Activation of the Wnt Pathway through AR79, a …...Oncogenes and Tumor Suppressors Activation of the Wnt Pathway through AR79, a GSK3b Inhibitor, Promotes Prostate Cancer Growth in

Oncogenes and Tumor Suppressors

Activation of the Wnt Pathway through AR79, a GSK3bInhibitor, Promotes Prostate Cancer Growth in SoftTissue and Bone

Yuan Jiang1,4, Jinlu Dai1, Honglai Zhang1, Joe L. Sottnik1, Jill M. Keller1, Katherine J. Escott5,Hitesh J. Sanganee5, Zhi Yao4, Laurie K. McCauley2,3, and Evan T. Keller1,3

AbstractDue to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and

sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3b) inhibits Wnt signaling by inducingb-catenin degradation, and a GSK3b inhibitor, AR79, is being evaluated as an osteoanabolic agent. However,Wntactivation has the potential to promote tumor growth; therefore, the goal of this study was to determine if AR79 hasan impact on the progression of prostate cancer. Prostate cancer tumors were established in subcutaneous and bonesites ofmice followed byAR79 administration, and tumor growth,b-catenin activation, proliferation, and apoptosiswere assessed. Additionally, prostate cancer and osteoblast cell lines were treated with AR79, and b-catenin status,proliferation (with b-catenin knockdown in some cases), and proportion of ALDHþCD133þ stem-like cells weredetermined. AR79 promoted prostate cancer tumor growth, decreased phospho-b-catenin, increased total andnuclear b-catenin, and increased tumor-induced bone remodeling. Additionally, AR79 treatment decreasedcaspase-3 and increased Ki67 expression in tumors and increased bone formation in normal mouse tibiae.Similarly, AR79 inhibited b-catenin phosphorylation, increased nuclear b-catenin accumulation in prostate cancerand osteoblast cell lines, and increased proliferation of prostate cancer cells in vitro throughb-catenin. Furthermore,AR79 increased the ALDHþCD133þ cancer stem cell–like proportion of the prostate cancer cell lines. Inconclusion, AR79, while being bone anabolic, promotes prostate cancer cell growth through Wnt pathwayactivation.

Implications: These data suggest that clinical application of pharmaceuticals that promote Wnt pathwayactivation should be used with caution as they may enhance tumor growth.Mol Cancer Res; 11(12); 1597–610.�2013 AACR.

IntroductionBone loss is a common pathology that accompanies

multiple diseases. Therefore, extensive research efforts havebeen aimed toward minimizing bone loss and have resultedin therapies that effectively decrease bone loss throughinhibition of bone resorption. In addition to minimizingbone resorption, methods to promote bone formation (i.e.,bone anabolic activity) have received great attention but

clinical therapeutics in this area lag behind antiresorptives(1). For example, in the United States, teriparatide (para-thyroid hormone, PTH 1-34) is the only U.S. Food andDrugAdministration (FDA)–approved bone anabolic agent.However, its use is limited to 2 years due to preclinicalfindings of osteosarcoma in the rat model. Thus, continuedefforts to identify bone anabolic agents are necessary toenhance efficacy and minimize their potential.The Wnt pathway mediates bone development (2) and

thus its manipulation serves as a potential bone anabolictherapy. Wnts mediate signaling through binding cell sur-face transmembrane low-density lipoprotein receptor-relat-ed (LRP5/6) and frizzled proteins, which results in b-cateninnuclear translocation and target gene activation (3). In theabsence of Wnts, b-catenin is degraded in the cytoplasmby interacting with a protein complex consisting of axin,adenomatous polyposis coli (APC), and glycogen synthasekinase 3b (GSK3b). Axin and APC are scaffold proteins thatfacilitate binding of b-catenin to GSK3b. GSK3b thenphosphorylatesb-catenin, which targets it for ubiquitinationand degradation. Due to the key role of GSK3b in degrading

Authors' Affiliations: Departments of 1Urology, 2Periodontics and OralMedicine, and 3Pathology, University of Michigan, Ann Arbor, Michigan;4Department of Immunology, TianjinMedical University, Tianjin, China; and5Emerging Innovations Unit, AstraZeneca R&D, Cheshire, United Kingdom

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

Corresponding Author: Evan T. Keller, Department of Urology, Universityof Michigan, 5308 CCGC, 1500 E. Medical Center Drive, Ann Arbor, MI48109-8940. Phone: 734-615-0280; Fax: 734-936-9220; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-13-0332-T

�2013 American Association for Cancer Research.

MolecularCancer

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b-catenin, inhibiting its activity should result in increasedb-catenin activity. Thus, GSK3b has been considered as atherapeutic target for induction of b-catenin activity, whichcould ultimately result in bone anabolic activity. Along theselines, other compounds that promote Wnt activity throughblocking inhibitors of Wnt signaling, such as sclerostin anddickkopf-1 (DKK1; ref. 4), are being evaluated for their boneanabolic effects, including the use of an antisclerostin anti-body in clinical trials (5).Although induction of bone anabolic activity through

increased Wnt pathway signaling has potential therapeuticbenefit, a major concern that should be considered is theimpact of chronic Wnt activation on cancer progression.Increased b-catenin expression has been associated withmultiple cancers (6, 7). Salmon calcitonin, which mediatesdegradation of GSK3b (8) and has been used therapeuticallyfor bone anabolic activity, has been associated with increasedcancer incidence, leading advisory panels of both the FDAto European regulatory committees to recommend dis-continuing its use for osteoporosis. The majority of Wntcancer-related action has been associated with early tumor-igenesis. However, Wnt signaling may also play a rolein established tumors, including maintenance of cancerstem cells (CSC; ref. 9). We have previously shown thatmodulation of the Wnt pathway with Wnt inhibitors,such as DKK1, promotes prostate cancer growth in softtissue and bone (10). Because of the potential for mod-ulation of the Wnt pathway to promote cancer, it is criticalto evaluate any Wnt pathway therapies for the impact oncancer biology.AR79 (AstraZeneca) is a potent inhibitor of GSK3b that

was developed as a potential bone anabolic agent. AR79upregulates b-catenin and bone mineralization in cell assaysand produces an increase in bonemass in vivo (11). As AR79modulates the Wnt pathway, we sought to determinewhether it could impact the progression of prostate cancerin soft tissue and bone.

Materials and MethodsCell cultureHuman prostate cancer cell lines DU145 and PC3 were

obtained from the American Type Culture Collection(ATCC) and cultured in RPMI-1640 (Invitrogen Co.)supplemented with 10% FBS and 1% penicillin–strepto-mycin (Life Technologies). The C4-2B cell line, which is anLNCaP subline (kindly provided by Dr. Leland Chung,Cedars Sinai, Hollywood, CA), was maintained in T medi-um [80% Dulbecco's Modified Eagle Medium (Life Tech-nologies), 20% F12 (Invitrogen), 100 U/L penicillin G, 100Ag/mL streptomycin, 5 mg/mL insulin, 13.6 pg/mL triio-dothyronine, 5 mg/mL transferrin, 0.25 mg/mL biotin, and25 mg/mL adenine] supplemented with 10% FBS. Thehuman colorectal adenocarcinoma cell line HCT116 waspurchased from ATCC and maintained in McCoy's 5aMedium (Gibco Technology) supplemented with 10%heat-inactivated FBS (HyClone), 100 U/mL penicillin,100 mg/mL streptomycin (Invitrogen Life Technologies),

2mmol/L L-glutamine (Invitrogen). TheMC3T3-E1 (cloneMC-4) cell line (kindly provided by Dr. Renny Franceschi,University ofMichigan, Ann Arbor,MI), a preosteoblast cellline derived from murine calvariae that, when treated withascorbate, expresses osteoblast-specific markers and pro-duces a mineralized matrix, was routinely maintained inMinimal Essential Medium Alpha (a-MEM) containing10% FBS and 1% penicillin–streptomycin (Life Technol-ogies). The ST2 cell line, a mouse bone marrow stromal cellline, was obtained from RIKEN Cell Bank and maintainedin a-MEM (Invitrogen) supplemented with 10% FBS, 1%penicillin–streptomycin (Life Technologies), and 2 mmol/LL-glutamine (Invitrogen). All cultures were maintained at37�C, 5% CO2, and 100% humidity. Luiferase containingvariants of the prostate cancer cell lines were made aspreviously described (12). Briefly, C4-2B and DU145 weretransduced with retrovirus encoding the luciferase gene andselected using G418. Stable expression of luciferase wasconfirmed using bioluminescent imaging (BLI). Cell iden-tities were confirmed using short tandem repeat mapping(Supplementary Table S1).

siRNA transfectionC4-2B and DU145 were plated at a density of 5� 105 on

100 mm plates and then transfected with 100 nmol/L of twodifferent sequences of b-catenin siRNAs (Cell SignalingTechnology; signalSilence b-catenin siRNAI&II,6225,6238)or scrambled control siRNA (Cell Signaling Technology;signalSilence control siRNA,6568) using LipofectamineRNAiMAX Reagent (Invitrogen, 13778). Transfection con-ditions were adjusted according to the manufacturer's guide-lines. After transfection for 72 hours, the cells were treatedwith AR79 (3 mg/mL) and rhWnt3a (60 ng/mL; R&DSystems) for 4 hours. Nuclear and cytoplasmic protein wasextractedusingNE-PERNuclear andCytoplasmicExtractionReagents (Thermo Scientific, 78835) following the manu-facturer's instructions.

Cell viability assayDU145 and C4-2B cells and cells transfected with b-cate-

nin siRNA were cultured in 96-well plates for overnight andthen cells were treated with AR79 (3 mg/mL) and rhWnt3a(60 ng/mL; R&D Systems) or vehicle [dimethyl sulfoxide(DMSO) or PBS] for 24, 48, and 72 hours. Cell proliferationreagentWST-1 (Roche, 1644807) was added and incubatedat 37�C and 5% CO2 for 4 hours. Absorbance was thenmeasured at 440 nm with a plate reader (Multi-ModeMicroplate Reader, SpectraMax M5, Molecular Devices;MDS Analytical Technologies).

TRACP 5b and osteocalcin assaysWhole blood was obtained and centrifuged to obtain

serum that was frozen at �80�C until assayed. MouseTRACP 5b (tartrate-resistant acid phosphatase 5b) andosteocalcin inmouse serumweremeasured using theMouse-TRAP Assay (Immunodiagnostics Systems Inc.) and theMouse Osteocalcin Assay (Biomedical Technologies Inc.),respectively, as recommended by the manufacturer.

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Caspase-3/7 assayC4-2B and DU145 were cultured into black 96-well

plates at a concentration of 1 � 104 (2 � 105 cell/mL,50 mL) per well. Cells were treated with 50 mL AR79 (0.03,0.3, and 3 mg/mL) or vehicle (DMSO) or etoposide (100nmol/L; Sigma) as a positive control after 12 hours and wereallowed to grow for 24 hours, then 100 mL of Apo-ONECaspase-3/7 Reagent (Promega) was added to each well andthe cells were incubated for extended periods (>4 hours).Absorbance was determined at 499 and 521 nmusing a platereader (Multi-Mode Microplate Reader, SpectraMax M5,Molecular Devices; MDS Analytical Technologies).

Bioluminescence imaging in vitroC4-2Bluc and DU145luc cells were plated into black,

clear bottom 96-well plates (Costar) at a concentration of3� 103 (for 24 and 48 hours test) or 2� 103 (for 72 hourstest) per well in RPMI medium with 3% FBS. In someinstances, after 12 hours, ST2 cells were added and platedwith C4-2Bluc or DU145luc at a 1:6 or 1:3 ratio andcocultured for 12 hours. C4-2Bluc or DU145luc withoutST2was used as vehicle control group. The cells were treatedwith 0.03, 0.3, and 3 mg/mL AR79 and vehicle (DMSO),andwere allowed to grow for 24, 48, and 72 hours, then 5mLluciferin (40 mg/mL; Regis Technologies Inc.) was added toeach well 5 to 10 minutes before imaging. BLI signal wasquantified (IVIS System; Caliper Life Sciences). To deter-mine the correlation between cell number and BLI signal,C4-2Bluc and DU145luc were seeded at the concentrationfrom 5 � 103 to 2 � 104 into black, clear bottom 96-wellplates in RPMI with 3% FBS for 24 hours, and BLI signalwas measured.

Protein extraction and Western blot analysisHT116, C4-2B, ST2, andMC3T3-E1 cell lines (1� 106

or 5� 106 cells/dish) were cultured in 100 mm dishes (1�106 cells/dish for whole-cell protein, or 5 � 106 for nuclearprotein extraction). After 12 hours, the culture medium waschanged to FBS-free medium for 12 hours and then the cellswere treated with AR79 (0.03, 0.3, and 3 mg/mL) for 0, 15,30, 60, and 120 minutes. Also, rhWnt3a (60 ng/mL) wasused as positive control (R&D Systems). Whole-cell proteinwas isolated as described previously (13). Nuclear proteinwas extracted using NE-PER Nuclear and CytoplasmicExtraction Reagents (Thermo Scientific, 78835) followingthe manufacturer's instructions. For tissue protein extrac-tion, the tumor tissues were homogenized in ice-cold RIPA(radioimmunoprecipitation assay) lysis buffer (Millipore).The tissue lysates were incubated at 4�C for 1 hour withrotation, followed by clarification of tissue debris by centri-fugation at 12,000 �g rpm for 10 minutes. The proteinconcentration of tumor extracts was determined using theBCA Protein Assay Kit (Pierce). The level of phosphorylatedb-catenin and b-catenin was analyzed using Westernblot analysis, which was done as previously described (13),with rabbit anti-human phospho-b-catenin (p-b-catenin)monoclonal antibody (mAb; 1:1,000; Invitrogen), mouseanti-human b-catenin mAb (1:1,000; Santa Cruz Bio-

technology), mouse anti-human a-tubulin mAb (1:2,500;Sigma-Aldrich), rabbit anti-hnRNP polyclonal antibody(1:1,000; Santa Cruz Biotechnology). The antibody bind-ing was revealed using an HRP-conjugated anti-rabbit IgG(1:3,000; Cell Signaling Technology), or anti-mouse IgG(1:3,000; Amersham Pharmacia Biotech). Antibody com-plexes were detected using SuperSignal West Pico Chemi-luminescent Substrate or SuperSignal West Dura Extend-ed Duration Substrate (Thermo Scientific) and exposureto X-Omat film (Kodak). The densities of the Westernblot analysis bands were quantified using ImageJ software(version 1.40; NIH, Bethesda, MD).

HIPK2 analysisFor immunoprecipitation of homeodomain-interacting

protein kinase 2 (HIPK2), total protein (500 mg) wasincubated with a polyclonal rabbit anti-human HIPK2antibody (1 mL; Abcam) for a minimum of 1 hour at 4�C.Antibody-bound protein complexes were recovered viabinding to mixed 50 mL protein A and protein G-Sepharosebeads (Millipore) and subsequently incubated overnight at4�C with gentle agitation. Afterward, the sepharose-boundimmunoprecipitates were centrifuged at 8,000�g rpm for 2minutes followed by several washing steps with immuno-precipitation buffer. Immune complexes were eluted frombeads into 30 mL of PBS and subsequent heating to 95�C for10minutes. After centrifugation, the supernatant containingthe immunoprecipitated proteins was separated by SDS–PAGE (8%) and blotted onto polyvinylidene difluoridemembrane (Bio-Rad). After blocking for 1 hour in 5%bovine serum albumin in TBS-T (TBS containing 0.05%Tween 20), blots were incubated with monoclonal mouseanti-human phosphortyrosine antibody (1:1,000; Cell Sig-naling Technology) overnight at 4�C followed by detectionof binding by anti-mouse IgG (1:3,000; Amersham Phar-macia Biotech). Antibody complexes were detected usingSuperSignal West Pico Chemiluminescent Substrate orSuperSignal West Dura Extended Duration Substrate(Thermo Scientific) and exposure to X-Omat film (Kodak).

In vivo prostate cancer modelsSeven-week-old male NOD.CB17-Prkdcscid/NCrCrl

(nonobese diabetic/severe combined immunodeficient,NOD/SCID) mice (Charles River Laboratories) were usedand housed under pathogen-free conditions. All experimen-tal protocols were approved by the University of MichiganAnimal Care and Use Committee (Ann Arbor, MI). Forsubcutaneous injection, single-cell suspensions (1 � 106

cells) of C4-2Bluc cells in T medium were injected in theflank at 100 mL per site using a 27-G3/8-inch needle underanesthesia with 2.5% isofluorane/air. Single-cell suspensions(3 � 105 cells) of DU145luc cells with Matrigel (BDBiosciences) were injected in the flank at 100 mL per siteusing a 27-G3/8-inch needle. Subcutaneous tumor growthwas monitored by palpation. Subcutaneous tumors weremeasured using BLI weekly. The tumor weights of theDU145luc injection group were measured at the end of thestudy. Animals in the C4-2Bluc injection group were

GSK3b Inhibitor Promotes Prostate Cancer Growth

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sacrificed at the end of 6 weeks and animals in theDU145lucinjection group were sacrificed at the end of 9 weeks whentumors were harvested. Half of each tumor was kept forhistologic analysis and the other half flash-frozen for molec-ular analysis. For intratibial injection, mice were anesthe-tized with 2.5% isofluorane/air, and both legs were cleanedwith betadine and 70% ethanol. The knee was flexed, and a27-G3/8-inch needle was inserted into the proximal end ofright tibia followed by injection of 20 mL single-cell suspen-sions of C4-2Bluc cells (5� 105 cells). Tumor developmentin bone was evaluated weekly using BLI (methodologydescribed in BLI section below) and radiography. All animalswere sacrificed at the end of 9 weeks. At the time ofeuthanasia, serumwas collected and necropsy was performedto evaluate for gross tumor lesions. Both tumor-injected andcontralateral tibiae were collected, placed in formalin, radio-graphed and subject to dual-energy X-ray absorptiometry(DEXA), followed by processing for histologic analysis.

ImmunohistochemistryBoth tibiae and subcutaneous tumors were fixed in 10%

neutral buffered formalin for 24 hours. The tibiae were thendecalcified for 48 hours in 10% EDTA and then both tibiaeand subcutaneous tumors were processed for paraffinembedding. Of note, 5 mmol/L sections were used forHematoxylin and eosin staining and immunohistochemis-try. Nonstained sections were deparaffinized and rehydratedthen stained for Ki67 (1:500; Santa Cruz Biotechnology),caspase-3 (1:200; Cell Signaling Technology), andb-catenin(1:500; BD Biosciences).

Bioluminescence imaging in vivoMice were injected intraperitoneally with 100mL luciferin

(40 mg/mL in PBS), anesthetized with 1.5% isoflurane, andimaged 15 minutes after luciferin injection on the IVIS BLIsystem (Caliper Life Sciences) as previously described (14).Mice were imaged weekly after tumor injection to monitortumor development. Bioluminescence generated by theluciferin/luciferase reaction served as a locator for cancergrowth and was used for quantification using the LivingImage software on a red (high intensity/cell number) to blue(low intensity/cell number) visual scale. A digital grayscaleanimal image was acquired followed by acquisition andoverlay of a pseudocolor image representing the spatialdistribution of detected photon counts emerging from activeluciferase within the animal. Signal intensity was quantifiedas the sum of all detected photons within the region ofinterest during a 1-minute luminescent integration time.

Dual-energy X-ray absorptiometry measurementBone mineral content (BMC) and bone mineral density

(BMD) of the excised tibiae were measured using DEXA onan Eclipse peripheral DEXA as previously described (13).Briefly, excised bones were subject to DEXA using pDEXASabre software version 3.9.4 in research mode (NorlandMedical Systems). Bones were scanned at 2 mm/s with aresolution of 0.1� 0.1 mm. Three 0.5 cm regions of interestwere randomly selected for each fragment to determine BMC

and BMD. Short-term precision (percentage of coefficient ofvariation) was approximately 3% for this technique.

Radiographic analysisMagnified flat radiographs of hind limbs were obtained

using a Faxitron machine (Faxitron X-ray Corp.).

Flow cytometry analysis of ALDH and CD133expressionC4-2B and DU145 cells were dissociated with Accutase

(Innovative Cell Technologies, Inc.) For aldehyde dehydro-genase (ALDH) staining, cells were subjected to the ALDE-FLUOR Assay Kit (STEMCELL Technologies, Inc.)according to the manufacturer's protocol. For CD133 stain-ing, the cells were incubated with CD133/1 (AC133)-PEantibody (Miltenyi Biotec). One microgram per milliliter of7-AAD (Sigma-Aldrich) was added to the samples beforeflow analysis to facilitate dead cell discrimination. Sampleswere analyzed on MoFlo XDP flow cytometer (BeckmanCoulter, Inc.).

AR79 treatment in vivoAll animal studies were approved by the University of

Michigan Animal Use and Care Committe. AR79 (Astra-Zeneca) was used at 15 and 50mg/kg orally once daily basedon in vivo studies in mice that demonstrated its inhibitedGSK3 activity and increased bone mass in femurs (11).AR79 was formulated fresh daily in 0.5% hydroxypropyl-methyl cellulose (HPMC; Sigma-H7509) with 0.1%Tween80 (Sigma-P8074) in water. Vehicle consisted of the carrier(0.5% HPMC/0.1% Tween 80 in water).

Statistical analysesAll in vitro experiments were performed at least three

times. Numerical data are expressed as mean � SD. Statis-tical analysis was performed by analysis of one-way ANOVAand/or Student t test for independent analysis. The value P <0.05 was considered statistically significant.

ResultsTo determine whether Wnt activation promotes prostate

cancer cell growth in soft tissue in vivo, we injected C4-2Bcells into the subcutaneous tissue of mice and treated themice with either vehicle or AR79 using low (15 mg/kg) andhigh (50 mg/kg) doses. Both doses of AR79 promotedprostate cancer growth over a 42-day period (Fig. 1A andB). We also evaluated Du145 prostate cancer cells usingAR79 (50mg/kg). Similar to the C4-2B cells, AR79 inducedDu145 cell growth (Fig. 1C–F). In agreement with the BLIfindings, individual tumor weights were greater in theDu145 tumors from mice that received AR79 (Fig. 1E andF). Mouse body weights were not impacted by treatment(Supplementary Fig. S1).In the basal state, GSK3 promotes phosphorylation and

degradation of b-catenin. WhenWnts are present, b-catenindissociates from the GSK3 complex and is not phosphory-lated, thus avoiding degradation resulting in increased b-cate-nin levels. AR79 mediates its activity through inhibiting

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GSK3, which results in decreased b-catenin phosphorylationand increased nuclear b-catenin. Consequently, we nextassessed whether AR79 impacted b-catenin expression intumor tissues by measuring total and phospho-b-catenin.AR79 increasedb-catenin in bothC4-2B (Fig. 2A andB) andDu145 cells (Fig. 2D and E) and decreased total phospho-b-catenin expression in both C4-2B (Fig. 2A and C) andDu145 (Fig. 2D and F) cells. We evaluated additionalprostate cancer cell lines (PC-3, ARCaP, and LNCaP) andin all instances, AR79 reduced phospho-b-catenin andincreased total b-catenin (Supplementary Fig. S2).In addition to impacting Wnt signaling, in vitro chemical

pharmacology screening studies with AR79 have shown thatthis compound also inhibits HIPK2 with equipotencycompared with GSK3b (11). To determine whether AR79impacted HIPK2 in prostate cancer, we exposed multipleprostate cancer cell lines to AR79 and measured changes inphospho-HIPK2 expression. AR79 had no impact on phos-pho-HIPK2 expression, whereas it decreased GSK3 and

phospho-b-catenin and increased total b-catenin (Supple-mentary Fig. S2).To determine whether the observed effects of Wnt acti-

vation on tumor in soft tissue also extended to bone, themost common site of prostate cancer metastases, we injectedC4-2B cells into the tibiae of mice and treated the mice witheither vehicle or AR79. Similar to tumors in soft tissue sites,AR79 at both 15 and 50 mg/kg induced C4-2B tumorgrowth in bone (Fig. 3A andB). The increased tumor growthwas accompanied by increased tumor-induced radiographicchanges in the tibiae (Fig. 3C). AR79 in nontumor-bearingtibiae had no impact on the radiographic appearance of thetibiae (Supplementary Fig. S3A). To better define the impactof AR79 on tumor-induced bone changes, we subjected thetibiae to DEXA scan to measure bone mineral changes. Theadministration of AR79 had no impact on BMD but wasassociated with decreased BMC in the tibiae containingtumors (Fig. 3D and E), indicating an overall loss of bone. Incontrast, AR79 (50 mg/kg) induced an increase in both

Figure 1. AR79 promotes prostate cancer cell growth in soft tissue in vivo. NOD/SCIDmicewere injected subcutaneously (SQ)withC4-2Bluc (1�106, in 100 mLT medium) and DU145luc (3 � 105 with Matrigel, in 100 mL RPMI). After establishment of tumor growth, treatment with either AR79 or vehicle was initiatedand continued 6 weeks for C4-2Bluc and 9 weeks for DU145luc. Mice were imaged weekly using BLI after tumor injection to monitor tumor development.There were 15 NOD/SCID mice in the C4-2Bluc injection group (50 mg/kg of AR79, n ¼ 5; 15 mg/kg of AR79, n ¼ 5; vehicle, n ¼ 5) and 20 NOD/SCIDmice in the DU145luc injection group (50 mg/kg of AR79, n ¼ 10; vehicle, n ¼ 10). A and C, representative images of BLI. Note the increased signals in theAR79-treated SQ implanted compared with vehicle-treated mice. B and D, quantification of tumor volume as measured using BLI. �, P < 0.05 versus vehicle.E, pictures of the SQ DU145luc tumors. F, tumor weight of the DU145luc injection group.






BMD and BMC in tibiae without tumor (SupplementaryFig. S3B and S3C), indicating an increase in bone mineralwithin the bone itself. The AR79-induced tumor-mediatedbone loss was associated with an overall increase of serumTRACP 5b, amarker of osteoclast activity (Fig. 3F), and hadno impact on serum osteocalcin, a marker of bone turnover(Fig. 3G).To explore cellular mechanisms that could account for

AR79-mediated promotion of tumor growth, we assessedthe tumor tissues for the expression of Ki67, a marker ofcellular proliferation; caspase-3, a marker of apoptosis; andnuclear b-catenin. In both the C4-2B and Du145 subcu-taneous tumors and the C4-2B intratibial tumors, theadministration of AR79 increased Ki67 and decreased cas-pase-3, indicating that it overall promoted tumor growththrough both increased proliferation and decreased apopto-sis (Fig. 4A and B). In addition, AR79 induced nuclearb-catenin expression in all of these tumors, indicating thatthe Wnt pathway was activated (Fig. 4A and B).The in vivo results suggested that AR79 stimulated pro-

liferation of prostate cancer cells. To determine whetherthere was a direct effect of AR79 on prostate cancer orwhether bone stromawas required for the proliferative effect,we exposed prostate cancer cells alone or in coculture withST2 stromal cells. AR79 at 0.03 and 0.3 mg/mL had noimpact; whereas, at 3.0 mg/mL induced C4-2B cell growthnearly 100% in vitro at both 48 and 72 hours (Fig. 5); buthad no effect at any dose at 24 hours (not shown). Theaddition of ST2 cells at ratios of 3:1 or 6:1 (ST2:C4-2B) inthe culture had no impact on cell number or AR79 effects

(Fig. 5). To ensure that the effect of AR79 was not specific toC4-2B cells, the study was repeated with Du145 cells.Similar to the results observed with C4-2B cells, AR79promoted Du145 cell growth (albeit only at 0.3 mg/mL)after 48 and 72 hours (Supplementary Fig. S4), but had noimpact at 24 hours (not shown). These results confirm thatAR79 directly induces prostate cancer growth.We next determined whether the impact of AR79 on the

Wnt pathway observed in vivo extended to C4-2B prostatecancer cells and the ST2 and MC3T3-E1 bone stromal celllines in vitro. AR79 (3 mg/mL, but not lower doses)decreased phospho-b-catenin and increased total b-cateninin all cell lines evaluated, including the colon cancerHCT116 cell line that was used as a positive control (Fig.6A). This effect was not observed until 60 minutes aftertreatment and continued through at least 120 minutes aftertreatment (Fig. 6B). A corresponding increase in nuclearb-catenin was observed in all cell lines evaluated, which wassimilar to the increase observed when the cells were treatedwith Wnt3a as a positive control (Fig. 6C and D; see NE fornuclear extracts).As kinase inhibitors can have off-target effects, we further

evaluated whether the proliferation-inducing activity ofAR79 was dependent on upregulation of b-catenin. Weused two different siRNA constructs to knockdown b-cate-nin expression in Du145 and C4-2B cells. Both constructsmediated more than 80% decreased in b-catenin proteinexpression in both cell lines compared with scrambledcontrol (Supplementary Fig. S5). To ensure that knockdownwas functional during administration of AR79, we assessed

Figure2. AR79downregulates phosphorylation ofb-catenin andupregulates total b-catenin in soft tissue prostate cancer tumors. Tumor tissues from themicedescribed in Fig. 1 were harvested, total protein extracted and subjected to Western blot analysis for phospho-b-catenin and total b-catenin. A, C4-2Bluc

Western blot analysiswith four different samples for each treatment shown. B, phospo-b-catenin and (C) total b-catenin band densities from theC4-2Bluc cellswere quantified using ImageJ software. Results were normalized to tubulin band density. D, Du145luc Western blot analysis with four different samplesfor each treatment shown. E, phospo-b-catenin and (F) total b-catenin band densities from the Du145luc cells were quantified using ImageJ software. Resultswere normalized to tubulin band density.

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the expression of nuclear b-catenin in the siRNA transfectedcells. Administration of Wnt3a or AR79 increased nuclearb-catenin in the scrambled siRNA control cells lines com-pared with vehicle-treated cells; whereas neither Wnt3a norAR79 increased nuclear b-catenin in the b-catenin siRNAcells compared with vehicle (Supplementary Fig. S3B).These results demonstrate that knockdown of b-cateninabrogates the ability of AR79 to increase nuclear b-catenin.To next assess whether blocking the ability of AR79 toinduce b-catenin expression impacts its ability to promoteprostate cancer proliferation, we evaluated the effect of AR79on the cells with knockdown of b-catenin. At 24 hours,neither Wnt3a nor AR79 affected cell growth, as previouslyobserved; however, at 48 and 72 hours, both Wnt3a andAR79 induced cell growth in the parental and scrambled

siRNA cell lines and this effect was completely inhibited byknockdown of b-catenin (Fig. 7).Wnts have been shown to promote maintenance of CSC-

like cells (15–17; also called tumor initiating cells), indicat-ing that modulation of Wnt activity may impact CSC. Thedefinition of prostate cancer CSC is constantly being refined;however, the presence of positive staining for ALDH (18,19) and CD133 (20, 21) has been associated with CSC-likephenotypes. Therefore, to determine whether AR79 mod-ulated stemness of the prostate cancer cell population,we assessed for the impact of AR79 on the proportion ofALDHþ/CD133þ cells in the C4-2B and Du145 popula-tions. AR79 increased the proportion of ALDHþ/CD133þ

cells by approximately 100% and 200% in C4-2B (Fig. 8Aand C) and Du145 (Fig. 8B and D), respectively.

Figure 3. AR79 promotes the development of prostate cancer cell growth in bone. C4-2Bluc cells (5 � 105, in 20 mL T medium) were injected into the tibiae ofNOD/SCID mice (n¼ 40). After establishment of tumors, treatment with either AR79 (50 mg/kg of AR79, n¼ 14; 15 mg/kg of AR79, n¼ 14) or vehicle (n¼ 12)was initiated and continued for 9 weeks. Mice were euthanized, bones were subjected to Faxitron X-ray analysis and DEXA, and blood was collectedand separated into serum that was subjected to bonemarker analysis (TRACP 5b and osteocalcin). A, representative BLI pictures. Note the increased signalsin the tibiae of the AR79-treated mice compared with vehicle-treated mice. B, total photons were measured using BLI weekly after tumor injection tomonitor tumor development. �,P < 0.05 AR79 versus vehicle;P valuewas determined by aStudent t test. C,microradiographs revealed increased presence oftumor-induced bone changes in the tibiae from mice treated with AR79. D, representative DEXA scans. Note decreased bone mineral in the AR79-treatedmice. E, BMD and BMC of tibiae with tumors. F, serum TRACP 5b and (G) serum osteocalcin were measured using ELISA.






DiscussionInduction of bone anabolic activity is a major therapeutic

goal to diminish bone mineral loss. Toward that end,systemic administration of PTH (22, 23) and local admin-istration of bone morphogenetic proteins with boneimplants (24) have been used for their bone anabolicproperties. However, because these therapies have limita-tions, including label warnings of cancer, there is a need toidentify additional bone anabolic agents. In this light,manipulation of the Wnt pathway is being explored as apotential therapy due to the ability of Wnts to induce boneformation.

Wnt signaling plays a central role in osteoblast develop-ment and bone formation (25–27). Wnts promote thelineage commitment of mesenchymal precursor cells andthe differentiation of progenitor cell lines into osteoblasts.They also stimulate direct effects on the formation andturnover of the mature skeleton. Mutations in componentsof the Wnt pathway are associated with human skeletaldiseases and variation of BMD (28–31), demonstrating thefunctional relevance of Wnt pathway for human bonebiology.Canonical Wnt signaling is mediated through increas-

ing nuclear b-catenin levels (3). GSK3b is a key regulator

Figure 4. AR79 increases Ki67, decreases caspase-3, and induces nuclear b-catenin expression in C4-2B and DU145 subcutaneous (SQ) tumors andC4-2B intratibial tumors. A, immunhistochemical analysis of paraffin sections from the SQ and intratibial tumors was performed using antibodiesto Ki67, caspase-3, and b-catenin. B, positive staining cells were counted in four random�40 fields from each section. Results are reported as percentage ofpositive cells compared with control.

Jiang et al.





of Wnt signaling through its ability to phosphorylate andtarget b-catenin for ubiquitination and degradation. Thus,inhibition of GSK3b is considered to be a viable andpromising strategy to increase Wnt pathway activitythrough promotion of nuclear b-catenin expression. Inagreement with the role of Wnts on bone production andinhibition of GSK3b on Wnt signaling, an orally availableGSK3b inhibitor was shown to increase bone mass in rats(32). AR79 was developed to block GSK3b activity andhence induce bone anabolic activity as demonstrated bythe stimulation of osteoblastogenesis with AR79 in vitroand a bone anabolic phenotype in rodents in vivo (11).However, before clinical evaluation of its bone anabolicpotential, it was deemed important to determine itsimpact on prostate cancer, which is known to be influ-enced by Wnt signaling (33, 34).Wnts may play a role in promotion of prostate cancer

progression through several mechanisms. For example,Wnts, through b-catenin activation, have been shown toinfluence expression of the androgen receptor, a key regu-lator of prostate cancer (35). Furthermore, GSK3b has been

shown to regulate the progression of prostate cancer to theadvanced castration resistant form (36). In addition, it hasbeen suggested that the activation of GSK3bmay inhibit theproliferative effects of Wnt and b-catenin in prostate cancer(36). We have observed that as prostate cancer progresses tothe bone metastatic phenotype, expression of the endoge-nousWnt inhibitor DKK1 decreases, allowing for enhancedWnt activity (37). This in turn can account for the osteo-blastic phenotype of prostate cancer as demonstrated by theobservation that the prostate cancer mediates bone produc-tion through activation of the Wnt receptor LRP5 (38).Taken together, these findings indicate that any therapy thatmodulates Wnt signaling could have an impact on prostatecancer progression.Our results clearly demonstrate that AR79 promoted

prostate cancer growth in soft tissue and bone in a murinemodel of prostate cancer. Furthermore, the increase ofprostate cancer growth was associated with decreased phos-pho-b-catenin expression and increased total b-cateninexpression. In addition, in vitro exposure of prostate cancercells to AR79 decreased phospho-b-catenin and increased

Figure 5. AR79 increases numbers of C4-2Bluc cells in the presence or absence of ST2 coculture. C4-2Bluc cells were grown alone or cocultured withST2 at 6:1 and 3:1. Then cells were treatedwith different concentrations (0.03, 0.3, and 3 mg/mL) of AR79 or vehicle (DMSO). Luciferase activity wasmeasuredafter 24 (not shown), 48, and 72 hours by BLI. Data are presented as the mean � SD from triplicate determinations.






nuclear b-catenin, providing further evidence that AR79effectively activates theWnt pathway. Taken together, theseresults suggest that AR79 promotes prostate cancer growththrough activation of the canonical Wnt pathway due to itsability to block GSK3b activity. Our findings seem toconflict with an earlier report in which the GSK3 inhibitorCHIR99021 decreased growth of the 22Rv1 human pros-tate cancer cell line in vitro (39). However, differences in celllines and compounds could account for differences. Inaddition, that report did not evaluate the in vivo impact ofCHIR99021, which is critical for a thorough assessmentdue to the importance of microenvironment on cancergrowth.To determine cellular mechanisms through which

AR79 promoted prostate cancer growth, we evaluatedKi67expression, a marker of proliferation, and caspase-3/7, markers of apoptosis. The fact that AR79 adminis-

tration was associated with both an increase of Ki67 and adecrease of caspase-3/7 expression in the tumor tissueindicated that it promoted overall tumor growth throughboth induction of cellular proliferation and inhibition ofapoptosis. This finding is consistent with activation of theWnt pathway, as Wnts are known to induce proliferationand inhibit apoptosis in a variety of cell systems (40). Itshould be pointed out that we measured these parametersonly at the time of sacrifice; thus, this may not have beenthe optimal to time to see the maximal changes. To furtheridentify whether AR79 modified prostate cancer cellproliferation and apoptosis directly, we evaluated itsimpact on prostate cancer cells in vitro. However, weonly identified an increase in cell proliferation and noimpact on apoptosis. This finding suggests that AR79modulates apoptosis in vivo through an indirect mecha-nism in the tumors. However, it may be that the basal

Figure 6. AR79 downregulates phosphorylation of b-catenin and upregulates total b-catenin nuclear translocation in prostate cancer and bone stromal celllines. A, cells were treated with the indicated concentration of AR79 or Wnt3a (60 ng/mL). After 3 hours, total protein was isolated and subjected toWestern blot analysis. B, cells were treated with 3 mg/mL AR79 for the indicated times at which time total protein was isolated and subjected toWestern blot analysis. C, cells (5 � 106) were serum-starved for 12 hours and then treated with AR79 at 3 mg/mL. Nuclear protein was extracted usingNE-PER Nuclear and Cytoplasmic Extraction Reagents and analyzed for b-catenin by Western blot analysis. CE, cytoplasmic extract; NE, nuclear extract.D, the densities of Western blot analysis bands were quantified with ImageJ software (version 1.40; NIH).

Jiang et al.





apoptosis rate in a two-dimensional culture in vitro, whichis well oxygenated, is much lower than that in a three-dimensional tumor in vivo, which will have areas ofhypoxia and presumably more endogenous apoptosis.Thus, it is possible we could not identify an antiapoptoticeffect in vitro due to differences in the growth environ-ment of the prostate cancer cells.Because in vitro pharmacology studies with AR79

indicated that this compound also inhibits HIPK2 withequipotency compared with GSK3b (11), we wantedto determine whether off-target effects of AR79 onHIPK2 could account for the impact on growth. HIPK2is known to act as a tumor suppressor through activationof p53, which both (i) mediates induction of apoptosis(41) and (ii) inhibits the wnt/b-catenin pathway throughmiRNA-34 (42). Therefore, HIPK2 may have contrib-uted to some of the responses observed in prostate cancerstudies with AR79. However, our results indicated thatAR79 had no impact on HIPK2 activation in prostatecancer cells. These data indicate that the responseobserved was not due to AR79 activity on HIPK2.Furthermore, the studies demonstrating that AR79 lost

its ability to induce prostate cancer cell growth in vitroupon knockdown of b-catenin provides strong evidencethat AR79 mediates its proliferative effect through activa-tion of canonical Wnt signaling. The mechanism throughwhich Wnt pathway activation mediates prostate cancercell growth is not clear at this time; however, it has beenpreviously demonstrated that there is cross-talk between theWnt pathway and the androgen receptor axis in prostatecancer (43, 44), which could contribute to the growthresponse.In addition to a proproliferative effect, AR79 increased

the proportion of CSC-like cells in two of the prostatecancer cell lines. This is consistent with the ability ofWnts to promote normal and CSC self-renewal andpluripotency (15–17, 45). This finding underscores thattherapies that promote Wnt signaling could result inenhancing CSC-like activity. As CSCs are consideredgain resistance to immunosurveillance (46), gain resis-tance to apoptosis and chemotherapy (47, 48), and havediminished induction of senescence (49), they may estab-lish a nidus of cancer recurrence. Thus, therapies thatresult in the overall promotion of Wnt pathway activity

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Figure 7. AR79 increases cell growth of DU145 and C4-2B cell lines via activating canonical Wnt signaling. DU145 and C4-2B parental cells (control) andcells transfected with scrambled or b-catenin siRNAs were grown in 96-well plates. Cells were then treated with AR79 (3 mg/mL) and rhWnt3a (60 ng/mL) orvehicle (DMSO or PBS) for 24, 48, and 72 hours. Cell numbers were measured using WST-1. Data are presented as the mean � SD from triplicatedeterminations.






should be carefully considered before their use. A caveatto this concern is that it is not absolutely clear that theALDHþ/CD133þ cell population that was increased aredefinitively CSCs. Although there are reports that indi-cate that cells with these markers contribute to the CSC-like cell population (18–21), there are also indicationsthat these markers may indicate the ability for these cellsto have a greater proliferative potential as opposed to clearstem cell properties (50, 51). Even if these markersrepresent more of a proproliferative capability as opposedto a clear CSC-like phenotype, the ability of AR79 toincrease this component of the population would still be amajor concern as they could contribute to increasedtumor growth.A key reason for the development of AR79 was to

promote bone anabolic activity through activation ofthe canonical Wnt pathway. Along these lines, AR79increased both BMD and BMC in normal bone, indi-cating an overall increase in bone production. Further-more, AR79 activated the Wnt signaling pathway in

osteoblast-like cells based on its ability to decreasephospho-b-catenin and increase total and nuclear b-cate-nin. Taken together, these results indicate that AR79 isan effective bone anabolic agent. However, in apparentcontrast with its ability to induce bone production, theadministration of AR79 decreased BMC in the bonecontaining tumor. The likely reason for this is that asAR79 induced prostate cancer cell growth, tumor-asso-ciated bone destruction occurred. This was evident fromradiographs and paralleled by the measureable decreasein BMC.A limitation of these experiments was the use of sub-

cutaneous injection to model soft tissue growth. Ideally,orthotopic injection may have provided a model that moreclosely recapitulates human prostate cancer than subcu-taneous injection. A challenge to orthotopic injection inthe murine model is the presence of four biologicallydifferent and anatomically separated lobes of the prostate.Another limitation to this model is that the cell lines useddo not completely recapitulate the osteoblastic bone

Figure 8. AR79 increases the proportion of ALDHþCD133þ cells in C4-2Bluc and DU145luc cell lines. C4-2Bluc and DU145luc (5� 106) were seeded into 10mmplates and treated with indicated concentrations of AR79 or vehicle (DMSO) for 48 hours. Cells were dissociated with Accutase and flow cytometricanalysis was performed for ALDH and CD133. A, flow cytometric analysis showing ALDH and CD133 expression in C4-2Bluc. B, flow cytometric analysisshowing ALDH and CD133 expression in DU145luc. C and D, proportion of ALDHþ:CD133þ in C4-2Bluc and DU145luc, respectively. Data are shown asthe mean � SD from two independent experiments. �, P < 0.05 versus vehicle.

Jiang et al.





response that is typical of clinical prostate cancer. Specif-ically, C4-2B cells induce mixed osteoblastic/osteolyticlesions and Du145 cells produce osteolytic lesions. It isunknown at this time whether the type of bone responseinduced by prostate cancer could influence the activity ofAR79.In summary, this research identified that inhibition of

GSK3b using AR79 had a bone anabolic effect, but alsostimulated prostate cancer growth in vivo a murine model.These findings suggest that therapeutic agents that modulatethe Wnt pathway should be used with caution, particularlyin the presence of prostate cancer. It is likely these effectswould extend to other cancers asWnts play a role inmultiplecancers.

Disclosure of Potential Conflicts of InterestE.T. Keller has received commercial research grant from AstraZeneca. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. Jiang, K.J. Escott, Z. Yao, L.K. McCauley, E.T.KellerDevelopment of methodology: Y. Jiang, H. Zhang, K.J. Escott, Z. Yao,L.K. McCauley, E.T. KellerAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Y. Jiang, J. DaiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): Y. Jiang, J.L. Sottnik, J.M. Keller, E.T. KellerWriting, review, and/or revision of the manuscript: Y. Jiang, J.L. Sottnik,K.J. Escott, H.J. Sanganee, Z. Yao, L.K. McCauley, E.T. KellerAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Y. JiangStudy supervision: H. Zhang, E.T. Keller

Grant SupportThis work was supported by the NIH grant P01 CA093900 and funding by

AstraZeneca.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accord-ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 20, 2013; revised August 28, 2013; accepted September 7, 2013;published OnlineFirst October 2, 2013.

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2013;11:1597-1610. Published OnlineFirst October 2, 2013.Mol Cancer Res Yuan Jiang, Jinlu Dai, Honglai Zhang, et al. Promotes Prostate Cancer Growth in Soft Tissue and Bone

Inhibitor,βActivation of the Wnt Pathway through AR79, a GSK3

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