Phosphorylation and Activation of Cell Division Cycle ... · Phosphorylation and Activation of Cell...

Phosphorylation and Activation of Cell Division Cycle

Associated 8 by Aurora Kinase B Plays a Significant

Role in Human Lung Carcinogenesis

Satoshi Hayama,1,2Yataro Daigo,

1Takumi Yamabuki,

1Daizaburo Hirata,

1Tatsuya Kato,

1

Masaki Miyamoto,2Tomoo Ito,

3Eiju Tsuchiya,

4Satoshi Kondo,

2and Yusuke Nakamura

1

1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;Departments of 2Surgical Oncology and 3Surgical Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Japan;and 4Kanagawa Cancer Center Research Institute, Kanagawa, Japan

Abstract

Through genome-wide gene expression analysis of lungcarcinomas, we detected in the great majority of lung cancersamples cotransactivation of cell division cycle associated8 (CDCA8) and aurora kinase B (AURKB), which wereconsidered to be components of the vertebrate chromosomalpassenger complex. Immunohistochemical analysis of lungcancer tissue microarrays showed that overexpression ofCDCA8 and AURKB was significantly associated with poorprognosis of lung cancer patients. AURKB directly phosphor-ylated CDCA8 at Ser154, Ser219, Ser275, and Thr278 and seemedto stabilize CDCA8 protein in cancer cells. Suppression ofCDCA8 expression with small interfering RNA against CDCA8significantly suppressed the growth of lung cancer cells. Inaddition, functional inhibition of interaction between CDCA8and AURKB by a cell-permeable peptide corresponding to 20-amino acid sequence of a part of CDCA8 (11R-CDCA8261–280),which included two phosphorylation sites by AURKB, signifi-cantly reduced phosphorylation of CDCA8 and resulted ingrowth suppression of lung cancer cells. Our data imply thatselective suppression of the CDCA8-AURKB pathway could bea promising therapeutic strategy for treatment of lung cancerpatients. [Cancer Res 2007;67(9):4113–22]

Introduction

Lung cancer is one of the most common cancers in the world,and non–small cell lung cancer (NSCLC) accounts for 80% of thosecases (1). Many genetic alterations involved in development andprogression of lung cancer have been reported, but the precisemolecular mechanisms still remain unclear (2). Over the lastdecade, newly developed cytotoxic agents, including paclitaxel,docetaxel, gemcitabine, and vinorelbine, have emerged to offermultiple therapeutic choices for patients with advanced NSCLC.However, those regimens provide only modest survival benefitscompared with cisplatin-based therapies (3, 4). In addition to thesecytotoxic drugs, several molecular targeted agents, such asmonoclonal antibodies (mAb) against vascular endothelial growthfactor (VEGF; i.e., bevacizumab/anti-VEGF) or epidermal growthfactor receptor (EGFR; i.e., cetuximab/anti-EGFR) as well as

inhibitors for EGFR tyrosine kinase (i.e., gefitinib and erlotinib),were developed and are applied in clinical practice (5). However,each of the new regimens can provide survival benefits to a smallsubset of the patients (6, 7). Hence, new therapeutic strategies, suchas development of more effective molecular targeted agentsapplicable to the great majority of patients with less toxicity, areeagerly awaited.Systematic analysis of expression levels of thousands of genes

using cDNA microarrays is an effective approach for identificationof unknown molecules involved in carcinogenic pathways and caneffectively screen candidate target molecules for development ofnovel therapeutics and diagnostics. We have been attempting toisolate potential molecular targets for diagnosis and/or treatmentof NSCLC by analyzing genome-wide expression profiles of 101lung cancer tissue samples on a cDNA microarray containing27,648 genes (8–12). To verify the biological and clinicopathologicsignificance of the respective gene products, we have establisheda screening system by a combination of the tumor tissuemicroarray analysis of clinical lung cancer materials and RNAinterference (RNAi) technique (13–24). This systematic approachrevealed that a cell division cycle associated 8 (CDCA8) wasfrequently overexpressed in primary lung cancers and wasessential for growth/survival and malignant nature of lung cancercells.Recently, CDCA8 was identified as a new component of the

vertebrate chromosomal passenger complex. CDCA8 was sug-gested to be phosphorylated in vitro by aurora kinase B (AURKB),but the precise phosphorylated sites and its functional importancein cancer cells as well as in normal mammalian cells remainunclear (25, 26). The chromosome passenger complex consists of atleast four proteins: AURKB, inner centromere protein (INCENP)antigens 135/155 kDa, BIRC5/survivin, and CDCA8 (27), each ofwhich shows a dynamic cellular localization pattern during mitosis(28). Because several mitotic functions of the chromosomalpassenger complex have been reported, such as the regulation ofmetaphase chromosome alignment, sister chromatid resolution,spindle checkpoint signaling, and cytokinesis (29), this complexwas likely to be categorized in a group of mitotic regulators.Activation of AURKB and BIRC5 was reported in some of humancancers (30, 31), and many other mitotic and/or cell cycleregulators are also aberrantly expressed in tumor cells and areconsidered to be targets for development of promising anticancerdrugs. In fact, cyclin-dependent kinase inhibitors ( flavopiridol,UCN-01, E7070, R-Roscovitine, and BMS-387032), specific KIF11inhibitor (monastrol), and histone deacetyltransferase inhibitorsrevealed anticancer activity and have applied in preclinical orclinical phases (32–34).

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

Requests for reprints: Yusuke Nakamura, Laboratory of Molecular Medicine,Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-4705

www.aacrjournals.org 4113 Cancer Res 2007; 67: (9). May 1, 2007

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We here describe the novel mechanism of oncogenic activationof CDCA8 by AURKB that is important for lung cancer growthand progression. We also show that functional inhibition of theCDCA8/AURKB interaction would lead to potential strategies fortreatment of lung cancer patients.

Materials and Methods

Cell lines and tissue samples. The human lung cancer cell lines used in

this study were as follows: lung adenocarcinomas, A427, A549, LC319, PC14,and NCI-H1373; lung squamous cell carcinomas (SCC), SK-MES-1, EBC-1,

and NCI-H226; and SCLC, DMS114, DMS273, SBC-3, and SBC-5. Human

lung-derived cells used in this study were as follows: MRC-5 ( fibroblast),

CCD-19Lu ( fibroblast), and BEAS-2B (lung epithelia: bronchus); these cellswere purchased from the American Type Culture Collection (Manassas, VA).

All cells were grown in monolayers in appropriate medium supplemented

with 10% FCS and were maintained at 37jC in atmospheres of humidifiedair with 5% CO2. Human small airway epithelial cells (SAEC) were grown in

optimized medium (SAGM) purchased from Cambrex BioScience, Inc.

Primary lung cancer samples had been obtained with written informed

consent as described previously (15). A total of 273 NSCLC and adjacentnormal lung tissue samples for immunostaining on tissue microarray

analysis were also obtained from patients who underwent surgery at

Hokkaido University and its affiliated hospitals (Sapporo, Japan). This study

and the use of all clinical materials were approved by individualinstitutional ethical committees.

Semiquantitative reverse transcription-PCR. Total RNA was extracted

from cultured cells and clinical tissues using Trizol reagent (LifeTechnologies, Inc.) according to the manufacturer’s protocol. Extracted

RNAs and normal human tissue polyadenylic acid RNAs were treated with

DNase I (Nippon Gene) and reversely transcribed using oligo(dT) primer

and SuperScript II reverse transcriptase (Invitrogen). Semiquantitativereverse transcription-PCR (RT-PCR) experiments were carried out with the

following synthesized CDCA8-specific primers, AURKB-specific primers,

E2F1-specific primers, or with h-actin (ACTB)–specific primers as an

internal control: 5¶-CATCTGGCATTTCTGCTCTCTAT-3¶ and 5¶-CTCAGG-GAAAGGAGAATAAAAGAAC-3¶ (CDCA8 ); 5¶-CCCATCTGCACTTGTCCT-CAT-3¶ and 5¶-AACAGATAAGGGAACAGTTAGGGA-3¶ (AURKB ); 5¶-GGAGTCTGTGTGGTGTGTATGTG-3¶ and 5¶-GAGGGAACAGAGCTGTTAG-GAAG-3¶ (E2F1); and 5¶-GAGGTGATAGCATTGCTTTCG-3¶ and 5¶-CAAGT-CAGTGTACAGGTAAGC-3¶ (ACTB). PCRs were optimized for the number

of cycles to ensure product intensity within the logarithmic phase of

amplification.Northern blot analysis. Human multiple-tissue blots containing 23

tissues (BD Biosciences Clontech) were hybridized with a 32P-labeled PCR

product of CDCA8 . The cDNA probe of CDCA8 was prepared by RT-PCR

using the primers described above. Prehybridization, hybridization, andwashing were done according to the supplier’s recommendations. The blots

were autoradiographed at room temperature for 30 h with intensifying BAS

screens (Bio-Rad Laboratories).

Antibodies. To obtain anti-CDCA8 antibody, we prepared plasmidsexpressing full-length of CDCA8 that contained His-tagged epitopes at their

NH2 termini using pET28 vector (Novagen). The recombinant proteins were

expressed in Escherichia coli , BL21 codon-plus strain (Stratagene), andpurified using TALON resin (BD Biosciences Clontech) according to the

supplier’s protocol. The protein, extracted on a SDS-PAGE gel, was

inoculated into rabbits; the immune sera were purified on affinity columns

according to standard methodology. Affinity-purified rabbit polyclonal anti-CDCA8 antibodies were used for Western blotting and immunostaining.

A rabbit polyclonal anti-AURKB antibody was purchased from Abcam, Inc.

On Western blots, we confirmed that the antibody was specific to CDCA8 or

AURKB, using lysates from NSCLC cell lines that either expressed CDCA8and AURKB endogenously or not or from cells transfected with CDCA8 or

AURKB expression vector.

Western blot analysis. Cells were lysed in lysis buffer [50 mmol/L Tris-

HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, 0.1%

SDS, protease inhibitor (Protease Inhibitor Cocktail Set III, Calbiochem)].We used an enhanced chemiluminescence Western blot analysis system (GE

Healthcare Biosciences) as described previously (15, 16).

Immunocytochemistry. Cultured cells were washed twice with PBS(�),fixed in 4% formaldehyde solution for 60 min at room temperature, and

rendered permeable by treatment for 1.5 min with PBS(�) containing 0.1%Triton X-100. Cells were covered with 3% bovine serum albumin in PBS(�)for 60 min to block nonspecific binding before the primary antibody

reaction. Then, the cells were incubated with antibody to human CDCA8 or

AURKB protein or with anti-FLAG mAb (Sigma-Aldrich Co.). The immune

complexes were stained with a goat anti-rabbit secondary antibody

conjugated to Alexa 594 (Molecular Probes) and viewed with a laser

confocal microscope (TCS SP2 AOBS, Leica Microsystems). To determine

the cell cycle–dependent expression and localization of wild-type (WT) or

mutant CDCA8 that was stably expressed in A549 cells, synchronization

at the G1-S boundary was achieved by aphidicolin block as described

previously (18). Cells were treated with 1 Ag/mL aphidicolin (Sigma-Aldrich)

for 24 h and released from the cell cycle arrest by washes in PBS for four

times. These cells were cultured in medium and harvested for analysis at

1.5 and 9 h after the release of the cell cycle arrest and were used for

immunoblotting and immunostaining.

Immunohistochemistry and tissue microarray analysis. To investi-

gate the CDCA8/AURKB protein in clinical materials, we stained tissuesections using EnVision+ kit/horseradish peroxidase (HRP; DakoCytoma-

tion). Affinity-purified anti-CDCA8 antibody or anti-AURKB antibody was

added after blocking of endogenous peroxidase and proteins, and each

section was incubated with HRP-labeled antirabbit IgG as the secondaryantibody. Substrate-chromogen was added and the specimens were

counterstained with hematoxylin.

Tumor tissue microarrays were constructed as published elsewhere, using

formalin-fixed NSCLCs (35–37). Positivity for CDCA8 and AURKB wasassessed semiquantitatively by three independent investigators without

prior knowledge of the clinical follow-up data. The intensity of histochemical

staining was recorded as absent (scored as 0), weak (1+), or strong (2+).

When all scorers judged as strongly positive, the cases were scored as 2+.Statistical analysis. We attempted to correlate clinicopathologic

variables, such as age, gender, and pathologic tumor-node-metastasis stage,

with the expression levels of CDCA8 and AURKB protein determined bytissue microarray analysis. Tumor-specific survival curves were calculated

from the date of surgery to the time of death related to NSCLC or to the last

follow-up observation. Kaplan-Meier curves were calculated for each

relevant variable and for CDCA8 or AURKB expression; differences insurvival times among patient subgroups were analyzed using the log-rank

test. Univariate analysis was done with the Cox proportional hazard

regression model to determine associations between clinicopathologic

variables and cancer-related mortality.RNAi assay. We had established previously a vector-based RNAi system,

psiH1BX3.0, which was designed to synthesize small interfering RNAs

(siRNA) in mammalian cells (13, 15, 16, 18–24). Ten micrograms of siRNAexpression vector were transfected using 30 AL LipofectAMINE 2000

(Invitrogen) into lung cancer cell lines, LC319 and SBC-5. The transfected

cells were cultured for 7 days in the presence of appropriate concentrations

of geneticin (G418), and the number of colonies was counted by Giemsastaining, and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) assay (cell counting kit-8 solution;

Dojindo), at 7 days after the G418 treatment. To confirm suppression of

CDCA8 protein expression, Western blotting was carried out with affinity-purified polyclonal antibody to CDCA8 according to the standard protocol.

The target sequences of the synthetic oligonucleotides for RNAi were as

follows: control 1 (enhanced green fluorescent protein (EGFP) gene, a mutant

of Aequorea victoria GFP), 5¶-GAAGCAGCACGACTTCTTC-3¶; control 2(Luciferase: Photinus pyralis luciferase gene), 5¶-CGTACGCGGAATACT-TCGA-3¶; si-CDCA8-#1 , 5¶-CAGCAGAAGCTATTCAGAC-3¶; and si-CDCA8-

#2 , 5¶-GCCGTGCTAACACTGTTAC-3¶.Luciferase assay. Human genomic DNA was extracted from LC319 cells

and used as templates for PCR. 5¶ Flanking region of the human CDCA8 or

AURKB gene was amplified by PCR with the following synthesized 5¶ region

Cancer Research

Cancer Res 2007; 67: (9). May 1, 2007 4114 www.aacrjournals.org



of CDCA8 -specific primers (5¶-CGGGGTACCCCGACAAGGCCTGCCGG-GAGTAGT-3¶ and 5¶-CCCAAGCTTGGGCGAATCTGTGCAGCTCGTGTC-3¶)and 5¶ region of AURKB -specific primers (5¶-AACGTAGGCATGTA-GAGGCTC-3¶ and 5¶-CGGGGAAGAAAGTGCTTAAAGGA-3¶). The fragments

of promoter region were excised with KpnI and HindIII restriction enzymes

and inserted into the corresponding enzyme sites of pGL3-Basic vector. Theentire coding sequence of E2F1 was cloned into the appropriate site of

pcDNA3.1/myc-His plasmid vector (Invitrogen) to achieve pcDNA3.1-E2F1 .

The plasmids containing the 5¶ flanking region of the CDCA8 gene and

phRL-SV40 (Promega) were cotransfected into LC319 cells with pcDNA3.1-E2F1 or mock vector using LipofectAMINE Plus (Invitrogen). Firely

luciferase activity values were normalized by comparing Firely luciferase

activity with Renilla luciferase activity, expressed from phRL-SV40 to allowvariation in transfection efficiency.

Recombinant CDCA8 proteins. We prepared 13 plasmids expressing

WT CDCA8 or various deletion mutant CDCA8 proteins (CDCA8D1–D12),each of which was mutated at serine/threonine to alanine and contained

His-tagged epitopes at their NH2 termini using pET28 vector. The

recombinant proteins were expressed in E. coli , BL21 codon-plus strain,

and purified using TALON resin according to the supplier’s protocol.In vitro kinase assay. Purified recombinant WT or mutated CDCA8

proteins were incubated with recombinant AURKB (rhAURKB; Upstate) and

[g-32P]ATP in kinase buffer [20 mmol/L Tris (pH 7.5), 10 mmol/L MgCl2,

2 mmol/L MnCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/LDTT] supplemented with a mixture of protease inhibitors, 10 mmol/L NaF,

5 nmol/L microcystin LR, and 50 Amol/L ATP. The reaction was terminated

by the addition of a 0.2 volume of 5� protein sample buffer and the proteinswere analyzed by SDS-PAGE.

Figure 1. Activation of CDCA8 andAURKB in lung tumor samples.A, expression of CDCA8 and AURKB inclinical samples of 14 NSCLC (T ) andcorresponding normal lung tissues (N ),examined by semiquantitative RT-PCR.We prepared appropriate dilutions of eachsingle-stranded cDNA prepared frommRNAs of clinical lung cancer samples,taking the level of h-actin (ACTB )expression as a quantitative control.B, expression of CDCA8 and AURKBproteins in 11 lung cancer cell lines,examined by Western blot analysis.C, subcellular localization of endogenousCDCA8 (top, red ) and endogenousAURKB (bottom, red ) in LC319 cells,detected by rabbit polyclonal antibodiesto CDCA8 or AURKB. Both weretriple-stained with a-tubulin (TUBA; green )and 4¶,6-diamidino-2-phenylindole (seemerged images).

Activation of CDCA8-AURKB in Lung Cancer




Inhibition of AURKB activity. To inhibit the AURKB activity inmammalian cells, we constructed siRNA oligonucleotides against AURKB

(si-AURKB-#1 and si-AURKB-#2), as well as control siRNA oligonucleotides

for EGFP. Cells seeded onto 10-cm dishes were incubated in mixtures of

both LipofectAMINE and control (5¶-GAAGCAGCACGACUUCUUC-3¶) orsi-AURKBs (si-AURKB-#1 , 5¶-CCAAACUGCUCAGGCAUAA-3¶; si-AURKB-#2 ,5¶-ACGCGGCACUUCACAAUUG-3¶) in a final concentration of 100 nmol/L.

At 4 h after transfection, the siRNA-LipofectAMINE mixtures were replaced

with fresh medium. The cells were collected and analyzed after additional72-h culture.

Synthesized cell-permeable peptide. Nineteen- or 20-amino acid

peptide sequences corresponding to a part of CDCA8 protein that

contained possible phosphorylation sites by AURKB were covalentlylinked at its NH2 terminus to a membrane transducing 11 poly-arginine

sequence (11R; refs. 21, 38). Three cell-permeable peptides were synthesized:

11R-CDCA8147–165, RRRRRRRRRRR-GGG-PSKKRTQSIQGKGKGKRSS; 11R-CDCA8209–228, RRRRRRRRRRR-GGG-ERIYNISGNGSPLADSKEIF; and 11R-

CDCA8261–280, RRRRRRRRRRR-GGG-NIKKLSNRLAQICSSIRTHK. Scramble

peptides derived from the most effective 11R-CDCA8261–280 peptides were

synthesized as a control: Scramble, RRRRRRRRRRR-GGG-TSAQIRHKC-SIKLSNIKNRL. Peptides were purified by preparative reverse-phase high-

pressure liquid chromatography.

LC319 and SBC-5 cells, as well as human lung-derived MRC-5, CCD19-Lu,

and BEAS-2B cells, were incubated with the 11R peptides at theconcentration of 2.5, 5, and 7.5 Amol/L for 7 days. The medium was

exchanged at every 48 h at the appropriate concentrations of each peptide

and the viability of cells was evaluated by MTT assay at 7 days after thetreatment. To confirm the transduction efficiency of the peptides, we

synthesized the 11R-CDCA8261–280 and its control peptides labeled with

FITC at NH2 terminus. Transduction of the peptides (2.5–7.5 Amol/L) was

monitored by fluorescence microscopic observation after a 3-h incubation

of the peptides with the cell lines at 37jC. The 11R-CDCA8261–280 as well asits control peptides was transduced into almost all of cultured cells as

reported elsewhere (38).

Results

Coactivation of CDCA8 and AURKB in lung cancers. Using acDNA microarray representing 27,648 genes of 101 lung cancertissues, we identified CDCA8 to be overexpressed in a largeproportion of lung cancers. Subsequently, we confirmed itstransactivation in 11 of 14 additional NSCLC cases ( four of sevenadenocarcinomas; all of seven SCCs) by semiquantitative RT-PCRexperiments (Fig. 1A, top). We further confirmed high levels ofendogenous CDCA8 expression in all of 11 lung cancer cell lines byWestern blot analysis using a rabbit polyclonal anti-CDCA8antibody (Fig. 1B, top). Northern blot analysis using CDCA8 cDNAas a probe identified a 2.5-kb transcript, exclusively in the testisamong 23 human tissues examined (Supplementary Fig. S1A).To determine the subcellular localization of endogenous CDCA8

in lung cancer cells, we did immunocytochemical analysis forLC319 cells using anti-CDCA8 antibody; CDCA8 proteins weremainly detected at nucleus in cells at the G1-S phase and at nucleusand contractile ring in cells at the G2-M phase (Fig. 1C, top). CDCA8was isolated previously as a new member of a vertebratechromosomal passenger complex (25), and phosphorylation ofCOOH-terminal region of glutathione S-transferase-tagged CDCA8by rhAURKB was shown by in vitro kinase assay (26). However,the precise phosphorylation site(s) and its functional significancein cancer cells remain unclear. To elucidate the biological role of

Figure 2. Association of CDCA8 andAURKB overexpression with poorclinical outcomes in NSCLC.A, immunohistochemical evaluation ofrepresentative samples from surgicallyresected SCC tissues, using anti-CDCA8(top ) and anti-AURKB (bottom ) polyclonalantibodies on tissue microarrays.Magnification, �100. B, Kaplan-Meieranalysis of tumor-specific survival timesaccording to expression of CDCA8 (left)and AURKB (right ) on tissue microarrays.

Cancer Research




CDCA8 activation in lung cancer cells, we first examined theexpression status of AURKB using our gene expression database(8–12) and found that AURKB was frequently overexpressed in lungcancers compared with normal lung cells. Furthermore, our dataindicated that the levels of AURKB expression seemed to becorrelated with those of CDCA8 . We subsequently reexaminedprimary lung cancer tissues and found increase of AURKBexpression in 12 of 16 NSCLC clinical samples ( five of sevenadenocarcinomas and all of seven SCCs) by semiquantitative RT-PCR experiments (Fig. 1A, middle). The expression patterns ofAURKB in lung cancers were very similar to those of CDCA8 . Wefurther confirmed by Western blot analysis that CDCA8 andAURKB proteins were coactivated in almost all of the lung cancercell lines examined (Fig. 1B, middle). Immunocytochemical analysisusing a rabbit polyclonal anti-AURKB antibody detected AURKBproteins to be located mainly at nucleus in cells at the G1-S phaseand at nucleus and contractile ring in cells at the G2-M phase;the subcellular localization of AURKB in lung cancer cells wasvery similar to that of CDCA8 as reported elsewhere (Fig. 1C,bottom ; ref. 25).

Association of CDCA8 and AURKB positivity with poorprognosis of NSCLC patients. Using tissue microarrays preparedfrom 273 surgically resected NSCLCs, we did immunohistochemicalanalysis with affinity-purified anti-CDCA8 and anti-AURKB poly-clonal antibodies. We classified patterns of CDCA8/AURKBexpression as absent (scored as 0), weak (scored as 1+), or strong(scored as 2+). Of the 273 NSCLC cases examined, 113 (41%) casesrevealed strong CDCA8 expression and 100 (37%) cases showedweak expression, although its expression was absent in 60 (22%)cases. For AURKB, strong expression was observed in 116 (42%)cases, weak expression in 94 (34%) cases, and no expression in 63(24%) cases. No staining was observed for either CDCA8 or AURKBin any of their adjacent normal lung tissues (Fig. 2A). Tumors (183of the 273) were positive (scored as 1+ to 2+) for both CDCA8 andAURKB, and 33 were negative for the both proteins. Cases (30 ofthe 273) were positive for only CDCA8 and 27 were positive for onlyAURKB. The expression pattern of CDCA8 protein was significantlyconcordant with AURKB protein expression in these tumors

(P < 0.0001, m2 test) as similar to the results by RT-PCR andWestern blotting. We found that strong expression (scored as 2+) ofCDCA8 in NSCLCs was significantly associated with tumor size(pT1 versus pT2-4; P = 0.0414, m2 test), lymph node metastasis (pN0

versus pN1-2; P = 0.0005, m2 test), and with shorter tumor-specific5-year survival times (P = 0.0009, log-rank test; Fig. 2B, left). Strongexpression (scored as 2+) of AURKB in NSCLCs was significantlyassociated with tumor size (pT1 versus pT2-4; P = 0.0361, m2 test),lymph node metastasis (pN0 versus pN1-2; P = 0.0004, m2 test), and5-year survival (P = 0.0001, log-rank test; Fig. 2B, right). NSCLCpatients without either CDCA8 or AURKB expression in theirtumors could reveal the longest survival period, whereas those withstrong positive staining for both markers showed the shortesttumor-specific survival (P < 0.0001, log-rank test; SupplementaryFig. S1B). Using univariate analysis, we found that lymph nodemetastasis (pN0 versus pN1 and N2; P < 0.0001, score test), tumorsize (pT1 versus pT2, T3, and T4; P < 0.0001, score test), and highCDCA8/AURKB expression (P = 0.0009 and = 0.0001, respectively,score test) were important correlative features for poor prognosesof patients with NSCLC.

Growth inhibition of lung cancer cells by specific siRNAagainst CDCA8. To assess whether CDCA8 is essential for growthor survival of lung cancer cells, we constructed plasmids to expresssiRNA against CDCA8 (si-CDCA8-#1 and si-CDCA8-#2), usingsiRNAs for EGFP and Luciferase as controls. Transfection of si-CDCA8-#1 or si-CDCA8-#2 into LC319 or SBC-5 cells significantlysuppressed expression of endogenous CDCA8 proteins in compa-rison with the two controls and resulted in significant decreases incell viability and colony numbers measured by MTT and colonyformation assays (representative data of LC319 were shown in Fig. 3).

Simultaneous activation of CDCA8 and AURKB regulated byE2F1. The concordant activation of CDCA8 and AURKB in lungcancers suggested that these two genes might be regulated bythe same transcription factor(s). To validate this hypothesis, weexamined the DNA sequences of the CDCA8 and AURKB promoterregions and found the cell cycle–dependent element (CDE) andcell cycle gene homology region (CHR) consensus sequences(CDE-CHR), by which transcription of AURKB as well as cyclin A

Figure 3. Inhibition of growth of lungcancer cells by siRNAs against CDCA8.Left, top, gene knockdown effect onCDCA8 protein expression in LC319 cellsby two si-CDCA8s (si-CDCA8-#1 andsi-CDCA8-#2 ) and two control siRNAs(si-EGFP and si-Luciferase ), analyzed byWestern blotting. Left, bottom and right,colony formation and MTT assays ofLC319 cells transfected with si-CDCA8sor control plasmids. Columns, relativeabsorbance of triplicate assays; bars, SD.





(CCNDA) and CDC25 is regulated (Supplementary Fig. S2A).Among the transcription factors that could bind to the CDE-CHRof the AURKB gene, we confirmed that E2F1 was coactivated withCDCA8 and AURKB as detected by semiquantitative RT-PCRanalysis of NSCLC cases (Supplementary Fig. S2B). To investigatethe direct transcriptional regulation of the CDCA8 gene promoterby E2F1, we transiently cotransfected LC319 cells with E2F1 ormock vector, along with CDCA8 or AURKB (positive control)promoter constructs containing putative regulatory elements(CDE-CHR) fused to a luciferase reporter gene. Expectedly, bothCDCA8 and AURKB promoter functions were activated by E2F1(Supplementary Fig. S2C). The induction of endogenous CDCA8and AURKB was further confirmed by introduction of exogenousE2F1 into LC319 cells (data not shown).

Phosphorylation of CDCA8 by AURKB in lung cancer cells.Western blot analysis detected two different sizes of CDCA8protein (Fig. 1B, top). To examine a possibility, the CDCA8phosphorylation, we incubated extracts from LC319 cells in thepresence or absence of protein phosphatase (Bio-Rad Laboratories)and analyzed the molecular weight of CDCA8 protein by Westernblot analysis. Because the measured weight of the majority ofCDCA8 protein in the extracts treated with phosphatase wassmaller than that in the untreated cells (Fig. 4A), we consideredthat CDCA8 was phosphorylated in lung cancer cells. We thenexamined whether AURKB could phosphorylate CDCA8 in vitro asreported previously (26). When full-length recombinant CDCA8(rhCDCA8) protein was incubated with rhAURKB protein in kinasebuffer, including [g-32P]ATP, CDCA8 was phosphorylated in anAURKB dose-dependent manner (Fig. 4B). To assess whether theabundant expression of endogenous AURKB is critical forphosphorylation of endogenous CDCA8 in cancer cells, weselectively knocked down with siRNA against AURKB (si-AURKB)the AURKB expression in LC319 cells, in which these two geneswere expressed abundantly. Reduction of AURKB protein by si-AURKBs (si-AURKB-#1 and si-AURKB-#2) dramatically decreasedthe amount of CDCA8 protein, whereas a level of CDCA8 trans-cripts in the same cells was not influenced by si-AURKBs (Fig. 4C).Hence, we hypothesized that endogenous CDCA8 protein may bestabilized when it is phosphorylated by endogenous AURKB and/orincorporated in some protein complex that are possibly regulatedby the AURKB signaling.Because our data imply that human CDCA8 and AURKB were

coactivated in lung cancer cells and that CDCA8 phosphorylationby AURKB might play a significant role in pulmonary carcinogen-esis, we then investigated the phosphorylation sites of CDCA8 byAURKB. We prepared six His-tagged CDCA8 proteins (CDCA8D1–D6), in which two or three serine/threonine residues weresubstituted to alanines (Fig. 5A, top). We did in vitro kinase assayusing these mutant CDCA8 proteins and detected a reduction ofphosphorylation levels in three mutated constructs (CDCA8D2,CDCA8D5, and CDCA8D6) compared with a WT, suggesting thatsix serine/threonines corresponding to the substituted sites couldbe putative phosphorylation ones (Fig. 5B, top). We furtherprepared six additional mutant CDCA8 constructs (CDCA8D7–D12), in which either of the six serine/threonines was substitutedto an alanine (Fig. 5A, bottom). In vitro kinase assay of these sixmutants revealed the reduction of phosphorylation levels in fourmutated constructs, including a substitution at either of fourserine/threonine residues, Ser154, Ser219, Ser275, and Thr278

(CDCA8D8, CDCA8D9, CDCA8D11, and CDCA8D12; Fig. 5B,bottom). We subsequently made a mutant construct (CDCA8D13),

in which all of these four serine/threonines were substituted to analanine, and did in vitro kinase assay using AURKB. The resultshowing complete disappearance of CDCA8 phosphorylation byAURKB (Fig. 5C) clearly showed that CDCA8 was phosphorylated

Figure 4. Phosphorylation of CDCA8 by AURKB. A, dephosphorylation ofendogenous CDCA8 protein in LC319 cells by treatment with E-phosphatase.White arrowhead, phosphorylated CDCA8; black arrowheads,nonphosphorylated form. B, in vitro phosphorylation of rhCDCA8 by rhAURKB.C, top, the expression levels of endogenous AURKB and CDCA8 proteins,detected by Western blot analysis in LC319 cells transfected with siRNAoligonucleotides against AURKB (si-AURKBs); bottom, the expression levels ofendogenous AURKB and CDCA8 transcripts, detected by semiquantitativeRT-PCR analysis in LC319 cells transfected with si-AURKBs.

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at four serine/threonine residues at Ser154, Ser219, Ser275, and Thr278

by AURKB.Growth inhibition of lung cancer cells by cell-permeable

peptides. To investigate the functional significance of interactionbetween CDCA8 and AURKB as well as CDCA8 phosphorylationfor growth or survival of lung cancer cells, we developed bioactivecell-permeable peptides that were expected to inhibit the in vivophosphorylation of CDCA8 by AURKB. We synthesized threedifferent peptides of 19- or 20-amino acid sequence that includedthe four CDCA8 phosphorylation sites (Ser154, Ser219, Ser275, and

Thr278). These peptides were covalently linked at its NH2 terminusto a membrane transducing 11 arginine-residues (11R). We firstinvestigated by in vitro kinase assay the effect of the three 11R-CDCA8 peptides on the phosphorylation of rhCDCA8 byrhAURKB. When full-length rhCDCA8 protein was incubated withrhAURKB protein with either of the three 11R-CDCA8 peptides ortheir scramble peptides in kinase buffer, including [g-32P]ATP, thephosphorylation level of rhCDCA8 was significantly suppressed bythe treatment with 11R-CDCA8261–280 peptides containing Ser275

and Thr278 compared with its scramble peptides (Fig. 6A).

Figure 5. Identification of the cognate phosphorylation sites on CDCA8 by AURKB. A, top, six full-length rhCDCA8 mutants that were substituted at putative serine/threonine phosphorylated sites to alanines; each construct contained two or three substitutions (CDCA8D1–D6); bottom, additional six full-length rhCDCA8 mutantsthat were substituted at either of six serine/threonine residues to an alanine residue (CDCA8D7–D12). B, top, in vitro kinase assays incubating WT and mutant CDCA8proteins with rhAURKB. CDCA8D2, CDCA8D5, and CDCA8D6 constructs resulted in a reduction of phosphorylation levels by AURKB (each of substituted residuewas indicated as bold character on underline), whereas CDCA8D1, CDCA8D3, and CDCA8D4 represented the same levels of phosphorylation compared with WTCDCA8. Bottom, CDCA8D8, CDCA8D9, CDCA8D11, and CDCA8D12 resulted in a reduction of phosphorylation, whereas CDCA8D7 and CDCA8D10 showed thesame levels of phosphorylation compared with WT, indicating that CDCA8 was phosphorylated at Ser154, Ser219, Ser275, and Thr278 (indicated as bold character withunderline) by AURKB. C, in vitro kinase assays incubating WT and mutant CDCA8 protein (CDCA8D13), in which all of the four serine/threonines were substituted to analanine, with rhAURKB. CDCA8D13 construct resulted in complete diminishment of CDCA8 phosphorylation by AURKB.





Figure 6. Inhibition of growth of lung cancer cells by cell-permeable CDCA8 peptides. A, reduction of the AURKB-dependent CDCA8 phosphorylation bycell-permeable CDCA8 peptides (11R-CDCA8261–280), detected by in vitro kinase assay. B, top, the expression levels of endogenous CDCA8 protein, detected byWestern blot analysis of LC319 cells transfected with the 11R-CDCA8261–280; bottom, the expression levels of endogenous CDCA8 transcript, detected bysemiquantitative RT-PCR analysis of LC319 cells transfected with the 11R-CDCA8261–280. C, top, MTT assay of LC319 cells, detecting a growth-suppressive effect of11R-CDCA8261–280. Columns, relative absorbance of triplicate assays; bars, SD. Bottom, cell cycle analysis of LC319 cells after the treatment with 11R-CDCA8261–280peptides or Scramble peptides. D, left, expression of CDCA8 protein in normal human lung fibroblast-derived MRC-5 and CCD19-Lu cells compared with lungcancer cell line LC319, examined by Western blot analysis; right, MTT assay, detecting no significant off-target effect of the 11R-CDCA8261–280 peptides on MRC-5cells that scarcely expressed CDCA8 and AURKB protein.

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Addition of the 11R-CDCA8261–280 into the culture medium ofLC319 cells inhibited the phosphorylation and decreased stabilityof endogenous CDCA8 protein, whereas no effect on a level ofCDCA8 transcript was observed (Fig. 6B). The 11R-CDCA8261–280treatment of LC319 cells resulted in significant decreases in cellviability as measured by MTT assay (representative data in Fig. 6C,top). To clarify the mechanism of tumor suppression by the 11R-CDCA8261–280 peptide, we did flow cytometric analysis of thetumor cells treated with these peptides and found the significantincrease in sub-G1 fraction at 48 h after the treatment of 11R-CDCA8261–280 (Fig. 6C, bottom). 11R-CDCA8261–280 revealed nosignificant effect on cell viability of human lung fibroblast-derivedMRC-5 or CCD19-Lu or human bronchial epithelia-derived BEAS-2B cells, in which CDCA8 and AURKB expression were hardlydetectable (representative data of MRC-5 and BEAS-2B cells wereshown in Fig. 6D ; Supplementary Fig. S3). The data indicate that11R-CDCA8261–280 could specifically inhibit an enzymatic reactionof AURKB for CDCA8 phosphorylation and have no or minimumtoxic effect on normal human cells that do not abundantly expressthese proteins.

Discussion

Toward the goal of developing novel therapeutic anticancerdrugs with a minimum risk of adverse reactions, we established apowerful screening system to identify proteins and their interactingproteins that were activated specifically in lung cancer cells. Thestrategy was as follows: (a) identification of up-regulated genes in101 lung cancer samples through the genome-wide cDNA micro-array system coupled with laser microdissection (8–12); (b)verification of very low or absent expression of such genes innormal organs by cDNA microarray analysis and multiple-tissueNorthern blot analysis (39, 40); (c) confirmation of the clinico-pathologic significance of their overexpression using tissue micro-array consisting of hundreds of NSCLC tissue samples (12, 14–24);and (d) verification of the targeted genes whether they are essentialfor the survival or growth of lung cancer cells by siRNA (13, 15, 16,18–24). By this systematic approach, we identified that CDCA8 andAURKB are cooverexpressed in the great majority of clinical lungcancer samples as well as lung cancer cell lines and that these twoproteins are indispensable for growth and progression of lungcancer cells.CDCA8 was shown to be phosphorylated in vitro by AURKB

previously (26), but its significance in development and/orprogression of human cancer had been never described. CDCA8was indicated recently to be one of new components of thevertebrate chromosomal passengers, such as AURKB, INCENP, andBIRC5 (25), which are considered to be key regulators of mitoticevents responsible for correcting the error of bipolar attachmentsthat inevitably occur during the ‘search-and-capture’ mechanism(41–43). In this study, we have shown that the CDCA8 protein islikely to be stabilized by its AURKB-dependent phosphorylation atSer154, Ser219, Ser275, and/or Thr278. Depletion of AURKB functionby RNAi or the 11R-CDCA8261–280 that could inhibit phosphoryla-tion of CDCA8 significantly decreased the level of endogenous

CDCA8 protein. Phosphorylation is an important posttranslationalmodification that regulates the protein stability, function, localiza-tion, and binding specificity to target proteins. For example, MKP-7phosphorylated at Ser446 or p27 phosphorylated at Ser10 has alonger half-life than unphosphorylated form; when at the sites weredephosphorylated, the amount of these proteins was promptlydecreased in cells (44, 45). The evidence suggests that the stabilityof CDCA8 protein could be tightly regulated by the AURKBsignaling in cancel cells. AURKB was shown to be overexpressed inmany tumor cell lines, and its overexpression was noted to beinvolved in chromosome number instability and tumor invasive-ness (30, 31). AURKB is one of the cancer-related kinases andtherefore was thought to be a promising target for anticancer drugdevelopment. Indeed, two AURKB inhibitors have been describedrecently: ZM447439 and Hesperadin (46, 47). Our study hasindicated that CDCA8 is a putative oncogene that is aberrantlyexpressed in lung cancer cells along with AURKB. We found bytissue microarray analysis that CDCA8 and AURKB were cooverex-pressed and that patients with NSCLC showing higher expressionof these proteins represented a shorter tumor-specific survivalperiod, doubtlessly suggesting that CDCA8, along with AURKB,plays a crucial role for progression of lung cancers. Furthermore,we showed at the first time that growth of lung cancer cellsoverexpressing CDCA8 and AURKB could be suppressed effectivelyby blocking the AURKB-dependent CDCA8 phosphorylation bymeans of the 20-amino acid cell-permeable peptide that corre-sponded to a part of CDCA8 protein and included twophosphorylation sites, Ser275 and Thr278, by AURKB. We detecteda significant increase in the sub-G1 fraction after the treatmentof 11R-CDCA8261–280 peptide, suggesting that the cell-permeablepolypeptides induced apoptosis of the cancer cells. Because thephosphorylation of CDCA8 at these sites was likely to beindispensable for the growth/survival of lung cancer cells andCDCA8 could belong to cancer-testis antigens, selective targetingof CDCA8-AURKB enzymatic activity could be a promisingtherapeutic strategy that is expected to have a powerful biologicalactivity against cancer with a minimal risk of adverse events.Further analyses of the mechanism of growth suppression byspecific inhibiting of CDCA8 phosphorylation by AURKB may be ofthe great benefit toward the development of new types ofanticancer agents.In summary, we have found that CDCA8 is coactivated with and

phosphorylated/stabilized by AURKB in lung cancer cells and thatphosphorylated CDCA8 plays a significant role in growth and/orsurvival of cancer cells. The data strongly imply the possibility ofdesigning new anticancer drugs targeting the CDCA8-AURKBassociation as a promising therapeutic strategy for lung cancer.

Acknowledgments

Received 12/21/2006; revised 2/13/2007; accepted 2/20/2007.Grant support: The Japan Society for the Promotion of Science ‘‘Research for the

Future’’ Program grant 00L01402 (Y. Nakamura).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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2007;67:4113-4122. Cancer Res Satoshi Hayama, Yataro Daigo, Takumi Yamabuki, et al. Human Lung CarcinogenesisAssociated 8 by Aurora Kinase B Plays a Significant Role in Phosphorylation and Activation of Cell Division Cycle

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