Therapeutics, Targets, and Chemical Biology Cancer ...As many cancer chemotherapeutic drugs and radiation therapy cause DNA damage, tumor cells defective in DNA repair pathways are

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Published OnlineFirst August 26, 2010; DOI: 10.1158/0008-5472.CAN-09-4521

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apeutics, Targets, and Chemical Biology

ergistic Chemosensitivity of Triple-Negative Breastcer Cell Lines to Poly(ADP-Ribose) Polymerase

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basal-like subtype of breast cancer is characterized by a triple-negative (TN) phenotype (estrogenor, progesterone receptor, and human epidermal growth factor receptor-2/neu negative). TN breast can-are similar gene expression profiles and DNA repair deficiencies with BRCA1-associated breast cancers.1-mutant cells exhibit sensitivity to gemcitabine, cisplatin, and poly(ADP-ribose) polymerase (PARP)ion; therefore, we hypothesized that TN cancer cells may also exhibit sensitivity to these drugs. In thiswe report that TN breast cancer cells are more sensitive to these drugs compared with non-TN breastcells. Moreover, combination treatments indicated that PARP inhibition by the small-molecule inhib-34 or siRNA knockdown synergized with gemcitabine and cisplatin in TN cells but not in luminal cancerN cells exhibited reduced repair of UV-induced cyclobutane pyrimidine dimers after PARP inhibition,ting that the synergistic effect of PJ34 and gemcitabine or cisplatin reflected inefficient nucleotiden repair. Mechanistic investigations revealed that in TN cells, PJ34 reduced the levels of ΔNp63α withurrent increase in p73 and its downstream target p21. Thus, the sensitivity to combination treatmentd to be mediated by sustained DNA damage and inefficient DNA repair triggering p63/p73–mediated

apoptosis. Our results suggest a novel therapeutic strategy to treat women with TN breast cancer, an aggres-sive disease that presently lacks effective treatment options. Cancer Res; 70(20); 7970–80. ©2010 AACR.

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ast cancer is the most common cause of malignancyhe second most common cause of cancer death inn (1). This heterogeneous disease is composed of fivebiological subtypes, which are based on microarraylassifications: luminal A, luminal B, normal breast-like,n epidermal growth factor receptor 2 (HER2), andlike breast cancers (1, 2). Whereas many of these sub-can be treated with much success, the basal-like carci-s are associated with high rates of relapse followingtherapy (3, 4). Basal-like breast tumors are largelyen receptor (ER), progesterone receptor (PR), andneu negative [triple negative (TN)] and express genesteristic of basal epithelial and normal breast myoe-al cells (5–7). However, the genes responsible for the

gressive phenotype of basal-like breast can-nown.

Theinvolvdoubltion (Hbase eRecenshownmeraswhenthis scin DNinhibi

: Division of Oncology, Stanford University School of, California

ry data for this article are available at Cancer Researchrres.aacrjournals.org/).

thor: James M. Ford, Stanford University School ofpus Drive, CCSR #1115, Stanford, CA 94305. Phone:: 650-725-1420; E-mail: [email protected].

5472.CAN-09-4521

ssociation for Cancer Research.

20) October 15, 2010

Research. on June 26, 202cancerres.aacrjournals.org aded from

many cancer chemotherapeutic drugs and radiationy cause DNA damage, tumor cells defective in DNApathways are predicted to be sensitive to their effects., cell lines deficient in BRCA1 (and BRCA2) have beento be sensitive to the DNA cross-linking agents cis-and mitomycin C (8, 9), the topoisomerase inhibitorside (10), and oxidative DNA damage (11). Recently,ve shown that BRCA1-deficient cells are sensitivecitabine (2′,2′-difluoro-2′-deoxycytidine; ref. 12), anue of cytosine arabinoside that exhibits anticancerrties due to potent inhibition of DNA synthesis (13).itabine is often used either alone or in combinationther drugs such as taxanes, vinorelbine, carboplatin,stuzumab in metastatic breast cancer. However, therereports of the efficacy of gemcitabine in the basal-likee of breast cancers.BRCA1 breast cancer susceptibility gene is known to beed in a number of DNA repair pathways, including DNAe-strand break repair through homologous recombina-R; ref. 14), nucleotide excision repair (NER; ref. 15), andxcision repair (BER) of oxidative DNA damage (11).tly, BRCA1- and BRCA2-deficient cells have also beento be sensitive to inhibitors of poly(ADP-ribose) poly-e (PARP; refs. 16, 17), an enzyme involved in BER, which,inhibited, leads to DNA strand breaks and cell death. Inenario, BRCA-mutant tumor cells with primary defects
A repair are particularly sensitive to small-moleculetors of BER, such as PARP inhibitors.
0. © 2010 American Association for Cancer

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ent studies indicate that sporadic basal-like or TN tu-ear a striking resemblance to breast tumors that ariseeditary BRCA1 mutation carriers. These similaritiesly suggest that sporadic basal-like tumors might bears in BRCA1-mediated DNA repair pathways (11) andt similar sensitivities to DNA-damaging agents andinhibitors.refore, using a panel of breast cancer cell lines, we ex-d the cytotoxic effects of gemcitabine, cisplatin, and ainhibitor alone and in combination. We show that, like1-mutant cells, basal-like TN breast cancer (TNBC)re sensitive to the PARP inhibitor PJ34, gemcitabine,splatin. We further show that PJ34 acts synergisticallyoth gemcitabine and cisplatin in TNBC cells, but not inal breast cancer cell lines. Moreover, we show that PJ34mcitabine disrupt NER, suggesting a novel mechanismsitivity to these drugs in TNBC cells.

rials and Methods

nes and reagentscell lines were used within 6 months of purchase.B468, hs578t, MCF7, BT549, and BT474 cell lines wereed from the American Type Culture Collection (ATCC)aintained in DMEM with 10% fetal bovine serum (FBS).and HCC1806 cells (ATCC) were maintained in RPMIith 10% FBS. ATCC provides molecular authentica-support of their collection through their genomics,

nology, and proteomic cores, as described, using DNAing and species identification, quantitative gene ex-on, and transcriptome analyses (18). SUM149PT cellsbtained from Asterand Plc, where they were authenti-through gene expression data through the use of Affy-GeneChips and biomolecular markers, such as ER andstatus. They were maintained in Ham's F-12 medium0% FBS. HCC1806 cells were transfected with themir retroviral vector in pSM2 against nonspecific

ing control, PARP1 or PARP2 (Thermo Fisher Scien-Stable clones were maintained with 1 μg/mL puro-and confirmed by Western blotting. The PARP

or PJ34 was purchased from EMD Biosciences. Methy-lue, MTT, and cisplatin were purchased from Sigma Che-. Gemcitabine was a gift from Eli Lilly Pharmaceuticals.

iability and colony formation assayviability was measured by an MTT assay. For the MTTcells were seeded in 96-well plates and treated withgemcitabine, and cisplatin as indicated. MTT reagentded to the cells, which was reduced to purple forma-ystals by the mitochondria of living cells. The reducedls were solubilized with DMSO, and the absorbanceeasured at 570 nm by spectrophotometry. All experi-were done in triplicate and also repeated three inde-nt times, and data were plotted as mean ± SD. Datarepresentative experiment are shown in the figures.

lony formation assay, HCC1806 and MCF7 were plated
al density and treated with 10 μmol/L PJ34, 0.6 nmol/Ltabine, or 4 μmol/L cisplatin for 72 hours. After treat-
PJ34,24 hou

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Research. on June 26, 202cancerres.aacrjournals.org Downloaded from

cells were counted and either 500 or 1,000 cells were. After 15 days, the cells were stained with methylenend individual colonies were counted.

combination studiescombination studies, BT474, MCF7, HCC1806, andB468 cells were seeded in triplicate in 96-well plateseated with PJ34, gemcitabine, or cisplatin alone or withmbination of PJ34 and gemcitabine or PJ34 and cisplat-he indicated doses. MTT assays were performed afterrs of treatment. The data were plotted using Calcusyn-t software (19, 20). Combination index (CI) and isobo-s were plotted using the CI equation of Chou-Talalay

A nonconstant ratio drug combination design wasnd normalized isobolograms were constructed usingsyn-Biosoft software. The experimental protocol andbologram plotted along with the equation for calculatingshown in Supplementary Fig. S1. CI < 1 was synergistic,was additive, and CI > 1 was antagonistic. Study

epeated three independent times and representativere shown.

X and Rad51 staining74, MDAMB468, HCC1806, HCC1806shP1, and806shP2 cells were plated overnight in chambered(1,500 cells per chamber). Cells were treated withol/L PJ34 or 5 nmol/L gemcitabine for either 1 tors for Rad51 staining or 24 hours for γH2AX. Controlsed primary alone, isotype control, and secondaryAfter treatment, cells were fixed in 4% paraformalde-nd stained overnight with primary antibody for Rad511:200 dilution; Santa Cruz Biotechnology) or γH2AX-(1:500 dilution; Cell Signaling). Cells were washedBS/bovine serum albumin and incubated for 1 hourm temperature with either Alexa 488 or Alexa 594ogen) secondary antibody for Rad51 or γH2AX, respec-Cells were fixed in Prolong gold antifade with 4′,6-dino-2-phenylindole (Invitrogen) and cured at roomrature for 24 hours before visualizing. For quantifica-f Rad51 foci and γH2AX foci, at least 100 cells fromroup were visually scored. Cells showing more thanfoci were counted as positive for γH2AX or Rad51.atio of Rad51 foci in the control versus the treateds was represented as fold change. For γH2AX, the folde was the ratio of γH2AX-positive cells in HCC1806l cells versus shPARP1 and shPARP2. Images fromm fields were taken using a Nikon Eclipse E 800 micro-with an attached camera and using Spot SoftwareDiagnostic Instruments (Diagnostic Instruments).s were taken with a 40× lens (40×/1.0 DIC H Planil immersion lens).

tosis detectionptosis was detected by staining HCC1806 cells forin V and cleaved caspase-3. Cells were plated overnightmbered slides followed by treatment with 10 μmol/L
0.6 nmol/L gemcitabine, or 4 μmol/L cisplatin forrs. For Annexin V-FITC, the standard manufacturer's
Cancer Res; 70(20) October 15, 2010 7971



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Hastak et al.

Cance7972


ol was followed (Sigma). For caspase-3, cells wered with a 1:250 dilution of cleaved caspase-3 antibody5, Cell Signaling) and visualized as mentioned above.

otide excision repair assayAMB468, HCC1806, HCC1806 control, HCC1806shP1,CC1806shP2 cells were grown overnight in six-well(in triplicate). Cells were rinsed with PBS and then irra-with 20 J/m2 UVC. MDAMB468 and HCC1806 cells wered with 10 μmol/L PJ34 or 5 nmol/L gemcitabine, where-dium without drugs was added to PARP1- and PARP2-down cells. Genomic DNA was extracted (QIAamp DNA
it, Qiagen) at 0 to 24 hours. Repair of cyclobutane pyrim- Tot
formation assay after 15 d. The numbers of colonies are shown; insets, represenate. Columns, average cell viability in log scale; bars, SD.

r Res; 70(20) October 15, 2010


using an ELISA. Briefly, genomic DNAwas distributed inate onto microtiter plates precoated with 0.003% prot-sulfate. DNA lesions were detected with either 1:5,0002 (for CPDs) or 1:5,000 64M-2 (for 6-4PPs; a gift fromshio Mori, Radioisotope Research Center, Nara Medicalrsity School ofMedicine, Nara, Japan; ref. 22). The signalsmplified and subsequently developed with 3,5,3′,5′-tet-hylbenzidine (Sigma Chemicals). Absorbance was mea-at 450 nm. Each experiment was repeated threendent times, and representative data are shown.

rn blot analyses
al cellular protein was isolated by lysing the cells in
imers (CPD) and 6-4 photoproducts (6-4PPs) was mea- modified radioimmunoprecipitation assay buffer. Proteins

1. Cell viability of breast cancer cells after PJ34, gemcitabine, or cisplatin treatment. A, cells were treated with 0.01 to 62.5 μmol/L PJ34 (i), 0.15 tool/L gemcitabine (ii), or 0.256 to 10 μmol/L cisplatin (iii) for 72 h, and cell viability was determined by an MTT assay. B, apoptosis in HCC1806shown by cleaved caspase-3 and Annexin V staining (inset, bright-field image). C, HCC1806 and MCF7 cells were stained with methylene blue for

tative figures. Experiments were done three independent times

Cancer Research



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Table 1. Synergistic effect of PJ34 with either gemcitabine or cisplatin in breast cancer cell lines

A B

Gemcitabine(μmol/L)

PJ34(μmol/L)

CI Effect PJ34(μmol/L)

Cisplatin(μmol/L)

Cl Effect

(i) HCC1806 (i) HCC18060.0016 1 0.88 Synergism 1.875 10 1.09 Additive0.008 1 0.95 Synergism 3.75 10 0.85 Synergism0.04 1 0.97 Synergism 7.5 10 0.83 Synergism0.2 1 0.98 Synergism 15 10 0.646 Synergism1 1 0.98 Synergism

(ii) MDAMB468 (ii) MDAMB4680.0016 1 0.74 Synergism 1.875 10 0.83 Synergism0.008 1 0.48 Synergism 3.75 10 0.76 Synergism0.04 1 0.25 Synergism 7.5 10 0.82 Synergism0.2 1 0.72 Synergism 15 10 0.91 Synergism1 1 0.90 Synergism

(iii) BT474 (iii) BT4740.0016 1 1.34 Antagonism 1.875 10 74 Antagonism0.008 1 3.51 Antagonism 3.75 10 1.31 Antagonism0.04 1 1.33 Antagonism 7.5 10 1.53 Antagonism0.2 1 18.30 Antagonism 15 10 1.54 Antagonism1 1 50.22 Antagonism

(iv) MCF7 (iv) MCF70.0016 1 1.29 Antagonism 1.875 10 1.71 Antagonism0.008 1 2.93 Antagonism 3.75 10 1.28 Antagonism0.04 1 7.79 Antagonism 7.5 10 1.20 Antagonism0.2 1 0.74 Synergism 15 10 0.89 Synergism1 1 1.19 Antagonism

NOTE: HCC1806, MDAMB468, MCF7, and BT474 cells were treated with PJ34, gemcitabine, or cisplatin alone or a combination ofthe drugs at the indicated concentrations for 72 h, and an MTT assay was performed. CI was calculated by Calcusyn-Biosoftsoftware. (A) CI values of PJ34 in combination with gemcitabine; (B) CI values of PJ34 in combination with cisplatin.


www.a


μg/lane) were separated by electrophoresis (10% SDS-) and electroblotted onto polyvinylidene difluorideranes (GE Healthcare). The membranes were probedntibodies against PARP, p63, p73, p21, actin (Santa Cruzhnology), or MCM (BD Biosciences). Protein levels fromts were evaluated using the gel analysis software Ima-IH), and the ratio of protein levels in the control versusated groups was represented as fold change.

lts

ivity of basal-like breast cancer cells to PJ34,tabine, and cisplatintest our hypothesis that PARP inhibitors that target1-pathway dysfunction might also be efficacious in theent of TNBCs, a panel of sporadic TNBC cells (BT549,
806, and MDAMB468) along with a BRCA1-mutant TNne (SUM149PT) and luminal breast cancer cell lines
luminHER2/

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4, MCF7, and T47D) were tested for their sensitivity to0–62.5 μmol/L), gemcitabine (0–4.8 nmol/L), and cis-(0–10 μmol/L). After 72 hours of treatment, cell viabil-s measured by an MTT assay. All TNBC cell lines werecantly more sensitive to PJ34, gemcitabine, and cisplat-tment than the luminal breast cancer cell lines (Fig. 1A,ementary Table S1). We confirmed the sensitivity ofcells to PJ34, gemcitabine, and cisplatin by colony for-n assay, and as shown in Fig. 1C, HCC1806 cells weresensitive to all the three drugs compared with the lu-MCF7 cells. Furthermore, all the drugs induced apopto-TNBC cells as evidenced by caspase-3 cleavage andin V staining (Fig. 1B). Therefore, we found that, similarA1-deficient cells, TNBC cells were selectively sensitiveP inhibition, gemcitabine, and cisplatin compared withher breast subtypes. Moreover, the resistance of the
al cell lines does not depend on HER2 status, as BT474 isneu positive whereas MCF7 is HER2/neu negative.



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Cance7974


gism between PJ34 and gemcitabine or cisplatinresults show that TNBC cells are selectively sensitive4, gemcitabine, and cisplatin when used individually.ver, to determine whether inhibition of PARP by PJ34a synergistic, additive, or antagonistic fashion with

tabine or cisplatin, we treated BT474, MCF7, HCC1806,DAMB468 cell lines with the agents alone or in combi-for 72 hours. Cells were treated either with 1 μmol/Lnd 0.0016 to 1 μmol/L gemcitabine or with 1.875 tool/L PJ34 and 10 μmol/L cisplatin. CalcuSyn softwareed to calculate the CI and plot normalized isobologramslementary Figs. S2 and S3; refs. 19, 21). CI < 1, CI = 1,I > 1 quantitatively indicate synergism, additivity,tagonism, respectively. We also calculated the linearient r value to estimate the accuracy of measurement,l our experiments had an r value >0.90 for the median-plot.hown in Table 1A (i and ii), TNBC cell lines HCC1806DAMB468 exhibited synergism for all the differentnations of PJ34 and gemcitabine doses, whereas theal BT474 and MCF7 cell lines (Table 1A, iii and iv)ted antagonistic effects for most dose combinations.rly, PJ34 and cisplatin had additive to synergisticin both HCC1806 and MDAMB468 cells (Table 1B,

ii), whereas antagonism was observed in the luminaland MCF7 cell lines (Table 1B, iii and iv).also treated the cells with varied concentrations ofnd kept the concentration of gemcitabine constant,nversely, we kept the concentration of PJ34 constantchanging the concentration of cisplatin. Again, TNBCes exhibited additive to synergistic effects, whereas
nism was observed in BT474 and MCF7 cell lines (dataown).
Fig. 3Athe Ra

r Res; 70(20) October 15, 2010


knockdown sensitizes cells to gemcitabineisplatiny PARP inhibitors are known to inhibit PARP1 and, both of which are involved in DNA repair pathways.fore, to investigate the role of PARP inhibition in sen-g TNBC cells to gemcitabine and cisplatin, we gener-table clones of PARP1- and PARP2-knockdown cells inHCC1806 cell line. Decreased protein expression of

1 and PARP2 protein was confirmed by immuno-g (Fig. 2A). We then treated HCC1806 nonsilencingl and HCC1806 shPARP1 and shPARP2 clones (twowith either 0.15 to 4.8 nmol/L gemcitabine or 0.25 tol/L cisplatin for 72 hours, measured cell viability by anssay, and calculated IC50 values by Prism software. Asin Fig. 2B and C, both PARP1 and PARP2 knockdownzed HCC1806 cells to gemcitabine and cisplatin. PARP2down significantly (P < 0.05) sensitized cells to gemcita-ompared with HCC1806 (Fig. 2B). Conversely, PARP1down significantly (P < 0.05) sensitized cells to cisplatinC).

amage in basal-like breast cancer cell lines afterand gemcitabine treatmentause PARP and gemcitabine play a major role in DNAand inhibition of DNA synthesis, respectively, we inves-d the effect of PJ34 and gemcitabine on DNA damageining for Rad51 foci and γH2AX, which accumulate atf broken DNA.study Rad51 foci formation, the luminal BT474 andDAMB468 cell lines were treated with either 10 μmol/Lr 5 nmol/L gemcitabine for 1 to 4 hours. As shown in
, 4 hours of PJ34 or gemcitabine treatment increasedd51 foci in MDAMB468 cells. BT474 cells had a higher
urePAr getmelysitrolARe trcencit

T ash. ICPrisgraper gCotri

s, SD; ★, P < 0.05.

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2. Cell viability of PARP1-RP2-knockdown cellsmcitabine and cisplatinnt. A, Western blots of PARP in HCC1806and shPARP1- andP2-knockdown cells. Cellseated with increasingtrations of eitherabine or cisplatin and ansay was performed after

50 values were determinedm and are representedhs for cells treated withemcitabine (B) or cisplatinlumns, average cell viabilityplicate experiments;

Cancer Research

ion for Cancer


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evel of Rad51 foci, but PJ34 and gemcitabine treatmentt further increase the number of foci. We next countedmber of Rad51 foci and observed a 2-fold increasePJ34 and gemcitabine treatment (Fig. 3C, top ande). Similar to PJ34 treatment, knockdown of PARP1ARP2 also increased the number of Rad51 foci com-with HCC1806 control cells, as seen and quantified. 3B and C (bottom).next treated BT474, MDAMB468, and HCC1806 cells0 μmol/L PJ34 or 5 nmol/L gemcitabine for 24 hoursained the cells for γH2AX. As shown in Fig. 4A, follow-eatment, there was no increase in the number ofX-positive cells in BT474 cell line. On the other hand,

tification of Rad51 foci after 1, 2, or 4 h of PJ34 (top) or gemcitabine (midci or more were counted as positive.

806 and MDAMB468 cell lines showed a significantse in the number of γH2AX-positive cells. PARP1- and

Recirrad

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-knockdown cells also exhibited 2-fold increases in theer of γH2AX-positive cells compared with HCC1806l cells, as shown and quantified in Fig. 4B and C. Thesevations suggest that TNBC cells accumulate DNAe due to inhibition of PARP activity or due to inhibitionA synthesis by gemcitabine. Moreover, cell cycle analy-ta not shown) illustrates that PJ34 treatment arrestedn the G2-M phase and gemcitabine induced an S-phasein TNBC cells, consistent with cell cycle arrest afteramage.

cient repair of UV-induced DNA damage afterand gemcitabine treatment

eatment or PARP1 and PARP2 knockdown (bottom). Cells with

3. Increase in Rad51 foci after PARP inhibition or gemcitabine treatment in TNBC cells. A, representative images of Rad51 foci in BT474 andB468 cells treated with either 10 μmol/L PJ34 or 5 nmol/L gemcitabine for 4 h. B, HCC1806 control and PARP1- and PARP2-knockdown cells.

ent studies have shown that PARP is activated by UViation (23, 24); however, the role of PARP after




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uced DNA damage is not clear. Previously, our laboratoryd that human and mouse BRCA1-mutant cells are defec-global genomic repair (GGR) compared with BRCA1pe and luminal breast cancer cells (15). Because TNBCear resemblance to BRCA-mutant cells, we investigatedduced DNA damage repair through the GGR pathwayC cells treated with either PJ34 or gemcitabine and in- and PARP2-knockdown cells.hown in Fig. 5A and B, both MDAMB468 and HCC1806reated with either PJ34 or gemcitabine were efficientairing 6-4PPs (top). However, treatment of cells with
r gemcitabine completely inhibited the repair of CPDs induce
1806 control and HCC1806 shPARP1- and shPARP2-knockdown cells. C, quan-knockdown cells.

r Res; 70(20) October 15, 2010


red the cells inefficient in repairing CPDs with noe in the ability to repair 6-4PPs (Fig. 5C). Therefore,nergistic effect observed in TNBC cells between PJ34isplatin or gemcitabine may be, in part, due to inhibi-f the GGR pathway along with defects in other DNApathways.

of p63 and p73 in sensitizing basal-like breastr cells to PJ34 and gemcitabinedies have suggested that 0% to 30% of invasive ductalcarcinomas express ΔNp63α protein (25–27). p73 can
apoptosis by p53-independent mechanisms, making it
m). Similarly, knockdown of both PARP1 and PARP2 particularly important for therapeutics in basal-like breast

4. DNA damage in cells after PARP inhibition or gemcitabine treatment. A, representative images of γH2AX in BT474, MDAMB468, and HCC1806ated with 10 μmol/L PJ34 or 5 nmol/L gemcitabine for 24 h. Inset, magnified image showing distinct punctate staining. B, γH2AX-positive cells

tification of γH2AX foci in HCC1806 control and PARP1- and

Cancer Research



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omas, which are most often null or mutant for p53. Wethat p63 is significantly overexpressed in TNBC celly analyzing gene expression data from a study pub-by Neve and colleagues (28). Because p63 can act ascogene (25), the correlation between p63 expressionJ34- and gemcitabine-mediated cell death becomesore important.hown in Fig. 6A, PJ34-treated hs568t, MDAMB486, and806 TN cells exhibit a more than 3-fold decrease in thesion of ΔNp63α. ΔNp63α binds to p73 and preventsapoptotic activity. We therefore analyzed the levels ofrotein and observed an increase in the expression ofPJ34-treated cells with a concurrent increase in thesion of p21 (Fig. 6B). A recent study showed thatnd p73, through p21, can repress minichromosomeenance (MCM) proteins (29). Because analysis of thehed microarray data (28) revealed that MCM proteinserexpressed in TNBC cells, we examined the expres-f MCM proteins after PJ34 treatment. As shown in, PJ34 treatment decreased the levels of MCM4 andin TNBC cells. We next treated PARP1- and PARP2-

down cells with gemcitabine for 24 or 48 hours, and

2. Bottom, repair of CPDs.

wn in Fig. 5D, gemcitabine treatment downregulatedotein level of ΔNp63α with a concurrent increase in

volvedstrand

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nd p21. Thus, the results suggest that DNA damagerigger the p63/p73 pathway to induce cell cycle arrestpoptosis.

ssion

discovery of molecular subclasses of breast cancerts that treatments may be targeted more selectivelymproved outcomes. Currently, a major challenge isntify such targets and more effective therapeutic regi-for TNBCs that are not responsive to endocrine ther-r trastuzumab. BRCA1 mutation carriers commonlyp basal-like breast tumors with defects in DNA repairave been shown to have altered sensitivity to certainxic DNA-damaging agents. In the current study, weed whether agents known to selectively target DNA-–deficient BRCA1-mutant cells would also be effectiveBC cells.viously, we have established that BRCA1-mutant cellre more sensitive to cisplatin and gemcitabine thaned BRCA1 wild-type cells (12). Mechanisms to explainbservation include defects in DNA repair pathways in-

5. Decreased NER in TNBC cells after PARP inhibition or gemcitabine treatment. Cells were irradiated with 20 J/m2 UVC (A–C) and then treated with

in HR, NER, and resolution of the intra- and inter-DNA cross-links induced by cisplatin. In our current




study,compathat Tbine, ctive tochemobreastPAR

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Hastak et al.

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we found that TNBC cell lines are sensitive to cisplatinred with luminal cells. We also report for the first timeNBC cell lines exhibit profound sensitivity to gemcita-ompared with the luminal types, which are not sensi-gemcitabine. These novel findings suggest a targetedtherapeutic approach to TN and BRCA1-deficientcancer.P1 and PARP2 are involved in various DNA repairnisms. They bind to the DNA damage sites and acti-hemselves by automodification. Recent studies haveted that decreasing PARP expression by RNA interfer-or by chemical inhibitors sensitizes BRCA1- and2-deficient cells to cell death through synthetic lethal-th their DNA repair defects (16, 17). Our finding thatcells share DNA repair defects with BRCA1-mutant11) suggests that PARP inhibitors may be effectiveting these tumors, as well. In fact, our results showNBC cells are more susceptible to PARP inhibitionhe luminal type of breast cancer cells. Moreover, theirvity to PARP inhibitors is similar to that of BRCA1-t cells.ause PARP plays a major role in the response to DNAe, we also wished to examine whether inhibitor ofacts synergistically with DNA-damaging cytotoxic. Therefore, to explore the effects of drug combinationsast cancer subtypes, we performed isobologram analy-
d found that the combination of PJ34 with gemcitabinelatin had a synergistic effect in TNBC cells. Remark-
foci reand/o

and p21 in hs578t, MDAMB468, and HCC1806 cells treated with PJ34 (B); and M). D, expression of PARP, ΔNp63α, p73, and p21 in HCC1806 and HCC1806 sh

r Res; 70(20) October 15, 2010


however, the combination proved antagonistic in-proficient luminal breast cancer cell lines.cent study (30) showed that PARP1 and PARP2 are re-to reactivate replication at stalled DNA forks. Thus,nergism observed between gemcitabine and PARPtion in TNBC cells may be attributed to stalled repli-forks caused by incorporation of gemcitabine into

ating DNA and failure to reactivate replication atalled fork due to inhibition of PARP activity. Thesehave clinical significance and suggest that a regimenning a platinum agent with gemcitabine and a PARPtor may have unique efficacy in TNBC but may noteffective in other subtypes of breast cancers. Interest-knocking down PARP2 further sensitized TN cells totabine, whereas PARP1-knockdown cells were sensi-o cisplatin. It is therefore possible that PARP1 and2 responded preferentially to DNA damage causedferent DNA-damaging agents, and this observation isfurther investigated.work shows that H2AX is phosphorylated and forms

ct nuclear foci in response to gemcitabine in TNBCes, similar to the effect of other deoxycytidine nucleo-nalogues, such as 1-h-D-arabinofuranosylcytosine anditabine (31). Additionally, consistent with previouss (32, 33), we found that gemcitabine treatment causedumulation of Rad51 nuclear foci. It is not clear if these
sult from gemcitabine-induced stalled replication forksr gemcitabine-induced accumulation of cells in S phase.
6. Modulation of p63, p73, p21, and MCMs in TNBC cells. Western blot analysis of ΔNp63α in MDAMB468 and hs578t cells treated with PJ34
CM4 and MCM7 in MDAMB468 and HCC1806 cells treated withPARP1 and shPARP2 cells treated with 0.6 nmol/L gemcitabine.
Cancer Research



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er, it is possible that stalled replication forks causedcitabine trigger HR repair of chemotherapy-inducedamage.ilarly, inhibition of PARP either by drugs or by RNAid to an increase in γH2AX foci and Rad51 foci, con-t with the idea that loss of PARP might increase thetion of DNA strand breaks that are repaired by HR). Recently, we (11) have shown that TNBC cells areive in BER; moreover, these cells may also be defective. Thus, inhibition of PARP along with defective DNAmechanisms may lead to synthetic lethality.ough UV-induced activation of PARP has been re-, the possible role of PARP in NER has not receivedattention. Studies have shown that repair of 8-oxoGulated by XPC (36) and CSB (37, 38), and becauseoratory has shown that BRCA1-mutant cells are defec-the GGR pathway of NER, but not in transcription-d repair (15) and BER (11), we investigated the roleP in GGR. We showed that PARP inhibition chemicallyRNAi decreased the capacity of TNBC cells to removenduced CPD lesions, a result consistent with previouss of PARP playing a role in GGR (39, 40). NER is knownnvolved in platinum-DNA adduct repair. Therefore, theism observed with PARP inhibition and cisplatin maytly due to inhibition of NER. However, whether the re-facilitated by the transcription-coupled repair path-f NER is not established by this study, and furtherigation into the mechanism is under way.erstanding the molecular mechanism behind drugent is critical to predict the clinical efficacy of treat-Meta-analyses of published microarray data (28) forexpression changes common to the basal-like and1-mutated cell lines identified p63 and MCM family
ers to be significantly overexpressed (P < 0.05) in both Rece
omycin-C resistance, and chromosome stability is restored withrrection of a Brca1 mutation. Cancer Res 2001;61:4842–50.attacharyya A, Ear US, Koller BH, Weichselbaum RR, Bishop DK.

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r gemcitabine resulted in a decreased expression of3α with a concurrent increase in the expression ofnd the cyclin-dependent kinase inhibitor p21, which,, repressed the MCM proteins (41). MCMs are requiredensing of origins, providing a signal for initiating repli-in S phase, and are frequently overexpressed in cancer42–44). Our data showed that MCM4/MCM7 are down-ted in TNBC cells treated with PJ34. Based on thesef evidence, we hypothesize that PJ34 treatment leadsnregulation of ΔNp63α with a concurrent increase inpression of p73 and p21, which, in turn, decreases thesion of MCM4/MCM7, leading to cell cycle arrest andath. Overall, we found that human TNBC cells, similarCA1-deficient cell lines, were more sensitive to PJ34,tabine, and cisplatin and exhibited synergistic re-es to combinations of these agents. This sensitivityto be dependent on inefficient DNA repair mechan-causing sustained DNA damage that may trigger the

t novel options for targeted treatment of TNBCs.

otential conflicts of interest were disclosed.

Support

grant R01 CA108794, the Breast Cancer Research Foundation (J.M. Ford),an G. Komen for the Cure Postdoctoral Fellowships (K. Hastak and

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

ived 12/21/2009; revised 07/12/2010; accepted 07/27/2010; published
s. Our study showed that tre atment of TNBC cells with OnlineFirst 08/26/2010.
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Cancer Research



2010;70:7970-7980. Published OnlineFirst August 26, 2010.Cancer Res Kedar Hastak, Elizabeth Alli and James M. Ford Gemcitabine, and CisplatinCell Lines to Poly(ADP-Ribose) Polymerase Inhibition, Synergistic Chemosensitivity of Triple-Negative Breast Cancer

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