Cell Cycle, Cell Death, and Senescence Molecular Cancer ......small molecule inhibitors phenocopies many of the effects seen with Aurora A overexpression (13, 14). MLN8054, a selective

Published OnlineFirst March 2, 2010; DOI: 10.1158/1541-7786.MCR-09-0300

Cell Cycle, Cell Death, and Senescence Molecular
Cancer
Research

MLN8054, an Inhibitor of Aurora A Kinase, InducesSenescence in Human Tumor Cells Both In vitro and In vivo

Jessica J. Huck, Mengkun Zhang, Alice McDonald, Doug Bowman, Kara M. Hoar,Bradley Stringer, Jeffery Ecsedy, Mark G. Manfredi, and Marc L. Hyer

Abstract

Authors'Massachus

CorresponLandsdowFax: 617-5

doi: 10.115

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Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes in-cluding centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors ofAurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despitethe progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fullyunderstood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevantcell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibitionof Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown inculture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence.These include increased staining of senescence-associated β-galactosidase, increased nuclear and cell body size,vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To de-termine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft–bearing animals were dosedorally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated β-galac-tosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining oftumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype.Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports theevaluation of senescence biomarkers in clinic samples. Mol Cancer Res; 8(3); 373–84. ©2010 AACR.

Introduction

Accelerated senescence is an irreversible terminal growtharrest that occurs as a result of cellular stress or DNA dam-age (1). Senescent cells can be identified by their hallmarkphenotype of an enlarged cellular size, increased number ofvacuoles, and the presence of senescence-associatedβ-galactosidase (SA-β-gal) activity (2). The onset of senes-cence is associated with increased levels of the tumor sup-pressor proteins p53 and p21 (3). In addition, the tumorsuppressor Rb protein is hypophosphorylated in senescentcells (3). A wide variety of anticancer agents have beenshown to induce accelerated senescence in tumor cells(reviewed in refs. 1, 4). Of these agents, the antimitoticshave been shown to be both strong (discodermalide;ref. 5) and weak (taxol, vincristine; refs. 4, 6) inducers ofsenescence. The role senescence plays in therapeutic out-come is controversial; however, preclinical studies have

Affiliation: Millennium Pharmaceuticals, Cambridge,etts

ding Author: Marc L. Hyer, Millennium Pharmaceuticals, 40ne Street, Cambridge, MA 02139. Phone: 617-551-8947;51-8906; E-mail: [email protected].

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shown that the immune system is capable of eradicatingsolid tumors by removal of senescent tumor cells (7).Aurora kinases are a family of serine/threonine mitotic ki-

nases responsible for regulating a diverse set of mitotic pro-cesses ranging from centrosome separation, spindle assembly,checkpoint activation, and monitoring of kinetochore-microtubule connections (8). Aurora A kinase maps to the20q13.2 amplicon, a chromosomal region that is frequentlyamplified in tumors and is associated with poor prognosis inpatients with breast and colon cancer (9, 10). Aurora A ki-nase localizes to the centrosomes and spindle poles, and isoverexpressed in many primary tumors. Cells overexpressingAurora A result in defects in themitotic spindle and chromo-some alignment, and experience transient delays in mitosisfollowed by inaccurate chromosome segregation (11, 12). In-hibition of Aurora A using short interfering RNA (siRNA) orsmall molecule inhibitors phenocopies many of the effectsseen with Aurora A overexpression (13, 14).MLN8054, a selective inhibitor of Aurora A kinase, has

shown similar effects associated with Aurora A knockdownusing either siRNA or antibodies directed against Aurora A(14, 15). Moreover, treatment with MLN8054 has beenshown to induce apoptosis both in vitro and in xenografttumor models after repeat dosing (15). In this article, weinvestigated the ability of MLN8054 to cause an alterna-tive cell fate that has been associated with classic antimitot-ic agents, i.e., cellular senescence.

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Here, we show that inhibition of Aurora A resulted inboth apoptosis and senescence in tumor cells. Culturedhuman tumor cells treated with MLN8054 display the fol-lowing senescent markers: hypophosphorylated Rb, stabi-lization and/or increased levels of both p53 and p21,increased cellular and nuclear size, and SA-β-gal staining.Moreover, SA-β-gal staining was observed in HCT-116(colon) tumor tissue harvested from tumor-bearing micetreated with MLN8054. To our knowledge, this is the firsttime that Aurora A inhibition has been directly connectedto senescence both in vitro and in vivo.

Materials and Methods

Cell Culture and ReagentsHCT-116, A549, DLD-1, NCI-H460, and SW480

cells were obtained from American Type Culture Collec-tion. HCT-116 cells were cultured in McCoy's 5A medi-um supplemented with heat-inactivated 10% fetal bovineserum. All other cell lines were cultured in RPMI 1640medium supplemented with heat-inactivated 10% fetal bo-vine serum. LysoTracker was obtained from MolecularProbes.

ImmunoblottingHCT-116 cells were plated in 10 cm dishes (1.1 × 106

cells/dish) and then incubated for 24-48 h at 37°C. Cells weretreated with either doxorubicin (100 nmol/L), MLN8054(0.25, 1, or 4 μmol/L), or bortezomib (0.03 μmol/L) forthe indicated times, then floating and adherent cells wereharvested and lysed in radioimmunoprecipitation assaybuffer containing a cocktail of protease inhibitors (Sigma).Cell lysates were stored on ice, sonicated for 10 s, centri-fuged at 15,000 × g for 20 min, and the supernatants wereassayed for protein concentration using the Dc Protein As-say (Bio-Rad). Protein (75 μg/lane) was loaded and sepa-rated on 8% to 12% gradient Nu Page Novex Bis-Tris Gelsor Novex Tris Glycine Gels according to the instructions ofthe manufacturer (Invitrogen). Gels were transferred ontonitrocellulose membranes for 1 h, and the membranes werethen blocked for 1 h in 1× TBS + 5% nonfat milk at roomtemperature. Membranes were incubated with the primaryantibody overnight at 4°C in antibody incubation solution(TBS containing 1% nonfat milk and 0.5% Tween 20).Membranes were rinsed thrice (10 min each) in TBS con-taining 0.5% Tween 20. Membranes were then incubatedwith a secondary antibody conjugated to horseradish per-oxidase in antibody incubation solution (either overnightat 4°C or for 1.5-2 h at room temperature). Membraneswere rinsed thrice in PBS containing 0.5% Tween 20.The secondary antibody was detected using the ECLWest-ern blotting Detection System (Amersham). Chemilumi-nescence was detected using Biomax MR film (Kodak) instainless steel autoradiography cassettes (Fisher Brand).The antibodies used were as follows: p53 (DO-1; SantaCruz Biotechnology), p21 (H-164; Santa Cruz Biotechnol-ogy), pRb (Ser795; Cell Signaling), caspase-8 (UpstateClone 1-1-37), caspase-9 (Medical and Biological Laborato-

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ries Clone 5B4), caspase-7 (Cell Signaling), cleaved caspase-3 (Asp175, 5A1; Cell Signaling), PARP [poly(ADP-ribose)polymerase; BD Biosciences Clone C2-10], α-tubulin(Abcam), pH3—Phospho-Histone H3 (Ser10; Cell Signal-ing), and β-actin (Abcam, ab8226).

Trypan Blue Dye Exclusion ExperimentsHCT-116 cells were plated in 12-well plates (1.4 × 105

cells/well) and allowed to incubate at 37°C for 24 h. Cellswere then treated with compound, as indicated, and incu-bated at 37°C for 72 h. Floating and adherent cells werecollected (trypsin was used to detach the adherent cells),centrifuged, and then counted using trypan blue dye (LifeTechnologies) exclusion to differentiate live and dead cellsaccording to the instructions of the manufacturer. Prior tocompound treatment, pretreatment samples were counted.

Annexin V Fluorescence-Activated Cell Sorting AssaysHCT-116 cells were plated in six-well plates (3 × 105

cells/well) and incubated overnight at 37°C. The nextday, cells were pretreated ±100 μmol/L Z-vad-fmk (orDMSO control) in fresh medium for 1 h (37°C) and thenchallenged with compound as indicated. At 48 and 72 h af-ter treatment, floating and adherent cells were harvestedand stained with Annexin V (Biovision AV-FITC Apopto-sis Detection Kit) according to the instructions of the man-ufacturer. Cells were analyzed by flow cytometry using aBecton Dickinson FACSCalibur.

β-Gal Staining of Cultured CellsCells were plated in 12-well plates (1.4 × 105 cells/well)

and incubated overnight at 37°C. The next day, cells weretreated with either doxorubicin (0.1 μmol/L) or MLN8054(0.25, 1, or 4 μmol/L). On the indicated days, cellswere fixed and stained for β-galactosidase expression us-ing the U.S. Biologicals staining kit according to the in-structions of the manufacturer. Cells were incubatedwith the β-galactosidase staining solution for 48 h at37°C to maximize the β-galactosidase signal. The stainingsolution was removed and the plates were stored at 4°C in1× PBS. To identify cell nuclei, cells were stained with4′,6-diamidino-2-phenylindole (DAPI; 100 ng/mL) in1× PBS for 30 min before imaging. The DAPI stainingwas useful in distinguishing individual cells during theβ-galactosidase quantification. Images were acquired on aNikon TE300 microscope using a 10× PlanFluor objectivelens, Spot Insight (Diagnostic Instruments) CCD camera,and MetaMorph (Molecular Devices) software. Five ran-dom fields of view were imaged for each time point andeach concentration. Cells were hand-scored as either β-galactosidase positive or negative (50-100 cells per fieldwere scored based on availability) and averaged for eachtreatment/time point. The results are reported as a percent-age of positive cells ± SD.

Aurora A Kinase siRNA ExperimentsSuspended HCT-116 tumor cells (2 × 105) were trans-

fected as described previously (14) at 0 h and again at 72 h.

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Cells were harvested at 24, 72, and 144 h after transfectionand processed for Western blot analysis as described previ-ously (14). A monoclonal antibody directed against AuroraA was used to detect Aurora A protein expression (Anti-IAK1; BD Transduction Laboratories). Aurora A KinasesiRNA transfected cells and scrambled siRNA control cellswere stained for the presence of β-galactosidase as de-scribed above. Images shown were taken as described aboveusing a 20× objective.

In vitro Crystal Violet StainingHCT-116, A549, DLD-1, NCI-H460, and SW480

cells were plated in six-well plates (600 cells/well) and in-cubated overnight at 37°C. The next day, cells were treatedwith MLN8054 (0.25, 1, or 4 μmol/L). Cells were treatedcontinuously for 6 or 12 d and then stained with crystalviolet, or for 12 d and then allowed to recover for 6 d indrug-free medium and stained on day 18. On the indicateddays, cells were fixed using ice-cold methanol and thenstained with a 0.5% crystal violet solution to identify the

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presence of cell colonies. Each well was imaged and thenumber of colonies was determined using Metamorph(Molecular Devices) software, in which colonies containingless than 20 cells were excluded. Results are reported as thenumber of colonies ±SD of three separate wells.

In vivo Efficacy StudyNCr female nude mice (Charles River) bearing HCT-116

xenograft tumors were dosed orally (p.o.) with vehicle orMLN8054 (30 mg/kg) for 21 d (n = 10 animals/group) us-ing a twice daily dosing schedule (0 and 8 h daily dosing).Tumor growth was measured using vernier calipers and tu-mor growth inhibition was calculated using the followingformula: tumor growth inhibition = 100 − (MTV treated /MTV control) × 100. Additional details have been describedpreviously (15). Statistical significance in the tumor growthtrends over time between pairs of treatment groups were as-sessed using linear mixed effects regression models. Thesemodels account for the fact that each animal was measuredat multiple time points. A separate model was fit for each

FIGURE 1. MLN8054 induces an apoptoticresponse in HCT-116 cells. A, cells were treatedwith increasing concentrations of MLN8054 or0.03 μmol/L of bortezomib as a positive control(48 h treatment). Antibodies used were asfollows: (a) caspase-8 and (b) caspase-9recognize only the full-length caspase, (c)caspase-7 recognizes both the full-length andcleaved fragment, (d) caspase-3 recognizesonly cleaved caspase-3 fragments, (e) PARPrecognizes both the full-length and cleavedfragments. B, cells were pretreated (1 h) with orwithout z-VAD-fmk (100 μmol/L) and thenchallenged with increasing concentrations ofMLN8054 or 0.2 μmol/L of staurosporin as apositive control. The percentage of AnnexinV–positive cells was determined using flowcytometry. Columns, mean; bars, SD.

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comparison, and the areas under the curve for each treat-ment group were calculated using the predicted values fromthe model. The percentage of decrease in areas under thecurve relative to the reference group were then calculated.A statistically significant P value suggests that the trendsover time for the two treatment groups were different.

In vivo ImmunohistochemistryHCT-116 tumor-bearing NCr female nude mice were

dosed with MLN8054 at 30 mg/kg using a twice daily dos-ing (0 and 8 h) schedule. Tumor tissue was harvested at theindicated times and placed in 10% neutral buffered forma-lin. Immunofluorescence was done on 5-μm paraffin-embedded tumor sections using the Discovery XT auto-mated staining system (Ventana Medical Systems). Sectionswere deparaffinized, followed by epitope unmasking withcell conditioning 1 solution (Ventana Medical Systems)for 20 min. Tumor sections were stained for pHisH3 (CellSignaling Technologies) as described previously (15). TheDNA stain DAPI (Vector Laboratories, Inc.) was used toestimate the total number of cells/field. One representativetissue section was used for each of the three animals in atreatment group. Images were acquired using a LeicaDMLB microscope (Leica Microsystems) with a Photo-metrics Cool SnapHQ camera. Five images from each slidewere captured using a 20× Leica Plan objective (Leica Mi-crosystems) and analyzed on Metamorph image processingsoftware using a custom image processing applicationmodule (Molecular Devices). The number of pHisH3-positive cells were counted and averaged in the five fieldsof view and DAPI staining was used to estimate the totalnumber of cells in the fields. Anti-alpha tubulin antibody(Cell Signaling Technologies, 2125; 0.18 μg/mL) was di-luted in Dako diluent and incubated with tissue sectionsfor 1 h at 37°C. Secondary goat anti-rabbit rhodaminered-X conjugate (Jackson ImmunoResearch, 111-295-144; 30 μg/mL) was added for 30 min at room temperature.



DAPI vectashield HardSet Medium (Vector Laboratories)was used as a chromatin counter stain. Images were capturedwith a Nikon Eclipse E800 (20× objective) and analyzed withMetamorph 6.3r7 software (Molecular Devices).

In vivo β-Gal Staining in HCT-116 Xenograft TumorsThroughout the efficacy study satellite tumors were ex-

cised and frozen in optimal cutting temperature compoundon days 3, 5, 10, 15, and 23. Five-micron tumor sectionswere stained for SA-β-gal expression using a kit from U.S.Biologicals. Staining was quantified using Metamorph soft-ware (Molecular Devices) and represented in the graph as atotal percentage of the positive area. Dunnett's multiplecomparison test was used to ascertain statistical significance.

Results

Aurora A Kinase Inhibition Leads to Apoptosis inCultured CellsA previous study showed that MLN8054 selectively in-

hibits Aurora A over Aurora B at concentrations of ≤1μmol/L, whereas a 4.0 μmol/L concentration inhibits bothAurora A and B kinases (15). Also in this study, MLN8054(0.25-4 μmol/L) induced an apoptotic response in theHCT-116 cell line as shown by PARP and caspase-3 cleav-age. Consistent with this previous report, HCT-116 cellsdisplayed PARP and caspase-3 cleavage 48 hours afterMLN8054 treatment (Fig. 1A). Expanding on this earlierwork, the following apoptotic markers were examined: (a)caspase-8, the most proximal caspase in the extrinsic celldeath pathway; (b) caspase-9, the most proximal caspasein the intrinsic cell death pathway; and (c) executioner cas-pase-7. The antibodies directed against caspase-8 and -9 inour studies recognize the full-length caspases, therefore, adiminution in signal is indicative of caspase activation.MLN8054 treatment resulted in no detectable decreasein either caspase-8 or caspase-9 after 48 hours, suggesting

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FIGURE 2. MLN8054 treatmentdoes not significantly reduce theHCT-116 cell number comparedwith baseline. After 72 h oftreatment, trypan blue dye wasused to determine the numberof live cells/well. Bortezomib(0.03 μmol/L) was used as apositive control. Inset, number oflive cells after 72 h withouttreatment. Columns, mean of fourseparate experiments; bars, SD.

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that these caspases were not activated following MLN8054treatment (Fig. 1A). In contrast, bortezomib treatment(positive control) induced the activation of both cas-pases-8 and -9. The caspase-7 antibody used in our studiesrecognized both the uncleaved (full-length) and cleaved



forms of caspase-7. Unlike the positive control (bortezo-mib), MLN8054 did not result in caspase-7 cleavage. Sim-ilar results were found at the 72-hour time point (data notshown). Summarizing the Western blot data, MLN8054treatment results in a modest level of apoptosis.

FIGURE 3. MLN8054 modulates the protein levels of p53, p21, and phosphorylated Rb in HCT-116 cells. A, MLN8054 stabilized and/or upregulatesp53 and p21. Doxorubicin (0.1 μmol/L) was used as a positive control. B, MLN8054 treatment results in hypophosphorylated Rb for the first 48 h.Longer time points were examined (out to 5 d) but phosphorylated Rb levels dropped below the detection limit. The antibody used in this experimentrecognizes only phosphorylated Rb (Ser795). Solid arrows, hypophosphorylated state of Rb.




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To better quantify the level of apoptosis associated withMLN8054 treatment, the Annexin V assay was used (re-viewed in ref. 16). As a positive control, HCT-116 cellswere treated with 0.2 μmol/L of staurosporin for 48 to72 hours, resulting in 54% to 67% Annexin V staining(Fig. 1B). Treating HCT-116 cells with MLN8054(0.25-4.0 μmol/L) resulted in 15% to 25% Annexin V–positive staining. Pretreating the cells with the pan spec-trum caspase inhibitor, z-VAD-fmk, completely blockedMLN8054-induced Annexin V staining, indicating thatthe cell death mechanism was indeed caspase-dependentand apoptotic.

Short-term Aurora A Kinase Inhibition Leads to aCytostatic Effect in Cultured Tumor CellsThe data above indicate that MLN8054 induces an ap-

optotic response in tissue culture cells. Next, we wanted todetermine if the apoptotic response was sufficient to causea net reduction in cell viability by comparing a pretreat-ment cell count to a posttreatment cell count. HCT-116cells were treated with increasing concentrations ofMLN8054 (0.1-10.0 μmol/L), and after 72 hours, floatingand adherent cells were trypsinized, treated with trypanblue, and counted to determine the number of viable cells.A baseline count was taken just prior to MLN8054 treat-ment to determine the number of viable cells prior to treat-ment. Comparing the number of viable cells in the 72-hour



MLN8054 treatment to the baseline count, no significantchanges were observed (Fig. 2). Control cells which wereseeded and left untreated increased in number by 5-foldcompared with the baseline count, and bortezomib(0.030 μmol/L) treatment reduced the cell number bygreater than half. These data suggests that althoughMLN8054 induces (albeit minor) apoptosis, this processis insufficient to reduce the total cell population belowthe baseline within 72 hours. It is possible that the apopto-tic cell loss is counterbalanced by low-level cell prolifera-tion, but the above results also suggest that alternatecellular outcomes exist.

Aurora A Kinase Inhibition via MLN8054 Leads toSenescence in Tissue Culture CellsThe above results stimulated our interest in exploring al-

ternate cellular outcomes associated with Aurora A inhibi-tion. Senescence is a terminal cellular outcome resulting ina cytostatic effect. To determine if Aurora A inhibition in-duced senescence, several biochemical senescent markerswere evaluated in MLN8054-treated HCT-116 cells.The tumor suppressors p53, p21, and Rb are particularlyimportant in regulating cellular senescence (17-20). Previ-ous work by Liu et al. (21) showed that Aurora A kinasephosphorylates p53 on Ser215, abrogating p53′s DNAbinding and transactivation activity. P53 has been shownto transcriptionally activate the p21 gene (22). Therefore,

FIGURE 4. Chronic MLN8054 treatment in HCT-116 cells results in SA-β-gal staining, enlarged cellular size and increased DNA content, and enlargedcytoplasmic vacuoles. Cells were chronically treated for 17 d (fresh medium and drug added every 2-3 d) with MLN8054 or vehicle. A, cells were stained forβ-galactosidase activity on day 17 (10× objective, 100 μm scale bar). B, points, percentage of β-galactosidase–positive cells over the 17-d experiment;bars, SD. Fifty cells/field ×5 fields (total 250 cells) were scored and 0.1 μmol/L of doxorubicin was used as a positive control. C, on day 17, cells werestained with LysoTracker (red) to identify lysosomes and DAPI (blue) to identify DNA content (20x objective from the 1 μmol/L sample). Black arrows,examples of cellular vacuoles devoid of LysoTracker staining; bar, 50 μm.






we hypothesized that inhibition of Aurora A kinase wouldinterfere with the stability of both p53 and p21. The pro-tein levels of both p53 and p21 were examined inMLN8054-treated HCT-116 cells using immunoblotting.Aurora A kinase inhibition (via MLN8054) resulted in thestabilization and/or upregulation of both p53 and p21(Fig. 3A) on days 1 to 5. Doxorubicin, a known inducerof senescence, yielded similar effects on both p53 and p21.Although no direct link has been found between Aurora Akinase and Rb, hypophosphorylated Rb has been associat-ed with SN-38–induced senescence (3). Based on this con-nection between Rb and senescence, the phosphorylationstatus of Rb was examined in MLN8054-treated HCT-116cells. MLN8054 treatment resulted in hypophosphoryla-tion of the Rb protein on days 1 and 2 (Fig. 3B).Decoy receptor 2 (DcR2) has also been associated with

senescence. Using immunohistochemistry, Collado et al.showed an increase in DcR2 staining in senescent adeno-mas (23). We measured DcR2 levels in MLN8054-treatedcells (using immunoblotting) and found no differences inDcR2 expression on days 1 to 5 (data not shown). Thetumor suppressor 16INK4a also had a strong connectionto senescence (24). However, HCT116 cells have a meth-ylated p16INK4a promoter and are p16INK4a null (25),therefore, p16INK4a levels were not examined. It is inter-esting to note that our data suggests that senescence couldoccur in the absence of p16INK4a.



To date, one of the best known markers for senescence isSA-β-gal expression (26). To determine if Aurora kinase in-hibition was linked to senescence, SA-β-gal activity was ex-amined in HCT-116 cells treated with MLN8054. Cellswere chronically treated for 3 to 17 days and stained forSA-β-gal activity on days 3, 5, 7, 10, 14, and 17. As a pos-itive control for the SA-β-gal activity doxorubicin (0.1μmol/L), a known inducer of senescence (6), was used.As expected, doxorubicin induced an increase in cellularsize and induced SA-β-gal staining (data not shown). Dosesof MLN8054 (0.25-1 μmol/L), which selectively inhibitonly Aurora A, induced SA-β-gal staining starting on day5 (Fig. 4A and B). The 4 μmol/L dose of MLN8054,which inhibits both Aurora A and B kinase, also inducedSA-β-gal staining beginning on day 5.After 17 days of MLN8054 treatment, many of the cells

remained viable, and the phenotype was characterized byenlarged cellular size and nucleus as well as enlarged cyto-plasmic vacuoles (Fig. 4C). These characteristics are all com-monly observed in senescent cells (reviewed in ref. 1).Senescent cells typically exhibit an increase in lysosomes,both in size and number (26). After staining the cells withLysoTracker, a dye which specifically accumulates in lyso-somes, many of the vacuoles did not stain positive, suggest-ing that many of these vacuoles were nonlysosomal.To determine if Aurora A inhibition induces SA-β-gal

staining in a variety of other cell lines, SW480, A549,

FIGURE 5. Chronic MLN8054 treatment in A549, DLD-1, NCI-H460, and SW480 cells results in SA-β-gal staining. Cells were chronically treated forup to 14 d (fresh medium and drug added every 2-3 d) with MLN8054 or vehicle (0.1 μmol/L doxorubicin was used as a positive control). Treatedcells were stained for β-galactosidase activity on days 5, 10, and 14. Fifty cells/field ×5 fields (total 250 cells) were counted and the percentage ofβ-galactosidase–positive ±SD is shown in the graph. No viable DLD-1 cells were present on days 10 and 14 in both the 4 μmol/L 8054 and 0.1 μmol/L Dox.




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DLD-1, and H460 cells were treated with MLN8054 andthen assayed for SA-β-gal activity. Data in Fig. 5 indicate adose-dependent SA-β-gal staining pattern in all lines exam-ined. In addition, MLN8054-treated cells displayed alarge, flat senescent phenotype (data not shown), similarto what was observed in the HCT116. In summary, Auro-ra A kinase inhibition via MLN8054 triggered the induc-tion of senescence in cultured tumor cells.

Aurora A Kinase siRNA Phenocopies the EffectsObserved Using the Small Molecule InhibitorMLN8054Previously, we used an Aurora A kinase siRNA construct to

show that the small molecule inhibitorMLN8054phenocop-ied the effects observed using the siRNA construct (14, 15).Using the same siRNA construct (scrambled siRNAwas usedas control), HCT116 cells were double transfected on days 0and 3, and SA-β-gal activity was assayed on day 6. Althoughthe transfection efficiency was <100%, Aurora A siRNA sig-nificantly knocked down the Aurora A protein levels (Fig.6A). In addition, Aurora A siRNA treatment resulted in large,flat, vacuolated cells which stained positive for SA-β-gal activ-ity (Fig. 6B). These data indicate that targeting Aurora A ki-nase with an siRNA construct phenocopied the effectsobserved using the small molecule inhibitor MLN8054.

MLN8054 Senescent Cells Do Not Re-enter the CellCycle and DivideColony-forming assays were used to determine if

MLN8054-induced senescent cells re-enter the cell cycleand divide. Cells were cultured in the presence ofMLN8054 for up to 12 days, drug was removed, and then



cells were cultured in drug-freemedium for 6 additional days.Cells were stained with crystal violet on days 6, 12, and 18 todetermine the number of colonies. Data shown in Fig. 7 in-dicate a reduction in the number of HCT116 andH460 col-onies (compared with untreated controls) when cultured inthe presence of 250 nmol/L of MLN8054. Few to no colo-nies were present after treatment with 1 and 4 μmol/L ofMLN8054, respectively. The number of colonies presenton day 18 inversely correlated with the percentage of senes-cent cells (SA-β-gal positive), indicating that senescent cellsdo not re-enter the cell cycle and divide. This correlation wasalso evident in the A549, DLD-1, and SW480 cell lines (datanot shown).

Aurora A Kinase Inhibition Induces Senescence In vivoThe HCT-116 xenograft tumor model was used to de-

termine if Aurora A kinase inhibition leads to senescencein vivo. First, we confirmed that dosing MLN8054 (twicedaily using 30 mg/kg) in vivo did not inhibit Aurora B ki-nase activity. Mice bearing HCT116 tumors were treatedorally with two 30 mg/kg doses (twice daily schedule) ofMLN8054 (8 hours apart), and tumor tissue was analyzedvia immunofluorescent chemistry for phosphorylated his-tone H3 (pHisH3), a direct substrate for Aurora B kinase.Dosing MLN8054 resulted in an increase in pHisH3staining over time (Fig. 8A), indicating that Aurora B ki-nase activity was not blocked. Previously, it was shown thata 30 mg/kg dose of MLN8054 inhibited Aurora A kinaseactivity in the HCT-116 tumor model, as evidenced by adecrease in the direct Aurora A substrate pT288 (15).HCT-116 tumor-bearing mice were treated orally for

21 days with MLN8054 (30 mg/kg dosed on a twice daily

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FIGURE 6. HCT-116 cells treated withAurora A kinase siRNA underwentsenescence. HCT-116 cells were double-transfected with an Aurora A siRNAconstruct (scrambled siRNA was used ascontrol) on days 0 and 3. A, Westernblotting indicates Aurora A kinase proteinknockdown on day 3. Day 6 samples werealso analyzed; however, Aurora A proteinlevels were difficult to detect because of theincreased cell confluency, i.e., few mitoticcells. B, on day 6, the Aurora A siRNA-treated cells were vacuolated, had anincrease in cellular size, and stainedpositive for SA-β-gal activity (50 μmol/Lscale bars are shown).


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schedule) to determine the antitumor effect associated withthis dose and schedule. Significant tumor growth inhibi-tion was observed (P = 0.0005) on day 21 with a tumorgrowth inhibition rate of 81% (Fig. 8B). Throughout this



study, satellite tumor samples were taken (days 3, 5,10, 15, and 23) and analyzed for the presence of senes-cence. A marked increase in tumor cell size, DNA con-tent/cell, and SA-β-gal expression was observed in the

FIGURE 7. MLN8054 treatment prevented/inhibited colony formation in both the HCT-116 and H460 cell lines. Cells were treated for up to 12 d(fresh medium and drug added every 2-3 d) with MLN8054 or vehicle. MLN8054 was removed on day 12 and cells were allowed to recover in the presenceof drug-free medium for 6 d (day 18). Cells were fixed and stained using a 0.5% crystal violet solution on days 6, 12, and 18 to identify colonies.Each individual well was imaged and the colonies were counted using Metamorph software (Molecular Devices). Graphs: on day 6, only control colonieswere counted; on day 18, control colonies could not be counted due to confluency; *, HCT-116 cells treated with 0.25 μmol/L were too confluent tocount on day 18, therefore day 12 counts are shown in the graph. Columns, average of three separate wells; bars, SD.




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MLN8054-treated tissues on days 15 and 23 (Fig. 8C andD). These data strongly suggest that Aurora A kinase inhi-bition leads to senescence as a terminal outcome in anin vivo setting.

Discussion

Although previous studies by us and others have shownthat Aurora A kinase inhibition leads to apoptosis, our ex-pectation, based on the findings from other antimitoticagents, was that alternative terminal outcomes (reviewedin ref. 27) might also be associated with Aurora A kinaseinhibition. Moreover, the following observations in solidtumor cells/models led us to hypothesize that Aurora A inhi-bition leads to outcomes other than apoptosis: (a) data sug-gest that MLN8054 induced a modest apoptotic response incultured cells (our report) and in in vivo model systems, and(b) in xenograft solid tumor models, treatment with



MLN8054 resulted mostly in a tumor-static effect (15).Taken together, these data suggest that Aurora A kinase in-hibition leads to terminal outcomes other than apoptosis.In tissue culture, using a concentration of MLN8054

which selectively inhibits Aurora A kinase activity, weshowed that MLN8054 induces senescence as character-ized by a senescent-like cellular morphology (enlarged cellbody/nucleus and increased vacuolization), SA-β-gal stain-ing, upregulation of p53 and p21, and a hypophosphory-lated Rb status. Higher concentrations of MLN8054 (4.0μmol/L) inhibit both Aurora A and B kinase activity, there-fore, it is unknown if the observed senescence at this con-centration is a result of inhibiting Aurora A or inhibitingAurora B kinase. To answer this question, a selective AuroraB kinase inhibitor is needed.Our findings indicate that MLN8054 selectively inhibits

Aurora A kinase activity in vivo when dosed at 30 mg/kg.At this dose in HCT116 tumor tissue, MLN8054 hasbeen shown to inhibit Aurora A autophosphorylation

FIGURE 8. In vivo, MLN8054 does not inhibit Aurora B activity, yet does reduce HCT-116 tumor burden in nude mice and induce senescence. Nudemice bearing HCT-116 xenografts were treated with 30 mg/kg of MLN8054 (dosed twice daily at 0 and 8 h) or with vehicle. A, HCT-116 tumor tissues werestained with a fluorescently labeled antibody directed against pHisH3 (Ser10) and the staining was quantified as described in Materials and Methods.B, HCT-116 tumor-bearing mice (n = 10/group) were orally treated for 21 d using 30 mg/kg/d of MLN8054 on a twice daily schedule. Tumor volumes weremeasured using vernier calipers and error bars represent SEM (*, P = 0.0005). C, day 23 tumor sections stained with DAPI, an antibody directed againsttubulin, and β-galactosidase expression. Tubulin/DAPI and β-gal images are from independent fields. Scale bars represent 50 μmol/L. MLN8054 treatmentresults in an increase in cell and nuclear size, and β-galactosidase expression. D, β-galactosidase expression was quantified in tumor sections during the21-d study using image analysis software. Data in the graph represent the mean ± SD of 15 fields (5 fields/tumor, i.e., three tumors). On days 15 (P = 0.0051)and 23 (P = 0.0099), a significant increase in β-galactosidase expression was detected in the MLN8054 group compared with the vehicle.






(pT288; ref. 15), and induce an increase in the Aurora Bsubstrate, pHisH3 (Fig. 8A). Our in vivo data show thatMLN8054 induces an increase in tumor cell/nuclear sizeand induces SA-β-gal staining, a phenotype consistent withsenescence. Because our in vivo experiments use concentra-tions of MLN8054 that selectively inhibit Aurora A kinase,we do not know if Aurora B inhibition would lead tosenescence. Again, a selective Aurora B inhibitor is neededto answer this question.The role p53 plays in the induction of senescence is

somewhat controversial, with some reports suggesting thatit is necessary for senescence (3) and others indicating thatit acts as a rheostat regulating the severity of senescence (6).There are at least two previous reports connecting the ac-tivity of Aurora A kinase to the stability and/or activity ofp53. In one report, Katayama et al. (28) showed that si-lencing Aurora A kinase activity leads to an increase inthe stability of p53 and cell cycle arrest at G2-M. In thesecond report, Liu et al. showed that silencing Aurora Akinase activity blocked it's ability to phosphorylate p53(Ser215), thereby stabilizing p53 (21). Our data are consis-tent with these two previous reports, in that, silencingAurora A activity, using MLN8054, leads to p53′s stabilityand/or upregulation. Currently, it is unknown if AuroraA's connection to p53 regulates the onset or severity of se-nescence. To answer these questions, it would be useful toassay for senescence in a p53-null setting, as all cell linesexamined herein were p53 wild-type.There is solid evidence connecting the activity of p53

to that of both p21 and Rb. El-Deiry et al. showed thatp53 transcriptionally regulates p21 (22) and both Xionget al. and Harper et al. showed that p21 inhibits thephosphorylation of Rb by cyclin D-CDK 4 and E-CDK2(29, 30). With the p53-p21-Rb and the Aurora A kinase-p53 connections in mind, it is reasonable to see how AuroraA inhibition (via MLN8054) could result in hypopho-sphorylated pRB.Interestingly, a recent report has linked Aurora A kinase

overexpression to senescence (31). In this report, Zhanget al. found that Aurora A overexpression resulted in senes-cence as a terminal outcome. Therefore, the effects ofAurora A overexpression and Aurora A inhibition seemto phenocopy each other. The similarity in effect betweenAurora A overexpression and inhibition is consistent withAurora A requiring a multisubunit complex for its func-tion. Such a multisubunit complex with Aurora A has pre-viously been suggested (32-36). Disruption of this complex



could occur by either inhibition of Aurora A, or by over-expression of Aurora A, the latter serving to sequester nec-essary components of the complex.Induction of tumor cell senescence in response to che-

motherapy is not a novel concept. In preclinical models,many laboratories have shown different classes of chemo-therapeutic agents and ionizing radiation induces senes-cent-like phenotypes in tumor cells both in vitro andin vivo (reviewed in ref. 27). A number of studies (usinga variety of different chemotherapeutics in preclinical mod-els) have shown that senescence induction contributes di-rectly to the antitumor effect of chemotherapeutics in vivo(37, 38). Two additional studies have directly shown thatsenescence induction leads to tumor regression (7, 39),with a likely candidate for this regression being the hostimmune system (7). In the clinic, existing data linking che-motherapy to senescence is rare; in fact, we could only findone report linking clinical samples to a senescent pheno-type (3). In this report, frozen archival breast tumors frompatients receiving neoadjuvant chemotherapies were foundto stain positive for SA-β-gal activity (41% compared with10% on nontreated tumors). Although SA-β-gal is argu-ably the best senescence biomarker, it is very unstable, per-haps lending to the difficulties in detecting senescence intissues. As our understanding of detectable senescentbiomarkers advance, senescence may become a morecommonly observed terminal outcome to monitor in theclinic.We believe the results shown here support senescence as

a terminal outcome associated with Aurora A kinase inhi-bition, and support the further development of senescencebiomarkers as indirect pathway indicators of tumor cell re-sponse to Aurora A inhibition.

Disclosure of Potential Conflicts of Interest

All authors are employees of Millennium Pharmaceuticals.

Acknowledgments

We thank Stephen Stroud and Todd Sells for the preparation of all MLN8054formulations and we especially thank Arijit Chakravarty for help writing andcritiquing this work and for the contribution to figure 8.

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

Received 07/07/2009; revised 01/08/2010; accepted 01/20/2010; publishedOnlineFirst 03/02/2010.

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2010;8:373-384. Published OnlineFirst March 2, 2010.Mol Cancer Res Jessica J. Huck, Mengkun Zhang, Alice McDonald, et al.

In vivo and In vitroin Human Tumor Cells Both MLN8054, an Inhibitor of Aurora A Kinase, Induces Senescence

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Cell Cycle, Cell Death, and Senescence Molecular Cancer ......small molecule inhibitors phenocopies many of the effects seen with Aurora A overexpression (13, 14). MLN8054, a selective