Phosphorylation and Activation of Cell Division Cycle Associated 8 by Aurora Kinase B Plays a Significant Role in Human Lung Carcinogenesis

Research Article

Satoshi Hayama,1,2 Yataro Daigo,1 Takumi Yamabuki,1 Daizaburo Hirata,1 Tatsuya Kato,1 Masaki Miyamoto,2 Tomoo Ito,3 Eiju Tsuchiya,4 Satoshi Kondo,2 andYusuke 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 inhibitors for EGFR tyrosine kinase (i.e., gefitinib and erlotinib), Through genome-wide gene expression analysis of lung were developed and are applied in clinical practice (5). However, carcinomas, we detected in the great majority of lung cancer each of the new regimens can provide survival benefits to a small samples cotransactivation of cell division cycle associated subset of the patients (6, 7). Hence, new therapeutic strategies, such 8(CDCA8) and aurora kinase B (AURKB), which were as development of more effective molecular targeted agents considered to be components of the vertebrate chromosomal applicable to the great majority of patients with less toxicity, are passenger complex.Immunohistochemical analysis of lung eagerly awaited. cancer tissue microarrays showed that overexpression of Systematic analysis of expression levels of thousands of genes CDCA8 and AURKB was significantly associated with poor using cDNA microarrays is an effective approach for identification prognosis of lung cancer patients.AURKB directly phosphor- of unknown molecules involved in carcinogenic pathways and can ylated CDCA8 at Ser154, Ser219, Ser275, and Thr278 and seemed effectively screen candidate target molecules for development of to stabilize CDCA8 protein in cancer cells.Suppression of novel therapeutics and diagnostics. We have been attempting to CDCA8 expression with small interfering RNA against CDCA8 isolate potential molecular targets for diagnosis and/or treatment significantly suppressed the growth of lung cancer cells.In of NSCLC by analyzing genome-wide expression profiles of 101 addition, functional inhibition of interaction between CDCA8 lung cancer tissue samples on a cDNA microarray containing and AURKB by a cell-permeable peptide corresponding to 20- 27,648 genes (8–12). To verify the biological and clinicopathologic significance of the respective gene products, we have established amino acid sequence of a part of CDCA8 (11R-CDCA8261–280), which included two phosphorylation sites by AURKB, signifi- a screening system by a combination of the tumor tissue cantly reduced phosphorylation of CDCA8 and resulted in microarray analysis of clinical lung cancer materials and RNA growth suppression of lung cancer cells.Our data imply that interference (RNAi) technique (13–24). This systematic approach selective suppression of the CDCA8-AURKB pathway could be revealed that a cell division cycle associated 8 (CDCA8) was a promising therapeutic strategy for treatment of lung cancer frequently overexpressed in primary lung cancers and was patients. [Cancer Res 2007;67(9):4113–22] essential for growth/survival and malignant nature of lung cancer cells. Introduction Recently, CDCA8 was identified as a new component of the vertebrate chromosomal passenger complex. CDCA8 was sug- Lung cancer is one of the most common cancers in the world, gested to be phosphorylated in vitro by aurora kinase B (AURKB), and non–small cell lung cancer (NSCLC) accounts for 80% of those but the precise phosphorylated sites and its functional importance cases (1). Many genetic alterations involved in development and in cancer cells as well as in normal mammalian cells remain progression of lung cancer have been reported, but the precise unclear (25, 26). The chromosome passenger complex consists of at molecular mechanisms still remain unclear (2). Over the last least four proteins: AURKB, inner centromere protein (INCENP) decade, newly developed cytotoxic agents, including paclitaxel, antigens 135/155 kDa, BIRC5/survivin, and CDCA8 (27), each of docetaxel, gemcitabine, and vinorelbine, have emerged to offer which shows a dynamic cellular localization pattern during mitosis multiple therapeutic choices for patients with advanced NSCLC. (28). Because several mitotic functions of the chromosomal However, those regimens provide only modest survival benefits passenger complex have been reported, such as the regulation of compared with cisplatin-based therapies (3, 4). In addition to these metaphase chromosome alignment, sister chromatid resolution, cytotoxic drugs, several molecular targeted agents, such as spindle checkpoint signaling, and cytokinesis (29), this complex monoclonal antibodies (mAb) against vascular endothelial growth was likely to be categorized in a groupof mitotic regulators. factor (VEGF; i.e., bevacizumab/anti-VEGF) or epidermal growth Activation of AURKB and BIRC5 was reported in some of human factor receptor (EGFR; i.e., cetuximab/anti-EGFR) as well as cancers (30, 31), and many other mitotic and/or cell cycle regulators are also aberrantly expressed in tumor cells and are considered to be targets for development of promising anticancer Note: Supplementary data for this article are available at Cancer Research Online drugs. In fact, cyclin-dependent kinase inhibitors (flavopiridol, (http://cancerres.aacrjournals.org/). Requests for reprints: Yusuke Nakamura, Laboratory of Molecular Medicine, UCN-01, E7070, R-Roscovitine, and BMS-387032), specific KIF11 Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 inhibitor (monastrol), and histone deacetyltransferase inhibitors Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: [email protected]. I2007 American Association for Cancer Research. revealed anticancer activity and have applied in preclinical or doi:10.1158/0008-5472.CAN-06-4705 clinical phases (32–34). www.aacrjournals.org 4113 Cancer Res 2007; 67: (9). May 1, 2007

We here describe the novel mechanism of oncogenic activation SDS, protease inhibitor (Protease Inhibitor Cocktail Set III, Calbiochem)]. of CDCA8 by AURKB that is important for lung cancer growth We used an enhanced chemiluminescence Western blot analysis system (GE and progression. We also show that functional inhibition of the Healthcare Biosciences) as described previously (15, 16). À CDCA8/AURKB interaction would lead to potential strategies for Immunocytochemistry. Cultured cells were washed twice with PBS( ), fixed in 4% formaldehyde solution for 60 min at room temperature, and treatment of lung cancer patients. rendered permeable by treatment for 1.5 min with PBS(À) containing 0.1% Triton X-100. Cells were covered with 3% bovine serum albumin in PBS(À) for 60 min to block nonspecific binding before the primary antibody Materials and Methods reaction. Then, the cells were incubated with antibody to human CDCA8 or Cell lines and tissue samples. The human lung cancer cell lines used in AURKB protein or with anti-FLAG mAb (Sigma-Aldrich Co.). The immune this study were as follows: lung adenocarcinomas, A427, A549, LC319, PC14, complexes were stained with a goat anti-rabbit secondary antibody and NCI-H1373; lung squamous cell carcinomas (SCC), SK-MES-1, EBC-1, conjugated to Alexa 594 (Molecular Probes) and viewed with a laser and NCI-H226; and SCLC, DMS114, DMS273, SBC-3, and SBC-5. Human confocal microscope (TCS SP2 AOBS, Leica Microsystems). To determine lung-derived cells used in this study were as follows: MRC-5 (fibroblast), the cell cycle–dependent expression and localization of wild-type (WT) or CCD-19Lu (fibroblast), and BEAS-2B (lung epithelia: bronchus); these cells mutant CDCA8 that was stably expressed in A549 cells, synchronization were purchased from the American Type Culture Collection (Manassas, VA). at the G1-S boundary was achieved by aphidicolin block as described All cells were grown in monolayers in appropriate medium supplemented previously (18). Cells were treated with 1 Ag/mL aphidicolin (Sigma-Aldrich) with 10% FCS and were maintained at 37jC in atmospheres of humidified for 24 h and released from the cell cycle arrest by washes in PBS for four air with 5% CO2. Human small airway epithelial cells (SAEC) were grown in times. These cells were cultured in medium and harvested for analysis at optimized medium (SAGM) purchased from Cambrex BioScience, Inc. 1.5 and 9 h after the release of the cell cycle arrest and were used for Primary lung cancer samples had been obtained with written informed immunoblotting and immunostaining. consent as described previously (15). A total of 273 NSCLC and adjacent Immunohistochemistry and tissue microarray analysis. To investi- normal lung tissue samples for immunostaining on tissue microarray gate the CDCA8/AURKB protein in clinical materials, we stained tissue analysis were also obtained from patients who underwent surgery at sections using EnVision+ kit/horseradish peroxidase (HRP; DakoCytoma- Hokkaido University and its affiliated hospitals (Sapporo, Japan). This study tion). Affinity-purified anti-CDCA8 antibody or anti-AURKB antibody was and the use of all clinical materials were approved by individual added after blocking of endogenous peroxidase and proteins, and each institutional ethical committees. section was incubated with HRP-labeled antirabbit IgG as the secondary Semiquantitative reverse transcription-PCR. Total RNA was extracted antibody. Substrate-chromogen was added and the specimens were from cultured cells and clinical tissues using Trizol reagent (Life counterstained with hematoxylin. Technologies, Inc.) according to the manufacturer’s protocol. Extracted Tumor tissue microarrays were constructed as published elsewhere, using RNAs and normal human tissue polyadenylic acid RNAs were treated with formalin-fixed NSCLCs (35–37). Positivity for CDCA8 and AURKB was DNase I (Nippon Gene) and reversely transcribed using oligo(dT) primer assessed semiquantitatively by three independent investigators without and SuperScript II reverse transcriptase (Invitrogen). Semiquantitative prior knowledge of the clinical follow-up data. The intensity of histochemical reverse transcription-PCR (RT-PCR) experiments were carried out with the staining was recorded as absent (scored as 0), weak (1+), or strong (2+). following synthesized CDCA8-specific primers, AURKB-specific primers, When all scorers judged as strongly positive, the cases were scored as 2+. E2F1-specific primers, or with h-actin (ACTB)–specific primers as an Statistical analysis. We attempted to correlate clinicopathologic internal control: 5¶-CATCTGGCATTTCTGCTCTCTAT-3¶ and 5¶-CTCAGG- variables, such as age, gender, and pathologic tumor-node-metastasis stage, GAAAGGAGAATAAAAGAAC-3¶ (CDCA8); 5¶-CCCATCTGCACTTGTCCT- with the expression levels of CDCA8 and AURKB protein determined by CAT-3¶ and 5¶-AACAGATAAGGGAACAGTTAGGGA-3¶ (AURKB); 5¶- tissue microarray analysis. Tumor-specific survival curves were calculated GGAGTCTGTGTGGTGTGTATGTG-3¶ and 5¶-GAGGGAACAGAGCTGTTAG- from the date of surgery to the time of death related to NSCLC or to the last GAAG-3¶ (E2F1); and 5¶-GAGGTGATAGCATTGCTTTCG-3¶ and 5¶-CAAGT- follow-upobservation. Kaplan-Meier curves were calculated for each CAGTGTACAGGTAAGC-3¶ (ACTB). PCRs were optimized for the number relevant variable and for CDCA8 or AURKB expression; differences in of cycles to ensure product intensity within the logarithmic phase of survival times among patient subgroups were analyzed using the log-rank amplification. test. Univariate analysis was done with the Cox proportional hazard Northern blot analysis. Human multiple-tissue blots containing 23 regression model to determine associations between clinicopathologic tissues (BD Biosciences Clontech) were hybridized with a 32P-labeled PCR variables and cancer-related mortality. product of CDCA8. The cDNA probe of CDCA8 was prepared by RT-PCR RNAi assay. We had established previously a vector-based RNAi system, using the primers described above. Prehybridization, hybridization, and psiH1BX3.0, which was designed to synthesize small interfering RNAs washing were done according to the supplier’s recommendations. The blots (siRNA) in mammalian cells (13, 15, 16, 18–24). Ten micrograms of siRNA were autoradiographed at room temperature for 30 h with intensifying BAS expression vector were transfected using 30 AL LipofectAMINE 2000 screens (Bio-Rad Laboratories). (Invitrogen) into lung cancer cell lines, LC319 and SBC-5. The transfected Antibodies. To obtain anti-CDCA8 antibody, we prepared plasmids cells were cultured for 7 days in the presence of appropriate concentrations expressing full-length of CDCA8 that contained His-tagged epitopes at their of geneticin (G418), and the number of colonies was counted by Giemsa

NH2 termini using pET28 vector (Novagen). The recombinant proteins were staining, and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)- expressed in Escherichia coli, BL21 codon-plus strain (Stratagene), and 2,5-diphenyltetrazolium bromide (MTT) assay (cell counting kit-8 solution; purified using TALON resin (BD Biosciences Clontech) according to the Dojindo), at 7 days after the G418 treatment. To confirm suppression of supplier’s protocol. The protein, extracted on a SDS-PAGE gel, was CDCA8 protein expression, Western blotting was carried out with affinity- inoculated into rabbits; the immune sera were purified on affinity columns purified polyclonal antibody to CDCA8 according to the standard protocol. according to standard methodology. Affinity-purified rabbit polyclonal anti- The target sequences of the synthetic oligonucleotides for RNAi were as CDCA8 antibodies were used for Western blotting and immunostaining. follows: control 1 (enhanced green fluorescent protein (EGFP) gene, a mutant A rabbit polyclonal anti-AURKB antibody was purchased from Abcam, Inc. of Aequorea victoria GFP), 5¶-GAAGCAGCACGACTTCTTC-3¶; control 2 On Western blots, we confirmed that the antibody was specific to CDCA8 or (Luciferase: Photinus pyralis luciferase gene), 5¶-CGTACGCGGAATACT- AURKB, using lysates from NSCLC cell lines that either expressed CDCA8 TCGA-3¶;si-CDCA8-#1,5¶-CAGCAGAAGCTATTCAGAC-3¶; and si-CDCA8- and AURKB endogenously or not or from cells transfected with CDCA8 or #2,5¶-GCCGTGCTAACACTGTTAC-3¶. AURKB expression vector. Luciferase assay. Human genomic DNA was extracted from LC319 cells Western blot analysis. Cells were lysed in lysis buffer [50 mmol/L Tris- and used as templates for PCR. 5¶ Flanking region of the human CDCA8 or HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, 0.1% AURKB gene was amplified by PCR with the following synthesized 5¶ region

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Figure 1. Activation of CDCA8 and AURKB in lung tumor samples. A, expression of CDCA8 and AURKB in clinical samples of 14 NSCLC (T) and corresponding normal lung tissues (N), examined by semiquantitative RT-PCR. We prepared appropriate dilutions of each single-stranded cDNA prepared from mRNAs of clinical lung cancer samples, taking the level of h-actin (ACTB) expression as a quantitative control. B, expression of CDCA8 and AURKB proteins in 11 lung cancer cell lines, examined by Western blot analysis. C, subcellular localization of endogenous CDCA8 (top, red) and endogenous AURKB (bottom, red) in LC319 cells, detected by rabbit polyclonal antibodies to CDCA8 or AURKB. Both were triple-stained with a-tubulin (TUBA; green) and 4¶,6-diamidino-2-phenylindole (see merged images).

of CDCA8-specific primers (5¶-CGGGGTACCCCGACAAGGCCTGCCGG- Recombinant CDCA8 proteins. We prepared 13 plasmids expressing GAGTAGT-3¶ and 5¶-CCCAAGCTTGGGCGAATCTGTGCAGCTCGTGTC-3¶) WT CDCA8 or various deletion mutant CDCA8 proteins (CDCA8D1–D12), and 5¶ region of AURKB-specific primers (5¶-AACGTAGGCATGTA- each of which was mutated at serine/threonine to alanine and contained ¶ ¶ ¶ GAGGCTC-3 and 5 -CGGGGAAGAAAGTGCTTAAAGGA-3 ). The fragments His-tagged epitopes at their NH2 termini using pET28 vector. The of promoter region were excised with KpnI and HindIII restriction enzymes recombinant proteins were expressed in E. coli, BL21 codon-plus strain, and inserted into the corresponding enzyme sites of pGL3-Basic vector. The and purified using TALON resin according to the supplier’s protocol. entire coding sequence of E2F1 was cloned into the appropriate site of In vitro kinase assay. Purified recombinant WT or mutated CDCA8 pcDNA3.1/myc-His plasmid vector (Invitrogen) to achieve pcDNA3.1-E2F1. proteins were incubated with recombinant AURKB (rhAURKB; Upstate) and 32 The plasmids containing the 5¶ flanking region of the CDCA8 gene and [g- P]ATP in kinase buffer [20 mmol/L Tris (pH 7.5), 10 mmol/L MgCl2, phRL-SV40 (Promega) were cotransfected into LC319 cells with pcDNA3.1- 2 mmol/L MnCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L E2F1 or mock vector using LipofectAMINE Plus (Invitrogen). Firely DTT] supplemented with a mixture of protease inhibitors, 10 mmol/L NaF, luciferase activity values were normalized by comparing Firely luciferase 5 nmol/L microcystin LR, and 50 Amol/L ATP. The reaction was terminated activity with Renilla luciferase activity, expressed from phRL-SV40 to allow by the addition of a 0.2 volume of 5Â protein sample buffer and the proteins variation in transfection efficiency. were analyzed by SDS-PAGE. www.aacrjournals.org 4115 Cancer Res 2007; 67: (9). May 1, 2007

Inhibition of AURKB activity. To inhibit the AURKB activity in of the peptides with the cell lines at 37jC. The 11R-CDCA8261–280 as well as mammalian cells, we constructed siRNA oligonucleotides against AURKB its control peptides was transduced into almost all of cultured cells as (si-AURKB-#1 and si-AURKB-#2), as well as control siRNA oligonucleotides reported elsewhere (38). for EGFP. Cells seeded onto 10-cm dishes were incubated in mixtures of ¶ ¶ both LipofectAMINE and control (5 -GAAGCAGCACGACUUCUUC-3 )or Results si-AURKBs(si-AURKB-#1,5¶-CCAAACUGCUCAGGCAUAA-3¶;si-AURKB-#2, 5¶-ACGCGGCACUUCACAAUUG-3¶) in a final concentration of 100 nmol/L. Coactivation of CDCA8 and AURKB in lung cancers. Using a At 4 h after transfection, the siRNA-LipofectAMINE mixtures were replaced cDNA microarray representing 27,648 genes of 101 lung cancer with fresh medium. The cells were collected and analyzed after additional tissues, we identified CDCA8 to be overexpressed in a large 72-h culture. proportion of lung cancers. Subsequently, we confirmed its Synthesized cell-permeable peptide. Nineteen- or 20-amino acid transactivation in 11 of 14 additional NSCLC cases (four of seven peptide sequences corresponding to a part of CDCA8 protein that adenocarcinomas; all of seven SCCs) by semiquantitative RT-PCR contained possible phosphorylation sites by AURKB were covalently experiments (Fig. 1A, top). We further confirmed high levels of linked at its NH terminus to a membrane transducing 11 poly-arginine 2 endogenous CDCA8 expression in all of 11 lung cancer cell lines by sequence (11R; refs. 21, 38). Three cell-permeable peptides were synthesized: Western blot analysis using a rabbit polyclonal anti-CDCA8 11R-CDCA8147–165, RRRRRRRRRRR-GGG-PSKKRTQSIQGKGKGKRSS; 11R- antibody (Fig. 1B, top). Northern blot analysis using CDCA8 cDNA CDCA8209–228, RRRRRRRRRRR-GGG-ERIYNISGNGSPLADSKEIF; and 11R- CDCA8261–280, RRRRRRRRRRR-GGG-NIKKLSNRLAQICSSIRTHK. Scramble as a probe identified a 2.5-kb transcript, exclusively in the testis peptides derived from the most effective 11R-CDCA8261–280 peptides were among 23 human tissues examined (Supplementary Fig. S1A). synthesized as a control: Scramble, RRRRRRRRRRR-GGG-TSAQIRHKC- To determine the subcellular localization of endogenous CDCA8 SIKLSNIKNRL. Peptides were purified by preparative reverse-phase high- in lung cancer cells, we did immunocytochemical analysis for pressure liquid chromatography. LC319 cells using anti-CDCA8 antibody; CDCA8 proteins were LC319 and SBC-5 cells, as well as human lung-derived MRC-5, CCD19-Lu, mainly detected at nucleus in cells at the G1-S phase and at nucleus and BEAS-2B cells, were incubated with the 11R peptides at the and contractile ring in cells at the G -M phase (Fig. 1C, top). CDCA8 concentration of 2.5, 5, and 7.5 Amol/L for 7 days. The medium was 2 was isolated previously as a new member of a vertebrate 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 the chromosomal passenger complex (25), and phosphorylation of treatment. To confirm the transduction efficiency of the peptides, we COOH-terminal region of glutathione S-transferase-tagged CDCA8 synthesized the 11R-CDCA8261–280 and its control peptides labeled with by rhAURKB was shown by in vitro kinase assay (26). However, FITC at NH2 terminus. Transduction of the peptides (2.5–7.5 Amol/L) was the precise phosphorylation site(s) and its functional significance monitored by fluorescence microscopic observation after a 3-h incubation in cancer cells remain unclear. To elucidate the biological role of

Figure 2. Association of CDCA8 and AURKB overexpression with poor clinical outcomes in NSCLC. A, immunohistochemical evaluation of representative samples from surgically resected SCC tissues, using anti-CDCA8 (top) and anti-AURKB (bottom) polyclonal antibodies on tissue microarrays. Magnification, Â100. B, Kaplan-Meier analysis of tumor-specific survival times according to expression of CDCA8 (left) and AURKB (right) on tissue microarrays.

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Figure 3. Inhibition of growth of lung cancer cells by siRNAs against CDCA8. Left, top, gene knockdown effect on CDCA8 protein expression in LC319 cells by two si-CDCA8s (si-CDCA8-#1 and si-CDCA8-#2) and two control siRNAs (si-EGFP and si-Luciferase), analyzed by Western blotting. Left, bottom and right, colony formation and MTT assays of LC319 cells transfected with si-CDCA8s or control plasmids. Columns, relative absorbance of triplicate assays; bars, SD.

CDCA8 activation in lung cancer cells, we first examined the (P < 0.0001, m2 test) as similar to the results by RT-PCR and expression status of AURKB using our gene expression database Western blotting. We found that strong expression (scored as 2+) of (8–12) and found that AURKB was frequently overexpressed in lung CDCA8 in NSCLCs was significantly associated with tumor size m2 cancers compared with normal lung cells. Furthermore, our data (pT1 versus pT2-4; P = 0.0414, test), lymph node metastasis (pN0 2 indicated that the levels of AURKB expression seemed to be versus pN1-2; P = 0.0005, m test), and with shorter tumor-specific correlated with those of CDCA8. We subsequently reexamined 5-year survival times (P = 0.0009, log-rank test; Fig. 2B, left). Strong primary lung cancer tissues and found increase of AURKB expression (scored as 2+) of AURKB in NSCLCs was significantly 2 expression in 12 of 16 NSCLC clinical samples (five of seven associated with tumor size (pT1 versus pT2-4; P = 0.0361, m test), 2 adenocarcinomas and all of seven SCCs) by semiquantitative RT- lymph node metastasis (pN0 versus pN1-2; P = 0.0004, m test), and PCR experiments (Fig. 1A, middle). The expression patterns of 5-year survival (P = 0.0001, log-rank test; Fig. 2B, right). NSCLC AURKB in lung cancers were very similar to those of CDCA8.We patients without either CDCA8 or AURKB expression in their further confirmed by Western blot analysis that CDCA8 and tumors could reveal the longest survival period, whereas those with AURKB proteins were coactivated in almost all of the lung cancer strong positive staining for both markers showed the shortest cell lines examined (Fig. 1B, middle). Immunocytochemical analysis tumor-specific survival (P < 0.0001, log-rank test; Supplementary using a rabbit polyclonal anti-AURKB antibody detected AURKB Fig. S1B). Using univariate analysis, we found that lymph node proteins to be located mainly at nucleus in cells at the G1-S phase metastasis (pN0 versus pN1 and N2; P < 0.0001, score test), tumor and at nucleus and contractile ring in cells at the G2-M phase; size (pT1 versus pT2,T3, and T4; P < 0.0001, score test), and high the subcellular localization of AURKB in lung cancer cells was CDCA8/AURKB expression (P = 0.0009 and = 0.0001, respectively, very similar to that of CDCA8 as reported elsewhere (Fig. 1C, score test) were important correlative features for poor prognoses bottom; ref. 25). of patients with NSCLC. Association of CDCA8 and AURKB positivity with poor Growth inhibition of lung cancer cells by specific siRNA prognosis of NSCLC patients. Using tissue microarrays prepared against CDCA8. To assess whether CDCA8 is essential for growth from 273 surgically resected NSCLCs, we did immunohistochemical or survival of lung cancer cells, we constructed plasmids to express analysis with affinity-purified anti-CDCA8 and anti-AURKB poly- siRNA against CDCA8 (si-CDCA8-#1 and si-CDCA8-#2), using clonal antibodies. We classified patterns of CDCA8/AURKB siRNAs for EGFP and Luciferase as controls. Transfection of si- expression as absent (scored as 0), weak (scored as 1+), or strong CDCA8-#1 or si-CDCA8-#2 into LC319 or SBC-5 cells significantly (scored as 2+). Of the 273 NSCLC cases examined, 113 (41%) cases suppressed expression of endogenous CDCA8 proteins in compa- revealed strong CDCA8 expression and 100 (37%) cases showed rison with the two controls and resulted in significant decreases in weak expression, although its expression was absent in 60 (22%) cell viability and colony numbers measured by MTT and colony cases. For AURKB, strong expression was observed in 116 (42%) formation assays (representative data of LC319 were shown in Fig. 3). cases, weak expression in 94 (34%) cases, and no expression in 63 Simultaneous activation of CDCA8 and AURKB regulated by (24%) cases. No staining was observed for either CDCA8 or AURKB E2F1. The concordant activation of CDCA8 and AURKB in lung in any of their adjacent normal lung tissues (Fig. 2A). Tumors (183 cancers suggested that these two genes might be regulated by of the 273) were positive (scored as 1+ to 2+) for both CDCA8 and the same transcription factor(s). To validate this hypothesis, we AURKB, and 33 were negative for the both proteins. Cases (30 of examined the DNA sequences of the CDCA8 and AURKB promoter the 273) were positive for only CDCA8 and 27 were positive for only regions and found the cell cycle–dependent element (CDE) and AURKB. The expression pattern of CDCA8 protein was significantly cell cycle gene homology region (CHR) consensus sequences concordant with AURKB protein expression in these tumors (CDE-CHR), by which transcription of AURKB as well as cyclin A www.aacrjournals.org 4117 Cancer Res 2007; 67: (9). May 1, 2007

(CCNDA) and CDC25 is regulated (Supplementary Fig. S2A). in which all of these four serine/threonines were substituted to an Among the transcription factors that could bind to the CDE-CHR alanine, and did in vitro kinase assay using AURKB. The result of the AURKB gene, we confirmed that E2F1 was coactivated with showing complete disappearance of CDCA8 phosphorylation by CDCA8 and AURKB as detected by semiquantitative RT-PCR AURKB (Fig. 5C) clearly showed that CDCA8 was phosphorylated analysis of NSCLC cases (Supplementary Fig. S2B). To investigate the direct transcriptional regulation of the CDCA8 gene promoter by E2F1, we transiently cotransfected LC319 cells with E2F1 or mock vector, along with CDCA8 or AURKB (positive control) promoter constructs containing putative regulatory elements (CDE-CHR) fused to a luciferase reporter gene. Expectedly, both CDCA8 and AURKB promoter functions were activated by E2F1 (Supplementary Fig. S2C). The induction of endogenous CDCA8 and AURKB was further confirmed by introduction of exogenous E2F1 into LC319 cells (data not shown). Phosphorylation of CDCA8 by AURKB in lung cancer cells. Western blot analysis detected two different sizes of CDCA8 protein (Fig. 1B, top). To examine a possibility, the CDCA8 phosphorylation, we incubated extracts from LC319 cells in the presence or absence of protein phosphatase (Bio-Rad Laboratories) and analyzed the molecular weight of CDCA8 protein by Western blot analysis. Because the measured weight of the majority of CDCA8 protein in the extracts treated with phosphatase was smaller than that in the untreated cells (Fig. 4A), we considered that CDCA8 was phosphorylated in lung cancer cells. We then examined whether AURKB could phosphorylate CDCA8 in vitro as reported previously (26). When full-length recombinant CDCA8 (rhCDCA8) protein was incubated with rhAURKB protein in kinase buffer, including [g-32P]ATP, CDCA8 was phosphorylated in an AURKB dose-dependent manner (Fig. 4B). To assess whether the abundant expression of endogenous AURKB is critical for phosphorylation of endogenous CDCA8 in cancer cells, we selectively knocked down with siRNA against AURKB (si-AURKB) the AURKB expression in LC319 cells, in which these two genes were expressed abundantly. Reduction of AURKB protein by si- AURKBs(si-AURKB-#1 and si-AURKB-#2) dramatically decreased the amount of CDCA8 protein, whereas a level of CDCA8 transcripts in the same cells was not influenced by si-AURKBs(Fig.4C). Hence, we hypothesized that endogenous CDCA8 protein may be stabilized when it is phosphorylated by endogenous AURKB and/or incorporated in some protein complex that are possibly regulated by the AURKB signaling. Because our data imply that human CDCA8 and AURKB were coactivated in lung cancer cells and that CDCA8 phosphorylation by AURKB might play a significant role in pulmonary carcinogenesis, we then investigated the phosphorylation sites of CDCA8 by AURKB. We prepared six His-tagged CDCA8 proteins (CDCA8D1– D6), in which two or three serine/threonine residues were substituted to alanines (Fig. 5A, top). We did in vitro kinase assay using these mutant CDCA8 proteins and detected a reduction of phosphorylation levels in three mutated constructs (CDCA8D2, CDCA8D5, and CDCA8D6) compared with a WT, suggesting that six serine/threonines corresponding to the substituted sites could be putative phosphorylation ones (Fig. 5B, top). We further prepared six additional mutant CDCA8 constructs (CDCA8D7– D12), in which either of the six serine/threonines was substituted Figure 4. Phosphorylation of CDCA8 by AURKB. A, dephosphorylation of to an alanine (Fig. 5A, bottom). In vitro kinase assay of these six endogenous CDCA8 protein in LC319 cells by treatment with E-phosphatase. White arrowhead, phosphorylated CDCA8; black arrowheads, mutants revealed the reduction of phosphorylation levels in four nonphosphorylated form. B, in vitro phosphorylation of rhCDCA8 by rhAURKB. mutated constructs, including a substitution at either of four C, top, the expression levels of endogenous AURKB and CDCA8 proteins, serine/threonine residues, Ser154,Ser219,Ser275, and Thr278 detected by Western blot analysis in LC319 cells transfected with siRNA oligonucleotides against AURKB (si-AURKBs); bottom, the expression levels of (CDCA8D8, CDCA8D9, CDCA8D11, and CDCA8D12; Fig. 5B, endogenous AURKB and CDCA8 transcripts, detected by semiquantitative bottom). We subsequently made a mutant construct (CDCA8D13), RT-PCR analysis in LC319 cells transfected with si-AURKBs.

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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 mutants that 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 CDCA8 proteins with rhAURKB. CDCA8D2, CDCA8D5, and CDCA8D6 constructs resulted in a reduction of phosphorylation levels by AURKB (each of substituted residue was indicated as bold character on underline), whereas CDCA8D1, CDCA8D3, and CDCA8D4 represented the same levels of phosphorylation compared with WT CDCA8. Bottom, CDCA8D8, CDCA8D9, CDCA8D11, and CDCA8D12 resulted in a reduction of phosphorylation, whereas CDCA8D7 and CDCA8D10 showed the same levels of phosphorylation compared with WT, indicating that CDCA8 was phosphorylated at Ser154, Ser219, Ser275, and Thr278 (indicated as bold character with underline) 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 an alanine, with rhAURKB. CDCA8D13 construct resulted in complete diminishment of CDCA8 phosphorylation by AURKB.

154 219 275 278 278 at four serine/threonine residues at Ser , Ser , Ser , and Thr Thr ). These peptides were covalently linked at its NH2 terminus by AURKB. to a membrane transducing 11 arginine-residues (11R). We first Growth inhibition of lung cancer cells by cell-permeable investigated by in vitro kinase assay the effect of the three 11R- peptides. To investigate the functional significance of interaction CDCA8 peptides on the phosphorylation of rhCDCA8 by between CDCA8 and AURKB as well as CDCA8 phosphorylation rhAURKB. When full-length rhCDCA8 protein was incubated with for growth or survival of lung cancer cells, we developed bioactive rhAURKB protein with either of the three 11R-CDCA8 peptides or cell-permeable peptides that were expected to inhibit the in vivo their scramble peptides in kinase buffer, including [g-32P]ATP, the phosphorylation of CDCA8 by AURKB. We synthesized three phosphorylation level of rhCDCA8 was significantly suppressed by 275 different peptides of 19- or 20-amino acid sequence that included the treatment with 11R-CDCA8261–280 peptides containing Ser the four CDCA8 phosphorylation sites (Ser154, Ser219, Ser275, and and Thr278 compared with its scramble peptides (Fig. 6A). www.aacrjournals.org 4119 Cancer Res 2007; 67: (9). May 1, 2007

Figure 6. Inhibition of growth of lung cancer cells by cell-permeable CDCA8 peptides. A, reduction of the AURKB-dependent CDCA8 phosphorylation by cell-permeable CDCA8 peptides (11R-CDCA8261–280), detected by in vitro kinase assay. B, top, the expression levels of endogenous CDCA8 protein, detected by Western blot analysis of LC319 cells transfected with the 11R-CDCA8261–280; bottom, the expression levels of endogenous CDCA8 transcript, detected by semiquantitative RT-PCR analysis of LC319 cells transfected with the 11R-CDCA8261–280. C, top, MTT assay of LC319 cells, detecting a growth-suppressive effect of 11R-CDCA8261–280. Columns, relative absorbance of triplicate assays; bars, SD. Bottom, cell cycle analysis of LC319 cells after the treatment with 11R-CDCA8261–280 peptides or Scramble peptides. D, left, expression of CDCA8 protein in normal human lung fibroblast-derived MRC-5 and CCD19-Lu cells compared with lung cancer 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-5 cells that scarcely expressed CDCA8 and AURKB protein.

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Addition of the 11R-CDCA8261–280 into the culture medium of CDCA8 protein. Phosphorylation is an important posttranslational LC319 cells inhibited the phosphorylation and decreased stability modification that regulates the protein stability, function, localiza- of endogenous CDCA8 protein, whereas no effect on a level of tion, and binding specificity to target proteins. For example, MKP-7 446 10 CDCA8 transcript was observed (Fig. 6B). The 11R-CDCA8261–280 phosphorylated at Ser or p27 phosphorylated at Ser has a treatment of LC319 cells resulted in significant decreases in cell longer half-life than unphosphorylated form; when at the sites were viability as measured by MTT assay (representative data in Fig. 6C, dephosphorylated, the amount of these proteins was promptly top). To clarify the mechanism of tumor suppression by the 11R- decreased in cells (44, 45). The evidence suggests that the stability CDCA8261–280 peptide, we did flow cytometric analysis of the of CDCA8 protein could be tightly regulated by the AURKB tumor cells treated with these peptides and found the significant signaling in cancel cells. AURKB was shown to be overexpressed in increase in sub-G1 fraction at 48 h after the treatment of 11R- many tumor cell lines, and its overexpression was noted to be CDCA8261–280 (Fig. 6C, bottom). 11R-CDCA8261–280 revealed no involved in chromosome number instability and tumor invasive- significant effect on cell viability of human lung fibroblast-derived ness (30, 31). AURKB is one of the cancer-related kinases and MRC-5 or CCD19-Lu or human bronchial epithelia-derived BEAS- therefore was thought to be a promising target for anticancer drug 2B cells, in which CDCA8 and AURKB expression were hardly development. Indeed, two AURKB inhibitors have been described detectable (representative data of MRC-5 and BEAS-2B cells were recently: ZM447439 and Hesperadin (46, 47). Our study has shown in Fig. 6D; Supplementary Fig. S3). The data indicate that indicated that CDCA8 is a putative oncogene that is aberrantly 11R-CDCA8261–280 could specifically inhibit an enzymatic reaction expressed in lung cancer cells along with AURKB. We found by of AURKB for CDCA8 phosphorylation and have no or minimum tissue microarray analysis that CDCA8 and AURKB were cooverex- toxic effect on normal human cells that do not abundantly express pressed and that patients with NSCLC showing higher expression these proteins. of these proteins represented a shorter tumor-specific survival period, doubtlessly suggesting that CDCA8, along with AURKB, Discussion plays a crucial role for progression of lung cancers. Furthermore, we showed at the first time that growth of lung cancer cells Toward the goal of developing novel therapeutic anticancer overexpressing CDCA8 and AURKB could be suppressed effectively drugs with a minimum risk of adverse reactions, we established a by blocking the AURKB-dependent CDCA8 phosphorylation by powerful screening system to identify proteins and their interacting means of the 20-amino acid cell-permeable peptide that corre- proteins that were activated specifically in lung cancer cells. The sponded to a part of CDCA8 protein and included two strategy was as follows: (a) identification of up-regulated genes in phosphorylation sites, Ser275 and Thr278, by AURKB. We detected 101 lung cancer samples through the genome-wide cDNA micro- a significant increase in the sub-G fraction after the treatment array system coupled with laser microdissection (8–12); (b) 1 of 11R-CDCA8 peptide, suggesting that the cell-permeable verification of very low or absent expression of such genes in 261–280 polypeptides induced apoptosis of the cancer cells. Because the normal organs by cDNA microarray analysis and multiple-tissue phosphorylation of CDCA8 at these sites was likely to be Northern blot analysis (39, 40); (c) confirmation of the clinico- indispensable for the growth/survival of lung cancer cells and pathologic significance of their overexpression using tissue micro- CDCA8 could belong to cancer-testis antigens, selective targeting array consisting of hundreds of NSCLC tissue samples (12, 14–24); of CDCA8-AURKB enzymatic activity could be a promising and (d) verification of the targeted genes whether they are essential therapeutic strategy that is expected to have a powerful biological for the survival or growth of lung cancer cells by siRNA (13, 15, 16, activity against cancer with a minimal risk of adverse events. 18–24). By this systematic approach, we identified that CDCA8 and Further analyses of the mechanism of growth suppression by AURKB are cooverexpressed in the great majority of clinical lung specific inhibiting of CDCA8 phosphorylation by AURKB may be of cancer samples as well as lung cancer cell lines and that these two the great benefit toward the development of new types of proteins are indispensable for growth and progression of lung anticancer agents. cancer cells. In summary, we have found that CDCA8 is coactivated with and CDCA8 was shown to be phosphorylated in vitro by AURKB phosphorylated/stabilized by AURKB in lung cancer cells and that previously (26), but its significance in development and/or phosphorylated CDCA8 plays a significant role in growth and/or progression of human cancer had been never described. CDCA8 survival of cancer cells. The data strongly imply the possibility of was indicated recently to be one of new components of the designing new anticancer drugs targeting the CDCA8-AURKB vertebrate chromosomal passengers, such as AURKB, INCENP, and association as a promising therapeutic strategy for lung cancer. BIRC5 (25), which are considered to be key regulators of mitotic events responsible for correcting the error of bipolar attachments that inevitably occur during the ‘search-and-capture’ mechanism Acknowledgments (41–43). In this study, we have shown that the CDCA8 protein is Received 12/21/2006; revised 2/13/2007; accepted 2/20/2007. likely to be stabilized by its AURKB-dependent phosphorylation at Grant support: The Japan Society for the Promotion of Science ‘‘Research for the Ser154, Ser219, Ser275, and/or Thr278. Depletion of AURKB function Future’’ Program grant 00L01402 (Y. Nakamura). The costs of publication of this article were defrayed in part by the payment of page by RNAi or the 11R-CDCA8261–280 that could inhibit phosphoryla- charges. This article must therefore be hereby marked advertisement in accordance tion of CDCA8 significantly decreased the level of endogenous with 18 U.S.C. Section 1734 solely to indicate this fact.

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