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Therapeutics, Targets, and Chemical Biology

Polo-like Kinase 1: A Potential Therapeutic Option inCombination with Conventional Chemotherapy for theManagement of Patients with Triple-Negative Breast Cancer

Virginie Maire1,3,4, Fariba N�emati1,3,5, Marion Richardson1,3,4,6, Anne Vincent-Salomon1,6,7,Bruno Tesson1,3,4,8,9, Guillem Rigaill1,2,3,4,8,9, El�eonore Gravier1,3,8,9,10, B�ereng�ere Marty-Prouvost1,3,4,Leanne DeKoning1,3,4,12, Guillaume Lang1,3,5, DavidGentien1,3,13, Aur�elie Dumont1,3,4, Emmanuel Barillot1,8,9,Elisabetta Marangoni1,3,5, Didier Decaudin1,3,5,11, Sergio Roman-Roman1,3, Alain Pierr�e14,Francisco Cruzalegui14, St�ephane Depil14, Gordon C. Tucker14, and Thierry Dubois1,3,4

AbstractBreast cancers are composed ofmolecularly distinct subtypeswith different clinical outcomes and responses to

therapy. To discover potential therapeutic targets for the poor prognosis-associated triple-negative breast cancer(TNBC), gene expression profiling was carried out on a cohort of 130 breast cancer samples. Polo-like kinase 1(PLK1) was found to be significantly overexpressed in TNBC compared with the other breast cancer subtypes.High PLK1 expressionwas confirmed by reverse phase protein and tissuemicroarrays. In triple-negative cell lines,RNAi-mediated PLK1 depletion or inhibition of PLK1 activity with a small molecule (BI-2536) induced an increasein phosphorylated H2AX, G2–M arrest, and apoptosis. A soft-agar colony assay showed that PLK1 silencingimpaired clonogenic potential of TNBC cell lines. When cells were grown in extracellular matrix gels (Matrigel),and exposed to BI-2536, apoptosis was observed specifically in TNBC cancerous cells, and not in a normal cell line.When administrated as a single agent, the PLK1 inhibitor significantly impaired tumor growth in vivo intwo xenografts models established from biopsies of patients with TNBC. Most importantly, the administration ofBI-2536, in combination with doxorubicin þ cyclophosphamide chemotherapy, led to a faster completeresponse compared with the chemotherapy treatment alone and prevented relapse, which is the major riskassociated with TNBC. Altogether, our observations suggest PLK1 inhibition as an attractive therapeuticapproach, in association with conventional chemotherapy, for the management of patients with TNBC. CancerRes; 73(2); 813–23. �2012 AACR.

IntroductionBreast cancer is a heterogeneous disease with different

subgroups characterized by specific clinical outcomes andresponses to therapy (1). Triple-negative breast cancers(TNBC) are characterized by a lack of expression of estrogenand progesterone receptors (ER/PR) and a lack of HER2 over-

expression (2). TNBCs account for 15% of breast cancers andare associated with African-American ethnicity, younger age,and poorer outcome when compared with the other breast can-cer subtypes: luminal A (LA), luminal B (LB), and HER2þ/ER�(HER2; ref. 2). TNBC show a high rate of TP53mutation (3), areassociated with a "BRCAness" phenotype (4), and are highlyproliferative, genetically unstable, poorly differentiated, andoften grade 3 carcinomas (5). In contrast to HER2 and luminalcarcinomas, which can be treated with targeted therapy suchas anti-HER2 monoclonal antibodies or hormonal therapies,respectively, there is no available targeted therapy for patientswith TNBC who are managed exclusively with conventionalchemotherapy. Although they show high rates of objectiveinitial response, the majority of patients do not display acomplete response and have a poorer prognosis than thosewithin other breast tumor subgroups due to high rates ofrecurrence (6). Because of this unfavorable prognosis and thelack of targeted therapy, there is currently an intensive searchfor identifying therapeutic targets for TNBC (7).

Polo-like kinase 1 (PLK1) is the best-characterized memberof the human PLK family (PLK1-5) of serine/threonine proteinkinases, which is involved in various functions such as mitoticentry, spindle assembly, and DNA damage response (8). In

Authors' Affiliations: 1Institut Curie, Centre de Recherche; 2AgroParis-Tech/INRA UMR MIA 518, Paris; 3Translational Research Department;4Breast Cancer Biology Group; 5Laboratory of Preclinical Investigation;6Tumor Biology Department, Institut Curie Hospital; 7INSERM U830;8INSERM U900; 9Mines ParisTech, Fontainebleau; 10Departments of Bio-statistics and 11Medical Oncology; 12Reverse Phase Protein Array Plat-form; 13Platform of Molecular Biology Facilities; and 14Oncology Researchand Development Unit, Institut de Recherches SERVIER, Croissy-sur-Seine, France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Thierry Dubois, D�epartement de RechercheTranslationnelle, Equipe "Biologie du Cancer du Sein", Centre deRecherche, Institut Curie, 26 rue d'Ulm, Paris 75248, France. Phone:0033-0153-197416; Fax: 0033-0153-194130; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-2633

�2012 American Association for Cancer Research.

CancerResearch

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on June 23, 2021. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 9, 2012; DOI: 10.1158/0008-5472.CAN-12-2633

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Figure 1. PLK1 is overexpressed in TNBC. PLK1 expression was measured in TNBC, HER2, LA, LB, and healthy breast tissues. A, mRNA PLK1 levels weredetermined by microarray analysis. PLK1 protein level was evaluated by reverse phase protein array (B) and immunohistochemistry (C). Protein andmRNA relative quantifications were logarithmic transformed and illustrated by box plots. C and D, PLK1 expression was analyzed on tissue microarray. Thegraph represents for each subtype the percentage of samples having more than a given percentage of stained cells (C). PLK1 displayed a cytoplasmic and a

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Cancer Res; 73(2) January 15, 2013 Cancer Research814




normal tissues, PLK1 is only found in actively proliferativetissues. PLK1 is overexpressed in diverse tumors includingbreast cancer, and is associated with poor prognosis (9–13).High PLK1 levels have been found in TNBC (9, 14), and PLK1has recently been proposed to be a potential therapeutic targetfor TNBC based on a protein kinase siRNA library screen and invitro assays (15). Besides proliferation, the overexpression ofPLK1 in mouse NIH3T3 fibroblasts transforms the cells, mak-ing them capable to form tumors when injected in mice (16).PLK1 depletion/inhibition triggers apoptosis in various cancercell lines, and impairs tumor growth in mice (17). PLK1depletion/inhibition preferentially kills cancer cells comparedwith normal cells (18–20) and p53-defective cancer cells com-pared with p53 wild-type cancer cells (21–23). Several PLK1inhibitors have been developed; some of them, such as BI-2536,have been evaluated in clinical trials (8, 24–27).We conducted RNA microarrays on a cohort composed of

tumor samples, healthy breast tissues, and triple-negative celllines. PLK1was overexpressed in TNBC comparedwith healthytissues and to the other breast cancer subtypes. We examinedthe effects of PLK1 depletion (RNAi) and inhibition (BI-2536)on the behavior of triple-negative cell lines and in humanTNBC-derived xenograft models. In vitro or in vivo, theseexperiments showed a beneficial effect of targeting PLK1 inTNBC.

Materials and MethodsHuman samples, clinical, and microarray dataOur cohort was composed of 35 LA, 40 LB, 46 TNBC, 33

HER2, 18 normal breast tissues, and 14 TNBC cell lines.Experiments were conducted in agreement with the BioethicLaw No. 2004–800 and the Ethic Charter from the FrenchNational Institute of Cancer (INCa), and after approval of theethics committee of our Institution. Immunohistochemistrywith PLK1 antibodies (Cell Signaling Technology) was con-ducted as described (28). Clinical data were collected anddisease-free interval was defined as the time from the diagnosisof breast cancer to the occurrence of a locoregional, distant, orcontrolateral recurrence. DNA (Affymetrix SNP6.0), RNA (Affy-metrix U133 plus 2.0), and RPPA microarrays were conductedas described (28).

Cell culture, RNA interference, PLK1 inhibitor, andcellular assaysCell lines were purchased in 2006 and 2008 from the Amer-

ican Type Culture Collection, and cultured as described (28).The cell lines have been characterized by DNA and RNAmicroarrays (year 2009). Cells were transfected with 20nmol/L of either a control RNAi (AllStars) or 2 distinct PLK1RNAi (PLK1#6, PLK1#7; all from Qiagen), or incubated with aPLK1 inhibitor (BI-2536; Selleck Chemicals). Cell proliferation

was determined by MTT (Sigma). Caspase 3/7 activity wasdetermined by the Caspase-Glo 3/7 luminescent assay (Pro-mega) or by Western blot analysis as described (28). Cell-cycleanalysis was carried out with FACScalibur (Becton Dickinson)using Cellquest software (Becton Dickinson) to determinecellular DNA content. For soft-agar colony formation, 0.35%agar containing RNAi-transfected MDA-MB-468 and HCC70cells was overlaid onto precast 0.5% bottom agar and incu-bated for 4 weeks. Colonies were visualized afterMTT staining.Three-dimensional (3D) cell culture was conducted withMatrigel (BD Biosciences) as described (29). Briefly, MDA-MB-468 and MCF10A were grown in the 3D on-top assay for7 and 10 days, respectively. Cells were then treated with BI-2536, and cell viability was determined 3 days later using theWST1 Cell Proliferation Assay Kit (Millipore).

Mice, compounds, treatment, and tumor growthmeasurement

Mice were purchased from Charles River. Their care andhousing were conformed to the institutional guidelines as putforth by the French Ethical Committee. Human TNBC breastcancer xenograft models (HBCx-10 and HBCx-24) were estab-lished as previously detailed (30–32). HBCx-10 represents axenograft model of doxorubicin þ cyclophosphamide (DC)-induced complete remission and tumor recurrence (30, 31),whereas HBCx-24 does not respond to DC (not shown). Mice-bearing human breast cancer xenografts were treated intra-peritoneally with BI-2536 (Selleck Chemicals, 20mg/kg) twice aweek alone or combined with doxorubicin (Teva Pharmaceu-ticals, 2 mg/kg) and cyclophosphamide (Baxter, 100 mg/kg) atdays 1, 22, and 43. Tumor growth was evaluated with a calipertwice a week.

Statistical analysisTheR softwarewas used for statistical analyses (33). Pearson

correlation was used to estimate an association between 2variables. For cellular assays and in vivo experiments, P valueswere calculated using the Student t test.

Additional experimental details are available in the Supple-mental Material.

ResultsPLK1 is overexpressed in triple-negative breast cancer

To discover potential therapeutic targets for the poor prog-nosis-associated TNBC, a gene expression profilingwas carriedout on a cohort of 130 breast cancers. PLK1 was expressed athigher levels in TNBC (mean log2¼ 4.36) compared with HER2(mean log2¼ 2.6;P¼ 2.8� 10�10), LA (mean log2¼ 1.56; P< 2�10�16), LB (mean log2 ¼ 2.16; P ¼ 7.6 � 10�13), and normaltissues (mean log2¼ 1.97; P¼ 1.1� 10�9; Fig. 1A). PLK1 proteinlevels, measured by reverse phase protein array, were higher in

nuclear localization. Scale bar, 30 mm (D). E, PLK1 analysis at the DNA level. The smoothed segmented copy number (CN) signal is presented in boxplots, withdashed lines indicating the thresholds retained to call CN gains and losses. F, correlation between PLK1 mRNA and Ki67 mRNA within the TNBCsubtype. Each tumor is represented by a solid circle. Kaplan–Meier curves of metastasis-free interval (G) and disease-free interval (H) for TNBC divided intotertiles according to their PLK1 expression by comparing the combined 2 top tertiles (>3.94) with the lowest tertile (�3.94) of PLK1mRNA expression. Being inthe lowest tertile was associated with a higher risk of developing metastasis (HR ¼ 4.05, 95%CI ¼ 1.17–14.05, P ¼ 0.028, log-rank test) and disease(HR ¼ 3.41, 95% CI ¼ 1.03–11.26, P ¼ 0.045, log-rank test).

PLK1 as a Therapeutic Target in Triple-Negative Breast Cancer

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TNBC compared with HER2 (P ¼ 2.2 � 10�5), LA (P ¼ 8.1 �10�8), and LB (P ¼ 4.5 � 10�8; Fig. 1B). These results wereconfirmed by immunohistochemistry showing a higher per-centage of cells expressing PLK1 in TNBCcomparedwithHER2(P ¼ 0.016), LA (P ¼ 2.5 � 10�5), and LB (P ¼ 0.001; Fig. 1C).PLK1 staining was observed both in the cytoplasm and thenucleus inTNBCsamples, andnot in healthy breast tissues (Fig.1D). The higher expression of PLK1 in TNBCdid not result froman increase in PLK1 DNA copy number (Fig. 1E). PLK1 mRNAlevels correlated positively with the proliferation marker Ki67mRNAwithin the TNBC subgroup (Pearson correlation¼ 0.45;

P ¼ 0.003; Fig. 1F). PLK2 was found to be expressed at lowerlevels in TNBCcomparedwith the other breast cancer subtypes(HER2: P¼ 8.9� 10�9, LA: P¼ 1.0� 10�11, LB: P¼ 6.6� 10�7)and normal tissues (P ¼ 2.0 � 10�4; Supplementary Fig. S1A),and this was associated with a loss of PLK2 DNA copy number(Supplementary Fig. S1B). PLK3 gene expression was notsignificantly different between the different groups (Supple-mentary Fig. S1C and D). PLK4 presented a pattern of expres-sion similar to that of PLK1, with higher expression in TNBCcompared with HER2 (P¼ 1.6� 10�5), LA (P¼ 6.0� 10�6), LB(P ¼ 1.2 � 10�3), and normal breast tissues (P ¼ 4.8 � 10�3;

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Figure 2. PLK1 depletion impairstriple-negative cell viability andaffects cell cycle. A, PLK1expression was analyzed byWestern blot analysis in BT20 (1),HCC38 (2), HCC1937 (3), HCC70(4), MDA-MB-468 (5), MDA-MB-231 (6), and MCF10A (7). Actin wasused as a control. B–G, cell lineswere transfected with control(black bars) or 2 different PLK1RNAi (#6 and #7) MDA-MB-468and HCC70 (B) were grown on softagar medium for 4 weeks. Colonieswere stained and counted. C–E,cell viability was assessed by MTTassay 24, 48, and 72 hours aftertransfection (C and D) or only72-hour posttransfection (E). B–E,data are mean� SD from at least 3independent experiments.P valuesare indicated (�, P < 0.05;��,P < 0.01; ��,P < 0.001). F andG,cell-cycle analysis. MDA-MB-468(F) andMCF10A (G)were incubatedwith propidium iodide 24 or 48 hourposttransfection. Flow cytometrywas carried out to determinecellular DNA content. Cells (%)within the different cell-cyclephases are presented. F and G, thedata are from a single experimentdone twice with similar results.

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Supplementary Fig. S1E and F). PLK5 was not detected inour study. In conclusion, transcriptomic data showed thatTNBC overexpressed both PLK1 and PLK4, and underex-pressed PLK2. These results suggest that PLK1 and PLK4may represent attractive therapeutic targets for this sub-group of breast cancer. We then focused on PLK1 becauseseveral PLK1 inhibitors have been evaluated in clinical trials(8, 27, 34, 35).

The lowest tertile of PLK1 expression is associated withpoor clinical outcome in triple-negative breast cancerWe investigated the relationship between PLK1 expression

and prognosis within our TNBC subcohort. As there was noconsensual prognostic threshold, patients with TNBC wereinitially divided into tertiles based on their PLK1 mRNAexpression levels. As the second and third top tertiles wereprognostically favorable (not shown), they were combined and

considered as high for PLK1 expression (PLK1 >3.94) andcompared with the low tertile (PLK1 �3.94). Being in thelowest tertile was significantly associated with a higher riskof developing metastasis (Fig. 1G) and disease (Fig. 1H) thanbeing in the 2 highest tertiles.

RNAi-mediated PLK1 depletion impairs clonogenicityand viability in triple-negative cell lines

PLK1 expression was analyzed in a panel of triple-negativecell lines including the "normal" MCF10A cells (Fig. 2A). PLK1silencing using 2 different RNAi significantly impaired HCC70andMDA-MB-468 to form colonies in a soft agar assay (Fig. 2B),indicating that PLK1 was required for anchorage-independentgrowth. Proliferation assays upon RNAi-mediated PLK1 deple-tionwere then conducted.MDA-MB-468 viabilitywas impairedafter RNAi transfection (Fig. 2C), andmore pronounced effectswere observed in MCF10A (Fig. 2D). PLK1-depletion inhibited

Figure 3. RNAi-mediated PLK1depletion induces apoptosis in triple-negative cell lines. MDA-MB-468 (A,C, and E) and MCF10A (B, D, and F)were transfected with control (ctrl)and PLK1 RNAi (#6 and #7). A and B,Annexin V binding assay. Percentageof viable, necrotic, and apoptoticcells were measured 48-hourposttransfection. C and D, caspase3/7 activity assay. E and F, Westernblot analysis was conducted withantibodies recognizing H2AX,phosphorylated H2AX, and thecleaved form of PARP, caspase 3,and caspase 7. PLK1 depletion wasverified using PLK1 antibodies. Actinwas used as a loading control. A–D,data are mean � SD from at least 3independent experiments. E and F,results are from a single experiment,representative of 3 (MDA-MB-468) or2 (MCF10A) different experiments.

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the proliferation of all tested TNBC cell lines (Fig. 2E). Our datawithMCF10Awere in contrast with those suggesting that these"normal" cells were less sensitive to PLK1 depletion (18, 19).However, MCF10A cells were the cells growing the fastest witha doubling time in 96-well plates as follows: MCF10A (16hours), MDA-MB-231 (25 hours), MDA-MB-468 (40 hours),HCC1937 (40 hours), HCC70 (50 hours), and BT20 (60 hours;not shown). Therefore, this result is not surprising, consideringthe function of PLK1 in mitosis.

RNAi-mediated PLK1 depletion induces DNA breaks,G2–M arrest, and apoptosis in triple-negative cell lines

We next analyzed by fluorescence-activated cell sorting(FACS) the effect of PLK1 depletion on cell-cycle progression.After RNAi transfection, MDA-MB-468 and MCF10A arrestedin G2–M, and the percentage of cells with sub-G1 DNA contentincreased suggesting that PLK1-depleted cells undergo apo-ptosis (Fig. 2F and G). Annexin V staining experiments showedthat PLK1 silencing induced apoptosis in MDA-MB-468 andMCF10A (30%–40%; Fig. 3A and B). Caspases 3/7 were acti-vated in both cell lines after PLK1 RNAi transfection (Fig. 3Cand D), and this was confirmed by Western blot analysis withthe appearance of their cleaved forms, and the cleaved form oftheir substrate Poly(ADP-ribose)polymerase (PARP; Fig. 3Eand F). PLK1 depletion induced H2AX phosphorylation,reflecting the presence of DNA breaks (Fig. 3E and F).

BI-2536–mediated PLK1 inhibition induces apoptosisand impairs viability in triple-negative cell lines

We next analyzed the effect of BI-2536, a PLK1 inhibitor, onthe behavior of triple-negative cell lines. BI-2536 impairedcell viability in the nanomolar range (Table 1). In contrast tothe results obtained after RNAi-mediated PLK1 depletion,MCF10A cells were less sensitive to PLK1 inhibition comparedwith the other cell lines. BI-2536 treatment of MDA-MB-468and MCF10A induced apoptosis in a dose-dependent manner(Fig. 4A and B). Caspase 3/7 activity was detected at 10 nmol/LBI-2536 in both cell lines, and at 5 nmol/L only inMDA-MB-468(Fig. 4A and B). Western blot analysis confirmed these results,with the detection of cleaved caspases 3, 7, and 8 and PARP

after cell treatment with 10 nmol/L BI-2536 (Fig. 4C and D).Again, 5 nmol/L BI-2536 induced the cleavage of PARP andcaspases only in MDA-MB-468 (Fig. 4C and D). Phosphory-lated H2AX was induced by BI-2536 at 5 and 10 nmol/L inMDA-MB-468, but only at 10 nmol/L in MCF10A (Fig. 4C andD). Overall, these results indicated that BI-2536 inducedapoptosis at lower concentrations in MDA-MB-468 comparedwith MCF10A.

BI-2536–mediated PLK1 inhibition specifically affectsmalignant cells in three-dimensional culture

BI-2536 was tested in triple-negative cell lines grown in amore physiologic context, in 3D culture in Matrigel (29).Under these conditions, nontransformed mammary epithe-lial cell lines, such as MCF10A, recapitulate epithelial mor-phogenesis by forming acinar structures within 10 days ofculture, and then stop to grow, whereas cancer cells, such asMDA-MB-468, exhibit disorganized structures and continueto proliferate (36). BI-2536, added to these structures onceformed, was effective on MDA-MB-468, and had no effect onMCF10A (Fig. 4E). This may be explained by the fact thatMCF10A does not express PLK1 once the acini are formed(Fig. 4F).

BI-2536–mediated PLK1 inhibition impairs tumorgrowth inhuman triple-negative breast cancer xenograftmodels

To evaluate the antitumoral effects of PLK1 inhibition invivo, BI-2536 was administrated in 2 breast cancer xenograftmodels established from patients with TNBC (30, 32). BI-2536,administrated alone, induced a dramatic tumor growth inhi-bition (TGI) in bothmodels (P < 0.0001) with a TGI of 99% (Fig.5A) and 85% (Fig. 5B). No body weight loss was observed (notshown). Three of 8 (37.5%) mice showed a complete response(CR) at day 24 in the HBCx-24 model (not shown). No CR wasobserved with the HBCx-10 model, and tumor relapsed afterBI-2536 withdrawal (Fig. 5B and Table 2).

BI-2536 in combination with chemotherapy impairstumor relapse in a human triple-negative breast cancer–derived xenograft model of tumor recurrence

We next addressed whether inhibition of PLK1 could impairtumor relapse after conventional chemotherapy, a major issuefor patients with TNBC. We had the opportunity to test in vivothis hypothesis using the HBCx-10 model, which represents amodel of chemotherapy-induced complete remission andtumor recurrence (31). A combination of DC, used for themanagement of TNBC patients, led first to CR; however, thetumor relapsed after stopping DC treatment (Fig. 5B, Table 2).The combination of DC with BI-2536 induced 100% CR, whichwere observed earlier than CR induced by DC treatment alone(Fig. 5B and Table 2). BI-2536 þ DC combination impairedtumor relapse in contrast to DC alone (Table 2). At day 200, 4mice were still tumor-free and could be considered as cured(Table 2). Altogether, these data suggested that, in vivo, PLK1inhibition in combination with conventional chemotherapy ismore efficient to achieve CR and, most importantly, impairedtumor relapse.

Table 1. BI-2536 impairs triple-negative cellviability

BI-2536 [IC50] ¼ nmol/LMCF10A 7.3 � 1.9MDA-MB-231 3.5 � 0.9MDA-MB-468 2.7 � 0.8HCC70 2.0 � 0.2HCC1937 1.7 � 1.0BT20 1.4 � 0.4NOTE: Cell lines were treated with various BI-2536 concen-trations and cell viability wasmeasured using anMTT assay.Half maximal inhibitory concentrations (IC50) were deter-mined from dose–response curves.

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DiscussionTreatment of patients with TNBC remains amajor challenge

for oncologists. Although they respond well to the currenttherapeutic strategies based on conventional chemotherapies,they represent a large proportion of breast cancer deathbecause of a high recurrence rate. Alternative treatments aretherefore needed to improve survival of these patients.

We found that both PLK1 and PLK4 are overexpressed inTNBC, and may represent potential therapeutic targets. Wefocused on PLK1 as small-molecule inhibitors have beenevaluated in clinical trials. We confirmed previous analysisshowing that PLK1 belongs to a list of 16 kinases overexpressedin TNBC compared with LA (14). We validated this TNBC-specific high PLK1 expression at a protein level using RPPA andimmunohistochemistry techniques. High PLK1 protein expres-sion has been reported in breast cancers (10, 13), specifically inTNBC (9), and shown to be associated with poor prognosis (9).We also found that high PLK1 expression is associated with

Table 2. In vivo efficacy of BI-2536 alone orcombined with DC in HBCx-10

CR, %

Treatment(CR mice/total mice)

Number of mice Day 33 Day 120 Day 200

Control 0% — —(n ¼ 9) (0/9)BI-2536 0% 0% —(n ¼ 9) (0/9) (0/7)DC 29% 0% —(n ¼ 7) (2/7) (0/6)DC þ BI-2536 100% 100% 100%(n ¼ 9) (9/9) (5/5) (4/4)NOTE: For abbreviations and legend, see Fig. 5B.

Figure 4. BI-2536–mediated PLK1inhibition induces apoptosis in triple-negative cell lines, and specificallyaffects malignant cells in 3D culture.MDA-MB-468 (A and C) andMCF10A (B and D) were treated withdimethyl sulfoxide (DMSO) or variousconcentrations of BI-2536 (1, 5, and10 nmol/L). Apoptosis was evaluatedby caspase 3/7 assay (A and B) orWestern blot analysis as in Fig. 3Eand F (C and D). A and B, data aremean � SD from at least 3independent experiments. C and D,results are from a single experiment,representative of 3 differentexperiments (apart for 5 nmol/L BI-2536: experiment done twice). E andF, MDA-MB-468 cells were grown inMatrigel for 7 days to form grape-likedisorganized structures andMCF10A for 10 days to form aciniround 3D well-organized structures.Cells were then treated with variousconcentrations of BI-2536 (orDMSO). Three days later, cells wereanalyzed directly in plate (E) orcollected for Western blot analysis(F). Cell viability was determinedusing a WST1 Cell ProliferationAssay Kit (E). Results are presentedas % cell growth compared withDMSO-treated cells. The data aremean � SD from 3 independentexperiments Western blot analysis(F), PLK1 expression in MDA-MB-468 (468) andMCF10A (10A) culturedin 3D (Matrigel) or 2D (plastic). Actinwas used as a loading control. Datashown are representative of 2different experiments.

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poor prognosis within the entire population; this could besimply reflecting the high expression of PLK1 in the poorprognosis-associated TNBC subtype. In fact, we found that,within the TNBC subtype, high PLK1 expression was associ-ated with a better prognosis. This may be explained by the factthat low PLK1-expressing tumors respond worse to conven-tional chemotherapy, possibly due to their lowest proliferativerates.

PLK1 is the most investigated PLK family member and hasbeen pointed out as an oncology target due to its overexpres-sion in several tumors (12, 17). Therefore, many studies haveanalyzed the effects of PLK1 inhibition/depletion in variouscancer cells (17). In agreement with those studies, we observedthat PLK1 depletion/inhibition had similar effects in triple-negative cell lines: increase of DNA breaks, G2–M arrest, andapoptosis as well as inhibition of cell viability and tumorige-nicity. We found that RNAi-mediated PLK1 silencing and BI-2536-mediated PLK1 inhibition promoted similar cellulareffects. Nevertheless, we noticed some differences. When com-pared with TNBC cell lines, the "normal" MCF10A were mostsensitive to PLK1 silencing (RNAi) but less sensitive to PLK1inhibition (BI-2536). BI-2536 inhibits the activity of all PLKs,

suggesting that the inhibition of several PLKs, comparedwith aspecific silencing of PLK1, could differentially affect cancercells. On the other hand, knocking down the expression mayhave different consequences compared with the inhibition ofthe kinase activity. Inhibiting the activity of PLK1may bemorespecific to cancer cells than knocking down its expression,which abolishes not only its kinase activity but also interac-tions with its cellular partners. PLK1 is essential for mitosisentry and it is not surprising that its inhibition/depletionimpairs viability of cultured cells. However, it is interestingto point out that the consequences of PLK1 knockdown are notonly related to the proliferative status of cells. Indeed, BT-20,the cancer cells of our panel with the lowest proliferative rateare the most sensitive to PLK1 inhibition and silencing. Thesecells may have some characteristics that render them moresensitive to PLK1 inhibition. The depletion of the tumorsuppressor PTEN (phosphatase and tensin homolog deletedon chromosome 10) induces an elevated expression of PLK1 inprostate cancer cells, and PTEN-null prostate cells are moresensitive to PLK1 inhibition compared with PTEN wt prostatecells (37). PTEN loss, which is a hallmark of TNBC (28, 38, 39),may therefore confer a higher sensitivity to PLK1 inhibition to

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Figure 5. BI-2536 in combinationwith chemotherapy impairs tumorrelapse in TNBC-derived xenograftmodel. A, BI-2536 (20 mg/kg) wasinjected intraperitoneally in a TNBCxenograft mice model (HBCx-24)twice a week until day 24 (n ¼ 8mice). Control group receiveddrug-formulating vehicle only(n ¼ 8 mice). B, BI-2536 wasadministrated intraperitoneally inanother TNBC xenograft micemodel (HBCx-10), alone at20 mg/kg (BI-2536), or incombination with DC (2 mg/kgdoxorubicin þ 100 mg/kgcyclophosphamide; DCþBI-2536)until day 75, and stopped becauseof 4 unexpected and unexplaineddeaths in the combined group(absence of body-weight changesbetween groups, not shown). DCwas administered at day 1, 22, and43. Control mice were treated withBI-2536 vehicle. BI-2536 (n ¼ 9mice), DC (n ¼ 7 mice), DC þ BI-2536 (n ¼ 9 mice), and control(n ¼ 9 mice). Tumor volume wasmeasured with calipers. Growthcurves were obtained by plottingrelative tumor volumemean versustime.

Maire et al.





this subgroup of breast cancer; however, this feature is sharedby triple-negative cell lines (28) and could not account for thehigh sensitivity of BT20 [which in fact express low PTEN levelscompared with the other triple-negative cell lines with nodetectable PTEN (28)] to PLK1 inhibition. PLK1 has beenfound to form a synthetic lethal pair with mutated Ras (40),but Ras is not mutated in human breast cancer and in thetriple-negative cell lines we analyzed in this article, with theexception of MDA-MB-231 (41, 42).Antitumoral effects of BI-2536 have been observed in vivo in

xenograft models derived from various cell lines (25). In morerelevant preclinical models established from human TNBC, weobserved that BI-2536, administrated alone, impaired tumorgrowth. However, we noticed that the tumor relapsed after thetreatmentwas stopped.Wehad the opportunity to evaluate theeffect of BI-2536 on tumor recurrence, the major issue forpatients with TNBC, by using a xenograft model characterizedwith high sensitivity to anthracycline-based chemotherapyfollowed by tumor relapse (31). In combination with DC usedin clinic for the management of patients with TNBC, BI-2536led to a faster CR compared with DC alone, and most impor-tantly impaired relapse after the treatments were stopped.Although BI-2536 has been shown to be well tolerated (25),some mice died during the combination treatment forunknown reasons. However, no relapse was observed still200 days after the beginning of the experiment for the survivingmice. Further experiments should be carried out to analyzewhether impaired relapse could still be observed at lower dosesof BI-2536, to avoid toxicity, in combination with DC. Our invivo results suggest that the BI-2536 þ DC association wascapable to eradicate all the cancer cells. The tumor-initiatingcells, which are resilient to chemotherapy and radiation, arethought to be responsible for tumor relapse. Recently, RNAiand small-molecule kinase inhibitors screens identified PLK1as a protein kinase that targets the tumor-initiating cells frombreast cancer (15) and neuroblastoma (43).Because of the high sequence homology between the dif-

ferent PLK, PLK1 inhibitors are not specific to PLK1 (44).Therefore, this may be a concern as these kinases seem tohave different expression patterns and nonoverlapping func-tions; the lack of selectivity of PLK1 inhibitors may result inundesired effects (8, 17). Indeed, PLK2 may act as a tumorsuppressor in vivo (17), and we show that the PLK2 gene isspecifically lost in TNBC. The inhibitor used in our study, BI-2536, inhibits PLK1 (IC50 ¼ 0.83 nmol/L), PLK2 (IC50 ¼ 3.5nmol/L), and PLK3 (IC50 ¼ 9.0 nmol/L; 25). New compoundstargeting specifically PLK1 within the PLK family may avoidthe undesired effects of the nonselective PLK1 inhibitors.Several PLK1 inhibitors have been evaluated in phase I/II

clinical trials (26, 27, 34, 35). BI-2536 phase I trials revealed thatthe drug was well tolerated with minor antitumor responses(27, 45). Two phase II trials have shown that BI-2536 mono-

therapy had limited antitumor activity (46, 47). Only 14 patientswith breast cancers were enrolled in one of the trials with noreported information regarding their molecular subtypes (46).Patients with TNBC have increased pathologic CR rates com-pared with non-TNBC, and those with pathologic completeresponse have excellent survival. However, patients with resid-ual disease after neoadjuvant chemotherapy have significantlyworse survival (6). Although clinical trialswith a PLK1 inhibitorused as a single agent did not show high antitumor activity indifferent types of cancers, we suggest that PLK1may representa promising therapeutic target in combination with conven-tional chemotherapy, in particular for the treatment of patientswith TNBC. Indeed, TNBC express high levels of PLK1, and theassociation of a PLK1 inhibitor with a conventional chemo-therapy treatment impairs tumor relapse in vivo in a recur-rence-prone TNBC xenograft model.

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

Authors' ContributionsConception and design: F. N�emati, A. Vincent-Salomon, D. Decaudin, A. Pierr�e,S. Depil, G.C. Tucker, T. DuboisDevelopment of methodology: V. Maire, F. N�emati, M. Richardson, T. DuboisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Maire, F. N�emati, M. Richardson, A. Vincent-Salomon, B. Marty-Prouvost, L. de Koning, G. Lang, D. Gentien, E. Marangoni,T. DuboisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Maire, F. N�emati, A. Vincent-Salomon, B. Tesson,G. Rigaill, E. Gravier, B. Marty-Prouvost, L. de Koning, D. Decaudin, G.C. Tucker,T. DuboisWriting, review, and/or revision of the manuscript: V. Maire, F. N�emati, A.Vincent-Salomon, G. Rigaill, E. Gravier, L. de Koning, G. Lang, A. Dumont, E.Marangoni, D. Decaudin, S. Roman-Roman, A. Pierr�e, F. Cruzalegui, S. Depil, G.C.Tucker, T. DuboisAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V. Maire, M. Richardson, E. Gravier, D.Gentien, A. Dumont, T. DuboisStudy supervision: F. N�emati, E. Barillot, S. Roman-Roman, A. Pierr�e, S. Depil, T.Dubois

AcknowledgmentsThe authors thank Martine Yann and Dr. Bernard Asselain for the clinical

data; Dr. Marika Pla and colleagues for animal facilities support; Dr. XavierSastre-Garau, his colleagues and patients for the human breast tumor samples;Dr. Fabien Reyal for normal breast tissues; Benoit Albaud, Caroline Hego, andCecile Reyes from the platform of Molecular Biology Facilities; and Am�elieBrisson and Drs. C�eline Baldeyron and Sylvie Maubant for critical reading of thismanuscript.

Grant SupportThis work was supported by Institut de Recherches Servier and Institut Curie.

L.D. Koning is supported by a grant from Canc�eropôle Ile de France.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 12, 2012; revised October 1, 2012; accepted October 15, 2012;published OnlineFirst November 9, 2012.

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2013;73:813-823. Published OnlineFirst November 9, 2012.Cancer Res Virginie Maire, Fariba Némati, Marion Richardson, et al. with Triple-Negative Breast Cancerwith Conventional Chemotherapy for the Management of Patients Polo-like Kinase 1: A Potential Therapeutic Option in Combination

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