA-damaging agents including PARP1 has been suggested to be involved in base excision radiation therapy (1). When PARP is inhibited, single-strand repair and SSB repair (13). Moreover, PARP-1 was reported breaks (SSB) degenerate to more lethal double-strand breaks to bind to DNA damages induced by platinum compounds, (DSB) that require repair by homologous recombination suggesting a direct role of PARP-1 in the repair of such (HR). Therefore, cells that are deficient in HR are highly damages (14, 15). The exquisite sensitivity of these cells to susceptible to PARP inhibitors (2–4), and this finding has the PARP inhibitor olaparib (AZD2281), alone or in com- – bination with cisplatin, provides strong support for olaparib been clinically validated (5 7). fi As many cancer chemotherapeutic drugs and radiation as a novel targeted therapeutic against BRCA-de cient therapy cause DNA damage, tumor cells defective in DNA cancers (16). Olaparib alone and in combination with repair pathways are predicted to be sensitive to their effects carboplatin greatly inhibit growth in BRCA2-mutated ovar- (8). Indeed, cell lines deficient in BRCA1 and BRCA2 have ian serous carcinoma (17). The exquisite sensitivity of BRCA1- or BRCA2-mutant cells to PARP inhibitors forms the rationale behind clinical trials that are now assessing the

Authors' Affiliations: 1Department of Hematology, Oncology, and Respi- potential of these agents (18). The preliminary results from ratory Medicine, Okayama University Graduate School of Medicine, Den- these clinical trials are promising, with favorable toxicity and tistry and Pharmaceutical Sciences; 2Department of General Internal Med- sustained tumor responses to the drug (3). icine, Kawasaki Medical School, Okayama; and 3Radiation Biology Center, Kyoto University, Kyoto, Japan Mutations in the phosphatase and tensin homolog (PTEN) gene and loss of PTEN expression have both been Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). associated with a wide range of human tumors (19). Approx- imately, 2% to 9% of non–small cell lung cancer (NSCLC) Corresponding Author: Nagio Takigawa, Department of General Internal PTEN PTEN Medicine 4, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, tumors are considered to have loss. loss and Okayama 700-8505, Japan. Phone: 81-86-225-2111; Fax: 81-86-232- EGF receptor (EGFR) mutation co-occurred in 1 of 24 8343; E-mail: [email protected] EGFR-mutant patients with lung adenocarcinoma (20, 21), doi: 10.1158/1541-7786.MCR-12-0401 and a recurrent gross mutation of the PTEN gene is iden- 2012 American Association for Cancer Research. tified in lung cancer with deficient DNA DSB repair. In

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other studies, 24% of early NSCLC samples lacked PTEN were treated with cisplatin and olaparib at the indicated expression, which correlated with PTEN promoter methyl- doses. After simultaneous exposure of the cells to 2 drugs ation (22) and PTEN protein expression was reduced or lost for 144 hours, growth inhibition was determined using an in 74% of lung tumors, and was associated with low or MTT assay and the multiple drug effect analysis (Sup- aberrant TP53 staining (23). In a later study, PTEN has plementary Method). Computer programs based on the novel nuclear functions, including transcriptional regulation median-effect plot parameters and combination index of the RAD51 gene, whose product is essential for HR repair (CI) equation have been used for data analysis in the of DNA breaks (24, 25). McEllin and colleagues reported present study (30). Experiments were repeated in that loss of PTEN in astrocytes resulted in increased sensi- triplicate. tivity to N-methyl-N0-nitro-N-nitrosoguanidine, a function- al analogue of temozolomide, and PARP inhibitor, due to Immunoblotting analysis inefficient repair (26). Cells were exposed to cisplatin (10 mmol/L) or/and We hypothesized that PTEN-deficient lung cancer cells olaparib (20 mmol/L) for 6 hours. The cells at the point suppressed DNA damage signaling and investigated whether of 6 hours after irradiation at a dose of 10 Gy using a the absence of PTEN could sensitize these cells to a con- Hitachi MBR-1520-R irradiator (150 kV; 20 mA; filter: current treatment of cisplatin and olaparib. 0.5-mm aluminum and 0.1-mm copper) were also used. They were lysed in radioimmunoprecipitation assay buffer Materials and Methods [1%TritonX-100,0.1%SDS,50mmol/LTris-HCl(pH Cell lines 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L Cells were cultured at 37 C with 5% CO2 in RPMI 1640 EGTA, 10 mmol/L b-glycerolphosphate, 10 mmol/L supplemented with 10% heat-inactivated FBS. H1650 is a NaF, 1 mmol/L sodium orthovanadate containing prote- lung adenocarcinoma cell line with co-occurrence of an ase inhibitor tablets (Roche Applied Sciences GmbH)]. EGFR mutation (in-frame deletion in exon 19) and homo- Proteins were separated by electrophoresis on polyacryl- zygous deletion of PTEN. PC-9 is a lung adenocarcinoma amide gels, transferred onto nitrocellulose membranes, cell line having the same in-frame deletion mutation of and probed with specific antibodies followed by detection EGFR with wild-type PTEN. PTEN transfected into with enhanced chemiluminescence plus (GE Healthcare þ H1650 cells (H1650PTEN ) and PTEN expression knocked Biosciences). down in the PC-9 cells (PC-9PTEN ) using shRNA, were established in our laboratory (27). H1299 and A549 are Reagents and antibodies NSCLC lines with wild-type EGFR and wild-type PTEN. Olaparib and cisplatin were kindly provided by AstraZe- PC-3 is a prostate cancer cell line with PTEN loss (Supple- neca and Nippon Kayaku Kogyo Co. Ltd., respectively. mentary Fig. S1). PC-9 was obtained from Immuno-Bio- Rabbit antisera against Akt, phosphorylated (p)Akt (Ser473; logical Laboratories. H1650, H1299, A549, and PC-3 were D9E), PTEN, pChk1 (Ser345), Mre11 (31H4), and b-actin purchased from the American Type Culture Collection. The were purchased from Cell Signaling Technology. Rabbit Mre11 expression vector was kindly provided by Drs. Kenshi antiserum against RPA70 was purchased from Abcam. Komatsu and Junya Kobayashi (Kyoto University, Kyoto, Mouse antisera against Chk1 (G-4) was purchased from Japan). The expression vector was transfected to H1650 cells Santa Cruz Biotechnology. using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Immunohistochemistry Formalin-fixed paraffin-embedded tissue blocks from the Sensitivity test samples were cut to a thickness of 5 mm, placed on glass Antiproliferative activity was determined by a modified 3- slides, then deparaffinized in xylene and graded alcohol for (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bro- 10 minutes. The antigen was incubated in 10 mmol/L mide (MTT) assay in terms of 50% inhibitory concentration sodium citrate buffer, pH 6.0, for 10 minutes in a 95C fl (IC50) values. Brie y, the cells were plated on 96-well plates water bath. The sections were then blocked for endogenous at a density of 1,000 to 3000 cells per well, and continuously peroxidase with 0.3% hydrogen peroxide in methanol. The exposed to each drug for 144 hours. Each assay was con- slides were rinsed with TBS containing 0.1% Tween 20 and ducted in triplicate or quadruplicate, and IC50 values were the sections were blocked with goat normal serum for 60 expressed as mean SD. minutes. The sections were incubated with 1:200 dilution of cleaved caspase-3 (Asp175; 5A1E; Cell Signaling) antibody Design for drug combination overnight at 4C. The sections were amplified using bioti- The constant-ratio design for the combination assay is nylated anti-rabbit antibodies and avidin–biotinylated highly recommended as it allows the most efficient data horseradish peroxidase conjugate for 10 minutes (LSAB 2 analysis (28). The multiple drug effect analysis of Chou Kit, Dako Cytomation) then reacted with 3,30-diaminoben- and Talaly, based on the median-effect principle, was used zidine. Finally, the sections were counterstained with hema- to calculate the combined drug effect (29). H1650, toxylin. Cleaved caspase-3 expression was scored as positive þ H1650PTEN ,PC-9,PC-9PTEN , H1299, A549, and if more than 10% of the tumor cells exhibited cytoplasmic PC-3 cells were seeded in triplicate in 96-well plates and staining (31).

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Immunofluorescence staining day of olaparib, and 5 mg/kg/week of cisplatin plus 50 We fixed cells 8 hours after exposure to cisplatin (10 mg/kg/day of olaparib. Vehicle and olaparib were adminis- mmol/L), or/and olaparib (20 mmol/L), or treatment with tered once a day, 5 times a week by intraperitoneal injection, 10 Gy. Cells were cultured on glass coverslips, fixed with and cisplatin was administered once a day, once a week by 4% formaldehyde, and permeabilized in PBS-0.25% Tri- intraperitoneal injection. Tumor volume (width2 length/ ton X-100. For DNA damage and repair analyses, cells 2) and body weight were determined periodically. Tumor were stained with 1:50 dilution of rabbit polyclonal anti- volume was expressed as mean SD. After the completion RAD51 (Santa Cruz Biotechnology) and 1:500 dilution of of the treatment, all mice per group were sacrificed and the mouse monoclonal anti-gH2AX (Millipore) for 2 hours at tumor specimens were obtained for analysis. All experiments room temperature. Cells were washed with PBS and involving animals were conducted under the auspices of incubated for 30 minutes at room temperature with either the Institutional Animal Care and Research Advisory Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen) second- Committee at the Department of Animal Resources, Oka- ary antibody for RAD51 or gH2AX, respectively. Nuclei yama University Advanced Science Research (Okayama, were visualized by staining with 40, 6-diamidino-2-phe- Japan). nylindole (DAPI). For quantification of RAD51 and gH2AX foci, at least 100 cells from each group were Results visually scored. Cells showing more than 5 foci were Olaparib synergizes cisplatin in PTEN-deficient lung counted as positive for g-H2AXorRAD51.Theseslides cancer cells fl were examined under a uorescence microscope (BIOR- Synergy between PARP inhibitors and platinum drugs EVO BZ-9000; Keyence). was expected (14, 15) in triple-negative breast cancer and BRCA2 ovarian cancer cells (8, 17). We expected that the Xenograft model combination of cisplatin with olaparib showed a syner- Female athymic mice at 7 weeks of age were purchased gistic effect in PTEN-deficient lung cancer cell lines. Cells from Japan Charles River Co. All mice were provided with were treated either with 10 mmol/L cisplatin and 10 to 50 sterilized food and water and housed in a barrier facility mmol/L olaparib or 20 mmol/L cisplatin and 20 to 100 6 under a 12-hour light/dark cycle. Cells (1 10 ) were mmol/L olaparib. Combination of cisplatin with olaparib injected bilaterally subcutaneously into the backs of 7-week- showed a synergistic effect according to the combination old female athymic mice. At 10 days after injection, mice index in the H1650 cells (Fig. 1A, 1B; Table 1). Com- were randomly assigned to 4 groups (5 mice/group) that bination indices were 0.23, 0.20, 0.57, and 0.29 when received either vehicle, 5 mg/kg/week of cisplatin, 50 mg/kg/ concentration ratios of cisplatin and olaparib were

Figure 1. Combination index and surviving fraction of H1650 and þ H1650PTEN cells treated with cisplatin in combination with olaparib simultaneously for 144 hours. Synergistic effects in H1650 cells are shown, designating to be molar ratios of 1:2 (A) and 1:5 (B). Antagonistic effects in H1650PTENþ cells are shown, designating to be molar ratios of 1:2 (C) and 1:5 (D).

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Table 1. Combination index Table 2. Drug sensitivity

Concentration ratio (molar) of Cell line Cisplatin (mmol/L) Olaparib (mmol/L) cisplatin to olaparib H1650 2.12 0.72 15.47 6.8 ]a Cell line 1:1 1:2 1:3 1:5 H1650PTENþ 1.65 0.97 50.83 7.7 H1650 0.23 0.20 0.57 0.29 PC-9 0.21 0.019 5.88 1.4 H1650PTENþ 1.76 1.59 1.77 1.07 PC-9PTEN 0.42 0.10 6.52 6.7 PC-9 4.38 5.16 10.8 0.55 PTEN NOTE: Values are presented as mean SD of 50% inhibitory PC-9 0.43 0.40 0.66 0.78 PTENþ concentration (IC50) of the drug. H1650 : PTEN- NOTE: Combination index according to various concentra- restored H1650; PC-9PTEN : PTEN-ablated PC-9; tion ratios of cisplatin and olaparib in each cell line is aP < 0.05. described. CI < 1, CI ¼ 1, and CI > 1 indicate synergism, additive effect, and antagonism, respectively. PTEN the IC50 values of olaparib in PC-9 and PC-9 cells were 5.88 1.4 mmol/L and 6.52 6.7 mmol/L, respectively. PC-9PTEN cells did not confer sensitization to designed to be molar ratios of 1:1, 1:2, 1:3, and 1:5, olaparib alone. IC50 values of cisplatin or olaparib alone in respectively. Restoration of PTEN in the H1650 cells PC-3, H1299, and A549 cells are shown in Supplementary decreased sensitivity to olaparib and cisplatin (CI Table S2. >1; Fig. 1C, 1D). Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin (CI <1), PTEN inactivation suppresses DNA damage signaling and PC-9 cells decreased sensitivity to olaparib and cis- Oncogenic activation of Akt frequently results from loss of platin (CI >1). Our results showed that PTEN-deficient PTEN expression or function leads to suppression of DNA PTEN damage signaling (32). In this study, immunoblotting assay lung cancer cell lines, H1650 and PC-9 , exhibited synergism for all combinations of olaparib doses, whereas revealed that H1650 and PC-9PTEN cells exhibited much þ þ H1650PTEN andPC-9cellsexhibitedantagonisticeffects higher levels of pAkt than H1650PTEN and PC-9 cells, respectively (Fig. 2A). In addition, pChk1 was not over- for most dose combinations. A synergistic effect was also shown in the PC-3 cells, whereas antagonistic effects at expressed in both H1650 and PC-9PTEN cells despite drug most dose combinations were shown in H1299 and A549 treatment. pChk1 was expressed after irradiation; however, cells (Supplementary Table S1). the increase was less in PTEN-deficient lung cancer cells Sensitivity of cisplatin or olaparib monotherapy is shown compared with their counterparts (Fig. 2B). Meanwhile, in Table 2. IC values of olaparib in H1650 and PTEN-deficient lung cancer cells exhibited lower levels of þ 50 H1650PTEN cells were 15.47 6.8 mmol/L and 50.83 Mre11 compared with their counterparts (Fig. 2C). To 7.7 mmol/L, respectively. PTEN-restored H1650 that address how lower expression levels of Mre11 affects the became resistant to olaparib (P < 0.05). On the other hand, synergism between cisplatin and olaparib, Mre11 expression

Figure 2. PTEN inactivation and DNA damage signaling. A, PTEN loss activated Akt and suppressed pChk1. pChk1 was not overexpressed in both H1650 and PC-9PTEN cells (PTEN-deficient lung cancer cells) despite drug treatment. B, pChk1 was expressed after irradiation; however, the increase was less in PTEN-deficient lung cancer cells compared with their counterparts. C, PTEN-deficient lung cancer cells exhibited lower levels of Mre11 compared with their counterparts.

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þ Figure 3. PTEN inactivation and expression of RAD51 and g-H2AX. A, subcellular localization of RAD51 and g-H2AX in H1650 or H1650PTEN cells after exposure of olaparib (20 mmol/L) or irradiation (10 Gy). B, PTEN deﬁciency resulted in signiﬁcant reduction in RAD51 focus formation after exposure of cisplatin þ (10 mmol/L) and/or olaparib (20 mmol/L), or irradiation (10 Gy) compared with H1650PTEN cells (P < 0.05); although g-H2AX was similarly increased in both cells.

þ vector was transfected into H1650 cells, and a stable trans- compared with H1650PTEN cells (P < 0.05); although formant in which Mre11 was overexpressed was obtained g-H2AX was similarly increased in both cells (Fig. 3B and þ (designated H1650Mre11 cells; Supplementary Fig. S2). In Supplementary Fig. S3B). this cell line, combination indices were 0.76, 0.91, 0.97, and 0.77 when concentration ratios of cisplatin and olaparib Effectiveness of the cisplatin with olaparib in a xenograft were designed to be molar ratios of 1:1, 1:2, 1:3, and 1:5, model þ respectively in H1650Mre11 (Supplementary Table S3). We examined xenograft tumors to determine the effec- Although these combination index values were somewhat tiveness of the cisplatin with olaparib in PTEN-deficient þ elevated compared with those observed in original H1650 lung cancer cells in vivo. H1650 and H1650PTEN xenograft cells, these results indicated that lower levels of Mre11 alone tumors grew at almost same rate. The immunostaining of could not be the sole reason for this synergism. cleaved caspase-3 is shown in Fig. 4A. The combination of On the other hand, PTEN has other nuclear functions, cisplatin and olaparib induced significant higher positive including transcriptional regulation of the RAD51 gene, cells than other groups in H1650 xenografts (P < 0.05). The whose product is essential for HR repair of DNA breaks positive cells were 43 3% for cisplatin plus olaparib, 10.6 (24, 25). Replication protein A (RPA) is displaced from 1.1% for cisplatin alone, 16 3.6% for olaparib alone, single-stranded DNA by RAD51 to initiate HR (32). In this and 3 2% for vehicle. However, the combination did not þ study, we investigated whether the formation of RAD51 and display synergistic effect in H1650PTEN xenografts. The RPA foci was reduced in PTEN-deficient lung cancer cell positive cells were 10 2.6% for cisplatin plus olaparib, 17.3 lines. Subcellular localization of RAD51, g-H2AX, and RPA 2.0% for cisplatin alone, 6.6 0.5% for olaparib alone, is shown in Fig. 3A and in Supplementary Fig. S3A. PTEN and 4.6 0.5% for vehicle (Fig. 4A). deficiency resulted in significant reduction in RAD51 and Treatment with cisplatin plus olaparib significantly sup- RPA focus formation after drug exposure or g-irradiation pressed growth of the H1650 tumors compared with

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Figure 4. A, cleaved caspase-3 staining (400) of tumor sections from each treatment group in H1650, þ H1650PTEN , PC-9, and PC-9PTEN cells, included as graphical representation. B, growth of xenograft tumors. Growth curves of H1650, H1650PTENþ, PC-9, and PC-9PTEN cells xenograft tumors in animals receiving the indicated drugs (50 mg/kg/d of olaparib, 5 mg/kg/wk of cisplatin, and both agents) or vehicle intraperitoneally were compared using Student t test. Bars, SD.

cisplatin alone, olaparib alone, and the untreated controls 9PTEN xenografts treated with the combination displayed (Fig. 4B). The tumor sizes (mm3) at day 22 were 124.8 signiﬁcantly higher number of positive cells than those of 33.1, 351.8 150.9, 413.1 66.0, and 847.8 98.5, other groups (44.3 4.0% for the combination, 7.3 3.2% þ respectively. H1650PTEN xenograft tumors did not show for cisplatin, 15.3 1.5% for olaparib, 1.0 1.7% for signiﬁcant response to the combination of cisplatin and vehicle). In PC-9 xenografts, there were no differences olaparib compared with cisplatin alone, olaparib alone, and among 4 groups: 10.6 2.0% for the combination, 7.3 the untreated controls. The tumor sizes (mm3) at day 22 1.5% for cisplatin, 8.3 1.5% for olaparib, 4.0 1.7% were 467.8 103.0, 373.9 113, 524.5 145.7, and for vehicle (Fig. 4A). Western blotting also indicated that the 801.4 113.2, respectively. combination of cisplatin with olaparib seemed to induce Next, we examined whether the combination of cisplatin more cleaved caspase-3 expressions than other treatments in with olaparib in PC-9 and PC-9PTEN xenografts was H1650 and PC-9PTEN xenografts, but not in PC-9 and þ effective or not. Cleaved caspase-3 expressions in PC- H1650PTEN xenografts (Supplementary Fig. S4).

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In the PC-9PTEN xenograft model, cisplatin plus ola- exposure to hydroxyurea (38). McEllin and colleagues parib inhibited tumor growth than other treatment (152.6 showed a significant decrease in mRNA expression on 8.06 mm3 for the combination, 336.6 45.7 mm3 for RAD51B, C, and D, and reduced HR-mediated repair cisplatin, 411.2 67.2 mm3 for olaparib, 774.3 95.8 in PTEN-null astrocytes (26). Meanwhile, Xu and collea- mm3 for vehicle). PC-9 xenograft tumors did not show gues observed PTEN knockdown in HCT116 cells atten- significant response to the combination compared with uated Mre11, which was a key gene in HR repair of DSBs cisplatin alone, olaparib alone, and the untreated controls (32). In addition, Fraser and colleagues reported PARP (279.1 69.5 mm3, 254.2 42.2 mm3, 296.0 57.5 inhibitor sensitivity associated with a defect in Mre11 mm3, and 642.3 133.2 mm3, respectively; Fig. 4B). expression (35). Our observation that PTEN-deficient In toxicity evaluation, all the treatment animals did not lung cancer cells exhibited lower levels of Mre11 com- show substantial loss of body weight (>10%) and the pared with their counterparts was in agreement with their addition of olaparib to cisplatin did not significantly increase studies. To investigate the effect of Mre11 itself on the weight loss compared with cisplatin single agent treatment synergy, we examined the combination effect of olaparib þ (data not shown). with cisplatin using H1650Mre11 cells. Unexpectedly, restored levels of Mre11 did not suppress the phenotypes Discussion observed in PTEN-deficient cells in this study (Table 1 We here showed that PTEN-deficient lung cancer cell and Supplementary Table S3). Further investigations lines suppressed DNA damage signaling, and were sensitive should be required to clarify whether and to what extent to the combination of olaparib with cisplatin. Synergy the molecular events including Chk1, Mre11, and RAD51 between PARP inhibitor and platinum drugs was expected are responsible for the synergic effect. Interestingly, Shen in triple-negative breast cancer and BRCA2 ovarian cancer and colleagues recently showed that PTEN is important cells (8, 17). In other study, addition of PARP inhibitor after for maintaining basal levels of transcription of the RAD51 alkylating agent, demethyl sulfate, treatment increased SSB gene in mouse embryonic fibroblasts (24). Although there levels indicating ongoing repair even at this late time-point were no significant differences of RAD51 and RPA levels (33). Recently, evidence suggested that PTEN was impor- by Western blotting (Supplementary Fig. S5), the forma- tant for the maintenance of genome stability (24, 25). The tion of RAD51 and RPA foci was reduced in PTEN- HR impairment caused by PTEN deficiency sensitized deficient lung cancer cells (Fig. 3 and Supplementary Fig. tumor cells to potent inhibitors of the DNA repair enzyme, S3). As RPA binds to single-stranded DNA, the RPA focus both in vitro and in vivo (34). Our results were in agreement could be a marker for end resection at the double-stranded with their studies. We showed that xenograft tumors bearing DNA ends (32). Thus, inactivation of PTEN might lead PTEN-deficient lung cancer cells were sensitive to the to suppression of DNA damage signaling, leading to the combination of cisplatin with olaparib, although this effect lower levels of end resection and, hence, less RPA focus was not observed in a xenograft model using PTEN wild- formation. As shown above, reduced levels of Mre11 alone type cells (Fig. 4B). However, it is possible that the results could not provide a sufficient explanation for this, though may be cell specific or have a different effect of PTEN loss on Mre11isinvolvedinthemolecularmechanismsoftheend HR capacity because PTEN-deficient prostate cancer cells resection (39). had only mild PARP inhibitor and DNA damaging agent's A number of clinical trials to treat triple-negative breast sensitivity (35). cancer, metastatic melanoma, malignant glioma, advanced Drug interaction between cisplatin and olaparib in PTEN- colorectal cancer, ovarian cancer, and lung cancer are now deficient lung cancer cells has not been elucidated. Our underway to test the efficacy of PARP inhibitors or PARP investigation revealed that PTEN deficiency caused a reduc- inhibitors in combination with DNA-damaging agents (40). tion of pChk1 (Fig. 2A) and decreased drug or radiation- Later, phase II studies using olaparib established proof-of- induced nuclear RAD51 and RPA focus formation (Fig. 3 concept of selectively killing of HR-deficient breast cancer and Supplementary Fig. S3). Oncogenic activation of Akt and ovarian cancer cells with BRCA1 or BRCA2 mutations, frequently resulted from loss of PTEN expression or func- resulting in a substantial clinical benefit with minimal tion (36). How PTEN loss affects DNA damage signaling toxicity (6, 7, 41). DNA repair biomarkers from multiple should be clarified. H1650 and PC-9PTEN cells exhibited DNA repair pathways on treatment response and cancer þ much higher levels of pAkt than H1650PTEN and PC-9 survival offers opportunity to evaluate patient tumor samples cells, respectively. Activation of Chk1 after irradiation was and determine their status of DNA repair pathways before attenuated in PTEN-deficient cells (Fig. 2B). Chk1 has a and during therapy for individual patients. In recent years, critical role in maintaining genomic stability by delaying S- our understanding of how to treat NSCLCs has undergone a fi and G2 phase progression of cells containing DNA damage paradigm shift by the identi cation of EGFR mutations to allow time for repair before mitosis and, the DSBs that (42, 43) and EML4–ALK translocation (44). In BRCA1- arise when Chk1 is inhibited are apparently related to a deficient lung cancer, PARP inhibition induced BAX/BAK- specific S-phase role whereby Chk1 suppresses aberrant independent synthetic lethality (45). Knowledge of the initiation of DNA replication that wound generate DNA status of multiple DNA repair profiling of patients and may lesions (37). Chk1 is reportedly required for HR repair and discriminate patients with likelihood to respond to PARP Chk1-depleted cells failed to form RAD51 nuclear foci after inhibitors.

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Olaparib with Cisplatin on PTEN-Deﬁcient Lung Cancer

We hypothesized that tumor cell with HR deficiency Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- PTEN tational analysis): D. Minami, N. Takigawa, H. Takeda, M. Takata, E. Ichihara, K. (such as -mutated cancer cells) were hypersensitive Kiura to PARP inhibitors in combination with cisplatin, resulting Writing, review, and/or revision of the manuscript: D. Minami, N. Takigawa, A. Hisamoto, K. Hotta, K. Kiura in killing of tumor cells based on the synthetic lethality Administrative, technical, or material support (i.e., reporting or organizing data, principle. A major important solution to these barriers is to constructing databases): N. Takigawa, E. Ichihara, K. Kiura build biomarker testing into patient tumor identification, Study supervision: N. Takigawa, M. Tanimoto, K. Kiura and to use the biomarker panels during treatment. The PTEN fi Acknowledgments combination of cisplatin with olaparib in -de cient The authors thank Drs. Takashi Ninomiya, Toshi Murakami, and Noboru Asada lung tumors might be further pursued in clinical trials. for expert technical support, Drs. Kenshi Komatsu and Junya Kobayashi, Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University (Kyoto, Japan) for providing the Mre11 expression vector and AstraZeneca for providing Disclosure of Potential Conflicts of Interest olaparib. N. Takigawa has a honoraria from speakers' bureau from AstraZeneca. K. Kiura has a honoraria from speakers' bureau from AstraZeneca. No potential conflicts of interest Grant Support were disclosed by other authors. This study was supported by the Ministry of Education, Culture, Sports, Science, and Technology, Japan grants 21590995 (to N. Takigawa) and 23390221 (to K. Kiura). Authors' Contributions The costs of publication of this article were defrayed in part by the payment of page advertisement Conception and design: D. Minami, N. Takigawa, E. Ichihara, K. Kiura charges. This article must therefore be hereby marked in accordance with Development of methodology: D. Minami, N. Takigawa, N. Ochi, E. Ichihara, K. 18 U.S.C. Section 1734 solely to indicate this fact. Kiura Acquisition of data (provided animals, acquired and managed patients, provided Received June 29, 2012; revised October 26, 2012; accepted November 16, 2012; facilities, etc.): D. Minami, N. Takigawa, K. Kiura published OnlineFirst December 13, 2012.

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148 Mol Cancer Res; 11(2) February 2013 Molecular Cancer Research

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

Daisuke Minami, Nagio Takigawa, Hiromasa Takeda, et al.

Mol Cancer Res 2013;11:140-148. Published OnlineFirst December 13, 2012.

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