Systematic Kinome shRNA Screening Identiﬁes CDK11 (PITSLRE ... · monoclonal antibody to human actin was purchased from Sigma-Aldrich. Goat anti-rabbit horseradish peroxidase (HRP)

Human Cancer Biology

Systematic Kinome shRNA Screening Identifies CDK11(PITSLRE) Kinase Expression Is Critical for OsteosarcomaCell Growth and Proliferation

ZhenfengDuan1, JianmingZhang3, EdwinChoy1,DavidHarmon1, XianzheLiu1, PeturNielsen2,HenryMankin1,Nathanael S. Gray3, and Francis J. Hornicek1

AbstractPurpose: Identification of new targeted therapies is critical to improving the survival rate of patients

with osteosarcoma. The goal of this study is to identify kinase based potential therapeutic target in

osteosarcomas.

Experimental Design:Weused a lentiviral-based shRNA kinase library to screen for kinases which play a

role in osteosarcoma cell survival. The cell proliferation assay was used to evaluate cell growth and survival.

siRNAassayswere applied to confirm theobservedphenotypic changes resulting from the loss of kinase gene

expression. CDK11 (PITSLRE) was identified as essential for the survival of osteosarcoma cells, and its

expressionwas confirmedbyWesternblot analysis and immunohistochemistry.Overall patient survivalwas

correlated with the CDK11 expression and its prognosis. The role of CDK11 expression in sustaining

osteosarcoma growth was further evaluated in an osteosarcoma xenograft model in vivo.

Results:Osteosarcoma cells display high levels of CDK11 expression. CDK11 expression knocked down

by either lentiviral shRNA or siRNA inhibit cell growth and induce apoptosis in osteosarcoma cells.

Immunohistochemical analysis showed that patients with osteosarcoma with high CDK11 tumor expres-

sion levels were associatedwith significantly shorter survival than patients with osteosarcomawith low level

of tumor CDK11 expression. Systemic in vivo administration of in vivo ready siRNA of CDK11 reduced the

tumor growth in an osteosarcoma subcutaneous xenograft model.

Conclusions: We show that CDK11 signaling is essential in osteosarcoma cell growth and survival,

further elucidating the regulatorymechanisms controlling the expression of CDK11 and ultimately develop

a CDK11 inhibitor that may provide therapeutic benefit against osteosarcoma. Clin Cancer Res; 18(17);

4580–8. �2012 AACR.

IntroductionOsteosarcoma is the most common primary malignant

tumor of bone. The standard treatment for osteosarcomaincorporates surgery and chemotherapy involving severalchemotherapeutic agents which include doxorubicin, cis-platin, ifosfamide, and methotrexate (1, 2). If these agentsare unable to lead to favorable tumor response, furtherchemotherapeutic options are very limited. Despite aggres-sive chemotherapy, more than 30% of patients with local-

ized osteosarcoma experience metastatic disease. Most ofthese patients will eventually develop multidrug resistancein late stages of osteosarcoma. The average survival periodafter metastases is less than one year (1–5). Therefore, thereis urgent need to improve the general condition and theoverall survival rate of patients with metastatic osteosarco-ma by identifying novel therapeutic strategies.

The discovery of oncogenic kinases and target-specificsmall-molecule inhibitors has revolutionized the treatmentof a selective group of cancers, such as chronic myeloidleukemia (CML) and gastrointestinal stromal tumors(GIST). Protein kinases play important roles in regulatingtumor cellular functions—proliferation/cell cycle, cellmetabolism, survival/apoptosis, DNA damage repair, cellmotility, and drug resistance—so it is not surprising thatprotein kinases are often oncogenic genes. Kinases such as c-Src, c-ABL, PI3K/AKT, EGFR, MAP, IGF-1R, and JAK arecommonly activated and highly expressed in cancer cellsand are known to contribute to cancer progression (6, 7).Kinases are now firmly established as a major class of anti-cancer drug targets. Significant progress has been made inunderstanding kinases and their functions. There has been

Authors' Affiliations: 1Center for Sarcoma and Connective Tissue Oncol-ogy and 2Department of Pathology, Massachusetts General Hospital; and3Dana Farber Cancer Institute, Harvard Medical School, Boston,Massachusetts

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

Corresponding Author: Zhenfeng Duan, Center for Sarcoma and Con-nective Tissue Oncology, Massachusetts General Hospital, 100 BlossomSt. Jackson 1115, Boston, MA 02114. Phone: 617-724-3144; Fax: 617-726-3883; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1157

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(17) September 1, 20124580

Research. on September 27, 2020. © 2012 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 12, 2012; DOI: 10.1158/1078-0432.CCR-12-1157

http://clincancerres.aacrjournals.org/

an explosion in thenumber of kinase inhibitors entering theclinic, and many more are in preclinical development. Thebest knownkinase inhibitor isGleevec used in the treatmentof patients with CML and GIST. Gleevec inhibits the kinaseactivity of BCR-ABL in CML and c-Kit in GIST (6, 8).However, the question remains as to whether other highlyexpressed and activated kinases in tumors, including oste-osarcoma, can be targeted in a similar manner.To identify kinases based potential therapeutic target in

osteosarcoma, we conducted a comprehensive kinome-wide shRNA screening and found that CDK11 (cyclin-dependent kinase 11, also knownasCDC2L for cell divisioncycle 2-like or PITSLRE) is an essential kinase for osteosar-coma cell line KHOS growth and proliferation. Cyclin-dependent kinases are protein kinases that are critical forcellular processes such as transcription (9, 10). However, toour knowledge, the oncogenic role of CDK11 has not beenreported in osteosarcoma previously. Further CDK11 pro-tein expression profiling indicated that the expression levelsof CDK11 in tumor tissues is closely correlated with clinicaloutcome.

Materials and MethodsCell lines and cell cultureHuman osteosarcoma cell line KHOS was kindly pro-

vided by Dr. Efstathios Gonos (Institute of BiologicalResearch & Biotechnology, Athens, Greece). Ewing sarcomacell line TC-71was provided byDrKatia Scotlandi (InstituteOrthopedics Rizzoli, Italy). The human osteosarcoma celllines, U-2OS and Saos, human ovarian cancer cell lineSKOV-3, and uterine sarcoma cell line MES-SA were pur-chased from the American TypeCulture Collection (ATCC).Osteosarcoma cell line OSA344 was established from pri-

mary osteosarcoma tissue. Human osteoblast cells HOB-cwere purchased from PromoCell GmbH, osteoblast cellsNHOst were purchased from Lonza Wallkersville Inc., andosteoblast cells hFOB were purchased from ATCC. Osteo-blast cells were cultured in osteoblast growth medium(PomoCell) with 10% FBS. All other cell lines were culturedin RPMI-1640 (Invitrogen) supplemented with 10% FBS,100 U/mL penicillin and 100 mg/mL streptomycin(Invitrogen).

Lentiviral human kinase shRNA library screenThe roles of protein kinases inmaintaining osteosarcoma

cell growth were examined using MISSION LentiExpressHuman Kinases shRNA library (Sigma). This library con-tains 3109 lentiviruses carrying shRNA sequences targeting673 human kinase genes. Screening was carried out byfollowing the manufacturer’s protocol as previouslydescribed (11, 12).

Proliferation assayThe initial cellular proliferation after lentiviral shRNA

infection was assessed using the CellTiter 96 AQueous OneSolution Cell Assay (Promega) by following the manufac-turer’s protocol as previously described (11).

Synthetic CDK11 siRNA and trasfectionFurther validation of CDK11 knockdown phenotype in

osteosarcoma cell lines was carried out with synthetichuman CDK11 siRNA purchased from Ambion at AppliedBiosystems. The siRNA sequence targeting CDK11 corre-sponded to coding regions (50- AGAUCUACAUCGUGAU-GAAtt-30, antisense 50-UUCAUCACGAUGUAGAUCUtg-30)of the CDK11 gene. The siRNA oligonucleotides were dis-solved in nuclease-free water at a concentration of 100mmol/L and kept at �20�C until the following transfectionexperiment. The nonspecific siRNA oligonucleotides(Applied Biosystems) were used as negative controls. Oste-osarcoma KHOS or U-2OS cells were either plated on 96-well plates for cell proliferation assays or plated on dishesfor Western blot protein isolation. Transfections were con-ducted with Lipofectamin RNAiMax reagent (Invitrogen)according to the manufacturer’s instruction. Medium wasreplaced with RPMI-1640 supplemented with 10% FBS 24hours after transfection. Total protein was isolated withRIPA Lysis Buffer (Upstate Biotechnology) 48 hours afterCDK11 siRNA transfection.

MTT assayEffects of CDK11 siRNA on cellular growth and prolifer-

ation were assessed in vitro using theMTT assay as describedpreviously. KHOS or U-2OS were transfected with CDK11siRNA as described above. After 72 hours of culture, 20mLofMTT (5 mg/mL in PBS, obtained from Sigma-Aldrich) wasadded to each well and the plates were incubated for 3hours. The resulting formazan product was dissolved withacid isopropanol and the absorbance at awavelength of 490nm (A490) was read on a SPECTRAmax Microplate Spec-trophotometer (Molecular Devices).

Translational RelevanceKinases play an essential role in cancer cell growth and

survival; however, the roles of most kinases in osteosar-coma cell growth are largely uncharacterized. In thesearch for kinases required for osteosarcoma cell growth,we identified CDK11 (PITSLRE) as a potential target by acomprehensive human kinome-wide shRNA screeningin osteosarcoma cell lines. Furthermore, knockdown ofCDK11 either by lentiviral shRNA, or by syntheticsiRNA-independent confirmation, can inhibit cellgrowth or induces apoptosis in osteosarcoma cells.Immunohistochemical analysis indicated that osteosar-coma patients with high CDK11 tumor expression levelswere associated with significantly shorter survival thanpatients with osteosarcoma low level of CDK11 expres-sion. Systemic in vivo administration of in vivo readysiRNA of CDK11 reduced tumor growth in an osteosar-coma s.c. xenograftmodel. These observations show thatCDK11 signaling is essential in osteosarcoma cell growthand survival, CDK11 may become a promising thera-peutic target in the management of osteosarcoma.

CDK11 in Osteosarcoma

www.aacrjournals.org Clin Cancer Res; 18(17) September 1, 2012 4581




Apoptosis assayCaspase-cleaved keratin 18-based quantification of apo-

ptosis was evaluated using the M30-Apoptosense ELISAassay kit, as per manufacturer’s instructions (Peviva AB).The ELISA apoptosis detects a 21-kDa fragment of cytoker-atin 18 that is only revealed after caspase cleavage of theprotein. KHOS or U-2OS cell lines reverse transfected withsynthetic CDK11 siRNA were seeded at 2,000 cells per wellin a 96-well plate. After 48 hours incubation, the cells werethen lysed by adding 10 mL of 10%NP-40 per well, and themanufacturer’s instructions for the apoptosis assay werethen followed. Apoptosis was also evaluated by Westernblot analysis using whole-cell lysates immunoblotted withspecific antibodies to PARP (Cell Signaling Technologies)and its cleavage products.

Western blottingThe concentration of the protein was determined by

Protein Assay Reagents (Bio-Rad)with a spectrophotometer(Beckman Du-640, Beckman Instruments, Inc.). The rabbitpolyclonal antibody (sc-928) to human CDK11 (PITSLRE)was purchased from Santa Cruz Biotechnology. The mousemonoclonal antibody to human actin was purchased fromSigma-Aldrich. Goat anti-rabbit horseradish peroxidase(HRP) and goat anti-mouse antibodies were purchasedfrom Bio-Rad. All other antibodies used in this study werepurchased from Cell Signaling Technologies. Western blotanalysis was conducted as previously reported (13).

Immunofluorescence assayFor immunostainings of cultured osteosarcoma cells,

KHOS or U-2OS cells were grown in 8-well chambers for24 hours and fixed in 3.7% buffered paraformaldehyde.Immunostainings were carried out using antibodies againstCDK11 and b-actin. After washing the cells, they wereincubated with Alexa Fluor secondary antibodies. Specifi-cally, the slides were stained with Alexa Fluor 488 (Green)conjugated goat anti-rabbit antibody (Invitrogen) forCDK11, and Alexa Fluor 594 (Red) conjugated for goatanti-mouse antibody for b-actin.

Human sarcoma tumor tissuesSix of the osteosarcoma tissue samples (OST1–OST6)

were obtained from Massachusetts General Hospital sarco-ma tissue bank (Boston, MA) and were used in accordancewith the policies of the institutional review board of thehospital. All diagnoses were confirmed histologically.

Osteosarcoma tissue microarray andimmunohistochemistry

Osteosarcoma tissue microarray was purchased fromImgenex Corp, which contains 57 tumor tissues. Immuno-histochemistry was conducted by following the manufac-turer’s instructions with HRP-DAB System Cell and TissueStaining Kit (R&D Systems). In brief, primary antibody ofCDK11 (1:50 dilution) in 1%bovine serumalbumin (BSA)was applied to the deparaffinized slide overnight at 4�C.After incubation with the HRP-conjugated goat anti-rabbit

antibody, and rinses in PBS thrice, bound antibody wasdetected with the substrate reagents fromHRP-DAB SystemCell and Tissue Staining Kit. Finally, slides were counter-stained with Hematoxylin QS (Vector Laboratories) andmounted with VectaMount AQ (Vector Laboratories).

Evaluation of immunohistochemical stainingCDK11-positive samples were defined as those showing

nuclear staining pattern of tumor tissue. CDK11 stainingpatterns were categorized into 6 groups: 0, no nuclearstaining; 1þ, <10% of cells nuclear stained–positive; 2þ,10%–25% positive cells; 3þ, 26%–50% positive cells; 4þ,51%–75% positive cells; and 5þ, >75% positive cells. Thepercentage of cells showing positive nuclear staining forCDK11 was calculated by reviewing the entire spot. Cate-gorizing of CDK11 staining was completed by 2 indepen-dent investigators. Discrepant scores between the 2 inves-tigators were rescored to get a single final score. CDK11images were obtained by using a Nikon Eclipse Ti-U fluo-rescencemicroscope (Nikon Corp) with a SPORT RT digitalcamera (Diagnostic Instruments Inc.).

Statistical analysisKaplan–Meier survival analysis (GraphPad PRISM Soft-

ware; GrahPad Software) was used to analyze the correla-tion between the level of CDK11 expression and prognosis.The Student t test was used to compare the differencesbetween groups. Results are given as mean� SD and valueswith P < 0.05 were considered as statistically significant.

CDK11 siRNA osteosarcoma tumor therapyAmbion In Vivo Ready CDK11 siRNA and nonspecific

siRNA were purchased from Applied Biosystems. These InVivo Ready, validated siRNAwere designed using the Silenc-er Select algorithm and incorporated with additional chem-ical modifications for superior serum stability with in vivoapplications (14–17). The Crl:SHO-PrkdcSCIDHrhr nudefemale mice at approximately 3 to 4 weeks of age werepurchased from The Charles River Laboratories. To deter-mine the effect of CDK11 siRNA on osteosarcoma cellgrowth in xenograft model, KHOS cells (1 � 106) wereinoculated subcutaneously with Matrigel from BD Bios-ciences into the right flank of the nude mouse. Two weeksafter injection, the mice were randomized into 3 groups (6mice/group). Group 1 received injection with sterile saline(0.9%NaCl), group2with InVivoReadynonspecific siRNA,and group 3 with In Vivo Ready CDK11 siRNA. For intra-tumoral injections, each animal was injected with 20 mL ofPBS containing 10 nmol/L of siRNA. All 3 groups weretreated twice a week for 2 weeks. The health of the mice andevidence of tumor growth were examined daily. Tumorvolumes were measured at a regular interval of for up to4 weeks with a digital calipers, Tumor volume (mm3) wascalculated as (width)2� length/2 whereW is width and L islength. Data are presented as mean � SD. Tumor tissuesfrom the above treated animals were collected and placed in10% formalin and embedded in paraffin for histologyanalysis. The silence efficiency CDK11 siRNA on CDK

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Clin Cancer Res; 18(17) September 1, 2012 Clinical Cancer Research4582




proteins were determined by immunohistochemical stain-ing, as described above. Animal experiments in the presentstudy were carried out in compliance with the protocol,whichwas approved by theMassachusetts General HospitalSubcommittee on Research Animal Care (SRAC) under theprotocol number 2009N000229.

ResultsCDK11 expression is critical for osteosarcoma cellgrowth and survivalTo identify the potential therapeutic kinase targets in

osteosarcoma cells, we conducted a comprehensivekinome-wide screening of lentiviral shRNA kinase libraryin osteosarcoma cell line KHOS. Among the targeted kinasegenes, we found that knocking down the expression ofCDK11, PLK1, DYRK1B, and ROCK1 led to inhibitorygrowth effects. Of those kinases that we found critical toosteosarcoma cell growth, the elevated expression of PLK1,DYRK1B, and ROCK1 have been previously reported invarious human tumors (6, 12, 18–24). However, directevidence regarding the relationship between expression ofCDK11 and cancer cell growth and survival is lacking.WhenLentiviral shRNA targeting CDK11 was transduced intoosteosarcoma cell lines KHOS and U-2OS, it led to signif-icantly reduced tumor cell growth and eventual cell death as

shown by cellular proliferation assay (Fig. 1A). To furthercharacterize the functional role of CDK11 in osteosarcoma,we rigorously validated the results using multiple indepen-dent experiments, including multiple shRNA per CDK11gene and extensively tested with control nonspecific shRNA(The sequence of 5 shRNA target different sites of CDK11 inSupplementary Table S1). The results revealed that 4 out of5 CDK11 shRNA inhibit osteosarcoma cell growth. Wefurther validated these resultswith synthetic humanCDK11siRNA. Consistent observations of the dose-dependentCDK siRNA inhibition of osteosarcoma cell growth andsurvival was established by MTT assay (Fig. 1B). We sub-sequently measured the expression of CDK11 in siRNAtransfected cells. Western blot analysis suggested thatdown-regulated expression of CDK11 protein by CDK11siRNA associated with the inhibition of cell growth (Fig. 1Band C).

CDK11knockdown induces apoptosis inosteosarcomacell lines

To investigate how CDK11 sustains tumor cell growthand survival, we investigated cellular events during the celldeath caused by CDK11 knockdown in osteosarcoma celllines KHOS and U-2OS. We investigated the potential forthe induction of apoptosis using the M-30-Apoptosense

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Figure 1. Effects of CDK11 inhibition by shRNA and siRNA in osteosarcoma cell lines. A, results of lentiviral shRNA targeting CDK11 in KHOS and U-2OS celllines. Proliferation was determined by the CellTiter 96 Aqueous One Solution Cell Assay. The data represent a single well of the 96-well plate of MISSIONLentiExpress Human kinases shRNA library as described. B, results of synthetic siRNA against CDK11 in KHOS and U-2OS cell lines. Proliferation wasassessed byMTT as described in theMaterials andMethods. The data represent themean�SE of 2 experiments carried out in triplicate. C, dose-dependentCDK siRNA downregulated the expression of CDK11. KHOS or U-2OS cells were transfectedwith CDK siRNA in a dose-dependentmanner. ForWestern blotanalysis, 25 mg of total cellular proteins was used for immunoblotting with specific antibody to CDK11. The results were detected by a chemiluminescencedetection system as described in Materials and Methods.






ELISA and by Western blotting for the cleavage of PARP.Depletion ofCDK11by siRNA resulted in a dose-dependentcell death in KHOS and U-2OS osteosarcoma cell lines,which was not observed with the nonspecific siRNA trans-fection (Fig. 2A). Consistent with these results, there wasalso a dose-dependent cleavage of PARP for siRNA-medi-ated depletion of CDK11 (Fig. 2B). CDK11 has beenreported to play an important role in the regulation of genetranscription and pre-mRNA splicing. We hypothesizedthat inhibition of apoptosis induced by CDK11 may beassociated with the deregulation of RNA processing asso-ciated with a subsequent decrease of antiapoptotic-depen-dent proteins. Therefore, we examined whether the inhi-bition of CDK11 by siRNA could result in decreasedexpression of antiapoptotic proteins MCL-1, Bcl-XL, survi-vin, cytochrome C, and cyclin D1. Most of these antiapop-totic proteins such as MCL-1, BcL-XL, and survivin arehighly expressed in osteosarcoma, and downregulation bysiRNA can inhibit cell growth and induce apoptosis inosteosarcoma cells. Western blot confirmed that transfec-tion of CDK11 siRNA significantly downregulated theexpression of several of these antiapoptotic proteins inboth in KHOS and U-2OS cells (Fig. 2C), whereas CDK11knockdown did not alter actin expression. Thus, theseresults indicate that CDK11 can control several aspects ofapoptosis signaling.

CDK11 is highly expressed in osteosarcoma cell linesand in tumor tissues

To further confirm the expression of CDK11 and deter-mine CDK11 protein subcellular localization in osteosar-coma cell lines,immunofluorescence assay was used inKHOS and U-2OS cell lines. Previous studies have reportedthat the CDK11 protein localizes both to the nucleus andcytoplasm. Our results showed that the CDK11 protein ismainly localized in the nucleus of osteosarcoma cells (Fig.3A). We also extended our evaluation of the expression ofCDK11 in other types of human cancer cell lines includ-ing uterine sarcoma (MES-SA), chondrosarcoma (CS-1),synovial sarcoma (SS-1), Ewing sarcoma (TC-71), andovarian cancer (SKOV-3, 3A, 2008). These tumor celllines exhibited a variety of expression levels of CDK11protein as evaluated by Western blot analysis (Fig. 3B). Toconfirm these data in primary cancer, 6 freshly isolatedprimary osteosarcoma specimens were also examined byWestern blot analysis to exclude the possibility of CDK11expression being an artifact induced by in vitro propaga-tion. Different levels of CDK11 expression were onceagain observed in these osteosarcoma patient samples,indicating the endogenous expression of CDK11 in tumorcells. In the normal human cells, the expression of CDK11is tightly regulated, for example, in normal human oste-oblast cell lines, HOB-c, NHOst, and hFOB there are

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Figure 2. Synthetic siRNA targeting CDK11 induces apoptosis in osteosarcoma cells. A, KHOS or U-2OS cells were transfected with CDK siRNA as dose-dependentmanner. The cells were lysedwith 10%NP40 after 48 hours transfection and the apoptosis was determined byM30-Apoptosense ELISA assay asdescribed in Materials and Methods. B, confirmation of CDK11 siRNA induced apoptosis of PARP cleavage by Western blot analysis. Total cellular proteinswere subjected to immunoblottingwith specific antibody toPARP asdescribed inMaterials andMethods.C,CDK11 siRNAdecreasesCDK11expression anddownregulated antiapoptotic proteins expression. For Western blot analysis, 25 mg of total cellular proteins was subjected to immunoblotting with specificantibody to CDK11, MCL-1, BcL-XL, survivin, cytochromeC, cyclin D1, and b-actin. The results were detected by a chemiluminescence detection system asdescribed in Materials and Methods.

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extremely low and almost undetectable levels of CDK11(Fig. 3C).

CDK11 expression levels correlates with clinicalprognosis in patients with osteosarcomaTo further validate the clinical relevant of CDK11 expres-

sion in patients with osteosarcoma, we analyzed CDK11protein levels by using osteosarcoma tissuemicroarray. Theresults showed the majority of tumors present on the tissuemicroarray had positive staining for CDK11. CDK11 nucle-ar staining percentage was graded into 6 groups. By com-paring the clinical characteristics of low-staining (<¼3) andhigh-staining (>¼ 4) osteosarcomas, no correlation existedbetween CDK11 expression and age or tumor location (P >0.05, Supplementary Table S2). Kaplan–Meier survivalanalysis showed that the outcome for patients in theCDK11high-staining group was significantly worse than for thosein the CDK11 low-staining group (Fig. 4A). On the basis of60 months survival rates, patients were grouped into survi-vors (survived up to 60 months post follow up) and non-survivors (deceasedwithin 60months of follow-up). A totalof 30 (67%) samples from survivors and 15 (33%) samplesfrom nonsurvivors were collected. Comparison of CDK11staining intensity between 2 group patients revealed thatCDK11 staining for samples from nonsurvivors were sig-nificantly higher than that of survivors. The average CDK11expression levels for survivors and nonsurvivors were 2.8 to

4.3, respectively (Fig. 4B). The immunohistochemical stain-ing of CDK11 protein indicated its location in the nucleus(Fig. 4C)which is consistentwith that of osteosarcoma cells,as measured by immunofluorescence assay (Fig. 3A).

CDK11 siRNA inhibit tumor growthThe significant association of CDK11 expression with

clinical outcome led us to further verify the essential roleof CDK11 in sustaining osteosarcoma growth in vivo.KHOS osteosarcoma cells (1 � 106) were injected subcu-taneously into the flank of nude mice. By 2 weeks, visibletumors had developed at injection sites (mean tumor vol-ume ¼ 52 mm3). CDK11 siRNA was then intratumorallyinjected, twice a week for 2 weeks. As shown in Fig. 5A,CDK11 In Vivo Ready siRNA significantly suppressed tumorgrowth as compared with vehicle (saline) and nonspecificsiRNA treatment. The immunohistochemical staining indi-cated a significant decrease of CDK11 expression in tumortreatedwith CDK11 In VivoReady siRNA (Fig. 5B). No grossadverse effects, i.e.the loss of body weight, were observedduring the experimental period.

DiscussionTo identify essential kinases which are responsible for

osteosarcoma growth, we first conducted a kinome-wideshRNA screen. CDK11, PLK1, DYRK1B, and ROCK1 werethe primary hits with loss of expression of these kinases

Figure 3. Expression of CDK11 inosteosarcoma cell lines andosteosarcoma tissues. A, expressionof CDK11 in KHOS or U-2OScells was assessed byimmunofluresecencewith antibodiesto CDK11 and b-actin. Cells werevisualized under a fluorescencemicroscope after incubated withsecondary fluorescent conjugatedantibodies Alexa Fluor 488 goat anti-rabbit IgG (green) and Alexa Fluor594 goat anti-mouse IgG (red) asdescribed in the Materials andMethods. B, levels of CDK11expression were determined byWestern blot analysis inosteosarcoma (U-2OS and KHOS),and other types of human cancer celllines, including uterine sarcoma(MES-SA), chondrosarcoma (CS-1),synovial sarcoma (SS-1), Ewingsarcoma (TC-71), and ovarian cancer(SKOV-3, 3A, 2008). C, levels ofCDK11 expression were determinedby Western blot analysis inosteosarcoma tissues [OST1 toOST6 and in normal osteoblast celllines (HOB-c, NHOst, and hFOB).OST1 to OST6 represents tissuessamples from 6 patients.

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significantly reduced cell growth and survival. We andothers had previously found that targeting PLK1, DYRK1B,and ROCK1 kinases inhibits osteosarcoma cell growth and

survival by using similar shRNA or siRNA kinase libraryscreenings (11, 12, 18, 25–27). However, the relationshipbetween the expression of CDK11 and osteosarcoma

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B Figure 4. Association of CDK11expression with clinical outcome inosteosarcoma. A, Kaplan–Meiersurvival curve of patients withosteosarcoma are subgrouped aseither CDK11 low staining (CDK11staining � 3) or high staining(CDK11 staining � 4). B,distribution of CDK11 stainingscores among the survivors andnonsurvivors. C, representativeimages of differentimmunohistochemical stainingintensities of CDK11 are shown inosteosarcoma tissues. For CDK11immunohistochemical staining, thepercentage of cells showingpositive nuclear staining for CDK11was calculated by reviewing theentire spot. On the basis of thepercentage of cells with positivenuclear staining, the stainingpatterns were categorized into 6groups: 0, no nuclear staining; 1þ,<10% of cells stained positive; 2þ,10% to 25% positive cells; 3þ,26% to 50% positive cells; 4þ,51% to 75% positive cells; 5þ,>75% positive cells.

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Figure 5. Inhibition oftumor growthby In VivoReady CDK11 siRNA in axenograft mouse model. A, CDK11In Vivo Ready siRNA, vehicle(saline) control, and nonspecificsiRNA were injected into the tumorregion. Day 1 corresponds to 2weeks after inoculation of KHOScells when tumor volume was 50 to60 mm3. Tumor diameters weremeasured at a regular interval of 4days for up to 4weekswith a digitalcaliper, and the tumor volume wascalculated. B, Histologic analysisof effect of CDK11 siRNA onCDK11 staining in osteosarcomatumor tissues showdownregulation of CDK11compared with vehicle ornonspecific siRNA.

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growth and survival was lacking. The important functionalrole of CDK11 in osteosarcoma cells was further validatedby using CDK11 gene-specific synthetic siRNA knockdownendogenous CDK11. Both initial shRNA screenings andfollow-up siRNA validation assay results highlighted theimportance of CDK11 in osteosarcoma.CDK11 is a serine/threonine protein kinase and encoded

by the CDK11 gene on chromosome 1p36.3 (9, 10). ThefunctionofCDK11hasnot beendescribed inosteosarcoma.There is only one CDK11 gene in mouse, whereas inhumans, there are 2 CDK11 genes that encode 2 almostidentical protein kinases. There are 3 CDK11 protein iso-forms, p110, p58, and p46 (28). CDK11 p58 protein isspecifically translated from an internal ribosome entry siteand expressed only in the G2–M phase of the cell cycle.These different CDK11 isoforms seem to playmultiple rolesin transcription, RNA processing, regulating cell-cycle pro-gression, cytokinesis, and apoptosis (10, 28). CDK11knockout mice display an earlier phenotype and deathduring the blastocyst stage of embryonic development(29). The CDK11 null cells exhibit proliferative defects,mitotic arrest, and apoptosis, thus suggesting that CDK11kinase is critical for embryonic development and cellularviability (29). By kinome-wide siRNA screen for Hedgehog(Hh) regulators, CDK11 has been shown to directly partic-ipate in the Hh pathway. CDK11 is necessary and sufficientfor the activation of the Hh pathway, functioning down-stream of Smo and upstream of the glioma-associated (Gli)transcription factors (19). CDK11 is also a modulator ofautophagy in human cells (30). Although the function ofCDK11 has been examined in different model systems, itspotential role in tumors has not been fully investigated dueto a lack of data regarding CDK11 expression and tumor cellgrowth. CDK11 was initially proposed as a tumor suppres-sor candidate gene, as the CDK11 chromosomal locationregion 1p36.3 is frequently deleted or translocated in anumber of different human tumors, including neuroblas-toma, breast cancer, and melanoma (31–33). However, astudy of neuroblastoma by FISH analysis excludes theCDK11 genes as a tumor suppressor gene (34). On thecontrary, CDK11 knockdown by RNAi in HeLa cellsinduces abnormal spindle assembly, mitotic arrest bycheckpoint activation, and cell death (20, 35). An unbi-ased high-throughput RNAi screening showed thatCDK11 is a positive modulator of the Wnt/b-cateninpathway in colon cancer (36). Consistent with thesestudies, a more recent kinome-wide siRNA study inhuman multiple myeloma identified CDK11 as 1 out of15 survival kinases (including PLK1, AKT1, and GRK6)that was repeatedly vulnerable in myeloma cells (37).Interestingly, a more recent RNAi lethality screening ofthe druggable genome also found CDK11 is the survivalgene in multiple myeloma (21). Our results in osteosar-coma are compatible with HeLa and myeloma cells inwhich CDK11 inhibition leads to decreased cell growthand induces apoptosis.It has been reported that treatment of the Fas-activated T

cells with a serine protease inhibitor prevented apoptotic

death and led to the accumulation of CDK11 p110 isoform,but not the CDK11 p58 isoform (22). To establish themechanisms of CDK11 knockdown-induced cell growthinhibition and apoptosis in osteosarcoma cells, we exam-ined antiapoptotic protein expression. Several antiapopto-tic proteins were reduced by CDK11 knockdown suggestingthat CDK11 can control several aspects of apoptosissignaling

Many studies have found survival-promoting kinasegenes to be highly expressed in human cancer, especiallyin high-grade tumors (10, 38). For CDK11, we show thatosteosarcoma cell lines and tumors express high level ofCDK11 protein in comparison with normal osteoblasts.Most importantly, the levels of CDK11 expression aresignificantly associated with clinical outcome in osteosar-coma. Overexpression of CDK11 was correlated with poorprognosis. Furthermore, silencing ofCDK11 reduced tumorvolume in an osteosarcoma xenograft mouse model. Thesein vivo studies have confirmed the anticancer effects ofCDK11 inhibition in vitro and provide a rationale forpharmacologic investigation of CDK11 as a novel therapytarget.

Taken together, this study identified that CDK11 asessential for osteosarcoma cell growth and survival.Experiments are underway to understand the mechanismsbehind CDK11 signaling in human cancer. In turn, thesefindings may lead to targeting CDK11 through genetherapy or kinase-specific inhibitors in the treatment ofosteosarcoma.

Disclosure of Potential Conflicts of InterestE. Choy is a consultant/advisory board for Amgen, Sanofi Aventis,

and BioMed Valley Discoveries, Inc. No potential conflicts of interest weredisclosed by other authors.

Authors' ContributionsConception and design: Z. Duan, J. Zhang, X. Liu, N.S. Gray, F.J. HornicekDevelopment of methodology: Z. Duan, J. Zhang, F.J. HornicekAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Z. Duan, J. Zhang, D. Harmon, F.J. HornicekAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Z. Duan, J. Zhang, X. Liu, H. Mankin, F.J.HornicekWriting, review, and/or revision of themanuscript: Z. Duan, J. Zhang, E.Choy, D. Harmon, P. Nielsen, H. Mankin, N.S. Gray, F.J. HornicekAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Z. Duan, J. Zhang, F.J. HornicekStudy supervision: Z. Duan, H. Mankin, F.J. Hornicek

Grant SupportThis project was supported, in part, by grants from the Gattegno and

Wechsler funds. Support has also been provided by the Jeff Guyer Fundand the Kenneth Stanton Fund. Dr. Duan is supported, in part, through agrant from Sarcoma Foundation of America (SFA), a grant from NationalCancer Institute (NCI)/National Institutes of Health (NIH), UO1, CA151452-01, and a grant from an Academic Enrichment Fund of MGHOrthopaedics.

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

Received April 7, 2012; revised June 29, 2012; accepted June 30, 2012;published OnlineFirst July 12, 2012.






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2012;18:4580-4588. Published OnlineFirst July 12, 2012.Clin Cancer Res Zhenfeng Duan, Jianming Zhang, Edwin Choy, et al. ProliferationKinase Expression Is Critical for Osteosarcoma Cell Growth and Systematic Kinome shRNA Screening Identifies CDK11 (PITSLRE)

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