aurorabisregulatedbythemitogen-activatedprotein … · 2012. 8. 18. · aurora b belongs to the...

Aurora B Is Regulated by the Mitogen-activated ProteinKinase/Extracellular Signal-regulated Kinase (MAPK/ERK)Signaling Pathway and Is a Valuable Potential Target inMelanoma Cells*□S

Received for publication, April 13, 2012, and in revised form, June 29, 2012 Published, JBC Papers in Press, July 5, 2012, DOI 10.1074/jbc.M112.371682

Caroline Bonet‡§1, Sandy Giuliano‡§, Mickaël Ohanna‡§2, Karine Bille‡§, Maryline Allegra‡§3, Jean-Philippe Lacour¶,Philippe Bahadoran¶, Stéphane Rocchi‡§¶, Robert Ballotti‡§¶, and Corine Bertolotto‡§¶4

From ‡Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Equipe 1, Biologie et Pathologies des Mélanocytes de laPigmentation Cutanée au Mélanome, Nice F-06204, the §Université de Nice Sophia-Antipolis, UFR Médecine, Nice, F-06107,and the ¶Centre Hospitalier Universitaire de Nice, Service de Dermatologie, Nice F-06204, France

Background: BRAFV600Emelanoma cells develop resistance to vemurafenib. The BRAF/ERK axis controls melanoma cellproliferation. Aurora B is a key actor of mitosis.Results: The BRAF/ERK axis regulates Aurora B. Vemurafenib-resistant melanoma cells are sensitive to Aurora B inhibition.Conclusion: Aurora B is a valuable target in melanoma cells.Significance: Our findings provide insights into Aurora B regulation and on new druggable targets to overcome vemurafenibresistance.

Metastatic melanoma is a deadly skin cancer and is resistant toalmost all existing treatment. Vemurafenib, which targets theBRAFV600E mutation, is one of the drugs that improves patientoutcome, but the patients next develop secondary resistance and areturn to cancer. Thus, new therapeutic strategies are needed totreatmelanomas and to increase the duration of v-Rafmurine sar-coma viral oncogene homolog B1 (BRAF) inhibitor response. TheERK pathway controls cell proliferation, andAurora B plays a piv-otal role in cell division. Here, we confirm that Aurora B is highlyexpressed in metastatic melanoma cells and that Aurora B inhibi-tion triggers both senescence-like phenotypes and cell death inmelanoma cells. Furthermore, we show that the BRAF/ERK axiscontrols Aurora B expression at the transcriptional level, likelythrough the transcription factor FOXM1. Our results provideinsight into the mechanism of Aurora B regulation and the firstmolecular basis of Aurora B regulation in melanoma cells. Theinhibition of Aurora B expression that we observed in vemu-rafenib-sensitive melanoma cells was rescued in cells resistant tothis drug. Consistently, these latter cells remain sensitive to theeffect of theAuroraB inhibitor.Noteworthy,wild-typeBRAFmel-anoma cells are also sensitive to Aurora B inhibition. Collectively,our findings, showing that Aurora B is a potential target in mela-noma cells, particularly in those vemurafenib-resistant, may opennew avenues to improve the treatment ofmetastaticmelanoma.

Metastatic melanomas represent the most deadly form ofskin cancer. Malignant melanoma is curable by surgical exci-

sion when detected at an early stage, but once it has metasta-sized, the complete removal is not possible andmetastatic mel-anoma is highly refractory to all existing treatments. Patientswithmetastaticmelanoma have amedian survival that typicallyranges from 6 to 10 months. Dacarbazine was a standard ofmetastaticmelanoma treatment, but the response to this chem-otherapy drug was often incomplete and associated with dis-ease relapse. Recently, targeted therapies have shown their effi-ciency in melanoma treatment. The monoclonal antibodyagainst the negative T-cell regulator cytotoxic T-lymphocyteantigen 4 (Ipilimumab) improves the overall survival, but itsbest overall response rate is limited to only 10% of patients (1).Furthermore, targeted therapy against BRAFV600E with vemu-rafenib in a subset of melanomas has shown the potential toimprove the overall survival of patients. However, the patientsinvariably develop relapses and show progressive disease after aperiod free of progression. Thus, new therapeutic strategies areurgently needed to lengthen the response to Raf inhibitors.BRAFV600E mutation is found in about 40–60% of melanomas(2), and 48% of them respond to vemurafenib treatment (3).Thus, the search remains to find efficient treatment for themajority of patients who develop melanoma.Tumor progression depends on expansion of tumor cells

through mitotic cell division. This process requires numerousproteins and protein complexes that will ensure the accuratecontrol of the events involved in the mitotic cell cycle. Amongthem, the chromosomal passenger protein complex, composedof Aurora B kinase, Survivin, Borealin, and INCENP, is a criticalmitotic regulator (4). Chromosomal passenger protein complexfunction is essential to chromosome condensation, kineto-chore-chromosome attachment, chromosome alignment at themetaphase plate, and control of the spindle checkpoint. Giventheir function in the control of the G2/M phase, the expressionof the chromosomal passenger protein complex proteins iscontrolled in a cell cycle-dependent manner. However, in-

* This work was supported in part by INSERM, the “Fondation de France,” andthe “Société Française de Dermatologie.”

□S This article contains supplemental Figs. S1–S7.1 Supported by the Fondation pour la Recherche Médicale.2 Supported by La Ligue Nationale Contre le Cancer.3 Supported by Fondation de France.4 To whom correspondence should be addressed. Tel.: 33-4-89-06-43-33;

E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 35, pp. 29887–29898, August 24, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

AUGUST 24, 2012 • VOLUME 287 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 29887

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creased expression of some of these components such asAurora B and Survivin has been observed in a spectrum of can-cers, includingmelanoma (5, 6), andpredicts early tumor recur-rence and poor prognosis (7, 8).Their targeting is therefore being pursued as attractive can-

cer targets. Although Survivin function in melanoma cells iswell documented that of Aurora B is less well determined.Aurora B belongs to the mammalian Aurora Kinase familyAurora A (AURKA), Aurora B (AURKB), and Aurora C(AURKC). Inhibitors of Aurora kinases, ZM447439, PHA-680,hesperadin, PF-03814735, SNS-314,VE-465, andMK0457/VX-680, have shown promising anti-tumor effects in several celltypes (9, 10), including melanoma cells (5, 6). Recently,AZD1152, a highly potent and specific aurora kinase inhibitorwith 1000-fold selectivity for Aurora B over Aurora A kinaseactivity, has been developed (11). AZD1152 is a prodrug that ismetabolized in the serum to its active formAZD1152-hydroxy-quinazoline pyrazol anilide (HQPA),5 an ATP binding pocketcompetitor. Preclinical studies have shown the antineoplasicproperties of AZD1152 in acute myelogenous leukemia, multi-ple myeloma, colorectal and breast cancer, yet the effect ofAZD1152 remains to be explored in melanoma cells.Here, we confirm an increased expression ofAurora B during

melanoma progression that starts in cells from the verticalgrowth phasemelanomas.We show thatAurora B inhibition byAZD1152-HQPA triggers cell cycle arrest, cellular senescence,and cell death by mitotic catastrophe. Similar results wereobtained with Aurora B specific siRNA. Preclinical studiesshow that Aurora B inhibition by AZD1152-HQPA dramati-cally inhibits growth of human melanoma xenografts. Addi-tionally, we provide insights into the mechanisms of Aurora Bregulation inmelanoma cells by demonstrating that Aurora B isa target of the ERKpathway. Stimulation of the ERKpathway bygrowth factors enhances Aurora B expression, whereas its inhi-bition by vemurafenib strongly decreases Aurora B mRNA andprotein levels. Vemurafenib also triggered a decrease in thelevel of FOXM1, a factor reported to regulate the transcriptionof Aurora B. Collectively, these observations indicate that theBRAF/ERK axis controls Aurora B at the transcriptional level.The decreased expression of Aurora B upon vemurafenib in thevemurafenib-sensitive melanoma cells is recovered in cellsresistant to this drug. In these latter cells, we demonstrate thatAurora B inhibition promotes cell death of vemurafenib-resist-ant melanoma cells. This study may open new therapeuticstrategies targeting Aurora B to improve the management ofmetastatic melanoma.

EXPERIMENTAL PROCEDURES

Materials—AZD1152-HQPA and vemurafenib (PLX-4032)were obtained from Selleck Chemicals LLC (Houston, TX).LabrafilM1944Cs was purchased fromGattefosse (Saint Priest,France). Monoclonal antibodies to Aurora B and caspase-3were from BD Transduction Laboratories, and monoclonal

antibodies to ERK2 (D-2), AIF (E-1), and actin (9) and poly-clonal antibodies to Rb (C-15), FOXM1 (A-11), p53 (DO-1),and HSP60 were from Santa Cruz Biotechnology (Santa Cruz,CA). Monoclonal antibodies to phospho-histone H3 (Ser-10),phospho-Aurora B (Thr-232), procaspase-2, and phospho-ERK1/2 (Thr-202/Tyr-204) and polyclonal antibodies to phos-pho-RB (Ser-807/811), histone H3, AKT, pS473-AKT, pT68-CHK2, and PARP were from Cell Signaling Technology Inc.(Beverly, MA). Monoclonal antibody to �H2AX was fromMil-lipore. Polyclonal antibody to Smac-Diablo was from UpstateCell Signaling (Billerica, MA).Cell Cultures—Human melanocyte cultures were obtained

from the foreskins of Caucasian children as described previ-ously (12). The different melanoma cell lines were describedpreviously (13).Construction of Vemurafenib-resistant Melanoma Cells—

Fresh sterile tissues were obtained from surgical waste frompatients diagnosed with metastatic melanoma at the Nice Cen-tre Hospitalier Universitaire Hospital and were treated asreported previously (#1 and #2) (14). Informed consent wasobtained from the patients. Genomic DNA was prepared withthe DNeasy blood and tissue kit (Qiagen). DNA was amplifiedby PCRs with specific primers, and Sanger sequencing was per-formed on a 3730 DNA analyzer (Applied Biosystems). Weestablished vemurafenib-resistant melanoma cells by exposingvemurafenib-naive parental melanoma cells (#1) or the humanmelanoma cell line WM9 (a gift of Dr. M. Herlyn, The WistarInstitute, Philadelphia) to incremental increases of vemu-rafenib. Vemurafenib-resistant selection continued until themelanoma cells could sustain viability and proliferate whenchallenged with 20 �M vemurafenib. Sequencing of the vemu-rafenib-resistant melanoma cells indicated that they did notharbor secondary BRAFmutations or N-RASmutations.Transient Transfection of siRNA—Briefly, a single pulse of 50

nM siRNA was administered to the cells at 50% confluency bytransfection with 5 �l of LipofectamineTM RNAiMAX in Opti-MEM medium (Invitrogen). Control scrambled (siC) andAurora B specific-siRNA (siAURKB) was described previously(15).Subcellular Fractionation andWestern Blot Assays—Subcel-

lular fractionation was performed using proteoextract subcel-lular proteome extraction kit according to the manufacturer’sinstructions (Calbiochem).Western blotting experiments wereperformed as described previously (16). Western blot analysisdata were repeated at least three times.Cell Viability Test—Cell viability was assessed using the cell

proliferation kit II (XTT; Roche Applied Science) according tothemanufacturer’s protocol. Cell viability,measured at 490 nm,was expressed as the percentage of the value in DMSO-treatedcells.Flow Cytometry—Cells were stained with propidium iodide

(40�g/ml) containing ribonucleaseA (10�g/ml) andwere ana-lyzed using a fluorescence-activated cell sorter (MACSQuant�analyzer) and MACSQuantifyTM software.Caspase Activities—Proteins were extracted with a buffer

containing 50mMHEPES, pH 7.4, 150mMNaCl, 20mM EDTA,0.2% Triton X-100, and protease inhibitors.

5 The abbreviations used are: HQPA, hydroxyquinazoline pyrazol anilide;PARP, poly(ADP-ribose) polymerase; SA-�-Gal, senescence-associated�-galactosidase; AIF, apoptosis-inducing factor; HGF, hepatocyte growthfactor; BRAF, v-Raf murine sarcoma viral oncogene homolog B1.

Regulation of AURKB by the ERK Pathway

29888 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 35 • AUGUST 24, 2012


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Samples (50 �g) were incubated with or without 0.2 mM

N-acetyl-DEVD-amido-4-methylcoumarin (caspase-3) or 0.2mM N-acetyl-VDVAD-amido-4-methylcoumarin (caspase-2)in triplicate. To assess specific caspase activities, hydrolysis wasfollowed at different times at 37 °C in the presence or absence of1 mM N-acetyl-DEVD-CHO or N-acetyl-VDVAD-CHO (Vil-leurbanne, France). Caspase activities were expressed as arbi-trary units. Each experiment was done in triplicate.Senescence-associated �-Galactosidase Assay and Immuno-

fluorescence—The senescence�-galactosidase staining kit (CellSignaling Technology) and immunofluorescences were per-formed as reported before (14).mRNA Preparation, Real Time/Quantitative PCR—Quanti-

tative PCRswere performed as reported previously (17). Primersequences for each cDNA were designed using qPrimer depot(primerdepot.nci.nih.gov) and are available upon request.

In Vivo Murine Cancer Model—Animal experiments werecarried out in accordance with French law and were approvedby a local institutional ethical committee. Animals were main-tained in a temperature-controlled facility (22 °C) on a 12-hlight/dark cycle and were given free access to food (standardlaboratory chow diet from UAR, Epinay-S/Orge, France).Human A375 melanoma cells (2.5 � 106 cells) were inoculatedsubcutaneously into 6-week-old female immune-deficientAthymic Nude FOXN1numice (Harlan Laboratory). When thetumors became palpable, mice received a daily intratumoralinjection for 7 days of AZD1152-HQPA (30 mg/kg/day) dis-solved in a mixture of Labrafil M1944 Cs, dimethylacetamide,and Tween 80 (90:9:1, v/v/v). Control mice were injected withLabrafil alone.Statistical Analysis—Data are presented as the average �

S.D. andwere analyzed by Student’s t test usingMicrosoft Excel

FIGURE 1. Inhibition of metastatic melanoma cell proliferation by the Aurora B inhibitor AZD1152. A, human A375 melanoma cells were exposed toincreasing concentration of AZD1152-HQPA (250 and 500 nM), and lysates were analyzed with the indicated antibodies. B, normal human melanocytes, humanmelanoma cells (A375, 1205LU, 501mel, and SkMel28), and mouse B16 melanoma cells were exposed to increasing concentrations of AZD1152-HQPA for 48 h,and then cell proliferation was assessed using the colorimetric XTT assay. C, lysates of A375 cells exposed to increasing concentrations of AZD1152 (250 and 500nM) were analyzed by Western blotting with the indicated antibodies.




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software. A p value of 0.05 (*, p � 0.05) or less (**, p � 0.01, and***, p � 0.001) was interpreted as indicating statistical signifi-cance when comparing experimental and control groups.

RESULTS

Aurora B Inhibition Induces Melanoma Cell Growth Arrest—We first examinedAurora B expression in normal humanmela-nocytes and in melanoma cell lines of different stages of pro-gression. As reported previously, Aurora B was expressed athigh levels inmetastaticmelanoma (supplemental Fig. S1A) (6).We next conducted experiments to assess the effect of the

Aurora B-specific inhibitor, AZD1152-HQPA (herein referredto as simply AZD1152), on melanoma cell viability. Activationof Aurora B kinase occurs through its autophosphorylation of

Thr-232 in the kinase activation loop (18). Detection of thismodification with anti-phosphothreonine 232 Aurora B anti-body revealed a decreased Aurora B phosphorylation inresponse to AZD1152 treatment (Fig. 1A). Decreased Aurora Bphosphorylation was observed as early as 30 min post-AZD1152 treatment (data not shown). Chromosome conden-sation during mitosis requires phosphorylation of histone H3on serine 10 byAurora kinase B (19). In this regard, the reducedHistoneH3 serine 10 phosphorylation confirmed the efficiencyof AZD1152. AZD1152 also caused a reduction in Aurora Bexpression, in agreement with findings in breast cancer cells(20). Having shown the efficiency of AZD1152, normal humanmelanocytes andmelanoma cell lineswere treatedwith increas-ing concentrations of AZD1152 from 50 to 250 nM, and cell

FIGURE 2. Aurora B inhibition induces polyploidization and senescence entry. A, immunofluorescence experiments of A375 cells exposed to 250 nM

AZD1152-HQPA for 48 h were labeled with anti-�-tubulin antibody (btub). Nuclei were stained with DAPI. B, FACS analysis of A375 melanoma cells exposed toincreasing concentrations of AZD1152-HQPA for 48 h. C, A375 cells treated with AZD1152-HQPA for 24 h were analyzed by immunoblotting with antibodies tophospho-Ser-387 CHK2 (pCHK2) and �H2AX. HSP60 ensures the even loading of each lane. D, human A375 melanoma cells were exposed to increasingconcentrations of AZD1152-HQPA (250 and 500 nM), and lysates were analyzed with antibodies to p53 and p21. E, A375 cells were exposed to AZD1152-HQPA250 nM for 96 h and cells remained adherent were analyzed for the SA-�-Galactosidase (SA-�-Gal) reactivity. The percentage of means � S.D. was derived fromcounting 100 cells in duplicate plates.




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proliferation was assessed (Fig. 1B). Normal melanocytes werehighly resistant to the AZD1152 effect with only 30% reductionin cell proliferation at the highest concentration of 250 nM. Incontrast, all the melanoma cell lines tested displayed highersensitivity to AZD1152 effect (Fig. 1B). AZD1152 also showedpotent inhibition of melanoma cells in a colony-forming assay(supplemental Fig. S1B). The pro-apoptotic inducer staurospo-rine, used as positive control, also dramatically inhibited mela-noma cell growth. These results demonstrate that melanomacell lines of different genetic background and species are sensi-tive to the growth inhibitory activity of AZD1152.In agreement with a proliferation cessation, human A375

melanoma cells exposed to AZD1152 showed a decreasedphosphorylation of the retinoblastoma protein on Ser-807/811and a shift down of total Retinoblastoma protein (RB), signs ofhypophosphorylation (Fig. 1C). Cyclin B1 and cyclin-depen-

dent kinase 1 (CDK1) level also declined post-AZD1152 treat-ment and that of Cyclin E1 increased.Reduction of phosphorylated Ser-10 Histone H3 has been

associated with cytokinesis failure. Immunofluorescence withantibody to �-tubulin and DAPI staining revealed enlargementand polyploidization of A375 cells exposed to AZD1152 (Fig.2A). Flow cytometry analysis confirmed that A375 melanomacells exposed to AZD1152 became polyploid as illustrated bythe high proportion of 8N and 16N cells 48 h post-treatment(Fig. 2B). Note that a small proportion of 8N and 16N cells maybe observed in control conditions that may reflect the disrup-tion of the normal cell cycle control observed in transformedcells. The percentage of 8N A375 cells rose from 7% beforetreatment to 70% at 48 h with AZD1152. Karyokinesis failurewas associated with DNA damages and senescence entry.Indeed, AZD1152 triggered activation of the DNA damage

FIGURE 3. Aurora B inhibition triggers cell death. A, sub-G1 FACS analysis of A375 melanoma cells exposed to 250 nM AZD1152-HQPA using propidiumiodide. B, caspase-2 activity was assessed in lysates of A375 cells exposed to AZD1152-HQPA for 96 h. When indicated, the pan-caspase inhibitor QVD-OPH(QVD, 20 mM) was added to the reactions. The pro-apoptotic inducer staurosporine (1 �M) was used as a negative control. C, cytoplasmic (FI) and microsomal(FII) fractions of A375 cells exposed for the time indicated to 250 nM AZD1152-HQPA were analyzed by Western blotting with AIF, Smac/Diablo, and actinantibodies. D, caspase-3 activity was assessed in lysates of A375 cells exposed to AZD1152-HQPA for 96 h. When indicated, the pan-caspase inhibitor QVD-OPH(20 mM) was added to the reactions. The pro-apoptotic inducer staurosporine (1 �M) was used as a positive control. E, A375 cell lysates exposed for 96 h to 250nM AZD1152-HQPA with or without 20 mM QVD-OPH were analyzed by Western blotting with total PARP1, Aurora B, and HSP60 antibodies.




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response pathway, illustrated by the detection of activatedphospho-CHK2 (p-CHK2) and the increase in �H2AX levels(Fig. 2C). Senescence-like phenotypes such as senescence-asso-ciated �-galactosidase (SA-�-Gal) staining (Fig. 2E), increasedcell size (supplemental Fig. S2A), and cell granularity (supple-mental Fig. S2B) were observed in cells that remained attachedto the culture dishes in response to AZD1152. In these condi-tions, p53 and its target gene p21Cip were up-regulated (Fig.2D). Several other melanoma cell lines and the freshly isolatedmelanoma cells tested also underwent senescence-like pheno-types such as SA-�-Gal staining (supplemental Fig. S2C), indi-cating this process was not unique to a particular cell type.Inhibition of Aurora B by a genetic approach (siRNA) engen-

dered similar results. A dramatic reduction in the number ofA375 cells as a function of time was observed (supplementalFig. S3A) andwas associatedwith a time-dependent decrease inHistone H3 phosphorylation at serine 10, in the level of CDK1and Cyclin B1 and with the hypophosphorylation of the Reti-noblastoma protein (RB) (supplemental Fig. S3B). Conversely,the level of ;cyclin E1 increased. These observations therebydemonstrate that suppression ofAurora B activity triggers a cellcycle arrest.

Aurora B Inhibition Promotes Death by Mitotic Catastrophe—A sub-G1 fraction was detectable 72 h post-AZD1152 exposure(Fig. 3A). Cell death occurring during mitosis is referred to as amitotic catastrophe and often takes place in conjunction withapoptosis. Activation of caspase-2 and release of apoptosis-in-ducing factor (AIF) and SMAC/DIABLO from mitochondriawere suggested as characteristics of mitotic catastrophe (21).AZD1152-mediated caspase-2 activation was observed by

fluorometric assay and was abolished by the caspase inhibitorQVD (Fig. 3B). Kinetic experiments showed the disappearanceof the inactive procaspase-2 with the concomitant detection ofthe cleaved fragments in Western blot experiments (supple-mental Fig. S4A). These observations indicated that the inactiveprocaspase-2 was processed to its catalytically active form. Fur-thermore, cell fractionation assays showed the presence of AIFand SMAC/DIABLO in the cytoplasmic fraction of cellsexposed to AZD1152 relative to control cells, with a maximumrelease after 96 h (Fig. 3C). AZD1152 also triggered caspase-3activation (Fig. 3D) and the cleavage of PARP, one of the beststudied caspase-3 substrates (Fig. 3E). Both caspase-3 andPARP cleavage were prevented in the presence of the caspaseinhibitor QVD. Staurosporine, a mitotic catastrophe-indepen-

FIGURE 4. In vivo anti-tumor effect of the Aurora B inhibitor AZD1152. A, A375 melanoma cells (2.5 � 106) were subcutaneously engrafted in athymic nudemice. Mice were injected with the vehicle alone (Ct) or with AZD1152-HQPA. The growth tumor curves were determined by measuring the tumor volume usingthe equation V � (L � W2)/2. Results are presented as mean (�S.E.) tumor volumes (mm3), and p values are from Student’s t test (*, p � 0.05; **, p � 0.01; ***,p � 0.001) comparing tumor size in AZD1152-HQPA-treated versus vehicle at each point. B, representative images and means of subcutaneous tumor weightfrom control (Ct) and AZD1152-HQPA treated mice are shown. C, TUNEL assay on 7-�m tumor sections.




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dent inducer of apoptotic cell death, did not promote pro-caspase-2 processing nor the generation of active fragments,but as expected, it enhanced caspase-3 activity and cleavage ofPARP. These events were counteracted by QVD (Fig. 3, B, D,and E). AZD1152, the efficiency of which wasmonitored by thedisappearance of Aurora B in Western blot, promotedcaspase-2 activation as evidenced by the disappearance of theinactive procaspase-2 in several cell lines and freshly isolatedmelanoma cells (supplemental Fig. S4B). Similarly, Aurora Binhibition by siRNA elicited cell death in all themelanoma cellstested. An increase in the sub-G1 population (supplemental Fig.S5) and a time-dependent disappearance of procaspase-2 withthe concomitant appearance of its cleaved fragments (supple-mental Fig. S6A) were indeed observed. Cell fractionationassays also showed the presence of AIF and SMAC/DIABLO inthe cytoplasmic fraction in Aurora B-silenced cells relative tocells transfected with scrambled siRNA (supplemental Fig.S6B). Aurora B knockdown and staurosporine enhancedcaspase-3 enzymatic activity (supplemental Fig. S6C) and cleav-age of PARP (supplemental Fig. S6D), and both were impairedby QVD treatment. Thus, Aurora B inhibition either by phar-macological (AZD1152) or genetic (siRNA) approaches ends inapoptosis of melanoma cells.AZD1152 Prevents Growth of Human Melanoma Tumor

Xenografts—The antineoplasic effect of AZD1152 was nextinvestigated in vivo. To this aim, the highly aggressive A375melanoma cells were engrafted subcutaneously into 6-week-old female athymic nude mice. When the tumors became pal-

pable (0.2–0.5 cm3), mice were treated with the Aurora kinaseinhibitor at a dose of 30 mg/kg or with its vehicle (labrafil) for 7days. Tumors were measured over a period of 15 days. Dose ofAZD1152 was based on a previous report (20). Treatment withthe inhibitor dramatically impaired melanoma tumor growth(Fig. 4A) comparedwith tumors that received the vehicle alone.Excised tumors in the AZD1152 group weighed significantlyless than those in the control group and appeared less vascular-ized (Fig. 4B). Consistently, a section of AZD1152-treatedtumors revealed the presence of cell death as illustrated by thehigh percentage of TUNEL-positive cells compared with con-trol of tumor sections where almost no TUNEL-positive cellscould be observed (Fig. 4C). Thus, Aurora B inhibition byAZD1152 displays clear anti-melanoma abilities also in vivo.Aurora B Is Controlled by the ERK Signaling Pathway—

BRAF,which controls the activity of the ERKpathway, is criticalfor mitotic spindle formation and activation (22, 23). Thus, wewere wondering whether Aurora B, which is a critical mitoticregulator, could be a target of the ERK pathway. InMeWomel-anoma cells that do not harbor the BRAFV600E activatingmuta-tion, ERK activity stimulation byHGF, EGF, or PDGFwas asso-ciated with increased Aurora B levels (Fig. 5, A–C). However,inhibition of the ERK pathway by U0126 or vemurafenib, cur-rently used in clinics for the treatment of melanoma, impairedAurora B expression in A375 cells (Fig. 6, A and C). In agree-ment with these observations, the increased Aurora B expres-sion was associated with an increased level of phospho-ERK1/2during melanoma progression, although we failed to detect a

FIGURE 5. Aurora B level is up-regulated upon growth factor ERK pathway. MeWo cells were treated with increasing duration of HGF (10 ng/ml) (A), EGF (10ng/ml) (B), or PDGF (20 ng/ml) (C). Western blots were performed for Aurora B, p-ERK1/2, and ERK2. The densitometric scanning of the AURKB immunoblotbands was performed digitally using the ImageJ software program (National Institutes of Health) and was normalized to the total ERK expression levels.




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perfect correlation (supplemental Fig. S1A). Contrastingly,inhibition of the PI3K pathway by LY294002 modestly reducedthe Aurora B level (Fig. 6A). Furthermore, our results showedthat vemurafenib decreased the mRNA level of Aurora B, indi-cating that the ERK signaling pathway controls Aurora B at thetranscriptional level (Fig. 6B). Interestingly, we found thatvemurafenib andUO126 also diminished to a similar extent themRNA level of FOXM1, a known regulator of Aurora B (24).This inhibitionwas also seen at the protein level (Fig. 6C). Addi-tionally, FOXM1 silencing by siRNA decreased Aurora Bexpression (Fig. 6D). Altogether, our results indicate that theBRAF/ERK axis controls Aurora B expression at the transcrip-tional level through FOXM1.Aurora B Is a Potential Target in Vemurafenib-resistantMel-

anoma Cells—Acquired resistance to vemurafenib is a majorobstacle to the effectiveness of this treatment, renderingurgent the identification of alternate targets to treat mela-noma in these patients. Melanoma cells, isolated from a

human biopsy, and the WM9 melanoma cell line, both har-boring the BRAFV600E mutation (supplemental Fig. S7A)(25), were rendered resistant to vemurafenib. XTT-basedproliferation assays indicate that vemurafenib diminishedthe viability (80%) of the parental cells (Fig. 7, A and B). Incontrasting, the two types of melanoma cells resistant tovemurafenib displayed almost no decrease in viability or inERK activity (Fig. 7, C and D) in response to vemurafenib,demonstrating that they escaped the effect of the drug. Inter-estingly, vemurafenib triggered a diminution of Aurora Blevels in the parental cells, whereas Aurora B expression wasrescued in vemurafenib-resistant cells (Fig. 7, C and D). Thisobservation agrees with a return to cell proliferation of theresistant cells and the disease relapse in patients. In MeWomelanoma cells expressing wild-type BRAF, vemurafenib didnot decrease the proliferation nor induced PARP cleavage(Fig. 7, E and F). Noteworthy, in BRAF-wild-type MeWocells, vemurafenib induced a stimulation of ERK (increase in

FIGURE 6. Aurora B mRNA and protein levels are decreased in response to ERK inhibition. A, A375 melanoma cells were exposed to pharmacologicalinhibitors of the ERK (U0126, 10 �M) and PI3K (LY294002, 10 �M) signaling pathways for 48 h and were analyzed by Western blotting. B, quantitative PCR analysisof Aurora B and FOXM1 mRNA expression in A375 (upper panel) and 501mel (bottom panel) melanoma cells treated with vemurafenib (V, 10 �M) or UO126 (UO,10 �M) for 48 h. C is control. C, A375 (left panel) or 501mel (right panel) melanoma cells were exposed to vemurafenib (V, 10 �M) or UO126 (UO, 10 �M) for 48 h.Cell lysates were analyzed by Western blotting. C is control. D, Western blot analysis of lysates from A375 (left panel) or 501mel (right panel) transfected withscrambled siRNA control (siC) or FOXM1 siRNA (siFOXM1).




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phosphorylated ERK1/2), rather than an inhibition. Conse-quently, an increase in Aurora B level could be observed.Thus, these observations prompted us to investigate

whether the Aurora B inhibitor, AZD1152, could be a ther-apeutic option. Interestingly, XTT tests showed that vemu-rafenib-resistant cells remained sensitive to AZD1152.AZD1152 was able to trigger death of the resistant cells, evenmore efficiently than in the parental vemurafenib-sensitivecells (Fig. 8, A and B). Cell death was evidenced by the pro-cessing of procaspase-2 and by the cleavage of PARP (sup-plemental Fig. S7B), as well as by an increase of dead cellsthat stained positive for the propidium iodide (supplementalFig. S7, C and D). In agreement with our previous results(supplemental Fig. S6D), inhibition of Aurora B by specificsiRNA led to a processing of PARP as a readout of cell death

(Fig. 8, C and D). A processing of PARP was also observed inthe resistant cells. Noteworthy, PARP cleavage was higher inthe resistant cells relative to the sensitive parental cells. Col-lectively, these observations indicate that Aurora B inhibi-tion may be a valuable therapeutic option in patients withBRAFV600E metastatic melanoma that developed secondaryresistance to vemurafenib and in patients with wild-typeBRAF (Fig. 1B and data not shown).

DISCUSSION

This study has focused upon Aurora B as a potential ther-apeutic target in melanoma treatment. More particularly,our results indicate that Aurora B inhibition could be a ther-apeutic option not only in individuals whose melanoma is awild type for BRAF and for whom there is a current lack of

FIGURE 7. Aurora B is re-expressed in vemurafenib-resistant melanoma cells. Freshly isolated human melanoma cells (#1) (left panel) (A) and WM9 cell line(right panel) (B) were rendered resistant to vemurafenib by continuous in vitro culture in increasing concentrations of the drug. Cell viability of the parental(vemurafenib-sensitive) (S) and of the resistant (R) melanoma cells treated with vemurafenib 5 and 10 �M for 48 h was assessed by XTT. C and D, vemurafenib-sensitive (S) and -resistant (R) melanoma cells (#1, left panel and WM9, right panel) were treated with 5 �M vemurafenib. Cell lysates were analyzed by Westernblotting with the indicated antibodies. E, effect of vemurafenib 5 and 10 �M on the number of MeWo melanoma cells by cell count. F, MeWo melanoma cellswere exposed to 5 and 10 �M vemurafenib for the time indicated on the figure and analyzed by Western blot with the indicated antibodies.




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therapies but it also could be seen as an option to lengthenthe initial response to Raf inhibitor of BRAFV600E melanomacells.Aurora kinase B plays a key role during mitosis, where it is

involved in the acute regulation of karyokinesis and cytokinesis,two processes with fundamental consequences on cell prolifer-ation and survival. In contrast to normal cells (26), the cycle ofactivation/deactivation (degradation) of Aurora B is compro-mised in tumor cells. Sustained over-representation of AuroraB has been shown to perturb mitotic checkpoints leading tochromosomal aberrations and to result in genomic instabilityand aneuploidy (27). In the context of melanoma cells,Wang etal. (6) previously reported that Aurora B increased with mela-noma progression and that its inhibition could be an anti-melanoma strategy. Here, we confirm these observations inanother set of melanoma cells, reinforcing the notion thatAurora B is a valuable therapeutic option.We show that inhibition of Aurora B by pharmacological

(AZD1152) or genetic loss of function (siRNA) approaches elic-its melanoma senescence and death. Senescence-like pheno-types are illustrated by polyploidization, enlarged and granularcell shape, and detection of the �-galactosidase activity and ofmarkers of DNA damages. Cell death by mitotic catastrophe isevidenced by polyploidization, caspase-2 activation, and detec-tion of dead cells. Previousworks with pan-Aurora inhibitors inmelanoma cells (5, 28) and in other tumor types (10, 20)reported similar effects. The progressive disappearance of bothCyclin B1 and CDK1 after Aurora B suppression is consistentwith cells that exit mitosis without completion of cytokinesisand with cells that enter a new round of cell division at a higherDNA content. An increased level in Cyclin E, which is impli-cated in the G1/S transition, also accords well with this dereg-

ulated mitotic progression. Consistently, Cyclin E up-regula-tion was also associated with cell death (29).In addition to inhibiting Aurora B activity, AZD1152 treat-

ment also dramatically decreases the Aurora B level in mela-noma cells. This is reminiscent of a previous report indicatingthat AZD1152 increased polyubiquitination and proteosomaldegradation of Aurora B (20). Aurora B functions in complexwith Survivin, Borealin, and INCENP allowing the docking ofAurora B at the right place at the right time duringmitosis (30).Depletion of any one subunit of the Aurora B-INCENP-Borea-lin-Survivin complex caused a substantial reduction not only inthe corresponding target but also in the other subunits andmaypromote cell death (31, 32). Therefore, by using AZD1152, sev-eral actors and tumor signaling circuitries may be simultane-ously disabled, amplifying the anti-tumor effect of the Aurora Binhibitor.Little is known about the mechanisms of Aurora B regula-

tion, although the product of the EWS-fli1 translocation genesand FOXM1 were reported to control Aurora B transcription(24, 33, 34).We find that inhibition of BRAF/ERK axis by eithervemurafenib orUO126 inBRAFV600Emelanoma cells decreasesFOXM1 and Aurora B mRNA and protein levels. Additionally,FOXM1 inhibition by siRNA decreases Aurora B expression.However, growth factors, such as HGF, EGF, or PDGF, that areimportant in melanoma development and progression (35–37)stimulate ERK activity and increase the protein level of AuroraB. Aurora B-regulation by EPS8, a substrate of the EGF receptorpathway (6), or by HGF (38) in HEK293 cells and hepatocytes,respectively, was previously reported. Therefore, although itremains to clearly determine how the BRAF/ERK signalingpathway controls the transcription of Aurora B, our results

FIGURE 8. Aurora B is a potential therapeutic target in vemurafenib-resistant melanoma cells. A and B, XTT proliferation assay was performed on thevemurafenib-sensitive (S) and -resistant (R) melanoma cells (#1, left panel and WM9, right panel) treated with increasing concentrations of vemurafenib orAZD1152 for 48 h. Data are significantly different from the sensitive cells at **, p � 0.01, and *, p � 0.05. C and D, Western blot experiment of cell lysates fromvemurafenib-sensitive (S) and -resistant (R) melanoma cells (#1, left panel, and WM9, right panel) transfected with Aurora B-specific inhibitor for 96 h.




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indicate that this transcriptional regulation could be mediatedby FOXM1.Of note, vemurafenib can elicit senescence-like phenotypes

in BRAFV600E melanoma cells such as a SA-�-Gal reactivity(data not shown), but it does not trigger polyploidization, indi-cating that it does not recapitulate all the phenotypes of AuroraB inhibition. The discrepancies between the phenotypes trig-gered by the two drugs are consistent with the notion thatBRAF/ERK signaling pathway has likely several other down-stream targets in addition to Aurora B.As reported previously, wild-type BRAF melanoma cells are

resistant to the cytostatic/cytotoxic effect of vemurafenib, andvemurafenib stimulates the ERK activity, rather than inhibitingit (39, 40). Consistently, an increased level of Aurora B could beobserved after 24 h.Interestingly, we observe that vemurafenib resistance ofmel-

anoma cells is accompanied by Aurora B re-expression, whichlikely drives their return to proliferation. Aberrant PDGF sig-naling has been previously associated with vemurafenib resist-ance (41), and our results demonstrate that PDGF increases theAurora B level. Thus, the PDGF-PDGF receptor � axis couldmediate resistance by favoring Aurora B recovery. Dysregula-tion of other signaling molecules, such as HGF or EGF thatenhances the Aurora B level, could also contribute to vemu-rafenib resistance. More importantly, AZD1152 preventsgrowth of both BRAFV600E-sensitive and BRAFV600Emelanomacells resistant to vemurafenib. Noteworthy, theAZD1152 effectis slightly but significantly higher in vemurafenib-resistantmel-anoma cells compared with the sensitive parental cells. UsingPARP cleavage as a readout of cell death, similar observationshave been made with Aurora B-specific siRNA. These findingsare important for the management of BRAFV600E mutant mel-anomas but also for those that are BRAF wild type. Indeed,there are no efficient cures for non-BRAFV600E-mutated mela-nomas, which represent half of all the cases. Collectively, ourdata disclose new therapeutic options for metastatic mela-noma, using the targeting of Aurora B.

Acknowledgments—We acknowledge the C3M imaging core facility(Microscopy and Imaging Platform Côte d’Azur) and the C3M ani-mal room facility.

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BertolottoCorineJean-Philippe Lacour, Philippe Bahadoran, Stéphane Rocchi, Robert Ballotti and

Caroline Bonet, Sandy Giuliano, Mickaël Ohanna, Karine Bille, Maryline Allegra,Potential Target in Melanoma Cells

Signal-regulated Kinase (MAPK/ERK) Signaling Pathway and Is a Valuable Aurora B Is Regulated by the Mitogen-activated Protein Kinase/Extracellular

doi: 10.1074/jbc.M112.371682 originally published online July 5, 20122012, 287:29887-29898.J. Biol. Chem.

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