identiﬁcation of an aurora kinase inhibitor speciﬁc for the ......2013/01/10 · molecule...

Molecular and Cellular Pathobiology

Identification of an Aurora Kinase Inhibitor Specific for theAurora B Isoform

Hua Xie1, Mee-Hyun Lee1, Feng Zhu1, Kanamata Reddy1, Cong Peng1, Yan Li1, Do Young Lim1,Dong Joon Kim1, Xiang Li1, Soouk Kang1, Haitao Li1, Weiya Ma1, Ronald A. Lubet2, Jian Ding3, Ann M. Bode1,and Zigang Dong1

AbstractAurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis.

In the present study, we used a ligand docking method to explore the novel scaffold of potential Aurora Binhibitors. One thousand compounds from our in-house compound library were screened against the Aurora Bstructure and one compound, (E)-3-((E)-4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one(designated herein as HOI-07) was selected for further study. HOI-07 potently inhibited in vitro Aurora B kinaseactivity in a dose-dependent manner, without obvious inhibition of another 49 kinases, including Aurora A. Thiscompound suppressed Aurora B kinase activity in lung cancer cells, evidenced by the inhibition of thephosphorylation of histone H3 on Ser10 in a dose- and time-dependent manner. This inhibition resulted inapoptosis induction, G2–Marrest, polyploidy cells, and attenuation of cancer cell anchorage-independent growth.Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of cancer cells to HOI-07.Results of an in vivo xenograft mouse study showed that HOI-07 treatment effectively suppressed the growth ofA549 xenografts, without affecting the body weight of mice. The expression of phospho-histone H3, phospho-Aurora B, andKi-67was also suppressed in theHOI-07 treatment group. Taken together, we identifiedHOI-07 as aspecific Aurora B inhibitor, which deserves further investigation. Cancer Res; 73(2); 1–9. �2012 AACR.

IntroductionTargeting the progression of mitosis is a highly successful

strategy for anticancer treatment (1). A closely related sub-group of 3 serine/threonine kinases, the Aurora kinases, arebelieved to play a key role in protein phosphorylation inmitosisand have been shown to contribute in the development andprogression of cancer. In mammals, the Aurora kinase familycomprises 3 members: Aurora A, B, and C (2). They displaydistinct roles during mitosis, which are reflected in theirsubcellular locations and functions. Aurora A is localized atthe centrosome from the time of centrosome duplicationthrough mitotic exit. It has been implicated in several pro-

cesses required for the generation of bipolar spindle apparatus,including centrosomematuration and separation (3, 4). Small-molecule inhibition of Aurora A kinase activity causes defectsin centrosome separation with the formation of characteristicmonopolar spindles (5). Aurora B is localized to the centro-meres from the prophase to the metaphase–anaphase transi-tion. Thereafter, it is localized to midzone spindle microtu-bules during the telophase and subsequently to midbodyduring cytokinesis (2, 3). Aurora B is a chromosomal passengerprotein in complex with the inner centromere proteins(INCENP), survivin, and borealin. During mitosis, as the "equa-torial kinase," Aurora B is required for histone H3 phosphor-ylation, chromosome biorientation, the spindle assemblycheckpoint, and cytokinesis (6, 7). Inhibition of Aurora B kinaseactivity with small molecules leads to failure in cytokinesis andabnormal exit from mitosis, resulting in endoreduplication,polyploidy cells, and ultimately apoptosis (8, 9). Aurora C is alsoa chromosomal passenger protein considered to have a similarsubcellular location as Aurora B. It has been described only inmammals, where it is expressed in testis and certain tumor celllines and localizes to spindle poles during late mitosis (2, 10).

Inhibition of Aurora kinases had been shown to be aneffective strategy for anticancer therapy, and several Aurorainhibitors have been described, including VX-680 (11), Hesper-adin (8), AZD1152 (12), andMLN8237 (13). More than 30 small-molecule Aurora kinase inhibitors are currently in differentstages of preclinical and clinical development (14); however,none have yet been approved by the U.S. Food and DrugAdministration for clinical use.

Authors' Affiliations: 1The Hormel Institute, University of Minnesota,Austin, Minnesota; and 2National Cancer Institute, Bethesda, Maryland;and 3State Key Laboratory of Drug Research, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Beijing, China

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

H. Xie, M.-H. Lee, F. Zhu, and K. Reddy contributed equally to this work.

Current address for H. Xie: State Key Laboratory of Drug Research,Shanghai Institute of Materia Medica, Chinese Academy of Sciences,Beijing, China.

Corresponding Author: Zigang Dong, The Hormel Institute, University ofMinnesota, 801 16thAvenueNE, Austin,MN55912. Phone: 507-437-9600;Fax: 507-437-9606; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-2784

�2012 American Association for Cancer Research.

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Published OnlineFirst November 1, 2012; DOI: 10.1158/0008-5472.CAN-12-2784

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Herein, we used a ligand docking computational method toidentify (E)-3-((E)-4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one (i.e., HOI-07) as a novel Aurora Bkinase inhibitor. Biologic testing further confirmed thatHOI-07 selectively and potently inhibited Aurora B activityand exhibited antitumor activity in vitro and in vivo.

Materials and MethodsReagents and materials

Compound HOI-07 was synthesized in-house followingthe reported protocol of similar compounds but with somemodifications (15). All cell lines were purchased from Amer-ican Type Culture Collection (ATCC) and were cultured inmonolayers at 37�C in a 5% CO2 incubator according toATCC protocols. Cells were cytogenetically tested andauthenticated before the cells were frozen. Each vial offrozen cells was thawed and maintained for about 2 months(10 passages). For transfection experiments, the jetPEI(Qbiogene, Inc.) transfection reagent was used following themanufacturer's instructions.

Anchorage-independent cell transformation assayTumor cells were suspended in Basal Medium Eagle media

and added to 0.6% agar, with different concentrations of HOI-07 in a base layer and a top layer of 0.3% agar. The cultureswere maintained at 37�C in a 5% CO2 incubator for 1 to 2weeks, and then colonies were counted under a microscopeusing the Image-Pro Plus software (v.4) program (MediaCybernetics).

Cell-cycle and apoptosis analysesCells were plated in 60-mm plates and treated or not treated

with HOI-07 for the indicated time. At each time point, cellswere fixed in 70% ethanol and stored at �20�C for 24 hours.After staining, cell-cycle distribution or apoptosis was deter-mined using a BD FACSCalibur Flow Cytometer (BD Bio-sciences).

MTS assayTo estimate the cytotoxicity of HOI-07, cells were seeded

(8� 103 cells per well) in 96-well plates and cultured overnight.

Figure 1. Computational predicted binding mode of HOI-07 with Aurora B and the cytotoxicity of HOI-07. A, chemical structure of HOI-07. B, computationalpredicted binding mode of HOI-07 with Aurora B. Left, HOI-07 is docked, using Glide, into the binding pocket of Aurora B. Aurora B is shown as light bluecartoon and HOI-07 is shown as sticks. Right, interaction between HOI-07 and Aurora B. HOI-07 forms a hydrogen bondwith Ala173. C and D, cytotoxicity ofHOI-07 on MRC-5 normal lung cells (C) and A549 lung cancer cells (D). No obvious cytotoxicity by HOI-07 was observed in either MRC-5 and A549 cells atconcentrations below 1 mmol/L.

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Cells were then fed with fresh medium and treated withdifferent doses of HOI-07. After culturing for various times,the cytotoxicity of HOI-07 was measured using an MTS assaykit (Promega) according to the manufacturer's instructions.

Western blot analysisProteins were resolved by SDS-PAGE and transferred onto

polyvinylidene difluoride membranes (Millipore), which wereblocked with nonfat milk and hybridized with specific primaryantibodies. The protein bands were visualized using anenhanced chemiluminescence reagent (GE Healthcare) afterhybridizationwith a horseradish peroxidase (HRP)-conjugatedsecondary antibody.

Aurora B and Aurora A in vitro kinase assaysInactive histone 3 proteins (1 mg) were used as the substrate

for an in vitro kinase assay with 100 ng of active Aurora B orAurora A kinase. Reactions were carried out in1� kinase buffer[25 mmol/L Tris-HCl pH 7.5, 5 mmol/L b-glycerophosphate, 2mmol/L dithiothreitol (DTT), 0.1 mmol/L Na3VO4, 10 mmol/LMgCl2, and 5 mmol/L MnCl2] containing 100 mmol/L ATP at30�C for 30 minutes. Reactions were stopped and proteinsdetected by Western blotting.

Immunofluorescence microscopyA549 cells were seeded in 4-chamber slides and cultured

overnight. The cells were then treated with dimethyl sulfoxide(DMSO) or HOI-07 (1 mmol/L) for 48 hours at 37�C. Aftertreatment, the cells were washed with PBS and fixed withmethanol for 12 hours, followed by blocking with 3% PBS for1 hour. The cells were then incubated with an a-tubulinantibody (1:100) overnight, and DNA was stained with 40,6-diamidino-2-phenylindole (DAPI, Pierce) for 30 minutes atroom temperature. The cells were evaluated by fluorescentmicroscopy.

Hematoxylin–eosin staining and immunohistochemistryTumor tissues frommice were embedded in a paraffin block

and subjected to hematoxylin and eosin (H&E) staining andimmunohistochemistry. Tumor tissues were deparaffinizedand hydrated, then permeabilized with 0.5% Triton X-100/1PBS for 10minutes, hybridizedwith phospho-histoneH3 (1:50),phospho-Aurora B (1:50), and Ki-67 (1:500) as the primaryantibodies and an HRP-conjugated goat anti-rabbit antibodywas used as the secondary antibody. After developing with 3,30-diaminobenzidine, the sections were counterstained withhematoxylin. All sections were observed by microscope(�400 magnification) and the Image-Pro Plus software (v.4)program (Media Cybernetics).

Xenograft mouse modelAthymic nudemice [Cr:NIH (S), NIHSwiss nude, 6-week-old]

were obtained from Harlan Laboratories and maintainedunder "specific pathogen-free" conditions based on the guide-lines established by the University of Minnesota (Austin, MN)Institutional Animal Care and Use Committee. Mice weredivided into different groups (n ¼ 10 in each group). A549lung cancer cells (3 � 106/0.1 mL) were injected subcutane-

ously into the right flank of each mouse. HOI-07 was preparedonce a week and protected from light and kept at 4�C.Compound or vehicle control was administered by intraper-itoneal injection twice a week. Tumor volumes and bodyweights were measured.

Statistical analysisAll quantitative data are expressed as mean values � SD or

SE, and significant differences were determined by the Studentt test or by one-way ANOVA. P < 0.05 was used as the criterionfor statistical significance.

Figure 2. HOI-07 inhibits the anchorage-independent growth of a panel ofNSCLC cell lines, including A549 cells (A), H1650 cells (B), andH520 cells(C). The asterisk indicates a significant (�, P < 0.05; ��, P < 0.01) decreasein colony formation in cells treated with HOI-07 compared with theDMSO-treated group. D, HCT116 p53�/� cells are more sensitive to theeffects of HOI-07 than wild-type HCT116 p53þ/þ cells. The asteriskindicates a significant (�, P < 0.05; ��, P < 0.01) difference in anchorage-independent growth inHCT116 p53þ/þ colon cancer cells comparedwithHCT116 p53�/� cells.

HOI-07 Is a Novel Aurora B Inhibitor

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ResultsThepredictedbindingmodeofHOI-07withAuroraBandcytotoxicity

With the purpose of identifying a novel Aurora B kinaseinhibitor, we conducted an intensive molecular docking anal-ysis using Glide v5.7 (16) to screen our in-house library ofcompounds against the structure of Aurora B. HOI-07 (Fig.1A) was identified as a potential Aurora B inhibitor based onits high docking score. HOI-07 is a novel compound syn-thesized in our laboratory. The predicted binding mode ofHOI-07 and Aurora B showed that HOI-07 occupies the ATP-binding site and forms a hydrogen bond with amino acidAla173 in the hinge linker region, which is quite similar tothe binding mode of other Aurora B kinase inhibitors(Fig. 1B). We then examined the toxicity of HOI-07 on both

MRC-5 normal lung cells (Fig. 1C) and A549 lung cancer cells(Fig. 1D). The result showed that HOI-07 possessed sub-stantial toxicity to both cell types at concentrations greaterthan 10 mmol/L. At a concentration of 1 mmol/L or less, noobvious cytotoxic effects were observed in either cell line. At5 mmol/L, HOI-07 treatment for 48 hours resulted in a weaktoxicity toward A549 cancer cells but not to MRC-5 normalcells.

HOI-07 inhibits anchorage-independent growth ofhuman lung cancer cells

We then examined the effect of HOI-07 treatment onanchorage-independent growth of human lung cancer cells,including A549 (Fig. 2A), H1650 (Fig. 2B), and H520 (Fig. 2C)cells. Treatment of these cells with HOI-07 potently inhibited

Figure 3. HOI-07 inhibits Aurora Bkinase activity both in vitro and exvivo. A, HOI-07 inhibits Aurora Bin vitro kinase activity in aconcentration-dependent manner.An inactive histone 3.3 protein wasused as the substrate for an in vitrokinase assay with active Aurora Band 100mmol/LATP. Proteinswereresolved by SDS-PAGE anddetected by Western blotting. B,HOI-07 has no effect on Aurora Ain vitro kinase activity. C, HOI-07inhibits phosphorylation of theAurora B downstream target,histone H3 (Ser10) in human lungcancer cells in a time- (left) anddose-dependent (right) manner.A549 cells were treated with HOI-07 at the indicated concentrationfor 24 hours or at 1 mmol/L andharvested at the indicated timepoints. Cells were harvested andhistone proteins were extractedand subjected to Western blotanalysis.

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their anchorage-independent growth in a concentration-dependent manner. HOI-07 at 0.5 or 1 mmol/L caused adecrease of more than 80 or 90% compared with control inall cell lines detected. The inhibition by HOI-07 was not dueto cytotoxicity because no toxicity was observed at 1 mmol/LHOI-07 (Fig. 1). Therefore, the results indicated that HOI-07 isa novel and very potent compound possessing antitumoractivity and deserves further investigation. We also exam-ined the effect of HOI-07 in a pair of colon cancer cell lines,p53 wild-type (HCT116 p53þ/þ) and p53-deficient (HCT116p53�/�) cells to determine whether the sensitivity of cells toHOI-07 directly correlates with the status of p53 in cells. Softagar assay results showed that HCT116 p53�/� cells aremore sensitive to HOI-07 than HCT116 p53þ/þ cells (Fig. 2D).HOI-07 at 0.05 mmol/L caused a 40% inhibition of growth ofHCT116 p53�/� cells, whereas no inhibition was observed inthe wild-type p53 cells. Moreover, 0.1 mmol/L HOI-07 inhib-

ited growth by more than 95% in HCT116�/� cells but only55% inhibition in HCT116 p53þ/þ cells. These data areconsistent with a previous report (9) showing that p53�/�

cells are more sensitive to Aurora B inhibitors comparedwith p53þ/þ cells.

HOI-07 is apotent inhibitor ofAuroraB, butnotAuroraAWe then used an in vitro kinase assay with a recombinant

Aurora B protein and various concentrations of HOI-07 todetermine whether HOI-07 could inhibit Aurora B kinaseactivity. Results indicated that the phosphorylation of histoneH3 on Ser10, an Aurora B substrate, was strongly inhibited byHOI-07 in a concentration-dependent manner (Fig. 3A). Forexample, 0.05 mmol/L HOI-07 caused a 22% inhibition ofAurora B kinase activity and 0.1 mmol/L HOI-07 resulted ina 50% inhibition. At a concentration of 1 mmol/L, only a weakhistone H3 (Ser10) band was observed. In addition, we also

Figure 4. HOI-07 inducespolyploidy, apoptosis, and G2–Mphase cell-cycle arrest in lung cancercells. A, HOI-07 induces polyploidy inA549 cells. Cells were treated withHOI-07 (1 mmol/L) for 48 hours andthen were evaluated byimmunofluorescence assay. Scalebar indicates 20 mm (�600). B and C,HOI-07 induces G2–M arrest in lungcancer cells. Cell-cycle analysis byflow cytometry of A549 cells (B) andH520 cells (C) treated with HOI-07. Dand E, HOI-07 induces apoptosis inlung cancer cells. Flow cytometricanalysis of apoptosis. A549 cells (D)and H520 cells (E) were incubatedwith the indicated concentration ofHOI-07 for 72 hours, then collectedand apoptosis was detected byAnnexin V and PI staining.






examined the effect of HOI-07 on Aurora A kinase activityusing an in vitro kinase assay, and no effect on histone H3(Ser10) phosphorylation was observed at 1 mmol/L HOI-07compared with control (Fig. 3B). These results continued tosupport HOI-07 as a potent inhibitor of Aurora B kinaseactivity.

HOI-07 blocks phosphorylation of histoneH3 on Ser10 inlung cancer cells

To provide evidence showing that HOI-07 is acting byinhibiting Aurora B in cancer cells, we examined its effectson Aurora B downstream signaling. Evidence has shown thathistone H3 is a direct downstream target of the Aurora kinases.Phosphorylation of a highly conserved serine residue (Ser10) inhistone H3 is thought to be crucial for entry into mitosis. Ourresults showed that HOI-07 suppresses histone H3 phosphor-

ylation on Ser10 in cancer cells in a dose- and time-dependentmanner (Fig. 3C), suggesting that the Aurora B kinases areinvolved in the antitumor activity of HOI-07.

HO1-07 induces polyploidy, cell-cycle arrest, andapoptosis in lung cancer cells

Aurora B inhibition leads to failure in cytokinesis andabnormal exit from mitosis, which could result in polyploidycells, cell-cycle arrest, and ultimately, apoptosis. The ability toinduce polyploidy cells was further examined in A549 cellstreated or not treated with HOI-07. Immunofluorescenceresults showed that treatment of A549 cells with 1 mmol/LHOI-07 caused the induction of polyploidy cells, whereas nopolyploidy cells were observed in control cells (Fig. 4A). Inaddition, HOI-07 treatment for 48 hours caused an increase inthe number of A549 (Fig. 4B) andH520 cells (Fig. 4C) occupyingthe G2–M phase. Moreover, exposure of these cells to HOI-07for 72 hours induced apoptosis as measured by Annexin V/propidium iodide (PI) staining (Fig. 4D and E). For example,exposure to 5mmol/LHOI-07 induced 48.6%or 83.4%apoptosisin A549 cells and H520 cells, compared with 8% or 41.3% inuntreated control cells, respectively. These results showed thatHOI-07, as an Aurora B inhibitor, induces polyploidy, apopto-sis, and G2–Mphase arrest in cancer cells and thus inhibits thegrowth of cancer cells.

Knockdown of Aurora B decreases the sensitivity ofcancer cells to HOI-07

We then examined whether knocking down Aurora Bexpression influences the sensitivity of A549 cancer cells toHOI-07. The efficiency of short hairpin RNA (shRNA) knock-down was examined, and the expression of Aurora B wasobviously decreased after shRNA transfection (Fig. 5A). More-over, the growth of cells in soft agar also decreased aftertransfection compared with the mock group (Fig. 5B). HOI-07 (0.5 mmol/L) inhibited anchorage-independent growth ofA549 cells transfected with mock shRNA by about 90%. Incontrast, the inhibition was less than 40% in A549 cellstransfected with Aurora B shRNA, indicating that A549 cellstransfected with Aurora B shRNAs were more resistant to HOI-07 treatment (Fig. 5B). These results suggested that Aurora Bplays an important role in the sensitivity of A549 cells to theantiproliferative effects of HOI-07.

Kinase profile resultTo identify other potential targets of HOI-07, a kinase profile

assay was conducted by Millipore, in which 49 kinases wereexamined. Only Akt2 activity was suppressed more than 50%after 5 mmol/L HOI-07 treatment, compared with an untreatedcontrol (Supplementary Table S1). In addition, the kinase acti-vity of Aurora A was decreased less than 25% at 5 mmol/LHOI-07.

HOI-07 inhibits Akt1 and 2 kinase activities in vitro, butnot in cells

According to the kinase profiler assay results, Akt2 alsomight be a potential target of HOI-07. We conducted Akt1 and

Figure 5. Knockdown of Aurora B in A549 cells decreases their sensitivityto HOI-07. A, efficiency of Aurora B shRNA in A549 cells. B, anchorage-independent growth of A549 cells transfected with mock shRNA orAurora B shRNA, treated or not treated with HOI-07.

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Akt2 in vitro kinase assays and results indicated that HOI-07inhibited both Akt1 (Supplementary Fig. S1A) and Akt2 (Sup-plementary Fig. S1B) in vitro kinase activities in a concentra-tion-dependent manner. Treatment with 5 mmol/L HOI-07caused a 32% or 52% inhibition of Akt1 or Akt2 activity,respectively, which is quite consistent with the kinase profilerassay result. However, Western blot analysis of A549 cells(Supplementary Fig. S1C) and H520 cells (Supplementary Fig.S1D) treated with HOI-07 showed that HOI-07 treatment hadno effect on the phosphorylation of Akt or its downstreamsignaling molecules, including Akt (Ser473), GSK3b (Ser9),p70S6K (Thr389), or S6 (Ser235,236). These results indicatedthat although HOI-07 can inhibit Akt1 and Akt2 in vitro kinaseactivity, it has no effect on Akt activity in cancer cells, suggest-ing that Akt1 and Akt2might not bemajor antitumor targets ofHOI-07.

HOI-07 suppresses the growth of A549 xenografts in vivoWe then evaluated the ability of HOI-07 to inhibit the growth

of human A549 lung cancer cell xenografts in athymic nudemice. Tumor volumes were measured twice a week, and mouseweights were determined once a week. HOI-07 caused amarkedreduction in tumor size in the human A549 xenograft model(Fig. 6A). Inmice treated with HOI-07 at 20mg/kg, twice a weekintraperitoneally, mean tumor volumes were reduced to 164mm3 in comparison with control group (408 mm3; P < 0.01). Inaddition, no obvious loss of body weight was observed (Fig. 6B),indicating that HOI-07 is well tolerated by the mice. Moreover,the effects of HOI-07 on phosphorylation of histone H3 andAuroraB andKi-67 a tumor proliferationmarkerwere evaluatedby immunohistochemistry and H&E staining of A549 tumortissues after the 31 days of treatment. The expression of all 3,including Ki-67, was markedly decreased by HOI-07 (Fig. 6C).

Figure 6. Effect of HOI-07 on lungcancer growth in an A549 xenograftmousemodel. A,HOI-07 significantlysuppresses cancer growth in anA549 xenograft mouse model. Theaverage tumor volume of vehicle-treated control mice (n ¼ 10) andHOI-07-treated mice (n ¼ 10 in eachgroup) plotted over 31 days aftertumor cell injection. The asterisksindicate a significant inhibition byHOI-07 on tumor growth (�, P < 0.05;��, P < 0.01). B, HOI-07 has no effecton mouse body weight. Bodyweights from treated or untreatedgroups of mice were measured oncea week. C, andimmunohistochemical analysis oftumor tissues. Treated or untreatedgroups of mice were euthanized andtumors were extracted. Tumor tissueslides were prepared with paraffinsections after fixation with formalinand then stained with H&E orindicated antibodies. Expressionswere visualized with a lightmicroscope. Stained cells werecounted from5separate areas on theslide and an average of 3 sampleswas calculated per group. Data areexpressed as mean percent ofcontrol�SD.Theasterisk indicatesasignificant decrease inphosphorylated histone H3 (Ser10;left), phosphorylated Aurora B(middle), andKi-67 expression (right).






These results indicated that HOI-07 suppressed tumor growthin vivo.

DiscussionCancer is a disease that is characterized by uncontrolled

proliferation of abnormal cells. Modulation of atypical cell-cycle regulation would therefore be a valuable therapeuticstrategy for different types of tumors. Aurora kinases reg-ulate many processes during cell division. Aurora B kinasesare essential for chromosome condensation, kinetochorefunction, cytokinesis, and the proper function of the spindleassembly checkpoint when spindle tension is perturbed(2, 6, 9, 17). Accumulating evidence has shown that AuroraB is implicated in cancer. For example, the expression ofAurora B is frequently elevated in various types of cancer,including non–small cell lung cancer (NSCLC), colon, andprostate (18–20). The evidence linking Aurora overexpres-sion and malignancy has generated significant interest in thedevelopment of small-molecule inhibitors against theseproteins.

Oxindoles (indolin-2-ones) are an important class of mole-cules, which are known to possess a wide variety of biologicproperties, and in particular, as protein kinase inhibitors (21).In the present study, HOI-07 was identified, using moleculardocking methods, as an Aurora B kinase inhibitor. The struc-ture of the molecule HOI-07 is oxindole-based; however, it isstructurally different from reported oxindole-containing Auro-ra inhibitors. Up until now, only one molecule, which is similarto HOI-07, has been reported in the literature, but without anyknown biologic activity (15).

The results of an Aurora B kinase assay clearly showedthat HOI-07 potently and dose dependently inhibited AuroraB kinase in vitro activity, indicating that this compound is apotent and novel Aurora B inhibitor. In addition, HOI-07 hadno effect on Aurora A kinase in vitro activity at the sameconcentration. A previous report showed that cells treatedwith an Aurora kinase inhibitor entered and exited mitosiswithout cell division and then proceeded to a second S-phase (9). Therefore, we further examined the effect of HOI-07 on cancer cells and results showed that HOI-07 sup-pressed cell growth in a panel of NSCLC cell line. Theinhibition was associated with induction of polyploidy cells,accumulation of G2–M cells, as well as apoptosis, which isconsistent with Aurora B inhibition. Moreover, knockingdown Aurora B expression in A549 cells decreased theirsensitivity to HOI-07, indicating that Aurora B plays animportant role in the antitumor activity of HOI-07. An invivo xenograft study also indicated that HOI-07 effectivelysuppressed tumor growth without affecting mouse bodyweight and was accompanied with a decrease in Ki-67expression, which is a marker of proliferation. HOI-07 treat-ment also decreased phosphorylation of histone H3 (Ser10)and Aurora B in tumor tissues.

According to our kinase profiling results, in which 49kinases treated or not treated with HOI-07 were examined,Akt might also be a potential target of HOI-07. Furtherexperimental results showed that HOI-07 inhibited Akt1 andAkt2 kinase activity in vitro at a higher concentration (1

mmol/L or more) than that required for Aurora B inhibition(1 mmol/L or lower). However, it had no effect on Aktdownstream signaling in cancer cells, indicating that Aktmight not be a major target of HOI-07. Together, theseresults indicated that HOI-07 is a potent and selectiveinhibitor of Aurora B. More than 30 Aurora kinase inhibitorsare in different stages of preclinical and clinical developmentcurrently (22). Most of the Aurora inhibitors are pan-Aurorainhibitors or Aurora A selective inhibitors, whereas only limitedAurora B selective inhibitors have been reported, includingcompound AZD1152, which also inhibits Aurora A at higherconcentrations (IC50 of 0.37 vs. 1,368 nmol/L for Aurora B andAurora A kinases, respectively; 22). Here, we identified HOI-07as an Aurora kinase inhibitor that is specific for the Aurora Bisoform. Previous reports showed that treatment of cells withdual inhibitors of Aurora A and B results in mitotic defectresembling inactivation of Aurora B. This might be caused bythe potential mechanism by which Aurora B is responsible formitotic arrest in the absence of Aurora A (17). Meanwhile,several studies showed that Aurora A and B should be distin-guished as 2 distinctive therapeutic objectives that can betargeted independently (23, 24). Thus, our identification ofHOI-07 as a specific Aurora B inhibitor provides one moreuseful tool to study the phenomenon of Aurora B–specificinhibition and to distinguish the potential differences betweeninactivation of Aurora A and B. Our work also presents a novelAurora B selective inhibitor, which suppresses tumor growthboth in vitro and in vivo and deserves further investigation anddevelopment.

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

Authors' ContributionsConception and design: H. Xie, M.-H. Lee, F. Zhu, R.A. Lubet, Z. DongDevelopment of methodology: H. Xie, M.-H. Lee, S. Kang, W.-Y. MaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H. Xie, M.-H. Lee, D.Y. Lim, H. Li, W.-Y. Ma, A.M. BodeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H. Xie, M.-H. Lee, F. Zhu, Y. Li, D.Y. Lim, X. Li, H. Li, A.M. Bode, Z. DongWriting, review, and/or revision of the manuscript: H. Xie, M.-H. Lee, R.A.Lubet, J. Ding, A.M. Bode, Z. DongAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Xie, C. Peng, D.-J. Kim, X. Li, W.-Y.Ma, A.M. BodeStudy supervision: Z. DongSynthesis of molecules: K. Reddy

AcknowledgmentsThe authors thank Todd Schuster and Alyssa Langerud at The Hormel

Institute, University of Minnesota (Austin, MA) for their technical support andTonyaM.PoormanatTheHormel Institute forhelp in submittingourmanuscript.

Grant SupportThis work was supported by The Hormel Foundation, NIH grants R37

CA081064, CA120388, ES016548, and CA0227501 and National Cancer InstituteContract No. HHSN-261200533001C-NO1-CN-53301.

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 July 13, 2012; revised October 16, 2012; accepted October 25, 2012;published OnlineFirst November 1, 2012.

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Published OnlineFirst November 1, 2012.Cancer Res Hua Xie, Mee-Hyun Lee, Feng Zhu, et al. Aurora B IsoformIdentification of an Aurora Kinase Inhibitor Specific for the

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