Bortezomib Sensitizes HCC Cells to CS-1008, an...

Preclinical Development

Bortezomib Sensitizes HCC Cells to CS-1008, an AntihumanDeath Receptor 5 Antibody, through the Inhibition of CIP2A

Kuen-Feng Chen1,4, Hui-Chuan Yu1,4, Chun-Yu Liu5,6,7, Hui-Ju Chen1,4, Yi-Ching Chen1,4,Duen-Ren Hou8, Pei-Jer Chen1,4, and Ann-Lii Cheng2,3,4

AbstractPreviously, we have shown that bortezomib overcame TRAIL resistance in hepatocellular carcinoma (HCC)

cells via the inhibition of Akt. Here, we report that bortezomib sensitizes these TRAIL-resistant cells, including

Huh-7, Hep3B, and Sk-Hep1, to CS-1008, a humanized agonistic antihuman death receptor 5 antibody.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) mediated the sensitizing effect of bortezomib to CS-

1008 through inhibiting protein phosphatase 2A (PP2A) activity. Combination treatment of bortezomib and

CS-1008 downregulated CIP2A in a concentration- and time-dependent manner, and increased PP2A activity

in HCC cells. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity, indicating that

CIP2A negatively regulates Akt-related PP2A activity in HCC cells. Moreover, silencing CIP2A by short

interfering RNA enhanced CS-1008–induced apoptosis in HCC cells and ectopic expression of CIP2A in HCC

cells abolished CS-1008–induced apoptosis, indicating that CIP2A plays an important role in the sensitizing

effect of bortezomib to CS-1008. Finally, our in vivo data showed that CS-1008 and bortezomib combination

treatment decreased tumor growth significantly. In conclusion, bortezomib sensitized HCC cells to CS-1008

through the inhibition of CIP2A. Mol Cancer Ther; 10(5); 892–901. �2011 AACR.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most com-mon solid tumor worldwide and remains a difficultmalignancy to treat (1). Major obstacles hindering effec-tive treatment for HCC include high recurrence aftercurative resection, underlying cirrhotic liver, and, impor-tantly, the frequent resistance of advanced HCC to con-ventional chemotherapy and radiotherapy (2). The factthat most patients with advanced HCC do not respondwell to current chemotherapeutic agents highlights theneed for the development of novel targeted therapy forHCC (3). And, the promising trial result shown by themultikinase inhibitor sorafenib in patients with advancedHCC showed the potential of molecular-targeted therapyfor advanced HCC (4).

Targeting apoptotic pathway is the foundation ofmanyanticancer strategies. Among the possibilities, the path-way involving TNF-related apoptosis inducing ligand(TRAIL) is currently the most promising, as preclinicalmodels suggest that apoptosis of tumor cells is achievablein vivo without lethal toxicities (5). TRAIL is a type IItransmembrane protein that belongs to the TNF super-family and functions through binding to death receptors(DR; ref. 6). Currently, 5 DRs that can be subdivided into 2distinctive functional groups are known to bind to TRAIL(7, 8). DR4 (TRAIL-R1) and DR5 (TRAIL-R2) contain aneffective cytoplasmic death domain that forms death-inducing signaling complex (DISC) on ligand bindingand transduce proapoptotic signals (7, 8). The binding ofTRAIL to DR4 or DR5 results in trimerization of the deathdomain-containing receptor, leading to the formation of amultiprotein complex designated the DISC, whichinvolves participation of an adaptor molecule, Fas-asso-ciated protein with death domain (FADD) and activationof the initiator caspase-8.

CS-1008, a novel humanized agonistic antihuman DR5antibody, was modified from a murine antihuman DR5mAb, TRA-8 (9). CS-1008 has shown selective cytotoxicitytoward tumor cells expressing DR5, such as colorectaladenocarcinoma cells, non–small-cell lung cancer cells,pancreatic carcinoma cells, and renal cell adenocarci-noma and has shown enhanced antitumor activity whenin combination with gemcitabine or docetaxel (9). In aphase I trial, CS-1008 exerted no dose limiting toxicity atdoses of up to 8 mg/kg weekly (10). CS-1008 is currently

Authors' Affiliations: Departments of 1Medical Research, 2Oncology, and3Internal Medicine, and 4National Center of Excellence for Clinical Trial andResearch, National Taiwan University Hospital; 5Division of Hematologyand Oncology, Department of Medicine, Taipei Veterans General Hospital;6School of Medicine and 7Institute of Biopharmaceutical Sciences,National Yang-Ming University, Taipei; and 8Department of Chemistry,National Central University, Jhongli City, Taoyuan County, Taiwan

Corresponding Author: Ann-Lii Cheng, Department of Internal Medicineand Department of Oncology, National Taiwan University Hospital, No 7,Chung-Shan S Rd, Taipei, Taiwan. Phone: 886-2-23123456 ext. 67251;Fax: 886-2-23711174. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0794

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 10(5) May 2011892

Research. on August 20, 2018. © 2011 American Association for Cancermct.aacrjournals.org Downloaded from

Published OnlineFirst March 10, 2011; DOI: 10.1158/1535-7163.MCT-10-0794

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undergoing phase II trials in combination with sorafenibfor the treatment of advanced HCC (NCI clinical trial:NCT01033240).Despite reports of selective TRAIL-induced apoptosis

in several types of tumor cells, many cancer cells areresistant to apoptosis induction by TRAIL and more andmore evidence suggest that TRAIL alone may not besufficient to efficiently induce apoptosis in many typesof cancers, including HCC (11–14). Therefore, it is impor-tant both to understand the mechanisms underlyingresistance and improve the potency of TRAIL-basedtherapeutic approaches, if TRAIL is going to be success-fully used for cancer therapy. Proteasome inhibitorsrepresent a highly promising class of anticancer agentsto overcome TRAIL resistance or resensitize tumors to theapoptotic effect of TRAIL.Bortezomib is the first proteasome inhibitor to be

approved clinically for multiple myeloma andmantel celllymphoma (15, 16). Bortezomib showed excellent antitu-mor activity against these 2 hematologic malignanciesthrough blocking proteasome degradation of IkB, an inhi-bitor of NF-kB (16). The fact that multiple cellular targetsare affected by bortezomib suggests its potential advan-tage in enhancing antitumor activities in combinationtreatment with TRAIL in HCC. Indeed, in our previousstudy, exploring the antitumor activity of bortezomibagainst HCC cells (17), we showed that downregulationofp-Aktdetermines the sensitivityof bortezomib, and thatbortezomib-induced apoptosis may not be associatedwith proteasome inhibition in HCC cells. Moreover, weconfirmed that bortezomib sensitizedHCC cells to TRAILthrough inhibition of p-Akt (14). Importantly, we foundthat proteinphosphatase 2A (PP2A), a phosphatasewhichdownregulates p-Akt, may mediate the effect of bortezo-mibonTRAIL sensitization (14). Themechanismbywhichbortezomib upregulates PP2A and, in turn, downregu-lates p-Akt was further delineated in our recent study onthe synergistic interaction between bortezomib and sor-afenib in HCC cells (18).PP2A is a complex of serine/threonine protein phos-

phatase with broad substrate specificity and diversecellular functions and consists of a dimeric core enzymecomprising of structural A and catalytic C subunits (theAC core enzyme), and heterogeneous regulatory B sub-units (19, 20). It is known that there are several cellularinhibitors of PP2A, including SET (21) and cancerousinhibitor of PP2A (CIP2A; ref. 22). Notably, CIP2A(KIAA1524, P90), first cloned from patients with HCC(23), has been shown to promote anchorage-independentcell growth and in vivo tumor formation by inhibitingPP2A activity toward c-myc (22). Moreover, CIP2A isoverexpressed in HCC and several other human malig-nancies (22, 24, 25) and is associated with clinical aggres-siveness in human breast cancer (24), suggesting its roleas an oncoprotein.In this study, we found that bortezomib sensitizes

TRAIL-resistant HCC cells to CS-1008 through the med-iatorCIP2A.We showed thatCIP2A, through inhibition of

PP2A-dependent p-Akt activity, mediates the sensitizingeffect of bortezomib to CS-1008. The combination of bor-tezomib and CS-1008 downregulated CIP2A and, in turn,increased Akt-related PP2A activity in these HCC cells.

Materials and Methods

Reagents and antibodiesBortezomib (Velcade) and CS-1008 were kindly pro-

vided by Millennium Pharmaceuticals and Daiichi San-kyo Pharmaceuticals, respectively. For in vitro studies,bortezomib or CS-1008 at various concentrations wasdissolved in dimethyl sulfoxide (DMSO) and then addedto cells in Dulbecco’s Modified Eagle’s Medium (DMEM)containing 5% FBS. The final DMSO concentration was0.1% after addition to the medium. Antibodies for immu-noblotting such as anti-Akt1, anti-PARP, and anti–PP2A-C, were purchased from Santa Cruz Biotechnology. Otherantibodies including anti–caspase-3, anti-CIP2A, andanti–p-Akt (Ser473) were from Cell Signaling.

Cell culture and Western blot analysisThe Sk-Hep1 and Hep3B cell lines were obtained from

the American Type Culture Collection. The Huh-7 HCCcell line was obtained from the Health Science ResearchResources Bank (Osaka, Japan; JCRB0403). All cellsobtained from the American Type Culture Collectionor the Health Science Research Resources Bank wereimmediately expanded and frozen down such that allcell lines could be restarted every 3 months from a frozenvial of the same batch of cells. No further authenticationwas done in our lab. Cells were maintained in DMEMsupplemented with 10% FBS, 100 units/mL penicillin G,100 mg/mL streptomycin sulfate, and 25 mg/mL ampho-tericin B in a 37�C humidified incubator and an atmo-sphere of 5%CO2 in air. Lysates of HCC cells treated withdrugs at the indicated concentrations for various periodsof time were prepared for immunoblotting of caspase-3,PARP, p-Akt, and Akt. Western blot analysis was done aspreviously reported (17).

Apoptosis analysisThe following 3 methods were used to assess drug-

induced apoptotic cell death: detection of DNA fragmen-tation with the Cell Death Detection ELISA kit (RocheDiagnostics), Western blot analysis of caspase activationand PARP cleavage, and measurement of apoptotic cellsby flow cytometry (sub-G1). ELISA was conductedaccording to the manufacturer’s instructions (8).

Gene knockdown using short interfering RNASmartpool short-interfering RNA (siRNA) reagents,

including a control (D-001810-10), and CIP2A (L-014135-01) were all purchased from Dharmacon and usedaccording to the procedure described previously (17).Briefly, cells were transfected with siRNA (final concen-tration, 100 nmol/L) in 6-well plates using the Dharma-FECT4 transfection reagent (Dharmacon), according to

Bortezomib Synergizes CS-1008 via CIP2A

www.aacrjournals.org Mol Cancer Ther; 10(5) May 2011 893




the manufacturer’s instructions. After 48 hours, themediumwas replaced and theHCC cells were incubatedwith bortezomib, harvested, and separated for Westernblot analysis and for apoptosis analysis by flow cytome-try as described previously (17).

Sk-Hep1 with constitutive active CIP2ACIP2A cDNA (KIAA1524) was purchased from Ori-

gene (RC219918). Briefly, following transfection, cellswere incubated in the presence of G418 (0.78 mg/mL).After 8 weeks of selection, surviving colonies, that is,those arising from stably transfected cells, were selectedand individually amplified. Sk-Hep1 cells, with stableexpression of CIP2A-myc, were then treated with borte-zomib, harvested, and processed for Western blot analy-sis as described previously.

Coimmunoprecipitation assayCells were harvested and lysed on ice for 30 minutes in

lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 100 mmol/LNaCl, 0.5% Nonidet P-40, 50 mmol/L NaF, 1 mmol/LNa3VO4, 5 mmol/L sodium pyrophosphate, and a pro-tease inhibitor tablet). The cell lysates were centrifuged at14,000 � g for 15 minutes, and the supernatants wererecovered. Supernatants containing equal amounts ofproteins were incubated with 2.5 mg of primary antibo-dies overnight at 4�C. The immunoprecipitates wereharvested using protein G PLUS-agarose beads (SantaCruz Biotechnology) that were washed once with regularwashing buffer (50 mmol/L Tris-HCl, 100 mmol/LNaCl,1 mmol/L EDTA, and 0.5% Nonidet P-40), twice withhigh salt washing buffer (50 mmol/L Tris-HCl, 500mmol/L NaCl, 1 mmol/L EDTA, and 0.5% NonidetP-40),and once again with regular washing buffer. Immu-noprecipitates were then eluted by boiling the beads for 5minutes in SDS-PAGE sample buffer and characterizedby Western blotting with appropriate antibodies.

PP2A phosphatase activityThe protein phosphatase activity in total cellular lysate

was determined by measuring the generation of freephosphate from threonine phosphopeptide using themalachite green–phosphate complex assay as describedby themanufacturer (Upstate Biotechnology). Cell lysateswere prepared in a low-detergent lysis buffer (1% Non-idet P-40, 10 mmol/L HEPES, 150 mmol/L NaCl, 10%glycerol, 1 mmol/L phenylmethylsulfonyl fluoride,5 mmol/L benzamidine, and 10 g/mL leupeptin). Thephosphatase assay was done in a PP2A-specific reactionbuffer (Upstate) containing 750 mmol/L phosphopeptidesubstrate. After 10 minutes of incubation at 30�C, themalachite dye was added, and free phosphate was mea-sured by optical density at 650 nm. To avoid variabilitydue to differences in the amounts of immunoprecipitatedprotein between samples, the phosphatase activities werenormalized to the amount of PP2A immunoprecipitated,as detected and quantified by immunoblot analysis foreach treatment group.

Immunofluorescent stainingSk-Hep1 cells (wild-type or CIP2A-myc) were seeded

on sterilized slides in a 10-cm dish overnight and thentreated with DMSO or okadaic acid (wild-type) or for-skolin (CIP2A-myc) for 24 hours. The cells were washedwith PBS, fixed with 4% formaldehyde for 10 minutes at37�C, and thenwashedwith PBS twice. Cells were treatedwith 0.1% Triton X-100 and then blocked with 0.5%bovine serum albumin (BSA) in PBS at 37�C for 1 hour.Then cells were treated with anti-CIP2A or anti–p-Aktantibody (1:50 in PBS containing 0.5% BSA) at 4�C over-night. Fluorescein isothiocyanate–conjugated goat anti-mouse IgG (CIP2A; 1:1,000) or anti-rabbit IgG (p-Akt;1:1,000) was applied for 1 hour at room temperature. Thecells were observed under a fluorescence microscope(Leica DM2500).

Xenograft tumor growthMale NCr athymic nude mice (5–7 weeks of age) were

obtained from the National Laboratory Animal Center(Taipei, Taiwan). The mice were housed in groups andmaintained under standard laboratory conditions on a12-hour light-dark cycle. They were given access tosterilized food and water ad libitum. All experimentalprocedures using these mice were conducted in accor-dance with protocols approved by the InstitutionalLaboratory Animal Care and Use Committee ofNational Taiwan University. Each mouse was inocu-lated subcutaneously in the dorsal flank, with 1 � 106

HCC cells suspended in 0.1 mL of serum-free mediumcontaining 50% Matrigel (BD Biosciences). Whentumors reached 150 to 200 mm3, mice were randomizeddivided into 4 groups (n ¼ 8) and received an intraper-itoneal injection of bortezomib (0.5 mg/kg body weight)twice weekly and/or intravenous injection of CS-1008200 mg 3 times a week. Controls received vehicle con-sisting of 0.5% methylcellulose and 0.1% polysorbate 80in sterile water. Data are representative of 3 indepen-dent experiments.

Statistical analysisTumor growth data points are reported as mean tumor

volume � SE. Comparisons of mean values were doneusing the independent samples t test in SPSS for Win-dows 11.5 software (SPSS Inc.).

Results

Bortezomib enhances CS-1008–induced apoptosisin HCC cells

To investigate the antitumor effect of CS-1008 on HCCcells, we first examined the apoptotic effect of CS-1008 ina panel of 3 human HCC cell lines, Huh-7, Sk-Hep1, andHep3B at the clinical relevant concentrations. Apoptoticcells (sub-G1) were determined by flow cytometry after 24hours treatment. As shown in Fig. 1, HCC cells were quiteresistant to CS-1008 and treatment with CS-1008 alonecould not efficiently induce apoptosis in these HCC cells,

Chen et al.

Mol Cancer Ther; 10(5) May 2011 Molecular Cancer Therapeutics894




even up to a concentration of 800 ng/mL. However,combining bortezomib at 50 nmol/L with CS-1008 over-came the resistance and sensitized cells to apoptosissignificantly in a concentration-dependent manner, start-

ing from CS-1008 at a concentration of 200 ng/mL. Wethen examined the effect of a fixed concentration of CS-1008 on escalating concentrations of bortezomib andfound that CS-1008 at a dose of 200 ng/mL increased

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Figure 1. Bortezomib enhances CS-1008–induced apoptosis in HCC cells. A, concentration-escalation effects of CS-1008 with 50 nmol/L bortezomibon apoptosis in 3 TRAIL-resistant HCC cells. B, concentration-escalation effects of bortezomib with 200 ng/mL CS-1008. HCC cells were exposed tobortezomib and/or CS-1008 at the indicated concentrations in DMEM, with 5% FBS in 6-well plates for 24 hours, and apoptotic cells were assessed by flowcytometry. Points, mean; bars, SD (n ¼ 3). C, effects of bortezomib on DNA fragmentation in HCC. Cells were exposed to CS-1008 (200 ng/mL) and/orbortezomib at the indicated concentrations (nmol/L) in DMEMwith 5% FBS for 24 hours. Cell lysates were prepared and analyzed for cell death by ELISA. Dataare representative of 3 independent experiments. D, the chemical structure of bortezomib.






bortezomib-induced apoptosis in a concentration-depen-dent manner (Fig. 1B).

We further examined these apoptotic effects using anELISA kit which detects DNA fragmentation. Our datashowed that combination of CS-1008 and bortezomibincreased DNA fragmentation in a dose- and time-depen-dent manner (Fig. 1C). These results indicate that acombination of CS-1008 and bortezomib overcame theresistance of HCC cells to the TRAIL receptor antibody,CS-1008.

Downregulation of CIP2A is associated withbortezomib and CS-1008 combination treatment inHCC cells

Our previous data suggested that the sensitizing effectof bortezomib on TRAIL resistance of HCC cells wasmediated by the PP2A-Akt signaling pathway (14).Because CIP2A inhibits PP2A, we further hypothesizedthat inhibition of CIP2A may be associated with thesensitizing effect of bortezomib on CS-1008–inducedapoptosis in HCC cells. As shown in Fig. 2A, combinedtreatment of bortezomib and CS-1008 reduced proteinlevels of CIP2A, in correlation with downregulation of

p-Akt expression and induced apoptosis in all testedHCC cells. Evidence for the association between CIP2Ainhibition and apoptosis induction was further strength-ened by the time-dependent PARP cleavage in the com-bined bortezomib- and CS-1008–treated HCC cells(Fig. 2B). These results suggest that inhibition of CIP2Aplays a key role in the mediating effects of the drugcombination.

Target validation of CIP2ATwo different approaches were used to validate the

role of CIP2A in the sensitizing effect of bortezomib onCS-1008–induced apoptosis in HCC cells. First, weknocked down protein expression of CIP2A by usingsiRNA. Sk-Hep1 cells were transfected with eitherscrambled siRNA as a control or CIP2A siRNA for 48hours and were then exposed to DMSO or CS-1008 (200ng/mL) for another 24 hours. As shown in Fig. 3A,depletion of CIP2A by siRNA significantly reduced theresistance of CS-1008 in Sk-Hep1 cells in tandem withdownregulation of p-Akt (Ser473) and enhanced apopto-tic cell death. Notably, knockdown of CIP2A alone or incombination with CS-1008 showed similar effects on

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Figure 2. Downregulation ofCIP2A is associated withsensitizing effects of bortezomibin HCC cells. A, effects ofbortezomib on protein levels ofCIP2A, p-Akt, Akt, and Bcl-2family in HCC cells. HCC cellswere treated with bortezomib(100 nmol/L) and/or CS-1008(200 ng/mL) for 24 hours and celllysates were prepared for Westernblot. Data are representative of 3independent experiments. B,time-dependent analysis of CIP2Alevel and apoptotic death withbortezomib and CS-1008combination treatment. Sk-Hep1cells were exposed to bortezomiband/or CS-1008 for the indicatedperiod of time. Cell lysates wereprepared and assayed for CIP2A,p-Akt, Akt, and PARP. CF, cleavedform (activated form).

Chen et al.





downregulation of p-Akt, suggesting that adding CS-1008 does not enhance the effect of CIP2A on p-Akt.Next, we generated Sk-Hep1 (CIP2A-myc) cells withstably expressed CIP2A to investigate the sensitizingeffect of bortezomib. Ectopic expression of CIP2A sig-nificantly abolished the sensitizing effect of bortezomibon CS-1008–induced apoptosis in comparison with wild-type Sk-Hep1 cells that underwent the same treatment(P < 0.05; Fig. 3B). Together, these results validated theimportance of CIP2A inhibition in mediating the effect ofbortezomib on CS-1008 sensitivity to HCC cells.

CIP2A inhibits Akt-associated PP2A activityOur previous work showed that bortezomib increased

PP2A activity in HCC cells (14, 18). We, thus, nextexamined how bortezomib and CS-1008 combinationtreatment affected PP2A activity. As shown in Fig. 4A,bortezomib plus CS-1008 significantly upregulated PP2Aactivity in Sk-Hep1 and Huh-7 cells. Moreover, we foundthat treatment with either of the drugs alone or in com-bination did not significantly affect the protein level ofPP2A complex including subunit A, B56g , and C in Sk-Hep1 and Hep3B cells, except the drugs combinationdecreased PP2A-B55y in Huh-7 cells (Fig. 4B). These datasuggested that the alterations of PP2A complex may notplay a major role in mediating the sensitizing effect ofbortezomib on CS-1008–treated HCC cells. Notably, CS-1008 and bortezomib combination treatment, comparedwith either drug alone, did not alter protein–proteininteractions between Akt and PP2A significantly, sug-gesting that CIP2A does not affect their binding affinitydirectly (Fig. 4C). In addition, previous study has indi-

cated that CIP2A inhibits c-myc–associated PP2A activitythereby stabilizing c-myc (22). We, therefore, similarlyexamined Akt-related PP2A activity by coimmunopreci-pitation of Akt in Sk-Hep1 cells. Our data indicated thatectopic expression of CIP2A reduced PP2A activity onAkt, suggesting that CIP2A plays a role in regulating Akt-related PP2A activity (Fig. 4D, left). In addition, the dataof immunofluoresence staining also showed that theCIP2A-myc cells had higher p-Akt than the wild-typecells (Fig. 4D, right). The treatment of okadaic acid (aPP2A inhibitor) in wild-type cells enhanced the expres-sion of p-Akt, whereas forskolin (a PP2A agonist)reduced the expression of p-Akt in CIP2A-myc cells(Fig. 4D, right). These data indicate that CIP2A reducesthe PP2A activity on Akt in HCC cells.

Effect of bortezomib on HCC xenograft tumorTo confirm whether the sensitizing effect of bortezo-

mib on CS-1008 has potentially relevant clinical impli-cations, we assessed the in vivo effect of bortezomib andCS-1008 on Huh-7 xenograft tumors. Tumor-bearingmice were treated with vehicle or bortezomib at 0.5mg/kg i.p. twice a week and/or CS-1008 200 mg 3 timesa week for the duration of the in vivo experiment.Throughout the course of treatment, all mice toleratedthe treatments well without observable signs of toxicityand had stable body weights. No gross pathologicabnormalities were noted at necropsy. As shown inFig. 5A, tumor growth was significantly inhibited bytreatment with bortezomib plus CS-1008 for 2 weeks (vs.control, P < 0.05), and tumor size in the treatment groupwas only half that in the control group at the end of the

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Figure 3. In vitro target validation of CIP2A. A, downregulation of CIP2A by siRNA overcomes the resistance to CS-1008 in Sk-Hep1 cells. Bottom,protein levels of CIP2A. Top, analysis of apoptotic cells. Cells were transfected with either control siRNA or CIP2A siRNA for 48 hours and then exposedto CS-1008 (200 ng/mL) for 24 hours. For analysis of apoptotic cells (sub-G1), cells were analyzed by flow cytometry. Columns, mean; bars, SD (n ¼ 3).B, protective effects of ectopic expression of CIP2A on apoptosis induced by the combination of CS-1008 and bortezomib in Sk-Hep1 cells. Right,protein levels of Akt in both wild-type and Huh7-Akt cells. WT, wild-type. Left, analysis of apoptotic cells. Columns, mean; bars, SD (n ¼ 3). Sk-Hep1cells were transfected with CIP2A with myc-tag and were selected for 8 weeks by G-418. Analysis of apoptotic cells was done by flow cytometryafter cells were exposed to the combination of bortezomib 100 nmol/L and CS-1008 200 ng/mL for 24 hours.






study. Treatment with CS-1008 alone had no significanteffect on Huh-7 tumor growth (vs. control, P > 0.05) andbortezomib alone showed modest effect. Moreover, asshown in Fig. 5B, CIP2A protein levels decreased sig-nificantly in Huh-7 tumors treated with bortezomib incombination with CS-1008. Finally, an analysis of the

effect of bortezomib on PP2A activity (Fig. 5C) showed asignificant increase inPP2Aphosphataseactivity (P<0.05)in Huh-7 tumors treated with bortezomib alone and bor-tezomib plus CS-1008. However, the treatment of CS-1008alone did not show significant effects on CIP2A and thePP2A activity (vs. control, P > 0.05), which is consistent

DMSO60

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2A a

ctiv

ity(%

of c

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– –+ +

– – + +

– –+ +

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8 h 16 h 24 h

– –+ +

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– –+ +

– – + +

– –+ +

Figure 4. CIP2A inhibits Akt-associated PP2A activity. A, analysis of PP2A activity in HCC cells. Columns, mean; bars, SD (n ¼ 3). Cells (Sk-Hep1 orHuh-7) were treated with DMSO or bortezomib (Btz; 100 nmol/L) and CS-1008 (200 ng/mL) for 24 hours. Cell lysates were prepared for detecting PP2A activity.B, effects of bortezomib and/or CS-1008 on protein levels of PP2A complex. Cells were exposed to bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for24 hours. C, effects of bortezomib on the dynamic PP2A–Akt interactions in Sk-Hep1 cells. Cells were treated with bortezomib (100 nmol/L) and/orCS-1008 (200 ng/mL) for 24 hours, and cell lysates were immunoprecipitated with anti–PP2A-C antibodies. The immunoprecipitates were probed with Akt1and PP2A-C by Western blot. D, left, overexpression of CIP2A decreased Akt-associated PP2A activity. Cell lysates from Sk-Hep1 cells (wild-type orCIP2A-myc) were immunoprecipitated with anti-Akt1 antibodies. Cell lysates were prepared for detecting PP2A activity. Columns, mean; bars, SD (n ¼ 3).Right, CIP2A and p-Akt staining. Top, Sk-Hep1 wild-type cells; bottom, CIP2A overexpression cells. OA, okadaic acid (a PP2A inhibitor). Forskolin, a PP2Aagonist. Cells were treated with DMSO or OA (wild-type) or forskolin (CIP2A-myc) for 24 hours then stained with CIP2A (red) or p-Akt (green) and examinedunder a fluorescence microscope.

Chen et al.





with previous in vitro findings (Fig. 2A andFig. 3A). Thesedata indicate mediation of the effect via PP2A-dependentAkt inactivation in vivo. Further clinical investigation iswarranted.

Discussion

In this study, we confirmed that CS-1008, a novel anti-human DR5 antibody with promising TRAIL-like antic-ancer activity, is not sufficient to kill HCC cells as amonotherapy. However, we found that bortezomib effec-tively sensitizes HCC cells to this novel anti-DR5 anti-body. Importantly, we delineated the mechanism bywhich bortezomib sensitizes these resistant cells to CS-1008. Our results have several important implications.First, CIP2Amay serve as a potential drug target in HCC.We identified CIP2A inhibition as a major determinant ofsensitizing effects of bortezomib which is dissociatedfrom proteasome inhibition. This suggests that othernovel agents that are capable of downregulating CIP2Aprotein expression may also be good sensitizers ofTRAIL-resistant cells. Indeed, knockdown of CIP2A bysiRNA was able to restore CS-1008–induced apoptosis inresistant Sk-Hep1 cells (Fig. 3A). Moreover, Sk-Hep1 cellswith ectopic overexpression of CIP2A were more resis-tant to the effect of bortezomib and CS-1008 combinationtreatment (Fig. 3B). As an oncoprotein, CIP2A may pro-mote cancer cell aggressiveness, thereby facilitating resis-tance of cancer cells to apoptotic-inducing agents.Currently, there are no known specific CIP2A inhibitors

and whether CIP2A is suitable as predictive markers fordrug therapy remain to be determined. Future preclinicalresearch exploring agents that inhibit CIP2A and clini-copathologic studies correlating CIP2A expression anddrug sensitivity in HCC patients are warranted to furtherconsolidate CIP2A as a good drug target. Notably, thedata from a phase I study in human indicate that CS-1008is well tolerated and the Cmin of CS-1008 is 14 mg/mL(1 mg/kg/wk; ref. 26).

Various mechanisms contribute to TRAIL resistance incancers (12). Resistance to TRAIL can occur at any step inthe apoptosis signaling cascade. For example, receptorlevel mutations or overexpression of DR4 or DR5 can leadto resistance (12). Defects in members participating inDISC assembly, such as FADD, caspase-8, and cellularFADD-like interleukin-1b–converting enzyme inhibitoryprotein can also result in resistance to TRAIL (27, 28).Furthermore, overexpression of antiapoptotic Bcl-2family proteins (Bcl-2, Bcl-XL, Mcl-1, etc; refs. 29, 30),loss of proapoptotic protein function (Bax or Bak), and aninability to activate mitochondria during apoptosis,such as reduced release of mitochondria-derived acti-vator of caspases (SMAC/DIABLO), have been shownto cause TRAIL resistance in mitochondria-dependenttype II cancer cells (12, 31). Finally, aberrantly activatedantiapoptotic pathways in various tumor cells, such asphosphoinositol-3-kinase (PI3K)/Akt signaling, mito-gen-activated protein kinases pathway and NF-kBmay contribute to development of TRAIL resistance(12, 32, 33). Interestingly, we discovered that through

Figure 5. In vivo effect ofbortezomib on Huh-7 xeonograftnude mice. A, effects ofbortezomib and/or CS-1008 ontumor growth. Points, mean(n ¼ 6); bars, SE. Each mouse wasinoculated subcutaneously in thedorsal flank with 1 million Huh-7cells suspended in Matrigel. Whenthe tumor reached a volume of200 mm3, mice receivedtreatments of vehicle or 0.5 mg/kgbortezomib i.p. twice weekly and/or CS-1008 200 mg i.v. 3 times aweek for the duration of the study.B, Western blot analysis of CIP2Ain Huh-7 and PLC5 tumors.C, analysis of PP2A activity intumors. Values are means � SD(n¼ 3). The figure is representativeof 3 independent experiments.

Vehicle

P = 0.28

P = 0.18

P = 0.16

P = 0.04

P = 0.07

P = 0.10

P = 0.035P = 0.024

P = 0.005

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Bortezomib + CS-1008

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B C

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1,000

500

00 5 10

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3 )

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V Btz Btz + CsCs

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2A a

ctiv

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of c

ontr

ol)

100

120

140 P = 0.036P = 0.25

P = 0.011

CIP2A

Tumor treated with

p-Akt

Akt

Actin

Vehicle Bortezomib Btz + CSCS-1008






CIP2A-PP2A-p-Akt regulatory mechanism, bortezomibovercomes CS-1008 resistance, supporting the hypoth-esis that constitutively active Akt signaling confersresistance of HCC cells to TRAIL and anti-DR5 CS-1008.

Previous literature, including our own work, hasshown that bortezomib and other investigational protea-some inhibitors are capable of sensitizing cancer cells toTRAIL-induced apoptosis (14, 34–38). However, themolecular mechanisms underlying the sensitizing effectof bortezomib seem to be complicated and may be spe-cific to cancer types. In certain cancer types, sensitizationto TRAIL resistance has been reported to be throughproteasome inhibition, including upregulation of TRAILreceptors (34, 38), activation of both extrinsic and intrinsicapoptosis pathways (39, 40), and probably, many othermultiple molecular machineries. For example, Hetschkoand colleagues (34) showed that the proteasome inhibi-tors MG132, via enhancement of transcription and sur-face expression of DR5, potentiates TRAIL sensitivity andreactivates apoptosis in TRAIL-resistant high-grade glio-mas. In addition, our group showedthat bortezomib isable to sensitize TRAIL resistance by its proteasomeinhibition independent effects in TRAIL-resistant HCCcells. Importantly, despite the fact that TRAIL can acti-vate NF-kB (41, 42) and that bortezomib effectively inhi-bits NF-kB signaling, it seems that in HCC cells,inhibition of NF-kB may not be the major determinantof TRAIL sensitization by bortezomib as shown by ourprevious work (14, 17).

Numerous studies have shown that a combination ofchemotherapy and targeted agents can enhance the anti-tumor activityof TRAILand its agonists throughcross talkbetween the intrinsic andextrinsic apoptoticpathways (8).Among the many novel agents with potential to sensitizeor overcome TRAIL resistance, the combination of borte-zomib and CS-1008 represents a fascinating strategy forseveral reasons. Bortezomib is approved for clinical useand exerts particularly tolerable toxicity when combinedwith other cytotoxic chemotherapeutic agents (such asdoxorubicin, melphalan, or vincristine), and even whenused in combinationwith intensive salvage chemotherapyin patients with refractory hematologic malignancies (43,

44). CS-1008, for its part, although still under clinicalinvestigation, has shown an excellent toxicity profile withlow hepatic toxicity (9, 10, 45), which is particularlysuitable for patients with HCC. In addition, as CS-1008isDR5 specific, the potencyofCS-1008maynot be affectedby problems with DR4 mutations or dysfunction. DR5mutations have been reported to be infrequent in HCCcells (46). However, the exact machinery by which borte-zomib downregulates CIP2A is still unknown. It is pos-sible that bortezomib affects the transcription ortranslation of CIP2A through an as yet unidentifiedmechanism or affects the degradation of CIP2A at post-translational level. Future work is needed in this area.

In summary, bortezomib sensitizes HCC cells to CS-1008–induced apoptosis through inhibiting a noveloncoprotein phosphatase interactive mechanism, theCIP2A-PP2A-p-Akt framework, and CIP2A may be apotential molecular target for HCC treatment. More-over, combination of bortezomib, an agent with multi-ple cellular targets, and a specific anti-DR5 TRAILagonist (CS-1008) is a promising anti-HCC–targetedtherapy that warrants clinical trials. Future studiesdetailing the clinical role of CIP2A in HCC, and themachinery by which bortezomib affects CIP2A expres-sion may lead to further progress in the development ofmolecular-targeted therapy for HCC.

Disclosure of Potential Conflicts of Interest

A-L. Cheng is a consultant for Daiichi-Sankyo.

Grant Support

This study was supported by grants NTUH99-P04 from National TaiwanUniversity Hospital (K-F. Chen), V99-B1-016 from Taipei Veterans GeneralHospital (C-Y. Liu), NSC98-2314-B-002-067-MY3 (K-F. Chen), and NSC99-2314-B-002-017-MY2 (K-F. Chen) from the National Science Council, Taiwan.

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 August 24, 2010; revised January 12, 2011; accepted January29, 2011; published OnlineFirst March 10, 2011.

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2011;10:892-901. Published OnlineFirst March 10, 2011.Mol Cancer Ther Kuen-Feng Chen, Hui-Chuan Yu, Chun-Yu Liu, et al. Death Receptor 5 Antibody, through the Inhibition of CIP2ABortezomib Sensitizes HCC Cells to CS-1008, an Antihuman

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