Targeting Aurora Kinase with MK-0457 Inhibits Ovarian Cancer Growth Yvonne G

CancerTherapy: Preclinical

Targeting Aurora Kinase with MK-0457 Inhibits Ovarian Cancer Growth Yvonne G. Lin,1Anand Immaneni,4 William M. Merritt,1Lingegowda S. Mangala,1SeungWook Kim,1 Mian M.K. Shahzad,1, 5 Yvonne T.M. Tsang,1Guillermo N. Armaiz-Pena,1, 6 Chunhua Lu,1 Aparna A. Kamat,1Liz Y. Han,1WhitneyA. Spannuth,1Alpa M. Nick,1Charles N. Landen, Jr.,1 Kwong K.Wong,1Michael J. Gray,2 Robert L. Coleman,1Diane C. Bodurka,1 William R. Brinkley,4 and Anil K. Sood1, 3

Abstract Purpose:TheAurora kinase family plays pivotal roles in mitotic integrity and cell cycle.We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457. Experimental Design: We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models. Results: In vitro cytotoxicity assays with HeyA8 and SKOV3ip1cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values < 0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P V 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P values < 0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by f3-fold (P < 0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis. Conclusions: Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer.The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials.

Authors’ Affiliations: Departments of 1Gynecologic Oncology, 2Surgical he Aurora family of serine/threonine kinases is essential for 3 T Oncology, and Cancer Biology,The University of Texas M. D. Anderson Cancer many cellular functions including high-fidelity progression Center; Departments of 4Molecular and Cellular Biology and 5Obstetrics and Gynecology, Baylor College of Medicine; 6Program in Cancer Biology, University through mitosis (1–4). Aurora-A is located on chromosome ofTexas Graduate School of Biomedical Sciences at Houston, Houston,Texas 20q13.2-q13.3 and is required for centrosome separation and Received 11/16/07; revised 2/27/08; accepted 3/4/08. maturation as well as proper mitotic spindle formation and Grant support: National Cancer Institute/Department of Health and Human function (1, 4, 5). Although the Aurora-A kinase activity is Services/NIH Training of Academic Gynecologic Oncologists GrantT 32-CA101642 cell cycle dependent and highest during G2-M, phosphorylation (Y.G. Lin, W.M. Merritt, W.A. Spannuth, and A.M. Nick); Reproductive Scientist 288 Development Program (C.N. Landen, Jr.); Dan Duncan Cancer Center at Baylor of human Aurora-A kinase, particularly at the Thr residue, College of Medicine and NIH grant CA41424 (W.R. Brinkley); and Marcus appears to be requisite for enhanced kinase activity (3). Foundation, NIH grants CA109298 and CA110793, The Betty Ann Asche Murray Following the G2-M phase of the cell cycle, degradation of Distinguished Professorship, and The University of Texas M. D. Anderson Cancer Aurora-A kinase is mediated by the ubiquitin-proteasome Center Specialized Program of Research Excellence in Ovarian Cancer grant P50CA083639 (A.K. Sood). pathway (5). The ability of Aurora-A kinase to induce The costs of publication of this article were defrayed in part by the payment of page multipolar spindles by overriding the mitotic spindle check- charges. This article must therefore be hereby marked advertisement in accordance points and transforming fibroblasts into aneuploid cells with 18 U.S.C. Section 1734 solely to indicate this fact. supports its role as a potential oncogene (1, 5). Aurora-B Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). kinase, located on chromosome 17p13.1, is a ‘‘chromosomal Y.G. Lin and A. Immaneni contributed equally to this project. passenger’’ protein that plays an important role in regulating Requests for reprints: Anil K. Sood, Ovarian Cancer Research, Department of mitosis, particularly cytokinesis. Aurora-C kinase is not as well Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center, understood but appears to have functions during mitosis that 1155 Herman Pressler, Unit 1352, Houston,TX 77030. Phone: 713-745-5266; Fax: overlap with Aurora-B kinase (3, 5). 713-792-7586; E-mail: [email protected]. F 2008 American Association for Cancer Research. We reported recently that Aurora-A is overexpressed in 83% doi:10.1158/1078-0432.CCR-07-4922 of human epithelial ovarian carcinomas and predicts poor

www.aacrjournals.org 5437 Clin Cancer Res 2008;14(17) September 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. CancerTherapy: Preclinical clinical outcome (6). Furthermore, the chromosome 20 ampli- Functional assays of Aurora kinase inhibition. Because Aurora-A 288 con corresponding to the Aurora-A gene location has been kinase autophosphorylation on Thr as well as phosphorylation of histone H3 on Ser10 and the centromeric histone variant, Cenp-A on reported in not only ovarian cancer cell lines but also in 54% to 7 100% of sporadic and hereditary human ovarian carcinomas Ser , are important indicators of Aurora kinase activity during mitosis, we assayed for these known mitosis-specific functions. Twenty- (7, 8). Although Aurora-A kinase overexpression has also been thousand HeyA8 and SKOV3ip1 ovarian carcinoma cells were plated correlated with centrosome amplification (6, 8), distinct poly- in 6 cm plates and allowed to adhere overnight. All plates were then morphisms within the Aurora-A kinase gene locus are also treated with the G2-M blocker, demecolcine solution (Sigma-Aldrich), associated with f20% increased risk of invasive ovarian cancer for 8 h. Mitotic cells were collected by mitotic shake-off, washed in fresh (9), further implicating Aurora-A kinase in tumor development. medium, and then released into new medium containing MK-0457

Through mechanisms including Akt activation and checkpoint (10, 100, 500, or 1,000 nmol/L) for 1 h at 37jC and 5% CO2.One dysregulation, Aurora-A kinase has also been implicated in plate maintained in demecolcine served as a control. Cells were protecting cells from apoptosis induced by traditional chemo- collected and washed in PBS. Cell pellets were lysed directly in NP-40 therapy agents, including mainstay cytotoxic agents against sample disruption buffer and separated on a 4% to 12% gradient ovarian cancer such as cisplatin and paclitaxel (10, 11). polyacrylamide gel electrophoresis (Invitrogen). Proteins were trans- ferred onto Immobilon-P membranes (Millipore) for 1 h using Moreover, Sun et al. have recently shown that inhibition of standard Western blot methods. Immobilized proteins were blocked Aurora kinase can sensitize SKOV3 cells to traditional chemo- in 5% milk/PBS with 0.1% Tween 20 and incubated overnight at 4jC n therapeutic agents via NF- B down-regulation, further support- with antibodies against phospho-Aurora-A (Thr288; 1:1,000; Abcam), ing the therapeutic role of Aurora kinase targeting in oncology phospho-histone H3 (Ser10; 1:1,000; Upstate), and phospho-Cenp-A (12). Recent reports have emerged highlighting the role of (Ser7; 1:1,000; Upstate). After washing and incubating with the Aurora-B in maintaining the spindle assembly checkpoint corresponding secondary antibodies, blots were developed using and supporting it as a valid and individual therapeutic target enhanced chemiluminescence (Amersham) reagents. (13–15). Western blot. Whole-cell lysates were used for Western blot analysis in vitro Given the high prevalence of Aurora kinase overexpression in to characterize the kinetics of Aurora kinase inhibition by MK- ovarian cancer and its diverse protumorigenic roles, inhibiting 0457. One million HeyA8 cells were plated onto 10 cm plates and allowed to adhere overnight. Cells were then treated with MK-0457 the Aurora kinase family appears to be an attractive therapeutic (10 nmol/L) for 5, 10, and 30 min and 1, 2, 4, 6, and 12 h. Cell lysates goal, particularly as ovarian cancer remains the leading cause of were prepared by incubating plates on ice for 20 min with 1Â modified death from gynecologic cancer (16). Based on the role Aurora radioimmunoprecipitation assay lysis buffer with 1Â protease inhibitor kinase plays during the cell cycle, we examined the effects of (Roche) supplemented with sodium orthovanadate. After centrifuging pan-Aurora kinase inhibition using a highly selective small- at 13,000 rpm for 20 min at 4jC, the supernatant was collected and molecule inhibitor, MK-0457 (formerly known as VX-680), stored at -80jC until ready for use. on ovarian cancer growth in preclinical orthotopic models Western blotting for phospho-Aurora-A (Thr288) and total Aurora-A of metastatic ovarian carcinoma using both chemotherapy- was done using 20 Ag total protein as determined by BCA Protein Assay sensitive and resistant cell lines. Kit (Pierce Biotechnology). After separation by 12% SDS-PAGE with wet transfer onto a nitrocellulose membrane, probing was done using an anti-phospho-Aurora-A (Thr288) antibody (1:1,000) and anti-total Aurora-A antibody (1:1,000, Bethyl Laboratories). Visualization was Materials and Methods achieved using a horseradish peroxidase–conjugated anti-rabbit (1:2,000; Amersham) antibody and enhanced chemiluminescence. Cell lines. To study the effects of Aurora kinase inhibition in Equal loading was verified using h-actin. ovarian carcinoma, we used two highly metastatic chemosensitive Cytotoxicity assay. The cytotoxic effects of Aurora kinase inhibition human ovarian cancer cell lines, HeyA8 and SKOV3ip1. Because most on tumor cells were determined using the 3-(4,5-dimethylthiazol-2-yl)- patients with recurrent ovarian cancer develop chemotherapy-resistant 2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) uptake method disease, we also used the taxane- and platinum-resistant human ovarian as described previously (20). Briefly, 1,000 HeyA8 or 2,000 SKOV3ip1 cancer cell lines, HeyA8-MDR and A2780-CP20, respectively. The cells in RPMI 1640 + 15% fetal bovine serum were seeded into each well derivation and source of these cell lines have been reported elsewhere of a 96-well plate and allowed to adhere overnight. Treatment (17–19). HeyA8, SKOV3ip1, and A2780-CP20 cells were maintained in conditions were conducted in replicates of 5. Cells were then treated monolayer cultures at 37jC in RPMI 1640 supplemented with 15% once with increasing concentrations of MK-0457 at 37jC for 96 h before fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts). 50 AL/well of 0.15% MTT solution were added. After incubation for The HeyA8-MDR cell line, a generous gift from Dr. Isaiah J. Fidler (The 2 h at 37jC, the medium/MTT solution was replaced with 100 AL/well University of Texas M. D. Anderson Cancer Center), was generated by DMSO (Sigma-Aldrich), and the absorbance was measured at 570 nm sequential exposure to increasing sublethal doses of paclitaxel and using a 96-well multiscanner (Dynex Technologies, MRX Revelation). grown in the same medium as the parental cells supplemented with The IC50 was determined by calculating the mean absorbance at 570 nm 300 Ag/mL paclitaxel (Bristol-Myers Squibb). All cell lines were and then identifying the corresponding MK-0457 concentration on the routinely screened for Mycoplasma using MycoAlert (Cambrex Bio dose-response curve using regression analysis. Science) as described by the manufacturer. In vitro and in vivo To characterize effects of combining MK-0457 with docetaxel on experiments were conducted with cell lines at 70% to 80% confluence. tumor cells, MTT assays were done. One thousand HeyA8 or 3,000 MK-0457. Inhibition of Aurora kinase was achieved using the SKOV3ip1 cells per well were seeded into a 96-well plate and allowed to small-molecule pan-Aurora kinase inhibitor, MK-0457, obtained from adhere overnight. Cells were then treated with either 1 or 0 nmol/L Merck & Co. The kinase specificity for this compound has been reported (fresh medium) of MK-0457 for 24 h. Sequential doses of docetaxel previously with reported activity against wild-type and mutated bcr-abl, mixed with medium and MK-0457 (0 or 1 nmol/L) were then including the T313I mutation, as well as JAK2 and FLT3 (1). In vitro administered to the cells for 72 h. MTT assay was then done as above, A experiments were conducted using dilutions from a 2 mmol/L stock of and IC50 levels were determined based on 570 readings. MK-0457 dissolved in DMSO. In vivo studies were conducted using MK- Cell cycle and apoptosis analysis by flowcytometry. Because of the 0457 dissolved in 1:1 PEG300/PBS for i.p. administration. role of Aurora kinase in cell cycle integrity, the ability of MK-0457 to

Clin Cancer Res 2008;14(17) September1,2008 5438 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. Inhibiting Aurora Kinases DecreasesTumorigenesis modulate the cell cycle and affect apoptosis in HeyA8 and SKOV3ip1 Orthotopic inoculation of tumor cells and necropsy. At f75% in vitro was evaluated using flow cytometry. Experimental conditions confluence, HeyA8, SKOV3ip1, HeyA8-MDR, and A2780-CP20 cells were done in replicates of 5. For each cell line, 1 Â 106 cells were seeded were collected from cultures using either 0.25% trypsin-EDTA (Life into 10 cm dishes and allowed to adhere overnight. Cell cultures were Technologies) or 0.1% EDTA depending on the cell line. Cells lifted washed with PBS and then treated with RPMI 1640 (negative control) with trypsin underwent trypsin-neutralization with fetal bovine serum– or medium containing MK-0457 or MK-0457 plus docetaxel. Then, 12, containing medium before being centrifuged and then resuspended in 24, and 48 h after treatment, cells were collected by trypsinization and the appropriate volume of serum-free HBSS (Invitrogen) for animal pooled with floating cells, which consisted of detached mitotic, inoculation. Cell lines not requiring trypsin-neutralization were directly apoptotic, and/or dead cells. After trypsin neutralization with fetal centrifuged at 1,000 rpm for 7 min at 4jC, washed with PBS, and then bovine serum–containing medium, cell suspensions were centrifuged resuspended in serum-free HBSS at the appropriate concentrations for for 5 min at 1,500 rpm at room temperature and then washed with PBS inoculation. HeyA8 cells were injected i.p. at 2.5 Â 105 per 200 AL twice before being fixed in 70% ethanol. Cells were stored at -20jC for HBSS. SKOV3ip1, HeyA8-MDR, and A2780-CP20 cells were injected at least 18 h after fixation. Immediately before analysis, fixed samples i.p. at 1 Â 106 per 200 AL HBSS. were washed with PBS and then resuspended in propidium iodide Long-term therapy experiments were done using all four cell lines. (50 Ag/mL) and RNase A (20 Ag/mL; Phoenix Flow System) for at least Mice were sacrificed when the control group appeared near moribund, 30 min at room temperature protected from light. Stained cells were f3 to 5 weeks after commencing therapy, depending on the cell line. analyzed on an EPICS XL flow cytometer (Beckman-Coulter) within Tumors were harvested from the peritoneal cavities of mice, tumor

2 h of staining. The low-level gate was set at the base of the G1 peak and nodules were quantified, and total tumor weight was determined. the percentages of cells within the G1, S, and G2-M phases of the cell Malignant ascites was aspirated and the volume was measured. cycle were determined by analysis with Multicycle (Phoenix Flow Additional tumor tissue for H&E staining and immunohistochemistry System). was formalin fixed at the time of tumor collection and then paraffin Immunohistochemistry. Phospho-histone H3 (Ser10) and prolife- embedded. Paraffin sections were uniformly cut at 5 Am thickness. rating cell nuclear antigen (PCNA) immunohistochemistry was done on Therapy experiments using MK-0457 in orthotopic murine models. 5-Am-thick, formalin-fixed, paraffin-embedded slides. Deparaffiniza- Dose-finding experiments were done by injecting HeyA8 tumor cells tion was achieved with xylene followed by descending grades of i.p. (2.5 Â 105) into athymic female mice. Nineteen days after tumor ethanol. Antigen retrieval was done by microwave-heated citrate buffer cell injection when i.p. tumors were palpable, the mice were rando- (pH 6.0) for 20 min. Endogenous peroxidases were blocked with 3% mized into three dosage groups: 0 mg (vehicle alone), 25 mg/kg, and

H2O2/methanol for 12 min at room temperature. Nonspecific epitopes 50 mg/kg. Twice daily doses of inhibitor or vehicle were administered were blocked with 10% normal goat serum (for phospho-histone H3) by i.p. injections for 2 days (total four doses, 12 h apart). Mice were or 5% normal horse serum/1% normal goat serum (for PCNA) for sacrificed at 24, 48, and 72 h after the final i.p. injection. Immuno- 30 min at room temperature. Slides were then incubated with anti- histochemistry for phospho-histone H3 was done on the tumors as phospho-histone H3 (Ser10) antibody (1:300) or PCNA clone PC10 described earlier. (1:50; DAKOCytomation) at 4jC overnight. Slides were then developed To determine the antitumor effects of Aurora kinase inhibition, we with either biotinylated goat anti-rabbit (BioCare Medical) for phospho- initiated treatment with MK-0457 and/or cytotoxic chemotherapy histone H3 detection followed by streptavidin-horseradish peroxidase injections 1 week after tumor cell inoculation using a minimal residual (1:300; DAKOCytomation) or rat anti-mouse IgG2a-horseradish disease model (20, 22–25). Docetaxel (Sanofi-Aventis; 35 Ag/mouse peroxidase (1:200; Serotec) for PCNA. Visualization was achieved with for SKOV3ip1 tumor-bearing mice or 50 Ag/mouse for HeyA8 and 3,3¶-diaminobenzidine (Open Biosystems), and counterstaining was HeyA8-MDR), cisplatin (160 Ag/mouse for A2780-CP20; Bristol-Myers done with Gill’s hematoxylin (Sigma-Aldrich). Phospho-histone H3 Squibb), or vehicle was injected i.p. once weekly. Docetaxel was the status was determined as the number of phospho-histone H3-positive chosen taxane given its favorable side-effect profile over paclitaxel in cells averaged over five ‘‘hotspot’’ high-power fields at Â100 per human studies (26–28). MK-0457 was administered twice daily for specimen. Proliferative index was calculated as the proportion of PCNA- 2 days, starting 1 day before treatment with docetaxel or cisplatin. Mice positive cells over five high-power fields at Â200 per specimen. were monitored daily for adverse effects and drug tolerance. All animals To quantify apoptosis, terminal deoxynucleotidyl transferase– were sacrificed and tumors were harvested at necropsy when the control mediated dUTP nick end labeling assay was done on 5-Am thick, mice began to appear moribund, f3 to 4 weeks after the initiation of paraffin-embedded tumor slides as described previously (20, 21). therapy, depending on the cell line used (19). Mouse weight, tumor Briefly, after deparaffinization, all slides were treated with proteinase K weight, tumor distribution, and ascites volume were recorded. (1:500). One DNase-treated specimen served as a positive control. To explore the therapeutic effect of the timing at which Aurora kinase

Then, 3% H2O2/methanol was applied to all specimens to block inhibition occurred relative to cytotoxic chemotherapy treatment, we endogenous peroxidases. After a terminal deoxynucleotidyl transferase employed the in vivo HeyA8 tumor model and initiated MK-0457 buffer (30 mmol/L Trizma, 140 mmol/L sodium cacodylate, 1 mmol/L treatment either 2 days before, 1 day before and with, concurrently and

CoCl2) rinse, all slides were incubated with terminal transferase (1:400; 1 day after, and 1 and 2 days after weekly docetaxel. Treatment Roche Diagnostics) and biotin-16-dUTP (1:200; Roche) and then continued until the vehicle-treated animals showed significant tumor blocked with 2% bovine serum albumin. Samples were then incubated burden and/or were moribund at which point all animals were in streptavidin-horseradish peroxidase (1:400; DAKO) for 40 min at sacrificed simultaneously. All tumor nodules were collected, counted, 37jC and rinsed with PBS. Visualization was done with 3,3¶-diamino- and weighed at necropsy. benzidine and counterstained with Gill’s hematoxylin. The apoptotic To compare the biological activity of i.v. versus i.p. aurora kinase index was quantified as the number of apoptotic tumor cells in five inhibition, we used the in vivo HeyA8 tumor model and initiated twice randomly selected Â100 high-power fields exclusive of necrotic areas. weekly either vehicle alone, i.v. MK-0457 therapy, or i.p. MK-0457. Animals. For all in vivo studies, female athymic mice (NCr-nu/nu) Dosages between the two treatment groups were matched and animals were purchased from the National Cancer Institute-Frederick Cancer were followed until animals in any group became moribund at which Research and Development Center. Mice were housed and maintained time all animals were sacrificed and tumors were harvested, weighed, under specific pathogen-free conditions in accordance with guidelines and recorded. from the American Association for Accreditation of Laboratory Animal Microarray analysis of tumors following MK-0457 treatment. Five Care and the NIH. All studies were approved and overseen by The vehicle-treated control mice and four MK-0457-treated experimental University of Texas M. D. Anderson Cancer Center Institutional Animal mice bearing orthotopic HeyA8 tumors were sacrificed 24 h after i.p. Care and Use Committee. treatment (50 mg/kg). Tumors were immediately removed and

www.aacrjournals.org 5439 Clin Cancer Res 2008;14(17) September 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. CancerTherapy: Preclinical preserved in RNAlater solution (Ambion) for subsequent RNA interrogate the phosphorylation status of Aurora-A (Thr288), extraction with RNeasy kit (Qiagen). The quality and purity were required for kinase activity, as well as additional downstream assessed by agarose gel electrophoresis and absorbance measurement at targets, phospho-histone H3 (Ser10) and phospho-Cenp-A A A 260/ 280. Commercially available high-density oligonucleotide micro- (Ser7) following treatment with MK-0457. In both HeyA8 and arrays (Human Genome U133 Plus 2.0; Affymetrix) were used for SKOV3ip1 mitotic cells, inhibition of autophosphorylation of expression analysis. Preparation of cRNA, hybridization, scanning, and 288 image analysis of the arrays were done according to the manufacturer’s Aurora-A kinase at Thr was observed within 1 h of treatment protocols (Affymetrix) as described previously (29). Microarray data with the inhibitor (10 nmol/L). Treatment with MK-0457 also were processed with dChip software (30) and differentially expressed resulted in a gradual decline in phosphorylation status of 10 genes were identified using SAM analysis (31). histone H3 (Ser ) and a dramatic down-regulation of Cenp-A Real-time PCR. cDNA was synthesized from total RNA using the (Ser7) phosphorylation (Fig. 1A). High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). To characterize the onset of Aurora kinase inhibition by Quantitative real-time PCR was done in a MX4000 multiplex quantitative MK-0457, we examined Aurora-A phosphorylation in plated PCR system (Stratagene) using predesigned TaqMan primers and probe HeyA8 cells by Western blot. Levels of phospho-Aurora-A rela- sets (Applied Biosystems) and the Brilliant QPCR kit (Stratagene). The tive to total-Aurora-A began to decrease 10 min after exposure conditions for the reaction were as follows: 1 cycle at 95jC for 10 min to the inhibitor. Inhibition of phospho-Aurora-A persisted and 40 to 50 cycles at 95jC for 15 s and 60jC for 1 min. Quantitative real- time PCR for each primer and probe set was done either in duplicate or through 6 h after exposure. At 12 h after exposure to the inhi- triplicate, and the means are reported. Samples were normalized to bitor, relative levels of phospho-Aurora-A began to increase cyclophilin A and the relative expression level was calculated by the toward baseline (Fig. 1B). In vitro comparative CT method using an untreated sample as the calibrator. cytotoxic effects of inhibiting Aurora kinase on ovarian Statistical analysis. In vivo therapy experiments were powered to carcinoma. Before in vivo experiments, we examined the detect a 50% reduction in tumor weight (b error = 0.2). The Mann- in vitro effects of targeting Aurora kinase on ovarian carcinoma Whitney rank-sum test was used to analyze nonparametric and cytotoxicity. The IC50 level of the Aurora kinase inhibitor was nonnormally distributed data sets. Comparison of dose-response curves f50 nmol/L for the HeyA8 cell line and 20 to 70 nmol/L for in the MTT analysis for single-agent and combination treatments was the SKOV3ip1 cell line (Fig. 2A). Treating HeyA8 cells with made by logistic regression analysis (Prism; GraphPad Software). Regres- the traditional cytotoxic agent, docetaxel, resulted in an IC50 sion and statistical analyses were done using SPSS 12.0 for Windows f (SPSS). A two-tailed P V 0.05 was deemed statistically significant. of 10 nmol/L; however, treating HeyA8 cells with varying concentrations of docetaxel after 24 h of pretreatment with MK-0457 resulted in at least a 10-fold improvement in the Results IC50 of docetaxel to 0.5 to 1 nmol/L (Fig. 2A). This enhanced effect was similar in the SKOV3ip1 cell line in which In vitro inhibition of Aurora kinase activity. Before initiating treating SKOV3ip1 cells with increasing concentrations of therapy experiments, we first did functional kinase assays to docetaxel after 24 h of MK-0457 pretreatment also resulted in

Fig. 1. A, Aurora-A kinase-mediated autophosphorylation in HeyA8 and SKOV3ip1ovarian cancer cells is selectively inhibited using MK-0457 (as low as 10 nmol/L) as shown by decreased levels of phospho-Aurora-A (Thr288). Inhibition of endogenous substrates, histone H3 (Ser10) and Cenp-A (Ser7), is also elicited by the Aurora-A kinase inhibitor, MK-0457, using doses as low as 10 nmol/L. B, time of onset of Aurora-A kinase inhibition is examined in a time-kinetic experiment using the lowest dose necessary to block phosphorylation of Aurora-A kinase as determined above. MK-0457 decreases Aurora-A phosphorylation (relative to the total amount of Aurora-A) in HeyA8 cells.

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Fig. 2. A, cytotoxicity (MTT) graphs depicting the inhibitory concentration at 50% of MK-0457 for HeyA8 and SKOV3ip1cell lines in vitro. HeyA8 (1,000 cells per well) and SKOV3ip1 (2,000 cells per well) were treated with MK-0457 for 96 h. Cell viability was determined using MTT method. Cell viability assay for HeyA8 and SKOV3ip1 cells treated with docetaxel alone and after pretreatment with MK-0457. B, cell cycle analysis by flow cytometry of HeyA8 (solid columns)andSKOV3ip1(open columns)cells shows profound and sustained G2-M arrest as soon as12h after inhibition of Aurora-A kinase. Bars, SD. *, P < 0.02, compared with controls. C, marked increases in z4N cells wereseen24hafterMK-0457exposureinHeyA8cells.D, inhibition of Aurora-A kinase in HeyA8 and SKOV3ip1cells with MK-0457 treatment also results in marked increases in apoptosis relative to untreated controls as indicated by the sub-G1fraction on flow cytometry. Bars, SD. *, P = 0.02, compared with controls.

a significant improvement compared with treatment with SKOV3ip1 cell line, this 3-fold increase in G2-M arrest was docetaxel alone (IC50 = 0.2-0.5 nmol/L with pretreatment present through 48 h after exposure to the Aurora kinase versus 5-10 nmol/L without pretreatment; P = 0.001; Fig. 2A). inhibitor (P = 0.02; Fig. 2B). Endoreduplication, a phenotype Based on the role of Aurora kinases in cell cycle progression, of Aurora-B inhibition, has been identified as a hallmark of we did flow cytometry to determine the effect on cell cycle after aberrant cytokinesis (32); therefore, we did flow cytometry to inhibition with the pan-Aurora kinase inhibitor. In the HeyA8 examine cell ploidy. Twenty-four hours after treatment with the and SKOV3ip1 cell lines, a one-time exposure of cells to inhibitor, 71% of the HeyA8 cells showed aneuploidy or z4N MK-0457 alone resulted in >3-fold increase in G2-M arrest (Fig. 2C). Because an important consequence of G2-M arrest is within 12 h of treatment (P = 0.02). A trend of persistent G2-M apoptosis, we used flow cytometry to determine the apoptotic arrest was evidenced in the HeyA8 cell line through 48 h after fraction of cells treated with the Aurora kinase inhibitor as exposure to the inhibitor (P =0.08).However,inthe represented by the sub-G1 cell population. Within 48 h after

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Aurora kinase inhibition, a 30-fold increase in apoptotic HeyA8 increase in apoptosis beginning 12 h after treatment and lasting cells was seen (P = 0.02) compared with controls (Fig. 2D). through 48 h compared with controls (P = 0.05; data not In the SKOV3ip1 cell line, treatment with the inhibitor elicited shown). a 3.5-7-fold increase in apoptosis by 48 h after exposure In vivo effects of Aurora kinase inhibition on ovarian (P = 0.02) compared with controls (Fig. 2D). Based on the carcinoma. To establish the optimal dose and frequency of induction of G2-M arrest by MK-0457, we next asked whether dosing to effectively inhibit Aurora kinase in vivo, we initiated docetaxel-induced apoptosis would be further enhanced by this dose-finding experiments using phospho-histone H3 status as a inhibitor. Combining MK-0457 with docetaxel in the SKO- biological indicator of Aurora kinase activity. Four twice-daily V3ip1 cell line resulted in a rapid and sustained 25- to 40-fold doses of MK-0457 (25 or 50 mg/kg) or vehicle alone were

Fig. 3. A, Aurora-A kinase inhibition with MK-0457 decreases phospho-histone H3 (P-HH3 ) in formed ovarian tumors (HeyA8) in a time- and dose-dependent manner. Compared with treatment with vehicle alone, MK-0457 treatment (25 or 50 mg/kg) decreased the number of cells with phospho-histone H3 by 40% to 52% in xenograft ovarian tumors collected from athymic mice as determined by immunohistochemical analysis (original magnification, Â100). *, P < 0.001, versus control within the same time point. B, mean tumor weights and distribution from MK-0457 therapy experiments. Mice inoculated with chemosensitive tumors, HeyA8 and SKOV3ip1, as well as taxane- and platinum-resistant tumors, HeyA8-MDR and A2780-CP20, respectively, were randomized into one of four treatment groups: (a) vehicle alone (control), (b) MK-0457 alone (50 mg/kg), (c) traditional cytotoxic docetaxel (35 Agor 50 Ag once weekly, depending on cell line) or cisplatin (160 Ag once weekly), or (d) MK-0457 plus docetaxel or cisplatin depending on the cell line. Animals from all groups were sacrificed when control animals were moribund (f3-4 wk after initiating therapy, depending on the cell line used). All tumors were harvested and the pattern of spread was noted.Tumor weights and the number of nodules were recorded. Columns, mean tumor weights; bars, SE. *, P < 0.01, versus control; c, P < 0.05, versus control; b, P V 0.01, versus docetaxel or cisplatin, depending on tumor model.

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Table 1. Tumor characteristics after MK-0457-based therapy

Cell line Treatment Nodules, median no. (range) P (vs control) HeyA8 Control 8.5 (1-31) MK-0457 3 (1-7) 0.01 Docetaxel 3 (2-10) 0.04 MK-0457/docetaxel 2 (1-6) 0.01 SKOV3ip1 Control 22 (3-60) MK-0457 5 (2-9) 0.006 Docetaxel 4.5 (2-40) 0.02 MK-0457/docetaxel 2 (1-9) 0.002 HeyA8-MDR Control 10 (2-21) MK-0457 4 (2-9) 0.02 Docetaxel 11 (2-30) NS MK-0457/docetaxel 4.5 (1-7) 0.003 A2780-CP20 Control 48 (4-63) MK-0457 3.5 (0-16) 0.01 Cisplatin 21 (7-36) NS MK-0457/cisplatin 2 (0-13) 0.008

administered by i.p. injection to athymic female mice bearing and platinum agents, we studied the effects of Aurora kinase HeyA8 i.p. tumors 19 days after tumor cell inoculation when inhibition in the taxane-resistant, HeyA8-MDR, and platinum- the tumors were palpable. Animals were sacrificed 24, 48, resistant, A2780-CP20, tumor models. Consistent with their and 72 h after the last dose, and tumors were harvested. resistance profiles, docetaxel monotherapy in the HeyA8- Examination of the tumors by immunohistochemistry revealed MDR model and cisplatin in the A2780-CP20 model did 40% to 50% lower levels of phospho-histone H3 in the 25 and not alter tumor growth compared with vehicle treatment 50 mg/kg groups, respectively, within 24 h after the last dose of (P values z 0.9). MK-0457 monotherapy and combination inhibitor (P values < 0.001, compared with controls; Fig. 3A). therapy with docetaxel in the HeyA8-MDR tumor model Although some degree of reduced phospho-histone H3 levels resulted in significant reductions in tumor burden (70% and was seen at 48 h after the last dose of MK-0457, the most 90%, respectively; P values < 0.01) compared with controls consistently observed response was at 24 h post-treatment; (Fig. 3B). Similarly, in the A2780-CP20 tumor model, treat- therefore, subsequent in vivo therapy experiments used MK- ment with either MK-0457 monotherapy or combined with 0457 dosed at 50 mg/kg starting 24 h before taxane-based cisplatin produced 78% and 92% reductions in tumor weight chemotherapy. compared with vehicle (P values < 0.05) and 80% and In vivo experiments with diverse cell lines in an orthotopic 91% reductions compared with cisplatin-treated animals murine model for metastatic ovarian cancer were employed to (P values = 0.001), respectively. characterize the antitumor effects of Aurora kinase inhibition. To further characterize the effects of Aurora kinase inhibition Aurora kinase inhibition using MK-0457 was initiated 1 week on tumor growth inhibition, we examined tumor nodule after tumor cell inoculation to model the clinical scenario of formation (Table 1). In the chemosensitive (HeyA8 and minimal residual disease as described previously (22). The four SKOV3ip1) and chemoresistant (HeyA8-MDR and A2780- treatment groups consisted of (a) vehicle alone, (b) MK-0457 CP20) tumor models, MK-0457 treatment alone resulted in twice daily for 2 days weekly, (c) docetaxel (or cisplatin for significantly fewer tumor nodules compared with controls the A2780-CP20 tumor model) i.p. once weekly, and (d) (65%, 77%, 60%, and 93% reduction, respectively; all P values MK-0457 twice daily for 2 days weekly starting 1 day before V 0.01). Combining MK-0457 with either docetaxel or cisplatin weekly docetaxel or cisplatin (A2780-CP20). Compared with also elicited marked reductions in tumor nodule formation therapy with vehicle alone, treatment with the inhibitor alone compared with controls (76%, 91%, 55%, and 96%, respec- resulted in highly significant 80% and 90% reductions in tumor tively; all P values V 0.01). In the two chemoresistant tumor weight (P values = 0.002 versus controls) in the HeyA8 and models, both MK-0457 monotherapy and combination therapy SKOV3ip1 tumor models, respectively (Fig. 3B). As expected in resulted in significant reductions in tumor nodule formation these chemosensitive tumor models, docetaxel effectively compared with treatment with docetaxel or cisplatin alone reduced tumor growth. Combining MK-0457 and docetaxel (P values V 0.01). resulted in the greatest efficacy in reducing tumor burden, To examine the therapeutic effect of altering the timing of eliciting a >90% reduction in tumor weight in both HeyA8 and Aurora kinase inhibition, we varied the time at which MK-0457 SKOV3ip1 tumor models (P values = 0.002 versus controls). was administered relative to the time of docetaxel treatment in Furthermore, MK-0457 plus docetaxel exhibited significantly the HeyA8 tumor model. No statistically significant difference improved efficacy in decreasing tumor burden compared with was observed among the four treatment groups relative to each docetaxel monotherapy in both HeyA8 (68% reduction; other (data not shown). We also asked whether the route of P < 0.01) and SKOV3ip1 (85% reduction; P = 0.01) tumor MK-0457 delivery could alter efficacy. No significant differences models. were noted in tumor weight between animals treated via i.v. Because recurrent and advanced ovarian cancer is typically versus i.p. routes (0.22 versus 0.27 g; P = nonsignificant). Both refractory to traditional cytotoxic agents, particularly taxanes routes of administration (i.v. and i.p.), however, yielded robust

www.aacrjournals.org 5443 Clin Cancer Res 2008;14(17) September 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. CancerTherapy: Preclinical reductions in tumor weight compared with animals treated with vehicle alone was 71%; however, treatment with MK-0457 with vehicle alone (85% and 82%; P < 0.001; Supplementary alone and in combination with docetaxel resulted in signifi- Fig. S1). cantly lower proliferation indices (23% and 36% reductions, Daily observation of all mice in long-term therapy expe- respectively, P values < 0.001; Fig. 4A). Notably, treatment with riments revealed no overt signs of toxicity such as alterations combination MK-0457 and docetaxel was superior to treatment in bowel or feeding habits, posture, or mobility. No significant with docetaxel alone in decreasing tumor cell proliferation differences in body weight were observed among treatment (27% reduction; P = 0.004). The SKOV3ip1 tumor model also groups in the chemosensitive HeyA8 and SKOV3ip1 models showed significant 15% and 34% decreases in proliferation as well as the taxane-resistant, HeyA8-MDR tumor model. indices compared with controls after treatment with MK-0457 Within the A2780-CP20 experiment, mice treated with cisplatin both as a single agent and combined with docetaxel, respec- alone weighed 13% and 10% less than mice receiving vehicle tively (P values < 0.001; data not shown). Similar to the HeyA8 alone (P = 0.01) or MK-0457 alone (P = 0.01), respectively. tumor model, the antiproliferative effect of combining MK- Mice receiving combination MK-0457/cisplatin displayed 21% 0457 with docetaxel was significantly better than the effect of and 12% reduced body weights compared with both MK-0457 docetaxel alone (P < 0.05; data not shown). monotherapy and single-agent cisplatin groups (P V 0.001), In the taxane-resistant model, treatment with docetaxel did respectively. Of note, mice receiving MK-0457 alone and not decrease the proliferative index (data not shown); however, vehicle alone showed no difference in body weight in our treatment with either MK-0457 alone or in combination with A2780-CP20 tumor model, further implicating cisplatin as the docetaxel significantly reduced the proliferative indices more agent primarily responsible for this side effect. These observa- than 20% from controls, respectively (P values < 0.001). In the tions are also consistent with observed cachexia and weight loss cisplatin-resistant model, treatment with MK-0457 alone and seen with cisplatin in the clinical setting (33, 34). combined with cisplatin resulted in similarly significant Effects of MK-0457 on proliferation and apoptosis. To explore reductions in tumor cell proliferation (P values < 0.001) com- possible mechanisms underlying the tumor growth inhibition pared with controls. Furthermore, treatment with combination evidenced in the MK-0457 therapy experiments, we first MK-0457 and cisplatin offered a significant 14% reduction in examined its effects on tumor cell proliferation by calculating proliferation index beyond that offered by MK-0457 mono- the proliferative index after PCNA immunohistochemistry on therapy (P = 0.02). tumors collected at necropsy from all therapy experiments. In Due to the proposed role of Aurora kinases in apoptosis, we the HeyA8 model, the proliferation index for animals treated examined tumor cell apoptosis using terminal deoxynucleotidyl

Fig. 4. A, proliferative index and tumor cell apoptosis of HeyA8 tumors with representative PCNA immunohistochemistry (original magnification, Â100) and terminal deoxynucleotidyl transferase ^ mediated dUTP nick end labeling (original magnification, Â100) sections, respectively, collected at necropsy. *, P < 0.01, versus control; c, P < 0.05, versus docetaxel. B, real-time reverse transcription- PCR analysis of genes induced by MK-0457 using both human and mouse gene-specificTaqMan probes. Fold induction (in log10) represents the ratio between the means of the expression values of four MK-0457-treated mice and five untreated vehicle controls. Mouse, mouse-specificTaqMan probes; Human, human-specificTaqMan probes;H, human; M, murine.

Clin Cancer Res 2008;14(17) September1,2008 5444 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. Inhibiting Aurora Kinases DecreasesTumorigenesis transferase–mediated dUTP nick end labeling staining in treatment with this inhibitor, the use of a nonorthotopic in vivo animals from therapy experiments. In the HeyA8 tumor system may not consider the influence of the relevant tumor model, inhibition of Aurora kinases using MK-0457 both as a microenvironment, an important factor in tumor growth and single-agent and combined with docetaxel yielded significant metastasis (38). This current study extends the available body 3-fold increases in the number of apoptotic tumor cells of knowledge by demonstrating antitumor effects and mecha- (P < 0.01) compared with untreated animals (Fig. 4A). In the nisms of activity of MK-0457, a highly potent pan-Aurora taxane-resistant tumor model (HeyA8-MDR), MK-0457 alone kinase inhibitor, in an orthotopic in vivo model of metastatic and with docetaxel also resulted in statistically significant >77% ovarian cancer. increases in the number of apoptotic tumor cells compared In addition to the fundamental role Aurora kinases play in with tumors from vehicle-treated animals (P values < 0.03; data cell cycle regulation, increasing interest exists in examining its not shown). potential role in chemoresistance. In ovarian cancer, chemo- To gain additional insight into downstream effects of MK- resistant recurrence is a significant clinical problem and second- 0457, we did expression profile studies on HeyA8 tumors line therapies have limited efficacy; therefore, the potential harvested from either vehicle- or MK-0457-treated animals. The clinical role for Aurora kinase manipulation in reversing drug expression profiles of tumors from MK-0457-treated animals resistance may be useful clinically. In vitro, HeLa cells stably differed from the vehicle controls as revealed by unsupervised overexpressing Aurora-A kinase were shown to be more clustering analysis (Supplementary Fig. S2). Tumors from four resistant to taxane-induced apoptosis (10). Similarly, Noguchi treated mice formed a single cluster, whereas the five vehicle showed that patients with breast tumors with high Aurora-A controls formed another cluster. Using SAM analysis with mRNA levels exhibited a lower response rate to docetaxel permutation to estimate the false discovery rate, we identified treatment than patients with low Aurora-A mRNA breast 174 significant probe sets (false discovery rate < 0.25%) that tumors (41% versus 71%; ref. 39). Hata et al. showed that were up-regulated z2-fold in tumors from MK-0457-treated down-regulation of Aurora-A kinase in pancreatic cancer cell mice (Supplementary Table S1). Several protease gene family lines using small interfering RNA–based targeting resulted in members including chymotrypsin, elastase, and carboxypepti- increased sensitivity to paclitaxel (40). Although the specific dase were found to be highly induced. To validate the up- mechanism for taxane sensitization is not thoroughly elucidat- regulation of these protease genes, real-time reverse transcrip- ed and is likely multifactorial, evidence suggests that apoptosis tion-PCR was done using both human- and mouse-specific inhibition plays an important role (11). Our study shows that TaqMan probes. The results indicated that the up-regulation of therapeutic inhibition of Aurora kinases in our taxane-resistant CPB1 (carboxypeptidase B1), CTRB1 (chymotrypsinogen B1), tumor model results in decreased tumor growth with a con- and ELA2A (elastase2A)aremousespecific(Fig.4B), comitant increase in apoptosis, further emphasizing apoptosis suggesting stromal effects of therapy. as an important antitumor mechanism of Aurora kinase inhibition. Remarkably, we discovered and validated that several protease-related genes (CPB1, CTRB1, and ELA2A) were highly Discussion up-regulated in the stroma (mouse-derived tissue within the HeyA8 tumor). Expression of these degradative genes within Notable findings from this current study are that inhibiting the stroma may be related to the decrease in tumor growth. Aurora kinases using an engineered small-molecule inhibitor Further work to gain mechanistic insights regarding stromal shows marked antitumor efficacy in ovarian carcinoma effects following Aurora kinase inhibition is being actively models. These effects were mediated, in part, by significantly pursued. decreasing tumor cell proliferation and increasing apoptosis. Based on their important roles in the cell cycle, Aurora Furthermore, these mechanisms were consistently shown in kinases represent an intriguing therapeutic target. In fact, both chemosensitive tumor models and two additional several groups have identified small-molecule inhibitors of models resistant to taxane- and platinum-based chemo- Aurora kinases, each with different degrees of selectivity for therapies. Aurora-A or B. Although other pathways such as the JAK/STAT To date, several studies support the important role Aurora have recently been implicated in increased aggressiveness and kinases play in cell cycle regulation and high-fidelity mitosis drug sensitivity of ovarian cancer (41, 42), the specificity of (35). Aurora-A kinase is necessary for mitotic spindle assembly MK-0457 for Aurora kinases is significantly greater for Aurora and balanced chromosome segregation between daughter cells. kinases (43), thereby supporting Aurora kinase inhibition as Overexpression results in a tumorigenic phenotype (3); how- the predominant mechanism for the therapeutic effects ever, the specific mechanisms of regulating Aurora-A expression observed in these experiments. Here, we describe the antitumor are still being elucidated. Recently, Kiat et al. identified Aurora- activity of MK-0457 in orthotopic advanced ovarian cancer A kinase interacting protein, an endogenous negative regulator models that implicates the Aurora kinase family as an impor- of Aurora-A kinase, from a yeast dosage suppressor screen (36). tant therapeutic target in ovarian cancer. In conclusion, our Although they were able to show specificity and efficacy in findings support pan-Aurora kinase targeting using the potent Aurora-A down-regulation via proteasome-dependent path- small-molecule inhibitor, MK-0457, alone or in combination ways, the clinical potential of degrading Aurora-A kinase was with traditional cytotoxic agents, for the treatment of ovarian not exploited. Manfredi et al. reported using a small molecule cancer. Although a recent industry newswire by manufacturers to exogenously inhibit Aurora-A kinase to elicit tumor cell Merck/Vertex (November 20, 2007) reporting QTc prolonga- apoptosis and tumor growth inhibition in colorectal and tion has placed ongoing clinical trials with MK-0457 on hold, prostate nonorthotopic xenograft models (37). Although they the antitumorigenic and therapeutic benefits of targeting the were able to show reductions in tumor growth after long-term Aurora kinase family in ovarian cancer remain the fundamental

www.aacrjournals.org 5445 Clin Cancer Res 2008;14(17) September 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. CancerTherapy: Preclinical findings from our investigations and support additional Disclosure of Potential Conflicts of Interest development of Aurora kinases as a therapeutic target. Our study extends previous work by showing potent antitumor No potential conflicts of interest were disclosed. activity in both taxane-resistant and platinum-resistant orthotopic tumor models of metastatic ovarian carcinoma. The robust antitumor effects, including cell cycle disruption and Acknowledgments apoptosis induction, seen in our studies provide preclinical We thank Derek J. Fiterman, NicholasJennings, Karen Ramirez, DonnaReynolds, rationale for upcoming clinical trials targeting Aurora kinase in Diana Urbauer, and Drs. Robert Langley and Yun-Fang Wang for insightful discus- ovarian cancer. sions and expertise.

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Yvonne G. Lin, Anand Immaneni, William M. Merritt, et al.

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