Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA, and MK-5108 on SW-872 and 93T449 human liposarcoma cells

Sandhya Noronha & Lauren A. C. Alt & Taylor E. Scimeca & Omran Zarou &
Justyna Obrzut & Brian Zanotti & Elizabeth A. Hayward & Akhil Pillai &

Shubha Mathur & Joseph Rojas & Ribhi Salamah & Nalini Chandar & Michael J. Fay

Received: 30 March 2017 / Accepted: 10 October 2017 / Editor: Tetsuji Okamoto
#The Society for In Vitro Biology 2017
2,4

Abstract Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequent-

in the total viable cell number. Based upon the EC50 values, the potency of the three Aurora kinase inhibitors in the SW-

ly diagnosed soft tissue sarcomas in humans. There is great
872 cells was as follows: AMG 900 (EC
50
= 3.7 nM) >

interest in identifying novel chemotherapeutic options for
A Z D 11 5 2 – H Q PA (E C
5 0
= 4 3. 4 nM ) > MK – 5 10 8

treating liposarcoma based upon molecular alterations in the
(EC50
= 309.0 nM), while the potency in the 93T449 cells

cancer cells. The Aurora kinases have been identified as prom-
was as follows: AMG 900 (EC50
= 6.5 nM) > AZD1152-

ising chemotherapeutic targets based on their altered expres-
HQPA (EC50
= 74.5 nM) > MK-5108 (EC50
= 283.6 nM).

sion in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhib- itor (AMG 900) on undifferentiated SW-872 and well- differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0 – 1000 nM), AZD1152-HQPA (0–1000 nM), and AMG 900 (0–1000 nM) for 72 h resulted in a dose-dependent decrease

*Michael J. Fay
[email protected]
The percentage of polyploidy after 72 h of drug treatment (0– 1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant in- crease in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant in- crease starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipo-

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Physician Assistant Program, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA
Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA
Department of Microbiology and Immunology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA
Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA
Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA
cytes and human mesenchymal stem cells. Western blot anal- ysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immuno- reactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differenti- ated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expres- sion of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of

. .
. . .

NORONHA ET AL.

Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.

Keywords Aurora kinase inhibitors Liposarcoma AMG

and cytokinesis and their altered expression in many different types of cancer (Lin et al. 2006). The aurora serine-threonine protein kinase gene was isolated from Drosophila melanogaster as part of a screen for mutations affecting the centrosome cycle (Glover et al. 1995). Three human homo-

900 AZD1152-HQPA MK-5108 SW-872 cell line 93T449 cell line

Introduction
.
logs have been identified which are now designated as Aurora kinase A (AURKA), B (AURKB), and C (AURKC) (Giet and Prigent 1999). The Aurora kinases consist of a conserved C- terminal catalytic domain, a conserved ATP binding site, and a variable N-terminal regulatory domain (Kollareddy et al. 2008). AURKA localizes near the centrosome and recruits

⦁ oft tissue sarcomas are malignant tumors that arise from mesenchymal tissues. For 2017, the American Cancer Society estimates that 12,390 new cases of soft tissue sarcoma will be diagnosed in the USA and 4,990 individuals in the USA will die due to soft tissue sarcoma (American Cancer Society, Cancer Facts and Figures 2017). Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue, and represents one of the most common types of soft tissue sarcomas that occur in humans (Henze and Bauer 2013). There are four histological subtypes of liposarcoma that are categorized into the following groups: well-differentiated liposarcoma/atypical lipomatous tumor and dedifferentiated liposarcoma (40–45%), myxoid liposarcoma (30%), and pleo- morphic liposarcoma (5%) (De Vita et al. 2016). There are also case reports in the veterinary literature of liposarcoma occurring in other species (Doria-Torra et al. 2015).
⦁ reatment modalities for liposarcoma patients include sur- gery, postoperative radiation therapy, and chemotherapy (Kollár and Benson 2014). Traditional chemotherapy regi- mens that include doxorubicin and/or ifosfamide have been used in liposarcoma patients with some level of limited suc- cess; however, poor patient outcomes indicate that more ef- fective chemotherapeutic options are still needed (Karavasilis et al. 2008). There is great interest in identifying targeted therapies for liposarcoma based upon molecular alterations in the cancer cells (Verweij and Baker 2010 ; Crago and Singer 2011; Abbas Manji et al. 2015; Bill et al. 2016a; Crago and Dickson 2016; De Vita et al. 2016). An example of such a genetic alteration in well-differentiated/ dedifferentiated liposarcoma involves amplification of onco- genes on chromosome 12q13-15, which includes MDM2 (mouse double minute 2 homolog) and CDK4 (cyclin- dependent kinase 4) (Sandberg 2004). Alterations in the ex- pression of Aurora kinases have also been identified in liposarcoma. Gene expression analysis by microarray has demonstrated that Aurora kinase A expression is upregulated in dedifferentiated liposarcoma (Puzio-Kuter et al. 2015), and the results of a pilot study demonstrated that Aurora kinase B is overexpressed in intermediate- and high-grade sarcoma tis- sues compared to normal tissues (Noronha and Volin 2012).
The Aurora kinases have been identified as promising che- motherapeutic targets based on their important roles in mitosis
substrate proteins to promote bipolar spindle formation and centrosome maturation (Berdnik and Knoblich 2002 ; Marumoto et al. 2003). AURKB interacts with the proteins Survivin and Borealin and inner centromere protein (INCENP) to form the chromosomal passenger complex, and the localization of this complex changes during mitosis to phosphorylate substrates that promote chromosome con- densation, proper kinetochore and microtubule association, and cytokinesis (Carmena et al. 2012). The cellular functions of AURKC are not as well understood; however, AURKC does share a high level of sequence homology with AURKB and is abundantly expressed in germ cells (Quartuccio and Schindler 2015).
Since altered expression of Aurora kinases has been report- ed in liposarcoma, we investigated the preclinical utility of a pan-Aurora kinase inhibitor (AMG 900), an AURKB inhibi- tor (AZD1152-HQPA), and an AURKA inhibitor (MK-5108) for liposarcoma using the SW-872 and 93T449 cell lines. The expression of AURKA and AURKB mRNA and protein ex- pression was also examined in SW-872 cells, 93T449 cells, differentiated adipocytes, and human mesenchymal stem cells (hMSCs).

Materials and Methods

Cell culture The undifferentiated SW-872 human liposarcoma cell line (ATCC® HTB-92 ™ ), the well- differentiated 93T449 human liposarcoma cell line (ATCC® CRL-3043™), and the HCT-116 human colorectal cancer cell line (ATCC® CCL-247™) were obtained from the American Type Culture Collection (Manassas, VA). The SW-872 cell line is an established in vitro research model for studying undifferentiated liposarcoma (Mills et al. 2009; Stratford et al. 2012), and the 93T449 cell line is an established in vitro research model for well-differentiated liposarcoma (Italiano et al. 2009). The HCT-116 cell line was used as a positive control since it has previously been shown to be re- sponsive to Aurora kinase inhibitors (Tao et al. 2008; Tao et al. 2009; Payton et al. 2010). The SW-872 cells were main- tained in DMEM high-glucose medium (Gibco® Life Technologies, Grand Island, NY) supplemented with 10%

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AURORA KINASE INHIBITORS AND LIPOSARCOMA

heat-inactivated FBS (Hyclone™, Logan, UT), and penicillin (100 units/mL)/streptomycin (100 μg/ml) (Gibco® Life Technologies). The 93T449 cells were maintained in RPMI 1640 medium (Gibco® Life Technologies) supplemented with 10% heat-inactivated FBS and penicillin-streptomycin. The HCT-116 cells were maintained in McCoy’s5a Modified Medium (Gibco® Life Technologies) supplemented with 10% heat-inactivated FBS and penicillin-streptomycin. All cell lines either received fresh medium or were subcultured three times per week. The identity of the SW-872, 93T449, and HCT-116 cell lines was confirmed by short tandem repeat (STR) analysis. Human mesenchymal stem cells were obtain- ed from Lonza (Walkersville, MD) and maintained in an un- differentiated state using the MSCGM™ Mesenchymal Stem Cell Growth Medium Bulletkit™ (Lonza). To differentiate the hMSC into adipocytes, cells were switched to an induction and maintenance protocol using hMSC Adipogenic Differentiation BulletKit™ Medium (Lonza). All cells were maintained in a humidified cell culture incubator at 37°C with 5% CO2/95% air.

Drug treatment and determination of viable cell number Cells were treated with a pan-Aurora kinase inhibitor (AMG 900, 0.5–1000 nM, Selleckchem, Houston, TX), an AURKB inhibitor (AZD1152-HQPA, 0.5–1000 nM, Selleckchem), an AURKA inhibitor (MK-5108, 0.5–1000 nM, Selleckchem), or DMSO (Mediatech, Inc., Manassas, VA) as the vehicle control, or received medium only with no drug. SW-872 (4.5 × 10 cells/plate), 93T449 (5 × 10 cells/plate), and HCT-116 (3.5 × 10 cells/plate) cells were plated into 100 mm dishes, and after 24 h, the cells were treated once with drug and were incubated for 72 h. Subconfluent prolif- erating cells were used for the drug treatments. Preliminary experiments were performed where cells were incubated with drug for 24, 48, and 72 h, and since the 72 h treatment had the greatest effect on viable cell number, this time point was used for subsequent studies. Viable cell counts were performed using trypan blue (Fisher Scientific, Pittsburgh, PA) and a BioRad TC-20 automated cell counter. Triplicate independent experiments were performed with duplicate or triplicate repli- cates per experiment, and each replicate was counted three times. The data is presented as the mean ± SEM. Statistical analysis and EC 50 determinations were performed with GraphPad Prism v.7 using ANOVA followed by Dunnett’s post hoc test (N = 3, p ≤ 0.05).

Analysis for polyploidy SW-872 and 93T449 cells were treated with Aurora kinase inhibitors for 72 h, as described above. The nuclei were stained with propidium iodide accord- ing to published methodology (Vindeløv et al. 1983 ; Robinson et al. 1996 ). After treatment, the cells were trypsinized (0.25% Trypsin-EDTA, Gibco® Life Technologies) and fixed in ice-cold 70% ethanol. Samples

were stored at − 20°C for no more than 1 wk. Prior to analysis, cells were washed twice with PBS. The cell pellet (1 × 10 cells) was resuspended in 1.0 mL of propidium iodide (Sigma, St. Louis, MO) staining solution at 37°C for 20 min. Cells were then placed in propidium iodide saline solution and an- alyzed using a BD FACSCalibur Flow Cytometer. The CellQuest Pro v. 5.2.1 software was used to determine the percentage of polyploid cells. The data is presented as the mean ± SEM. Statistical analysis was performed on trip- licate independent experiments, with duplicate or tripli- cate replicates per independent experiment, using ANOVA followed by Dunnett’s post hoc test ( N = 3, p ≤ 0.05, GraphPad Prism v. 7).
As an independent method to confirm polyploidy, SW-872 and 93T449 cells were plated on coverslips and treated for 72 h with DMSO vehicle control or 1000 nM of AMG 900. Cells were fixed with 3.6% formaldehyde (Sigma) and stained with Alexa Fluor® 488 Phalloidin (Cell Signaling Technologies, Danvers, MA) and ProLong® Gold Antifade Reagent with DAPI (Cell Signaling Technologies). Cells were visualized on the EVOS® FL Auto Imaging Station (Life Technologies) with a × 40 objective.

Evaluation of hMSC adipocytic differentiation by Oil Red O staining The hMSCs that had undergone the adipogenic differentiation protocol were fixed with 10% formalin (Fisher, Fair Lawn, NJ) and stained at room temperature with an Oil Red O (aMRESCO, Solon, OH) working solution followed by hematoxylin (Fisher Scientific, Pittsburgh, PA). Cultures were viewed and photographed using a Nikon TiE inverted phase contrast microscope with a × 20 objective.

Evaluation of hMSC adipocytic differentiation by RT- PCR Total RNA was isolated using TRI reagent® (Molecular Research Center, Cincinnati, OH). Semi- quantitative RT-PCR for PPAR- γ and A-FABP was per- formed using the SuperScript ™ One-Step RT-PCR with Platinum® Taq kit (Life Technologies, Carlsbad, CA). Both PPAR-γ and A-FABP are markers of adipocyte differentiation and were used to confirm adipocytic differentiation of the hMSCs. The PPAR- γ primers used were as follows, PPAR-γ sense: 5′-AAACTCTGGGAGATTCTCCT-3′,and PPAR- γ anti-sense: 5′-TCTTGTGAATGGAATGTCTT-3′. The A-FABP primers were as follows, A-FABP sense: 5′- AAAACTGCAGCTTCCTTCTCACCT-3′,and A-FABP an- ti-sense: 5′-ACGCATTCCACCACCAGTTTATC-3′.β-Actin was used as an endogenous housekeeping control, and the primers used were as follows, β -Actin sense: 5 ′ GATATCGCCGCGCTCGTCGTC-3 ′,and β-Actin anti- sense: 5′-GTCCCGGCCAGCCAGGTCCAG-3′.The primers used for semi-quantitative RT-PCR were obtained from IDT (Coralville, IA). The RT step consisted of 50°C for 30 min followed by 94°C for 2 min. PCR consisted of a denaturation

−ΔCT

NORONHA ET AL.

step of 94°C for 15 s, an annealing step of 56°C (PPAR-γ) or 55°C (A-FABP) or 63.4°C (β-Actin) for 30 s, and an exten- sion step of 68°C for 1 min for 30 cycles (PPAR-γ), or 35 cy- cles (A-FABP), or 25 cycles (β -Actin), with a final extension step of 68°C for 5 min. Ethidium bromide-stained gels were visualized using a Kodak Gel Logic 1500 Imaging Station (Kodak, Rocheser, NY) and Kodak molecular imaging soft- ware (v. 4.0).

Real-time PCR analysis of AURKA and AURKB mRNA expression Real-time PCR using Taqman® gene expression assays (Life Technologies) and the 7300 real-time PCR sys- tem or the QuantStudio ™ 5 real-time PCR system (Applied Biosystems, Grand Island, NY) were used to examine AURKA and AURKB mRNA expression using the compar- ative CT method (Schmittgen and Livak 2008) and detailed methodology as previously described by Baxter et al. (2009). Total RNA (100 ng) was reverse transcribed into single- stranded cDNA using the High-Capacity cDNA Reverse Transcription kit (Life Technologies) as recommended by the manufacturer. Taqman® gene expression assays for AURKA (ID No. Hs01582072_m1), AURKB (ID No. Hs00177782_m1), and β-Actin (ID No. Hs99999903_m1) were used. For the PCR assay, each plate contained triplicate replicate wells for each independent sample (N = 3), and data was analyzed using the 7300 Real-Time PCR system RQ study software v. 1.4 or QuantStudio™ Design and Analysis Software v. 1.4. Statistical analysis was performed using either ANOVA followed by Tukey’spost hoc test (N = 3, p ≤ 0.05) or unpaired two-tailed t test (N = 3, p ≤ 0.05) on the 2 values.

Western blot analysis of AURKA and AURKB expression Total protein was extracted using RIPA buffer (Thermo Scientific) supplemented with Halt ™ Protease Inhibitor Cocktail (Thermo Scientific). The protein concentration was determined using the Pierce® BCA Protein Assay kit (Thermo Scientific). Protein (100 μg) was loaded into a 15% Ready Gel® Tris-HCl gel (BioRad, Hercules, CA) or a Novex™ Bolt™ 4–12% Bis-Tris gel (Thermo Scientific), and samples were electrophoresed at 150 V for 1 h and transferred to a PVDF membrane overnight. Blots were blocked for 1 h with SuperBlock T20 (TBS) Blocking Buffer (Thermo Scientific) followed by incubation with the AURKA Rabbit mAB primary antibody (1:1000, Cell Signaling) overnight at 4°C, or with the AURKB Rabbit polyclonal primary antibody (1:1000, Bethyl, Montgomery, TX) for 1 h at room tempera- ture. The blots were then washed and incubated with Goat anti-Rabbit IgG-HRP conjugated secondary antibody (1:10,000, Thermo Scientific) for 1 h at room temperature. The blots were then washed and developed using the SuperSignal® west pico Chemiluminescence Substrate kit (Thermo Scientific) and visualized with a Kodak Gel Logic

Imaging Station. The blots were then stripped with Restore™ Western blot stripping buffer (Thermo Scientific) and re- probed using Rabbit anti-Actin (1:200, Sigma) as an endog- enous control to ascertain equal loading.

Results

Effect of Aurora kinase inhibitors on SW-872, 93T449, and HCT-116 viable cell number As shown in Fig. 1, all three of the Aurora kinase inhibitors caused a dose- dependent decrease in the total viable cell number, as com- pared to the DMSO vehicle control, with 72 h of drug treat- ment. Drug concentrations that caused a statistically signifi- cant decrease in the total viable cell number compared to the DMSO vehicle control are indicated with an asterisk (*) (N = 3, p ≤ 0.05, ANOVA followed by Dunnett’spost hoc test). The EC50 values for AMG 900 were 3.7 nM for SW-872 cells (Fig. 1A), 6.5 nM for 93T449 cells (Fig. 1B), and 1.4 nM for HCT-116 cells (Fig. 1C). The EC50 values for AZD1152- HQPA were 43.4 nM for SW-872 cells (Fig. 1D), 74.5 nM for 93T449 cells (Fig. 1E), and 27.3 nM for HCT-116 cells (Fig. 1F). The EC50 values for MK-5108 were 309.0 nM for SW- 872 cells (Fig. 1G), 283.6 nM for 93T449 cells (Fig. 1H), and 135.1 nM for HCT-116 cells (Fig. 1I). The amount of DMSO used for the vehicle control (0.01%) represents the amount in the highest drug concentration (1000 nM), and this level of DMSO did not cause a statistically significant change in the total viable cell number compared to cells that received medi- um only without drug (data not shown).

Effect of Aurora kinase inhibitors on polyploidy To evalu- ate the ability of the 72 h treatment with Aurora kinase inhib- itors to induce polyploidy in SW-872 and 93T449 cells, the cells were stained with propidium iodide and the percentage of cells with a DNA content greater than 4N was determined by flow cytometry. Representative DNA histograms for cells treated for 72 h with AMG 900, AZD1152-HQPA, and MK- 5108 are shown in Fig. 2A for SW-872 cells and Fig. 3A for 93T449 cells. A gate was placed after the G2/M peak to deter- mine the percentage of cells with a DNA content greater than 4N. As shown in Fig. 2B for the SW-872 cells, AMG 900 caused a significant increase in the percent polyploidy starting with the 25 nM dose, and this dose of drug demonstrated a statistically significant ~4.8-fold increase in the percentage of polyploid cells versus the DMSO vehicle control ( N = 3, p ≤ 0.05, ANOVA followed by Dunnett’spost hoc test). The higher doses of AMG 900 (50–1000 nM) caused a similar statistically significant increase in the percentage of polyploid cells. The AURKB inhibitor AZD1152-HQPA caused a sta- tistically significant increase in the percentage of polyploid cells starting at 100 nM (Fig. 2C), and this dose caused a 3.1-fold increase in the percentage of polyploid cells versus

AURORA KINASE INHIBITORS AND LIPOSARCOMA

Figure 1. Effect of Aurora kinase inhibitors on SW-872, 93T449, and

with AMG 900, AZD1152-HQPA, and MK-5108 in panels A, D, and G,

HCT-116 viable cell number. The SW-872 and 93T449 human
respectively. Dose response curves and EC50
values are shown for the

liposarcoma cells, and HCT-116 human colorectal cancer cells were treat- ed for 72 h with the pan-Aurora kinase inhibitor (AMG 900, 0.5– 1000 nM), Aurora kinase B inhibitor (AZD1152-HQPA, 0.5 – 1000 nM), Aurora kinase A inhibitor (MK-5108, 0.5–1000 nM), 0.01% DMSO vehicle control, or medium-only control (data not shown). The viable cell number was determined using trypan blue and a TC-20 auto- mated cell counter, and the data is shown as the mean (± SEM) percent total viable cell number compared to the DMSO vehicle control. Dose
93T449 cells treated with AMG 900, AZD1152-HQPA, and MK-5108 in panels B, E, and H, respectively. Dose response curves and the EC50 values are shown for the HCT-116 cells treated with AMG 900, AZD1152-HQPA, and MK-5108 in panels C, F, and I, respectively. Triplicate independent experiments were performed with duplicate or triplicate replicates per experiment, and each replicate was counted three times. Statistical analysis was performed using ANOVA followed by Dunnett ’spost hoc test (N = 3, p ≤ 0.05), and an asterisk (*) indicates

response curves and EC50
values are shown for the SW-872 cells treated
statistical significance from the DMSO vehicle control.

the DMSO vehicle control (N = 3, p ≤ 0.05, ANOVA followed by Dunnett’spost hoc test). The 250, 500, and 1000 nM doses of AZD1152-HQPA also caused a significant 6.2-, 10.3-, and 12.9-fold increase in the percentage of polyploid cells versus the DMSO vehicle control, respectively (Fig. 2C). Treatment with the AURKA inhibitor MK-5108 did not significantly increase the percentage of polyploid cells compared to the DMSO vehicle control at any of the doses tested (Fig. 2D). As shown in Fig. 3B for the 93T449 cells, AMG 900 caused a significant increase in the percent polyploidy starting with the 25 nM dose, and this dose of drug demonstrated a statistically significant ~1.9-fold increase in the percentage of polyploid cells versus the DMSO vehicle control (N = 3, p ≤ 0.05, ANOVA followed by Dunnett’spost hoc test). The 50, 100, 250, 500, and 1000 nM doses of AMG 900 also caused a

significant 2.2-, 2.3-, 2.7-, 2.8-, and 2.9-fold increase in the percentage of polyploid cells versus the DMSO vehicle con- trol, respectively (Fig. 3B). The AURKB inhibitor AZD1152- HQPA caused a statistically significant increase in the percent- age of polyploid cells starting at 250 nM (Fig. 3C), and this dose caused a 2.1-fold increase in the percentage of polyploid cells versus the DMSO vehicle control (N = 3, p ≤ 0.05, ANOVA followed by Dunnett’spost hoc test). The 500 and 1000 nM doses of AZD1152-HQPA also caused a similar statistically significant increase in the percentage of polyploid cells. Treatment with the AURKA inhibitor MK-5108 did not significantly increase the percentage of polyploid cells com- pared to the DMSO vehicle control at any of the doses tested (Fig. 3D). The amount of DMSO used for the vehicle control (0.01%) represents the amount in the highest drug

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AURORA KINASE INHIBITORS AND LIPOSARCOMA

ƒFigure 2. Effect of Aurora kinase inhibitors on polyploidy in SW-872 cells. The SW-872 human liposarcoma cells were treated for 72 h with the pan-Aurora kinase inhibitor (AMG 900, 0.5–1000 nM), Aurora kinase B inhibitor (AZD1152-HQPA, 0.5–1000 nM), Aurora kinase A inhibitor (MK-5108, 0.5–1000 nM), 0.01% DMSO vehicle control, or medium- only control. The percentage of cells with DNA content greater than 4N was determined using propidium iodide staining and flow cytometry. Panel A Representative DNA histograms for SW-872 cells treated with the Aurora kinase inhibitors. Panel B Percent polyploidy (mean ± SEM) for cells treated with AMG 900. Panel C Percent polyploidy (mean ± SEM) for cells treated with AZD1152-HQPA. Panel D Percent polyploi- dy (mean ± SEM) for cells treated with MK-5108. Triplicate independent experiments were performed with triplicate replicates per independent experiment. Statistical analysis was performed using ANOVA followed by Dunnett ’spost hoc test (N = 3, p ≤ 0.05), and an asterisk (*) indicates statistical significance from the DMSO vehicle control. Polyploidy was also confirmed in Alexa Fluor® 488 Phalloidin and DAPI-stained SW- 872 cells treated for 72 h with DMSO vehicle control (panel E) or 1000 nM AMG 900 (panel F) using the EVOS® FL Auto Imaging Station with a × 40 objective (size bar = 100 μm).

concentration (1000 nM), and this level of DMSO did not cause a statistically significant change in the percentage of polyploid cells compared to cells that received medium only without drug for either the SW-872 or 93T449 cells. When the hMSCs were treated with the three Aurora kinase inhibitors at selected doses (0, 1, 100, 500, and 1000 nM) for 72 h, there was no significant effect on polyploidy (data not shown). AMG 900-induced polyploidy was also confirmed by exam- ining DAPI and Alexa Fluor® 488 Phalloidin-stained cells, and treatment of the SW-872 and 93T449 cells with AMG 900 (1000 nM) resulted in large polyploid cells (Figs. 2F and 3F) compared to the DMSO vehicle control-treated cells (Figs. 2E and 3E).

Real-time RT-PCR analysis of AURKA and AURKB mRNA expression The expression of AURKA and AURKB mRNA in SW-872 cells, HCT-116 cells, hMSCs, and differentiated adipocytes was determined by real-time RT-PCR using the comparative CT method. Compared to the differentiated adipocytes, the SW-872 cells demonstrated a 32-fold increase and the HCT-116 cells demonstrated a 40- fold increase in AURKA mRNA expression (Fig. 4A). The increased AURKA mRNA expression in the SW-872 and HCT-116 cells was statistically significant from both the adi- pocytes and hMSCs (N = 3, p ≤ 0.05, ANOVA followed by Tukey’spost hoc test). A similar trend was observed for AURKB mRNA expression (Fig. 4B). With AURKB, there was a 202-fold increase in mRNA expression in SW-872 cells and a 468-fold increase in the HCT-116 cells compared to the adipocytes. The increased AURKB mRNA expression in the SW-872 and HCT-116 cells was statistically significant from both the adipocytes and hMSCs, and HCT-116 expressed sig- nificantly more AURKB mRNA compared to the SW-872 cells (N = 3, p ≤ 0.05, ANOVA followed by Tukey’spost hoc test). The undifferentiated SW-872 cells expressed 3.3-

fold more AURKA mRNA and 4.6-fold more AURKB mRNA than the well-differentiated 93T449 cells, and these differences were statistically significant (Fig. 4C) (N = 3, p ≤ 0.05, unpaired two-tailed t test). The differentiation of hMSCs (Fig. 4E) to adipocytes (Fig. 4F) was confirmed by Oil Red O staining and increased expression of mRNA for the adipocyte differentiation markers A-FABP and PPAR-γ (Fig. 4D).

Western blot analysis of AURKA and AURKB protein expression The expression of AURKA and AURKB protein was examined in SW-872, 93T449, HCT-116, differentiated adipocytes, and hMSCs using Western blot analysis. Both SW-872 and HCT-116 cells demonstrated expression of AURKA with an immunoreactive band at ~ 48 kDa (Fig. 5A); however, AURKA protein was not detected in the hMSCs or the differentiated adipocytes. As shown in Fig. 5B, an immunoreactive band for AURKB was found at ~ 39 kDa for SW-872, adipocytes, HCT-116, and hMSCs. In the SW- 872 and HCT-116 cells, a smaller immunoreactive band was also detected in the SW-872 and HCT-116 cells, and this band was not detected in the differentiated adipocytes or hMSCs. The well-differentiated 93T449 cells also express AURKA (Fig. 5C) and the two bands for AURKB (Fig. 5D), although the expression of both AURKA and AURKB appeared to be decreased compared to the undifferentiated SW-872 cells.

Discussion

There are a number of published preclinical in vitro studies that have examined the chemotherapeutic effects of Aurora kinase inhibitors on cancer cell lines of different tissue origin (Ulisse et al. 2006; Payton et al. 2010; Baldini et al. 2014; Chinn et al. 2014; Zekri et al. 2015; Helfrich et al. 2016; Tuccilli et al. 2016). Aurora kinase inhibitors are also being evaluated as chemotherapeutic agents in human clinical trials (Cheung et al. 2014; Falchook et al. 2015), and some of the cancers included in these trials include ovarian cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lympho- cytic leukemia, prostate cancer, and soft tissue sarcomas (Giles et al. 2007; Löwenberg et al. 2011; Tsuboi et al. 2011; Dennis et al. 2012; Matulonis et al. 2012; Giles et al. 2013; Meulenbeld et al. 2013; Dickson et al. 2016; DuBois et al. 2016). However, there is limited published information in re- gard to the effects of Aurora kinase inhibitors on cultured human sarcoma cells such as liposarcoma. Since previous research demonstrated that AURKB is overexpressed in intermediate- and high-grade sarcoma cases compared to nor- mal tissues (Noronha and Volin 2012 ) and AURKA is overexpressed in dedifferentiated liposarcoma (Puzio-Kuter et al. 2015), we investigated the effects of Aurora kinase in- hibitors on undifferentiated SW-872 and well-differentiated

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AURORA KINASE INHIBITORS AND LIPOSARCOMA

ƒFigure 3. Effect of Aurora kinase inhibitors on polyploidy in 93T449 cells. The 93T449 human liposarcoma cells were treated for 72 h with the pan-Aurora kinase inhibitor (AMG 900, 0.5–1000 nM), Aurora kinase B inhibitor (AZD1152-HQPA, 0.5–1000 nM), Aurora kinase A inhibitor (MK-5108, 0.5–1000 nM), 0.01% DMSO vehicle control, or medium- only control. The percentage of cells with DNA content greater than 4N was determined using propidium iodide staining and flow cytometry. Panel A Representative DNA histograms for 93T449 cells treated with the Aurora kinase inhibitors. Panel B Percent polyploidy (mean ± SEM) for cells treated with AMG 900. Panel C. Percent polyploidy (mean ± SEM) for cells treated with AZD1152-HQPA. Panel D Percent polyploi- dy (mean ± SEM) for cells treated with MK-5108. Triplicate independent experiments were performed with duplicate replicates per independent experiment. Statistical analysis was performed using ANOVA followed by Dunnett ’spost hoc test (N = 3, p ≤ 0.05), and an asterisk (*) indicates statistical significance from the DMSO vehicle control. Polyploidy was also confirmed in Alexa Fluor® 488 Phalloidin- and DAPI-stained 93T449 cells treated for 72 h with DMSO vehicle control (panel E) or 1000 nM AMG 900 (panel F) using the EVOS® FL Auto Imaging Station with a × 40 objective (size bar = 100 μm).

93T449 human liposarcoma cells. Both the SW-872 and 93T449 cells, and positive control HCT-116 cells demonstrat- ed a dose-dependent decrease in the total viable cell number after treatment for 72 h with AMG 900, AZD1152-HQPA, and MK-5108. The pan-Aurora kinase inhibitor (AMG 900) demonstrated the greatest potency followed by the AURKB inhibitor (AZD1152-HQPA), and the AURKA inhibitor (MK- 5108) was the least potent of the three drugs. A similar trend was seen with the polyploidy data, where AMG 900 caused a significant increase in the percentage of cells with a DNA content >4N starting at the 25 nM dose for both SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase in the percentage of cells with a DNA content >4N starting at the 100 nM dose for SW-872 cells and 250 nM dose for 93T449 cells. While AMG 900 and AZD1152-HQPA caused polyploidy in both the SW-872 and 93T449 cells, the SW-872 cells exhibited a higher percentage of polyploid cells after drug treatment compared to the 93T449 cells. In contrast, AMG 900 and AZD1152-HQPA had no effect on increasing the percentage of polyploid cells when the hMSCs were treat- ed with drug. The AURKA inhibitor (MK-5108) did not sig- nificantly increase the percentage of cells with a DNA content >4N at any of the doses tested in the SW-872 cells, 93T449 cells, or hMSCs. The pan-Aurora kinase inhibitor used in these studies, AMG 900, is a potent and selective inhibitor of AURKA, AURKB, and AURKC with reported IC50 values of 5, 4, and 1 nM for the three Aurora kinases, respectively (Payton et al. 2010). AZD1152-HQPA is the active form of the pro-drug AZD1152 (barasertib) (Mortlock et al. 2007), and preferential inhibition of AURKB versus AURKA has

concentrations for efficacy and at higher doses (> 500 nM) may exhibit non-specific activity (Shimomura et al. 2010; de Groot et al. 2015). The similar growth inhibitory and polyploi- dy effects observed with AMG 900 and AZD1152-HQPA- treated SW-872 cells are consistent with inhibition of AURKB which causes DNA endoreduplication without cyto- kinesis (Yang et al. 2005). In support of this finding, previous research indicates that inhibition of AURKA and AURKB bypasses the mitotic requirement for AURKA and produces cellular effects, such as polyploidy, that are consistent with inactivation of AURKB (Yang et al. 2005). The MK-5108 was the least potent of the three Aurora kinase inhibitors that was tested with both the SW-872 and 93T449 liposarcoma cells. Blocking AURKA activity leads to chromosome mis- alignment which initiates the spindle assembly checkpoint with an amassing of cells in the G2/M phase of the cell cycle (Yang et al. 2005). At the higher doses of MK-5108 (500 and 1000 nM), there was an apparent increase in the G2/M peak in the SW-872 and 93T449 cells without a statistically signifi- cant increase in polyploidy which is consistent with AURKA inhibition. The DNA histograms for the SW-872 and 93T449 cells treated with 1000 nM MK-5108 indicates a small 8N peak that was not statistically significant from the DMSO vehicle control for SW-872 cells (p = 0.1812) or 93T449 cells (p = 0.0839); however, this trend for inducing polyploidy may represent MK-5108 inhibiting AURKB at a high dose of the drug.
Our results demonstrating susceptibility of SW-872 and 93T449 human liposarcoma cells to Aurora kinase inhibitors are consistent with the published results of Nair and Schwartz (2016) who demonstrated that MLN-8237 (alisertib), which preferentially inhibits AURKA, caused growth inhibition of a number of sarcoma cell lines, including the LS141 and DDLS dedifferentiated liposarcoma cell lines. MLN-8237 has under- gone phase I and phase II clinical testing, and the results indi- cate that the drug was well tolerated with a favorable progression-free survival in liposarcoma patients (DuBois et al. 2016; Dickson et al. 2016). It should also be noted that ABT-348, a kinase inhibitor with activity against several ki- nases, including Aurora kinases, demonstrated inhibitory ac- tivity against HT 1080 human fibrosarcoma xenografts (Glaser et al. 2012).
The SW-872 liposarcoma cells are an undifferentiated cell line that have been used as an in vitro research model of pleomorphic liposarcoma (Bill et al. 2016b). One of the ge- netic alterations identified in pleomorphic liposarcoma is a high occurrence of p53 mutations (Ghadimi et al. 2011), and SW-872 cells also have mutated p53 (Müller et al. 2007; Mills et al. 2009).

been demonstrated with reported K
i
values of 0.36 and
Therefore, having a chemotherapeutic approach for pleo-

1369 nmol/L, respectively (Wilkinson et al. 2007; de Groot et al. 2015). MK-5108 demonstrates preferential activity for AURKA; however, MK-5108 is known to require higher drug
morphic liposarcoma that is independent of p53 may be ad- vantageous. There are reports that cell lines with p53 dysfunc- tion may be more susceptible to Aurora kinase inhibitor

−ΔΔCT
−ΔCT
−/−
WT

NORONHA ET AL.

Figure 4. Real-time RT-PCR analysis of Aurora kinase A and B mRNA expression. Taqman® as- says were used to determine the relative expression of Aurora ki- nase A (panel A) and Aurora ki- nase B (panel B) mRNA in hu- man adipocytes, hMSCs, SW-872 cells, and HCT-116 cells. The data is shown as the fold change cal- culated from the equation 2 ± SD. Statistical analysis was performed using ANOVA followed by Tukey’spost hoc test (p ≤ 0.05) on the 2 values (N = 3). A single asterisk (*) in- dicates a statistically significant
difference compared to adipo- cytes and hMSCs. Two asterisks (**) indicate a statistically signif- icant difference between SW-872 and HCT-116 cells. Aurora kinase A and B mRNA expression was compared between the well-
differentiated 93T449 cells and
the undifferentiated SW-872 cells (panel C, an asterisk (*) indicates a statistically significant differ- ence in expression in the SW-872 cells compared to the 93T449 cells, unpaired two-tailed t test, N = 3, p ≤ 0.05). Differentiation of the hMSCs (panel E) into adipo- cytes (panel F) was confirmed by Oil Red O staining (size
bar = 100 μm) and by RT-PCR analysis of A-FABP and PPAR- γ mRNA expression (panel D).

therapy (Kalous et al. 2013; Marxer et al. 2014). In one study, it was found that breast cancer cell lines that were highly sensitive to AMG 900 had a statistically significant higher frequency of p53 loss of function mutations compared to the cell lines that were less sensitive (Kalous et al. 2013). Another study found that p53 HCT-116 cells are more susceptible to AURKB inhibition, as demonstrated by a greater percentage of cells undergoing mitotic slippage and genome reduplication versus p53 HCT-116 cells, and depletion of p53 using an siRNA approach had a similar effect (Marxer et al. 2014). The well-differentiated 93T449 human liposarcoma cell line was also responsive to the Aurora kinase inhibitors used in this study. The 93T449 cells are reported to contain wild-type

p53 and amplification of MDM2 which negates p53 function (Italiano et al. 2009; Laroche et al. 2017).
Since the SW-872 cells were responsive to Aurora kinase inhibitor drug therapy, we evaluated the expression of AURKA and AURKB at the mRNA and protein levels in comparison to hMSCs, differentiated adipocytes, and HCT- 116 cells. At the mRNA level, we found that AURKA and AURKB expression were significantly increased in the SW- 872 human liposarcoma cells compared to the differentiated adipocytes and hMSCs. At the protein level, AURKA was expressed in the SW-872 cells but was not detected by Western blot analysis in the differentiated adipocytes or hMSCs. Western blot analysis of AURKB demonstrated a

AURORA KINASE INHIBITORS AND LIPOSARCOMA

Figure 5. Western blot analysis of AURKA and AURKB protein expression. Western blot analysis was used to evaluate AURKA (panel A) and AURKB (panel B) protein expression in human differentiated adipocytes, hMSCs, SW-872 cells, and HCT-116 cells ( N = 3).

~ 39 kDa band in the SW-872 cells, differentiated adipocytes, HCT-116 cells, and hMSCs. The expression of the ~ 39 kDa band for AURKB in the differentiated adipocytes and hMSCs in relation to the low level of mRNA expression may be due to attenuated ubiquitin-mediated degradation of the protein, as protein levels of Aurora kinases are regulated by this mecha- nism (Lindon et al. 2016). A smaller immunoreactive band was detected in the SW-872 and HCT-116 cancer cell lines that was not present in the differentiated adipocytes or hMSCs. The product information for the AURKB antibody used in this research indicates that the antibody detects AURKB and a smaller truncated splice variant. Truncated splice variants of AURKB have been described in hepatocel- lular carcinoma (Sistayanarain et al. 2006), and one of these variants (AURKB-Sv2) was not detected in normal liver but was expressed in 33.6% of the hepatocellular carcinoma cases examined and in 61.1% of the metastatic hepatocellular carci- noma cases examined (Yasen et al. 2009). In addition, the expression of this splice variant in hepatocellular carcinoma was associated with advanced cancer stage and poor patient prognosis (Yasen et al. 2009). Investigating how expression of splice variants of AURKB affect susceptibility to Aurora

AURKA (panel C) and AURKB (panel D) expression was also examined in the 93T449 well-differentiated liposaracoma cells versus the undiffer- entiated SW-872 cells

kinase inhibitor chemotherapy is an area of future research. It should be noted that AZD1152-treated HeLa cells demon- strated decreased expression of both activated phosphorylated AURKB and a smaller immunoreactive band, and this smaller immunoreactive band may represent a truncated version of AURKB (Marxer et al. 2014). Our findings for AURKA and AURKB expression in the SW-872 liposarcoma cell line are consistent with previous findings that demonstrated differential expression of Aurora kinases in cancer cells versus normal cells (Smith et al. 2005; Lin et al. 2006; Ulisse et al. 2006 ; Noronha and Volin 2012 ; Puzio- Kuter et al. 2015 ). The 93T449 well-differentiated liposarcoma cells expressed AURKA and AURKB mRNA and protein; however, the expression level was decreased compared to the undifferentiated SW-872 cells. Like the other cancer cell lines examined in this study, the 93T449 cells express both the ~ 39 kDa band and the smaller immunoreactive band for AURKB. We did not examine AURKC expression, as previous re- search indicates that AURKC is most abundantly expressed in reproductive tissue (Gopalan et al. 1997 ; Tseng et al. 1998; Assou et al. 2006 ), and many non-

NORONHA ET AL.

reproductive human tissues and cells are reported to express AURKA and AURKB more abundantly than AURKC (Lin et al. 2006 ).

Conclusions

The results of these in vitro studies using the SW-872 and 93T449 human liposarcoma cell lines indicate that Aurora kinases should be further investigated as a chemotherapeutic drug target for human liposarcoma. Previous research has ex- amined the chemotherapeutic effect of the Aurora kinase A inhibitor, MLN-8237 (alisertib), on the LS141 and DDLS dedifferentiated liposarcoma cell lines in vitro (Dickson et al. 2016). Our in vitro studies with undifferentitated/ pleomorphic SW-872 cells and well-differentiated 93T449 cells indicate that Aurora kinase B inhibitors and pan-Aurora kinase inhibitors should also be investigated further as chemo- therapeutic agents for human liposarcoma.

Acknowledgments We thank Laura Phelps for assistance with the gen- eration of figures.

Funding information This research was supported by Midwestern University intramural research funds and by student research funds from the Biomedical Sciences Program at Midwestern University.

References

American Cancer Society, Cancer Facts and Figures. (2017). https:// www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-
statistics/annual-cancer-facts-and-figures/2017/cancer-facts-and- figures-2017.pdf. Cited Feb 2017
Abbas Manji G, Singer S, Koff A, Schwartz GK (2015) Application of molecular biology to individualize therapy for patients with liposarcoma. Am Soc Clin Oncol Educ Book 2015:213 –218
⦁ ssou S, Anahory T, Pantesco V, Le Carrour T, Pellestor F, Klein B, Reyftmann L, Dechaud H, De Vos J, Hamamah S (2006) The human cumulus-oocyte complex gene-expression profile. Hum Reprod 21: 1705–1719
⦁ axter SS, Carlson LA, Mayer AMS, Hall ML, Fay MJ (2009) Granulocytic differentiation of HL-60 promyelocytic leukemia cells is associated with increased expression of Cul5. In Vitro Cell Dev Biol Anim 45:264–274
Baldini E, Tuccilli C, Prinzi N, Sorrenti S, Antonelli A, Gnessi L, Morrone S, Moretti C, Bononi M, Arlot-Bonnemains Y, D’Armiento M, Ulisse S (2014) Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines. Endocr Relat Cancer 21:797–811
Berdnik D, Knoblich JA (2002) Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein lo- calization during mitosis. Curr Biol 12:640–647
Bill KL, Casadei L, Prudner BC, Iwenofu H, Strohecker AM, Pollock RE (2016a) Liposarcoma: molecular targets and therapeutic implica- tions. Cell Mol Life Sci 73:3711–3718
Bill KL, Garnett J, Meaux I, Ma X, Creighton CJ, Bolshakov S, Barriere C, Debussche L, Lazar AJ, Prudner BC, Casadei L, Braggio D, Lopez G, Zewdu A, Bid H, Lev D, Pollock RE (2016b) SAR405838: a novel and potent inhibitor of the MDM2:p53 axis

for the treatment of dedifferentiated liposarcoma. Clin Cancer Res 22:1150–1160
Carmena M, Wheelock M, Funabiki H, Earnshaw WC (2012) The chro- mosomal passenger complex (CPC): from easy rider to the godfa- ther of mitosis. Nat Rev Mol Cell Biol 13:789–803
Cheung CH, Sarvagalla S, Lee JY, Huang YC, Coumar MS (2014) Aurora kinase inhibitor patents and agents in clinical testing: an update (2011–2013). Expert Opin Ther Pat 24:1021–1038
Chinn DC, Holland WS, Mack PC (2014) Anticancer activity of the Aurora A kinase inhibitor MK-5108 in non-small cell lung cancer (NSCLC) in vitro as monotherapy and in combination with chemo- therapies. J Cancer Res Clin Oncol 140:1137–1149
Crago AM, Dickson MA (2016) Liposarcoma: multimodality and future targeted therapies. Surg Oncol Clin N Am 25:761–773
Crago AM, Singer S (2011) Clinical and molecular approaches to well differentiated and dedifferentiated liposarcoma. Curr Opin Oncol 23:373–378
de Groot CO, Hsia JE, Anzola JV, Motamedi A, Yoon M, Wong YL, Jenkins D, Lee HJ, Martinez MB, Davis RL, Gahman TC, Desai A, Shiau AK (2015) A cell biologist’sfield guide to Aurora kinase inhibitors. Front Oncol 5:285
Dennis M, Davies M, Oliver S, D’SouzaR, Pike L, Stockman P (2012) Phase I study of the Aurora B kinase inhibitor barasertib (AZD1152) to assess the pharmacokinetics, metabolism and excretion in patients with acute myeloid leukemia. Cancer Chemother Pharmacol 70: 461–469
De Vita A, Mercatali L, Recine F, Pieri F, Riva N, Bongiovanni A, Liverani C, Spadazzi C, Miserocchi G, Amadori D, Ibrahim T (2016) Current classification, treatment options, and new perspec- tives in the management of adipocytic sarcomas. Onco Targets Ther 9:6233–6246
Dickson MA, Mahoney MR, Tap WD, D’Angelo SP, Keohan ML, Van Tine BA, Agulnik M, Horvath LE, Nair JS, Schwartz GK (2016) Phase II study of MLN8237 (alisertib) in advanced/metastatic sar- coma. Ann Oncol 27:1855–1860
Doria-Torra G, Martínez J, Domingo M, Vidaña B, Isidoro-Ayza M, Casanova MI, Vidal E (2015) Liposarcoma in animals: literature review and case report in a domestic pig (Sus scrofa). J Vet Diagn Investig 27:196–202
DuBois SG, Marachelian A, Fox E, Kudgus RA, Reid JM, Groshen S, Malvar J, Bagatell R, Wagner L, Maris JM, Hawkins R, Courtier J, Lai H, Goodarzian F, Shimada H, Czarnecki S, Tsao-Wei D, Matthay KK, Mosse YP (2016) Phase I study of the aurora A kinase inhibitor alisertib in combination with irinotecan and temozolomide for patients with relapsed or refractory neuroblastoma: a NANT (New Approaches to Neuroblastoma Therapy) trial. J Clin Oncol 34:1368 –1375
⦁ alchook GS, Bastida CC, Kurzrock R (2015) Aurora kinase inhibitors in oncology clinical trials: current state of the progress. Semin Oncol 42:832–848
⦁ hadimi MP, Liu P, Peng T, Bolshakov S, Young ED, Torres KE, Colombo C, Hoffman A, Broccoli D, Hornick JL, Lazar AJ, Pisters P, Pollock RE, Lev D (2011) Pleomorphic liposarcoma: clin- ical observations and molecular variables. Cancer 117:5359–5369
Giet R, Prigent C (1999) Aurora/IpI1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J Cell Sci 112:3591– 3601
Giles FJ, Cortes J, Jones D, Bergstrom D, Kantarjian H, Freedman SJ (2007) MK-0457, a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the T315I BCR-ABL mutation. Blood 109:500 –502
Giles FJ, Swords RT, Nagler A, Hochhaus A, Ottmann OG, Rizzieri DA, Talpaz M, Clark J, Watson P, Xiao A, Zhao B, Bergstrom D, Le Coutre PD, Freedman SJ, Cortes JE (2013) MK-0457, an Aurora kinase and BCR-ABL inhibitor, is active in patients with BCR-ABL T315I leukemia. Leukemia 27:113–117

AURORA KINASE INHIBITORS AND LIPOSARCOMA

Glaser KB, Li J, Marcotte PA, Magoc TJ, Guo J, Reuter DR, Tapang P, Wei RQ, Pease LJ, Bui MH, Chen Z, Frey RR, Johnson EF, Osterling DJ, Olson AM, Bouska JJ, Luo Y, Curtin ML, Donawho CK, Michaelides MR, Tse C, Davidsen SK, Albert DH (2012) Preclinical characterization of ABT-348, a kinase inhibitor targeting the aurora, vascular endothelial growth factor receptor/platelet- derived growth factor receptor, and Src kinase families. J Pharmacol Exp Ther 343:617 –627
Glover DM, Leibowitz MH, McLean DA, Parry H (1995) Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81:95–105
⦁ opalan G, Chan CS, Donovan PJ (1997) A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators. J Cell Biol 138:643–656
⦁ elfrich BA, Kim J, Gao D, Chan DC, Zhang Z, Tan AC, Bunn PA Jr (2016) Barasertib (AZD1152), a small molecule Aurora B inhibitor, inhibits the growth of SCLC cell lines in vitro and in vivo. Mol Cancer Ther 15:2314 –2322
⦁ enze J, Bauer S (2013) Liposarcomas. Hematol Oncol Clin N Am 27: 939–955
⦁ taliano A, Maire G, Sirvent N, Nuin PA, Keslair F, Foa C, Louis C, Aurias A, Pedeutour F (2009) Variability of origin for the neocentromeric sequences in analphoid supernumerary marker chromosomes of well-differentiated liposarcomas. Cancer Lett 273:323 –330
Kalous O, Conklin D, Desai AJ, Dering J, Goldstein J, Ginther C, Anderson L, Lu M, Kolarova T, Eckardt MA, Langerød A, Børresen-Dale AL, Slamon DJ, Finn RS (2013) AMG 900, pan- Aurora kinase inhibitor, preferentially inhibits the proliferation of breast cancer cell lines with dysfunctional p53. Breast Cancer Res Treat 141:397–408
Karavasilis V, Seddon BM, Ashley S, Al-Muderis O, Fisher C, Judson I (2008) Significant clinical benefit of first-line palliative chemother- apy in advanced soft-tissue sarcoma: retrospective analysis and identification of prognostic factors in 488 patients. Cancer 112: 1585–1591
Kollár A, Benson C (2014) Current management options for liposarcoma and challenges for the future. Expert Rev Anticancer Ther 14:297– 306
⦁ ollareddy M, Dzubak P, Zheleva D, Hajduch M (2008) Aurora kinases: structure, functions and their associations with cancer. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 152:27–33
⦁ aroche A, Chaire V, Le Loarer F, Algéo MP, Rey C, Tran K, Lucchesi C, Italiano A (2017) Activity of trabectedin and the PARP inhibitor rucaparib in soft-tissue sarcomas. J Hematol Oncol 10:84
Lin YS, Su LJ, Yu CT, Wong FH, Yeh HH, Chen SL, Wu JC, Lin WJ, Shiue YL, Liu HS, Hsu SL, Lai JM, Huang CY (2006) Gene ex- pression profiles of the aurora family kinases. Gene Expr 13:15–26
Lindon C, Grant R, Min M (2016) Ubiquitin-mediated degradation of Aurora kinases. Front Oncol 5:307
⦁ öwenberg B, Muus P, Ossenkoppele G, Rousselot P, Cahn JY, Ifrah N, Martinelli G, Amadori S, Berman E, Sonneveld P, Jongen-Lavrencic M, Rigaudeau S, Stockman P, Goudie A, Faderl S, Jabbour E, Kantarjian H (2011) Phase 1/2 study to assess the safety, efficacy, and pharmacokinetics of barasertib (AZD1152) in patients with ad- vanced acute myeloid leukemia. Blood 118:6030–6036
⦁ arumoto T, Honda S, Hara T, Nitta M, Hirota T, Kohmura E, Saya H (2003) Aurora-A kinase maintains the fidelity of early and late mi- totic events in HeLa cells. J Biol Chem 278:51786–51795
Marxer M, Ma HT, Man WY, Poon RYC (2014) p53 deficiency enhances mitotic arrest and slippage induced by pharmacological inhibition of Aurora kinases. Oncogene 33:3550 –3560
Matulonis UA, Sharma S, Ghamande S, Gordon MS, Del Prete SA, Ray- Coquard I, Kutarska E, Liu H, Fingert H, Zhou X, Danaee H, Schilder RJ (2012) Phase II study of MLN8237 (alisertib), an inves- tigational Aurora A kinase inhibitor, in patients with platinum-

resistant or -refractory epithelial ovarian, fallopian tube, or primary peritoneal carcinoma. Gynecol Oncol 127:63–69
Meulenbeld HJ, Bleuse JP, Vinci EM, Raymond E, Vitali G, Santoro A, Dogliotti L, Berardi R, Cappuzzo F, Tagawa ST, Sternberg CN, Jannuzzo MG, Mariani M, Petroccione A, de Wit R (2013) Randomized phase II study of danusertib in patients with metastatic castration-resistant prostate cancer after docetaxel failure. BJU Int 111:44–52
Mills J, Matos T, Charytonowicz E, Hricik T, Castillo-Martin M, Remotti F, Lee FY, Matushansky I (2009) Characterization and comparison of the properties of sarcoma cell lines in vitro and in vivo . Hum Cell 22:85–93
Mortlock AA, Foote KM, Heron NM, Jung FH, Pasquet G, Lohmann JJ, Warin N, Renaud F, De Savi C, Roberts NJ, Johnson T, Dousson CB, Hill GB, Perkins D, Hatter G, Wilkinson RW, Wedge SR, Heaton SP, Odedra R, Keen NJ, Crafter C, Brown E, Thompson K, Brightwell S, Khatri L, Brady MC, Kearney S, McKillop D, Rhead S, Parry T, Green S (2007) Discovery, synthesis, and in vivo activity of a new class of pyrazoloquinazolines as selective inhibitors of aurora B kinase. J Med Chem 50:2213 –2224
⦁ üller CR, Paulsen EB, Noordhuis P, Pedeutour F, Saeter G, Myklebost O (2007) Potential for treatment of liposarcomas with the MDM2 antagonist Nutlin-3A. Int J Cancer 121:199 –205
⦁ air JS, Schwartz GK (2016) MLN-8237: a dual inhibitor of aurora A and B in soft tissue sarcomas. Oncotarget 7:12893 –12903
Noronha S, Volin M (2012) Aurora kinase B protein is overexpressed in high-grade sarcomas: a pilot study. Am J Clin Pathol 138:A117
Payton M, Bush TL, Chung G, Ziegler B, Eden P, McElroy P, Ross S, Cee VJ, Deak HL, Hodous BL, Nguyen HN, Olivieri PR, Romero K, Schenkel LB, Bak A, Stanton M, Dussault I, Patel VF, Geuns-Meyer S, Radinsky R, Kendall RL (2010) Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines. Cancer Res 70: 9846–9854
⦁ uzio-Kuter AM, Laddha SV, Castillo-Martin M, Sun Y, Cordon-Cardo C, Chan CS, Levine AJ (2015) Involvement of tumor suppressors PTEN and p53 in the formation of multiple subtypes of liposarcoma. Cell Death Differ 22:1785 –1791
⦁ uartuccio SM, Schindler K (2015) Functions of Aurora kinase C in meiosis and cancer. Front Cell Dev Biol 3:50
⦁ obinson JK, Rademaker AW, Goolsby C, Traczyk TN, Zoladz C (1996) DNA ploidy in nonmelanoma skin cancer. Cancer 77:284–291
⦁ andberg AA (2004) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: liposarcoma. Cancer Genet Cytogenet 155:1–24
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108
Shimomura T, Hasako S, Nakatsuru Y, Mita T, Ichikawa K, Kodera T, Sakai T, Nambu T, Miyamoto M, Takahashi I, Miki S, Kawanishi N, Ohkubo M, Kotani H, Iwasawa Y (2010) MK-5108, a highly selec- tive Aurora-A kinase inhibitor, shows antitumor activity alone and in combination with docetaxel. Mol Cancer Ther 9:157–166
Sistayanarain A, Tsuneyama K, Zheng H, Takahashi H, Nomoto K, Cheng C, Murai Y, Tanaka A, Tanako Y (2006) Expression of Aurora-B kinase and phosphorylated histone H3 in hepatocellular carcinoma. Anticancer Res 26:3585–3593
Smith SL, Bowers NL, Betticher DC, Gautschi O, Ratschiller D, Hoban PR, Booton R, Santibáñez-Koref MF, Heighway J (2005) Overexpression of aurora B kinase (AURKB) in primary non- small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability. Br J Cancer 93:719–729
Stratford EW, Castro R, Daffinrud J, Skårn M, Lauvrak S, Munthe E, Myklebost O (2012) Characterization of liposarcoma cell lines for preclinical and biological studies. Sarcoma 2012:148614

NORONHA ET AL.

Tao Y, Leteur C, Calderaro J, Girdler F, Zhang P, Frascogna V, Varna M, Opolon P, Castedo M, Bourhis J, Kroemer G, Deutsch E (2009) The aurora B kinase inhibitor AZD1152 sensitizes cancer cells to frac- tionated irradiation and induces mitotic catastrophe. Cell Cycle 8: 3172–3181
Tao Y, Zhang P, Girdler F, Frascogna V, Castedo M, Bourhis J, Kroemer G, Deutsch E (2008) Enhancement of radiation response in p53- deficient cancer cells by the Aurora-B kinase inhibitor AZD1152. Oncogene 27:3244–3255
Tseng TC, Chen SH, Hsu YP, Tang TK (1998) Protein kinase profile of sperm and eggs: cloning and characterization of two novel testis- specific protein kinases (AIE1, AIE2) related to yeast and fly chro- mosome segregation regulators. DNA Cell Biol 17:823–833
Tsuboi K, Yokozawa T, Sakura T, Watanabe T, Fujisawa S, Yamauchi T, Uike N, Ando K, Kihara R, Tobinai K, Asou H, Hotta T, Miyawaki S (2011) A phase I study to assess the safety, pharmacokinetics and efficacy of barasertib (AZD1152), and Aurora B kinase inhibitor, in Japanese patients with advanced acute myeloid leukemia. Leuk Res 35:1384 –1389
⦁ uccilli C, Baldini E, Prinzi N, Morrone S, Sorrenti S, Filippini A, Catania A, Alessandrini S, Rendina R, Coccaro C, D’Armiento M, Ulisse S (2016) Preclinical testing of selective Aurora kinase inhib- itors on a medullary thyroid carcinoma-derived cell line. Endocrine 52:287–295
⦁ lisse S, Delcros JG, Baldini E, Toller M, Curcio F, Giacomelli L, Prigent C, Ambesi-Impiombato FS, D’Armiento M, Arlot-Bonnemains Y

(2006) Expression of Aurora kinases in human thyroid carcinoma cell lines and tissues. Int J Cancer 119:275–282
Verweij J, Baker LH (2010) Future treatment of soft tissue sarcomas will be driven by histological subtype and molecular aberrations. Eur J Cancer 46:863–868
⦁ indeløv LL, Christensen IJ, Nissen NI (1983) A detergent-trypsin meth- od for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3:323–327
⦁ ilkinson RW, Odedra R, Heaton SP, Wedge SR, Keen NJ, Crafter C, Foster JR, Brady MC, Bigley A, Brown E, Byth KF, Barrass NC, Mundt KE, Foote KM, Heron NM, Jung FH, Mortlock AA, Boyle FT, Green S (2007) AZD1152, a selective inhibitor of Aurora B kinase, inhibits human tumor xenograft growth by inducing apopto- sis. Clin Cancer Res 13:3682 –3688
Yang H, Burke T, Dempsey J, Diaz B, Collins E, Toth J, Beckmann R, Ye X (2005) Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase. FEBS Lett 579:3385–3391
⦁ asen M, Mizushima H, Mogushi K, Obulhasim G, Miyaguchi K, Inoue K, Nakahara I, Ohta T, Aihara A, Tanaka S, Arii S, Tanaka H (2009) Expression of Aurora B and alternative variant forms in hepatocel- lular carcinoma and adjacent tissue. Cancer Sci 100:472–480
⦁ ekri A, Ghaffari SH, Ghanizadeh-Vesali S, Yaghmaie M, Salmaninejad A, Alimoghaddam K, Modarressi MH, Ghavamzadeh A (2015) AZD1152-HQPA induces growth arrest and apoptosis in androgen- dependent prostate cancer cell line (LNCaP) via producing aneugenic micronuclei and polyploidy. Tumour Biol 36:623–632 AMG-900