inhibition of mtor pathway sensitizes acute …...cell death and survival inhibition of mtor pathway...

Cell Death and Survival

Inhibition of mTOR Pathway Sensitizes Acute MyeloidLeukemia Cells to Aurora Inhibitors by Suppression ofGlycolytic Metabolism

Ling-Ling Liu1,2, Zi-Jie Long1,3, Le-Xun Wang1,3, Fei-Meng Zheng2, Zhi-Gang Fang1, Min Yan2,Dong-Fan Xu1,3, Jia-Jie Chen1,3, Shao-Wu Wang4,5, Dong-Jun Lin1,3, and Quentin Liu1,2,3,4

AbstractAurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In

this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) inducedpolyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantlyincreased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggestingthat polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway bymTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in thepolyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptakeand lactate production in polyploidy cells as well as the expression of p62/SQSTM1.Moreover, knockdown of p62/SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy.Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidycells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatmentin AML. Mol Cancer Res; 11(11); 1326–36. �2013 AACR.

IntroductionAnticancer drug that targets cell-cycle regulators has

been widely applied in the treatment of the hematologicmalignancies (1–3). Aurora kinases A–C, which playimportant roles in the process of mitosis, have beenimplicated in the tumorigenesis (1–3). Aurora A and Bproteins were overexpressed in a number of tumors,including leukemia cells, which were correlated with aneu-ploidy in tumor tissues and poor prognosis (4, 5). Previousreport showed that Aurora kinases inhibitors could sup-press the growth of leukemia cells (1). ZM447439 (ZM),the dual Aurora A and B inhibitor, inhibited the cellproliferation, induced polyploidization, and promoted

apoptosis of acute myeloid leukemia (AML) cells (6). Ourprevious study showed that VX-680 (MK-0457), a rela-tively specific Aurora A inhibitor, induced apoptotic celldeath in AML cells, especially in the primary leukemicblasts with high Aurora A expression (4, 7). AlthoughAurora kinases inhibitors have significant therapeuticpotential in AML, single-agent activity seems to be uni-formly modest and the efficacy needs improving (6).Recently, we also showed that Aurora A inhibition trig-gered drug resistance could be reversed by autophagyinhibitors in breast cancer cells (8). In addition, combi-nation of Aurora kinases inhibitors with vincristine ordasatinib presented synergistic effect to inhibit cell viabilityin human pre-B acute lymphoblastic leukemia (9). Thus,new strategy for drug combination is urgently required toovercome drug resistance induced by Aurora A inhibition.Polyploidy is a prominent characteristic of human cancers

and proposed as a driver of tumorigenesis (10). Studyshowed that somatic polyploidy was associated withreadjustment of main metabolic pathways. For example,mitochondrial process was depressed, and carbohydratedegradation and lipid biosynthesis were increased in thepolyploidy cells (11). Warburg reported that tumors pre-ferred aerobic glycolysis to mitochondrial oxidative phos-phorylation (12). Based on the reports, suppression of theaerobic glycolytic metabolism might help for restraining theproliferation of polyploidy cancer cells. Aurora kinases arecrucial for cytokinesis in a number of reports. Aurora kinasesdefection induced mitosis arrest and promoted polyploidyformation (13). Therefore, readjustment of the metabolic

Authors' Affiliations: 1Department of Hematology, the Third AffiliatedHospital, Sun Yat-sen University, Guangzhou, China; 2State Key Labora-tory of Oncology in South China, Cancer Center, Sun Yat-sen University,Guangzhou, China; 3Sun Yat-sen Institute of Hematology, Guangzhou,China; 4Institute of Cancer Stem Cell, Dalian Medical University, Dalian,China; and 5Department of Radiology, the First Affiliated Hospital, DalianMedical University, Dalian, China

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

L.-L. Liu and Z.-J. Long have contributed equally to this article.

Corresponding Authors: Quentin Liu, Department of Hematology, theThird Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road,Guangzhou 510630, China. Phone: 86-20-8525-3601; Fax: 86-20-87343171; E-mail: [email protected]; and Dong-Jun Lin,[email protected]

doi: 10.1158/1541-7786.MCR-13-0172

�2013 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 11(11) November 20131326

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Published OnlineFirst September 5, 2013; DOI: 10.1158/1541-7786.MCR-13-0172

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status in polyploidy cells induced by Aurora kinases inhibi-tion could also be a promise approach to cancer therapy.The mTOR, a well-conserved serine/threonine kinase

with 2 complexes, mTOR complex 1 (mTORC1) andmTOR complex 2 (mTORC2), is essential for cell growth,protein translation, autophagy, and metabolism (14). Toadapt the condition of hypermetabolic demand and thriverapidly, oncogenes in growth and nutrient-sensing signalingpathways was activated (15). In numerous cancer types,mTOR signaling pathway was activated by deregulation ofits upstream factors, such as Akt activation or deficiency oftuberous sclerosis complex 2 (TSC2; refs. 16 and 17).Activation of mTORC1, a metabolic hallmark of cancer,increased glycolysis, and lipid biosynthesis (15). AlthoughmTORC2 was not thoroughly studied in the metabolism oftumor, RNAi against one of mTORC2 components alsoimpaired the glucose and lipid metabolism in muscle andfat cells (18, 19). Furthermore, mTOR was associated withthe development of polyploidization. Ma and colleaguesshowed that S6K1, a downstream of mTOR, was involvedin polyploidization through its phosphorylation at Thr421/Ser424 (20). In addition, activation of mTOR pathway wasrequired for TSC2þ/EK [EKer rat model carrying a germlineinsertional deletion in one copy of the Tsc2 (tuberoussclerosis 2) gene] smooth muscle cells of pulmonary lym-phangioleiomyomatosis to undergo tetraploidization (21).Here, we found thatmTOR signaling pathway was critical

formaintaining the glycolyticmetabolism state of polyploidycancer cells induced by inhibition of Aurora kinases. More-over, we provided evidences that combination of mTORinhibitors and Aurora kinases inhibitors effectively elimi-nated leukemia cells by inducing apoptosis and autophagy.These results suggested a new approach of targeted thera-peutic drugs combination for AML treatment.

Materials and MethodsReagents and cell cultureReagents included MK-0457 (Selleck Chemicals, South

Loop West, Houston), ZM447439 (AstraZeneca, Canada),PP242, 2-deoxy-D-glucose (2DG), and rapamycin (Sigma).U937 cell line was obtained from the American TypeCulture Collection (ATCC). 293FT cell line was obtainedfrom Invitrogen.NB4 andHL-60 cell lines were provided byShanghai Institute of Hematology, Ruijin Hospital. U937,NB4, and HL-60 cell lines were cultured in RPMI 1640(Gibco; Invitrogen) supplemented with 10% (v/v) FBS(Hyclone) at 37�C in 5% CO2 incubator. 293FT cell linewas cultured in DMEM supplemented with 10% (v/v) FBSat 37�C in 5% CO2 incubator. KG1a cell line was obtainedfrom Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ; Braunschweig) and culturedin RPMI 1640 supplemented with 20% (v/v) FBS at 37�Cin 5% CO2 incubator. The RPMI 1640 medium withoutglucose and DMEM were also purchased from Gibco(Invitrogen). Cell lines were authenticated by comparingthe short tandem repeat (STR) site and sex genes of theextracted DNA with that of cell lines in ATCC in 2012.

Peripheral blood mononuclear cells (PBMC) were enrichedby Ficoll–Hypaque density gradient centrifugation.

Cell-proliferation assayCell viability was assessed by Cell counting kit-8

(CCK8; Dojindo Biotechnology) according to the man-ufacturer's instructions. The optical density at 450 nm wasmeasured using a multiwell plate reader (Micro-plateReader; Bio-Rad).

Cell-cycle analysisCell cycle was analyzed by flow cytometer. Cells were

treated with drugs for 24 hours. Then, cells were harvestedand stained with mixture of 50 mg/mL propidium iodide(PI), 0.2% Triton X-100, and 100 mg/mL RNAase inhib-itor. Cell-cycle analysis was conducted using a FACS flowcytometer equipped with Modfit LT for Mac V2.0 software(BD Biosciences).

Microscopic analysis of cells and nuclear morphologyCells were treated with Aurora kinases inhibitors for 24

hours, followed by PP242 or 2DG for another 24 hours.After 48 hours, the cells were photographed under aninvertedmicroscopy (Olympus). Nuclear fragmentation wasstained by Hoechst 33342 (Sigma) with 10 mg/mL for 15minutes at 37�C. Slides were viewed using a fluorescencemicroscope.

Monodansylcadaverine stainingCells were incubated with the monodansylcadaverine

(MDC; Sigma) with a final concentration of 0.05 mmol/Lfor 10 minutes at 37�C. Then, cells were viewed on slidesunder a laser scanning confocal microscope (ZEISS,Germany), and tested by flow cytermetry. MDC dye wasapplied to stain the late autophagic vacuoles.

Western blotting analysisTotal cellular proteins were isolated with RIPA buffer.

Lysates were subjected to SDS-PAGE gel electrophoresis,and were transferred to nitrocellulose membranes. Themembranes were blocked with 5% bovine serum albuminand then incubated overnight with LC3 (Novus), p62 (SantaCruz), Aurora A (Upstate), p-mTOR (Ser2448, Ser2481),mTOR, p-Aurora A (Thr288), cleaved caspase 3 (Asp175),cleaved PARP (Asp214), and b-actin antibodies (Cell Sig-naling Technology). p-4EBP1/2/3 (Thr45) and 4EBP1/2/3antibodies were purchased from Epitomics, Inc. Subse-quently, the membranes were incubated with an HRP-conjugated secondary antibody (Protein Tech Group) atroom temperature for 1 hour. The protein bands weredetected with an enhanced chemiluminescence reagent(Sigma), according to the manufacturer's instructions.

Measurement of glucose uptake and lactate productionof cells in the mediaCells were counted using trypan blue staining before

measurement of the glucose uptake and lactate production.The media were collected, and the glucose and lactate levels

mTOR Inhibition Enhances the Efficacy of Aurora Inhibitors

www.aacrjournals.org Mol Cancer Res; 11(11) November 2013 1327




were immediately measured using Automatic BiochemicalAnalyzer (Olympus AU5400). The glucose consumptionand lactate production were normalized to the number ofcells and results were presented as pmol/cell (22).

Plasmid construction and retrovirus packagingThe coding sequence of p62/SQSTM1 was amplified

from human leukemia cells. The primers were as followed:50 Bgl II: TTAGATCTATGGCGTCGCTCACCGTGA-AGG; 30 Sal I: GTGTCGACTGGGCAAAAGTGGTCA-CAACG. The cDNA was cloned into MSCV-IRES-GFPplasmid. 293FT cells were transfected with packagingvectors and MSCV-p62-IRES-GFP or MSCV-IRES-GFPusing Lipofectamine 2000 (Invitrogen). Media containingretrovirus were collected 48 hours after transfection forU937 cells infection. The short hairpin RNA (shRNA)targeting p62 (NM_003900), Aurora A (NM_003600),and the nontarget shRNA (SHC002) were obtained fromSigma. 293FT cells were used for packaging lentivirus. Afterinfection with the lentivirus, infected U937 cells wereselected with puromycin.

StatisticsData were expressed as mean � SD of 3 determinations

with GraphPad Prism software. Statistical analysis wasanalyzed using the Statistical Package for Social Sciences(SPSS) software (version 16.0). P-value < 0.05 was consid-ered statistically significant.

ResultsAurora kinases inhibitors induce polyploidization ofAML cell linesTo study the effect ofAurora kinases inhibitors onAMLcell

lines, the proliferation of cells was tested.U937,NB4,HL-60,and KG1a cell lines treated with Aurora kinases inhibitors(MK or ZM) for 24 and 48 hours were subjected to CCK8assay (Fig. 1A and Supplementary S1A). The 2 drugs hadslight effects on leukemia cell lines viability. For example,MK1mmol/L suppressed the proliferation ofU937 cells by 17.3%� 5.4% and 22% � 6.3% at 24 and 48 hours respectively,and inhibited the proliferation ofNB4 cells by 18.7%� 1.8%and 20.4%� 1.8%at 24 and 48hours, respectively (Fig. 1A).As Aurora kinases were critical for the regulation of mitosis

Figure 1. The effect of Aurorakinases inhibitors onAMLcell lines.A, U937 and NB4 cells treated withdifferent concentrations of MK orZM for 24 and 48 hours weresubjected to CCK8 assay. Datawere mean � SD (n¼ 3). �P < 0.05,��P < 0.01. B, U937 and NB4 cellswere treated with differentconcentrations of MK or ZM for 24hours. Flow cytometry analysiswas conducted for the cell-cycledistribution and the percentageof polyploidy cells was displayed.Western blotting was alsoconducted for the phosphorylatedand total Aurora A proteinexpression (C).

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process, the cell-cycle distribution of these cell lines wasanalyzed. Flow cytometry analysis showed that treatment ofMK 1 mmol/L or ZM 2 mmol/L could induce polyploidiza-tion of bothU937 andNB4 cells, as well asHL-60 andKG1acells (Fig. 1B and Supplementary Fig. S1B).Western blottingconfirmed the effect of Aurora kinases inhibition by testingthe level of T288 phosphorylated Aurora A protein, and theresults showed that the phosphorylated Aurora A decreased ina dose-dependent manner (Fig. 1C). Moreover, knockdownof Aurora A in U937 cells reduced the cell viability (Supple-mentary Fig. S2), indicating that the antiproliferation effect ofMK and ZM was largely due to Aurora A inhibition.

Polyploidy cells induced by Aurora inhibition have highlevel of glycolytic metabolismAfter 24hours incubatingwithMKorZM,U937 andNB4

cells converted to polyploidy cells. Study showed that metab-olism was altered in polyploidy cells (23). So next, glucoseuptake and lactate production in culturemediaweremeasuredto evaluate the level of glycolyticmetabolism. Compared withthat of control cells, the level of glucose uptake and lactateproduction ofU937 polyploidy cells was increased [Fig. 2A(a,b); glucose uptake, 8.3� 0.8 pmol/cell for control vs. 14.0�0.4 pmol/cell for MK, 13.3 � 2.8 pmol/cell for ZM andlactate production, 15.1� 2.2 pmol/cell for control vs. 25.4

Figure 2. Polyploidy cells inducedby Aurora inhibition have highermetabolism level than control cells.A, U937 andNB4cellswere treatedwith MK 1 mmol/L or ZM 2 mmol/Lfor 24 hours, and the level ofglucose uptake (a, c) and lactateproduction (b, d) in the culturemedia was examined. The resultswere normalized to the cellsnumber, and conducted aspmol/cell. B, U937 and NB4 cellswere treated with MK 1 mmol/L orZM 2 mmol/L for 24 hours, followedby 2DG for another 24 hours, andCCK8 assay was conducted. Datawere mean� SD (n¼ 3). �P < 0.05,��P < 0.01.






� 0.1 pmol/cell for MK, 24.4 � 1.7 pmol/cell for ZM].Similar results were observed in NB4 cells [Fig. 2A(c, d)].2DG, a glucose analog, was used to confirm whether thepolyploidy cells were sensitive to energy deprivation. 2DGtransiently inhibited glycolysis, which was considered as themainly energy source of tumor cells (24). Figure 2B(left)showed that 2DG inhibited the proliferation of U937 poly-ploidy cells by�30% at 2 mmol/L and�40% at 5 mmol/L.In Fig. 2B(right), the inhibition rate of NB4 polyploidy cellsby 2DG was �30% at 2 mmol/L and �50% at 5 mmol/L.Two drugs combination test was also conducted in normalPBMCs and we found minimal cell death in PBMCs witheither single drug or 2 drugs combination treatment, indi-cating the observed phenotypes were tumor specific (Supple-mentary Fig. S3). Our data suggested that the polyploidy cells

induced by Aurora kinases inhibitors required higher level ofglycolytic metabolism than control cells.

Inhibition of mTOR pathway decreases the level ofglycolytic metabolism in AML cell linesRapamycin is the specific inhibitor of mTORC1, and

PP242 inhibits mTORC1 and mTORC2. U937 and NB4cells were treated with different concentrations of rapamycinor PP242 for 24 hours, respectively. The activity of mTORand its substrate 4EBP1 was detected by Western blotting.As shown in Fig. 3A, rapamycin inhibited the mTORactivation, as well as phosphorylated 4EBP1. The sameresults were also detected in PP242 treated cells. Rapamycin0.05 mmol/L or PP242 1 mmol/L could inhibit mTORactivation, and the 2 concentrations were chosen for further

Figure 3. Inhibition of mTORpathway decreases the metaboliclevel in AML cell lines. A, differentconcentrations of rapamycin(Rapa) or PP242 were added toU937 and NB4 cells and the celllysates were subjected to Westernblotting analysis with indicatedantibodies. Three independentexperiments were conducted, andrepresentative data were shown.B,U937 andNB4cellswere treatedwith rapa 0.05 or PP242 1 mmol/Lfor 24 hours, and cells werecounted and the level of glucoseuptake and lactate production inthe culture media was immediatelytested. The results werenormalized to the cells number.Data were mean � SD (n ¼ 3).�P < 0.05. C, U937 and NB4 cellswere treated with differentconcentrations of MK or ZM. Celllysates were subjected to Westernblotting analysis with indicatedantibodies.

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study. In Fig. 3B, rapamycin decreased the level of glucoseuptake, whereas PP242 significantly downregulated theglucose uptake and lactate production in U937 and NB4cells. In addition, we also tested the inhibition of mTORpathway in U937 and NB4 cells with increasing dose ofAurora kinases inhibitors. However, there was no change forthe expression of phosphorylated mTOR and 4EBP1, sug-gesting that the mTOR pathway could not be suppressed byAurora inhibitors (Fig. 3C).

Polyploidy cells induced by Aurora inhibition aresensitive to the mTOR inhibitorsThen we examined the effect of mTOR inhibitor on

polyploidy cells. After exposed to MK 1 mmol/L or ZM 2mmol/L for 24 hours, U937 and NB4 cells were treatedwith PP242 for another 24 hours. As shown in Fig. 4A,PP242 significantly suppressed the viability of polyploidy

cells. Under the microscopy, the polyploidy cells exposedto PP242 broke into pieces [Fig. 4B(a)]. These morpho-logical changes were confirmed by flow cytometry. ForU937 cells treated with PP242, the sub-G1 rates ofpolyploidy cells induced by MK or ZM were 33.5 �3.0% or 43.4 � 2.5% versus 2.15 � 0.55% of controlcells. For NB4 cells treated with PP242, the sub-G1 ratesof polyploidy cells induced by MK or ZM were 24.3 �1.1% or 22.5 � 1.6% versus 2.0 � 0.3% of control cells[Fig. 4B(b)]. As mentioned in Fig. 3B, inhibition ofmTOR decreased the metabolism level of the controlcells. Moreover, the metabolism level significantlydeclined after the polyploidy cells were treated with PP242(Fig. 4C). In U937 cells, the glucose uptake decreasedfrom 23.1 � 2.5 to 14.7 � 1.1 pmol/cell, and lactateproduction decreased from 33.9 � 0.8 to 18.6 � 2.8pmol/cell. In NB4 cells, the glucose uptake decreased

Figure 4. Polyploidy cells inducedby Aurora inhibition are sensitive tothe mTOR inhibitors. U937 andNB4 cells were treated with MK1 mmol/L or ZM 2 mmol/L for24 hours, followed by differentconcentrations of PP242 foranother 24 hours. CCK8 assay wasconducted for the proliferationdetection (A). Data were mean� SD (n ¼ 3). �P < 0.05, ��P < 0.01.Cells were photographed undermicroscopy (B-a) and flowcytometry analysis for the sub-G1

percentage was shown (B-b).��, P < 0.001. C, the level ofglucose uptake and lactateproduction in the culture mediawas tested in polyploidy cellstreated with or without PP242 for24 hours. Data were mean � SD(n ¼ 3). �P < 0.05, ��P < 0.01.






from 14.8 � 2.0 to 8.3 � 1.1 pmol/cell, and lactateproduction decreased from 31.2 � 5 to 21.8 � 4.4 pmol/cell. To further investigate the combinational effect, U937and NB4 cells were treated with Aurora kinases inhibitors(MK or ZM) and mTOR inhibitors (rapamycin or PP242)simultaneously for 48 hours. The metabolism examina-tion also showed the same outcomes (Supplementary Fig.S4). Of note, as shown in Supplementary Figs. S5 and S6,the combination had synergistic effect on the proliferationinhibition of AML cells, but showed little effect on thenormal PBMCs. These results indicated that, comparedwith control cells, the polyploidy cells had higher metab-olism level and were more sensitive to mTOR inhibitors.

mTOR inhibitors or the glycolysis inhibitor induce notonly apoptosis but also autophagy in polyploidy cellsNext, control cells and polyploidy U937 cells induced

by Aurora inhibitors were treated with PP242 or 2DG,respectively. After 24 hours, the cells were stained withHoechst 33342 dye and observed under fluorescence micro-scopy. Figure 5A showed that much more apoptotic bodiesin polyploidy cells than that in control cells, and thepercentage of apoptotic bodies was presented (MK,14.0% � 5.1% for control, 56.3% � 5.7% for PP242,47.3%� 11.0% for 2DG; ZM, 15.7%� 5.5% for control,56.0% � 15.6% for PP242, 46.7% � 10.7% for 2DG).MDC dye was applied to stain the late autophagic vacuoles.

Figure 5. PP242 or 2DG induces notonly apoptosis but also autophagyin polyploidy cells. U937 cells weretreated with MK 1 mmol/L or ZM2 mmol/L for 24 hours, followed byPP242 1 mmol/L or 2DG 5 mmol/Lfor another 24 hours. The apoptoticbodies in the cells were stained byHoechst 33342 dye and thepercentage of apoptosis cells wastested by flow cytometry(A). Autophagic vacuoles in thecells were detected by MDC dyeand the percentage of MDCstaining in cells was quantified byflow cytometry (B). The apoptosisand autophagy-related proteinsin U937 cells were determinedby Western blotting (C). �, P < 0.05;��, P < 0.01; ��, P < 0.001.

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As shown in Fig. 5B, inhibition of mTOR or the glycolysisin polyploidy cells increased the appearance of autophagicvacuoles, which seemed as distinct dot-like structures dis-tributed within the cytoplasm or localized in the perinuclearregions in cells. Moreover, the percentage of autophagicvacuoles in cells tested by flow cytometry also showed thesame results (MK, 36.5% � 17.3% for control, 79.2% �10.0% for PP242, 77.7% � 18.7% for 2DG; ZM, 64.8%� 6.0% for control, 80.4% � 9.6% for PP242, 85.3% �6.9% for 2DG; Fig. 5B).The expression of apoptosis-associated proteins and

autophagy markers was further determined. As shownin Fig. 5C, PP242 or 2DG inhibited mTOR activation atserine 2448 and 2481 phosphorylated sites in polyploidycells. Either PP242 or 2DG increased the expression ofcleaved caspase 3 and PARP (poly ADP-ribose polymerase)in polyploidy cells. P62/SQSTM1 was downregulated andLC3II were upregulated in polyploidy cells with PP242 or2DG treatment, indicating autophagy occurred (Fig. 5C).Moreover, in Supplementary Fig. S7, the expression of p62decreased and cleaved caspase 3 expression increased in cellswith drug combination simultaneous for 48 hours.

P62/SQSTM1 acts as a regulator in the metabolism ofpolyploidy cells induced by Aurora kinase inhibitorsP62 is a key regulator of nutrient sensing in the mTOR

pathway (25). The glucose deprivation was applied to furtherstudy the function of p62 in polyploidy cells. We found thatthe expression of p62 in polyploidy cells was significantlydecreased when cultured in glucose-free media, suggestingthat p62 was correlated with the metabolism of polyploidy

cells (Fig. 6A). Expression of cleaved caspase 3 and PARP wassignificantly increased when compared with cells culturedwith glucose-containing media (Fig. 6A). Furthermore, wechose one plasmid which showed great efficiency to knock-down p62 expression for virus packaging and U937 infection(Supplementary Fig. S8). P62 knockdown-U937 cells weretreated with MK and subjected to Western blotting. Theresult showed that MK increased PARP cleavage anddecreased more p62 expression in the p62 knockdown-U937 cells (Fig. 6B). On contrast, overexpression of p62 inU937 cells with treatment of MK partly reversed the MK-induced apoptosis with decreased expression of cleaved PARP(Fig. 6B). These results implied that p62 might act as aregulator involving in polyploidy cells metabolism pathway.

DiscussionIn this study, we found that Aurora kinases inhibitors

could induce polyploidization in various AML cell lines. Theinduced polyploidy cells had higher level of glycolyticmetab-olism than control cells. Importantly, mTOR inhibitorscould target p62 to promote apoptotic and autophagic deathin Aurora kinases inhibitors induced polyploidy cells. Ourdata suggested that suppression themetabolismof polyploidycells by mTOR inhibition was a novel approach to improvethe therapeutic effect of Aurora kinases inhibitors in AML.Aurora kinases were participated in a wide range of

cell-cycle events and overexpressed in a numerous of tu-mors. Aurora kinases inhibitors have been considered as theattractive anticancer regents (4, 6, 26). In our study, MKand ZM inhibited Aurora A phosphorylation in a dose-

Figure 6. P62/SQSTM1 acts as aregulator in the metabolism ofpolyploidy cells induced by Aurorakinases inhibitors. A, U937 cellswere treated with MK 1 mmol/Lor ZM 2 mmol/L for 24 hours. Afterwashed with PBS, cells werereseeded in culture media withor without glucose. The expressionof cleaved caspase 3 and PARPwas conducted. B, P62 wasknocked down (shp62-1#) oroverexpressed (Oep62) in U937cells, and the cells were treatedwith MK 1 mmol/L for 24 hours. Celllysates were subjected to Westernblotting analysis with indicatedantibodies.






dependent manner and showed modest inhibition on theproliferation of AML cell lines (Fig. 1A and C and Supple-mentary Fig. S1A), which was consistent with the result inAurora A knockdown cells (Supplementary Fig. S2). Thesedata indicated the antitumor effect of Aurora kinases inhi-bitors was largely related to Aurora A inhibition. Further-more, the cells treated with MK and ZM showed polyploi-dization (Fig 1B and Supplementary Fig. S1B). P53 played avital role during mitosis and loss of p53 supported thesurvival of polyploidy cells (27, 28). However, our datasuggested that Aurora kinases inhibitors induced poly-ploidy might be independent of p53 pathways (data notshow). The study of ZMon leukemia cells also indicated thatpolyploidy induced by Aurora kinases inhibitors did notinevitably result in apoptosis (29). Thus, polyploidizationinduced by Aurora kinases inhibitors seemed to be the causeof modest effect on proliferation. Eliminating polyploidycells might enhance the effect of Aurora kinase inhibitors inAML treatment.Metabolic pathways are activated in tumor cells, as well as

polyploidy tumor cells (30). In the study of comparison inthe whole genomic transcripts of polyploidy and nonpoly-ploidy decidual cells in mouse, genes involved in metabolicprocess were upregulated (23). In addition, immortalizednontumorigenic human prostate and mammary epithelialcells which overexpressed pim-1, could gradually convert topolyploidy cells, with increase of reactive oxygen species(ROS) level (31). In aneuploid cells, themetabolic propertieswere also altered with increased glucose and glutamineconsumption (32). Anatskaia and colleagues reported thatthe rearrangement of the mainly metabolic pathways such asdepression of mitochondrial processes and increase of car-bohydrate degradation and lipid biosynthesis in polyploidycells, triggered pentose–phosphate pathway and depressedoxidative stress (33). Furthermore, these metabolic changescould increase metabolic plasticity and protect replicatingDNA from oxidative damage (33), suggesting that polyploi-dy cells might be sensitive to metabolic inhibitors. Polyploi-dy induced by Aurora kinases inhibitors had been showedwith more than 4N chromosomes (Fig. 1B and Supplemen-tary Fig. S1B). The most obvious phenomenon of polyploi-dy-related changes was the decrease of nuclear surface-to-volume ratio, which modified chromatin architecture andexerted spectacular effects on genes activity (33, 34). A greatof ATPase and energy were required for stemming from themetabolic shift from tricarboxylic acid cycle to glycolysis(35). Therefore, polyploidy induced by Aurora kinasesinhibitors might display elevated glycolysis. Consistent withthe previous reports, we showed that polyploidy cellsinduced by Aurora kinases inhibitors had higher level ofglycolytic metabolism than control cells (Fig. 2A). 2DG,glucose analog, had been reported to inhibit hexokinase,decrease intracellular ATP, and induce autophagy (36). Inour study, 2DG could inhibit the proliferation of thepolyploidy cells (Fig. 2B), suppress the mTOR pathway,and result in apoptotic and autophagic death (Fig. 5),indicating that the polyploidy cells with high metabolismwere susceptible to 2DG.

The mTOR signaling pathway integrates various meta-bolic pathways in cells. mTORC1 positively regulates thegenes encompassed glycolysis, pentose phosphate pathway,lipid/sterol biosynthesis, and mitochondrial function (37,38). For glycolysis, mTORC1 signaling stimulates glucoseuptake, promotes glycolysis flus, and increases lactate secre-tion (37, 38).mTORC2modulates cellsmetabolism throughphosphorylating Akt/PKB and activating the downstreamsuch as FoxO3a (19, 39). Furthermore, knockout of rictor(one of the mTORC2 components) in mice liver (40),adipose (19), or muscle (20) cells impaired glycolysis andlipogenesis. These studies suggested that mTOR pathwayplayed an important role in activating cellular bio-energeticprocesses contributed to cancer. In this study, mTOR inhi-bitors decreased the glucose uptake and lactate production ofAML cell lines (Fig. 3B). Numerous studies reported thatrapamycin activated Akt signaling pathway via a negativefeedback loop by inhibiting mTORC1 activation (41, 42).Low concentrations of rapamycin increased Akt and Erkphosphorylation through an mTORC1-dependent mecha-nism, but high doses of rapamycin inhibited Akt and Erkphosphorylation mainly via mTORC2 signaling pathway(43). Compared with mTORC1 inhibitor rapamycin, thedual mTORC1/2 inhibitor PP242 showed more effective toreduce cellular metabolic level in cancer cells (Fig. 3B).Furthermore, PP242 significantly decreased the proliferationof polyploidy cells, as well as the glucose uptake and lactateproduction, compared with that of control cells (Fig. 4),providing evidence that mTOR pathway regulated themetabolism of polyploidy cells. Thus, mTORC1/2 inhibi-tionmight be a potential approach to eliminate the polyploidycells. Indeed, the improved anticancer effect of mTORinhibitors and Aurora kinases inhibitors combination wasreported in HCT116 cells recently (44). In our study, weelucidated a novel mechanism of the synergistic combinationby suppression of cell metabolism.Previous study reported T-cell lymphomas that overex-

pressed Myc were sensitive to VX-680 (MK), and thesynthetic lethal interaction was executed by sequential apo-ptosis and autophagy. Exposed to VX-680 (MK) for 3 days,�30% of the cells were in apoptosis, and the survived cellsentered a new killing phase induced by autophagy after 3days (45). In our study, apoptosis slightly increased andautophagy elevated in AML cells incubating with Aurorakinases inhibitors for 2 days. Significantly, PP242 and 2DGincreased the apoptosis and autophagy of the polyploidy cells(Fig. 5). The results implied that inhibition the metabolismof polyploidy cells provoked not only apoptotic cell death,but also significant autophagy.Autophagy, an evolutionary highly conserved process, is

involved in degrading proteins and organelles in response tocellular stress (46). However, the function of autophagy intumorgenesis is complex and paradoxical. On the one hand,autophagy protects tumor cells from stress including nutrientstarvation, radiotherapy, and certain cytotoxic drugs (47). Onthe other hand, autophagy plays a vital role in suppressingtumorgenesis. Overexpression Beclin-1 gene exhibitedincreased susceptibility to cancer (48). Prolonged stress and

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excessive cellular damage might lead to cell death by over-stimulating autophagy (49). In this study, AMLcell lines wereshowed increased autophagy level with polyploidization aftertreating with Aurora kinases inhibitors (Fig. 5B). Further-more, either mTOR inhibitor PP242 or glycolysis inhibitor2DG promoted the polyploidy cells death by significantlyinducing apoptosis and autophagy (Fig. 5). We previouslyshowed that Aurora inhibition induced autophagy andtriggered drug resistance, which could be reversed byautophagy inhibitors (8). These paradoxical data suggestedthat cell death or survival promoted by autophagy dependedon the physiologic state of the cell (47). In our presentsystem, mTOR inhibition might overstimulate autophagyin polyploidy cells and eventually induce the polyploidycells death.The adaptor p62 is initially identified as a partner of the

atypical PKC (protein kinase C), interacting with varioussignaling proteins to regulate multiple cellular functions,including apoptosis and autophagy (50–52). UBA and LIRdomains of p62 confer the protein to function as an adaptorbetween autophagy and ubiquitinated proteins (53). As anautophagy substrate, p62 is recruited to the autophagosomalmembrane dependent or independent of LC3 for degrada-tion (54, 55). Upregulation of p62 by defective autophagywas a key factor in tumorigenesis, and elimination of p62might benefit to cancer therapy (56, 57). Deficiency of p62increased ROS levels, enhanced cell death, and reducedtumorigenicity of Ras (58). mTORC1 activation inhibitedautophagy by abolishing phosphorylated ATG13 proteinbinding to ATG1, promoting the accumulation of p62protein via reducing its degradation (59). Indeed, decreasethe p62 expression by mTOR inhibitor was showed in somereports (8, 60). Similarly, we found that expression of p62decreased in mTOR inhibitors treated polyploidy cells (Fig.5C), implying that p62 might be a target for oncotherapy.In addition, our data showed that p62 knockdown U937cells treated with MK were observed with more apoptosis,whereas p62 overexpressed U937 cells reversedMK induced

apoptosis, indicating that p62 might act as a potential targetfor cancer treatment (Fig. 6B).In summary, we found that polyploidy cells induced by

Aurora inhibitors required more energy than that of thecontrol cells. Suppression ofmetabolism bymTORpathwayinhibition increased the apoptosis and autophagy of thepolyploidy cells. Moreover, p62 expression was decreased bymTOR inhibitor or glucose deprivation in polyploidy cellsand might act as an attractive target. Thus, inhibition ofmTOR pathway could increase the potential efficiency ofAurora kinases inhibitors in AML treatment.

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

Authors' ContributionsConception and design: L.-L. Liu, Z.-J. Long, D.-J. Lin, Q. LiuDevelopment of methodology: L.-L. Liu, F.-M. Zheng, D.-F. Xu, J.-J. Chen, Q. LiuAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): L.-L. Liu, Z.-J. Long, L.-X. Wang, Q. LiuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): L.-L. Liu, L.-X. Wang, F.-M. Zheng, Z.-G. Fang, D.-F. Xu,J.-J. Chen, Q. LiuWriting, review, and/or revision of the manuscript: L.-L. Liu, Z.-J. Long,F.-M. Zheng, D.-J. Lin, Q. LiuAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): L.-L. Liu, Z.-J. Long, F.-M. Zheng, M. Yan, S.-W. Wang,D.-J. Lin, Q. LiuStudy supervision: D.-J. Lin, Q. Liu

AcknowledgmentsThe authors thank the members of LiuQ lab and Medical Research Center of the

Third Affiliated Hospital of Sun Yat-sen University for their critical comments andtechnical support.

Grant SupportThis work was supported by the National Natural Science Foundation of China (No.

81130040 to Q. Liu; No. 81000217 to Z.-J. Long), the Science and TechnologyFoundation of Guangzhou (No. 2012J2200077 to Z.-J. Long), the FundamentalResearch Funds for the Central Universities (No. 11ykpy37 to Z.-J. Long), andNationalBasic Research Program of China (973 Program; No. 2012CB967000 to Q. Liu).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received April 9, 2013; revised July 29, 2013; accepted August 1, 2013;published OnlineFirst September 5, 2013.

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2013;11:1326-1336. Published OnlineFirst September 5, 2013.Mol Cancer Res Ling-Ling Liu, Zi-Jie Long, Le-Xun Wang, et al. Cells to Aurora Inhibitors by Suppression of Glycolytic MetabolismInhibition of mTOR Pathway Sensitizes Acute Myeloid Leukemia

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