p53 family members regulate phenotypic response to ......small molecule therapeutics p53 family...

Small Molecule Therapeutics

p53 Family Members Regulate PhenotypicResponse to Aurora Kinase A Inhibition inTriple-Negative Breast CancerJohn J. Tentler1, Anastasia A. Ionkina1, Aik Choon Tan1, Timothy P. Newton1,Todd M. Pitts1, Magdalena J. Glogowska1, Peter Kabos1, Carol A. Sartorius2,Kelly D. Sullivan3, Joaquin M. Espinosa3, S. Gail Eckhardt1, and Jennifer R. Diamond1

Abstract

Triple-negative breast cancer (TNBC) is an aggressive diseasewith a poor prognosis. Advances in the treatment of TNBC havebeen hampered by the lack of novel effective targeted therapies.The primary goal of this study was to evaluate the efficacy oftargeting Aurora kinase A (AurA), a key regulator of mitosis, inTNBC models. A secondary objective was to determine the roleof the p53 family of transcriptional regulators, commonlymutated in TNBC, in determining the phenotypic response tothe AurA inhibitor alisertib (MLN8237). Alisertib exhibitedpotent antiproliferative and proapoptotic activity in a subsetof TNBC models. The induction of apoptosis in response to

alisertib exposure was dependent on p53 and p73 activity. Inthe absence of functional p53 or p73, there was a shift in thephenotypic response following alisertib exposure from apo-ptosis to cellular senescence. In addition, senescence wasobserved in patient-derived tumor xenografts with acquiredresistance to alisertib treatment. AurA inhibitors are a promis-ing class of novel therapeutics in TNBC. The role of p53 andp73 in mediating the phenotypic response to antimitotic agentsin TNBC may be harnessed to develop an effective biomarkerselection strategy in this difficult to target disease. Mol CancerTher; 14(5); 1117–29. �2015 AACR.

IntroductionTriple-negative breast cancer (TNBC) is an aggressive breast

cancer subtype defined by the lack of estrogen and progesteronereceptor expression and lack of HER2 amplification (1). Patientswith TNBC have a higher risk of cancer recurrence and deathrelated to breast cancer as compared with other breast cancersubtypes (1, 2). Systemic therapy is currently limited to cytotoxicchemotherapy and effective targeted anticancer therapy remainsto be proven (3). TNBC is a biologically heterogeneous diseaseand the development of active targeted therapies in conjunctionwith effective biomarker selection strategies is an area of unmetclinical need (3, 4).

Aurora kinase A (AurA) is a serine/threonine kinase integral tomitotic cell division (5, 6). AurA is essential in chromosomealignment, the formation of a bipolar mitotic spindle, and cyto-kinesis during mitosis (7, 8). It may function as an oncogene

through the promotion of genetic instability (9) and overexpres-sion of AurA has been linked to inferior outcomes in patientswith early-stage breast cancer (10). Aurora kinase inhibitors (AKI)are a promising class of drugs for the treatment of TNBC as theyare mitotic inhibitors and TNBC as a subtype has a high mitoticindex (11).

TP53 is the most commonly mutated gene in TNBC with anincidence of approximately 85% (12). Although the majority ofmutations are missense mutations in the DNA-bindingdomain, more complex mutations (i.e., frameshift and non-sense mutations) occur at a higher frequency in TNBC ascompared with luminal breast cancers (13). Mutations inp53 may abrogate its tumor suppressor function resulting inimpairment of cell-cycle arrest, DNA repair, and apoptosis (14).AurA overexpression may lead to increased p53 degradation viaphosphorylation of p53 at Ser315, leading to increased ubi-quitination by MDM2 (15). Furthermore, silencing of AurAresults in stabilization of p53 and a characteristic G2–M cell-cycle arrest (15). The role of p53 in mediating sensitivity toAKIs in TNBC is critical due to its high mutation rate in TNBCand the potential for p53 to affect terminal cellular outcomefollowing drug exposure.

Alisertib (MLN8237) is an orally bioavailable, second-gener-ation selective inhibitor of Aurora kinases which binds to AurAand prevents its phosphorylation and activation (16). We havepreviously shown that p53-mutated TNBC cell lines withincreased p53 protein and mRNA expression had increased sen-sitivity in vitro to the antiproliferative effects of the multitargetAurA and angiogenic kinase inhibitor, ENMD-2076 (17). Thepurpose of this study was to evaluate the antiproliferative activityof alisertib against preclinical TNBC models and investigate the

1Division of Medical Oncology, Department of Medicine, University ofColorado Anschutz Medical Campus, Aurora, Colorado. 2Departmentof Pathology, University of Colorado Anschutz Medical Campus,Aurora, Colorado. 3Molecular, Cellular and Developmental Biology,University of Colorado Boulder, Boulder, Colorado.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Jennifer R. Diamond, Division of Medical Oncology,University of Colorado Anschutz Medical Campus, Mailstop 8117, 12801 East 17thAvenue, Room L18-4100, Aurora, CO 80045. Phone: 303-724-5499; Fax:303-724-3889; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0538-T

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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Published OnlineFirst March 10, 2015; DOI: 10.1158/1535-7163.MCT-14-0538-T

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role of p53 and the p53 family member, p73, in mediatingresponse to selective AurA inhibition.

Materials and MethodsCell culture and reagents

Human TNBC cell lines were obtained and cultured as previ-ously described (17). In addition, SW527 and HCC1395 wereobtained from ATCC. CAL-51 was obtained from the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ). Cells were passaged for less than 6 months.

MLN8237 was prepared in DMSO for in vitro experimentationand suspended in a 1:1 mixture of 10% hydroxypropyl b-cyclo-dextran (HPBCD) and 2% NaHCO2 for in vivo use. Nutlin-3(Sigma-Alrich) was prepared as a 10 mmol/L stock solution inDMSO.

Cell viability experimentsThe sulforhodamine B (SRB) proliferation assay was per-

formed as previously described to evaluate the cytotoxic effectof MLN8237 on TNBC cell lines at least in triplicate (18, 19).The CyQuant measurement of cellular DNA content via fluo-rescent dye was performed using the CyQuant NF Cell Prolif-eration Assay Kit and Protocol (Invitrogen). In brief, cells wereharvested during the logarithmic growth phase and plated in96-well flat-bottomed plates with lids. Cells were allowed toadhere overnight and then exposed to increasing doses ofMLN8237 from 0 mmol/L to 0.1 mmol/L for 96 hours. For theSRB assay, the incubated cells were fixed, stained with 0.4% SRB(MP Biomedicals), and intensity read using a plate reader(Biotek Synergy 2) at an absorbance wavelength of 565 nm.For the CyQuant assay, cellular growth media were removedfollowed by incubation with the CyQuant dye for 30 minute at37�C. Next, the fluorescence intensity of each plate was mea-sured using a plate reader (Biotek Synergy 2) with excitation atapproximately 485 nm and emission detection at approximate-ly 530 nm.

CAL-51 p53 and p73 shRNA knockdown modelsThe CAL-51 cell line was transduced with several clones of

shRNA GFP-tagged constructs targeting p53 or p73 and grown inpuromycin (2.5%) supplementedmedia for at least 21 days. qRT-PCR was used to confirm adequate KD using TaqMan microRNAAssay kit (Applied Biosystems).

Analysis of apoptosisThe CAL-51, CAL-51 scramble control (SCR), and p53/p73

knockdown (KD) clones were seeded in 6-well plates (3� 105 perwell) and allowed to adhere for 24 hours at 37 �C.Next, they wereexposed to MLN8237 (50 or 100 nmol/L) for 24 to 96 hours at37 �C and prepared using the Annexin V/Dead Cell Apoptosis Kit(Invitrogen). The CAL-51 cells were stained with propidiumiodide (PI) and FITC, whereas the CAL-51 SCR and KD cell lineswere stained with PI and allophycocyanin conjugate (APC), dueto theGFP tag present in their plasmids. Unstained, FITC andAPCcontrols were used as standards. Samples were analyzed using theCyAn ADP Analyzer at the UCCC Flow Cytometry Core Facility(FCCF) for viable, early apoptotic, late apoptotic, and dead cells.Apoptosiswas also assessed using a luminometric Caspase-Glo-3/7 assay (Promega).

Flow-cytometric analysis of cell-cycle distributionCells were seeded in 6-well plates (2 � 105 per well) and

allowed to adhere for 24 hours at 37�C before being treated withMLN8237 (20 or 80 nmol/L) for 2 to 10 days. Analysis wasperformed as previously described (17).

SenescenceCells were seeded in 24-well plates, allowed to adhere over-

night at 37�C, and treated with vehicle or MLN8237 (50 or 100nmol/L) for 5 to 15 days. Cells were fixed and stained forsenescence-associated b-galactosidase (SA-b-gal) using the Senes-cence b-galactosidase Staining Kit (Cell Signaling Technology).Images were acquired using a Nikon inverted microscope at �20magnification. Images from three different representative fields ofview were taken at each time point. The number of cells in eachfield was counted and an average with SD calculated. The averagecellular size was obtained by selecting a representative total of 10cells from each of the three fields of view followed by calculatingthe average with SD.

For analysis of senescence onxenograft tumor samples, stainingfor SA-b-gal activity was performed as previously described byDebacq-Chainiaux and colleagues (20). Hematoxylin and eosin(H&E) staining was performed on matched slides by the UCCCPathology Core Facility and reviewed by a pathologist to confirmthe presence of tumor cells.

Nutlin combination studiesCAL-51 cells were plated in 96-well plates, allowed to adhere

overnight then exposed to increasing doses of MLN8237 (0–200nmol/L), Nutlin-3 (0–20.0 mmol/L), or the combination for 96hours. Proliferation was assessed using the SRB method asdescribed above and synergy was determined using the Chouand Talalay method using the Calcusyn software program(Biosoft; ref. 21). For each combination, the combination index(CI) was calculated with synergy indicated by a CI < 1, additivityby a CI ¼ 1, and antagonism by a CI > 1.

Immunoblotting analysisCellswere seeded into 6-well plates and allowed to attach for 24

hours before exposure to MLN8237. Cells were harvested withtrypsin/EDTA and then lysed in Cell Lysis Buffer (Cell SignalingTechnology). Forty mg of total protein was loaded onto a 4% to20% gradient gel, electrophoresed, and then transferred to nitro-cellulose using the i-Blot system (Invitrogen). Membranes wereblocked in blocking buffer and incubated overnight at 4�C withprimary antibodies: PARP, p53, BAX, p21 (Cell Signaling Tech-nology), p16 (Santa Cruz Biotechnology), and b-actin (Sigma-Aldrich). Following primary antibody incubation, membraneswere washed in TBS-Tween (0.1%) and incubated with secondaryanti-rabbit or anti-mouse IgG1 horseradish peroxidase-linkedantibody at 1:15,000 (Jackson ImmunoResearch) for 1 hour atroom temperature (RT). Blots were developed using the OdysseyInfrared Imaging System (LI-COR Biosciences). Experiments wereperformed in triplicate.

ImmunofluorescenceCAL-51 SCR and p53/p73 KD cells were seeded onto glass

chamber slides and allowed to incubate overnight at 37�C thentreated with either vehicle or MLN8237 (50 or 100 nmol/L) for 4days then fixed with paraformaldehyde. Chamber slides were

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Mol Cancer Ther; 14(5) May 2015 Molecular Cancer Therapeutics1118




blockedwith 1%BSA inPBS for 30minutes at 37�Cand incubatedwith the primary antibody for 1 hour at RT [p53mousemAb (CellSignaling Technology), p16 (C-20 Santa Cruz Biotechnology), orBAX (ab10813 Abcam]. Slides were washed in PBS and incubatedwith secondary antibodies [AlexaFluor 555 (Invitrogen) or Alex-aFluor 488 (Life Technologies)] and washed with PBS. Counter-staining with 300 nmol/L DAPI in PBS for 5 minutes at RT wasperformed. Slides were mounted with Fluoromount-G mountingmedia (SouthernBiotech), cover slipped, and sealed with clearnail polish. Slides were imaged using the Olympus FV-1000confocal microscope at �60 magnification.

In vivo patient tumor-derived and TNBC cell line xenograftstudies

Five- to 6-week-old female athymic nude (nu/nu) mice (HarlanSprague Dawley) were housed in groups of five, allowed toacclimate for one week before handling, provided with sterilizedfood and water ad libitum and kept on a 12-hour light/dark cycle.Patient tumor tissue was obtained from consented patients underan Institutional Review Board-approved protocol and implantedand expanded initially in NOD/SCID/ILIIrg(�/�) mice (22).After expansion, tumor tissue was obtained, minced into5 mm3 sections and implanted subcutaneously into nu/nu miceusing a 12-gauge trocar as previously described (23, 24). CAL-51cells were harvested during logarithmic growth and resuspendedin a 1:1 mixture of serum-free DMEM and Matrigel (BD Bios-ciences). The cells were injected bilaterally into the flank using 2.5� 106 cells/injection in a volume of 100 mL. Mice weremonitoreddaily for toxicity andweighed twiceweekly. Tumormeasurementswere performed daily using calipers with the Study Directorsoftware package (Studylog Systems) and tumor volume wascalculated using the following formula: volume ¼ (length �width2) � 0.52. When tumors reached a mean volume of150 mm3, mice were randomized into vehicle and MLN8237groups with 10 tumors/group. MLN8237 (30 mg/kg) or vehiclecontrol was administered via oral gavage with continuous oncedaily dosing. At the end of treatment, mice were euthanized withCO2 and tumor samples were collected for gross anatomic andIHCanalyses. Tumor growth inhibition (TGI)was calculated fromthe average tumor volume of the treated (Vt) and vehicle control(Vvc) groups, with the equation: TGI¼ 1� (Vt/Vvc). All xenograftstudies were performed in accordance with theNIH guidelines forthe care and use of laboratory animals in a facility accredited bythe American Association for Accreditation of Laboratory AnimalCare with approval by the University of Colorado (Aurora, CO)Institutional Animal Care and Use review board before initiationof experiments.

IHCFormalin fixed, paraffin-embedded tumor samples were pro-

cessed and stained using Ki-67 Cline SP6 (Thermo Scientific)and cleaved caspase-3 (Cell Signaling Technology) by the UCCCHistology Core Facility. Images were acquired using a Zeissmicroscope at �10 magnification.

Statistical analysisTreatment groups were compared by ANOVA parametric

analysis of the means using a commercially available statisticalprogram (Prism 4.0, GraphPad). The comparisons betweensensitivity of treatment and vehicle groups in vivo were com-

pared by unpaired parametric t test with Welch's correctionsusing a commercially available statistical program (Prism 4.0,GraphPad). Specific tests applied are included in the figurelegends.

ResultsAntiproliferative activity of MLN8237 against 18 TNBC celllines in vitro

A panel of 18 TNBC cell lines representing the basal-like 1and 2 (BL1/2), immunomodulatory (IM), mesenchymal (M),and mesenchymal stem-like (MSL) subtypes was screened for invitro sensitivity to MLN8237 (4). Figure 1A depicts the in vitroresponse of MLN8237 treatment in breast cancer cell lineswhere IC50 values ranged from 23 nmol/L to > 5 mmol/L(Supplementary Table S1). We did not observe statisticallysignificant differences in sensitivity to MLN8237 by TNBCsubtype (mean IC50 IM 0.23 mmol/L, BL1/2 0.69 mmol/L,M/MSL 0.93 mmol/L, P ¼ 0.197). Within the MLN8237-sensi-tive TNBC cell lines (IC50 < 0.1 mmol/L), the IM, BL1, BL2, andmesenchymal (M) TNBC subtypes were represented (Fig. 1A).BL1 TNBC cell lines exhibited both sensitivity (MDA-MB-468IC50 0.036 mmol/L) and resistance (HCC1937 IC50 > 5 mmol/L)to MLN8237 exposure (Fig. 1A). Although in the Lehmann andcolleagues landmark study (4), the BL1 and BL2 subtypesexhibited increased expression of genes associated with cell-cycle pathways and proliferation (including AURKA andAURKB), we did not find that these subtypes were predictiveof in vitro sensitivity to MLN8237.

On the basis of the previously described interactions betweenp53 and AurA (15, 25), we investigated p53 mutation statusand mRNA expression of TP53 and AURKA in our TNBC breastcancer panel (Fig. 1A). Microarray gene expression data wereobtained from the Cancer Cell Line Encyclopedia (GSE36133).There was no correlation between TP53 mutation, AURKAexpression, or TP73 expression and sensitivity to MLN8237exposure; however, there was a trend toward increased sensi-tivity to MLN8237 in cell lines with increased TP53 expression(Pearson correlation coefficient r ¼ �0.49, P ¼ 0.066). Aninverse correlation between AURKA expression and p53 expres-sion was observed possibly explained by the ability of AURKAto phosphorylate p53 at Ser315 and accelerate its degradation(15).

Knockdown (KD) of p53 or p73 leads to resistance toMLN8237AurA expression in malignant cells is associated with an

abrogation of the DNA damage-induced apoptotic response, atleast in part due to functional inactivation of p53 (15, 25). Inaddition, AurA overexpression leads to increased phosphory-lation of the p53 family member p73. This results in cyto-plasmic sequestration of p73 with its chaperone protein,mortalin, thus diminishing its transactivation function (26).On the basis of the regulation of both p53 and p73 by AurAand reports in the literature supporting a role for both p53family members in determining cellular fate in response toAKI, we investigated the role of both in mediating sensitivity toMLN8237 in TNBC (26, 27). We hypothesized that sensitivityto MLN8237 in p53 WT TNBC is mediated through both p53and p73 and selected the p53 WT CAL-51 cell line forshRNA KD of p53 and p73. Expression of p53 was decreasedby 82% (P < 0.01) and 63.5% (P < 0.05) in the p53-10 andp53-12 clones, respectively, as compared with the SCR control

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(Fig. 1B). Expression of p73 was decreased by 71% (P < 0.05)and 77% (P < 0.05) in the p73-26 and p73-55 clones, respec-tively, as compared with the SCR control (Fig. 1C).

In the p53-10, p53-12, p73-26, and p73-55 clones, exposure toincreasing doses of MLN8237 resulted in an increase in the meanIC50 to > 1.5, 0.66, > 1.5, and 1.49 mmol/L, respectively, ascompared with the mean IC50 in the SCR control of 0.39mmol/L assessed using the SRB proliferation assay (Fig. 1D andSupplementary Table S2). There was a statistically significantincrease in residual cellular proliferation following exposure toMLN8237 0.1 mmol/L for 96 hours in the p53-10, p53-12, andp73-26 clones, compared with the SCR control (P < 0.05, Fig. 1E).These findings were confirmed using the CyQuant proliferationassay (Fig. 1F). In summary, KD of p53 or p73 resulted inincreased resistance to the antiproliferative effects of MLN8237in vitro.

Effect of p53 and p73 KD on MLN8237-induced apoptosis andcell-cycle arrest

Inhibition of AurA leads to a transient G2–M cell-cycle arrestfollowed by aberrant cell division and aneuploidy (28). Previousstudies have shown that AurA inhibition induces a modest proa-poptotic response, but can also lead to senescence as a terminaloutcome (29–31). To understand the mechanism of the antipro-liferative effects of MLN8237 in TNBC that are affected by p53 orp73 KD, we investigated alterations in cellular fate, includingapoptosis, cell-cycle arrest, and senescence. Beginning with theCAL-51 p53 WT cell line, we observed an increase in apoptosiswhich peaked at 96 hours as measured by Annexin V bindingdetected using flow cytometry (Fig. 2A). Annexin V is a specificbinder of phosphatidylserine which appears on a cell's surface asan indicator of apoptosis (32). On the basis of a peak in apoptosisat 96 hours in the CAL-51 cell line, this timepoint was selected for

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Figure 1.Antiproliferative effects of MLN8237 against TNBC cell lines in vitro. A, absolute IC50 values of a panel of 18 TNBC cell lines. Below, TNBC subtype, TP53mutation statusand heatmap of TP53, TP73, AURKA, and AURKB gene expression levels are depicted (TNBC subtypes as described in ref. 8. U, unknown; IF, in-frame deletion; MS,missense; FS, frameshift; NS, nonsense). B, relative expression levels of p53 RNA as measured by qRT-PCR in CAL-51 scrambled control (SCR) and p53 shRNAclones 10 and 12 (p53-10, p53-12). C, relative expression levels of p73 RNA as measured by qRT-PCR in CAL-51 SCR and p73 shRNA clones 26 and 55 (p73-26, p73-55). D,effect of increasing doses of MLN8237 on cellular proliferation in the CAL-51 SCR and p53, p73 shRNA knockdown clones as measured by the SRB assay. CAL-51scrambled control (SCR), CAL-51 p53 shRNA clones 10 and 12 (p53-10, p53-12), and CAL-51 p73 shRNA clones 26 and 55 (p73-26, p73-55) were exposed to increasingconcentrations of MLN8237 for 96 hours and proliferation was assessed by SRB. E, effect of MLN8237 at the top dose of 0.1 mmol/L on cellular proliferation in the CAL-51SCR, p53-10, p53-12, p73-26, and p73-55 shRNA knockdown clones as measured by SRB assay. F, effect of increasing doses of MLN8237 on cellular proliferationin the CAL-51 SCR and p53, p73 shRNA knockdown clones as measured by the CyQuant assay. �� , P < 0.001; �� , P < 0.01; �, P < 0.05, as compared with SCR control.

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further experimentation using the p53 and p73 KD clones. At 96hours, there was statistically significant increase in apoptosisfollowing exposure to MLN8237 in the CAL-51scramble con-trol as compared with the CAL-51 p53-10 and p73-55 KDclones (P < 0.001 and < 0.01, respectively; Fig. 2B and C). Inthe p53 and p73 KD cell lines, we observed a decrease in thedegree of apoptosis following exposure to MLN8237; however,there was not a complete abrogation of the proapoptoticresponse. These findings were confirmed using a caspase-3/7activity assay (Fig. 2D).

Next, we investigated the effect of MLN8237 exposure oncell-cycle distribution using flow cytometry in the CAL-51scramble, p53-10 KD and p73-55 KD clones. We observed

that in the CAL-51 scramble cell line, treatment withMLN8237 (20 or 80 nmol/L) for 48 hours led to a dose-dependent G2–M cell-cycle arrest (Fig. 3A and 3B). This resultis consistent with existing literature reporting a G2–M arrestfollowing exposure to AKI in vitro (31, 17). There was not adifference in the G2–M population following exposure toMLN8237 in the p73-55 KD cell line (Fig. 3A and 3B). Beforeinitiating treatment with MLN8237, we observed a markedincrease in the G2–M cell population in the p53-10 KD cells(Fig. 3A). We hypothesize that this may be a result of increasedgenomic instability related to the functional loss of p53. Giventhe increased G2–M population at baseline, we did notobserve further G2–M arrest with MLN8237 treatment for

Figure 2.A, induction of apoptosis in the CAL-51wild-type (WT) cell line when exposedto MLN8237 (100 nmol/L) for theindicated time points as measured byAnnexin V/FITC staining and flowcytometry. � , P < 0.05 compared withDMSO control. B, apoptotic responseof CAL-51scrambled control cell line(SCR) and CAL-51 p53 shRNAknockdown clone 10 (p53-10) andCAL-51 p73 shRNA knockdownclone12 (p73-55) as measured byAnnexin V/APC staining and flowcytometry. �� , P < 0.01; �� , P < 0.001,compared with SCR control.C, representative dot-plots of flow-cytometric analyses of CAL-51 SCRand p53-10 after 96-hour treatmentwith vehicle orMLN8237 (100nmol/L).D, caspase-3/7 activity followingexposure to MLN8237 at the indicateddoses for 48 hours in the CAL-51 WT,SCR, p53, and p73 knockdown clones.� , P < 0.05; �� , P < 0.01, compared withcorresponding DMSO (ND) control.






48 hours (Fig. 3A). There was, however, a small increase inaneuploidy following treatment with MLN8237 at the higherdose level (80 nmol/L; Fig. 3A).

On the basis of the above results and reports describingdelayed effects of AurA inhibition on cellular fate (29), weinvestigated the effect of MLN8237 exposure on cell-cycledistribution following a 10-day exposure. There were notsufficient numbers of CAL-51 SCR control cells viable afterprolonged exposure to MLN8237 for cell-cycle analysis due tothe potent antiproliferative effects. In both the p53-10 KD andthe p73-55 KD cell lines, however, we observed a continueddose-dependent increase in aneuploidy (Fig. 3A). In summary,we observed a dose-dependent G2–M arrest following exposureto MLN8237 for 48 hours in the CAL-51 WT and SCR controlwhich was diminished in the p73-55 KD and increased aneu-ploidy in the p53-10 and p73-55 KD clones following a 10-dayexposure. An increase in aneuploidy in the p53-10 KD cell linemay be related to the loss of p53-dependent elimination oftetraploid cells (33).

Resistance to Aurora kinase inhibition in vitro is associatedwithinduction of senescence

We have previously demonstrated that AKI leads to induc-tion of senescence as a terminal cellular fate in a subset of TNBCcell lines less sensitive to the antiapoptotic effects (17). The roleof p53 and p73 in mediating induction of senescence is notclearly defined in the literature, with evidence pointing bothtoward a role for p53 in mediating senescence and in itsparadoxical suppression of senescence (34, 35). Senescence isclassically described as an irreversible terminal growth arrestthat can be identified by changes in cellular morphology (large,flattened cells with increased vacuoles) and the presence ofSA-b-gal activity (36). To explore the role of p53 and p73 inmediating induction of senescence TNBC, we exposed the sameCAL-51 scramble, p53-10 KD and the p73-55 KD cell lines toMLN8237 (100 nmol/L) for 5, 10, or 15 days and evaluatedcells for morphologic changes and the presence of SA-b-galactivity. As shown in Fig. 4A, Supplementary Figs. S1 and S2and Supplementary Table S3, treatment with MLN8237

Figure 3.A, quantification of cell-cycle parameters using propidium iodide staining and flow cytometry in the CAL-51 SCR, p53-10, and p73-55 knockdown clonesfollowing exposure to MLN8237 at the indicated doses for 48 hours and 10 days. A statistically significant G2–M arrest was observed for CAL-51 SCR after48 hours of 80 nmol/L MLN8237 exposure. þþ, P < 0.01 indicates a statistically significant increase in G2–M as compared with the corresponding NDcontrol. ��, P < 0.001; �� , P < 0.01; � , P < 0.05 indicate a statistically significant increase in aneuploidy as compared with the corresponding ND control. B,representative flow-cytometric analysis of cell-cycle parameters in CAL-51 SCR and p73-55 cell lines. Peaks denoting G1, G2–M and aneuploidy are shown.

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resulted in increased SA-b-gal activity and cellular size at alltime points tested in the p53-10 and p73-55 KD cell lines ascompared with vehicle control. Of note, in the CAL-51 scram-ble cell line, exposure to MLN8237 100 nmol/L for 5 to 15 daysresulted in nearly complete cell death (Fig. 4A). These findingssupport the induction of senescence as a mechanism for resis-tance to the antiapoptotic effects of MLN8237 in cells lackingfunctional p53 or p73.

Nutlin is synergistic withMLN8237 in theCAL-51 p53wild-typecell line

Our results above support a functional role for p53 inmediating a proapoptotic response to MLN8237 exposurein vitro. On the basis of this, we hypothesized that increasingintracellular concentrations of p53 would lead to increasedsensitivity to MLN8237 in p53 WT cells. Nutlin-3 is a small-molecule inhibitor of p53 degradation. It exerts its effects

through inhibition of the interaction between p53 and MDM2,thus decreasing MDM2-mediated ubiquitin-dependent degra-dation of p53 (37). Figure 4B illustrates the antiproliferativeeffect of increasing concentrations of nutlin-3, MLN8237, orvarious combinations of both drugs. Synergistic antiprolifera-tive activity was observed for the combination at a variety ofdose levels using the Chou and Talalay method (Supplemen-tary Table S4; ref. 21). An increase in apoptosis was observedwith the combination at the highest dose level of MLN8237(Fig. 4C). These findings are consistent with a recent reportfrom Vilgelm and colleagues in melanoma (38).

Loss of p53 leads to alterations in known p53-downstreammediators of AKI-induced cellular fate

Immunofluorescence and immunoblotting studies were per-formed to investigate the mechanism of the observed shift fromapoptosis to senescence following MLN8237 exposure in cells

Figure 4.A, treatment with MLN8237 selectively induces markers of cellular senescence in CAL-51 p53 and p73 shRNA knockdown clones. CAL-51 scrambled control (SCR),p53-10, and p73-55 were treated with vehicle or MLN8237 (100 nmol/L) for the indicated time points and then analyzed for expression of senescence-associatedb-galactosidase activity (blue stain) as described in Materials and Methods. Note increase in cell size upon treatment with MLN8237 in p53 and p73 knockdownclones. Representative images were taken at �20 magnification. Scale, 10 mm. B, antiproliferative effects of the combination of MLN8237 and Nutlin-3 onthe p53WTCAL-51 cell line asmeasuredby SRBassay. Increasing doses of nutlin-3were combinedwith either vehicle orMLN8237 at the indicated doses for 96hours.C, percentage of apoptotic cells in the CAL-51WT cell line asmeasured byAnnexin V/FITC staining and flow cytometry following exposure toMLN8237, Nutlin-3, or acombination of the two agents at the indicated doses for 96 hours. A statistically significant difference was observed when comparing the MLN8237 (50 nmol/L and100 nmol/L) alone to the Nutlin-3 combination and Nutlin-3 alone to the MLN8237 100 nmol/L plus Nutlin-3 combination. � , P < 0.05; �� , P < 0.01.






lacking functional p53 or p73 by shRNA KD. In the CAL-51 SCR,exposure to MLN8237 led to a more robust increase in nuclearp53, cytoplasmic BAX, and nuclear BAX colocalized with p53 ascompared with the p53 and p73 KD cell lines (Fig. 5A–E andSupplementary Fig. S3A). Nuclear Bax/p53 complexes are asso-ciated with induction of apoptosis in response to DNA damage(39). We hypothesize that induction of BAX is mediated via themitotic arrest imposed byMLN8237 treatment in a p53-indepen-dent manner.

p16 is an established functional biomarker of senescencelikely due to its integral role in preventing cell-cycle progression(40). A dose-dependent increase in p16 staining was observedfollowing exposure to MLN8237 in the p53 and p73 KD celllines, but not in the CAL-51 SCR consistent with the observedincrease in SA-b-gal activity and phenotypic changes indicative

of cellular senescence (Figs. 4A, 5C and D and SupplementaryFig. S3B). A significant difference in p21 or p73 expression wasnot observed following exposure to MLN8237 in the CAL-51SCR or p53 KD cell lines (Supplementary Fig. S3C and S3D). Asexpected based on the p73 KD, expression of p73 was notobserved in CAL-51 p73-55 following exposure to MLN8237(Supplementary Fig. S3D).

Senescence is associated with acquired resistance to AurAinhibition in vivo

To confirm the in vitro antiproliferative activity of MLN8237in a more clinically relevant system, treatment studies wereperformed using a CAL-51 xenograft and 2 TNBC PDTXmodels. As depicted in Fig. 6A and B, MLN8237 treatmentresulted in statistically significant TGI (P < 0.0007) at day 35

CAL-51 p53-10DAPI p53 BAX DAPI/BAX p53/BAX

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Figure 5.Effects of MLN8237 on markers ofapoptosis and senescence. A–D,immunofluorescence analysis ofp53 wild-type CAL-51 TNBC cell linestably transfected with scrambled(SCR) or p53 shRNA constructs(p53-10). Cells were treated withvehicle or MLN8237 at 50 and 100nmol/L for 4 days and stained withthe indicated antibodies asdescribed inMaterials andMethods.Representative images wereacquired using confocalmicroscopy at �60 magnification.Scale, 10 mm. E, immunoblotanalysis of CAL-51 SCR and p53-10cell lines for apoptosis- andsenescence-associated proteinsafter treatment with MLN8237 atthe indicated doses and timepoints.

Tentler et al.





which was associated with a decrease in Ki-67 staining(Fig. 6C). This was accompanied by an increase in cleavedcaspase-3 staining (Supplementary Fig. S4A). Similar antitu-mor activity accompanied by an increase in cleaved caspase-3was observed in PDTX models [CU_TNBC_003, p53 WT, andCU_TNBC_002, p53 mutant (R249S); Fig. 6D–G and Supple-mentary Fig. S4B and S4C]. CU_TNBC_003 exhibited slowergrowth kinetics for the vehicle control (Fig. 6E); therefore,treatment for 65 days was required to show a statisticallysignificant TGI (P < 0.0001; Fig. 6D). CU_TNBC_003 andCU_TNBC_002 were both obtained from primary breasttumors either following neoadjuvant chemotherapy or beforetreatment, respectively.

Following 4 days of MLN8237 treatment (30 mg/kg daily),there was no significant difference in p53 expression as com-pared with vehicle control (Fig. 6H). We observed p53 expres-sion in the vehicle control tumors, consistent with the presenceof p53 cellular accumulation observed in cells with mutatedp53 (41). There was not a significant change in pHH3 follow-ing dosing with MLN8237 for 4 days (Fig. 6H). This is likelydue to its selective AurA inhibition at this dose. We observed adecrease in p16 following prolonged dosing in the respondingtreated tumors (Fig. 6H), suggesting that induction of senes-cence as a terminal outcome is not responsible for the observedcontinued TGI. Of note, the expression of p16 in the vehicle-treated control tumor likely represents a population of tumor

Figure 6.Efficacy of MLN8237 against TNBCxenografts in vivo. Tumor growthcurves (A) and tumor growth rates (B)for the CAL-51 TNBC cell linexenografts. There is a statisticallysignificant decrease in tumor volumesat day 35 in the treated animals ascompared with vehicle control;�� ,P<00007. C, histologic analysis ofCAL-51 xenograft tumors from animalsdepicted in A and B. Individual tumorswere harvested from vehicle controland MLN8237-treated animals at day35. Tumors were formalin fixed,paraffin embedded, and 5-micronsections were stained for Ki-67 andH&E. A decrease in Ki-67 staining wasobserved in the MLN8237-treatedtumors as compared with the vehiclecontrol. Representative images areshown at �10 magnification.Scale, 10 mm. D–G, tumor growthcurves and growth rates for TNBCpatient-derived tumor xenograft(PDTX)models CU_TNBC_003 (D andE), and CU_TNBC_002 (F and G). Astatistically significant decrease intumor volumes in the treated animalsas compared with the vehicle controlat the indicated dates was observedfor both models. H, immunoblotanalysis of two individualCU_TNBC_02 tumors treated withvehicle or MLN8237 (30 mg/kg QD)and harvested at day 4 and 40 oftreatment. A decrease in p16expression, a marker of senescence,is observed following treatment withMLN8237 at day 40 as comparedwith vehicle control.






cells exhibiting baseline senescence related to hypoxia due torapid tumor growth (42).

To investigate acquired resistance to prolonged AKI, wecontinued treatment of the responding CU_TNBC_002 tumorsuntil we observed progression in a subset of tumors (day115, Fig. 7A). As shown in Fig. 7B, we selected a tumorexhibiting continued TGI (right flank) and a tumor from thesame animal exhibiting rapid growth following approximately90 days of growth inhibition (left flank) for further experimen-tation. These tumors were excised and evaluated for phenotypicchanges consistent with senescence and the presence of SA-b-gal activity. Comparison was also made with a vehicle controltumor harvested earlier in the experiment (day 4, approximate-

ly 300 mm3). As depicted in Fig. 7C, we observed larger cellswith increased vacuoles consistent with senescent cells in theprogressing tumor not observed in the sensitive tumor. Adecrease in Ki-67 staining and an increase in cleaved cas-pase-3 staining were observed in the sensitive tumor as com-pared with the vehicle control and the tumor at the time ofacquired resistance (Fig. 7C).

DiscussionTNBC is a highly aggressive breast cancer subtype with

limited systemic treatment options. AKIs represent a promis-ing class of targeted agents for the treatment of TNBC based

Figure 7.Antitumor activity of MLN8237against TNBC patient-derived tumorxenografts (PDTX) in vivo. A, tumorgrowth curves for PDTX modelCU_TNBC_02 treated with vehicle orMLN8237 (30 mg/kg QD) forindicated time periods. Vehicle armwas terminated at 39 days due totumor burden. Animals in theMLN8237 arm were continued ontreatment until development ofresistance, as measured by a markedincrease in tumor growth rate,beginning at approximately day 90.�� , P < 0.01. B, individual tumorgrowth curves from a single animalharboring CU_TNBC_02 dual flanktumors. The left flank tumordeveloped resistance to MLN8237 atapproximately day 90, whereas theright flank tumor remained sensitive.C, senescence and histologic analysisof tumors from animals depicted in B.Individual CU_TNBC_02 tumors wereharvested from vehicle and aMLN8237-treated animal harboringdual flank tumors at day 115. Tumorswere formalin fixed, paraffinembedded, and 5-micron sectionswere stained for senescence-associated b-galactosidase(SA-b-gal), H&E, Ki-67, and cleavedcaspase-3. Note increased SA-b-galstaining and increased cellular size(arrows) in the resistant left-sidedtumor compared with the sensitiveright-sided tumor and vehicle control.A decrease in Ki-67 staining and anincrease in cleaved caspase-3 wereobserved in the sensitive right-sidedtumor, as compared with the vehiclecontrol and the resistant left-sidedtumor. In addition, apoptotic cellswere observed on H&E correspondingto areas of cleaved caspase-3 stainingin the sensitive right-sided tumor(arrows). Representative images ofSA-b-gal activity and H&E stainingwere taken at �20 magnification.Representative images of Ki-67,cleavedcaspase-3, andcorrespondingH&E staining were taken at �10magnification. Scale, 10 mm.

Tentler et al.





on promising preclinical and early clinical trial data(17, 43, 44). The purpose of this study was to determine theantitumor activity of MLN8237, a selective AurA inhibitor, inTNBC cell lines, and PDTX models. Another significant objec-tive was to investigate the role of p53 and its family memberp73 in mediating terminal cellular fates in response to AKI inTNBC.

Our studies showed that MLN8237 has antiproliferativeactivity against TNBC cell line-based and PDTX models. Thesensitivity of TNBC cell lines to MLN8237 in vitro was notdependent on TNBC subtype and both p53 mutated and WTcell lines exhibited sensitivity. We did observe a trend wherebyincreased p53 mRNA expression was associated with sensitivityto MLN8237, consistent with our previously published data forENMD-2076 which also target AurA (17). MLN8237 exposureresulted in induction of apoptosis in the CAL-51 p53 WT cellline, which was partially abrogated by shRNA KD of p53 or p73.Our observations are consistent with data reported by Nair andcolleagues demonstrating apoptosis following exposure to AKIin the HCT116 p53 WT colon cancer cell line (45). In theirstudy, the apoptotic response was abrogated by knockdown ofp53 where an increase in endoreduplication and polyploidywas observed following AKI exposure (45). Although ourresults are consistent with their findings, they did not investi-gate whether the polyploidy they observed was associated withsenescence (46).

We observed the classic AKI-induced G2–M cell-cycle arrestfollowing MLN8237 exposure and an increase in aneuploidcells with p53 shRNA KD. Our in vitro data indicate thatsensitivity to MLN8237 in the CAL51 p53 WT cell line can bereversed with shRNA KD of either p53 or p73 and that this shiftin sensitivity is accompanied by induction of senescence. Toour knowledge, these data are unique in supporting a role forp53 and its family member p73 in mediated sensitivity to AKIin TNBC. These data are consistent with our prior report ofmultitarget AKI leading to senescence in TNBC models resistantin vitro to the antiproliferative activity (17). Furthermore, thisstudy confirms these findings in vivo using a clinically relevantPDTX TNBC model where we observed induction of cellularsenescence in tumors as they developed acquired resistance toMLN8237 in vivo.

The p53 tumor suppressor gene is often referred to as "theguardian of the genome," regulating the expression of a broadpanel of genes responsible for DNA repair, cell-cycle arrest,apoptosis, and senescence in response to DNA damage (47).TAp73 is a p53 family member with structural and functionalsimilarity to p53 (48). A functional loss of p53 due to deletion ormutation is found inup to80%of TNBC; however, p53mutationsare quite heterogeneous with differing degrees of preservation ofp53 function (12, 49, 50). In this study, we demonstrate that lossofWTp53 leads to resistance to AKI in TNBC; however, it does notphenocopy the p53-mutated models with regards to response toAKI and terminal cellular outcome. Although loss of WT p53 byshRNA knockdown impairs induction of apoptosis in response toAKI in TNBC, the effect of individual p53mutations onmediatingsensitivity to AKI is unknown. To answer this question, mutantp53 knockdownmodels are needed. Possible mechanisms for theobserved sensitivity of multiple p53-mutated cell lines toMLN8237 include partially retained p53 function for individualp53 mutations, potential gain-of-function with regards to induc-tion of apoptosis, and overlapping apoptotic capabilities of p73

in response to AKI in TNBC. We have previously reported upre-gulation of p73 in response to AKI in p53-mutated TNBC;however, a global analysis of baseline p73 expression in ourpanel of TNBC cell lines did not correlate significantly withsensitivity to MLN8237 (17). Clearly, this is an area in need offuture research.

Regulation of senescence by p53 is somewhat controversial inthe literature. Evidence exists in support of p53-dependent senes-cence and its role in impairing a favorable drug response todoxorubicin in preclinical breast cancer models (34). There areother reports, however, supporting a role for p53 in suppressingp21-mediated cellular senescence (35, 40). Our data are consis-tent with these latter reports in that silencing p53 activity throughshRNA knockdown leads to a shift in terminal cellular outcomefrom apoptosis to senescence in response to AKI. AlthoughJackson and colleagues showed that MMTV-Wnt1 mammarytumors with WT p53 or a heterozygous p53 R172H missensemutation exhibited a senescent-like phenotype following inhibi-tion of DNA/RNA synthesis by doxorubicin, the mechanism ofcellular fate may be different with AKI (34). Our data are consis-tent with other studies reporting induction of apoptosis inresponse to AKI in p53-mutated cancer cell lines (17, 31, 43).The role of mutant p53 in the induction or suppression ofsenescence in response to AKI remains unknown. Mutant p53knockdownmodels would be useful in answering these questionsas the cell line subject to p53 knockdown in this study was p53WT.

Cellular senescence is a physiologic process leading to per-manent growth arrest in response to certain types of cellularstress (40, 51). Senescence is characterized by changes incellular morphology (i.e., enlarged and flattened cells withincreased vacuoles), high levels of p21 and p16, markers ofthe DNA damage response, and the senescence-associatedsecretory phenotype (SASP). In this study, we observed thatthe induction of senescence in TNBC cell lines was associatedwith resistance to the antiproliferative activity of MLN8237in vitro. Furthermore, using a TNBC PDTX model, we observedinduction of senescence at the time of acquired resistance toMLN8237. This is consistent with reports associating senes-cence with impaired response to chemotherapy, cancer recur-rence following treatment, and malignant transformation ofpreneoplastic cells (34). Our findings may be explained bycytokines and growth factors secreted by senescent cells (SASP)which are necessary for maintenance of the senescent state, butwhen secreted into the tumor microenvironment can stimulatetumor growth. The effect of induction of senescence on globaltumor progression deserves further investigation using preclin-ical in vivo modeling, but also using correlative tissue samplesobtained from patients enrolled on clinical trials receivingpotentially pro-senescent therapies.

TNBC represents a challenging breast cancer subtype clinicallydue to its aggressive disease course and limited systemic treatmentoptions. We believe that our data support the further clinicalinvestigation of AKIs, including MLN8237, in TNBC. Our obser-vation of senescence associated with acquired resistance to AKI inp53-mutated TNBC may support the rational combination ofAKIs with inhibitors of the mTOR pathway based on their abilityto decelerate cellular senescence (52). This study also supportsfurther investigation of the p53 landscape in TNBC and the roleof individual p53 mutations in mediating response to AKI,including terminal cellular fate.






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

Authors' ContributionsConception and design: J.J. Tentler, A. Ionkina, A.-C. Tan, T.M. Pitts,K.D. Sullivan, J.M. Espinosa, S.G. Eckhardt, J.R. DiamondDevelopment of methodology: J.J. Tentler, A. Ionkina, T.P. Newton, T.M. Pitts,P. Kabos, S.G. Eckhardt, J.R. DiamondAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.J. Tentler, A. Ionkina, T.P. Newton, M.J. Glogowska,P. Kabos, C.A. Sartorius, J.R. DiamondAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Ionkina, A.-C. Tan, T.P. Newton, T.M. Pitts,J.M. Espinosa, S.G. Eckhardt, J.R. DiamondWriting, review, and/or revision of the manuscript: J.J. Tentler, A. Ionkina,P. Kabos, J.M. Espinosa, S.G. Eckhardt, J.R. DiamondAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Ionkina, T.P. Newton, J.R. DiamondStudy supervision: J.J. Tentler, J.M. Espinosa, J.R. Diamond

AcknowledgmentsThe authors acknowledgeMillenniumPharmaceuticals, Inc., and theNation-

al Cancer Institute, NIH for providing MLN8237.

Grant SupportThisworkwas supported by theNIH and theNational Cancer Institute (NCI)

through 5P30CA046934-25 (University of Colorado Cancer Center SupportGrant), 1K23CA172691-01A1 (to J.R. Diamond), and 5K12CA086913-10(to J.R. Diamond), and Howard Hughes Medical Institute through HHMIEarly Career Science Award (to J.M. Espinosa).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 24, 2014; revised February 24, 2015; accepted February 24,2015; published OnlineFirst March 10, 2015.

References1. Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis

of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative,and HER2-negative invasive breast cancer, the so-called triple-negativephenotype: a population-based study from the California cancer Registry.Cancer 2007;109:1721–8.

2. Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, et al.Race, breast cancer subtypes, and survival in the Carolina Breast CancerStudy. JAMA 2006;295:2492–502.

3. Metzger-Filho O, Tutt A, de Azambuja E, Saini KS, Viale G, Loi S, et al.Dissecting the heterogeneity of triple-negative breast cancer. J Clin Oncol2012;30:1879–87.

4. LehmannBD, Bauer JA, ChenX, SandersME, Chakravarthy AB, Shyr Y, et al.Identification of human triple-negative breast cancer subtypes and pre-clinical models for selection of targeted therapies. J Clin Invest 2011;121:2750–67.

5. Nikonova AS, Astsaturov I, Serebriiskii IG, Dunbrack RL Jr, Golemis EA.Aurora A kinase (AURKA) in normal and pathological cell division.Cell Mol Life Sci 2013;70:661–87.

6. Lioutas A, Vernos I. Aurora A kinase and its substrate TACC3 are requiredfor central spindle assembly. EMBO reports 2013;14:829–36.

7. Marumoto T, Honda S, Hara T, NittaM, Hirota T, Kohmura E, et al. Aurora-A kinasemaintains the fidelity of early and latemitotic events inHeLa cells.J Biol Chem 2003;278:51786–95.

8. Kufer TA, SilljeHH, Korner R, GrussOJ,Meraldi P,Nigg EA.Human TPX2 isrequired for targeting Aurora-A kinase to the spindle. The Journal of cellbiology 2002;158:617–23.

9. Bischoff JR, Anderson L, Zhu Y,Mossie K, Ng L, Souza B, et al. A homologueof Drosophila aurora kinase is oncogenic and amplified in human colo-rectal cancers. EMBO J 1998;17:3052–65.

10. Nadler Y, Camp RL, Schwartz C, Rimm DL, Kluger HM, Kluger Y. Expres-sion of Aurora A (but not Aurora B) is predictive of survival in breast cancer.Clin Cancer Res 2008;14:4455–62.

11. BerradaN,Delaloge S, Andre F. Treatment of triple-negativemetastatic breastcancer: toward individualized targeted treatments or chemosensitization?Ann Oncol 2010;21:vii30–vii5.

12. Cancer Genome Atlas Network. Comprehensive molecular portraits ofhuman breast tumours. Nature 2012;490:61–70.

13. DumayA, Feugeas JP,Wittmer E, Lehmann-Che J, BertheauP, EspieM, et al.Distinct tumor protein p53 mutants in breast cancer subgroups. Int JCancer 2013;132:1227–31.

14. Joerger AC, Fersht AR. Structure-function-rescue: the diverse nature ofcommon p53 cancer mutants. Oncogene 2007;26:2226–42.

15. Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, Suzuki F, et al.Phosphorylation by aurora kinase A induces Mdm2-mediated destabili-zation and inhibition of p53. Nat Genet 2004;36:55–62.

16. Manfredi MG, Ecsedy JA, Chakravarty A, Silverman L, Zhang M, Hoar KM,et al. Characterization of Alisertib (MLN8237), an investigational small-

molecule inhibitor of aurora A kinase using novel in vivo pharmacody-namic assays. Clin Cancer Res 2011;17:7614–24.

17. Diamond JR, Eckhardt SG, Tan AC, Newton TP, Selby HM, Brunkow KL,et al. Predictive biomarkers of sensitivity to the aurora and angiogenickinase inhibitor ENMD-2076 in preclinical breast cancer models.Clin Cancer Res 2013;19:291–303.

18. Pitts TM, Tan AC, Kulikowski GN, Tentler JJ, Brown AM, Flanigan SA, et al.Development of an integrated genomic classifier for a novel agent incolorectal cancer: approach to individualized therapy in early develop-ment. Clin Cancer Res 2010;16:3193–204.

19. SkehanP, StorengR, ScudieroD,MonksA,McMahon J, VisticaD, et al.Newcolorimetric cytotoxicity assay for anticancer-drug screening. J Natl CancerInst 1990;82:1107–12.

20. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O. Protocols todetect senescence-associated beta-galactosidase (SA-betagal) activity, abiomarker of senescent cells in culture and in vivo. Nat Protoc 2009;4:1798–806.

21. ChouTC, Talalay P.Generalized equations for the analysis of inhibitions ofMichaelis-Menten and higher-order kinetic systems with two or moremutually exclusive and nonexclusive inhibitors. Eur J Biochem 1981;115:207–16.

22. Diamond JR, Finlayson CA, Thienelt C, Kabos P, Hardesty L, Barbour L,et al. Early-stage BRCA2-linked breast cancer diagnosed in thefirst trimesterof pregnancy associated with a hypercoagulable state. Oncology (WillistonPark) 2009;23:784–91.

23. Spreafico A, Tentler JJ, Pitts TM, Tan AC, Gregory MA, Arcaroli JJ, et al.Rational combination of a MEK inhibitor, selumetinib, and the Wnt/calcium pathway modulator, cyclosporin A, in preclinical models ofcolorectal cancer. Clin Cancer Res 2013;19:4149–62.

24. Tentler JJ, Tan AC, Weekes CD, Jimeno A, Leong S, Pitts TM, et al. Patient-derived tumour xenografts as models for oncology drug development. NatRev Clin Oncol 2012;9:338–50.

25. Liu Q, Kaneko S, Yang L, Feldman RI, Nicosia SV, Chen J, et al. Aurora-Aabrogation of p53 DNA binding and transactivation activity by phosphor-ylation of serine 215. J Biol Chem 2004;279:52175–82.

26. Katayama H, Wang J, Treekitkarnmongkol W, Kawai H, Sasai K, Zhang H,et al. Aurora kinase-A inactivates DNA damage-induced apoptosis andspindle assembly checkpoint response functions of p73. Cancer Cell2012;21:196–211.

27. Dar AA, Belkhiri A, Ecsedy J, Zaika A, El-Rifai W. Aurora kinase A inhibitionleads to p73-dependent apoptosis in p53-deficient cancer cells. Cancer Res2008;68:8998–9004.

28. Marumoto T, Zhang D, Saya H. Aurora-A - a guardian of poles. Nat RevCancer 2005;5:42–50.

29. Huck JJ, Zhang M, McDonald A, Bowman D, Hoar KM, Stringer B, et al.MLN8054, an inhibitor of Aurora A kinase, induces senescence in humantumor cells both in vitro and in vivo. Mol Cancer Res 2010;8:373–84.


Tentler et al.




30. ManfrediMG, Ecsedy JA,Meetze KA, Balani SK, BurenkovaO,ChenW, et al.Antitumor activity of MLN8054, an orally active small-molecule inhibitorof Aurora A kinase. Proc Natl Acad Sci U S A 2007;104:4106–11.

31. Zhou N, Singh K, Mir MC, Parker Y, Lindner D, Dreicer R, et al. Theinvestigational aurora kinase a inhibitor MLN8237 induces defects in cellviability and cell-cycle progression inmalignant bladder cancer cells in vitroand in vivo. Clin Cancer Res 2013;19:1717–28.

32. Hanshaw RG, Smith BD. New reagents for phosphatidylserinerecognition and detection of apoptosis. Bioorg Med Chem 2005;13:5035–42.

33. Aylon Y, Oren M. p53: guardian of ploidy. Mol Oncol 2011;5:315–23.34. Jackson JG, Pant V, LiQ,Chang LL,Quintas-CardamaA,GarzaD, et al. p53-

mediated senescence impairs the apoptotic response to chemotherapy andclinical outcome in breast cancer. Cancer Cell 2012;21:793–806.

35. Demidenko ZN, Korotchkina LG, Gudkov AV, Blagosklonny MV. Para-doxical suppression of cellular senescence by p53. Proc Natl Acad Sci U S A2010;107:9660–4.

36. Lawless C, Wang C, Jurk D, Merz A, Zglinicki T, Passos JF. Quantitativeassessment of markers for cell senescence. Exp Gerontol 2010;45:772–8.

37. Miyachi M, Kakazu N, Yagyu S, Katsumi Y, Tsubai-Shimizu S, Kikuchi K,et al. Restoration of p53 pathway by nutlin-3 induces cell cycle arrest andapoptosis in human rhabdomyosarcoma cells. Clin Cancer Res 2009;15:4077–84.

38. Vilgelm AE, Pawlikowski JS, Liu Y, Hawkins OE, Davis TA, Smith J, et al.Mdm2 and aurora kinase a inhibitors synergize to blockmelanoma growthby driving apoptosis and immune clearance of tumor cells. Cancer Res2015;75:181–93.

39. Raffo AJ, Kim AL, Fine RL. Formation of nuclear Bax/p53 complexes isassociated with chemotherapy induced apoptosis. Oncogene 2000;19:6216–28.

40. Salama R, SadaieM,HoareM,NaritaM.Cellular senescence and its effectorprograms. Genes Dev 2014;28:99–114.

41. LacroixM, ToillonRA, LeclercqG. p53 andbreast cancer, anupdate. EndocrRelat Cancer 2006;13:293–325.

42. LaPorta CA, Zapperi S, Sethna JP. Senescent cells in growing tumors:population dynamics and cancer stem cells. PLoS Comput Biol 2012;8:e1002316.

43. Romanelli A, Clark A, Assayag F, Chateau-Joubert S, Poupon MF,Servely JL, et al. Inhibiting aurora kinases reduces tumor growth andsuppresses tumor recurrence after chemotherapy in patient-derivedtriple-negative breast cancer xenografts. Mol Cancer Ther 2012;11:2693–703.

44. Bush TL, PaytonM,Heller S, Chung G,Hanestad K, Rottman JB, et al. AMG900, a small-molecule inhibitor of aurora kinases, potentiates the activityofmicrotubule-targeting agents in humanmetastatic breast cancermodels.Mol Cancer Ther 2013;12:2356–66.

45. Adams KF, Leitzmann MF, Albanes D, Kipnis V, Moore SC, Schatzkin A,et al. Body size and renal cell cancer incidence in a large US cohort study.Am J Epidemiol 2008;168:268–77.

46. Nair JS, Ho AL, Schwartz GK. The induction of polyploidy or apoptosis bythe Aurora A kinase inhibitor MK8745 is p53-dependent. Cell Cycle2012;11:807–17.

47. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW. Partici-pation of p53 protein in the cellular response to DNA damage. Cancer Res1991;51:6304–11.

48. Ozaki T, Nakagawara A. p73, a sophisticated p53 family member in thecancer world. Cancer Sci 2005;96:729–37.

49. Bertheau P, Lehmann-Che J, Varna M, Dumay A, Poirot B, Porcher R, et al.p53 in breast cancer subtypes and new insights into response to chemo-therapy. Breast 2013;22S2:S27–S9.

50. Chen X, Ko LJ, Jayaraman L, Prives C. p53 levels, functional domains, andDNAdamage determine the extent of the apoptotic response of tumor cells.Genes Dev 1996;10:2438–51.

51. Campisi J, d'Adda di Fagagna F. Cellular senescence: when bad thingshappen to good cells. Nat Rev Mol Cell Biol 2007;8:729–40.

52. Demidenko ZN, Zubova SG, Bukreeva EI, Pospelov VA, Pospelova TV,Blagosklonny MV. Rapamycin decelerates cellular senescence. Cell Cycle2009;8:1888–95.






2015;14:1117-1129. Published OnlineFirst March 10, 2015.Mol Cancer Ther John J. Tentler, Anastasia A. Ionkina, Aik Choon Tan, et al. Kinase A Inhibition in Triple-Negative Breast Cancerp53 Family Members Regulate Phenotypic Response to Aurora

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