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Molecular and Cellular Pathobiology

CIP2A Modulates Cell-Cycle Progression in Human CancerCells by Regulating the Stability and Activity of Plk1

Jae-Sung Kim1, Eun Ju Kim2, Jeong Su Oh3, In-Chul Park1, and Sang-Gu Hwang1

AbstractAbnormal cell-cycle control can lead to aberrant cell proliferation and cancer. The oncoprotein cancerous

inhibitor of protein phosphatase 2A (CIP2A) is an inhibitor of protein phosphatase 2A (PP2A) that stabilizesc-Myc.However, the precise role of CIP2A in cell division is not understood.Herein,we show thatCIP2A is requiredfor mitotic progression by regulating the polo-like kinase (Plk1). With mitotic entry, CIP2A translocated from thecytoplasm to the nucleus, where it was enriched at spindle poles. CIP2A depletion delayed mitotic progression,resulting in mitotic abnormalities independent of PP2A activity. Unexpectedly, CIP2A interacted directly withthe polo-box domain of Plk1 during mitosis. This interaction was required to maintain Plk1 stability by blockingAPC/C-Cdh1–dependent proteolysis, thereby enhancing the kinase activity of Plk1 during mitosis. We observedstrong correlation and in vivo interactions between these two proteins in multiple human cancer specimens.Overall, our results established a novel function for CIP2A in facilitating the stability and activity of the pivotalmitotic kinase Plk1 in cell-cycle progression and tumor development. Cancer Res; 73(22); 6667–78. �2013 AACR.

IntroductionProper cell-cycle progression involves coordination of mul-

tiple events, such as chromosome condensation, spindle for-mation, chromosome segregation, and cytokinesis, and istightly controlled by posttranslational modifications such asphosphorylation and ubiquitination (1, 2). For example, mitot-ic kinases, including cyclin-dependent kinase 1, polo-likekinases (Plk), and Aurora kinases, phosphorylate their sub-strates during mitotic progression. In contrast, components ofthe ubiquitin–proteasome system, such as the anaphase-pro-moting complex/cyclosome (APC/C), in turn directs theordered destruction of critical mitotic substrates (i.e., spindleassembly checkpoint proteins) andmitotic kinases (1, 2). Thus,the concerted effort of mitotic kinases and the APC/C isrequired for fine tuning of cell-cycle progression.Plk1 plays a crucial role in multiple steps of mitosis, includ-

ing the G2–M transition, centrosome maturation, bipolarspindle formation, chromosome segregation, and cytokinesis(3, 4). Plk1 contains an N-terminal kinase domain and aC-terminal polo-box domain (PBD) that have been implicatedin regulating kinase activity and subcellular localization (3, 4).

Plk1 activity begins to increase during G2-phase and peaks inmitosis. This temporal control is tightly regulated by phos-phorylation and ubiquitin-dependent proteolysis (4). In thelate G2-phase, Plk1 is activated by phosphorylation of Thr210 inthe T-loop by Aurora A kinase/bora (4). In contrast, down-regulation of Plk1 is mediated by the ubiquitin–proteasomesystem, involving APC/C-Cdh1 or -Cdc20, during the late MandG1 phases (2, 4). Consistent with the diverse roles of Plk1 inmitosis, inhibition of Plk1 leads to multiple mitotic defects,including aberrant spindle formation, misaligned chromo-somes, and improper chromosome condensation (5–7). Inaddition, Plk1 is highly upregulated in tumors and inhibitionof its activity induces apoptosis in cancer cells, but not innormal cells (4, 8). Thus, Plk1 is a promising drug target incancer therapy (8). However, the regulatory mechanisms oftumor-associated Plk1 are poorly understood.

Cancerous inhibitor of protein phosphatase 2A (PP2A;CIP2A), also namedKIAA1524 or p90 tumor-associated antigen,is a novel oncogene that is known to inhibit c-Myc–associatedPP2A activity and thereby stabilize the oncogenic c-Mycin human malignancies (9). Downregulation of CIP2A reducesmalignant cellular growth and in vivo tumor formation (9–11).In addition, overexpression of CIP2A has been found in severalcommon cancers and is associated with poor prognosis(9–14). Furthermore, recent reports have suggested that CIP2Ais a potential target for anticancer drugs in several cancers(11, 15–18). Although the oncogenic role of CIP2A in humanmalignancies has been suggested, the mechanisms throughwhich it exerts its oncogenic properties are still unclear.

In this study, we investigated the novel functions of CIP2A incell division.We showed that CIP2A bindswith Plk1 to regulatePlk1 stability and activity during mitosis. Furthermore, weprovided evidence of the clinical relevance of this regulatorymechanism in human cancers.

Authors' Affiliations: Divisions of 1Radiation Cancer Research and 2Radi-ation Effect, Korea Institute of Radiological and Medical Sciences, Seoul;and 3Department of Genetic Engineering, Sungkyunkwan University,Suwon, South Korea

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

Corresponding Author: Jae-Sung Kim, Division of Radiation CancerResearch, Korea Institute of Radiological and Medical Sciences, 215-4Gongneung-Dong, Nowon-Ku, Seoul 139-706, South Korea. Phone: 82-2-970-1669; Fax: 82-2-970-2417; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-0888

�2013 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst August 27, 2013; DOI: 10.1158/0008-5472.CAN-13-0888

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Materials and MethodsCell culture, synchronization, and treatment

HeLa, H1299, H460, Hs68, 293T, and 293 cells (AmericanType Culture Collection, ATCC) were cultured in Dulbecco'sModified Eagle Medium (Gibco-BRL) supplemented with 10%FBS and penicillin/streptomycin. Fluorescent ubiquitination-based cell-cycle indicator (FUCCI)-expressing HeLa cells wereobtained from the RIKEN Cell Bank. Cells were preserved andpassaged according to the ATCC protocol for no longer than2 months and tested monthly for Mycoplasma infection byHoechst 33258 staining. Cells were synchronized at G1–S orG2–M phase by a double thymidine block/release or nocoda-zole block protocol (19). Briefly, cells were treated with 2mmol/L thymidine (Sigma) for 16 hours. After releasing cellsfor 2 hours, cells were transfected with siRNAs. Eight hoursafter release from the first thymidine block, thymidine was re-added for an additional 14 hours. Cells were released from thesecond thymidine block into medium with or without 100 ng/mL nocodazole (Sigma), dependent on the experimentaldesign. To measure endogenous Plk1 stability, cells wereincubated with 100 mg/mL cycloheximide (Sigma) for theindicated times. For proteosome inhibition, cells were incu-bated with 10 mmol/LMG132 (Sigma) for 3 hours. Okadaic acid(50 nmol/L; Sigma) or forskolin (40mmol/L; Sigma)was used toinhibit or activate PP2A activity, respectively.

Plasmid constructsHuman CIP2A cDNA purchased from Origene was PCR-

amplified and cloned into pEXPR-IBA105 (Strep fusion) andpcDNA3.1-Myc (Myc fusion) vectors. CIP2A.1 siRNA–resistantcDNA, generated by inserting four silentmutations, was clonedinto the pcDNA3.1-Myc vector. The following mutagenizingoligonucleotides were used: 50-tcaccttgttggcccatagtagtttaactg-tcgtcgtcttcgcactttcaatattatcc-30 (sense), and 50-ggataatattgaa-agtgcgaagacgacgacagttaaactactatgggccaacaaggtgat-30 (anti-sense). PTEN, Flag-tagged CHFR, and Plk1 constructs (WT,FAA, N-terminus, and C-terminus) were kindly provided by Dr.J.K. Park (Korea Institute of Radiological andMedical Sciences,Seoul, South Korea), Dr. J. H. Seol (Seoul National University,Seoul, Korea), and Dr. H. S. Yim (Hanyang University, Seong-dong-gu, Korea), respectively.

RNA interferenceThe following sequences were used for RNA interference:

CIP2A.1, 50-cugugguuguguuugcacutt-30; CIP2A.2, 50-ggagug-guuugucggagca-30; CIP2A.3, 50-caaguguaccacucuuaua-30; Plk1,50-tgaagaagaucacccuccu-30; Cdh1, 50-ugagaagucucccagucag-30;Cdc20, 50-gaagaccugccguuacauu-30; PP2A-Aa, 50- gcaucaau-gugcugucauatt-30; PP2A-Ab, 50-cgacucaacaguauuaagatt-30; andMAD2, 50- aaagtggtgaggtcctggaaa-30. Nonsilencing siRNA (Bio-neer) was used as a negative control. Transfection of siRNAs(50 nmol/L CIP2A.1, CIP2A.2, Plk1, Cdc20, and MAD2; 100nmol/L CIP2A.3; and 20 nmol/L PP2A-Aa and PP2A-Ab) wasconducted using Lipofectamine 2000 (Invitrogen) according tothe manufacturer's protocol. For stable depletion of CIP2A,FUCCI–HeLa cells were transduced with control or CIP2Ashort hairpin RNA (shRNA) lentivirus (Santa Cruz Biotechnol-ogy, Inc.) according to the manufacturer's protocol.

AntibodiesThe following antibodies were used: mouse monoclonal

antibodies against CIP2A, cyclin B1 (GNS1), and p27 (SantaCruz Biotechnology, Inc.); Cdc20, Cdh1 (DH01), and Plk1(Abcam); Flag-tag and b-actin (Sigma); hemagglutinin (HA)-tag (Roche); phospho-MPM2 (Millipore); PP2Ac (BD Trans-duction Laboratories); and Strep-tag (Qiagen); rabbit mono-clonal antibodies against Plk1 (208G4; Cell Signaling Technol-ogy, Inc.) and phospho-Akt (Epitomics); rabbit polyclonalantibodies against c-Myc, phospho-Plk1 (Thr210), phospho-histone H3 (phospho-H3), PTEN, and Myc-tag (Cell SignalingTechnology, Inc.); pericentrin and MAD2 (Abcam); cyclin B1,cyclin A, and PP2A-Aa/b (Santa Cruz Biotechnology, Inc.); andCIP2A (Novus Biologicals).

ImmunofluorescenceCells grown on coverslips were fixed with 4% paraformal-

dehyde in PBS. The cells were permeabilized and blocked with0.2%Triton X-100 and 5% fetal calf serum in PBS. Thefixed cellswere consecutively incubated with primary antibodies againstphospho-H3 (Millipore; 1:500), cyclin B1 (Santa Cruz Biotech-nology, Inc.; 1:200), pericentrin (Abcam; 1:500), Plk1 (208G4;Cell Signaling Technology, Inc.; 1:200), or CIP2A (Santa CruzBiotechnology, Inc.; 1:200) and secondary antibodies such asanti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594(Molecular Probes). Slides were mounted in medium contain-ing 40,6-diamidino-2-phenylindole (DAPI), and images werethen obtained using a confocal laser-scanning microscope(LSM 710; Carl Zeiss, Inc.). All images were processed by usingZEN 2009 Light Edition (Carl Zeiss) and Adobe Photoshop CS4.

ImmunohistochemistryHuman tissue microarrays were purchased from two com-

mercially available tissue arrays [SuperBioChips (cat. numbers:CC, CCN, CD, CDN, CQ, CQN, CZA, and BB) and AccuMax (cat.numbers: A206III and A206V)]. Tissue arrays were able tocollect up to 142 lung cancer, 59 gastric cancer, 59 coloncancer, and 55 cervical cancer specimens, along with severalmatched normal tissues. Immunohistochemical staining wasconducted with anti-CIP2A rabbit polyclonal antibody (NovusBiologicals; 1:100), anti-Plk1 rabbit polyclonal antibody(Abcam; 1:100), or anti-phospho-Plk1 rabbit monoclonal anti-body (Abcam; 1:100). Immunostaining was detected by theavidin–biotin–peroxidase method according to the manufac-turer's instructions (Invitrogen). The staining scoring wasevaluated on a scale of 0–3. Staining intensity was scored asfollows: 0 (no visible staining), 1þ (faint staining), 2þ (mod-erate staining), and 3þ (strong staining; SupplementaryFig. S1A).

Immunoprecipitation and Strep pull-downImmunoprecipitation was conducted as previously

described (20). Briefly, cells were lysed by NP-40 lysis bufferand the lysates were then precipitated with negative controlmouse antibody (Santa Cruz Biotechnology, Inc.) or mousemonoclonal antibody against either CIP2A or Plk1 (Santa CruzBiotechnology, Inc.). Immune complexes were collected usingprotein G-Sepharose and washed three times and SDS sample

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buffer was then added. Strep-tagged CIP2A proteins werepulled down by using Strep-Tactin beads (IBA TAGnologies),washed five times, and eluted with desthiobiotin (IBA TAGnol-ogies) according to the manufacturer's protocol. The elutedsamples were size-fractionated by electrophoresis anddetected using immunoblotting.

Cell-cycle analysisHeLa cells released from double thymidine block/release or

nocodazole block were trypsinized, washed twice in PBS, andfixed with ice-cold 70% ethanol. Fixed cells were incubatedwith 50mg/mL propidium iodide and 100mg/mLRNase at 37�Cfor 30 minutes and then analyzed with a FACScan flowcytometer (Becton Dickson).

Live imaging of FUCCI-expressing cellsFUCCI-expressing HeLa cells (21) were transduced with

control or CIP2A shRNA lentivirus (Santa Cruz Biotechnology,Inc.) according to the manufacturer's protocol. Cells synchro-nized by a double thymidine block were released with freshmedium and time-lapse images were acquired at 30-minuteintervals using confocal and phase-contrast microscopy (CarlZeiss, Inc.).

In vivo ubiquitination assayCells transfected with HA-tagged ubiquitin and empty or

CIP2A-Myc vector were synchronized at the G1–S border by adouble thymidine block and then released. Eight hours afterrelease, the cells were treated withMG132 for 3 hours and thenanalyzed for ubiquitination in vivo, as previously described(22). Briefly, the cells were lysed by incubation with 2 volumesof TBS containing 2% SDS at 95�C for 10 minutes. After addingeight volumes of 1%TritonX-100 inTBS, lysateswere sonicatedfor 2 minutes and then precleared with protein G-Sepharose.The lysates were then immunoprecipitated with anti-Plk1antibody (Abcam) coupled to protein G-Sepharose. The beadswere washed with 0.5 mol/L LiCl in TBS, washed twice withTBS, and immunoblotted using anti-HA antibody.

Plk1 kinase assayRecombinant active His-Plk1 (produced in Sf-9 cells) was

purchased from R&D Systems. Strep-tagged CIP2A from 293cells expressing Strep-CIP2A was purified by the Strep-tagpurification method. Various concentration ratios of purifiedStrep-CIP2A protein (0.25, 0.5, or 1 mg) were subjected to invitro kinase assay with active His-Plk1 (0.1 mg). In vitro kinaseassays were conducted in kinase buffer (20mmol/L HEPES, pH7.5, 150 mmol/L KCl, 10 mmol/L MgCl2, 1 mmol/L EDTA, 2mmol/L dithiothreitol, 5 mmol/L NaF, and 0.2 mmol/L Na3VO4) supplemented with 50 mmol/L ATP and 2.5 mCi of [g-32P]ATP at 30�C for 30 minutes in the presence of 2 mg depho-sphorylated a-casein (Sigma). The reaction mixture wasanalyzed by SDS-PAGE and autoradiography. BI2536 (SelleckChemicals) was used as a positive control to inhibit Plk1activity.

In situ proximity ligation assayIn situ proximity ligation assay (PLA) was conducted as

previously described (23). Paraformaldehyde-fixed HeLa cells

were permeabilized with 0.2% Triton X-100, washed, andblocked with blocking solution (Olink Bioscience). Antigen-retrieved normal and cancer tissues (SuperBioChips) wereincubated with 3% hydrogen peroxide, washed, and blockedwith blocking solution. Mouse monoclonal anti-CIP2A anti-body (Santa Cruz Biotechnology, Inc.; 1:200) together withrabbit polyclonal anti–phospho-Plk1 (Thr210) antibody (CellSignaling Technology, Inc.; 1:100) or rabbit polyclonal anti-CIP2A antibody (Novus Biologicals; 1:200) togetherwithmousemonoclonal anti-Plk1 antibody (Abcam; 1:300) were used forthe proximity ligation reaction. The assay was conductedaccording to the manufacturer's protocol using the DuolinkDetection Kit with a pair of nucleotide-labeled secondaryantibodies (rabbit PLA probe MINUS and mouse PLA probePLUS; Olink Bioscience). Nuclei were stained with Hoechst,which was included in the PLA reagent kit. Amplified PLAsignals, represented as red fluorescent dots, were analyzedusing confocal microscopy and quantified using CellProfilersoftware (available at www.cellprofiler.org).

Statistical analysisThe correlation between CIP2A and Plk1 immunointensity

was analyzed using Spearman rank correlation test. The two-tailed Student t test was conducted to analyze statisticaldifferences between groups. P values of less than 0.05 wereconsidered statistically significant. Statistical analyses wereconducted using Excel and XLSTAT software.

ResultsRegulation of CIP2A during cell-cycle progression

To investigate the role of CIP2A in cell division, we firstexamined CIP2A protein expression during different phases ofthe cell cycle in HeLa cells synchronized by using a doublethymidine and thymidine–nocodazole block protocol (Fig. 1A).The expression of CIP2A increased during theG2–Mphase (Fig.1A, left) and decreased during the exit from mitosis (Fig. 1A,right). Moreover, most of the CIP2A protein expressed waslocalized in the cytoplasm during interphase, and, as cellspassed from the S-phase to theG2–Mphase, CIP2Awas initiallyconcentrated in the pericentrosomal region and then clearlylocalized at spindle poles during mitosis, as shown by immu-nofluorescence analysis (Fig. 1B). Notably, CIP2A was enrichedin the nucleus during entry into mitosis (Fig. 1B and C). Thesecell cycle–dependent expression and localization patterns ofCIP2A were also observed in two other cell lines (Supplemen-tary Fig. S2A and S2B).

To further understand the cell cycle–dependent regulationof CIP2A, we used cells that expressed high or very lowamounts of CIP2A, that is, H1299 lung cancer cells or Hs68normal fibroblasts, respectively (Supplementary Fig. S2C).Because it has been well established that PTEN induces G1

arrest in most types of cancer cells (24, 25) and that Plk1depletion causes G2–M arrest but not apoptosis in Hs68 cells(8), we overexpressed a WT PTEN construct in H1299 cells(PTEN null) or depleted Plk1 with siRNA in Hs68 cells. Ectopicoverexpression ofWT PTEN suppressed CIP2A expression andcaused G1 arrest in H1299 cells (Fig. 1D). On the other hand,CIP2A expression was obviously increased in Plk1-depleted

CIP2A Regulates Plk1 Stability and Activity during Mitosis

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and mitotic Hs68 cells (Fig. 1E and F). PTEN overexpression orPlk1 depletion did not affect cell viability (SupplementaryFig. S2D). Likewise, we also observed that irradiation, whichinduces G1 or G2–M arrest in a p53-dependent manner (26),triggered a reduction in CIP2A expression in H460 cells (p53WT), but not in H1299 cells (p53 null; Supplementary Fig. S2E).Taken together, these results indicated that the expressionand localization of CIP2A were associated with cell-cycleprogression.

CIP2Awas required formitotic progression independentof PP2A

Next, we designed three siRNAs against different regions ofCIP2A and found that two of them (#1 and #2 siRNAs) werehighly efficient in silencing the CIP2A gene, but #3 siRNAexhibited poor efficiency (Fig. 2A, top). In addition, we con-structed a CIP2A siRNA-resistant rescue vector (CIP2Arv)against the #1 siRNA to use in rescue experiments (Fig. 2A,bottom).

To examine the role of CIP2A during mitosis, cells werearrested at mitosis using nocodazole (Fig. 2B). Interestingly,mitotic arrest was clearly reduced in CIP2A-depleted cells,similar to MAD2 depletion or CHFR overexpression, which

are known to cause reduction of the mitotic index byinducing defects in the spindle checkpoint (1, 27) or theactivation of the prophase checkpoint (28) under nocodazoletreatment, respectively (Fig. 2B). Consistent with this, mitot-ic phosphoproteins, assessed by Western blotting with anti–phospho-MPM2 antibody, were downregulated in CIP2A-depleted (#1 siRNA), MAD2-depleted, or CHFR-overexpres-sing cells compared with cells transfected with controlsiRNA or poor-efficiency CIP2A siRNA (#3; Fig. 2C). Impor-tantly, the reduced mitotic index was restored by over-expression of CIP2Arv, excluding off-target effects of siRNA(Fig. 2D and E). A high percentage of MAD2-depleted cellscontained multilobed nuclei, whereas CHFR-induced pre-mitotic arrest did not affect nuclear morphology (Fig. 2F andG); these results are consistent with those of previousstudies (27, 28). Interestingly, the presence of normal inter-phase nuclei in CIP2A-depleted cells suggested that, similarto CHFR overexpression, CIP2A depletion induced premito-tic arrest (Fig. 2F and G).

Because CIP2A has been shown to inhibit c-Myc–associatedPP2A (9), we speculated that CIP2A-mediated cell-cycle reg-ulation may be dependent on PP2A. Unexpectedly, both inhi-bition of PP2A either by PP2A-specific siRNAs or okadaic

Figure 1. CIP2A is a cell-cycle regulated protein. A, HeLa cells were synchronized by either a double thymidine block or nocodazole block, released into freshmedium at indicated time points, and then analyzed by immunoblotting with antibodies against the indicated proteins. Synchronization and progressionthrough the cell cycle was confirmed by fluorescence-activated cell sorting analysis. B, HeLa cells were stained with anti-CIP2A antibody (green),anti-pericentrin antibody (red), and DAPI (DNA, blue). C, an enlarged single-cell image from HeLa cells stained with anti-CIP2A antibody (green)and DAPI (blue). D, H1299 cells were transfected with the PTEN-expressing construct or empty vector. E, Hs68 cells were transfected with eithercontrol (Ctrl) siRNA or Plk1 siRNA. D and E, after transfection for 48 hours, PTEN-induced G1 arrest or Plk1 depletion–induced G2–M arrest was analyzedby immunoblotting with antibodies against the indicated proteins or by fluorescence-activated cell sorting analysis, respectively. F, Hs68 cells werestained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) or anti-cyclin B1 antibody (red) with DAPI (blue). Data shown represent typicalresults from at least four independent experiments. Scale bars, 10 mm.

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acid treatment and activation of PP2A by forskolin treatmentwere not able to rescue the CIP2A-depletion phenotype(Supplementary Fig. S3A and S3C). Consistent with this, deple-tion or activation of PP2A did not rescue reduced mitoticphosphorylation due to CIP2A depletion (SupplementaryFig. S3B and S3D). Along with the observation that CIP2Adepletion induced premitotic arrest, these results indicatedthat CIP2A was required for mitotic progression indepen-dent of PP2A.

CIP2A was required for timely entry into mitosis andproper completion of mitosisTo further investigate the role of CIP2A during mitotic

progression, cells were transfected with CIP2A siRNAs in theinterval between the two thymidine blocks and then releasedinto the cell cycle with or without nocodazole (Fig. 3A, top).CIP2A depletion retarded the entry of synchronized cells intomitosis compared with the control cells, regardless of noco-

dazole treatment (Fig. 3A, bottom). Moreover, the accumula-tion of mitotic phosphoproteins and mitotic regulators, suchas cyclin B1 and Plk1, was delayed in CIP2A-depleted cells,whereas the expression of cyclin A, which ordinarily accumu-lates in S and G2 phases, was elevated (Fig. 3B, bottom).However, CIP2A depletion did not significantly affect theexpression of c-Myc protein (Fig. 3B, bottom) or the kineticsof DNA replication during cell-cycle progression (Fig. 3B, top).To distinguish cells in the S–G2–M (green) phase from the G1

-phase (red), we used a FUCCI. FUCCI can help visualize thedynamics of cell-cycle progression by harnessing the ubiqui-tination oscillators that control cell-cycle transitions (21).CIP2A was stably depleted by transducing FUCCI-expressingHeLa cells with a lentiviral vector expressing CIP2A shRNA(Fig. 3C, right). Knockdown of CIP2A delayed entry of the cellsinto mitosis and the G1-phase by approximately 2 to 3 hoursand resulted in a much more irregular entry into mitosiscompared with that in cells expressing control shRNA (Fig.

Figure 2. CIP2A depletion blocks nocodazole-induced mitotic arrest. A, HeLa cells were transfected with the indicated siRNAs (top) or transfectedwith control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNA and CIP2Arv (bottom) for 48 hours and then analyzed by immunoblotting withanti-CIP2A antibody. B, C, F, and G, HeLa cells were transfected with the indicated siRNAs or Flag-CHFR vector for 36 hours and then incubated with100 ng/mL nocodazole for 16 hours. MAD2 siRNA or the Flag-CHFR vector was used for a positive control of spindle checkpoint regulation orpremitotic regulation, respectively. Cells were analyzed by light microscopy (B, left), fluorescence-activated cell sorting analysis (B, right), orimmunoblotted with antibodies against the indicated proteins (C). D and E, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, orboth CIP2A.1 siRNA and CIP2Arv for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Nocodazole-treated cells werestained with anti-CIP2A antibody (green) and anti–phospho-H3 antibody (red) with DAPI (blue; D). E, the mitotic index was determined by thepercentage of phospho-H3–positive cells (bottom) or by FACS (top) and was quantified using CellProfiler software (�300 cells for each data point,n ¼ 3; �, P < 0.001). F, nocodazole-treated cells were fixed with DAPI (blue) and the percentage of nonmitotic cells with multilobed or interphasenuclei was quantified (�300 cells for each data point, n ¼ 3; ��, P < 0.01). G, representative confocal images of the cells that indicate thedifferent nuclear morphologies, including mitotic arrest, checkpoint bypass, or premitotic arrest. Arrows, multilobed nuclei. Data shown representtypical results from at least three independent experiments. Scale bars, 20 mm. p-H3–positive, phospho-H3–positive.






3C, left, and D). Although CIP2A depletion primarily preventedmitotic entry, prolonged culture allowed delayed entry intomitosis. Therefore, we next asked whether CIP2A depletioncaused mitotic defects. After 72 hours, CIP2A depletion sig-nificantly increased aberrant micronuclei, and this phenotypewas rescued by cotransfection with CIP2Arv (Fig. 3E and F). Inaddition, CIP2A depletion caused multiple mitotic abnormal-ities (Fig. 3G and H), including the formation of multipolarspindles (Fig. 3H, ii), monopolar spindles (Fig. 3H, iii), mis-aligned chromosomes (Fig. 3H, iv and v), and lagging chromo-somes (Fig. 3H, vi). Taken together, these results suggested that

CIP2A was required for timely entry into mitosis and theproper completion of mitosis.

CIP2A bound to Plk1 during mitosisBecause the stage-specific activity of mitotic kinases is

crucial for proper cell-cycle progression (1) and because CIP2Ais required formitotic entry, we screened for kinases capable ofrescuing CIP2A depletion mitotic delay. We overexpressedseveral mitotic kinases, including Aurora A, Aurora B, Plk1,and Nek2A, and among all of these kinases, only Plk1 rescuedthe CIP2A-depletion phenotype (Fig. 4A and B). Thus, we

Figure 3. CIP2A depletion results in delay of mitotic entry and mitotic abnormalities. A, schematic of cell synchronization protocol by doublethymidine block and for transfection with siRNA (top). A and B, HeLa cells were transfected with control (Ctrl) siRNA or CIP2A.1 siRNA, synchronized atthe G1–S phase by a double thymidine block and released from the secondary thymidine block into medium with or without 100 ng/mLnocodazole (treated 6 hours after release). A, the mitotic index of control- or CIP2A-depleted cells was expressed as the percentage of phospho-H3–positive cells (�500 cells at each time point; bottom). B, cells were analyzed by fluorescence-activated cell sorting (FACS) analysis (top) orimmunoblotted with the indicated antibodies (bottom). C and D, control (Ctrl) or CIP2A shRNA knockdown cells expressing FUCCI probes weresynchronized by double thymidine block and released with fresh medium. C, the time of mitotic entry was determined by observing mitotic cellrounding with signs of DNA condensation and was monitored by time-lapse microscopy (left). Stable knockdown of CIP2A was determined byimmunoblotting with anti-CIP2A antibody (right). D, representative time-lapse images of control (Ctrl) or CIP2A shRNA knockdown cellsexpressing FUCCI probes during cell-cycle progression. E–H, HeLa cells were transfected with either control (Ctrl) siRNA or CIP2A.1 siRNA for72 hours. E, CIP2Arv was cotransfected with CIP2A.1 siRNA for the rescue of CIP2A. The percentage of normal and aberrant nuclei was quantifiedusing fluorescence microscopy (�500 cells for each data point). F, representative images of CIP2A-depleted cells stained with anti-CIP2Aantibody (green) and DAPI (blue). Arrows indicate aberrant nuclei. G, CIP2A-depleted cells were scored for abnormal mitosis (�200 mitotic cells foreach data point, n ¼ 4, error bars, � SD). H, representative images of a normal mitotic cells (i) and CIP2A-depleted mitotic cells (ii–vi) stained withanti-CIP2A antibody (green), anti-pericentrin antibody (red), and DAPI (blue). The data shown represent typical results from at least three independentexperiments. Scale bars, 10 mm. �, P < 0.001. p-H3–positive, phospho-H3–positive; Noc, nocodazole.

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hypothesized that CIP2A regulated Plk1 activity during cell-cycle progression. To test this, we conducted coimmunopre-cipitation assays with CIP2A and Plk1. Interestingly, CIP2Ainteracted with endogenous Plk1 (Fig. 4C), and this interactionwas particularly apparent during mitosis (Fig. 4D). To furtherdissect the interaction between CIP2A and Plk1, cells weretransfected with Strep-tagged CIP2A with various Flag-taggedPlk1 constructs, including WT, polo-box–mutant (three pointmutations at W414F, V415A, and L427A, which are known todisrupt the PDB function (FAA; ref. 29), N-terminal–mutant(amino acids 1–305; N), and C-terminal–mutant (amino acids306–603; C) Plk1, followed by a pull-down assay using Strep-CIP2A. As shown in Fig. 4E, Strep-CIP2A associated with WT

Plk1, FAA Plk1, and C-terminal Plk1, but not with N-terminalPlk1. In addition, the interaction was reduced in FAA Plk1compared withWTor C-terminal Plk1 (Fig. 4E), indicating thatthe CIP2A–Plk1 interaction required the intact PBD of Plk1. Tofurther confirm this interaction, in situPLAswere conducted tovisualize the in vivo interactions between two proteins. Nota-bly, positive signals indicating interactions between CIP2A andPlk1 were clearly observed in mitotic cells compared withinterphase cells (Fig. 4F and G). Moreover, these signals beganto increase from prophase, peaked at prometaphase, and thengradually decreased in anaphase (Fig. 4H and I). Taken togeth-er, these results showed that CIP2A interacted directly withPlk1 during mitosis.

Figure 4. CIP2A binds to Plk1 during mitosis. A and B, HeLa cells were transfected with control (Ctrl) siRNA, CIP2A.1 siRNA, or both CIP2A.1 siRNAand Flag-Plk1 vector for 48 hours and then incubated with 100 ng/mL nocodazole for 16 hours. Cells were analyzed by light microscopy (A)or immunoblotted with antibodies against the indicated proteins (B). C, lysates of HeLa cells were immunoprecipitated with anti-CIP2A antibody,anti-Plk1 antibody, or their respective control immunoglobulin G (IgG) antibodies and immunoblotted with anti-CIP2A or anti-Plk1 antibody. D,lysates of HeLa cells released from thymidine block for the indicated times were immunoprecipitated with anti-CIP2A antibody and immunoblottedwith anti-Plk1 or anti-CIP2A antibody. E, lysates of 293T cells expressing Flag, Flag-tagged Plk1 (WT), polo-box–mutant (FAA) Plk1, N-terminal(N) Plk1, and C-terminal (C) Plk1 with Strep-CIP2A were pulled down with Strep-Tactin beads. The Flag-Plk1 protein associated with Strep-CIP2Awas detected by immunoblotting with anti-Flag antibody. F–I, HeLa cells were fixed and incubated with mouse anti-CIP2A antibody togetherwith rabbit anti-phospho-Plk1 (Thr210) antibody (top) or rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody (bottom), followedby in situ PLA analysis. Arrows indicate mitotic cells (F). G and I, dots per cell were counted using CellProfiler (�100 cells for each datapoint; G) or �20 single cells in each respective cell-cycle phase (I); error bars, � SD, �, P < 0.001). The variation in the number of signal dotsbetween G and I was due to the size difference between multiple- and single-cell images. H, representative confocal images of cells with PLA-positivesignals in each respective cell-cycle phase. Data shown represent typical results from at least three independent experiments. Scale bars, 20 mm.p-MPM2, phospho-MPM2.






The CIP2A–Plk1 interaction enhanced the stability andactivity of Plk1 during mitosis

Because Plk1 directly interacted with CIP2A (Fig. 4) and itsexpression was decreased in CIP2A-depleted cells (Fig. 3B;Supplementary Fig. S3B), we hypothesized that Plk1 stabilitymay be affected by CIP2A during mitosis. To investigate thispossibility, we examined the half-life of mitotic Plk1 in thepresence of protein synthesis (cycloheximide) or proteasome(MG132) inhibitors. CIP2A depletion significantly reduced thestability of Plk1 protein after cycloheximide treatment (Fig. 5Aand B). Interestingly, inhibition of the proteasome withMG132blocked Plk1 degradation in CIP2A-depleted cells (Fig. 5C),supporting that CIP2A has a role in the stabilization of Plk1.Indeed, ectopic overexpression of CIP2A reduced the ubiqui-tination of Plk1 in mitotic cells (Fig. 5D). Because Plk1 degra-dation is mediated by APC/C-Cdh1 or -Cdc20, we determinedwhich of these cofactors were involved in the regulation of Plk1stability in CIP2A-depleted cells. Notably, Plk1 levels in CIP2A-

depleted cells were restored by Cdh1 depletion, but not byCdc20 depletion (Fig. 5E), indicating that CIP2A stabilized Plk1by interferingwithAPC/C-Cdh1–mediated degradation duringmitosis.

Unexpectedly, we also observed that CIP2A depletion sig-nificantly reduced g-tubulin recruitment and microtubuleorganization during mitosis (data not shown). Because Plk1activity is critical for centrosome maturation during mitosis(6, 30), we considered the possibility that CIP2A depletion mayaffect Plk1 activity and stability. To test this possibility, wemeasured the level of Plk1 phosphorylation at Thr210, indic-ative of Plk1 activation, and Plk1 kinase activity. Notably, Plk1phosphorylation and kinase activity were dramaticallydecreased during mitosis in CIP2A-depleted cells comparedwith control cells, despite the increase in total Plk1 (Fig. 5F andSupplementary Fig. S4A and S4B). To further confirm this, weconducted in vitro kinase assays using Strep-CIP2A (purifiedfrom 293 cells) and His-Plk1 (obtained from Sf9 cells) protein.

Figure 5. CIP2A regulates the stability and activity of Plk1 during mitosis. A, HeLa cells were transfected with the indicated siRNAs, synchronized, and treatedwith cycloheximide (CHX), which was added 7 hours after release from G1–S. At the indicated times after the addition of cycloheximide, cells wereanalyzedby immunoblottingwith the indicatedantibodies. B, the levels of Plk1werequantifiedusing ImageJ software (n¼3; error bars,�SD, �,P<0.01). C, at7 hours after release fromG1–S, cells were treated withMG132 for 3 hours. D, cells cotransfected with vectors for HA-ubiquitin (Ub) and empty or CIP2A-Mycwere released fromG1–S for 1 hour (interphase) or 7 hours (mitosis) andMG132wasadded for 3 hours. Lysates of the cells were immunoprecipitatedwith anti-Plk1 antibody and immunoblotted with anti-HA antibody. E, HeLa cells transfected with the indicated siRNAs targeting CIP2A, Cdh1, or Cdc20 weresynchronized at the G1–S phase, released for 10 hours and then analyzed by immunoblotting with the indicated antibodies. F, HeLa cells transfected withcontrol (Ctrl) siRNAorCIP2A.1 siRNAwere synchronized by a double thymidine block and released into freshmedium.Cellswere analyzedby immunoblottingwith the indicated antibodies.G, purifiedStrep-CIP2Aprotein (1mg) incubatedwithorwithout recombinantHis-Plk1 (0.1mg)wasusedas thecontrol to excludethe possibility that purified Strep-CIP2A protein was associated with kinases that can phosphorylate casein (left). Recombinant His-Plk1 (0.1 mg) wasincubatedwith 50mmol/L BI2536 alone orwith serial concentrations of purifiedStrep-CIP2A proteins (0.25, 0.5, or 1mg) for 30minutes (right). Plk1 activity wasdetermined by in vitro kinase assay using casein as a substrate. Data shown represent typical results from at least three independent experiments.

Kim et al.





Consistent with the decrease in Plk1 activity by CIP2A deple-tion, CIP2A protein noticeably enhanced Plk1 activity andautophosphorylation (Fig. 5G). Furthermore, we observed thatCIP2A-depleted cells were less sensitive to BI2536, a pharma-cologic inhibitor of Plk1, than control cells (Supplementary Fig.S5), supporting that Plk1 possessed CIP2A-dependent activity.Taken together, these results suggested that the CIP2A–Plk1interaction increased both the activity and stability of Plk1during mitosis.

In vivo correlation between CIP2A and Plk1 in humancancersTo assess the physiologic relevance of CIP2A-mediated Plk1

regulation in human cancers, we evaluated CIP2A and Plk1 ex-pression using tissue microarrays derived from distinct humancancer tissues (see the Materials and Methods for details). Con-sistent with other reports, CIP2A and Plk1 were significantlyincreased in lung, gastric (stomach), colon, and cervical cancers(Fig. 6A). Importantly, the staining regions of these two proteinsin serial sections of the same tissues were strikingly similar (Fig.

6B–D). Similar staining patterns were also observed in variouscancer tissues (Supplementary Fig. S1B). Furthermore, we foundthat CIP2A expression was strongly correlated with Plk1 expres-sion in four common types of cancer tissues (SupplementaryTable S1; lung cancer, r¼ 0.682; cervical cancer, r¼ 0.77; gastriccancer, r ¼ 0.787; and colon cancer, r ¼ 0.735). Finally, we veri-fied the in vivo interactions of these two proteins in cancer andnormal tissues by in situ PLA. Remarkably, positive signals werepredominantly detected in cancer tissues compared with theirnormal counterparts (Fig. 6E and F). Of the normal tissues tested,positive signals were also detected in stomach and colon tissues,both highly proliferative tissues, but to a lesser extent than incorresponding cancer tissues (Fig. 6E). Collectively, our data pro-vided strong evidence for the physiologic relevance of CIP2A-mediated Plk1 regulation in a wide variety of human cancers.

DiscussionIn this study, we showed a novel regulatory function for

CIP2A during cell-cycle progression. Although CIP2A is a well-known tumor marker and potent oncogene in a variety of

Figure 6. Association betweenCIP2A andPlk1 in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. A, quantification of CIP2A andPlk1 staining intensities in lung, gastric, colon, and cervical cancers and their normal tissue counterparts. B and C, representative microscopic images oflung, gastric, colon, and cervical cancers and their normal tissue counterparts stained with anti-CIP2A antibody (B) or anti-Plk1 antibody (C). Scale bars,200 mm. D, representative high-magnification images of gastric cancer tissues stained with the indicated antibodies. Scale bars, 100 mm. E and F,tissue sections were incubated with rabbit anti-CIP2A antibody together with mouse anti-Plk1 antibody followed by in situ PLA analysis. Representativeconfocal images of lung, gastric, colon, and cervical cancers and their normal tissue counterparts (E, left and F). Scale bars, 20 mm. Four differentareas were obtained for each sample and 200 to 500 cells were quantified per area (mm2) using CellProfiler (E, right). Nuclei were stained with Hoechst (blue).The green signal represents autofluorescence. N, normal lung tissue. C, cancer tissue. A and E (left), data are presented as box-and-whisker plots.�, P < 0.001 compared with their normal tissue counterparts.






tumors, the regulatory functions of CIP2A during cell-cycleprogression have not been well studied. We showed for thefirst time that CIP2A controlled mitotic progression bydirectly interacting with the mitotic kinase Plk1, therebystabilizing and promoting Plk1 activity. We also showed thatCIP2A and Plk1 expression was tightly correlated in a largevariety of cancers. Therefore, these findings suggested thatCIP2A plays an important role in fine-tuning Plk1 expressionand ultimately contributes to malignant cell growth andtumor formation.

Our results revealed that CIP2A was associated with cell-cycle regulation. Similar to our observations, several lines ofevidence have indicated that cell cycle–arresting agents/con-ditions modulate CIP2A expression in various cancer cells. Forexample, serum starvation reduces CIP2A expression in gastriccancer cells and doxorubicin treatment reduces it in p53-proficient cells but not in p53-deficient cells (31, 32). Moreover,several cell cycle–promoting factors, such as c-Myc and E2F1,have been reported to stimulate CIP2A expression (12, 33). Inaddition to the cell cycle–dependent expression of CIP2A, wealso found that CIP2A exhibited nuclear and centrosomallocalization during mitosis. Consistent with this observation,a recent report has shown that cytoplasmic CIP2A shuttlesinto the nucleus upon treatment with leptomycin B, an inhib-itor of the nuclear export factor (34), suggesting that thenuclear-cytoplasmic shuttling of CIP2A may be importantfor mitotic progression. Furthermore, we showed that CIP2Ais required for mitotic progression. Depletion of CIP2A andoverexpression of CHFR, which regulates entry into mitosis byubiquitinating Plk1 upon microtubule poisoning (28), causedthe same phenotype of Plk1-dependent premitotic arrest,albeit via different mechanisms, thus supporting the occur-rence of CIP2A-mediated Plk1 regulation during mitoticprogression. Similarly, a recent report showed that depletionof CIP2A disrupted the cell cycle in human ovarian cancercells (35). In terms of c-Myc regulation, many reports haveobserved reduced levels of c-Myc in CIP2A-depleted cells(9, 12, 32, 35, 36). These studies were inconsistent with ourcurrent observation that CIP2A depletion did not affect c-Myclevels or the kinetics of DNA replication; however, it should benoted that these previous reports examined c-Myc levels at 72hours after CIP2A depletion, while we observed c-Myc expres-sion after relatively short-term depletion. In addition, a pre-vious report showed that S-phase synchronization in thymi-dine/aphidicolin-treated gastric cancer cells did not affectCIP2A expression compared with unsynchronized cells, sug-gesting that cell-cycle activity is not associated with c-Myc–mediated regulation of CIP2A expression in gastric cancer cells(12). Therefore, it is likely that CIP2A-dependent cell-cycleregulation is independent of c-Myc.

Our data indicated that CIP2A blocks ubiquitin-dependentproteolysis ofmitotic Plk1, similar to previous studies that haveshown the CIP2A-dependent proteolytic degradation of c-Myc(9). On a molecular level, R337, located near the C-terminalregion of Plk1, acts as a functional destruction box (4). Giventhat CIP2A interacted with the C-terminal region of Plk1 andthat ectopic overexpression of CIP2A inhibited Plk1 ubiquiti-nation, it is possible that the CIP2A–Plk1 interaction could

hinder the reaction between APC/C-Cdh1 and the destructionbox on Plk1, thereby decreasing Plk1 ubiquitination. APC/C-Cdh1 is thought to be active during late M and G1-phase (2).However, recent observations have indicated that APC/C-Cdh1is also active during early mitosis (37). Thus, the CIP2A–Plk1interaction could also protect Plk1 from APC/C-Cdh1–depen-dent proteolysis throughout mitosis.

In addition to the regulation of Plk1 stability, we also foundthat CIP2A regulated Plk1 activity during mitosis. Theincreased autophosphorylation and activity of Plk1 inducedby the addition of CIP2A protein may suggest that the inter-action of CIP2A–Plk1 via PBD could relieve the autoinhibitoryinteraction of PBD with the kinase domain of Plk1 (3, 4). ThePlk1 PBD plays a pivotal role in mediating both the kinaseactivity and localization of Plk1 (3, 4). Interestingly, we alsofound that CIP2A depletion increased Plk1 mislocalizationduring mitosis (Supplementary Fig. S6). Proper localization ofPlk1 is required for the phosphorylation and activation of Plk1during mitosis (3, 4). Thus, it is also likely that decreasedactivity of Plk1 in CIP2A-depleted cells may be caused bymislocalization of Plk1.

CIP2A is a clinically relevant prognostic marker in manytypes of cancers. For example, high CIP2A expression predictspoor survival in several types of cancers (10, 13, 14, 36, 38).Furthermore, recent reports suggest that CIP2A expression isassociated with resistance to several anticancer drugs (14,15, 31). Likewise, it has been well established that Plk1 isupregulated in tumors and has been validated as a therapeutictarget inmany types of cancers (8). In addition, Plk1 plays a keyrole tomitotic reentry followingDNAdamage-induced arrest, amechanism contributing to tumor resistance against antican-cer drugs (39–41). Our immunohistochemical and PLA anal-yses indicated that CIP2A tightly cooperated with mitotic Plk1in a wide variety of human cancers. Thus, our findings sug-gested that the CIP2A–Plk1 complex may serve as a potentialprognostic marker for poor survival and tumor resistanceagainst anticancer drugs. In addition, small molecules inter-fering with CIP2A–Plk1 binding or triggering CIP2A down-regulation could be effective as antimitotic drugs for cancertherapy.

How can CIP2A modulate mitotic Plk1 through multiplemechanisms? CIP2A has multiple predicated domains,including armadillo-like, transmembrane, leucine zipper,and coiled-coil domains (Supplementary Fig. S7A). Interest-ingly, under nonreducing conditions, the molecular mass ofendogenous CIP2A was approximately 360 kDa (Supplemen-tary Fig. S7B), suggesting that endogenous CIP2A forms amultimeric complex. Scaffold proteins, which usually formlarge complexes, are crucial regulator of particular signalingpathways, acting by tethering signal components, localizingthese components to specific areas, coordinating positive ornegative feedback signals, and protecting the signalingcomponents (42). This study suggested that CIP2A regulatedmitotic Plk1 via multiple mechanisms, including the regu-lation of stability, activity, and localization. On the basis ofthese aspects, one reasonable answer to the question statedearlier is that CIP2A is a transient mitotic scaffold for Plk1during mitosis.

Kim et al.





In conclusion, our results collectively indicated that CIP2Aacted as amitotic regulator by directly regulating Plk1 and wasrequired for mitotic progression in human cancer cells. Ourwork provides novel molecular insights into CIP2A-mediatedPlk1 regulation in proliferating cancer cells.

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

Authors' ContributionsConception and design: J.-S. KimDevelopment of methodology: J.-S. Kim, E.J. KimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-S. Kim, E.J. KimAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-S. Kim, E.J. Kim, J.S. Oh, I.-C. Park, S.-G. HwangWriting, review, and/or revision of the manuscript: J.-S. Kim, J.S. Oh

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.-S. KimStudy supervision: J.-S. Kim

AcknowledgmentsThe authors thank A. Miyawaki (RIKEN) for providing the HeLa cells

expressing the FUCCI probes.

Grant SupportThis work was supported by Basic Science research Program (Grant no.

2012R1A1A2002955) and the Nuclear Research and Development Programthrough a National Research Foundation of Korea (NRF; Daejeon, Korea) grantfunded by the Korean government.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 28, 2013; revised July 14, 2013; accepted August 12, 2013;published OnlineFirst August 27, 2013.

References1. Nigg EA. Mitotic kinases as regulators of cell division and its check-

points. Nat Rev Mol Cell Biol 2001;2:21–32.2. Nakayama KI, Nakayama K. Ubiquitin ligases: cell-cycle control and

cancer. Nat Rev Cancer 2006;6:369–81.3. Barr FA, Sillje HH, Nigg EA. Polo-like kinases and the orchestration of

cell division. Nat Rev Mol Cell Biol 2004;5:429–40.4. Archambault V, Glover DM. Polo-like kinases: conservation and diver-

gence in their functions and regulation. Nat Rev Mol Cell Biol2009;10:265–75.

5. LaneHA, Nigg EA. Antibodymicroinjection reveals an essential role forhuman polo-like kinase 1 (Plk1) in the functional maturation of mitoticcentrosomes. J Cell Biol 1996;135:1701–13.

6. McInnes C, Mazumdar A, Mezna M, Meades C, Midgley C, Scaerou F,et al. Inhibitors of Polo-like kinase reveal roles in spindle-pole main-tenance. Nat Chem Biol 2006;2:608–17.

7. Lera RF, Burkard ME. High mitotic activity of Polo-like kinase 1 isrequired for chromosome segregation and genomic integrity in humanepithelial cells. J Biol Chem 2012;287:42812–25.

8. Strebhardt K, Ullrich A. Targeting polo-like kinase 1 for cancer therapy.Nat Rev Cancer 2006;6:321–30.

9. Junttila MR, Puustinen P, Niemela M, Ahola R, Arnold H, Bottzauw T,et al. CIP2A inhibits PP2A in human malignancies. Cell 2007;130:51–62.

10. ComeC, Laine A, ChanrionM, EdgrenH,Mattila E, Liu X, et al. CIP2A isassociated with human breast cancer aggressivity. Clin Cancer Res2009;15:5092–100.

11. Ma L, Wen ZS, Liu Z, Hu Z, Ma J, Chen XQ, et al. Overexpression andsmall molecule-triggered downregulation of CIP2A in lung cancer.PLoS ONE 2011;6:e20159.

12. Khanna A, Bockelman C, Hemmes A, Junttila MR, Wiksten JP, LundinM, et al. MYC-dependent regulation and prognostic role of CIP2A ingastric cancer. J Natl Cancer Inst 2009;101:793–805.

13. Lucas CM, Harris RJ, Giannoudis A, CoplandM, Slupsky JR, Clark RE.Cancerous inhibitor of PP2A (CIP2A) at diagnosis of chronic myeloidleukemia is a critical determinant of disease progression. Blood 2011;117:6660–8.

14. TengHW,YangSH, Lin JK, ChenWS, Lin TC, Jiang JK, et al. CIP2A is apredictor of poor prognosis in colon cancer. J Gastrointest Surg2012;16:1037–47.

15. ChenKF, LiuCY, Lin YC, YuHC, Liu TH,HouDR, et al. CIP2Amediateseffects of bortezomib on phospho-Akt and apoptosis in hepatocellularcarcinoma cells. Oncogene 2010;29:6257–66.

16. Chen KF, Pao KC, Su JC, Chou YC, Liu CY, Chen HJ, et al. Devel-opment of erlotinib derivatives as CIP2A-ablating agents independentof EGFR activity. Bioorg Med Chem 2012;20:6144–53.

17. Tseng LM, Liu CY, Chang KC, Chu PY, Shiau CW, Chen KF. CIP2A is atarget of bortezomib in human triple negative breast cancer cells.Breast Cancer Res 2012;14:R68.

18. YuHC, HouDR, Liu CY, Lin CS, Shiau CW,Cheng AL, et al. Cancerousinhibitor of protein phosphatase 2A mediates bortezomib-inducedautophagy in hepatocellular carcinoma independent of proteasome.PLoS ONE 2013;8:e55705.

19. Ma HT, Poon RY. Synchronization of HeLa cells. Methods Mol Biol2011;761:151–61.

20. Kim JS, Park ZY, Yoo YJ, Yu SS, Chun JS. p38 kinase mediates nitricoxide-induced apoptosis of chondrocytes through the inhibition ofprotein kinase C zeta by blocking autophosphorylation. Cell DeathDiffer 2005;12:201–12.

21. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H,Osawa H, et al. Visualizing spatiotemporal dynamics of multicellularcell-cycle progression. Cell 2008;132:487–98.

22. Buschmann T, Fuchs SY, Lee CG, Pan ZQ, Ronai Z. SUMO-1 mod-ification of Mdm2 prevents its self-ubiquitination and increases Mdm2ability to ubiquitinate p53. Cell 2000;101:753–62.

23. Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ,Jarvius J, et al. Direct observation of individual endogenous proteincomplexes in situ by proximity ligation. Nat Methods 2006;3:995–1000.

24. Di Cristofano A, Pandolfi PP. The multiple roles of PTEN in tumorsuppression. Cell 2000;100:387–90.

25. Weng LP, Brown JL, Eng C. PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancermodel. Hum Mol Genet 2001;10:599–604.

26. Agarwal ML, Agarwal A, Taylor WR, Stark GR. p53 controls both theG2/M and the G1 cell cycle checkpoints and mediates reversiblegrowth arrest in human fibroblasts. Proc Natl Acad Sci U S A 1995;92:8493–7.

27. Stegmeier F, RapeM, Draviam VM, Nalepa G, SowaME, Ang XL, et al.Anaphase initiation is regulated by antagonistic ubiquitination anddeubiquitination activities. Nature 2007;446:876–81.

28. Sanbhnani S, Yeong FM. CHFR: a key checkpoint component impli-cated in a wide range of cancers. Cell Mol Life Sci 2012;69:1669–87.

29. Liu X, Zhou T, Kuriyama R, Erikson RL. Molecular interactions of Polo-like-kinase 1with themitotic kinesin-like protein CHO1/MKLP-1. JCellSci 2004;117:3233–46.

30. Lee K, Rhee K. PLK1 phosphorylation of pericentrin initiates cen-trosome maturation at the onset of mitosis. J Cell Biol 2011;195:1093–101.

31. Choi YA, Park JS, Park MY, Oh KS, Lee MS, Lim JS, et al. Increase inCIP2A expression is associated with doxorubicin resistance. FEBSLett 2011;585:755–60.

32. Li W, Ge Z, Liu C, Liu Z, Bjorkholm M, Jia J, et al. CIP2A is over-expressed in gastric cancer and its depletion leads to impaired clo-nogenicity, senescence, or differentiation of tumor cells. Clin CancerRes 2008;14:3722–8.






33. Laine A, Sihto H, Come C, Rosenfeldt MT, Zwolinska A, Niemela M,et al. Senescence sensitivity of breast cancer cells is defined bypositive feedback loop between CIP2A and E2F1. Cancer Discov2013;3:182–97.

34. Thakar K, Karaca S, Port SA, Urlaub H, Kehlenbach RH. Identificationof CRM1-dependent nuclear export cargos using quantitative massspectrometry. Mol Cell Proteomics 2013;12:664–78.

35. Fang Y, Li Z, Wang X, Zhang S. CIP2A is overexpressed in humanovarian cancer and regulates cell proliferation and apoptosis. TumourBiol 2012;33:2299–306.

36. Ren J, Li W, Yan L, Jiao W, Tian S, Li D, et al. Expression of CIP2A inrenal cell carcinomas correlates with tumour invasion, metastasis andpatients' survival. Br J Cancer 2011;105:1905–11.

37. Jeganathan KB, Malureanu L, van Deursen JM. The Rae1-Nup98complex prevents aneuploidy by inhibiting securin degradation.Nature 2005;438:1036–9.

38. Bockelman C, Hagstrom J, Makinen LK, Keski-Santti H, Hayry V,Lundin J, et al. High CIP2A immunoreactivity is an independentprognostic indicator in early-stage tongue cancer. Br J Cancer2011;104:1890–5.

39. Macurek L, Lindqvist A, Lim D, LampsonMA, Klompmaker R, Freire R,et al. Polo-like kinase-1 is activated by aurora A to promote checkpointrecovery. Nature 2008;455:119–23.

40. Bassermann F, Frescas D, Guardavaccaro D, Busino L, Peschiaroli A,Pagano M. The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-dam-age-response checkpoint. Cell 2008;134:256–67.

41. Gleixner KV, Ferenc V, Peter B, Gruze A, Meyer RA, Hadzijusufovic E,et al. Polo-like kinase 1 (Plk1) as a novel drug target in chronic myeloidleukemia: overriding imatinib resistancewith the Plk1 inhibitor BI 2536.Cancer Res 2010;70:1513–23.

42. Good MC, Zalatan JG, LimWA. Scaffold proteins: hubs for controllingthe flow of cellular information. Science 2011;332:680–6.

Kim et al.





2013;73:6667-6678. Published OnlineFirst August 27, 2013.Cancer Res Jae-Sung Kim, Eun Ju Kim, Jeong Su Oh, et al. Regulating the Stability and Activity of Plk1CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by

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