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Cancer Biology and Signal Transduction

Pharmacologic Suppression of JAK1/2 by JAK1/2 InhibitorAZD1480 Potently Inhibits IL-6–Induced ExperimentalProstate Cancer Metastases Formation

Lei Gu1, Pooja Talati1, Paraskevi Vogiatzi1, Ana L. Romero-Weaver1, Junaid Abdulghani1, Zhiyong Liao1,Benjamin Leiby4, David T. Hoang1, Tuomas Mirtti6,7,8, Kalle Alanen8, Michael Zinda5, Dennis Huszar5, andMarja T. Nevalainen1,2,3

AbstractMetastatic prostate cancer is lethal and lacks effective strategies for prevention or treatment, requiring novel

therapeutic approaches. Interleukin-6 (IL-6) is a cytokine that has been linked with prostate cancer patho-

genesis by multiple studies. However, the direct functional roles of IL-6 in prostate cancer growth and

progression have been unclear. In the present study, we show that IL-6 is produced in distant metastases of

clinical prostate cancers. IL-6–activated signaling pathways in prostate cancer cells induced a robust 7-fold

increase inmetastases formation in nudemice. We further show that IL-6 promotedmigratory prostate cancer

cell phenotype, including increased prostate cancer cell migration, microtubule reorganization, and hetero-

typic adhesionof prostate cancer cells to endothelial cells. IL-6–drivenmetastasiswaspredominantlymediated

by Stat3 and to lesser extent by ERK1/2. Most importantly, pharmacologic inhibition of Jak1/2 by AZD1480

suppressed IL-6–induced signaling, migratory prostate cancer cell phenotypes, and metastatic dissemination

of prostate cancer in vivo in nudemice. In conclusion, we demonstrate that the cytokine IL-6 directly promotes

prostate cancer metastasis in vitro and in vivo via Jak–Stat3 signaling pathway, and that IL-6–drivenmetastasis

can be effectively suppressed by pharmacologic targeting of Jak1/2 using Jak1/2 inhibitor AZD1480. Our

results therefore provide a strong rationale for further development of Jak1/2 inhibitors as therapy for

metastatic prostate cancer. Mol Cancer Ther; 13(5); 1246–58. �2014 AACR.

IntroductionDevelopment of metastatic disease is one of the key

challenges in clinical management of prostate cancer (1).Although localized prostate cancer can be effectivelymanaged by surgery and radiation, the single greatestcause of prostate cancer morbidity andmortality is due toformation of distant metastases (2–6). Identification of themolecular changes leading to metastatic dissemination of

prostate cancer is critical for development of therapeuticinterventions to prevent prostate cancer progression tothe lethal metastatic form of the disease.

Interleukin-6 (IL-6) is a multifunctional cytokine impli-cated in prostate cancer pathogenesis (7). IL-6 and its recep-tors are expressed in prostate cancer, with elevated expres-sion in high Gleason score prostate cancers (8, 9). Concom-itantly, IL-6 has been found to be elevated in the sera ofpatients with castrate-resistant prostate cancer (CRPC), andIL-6 serum levels correlate with markers of prostate cancerprogression (10–16). Furthermore, IL-6 is expressed at highlevels in theandrogen-independentprostate cancer cell linesPC3 and DU145 (15), while the androgen-dependent pros-tate cancer cell line LNCaP expresses lower IL-6 levels (17).IL-6binds to the IL-6 receptor, a complexwhich is composedof two subunits: IL-6R a (IL-6–specific) and gp130 (sharedby IL-6 and related cytokines; refs. 5, 18, 19). IL-6 first bindsto the IL-6R a subunit (20), which then leads to recruitmentof the signal-transducing gp130 subunit. Association ofgp130 with IL-6 and IL-6R a leads to formation of thehigh-affinity IL-6 receptor complex and homodimerizationof two gp130 subunits, resulting in activation of down-stream Jak1/2–Stat3, ERK1/2–MAPK, and/or PI3K–Aktsignaling in prostate cancer cells (5, 18, 19, 21, 22). Inandrogen receptor (AR)–positive prostate cancer cell lines,it has been suggested that some of the biologic effects of IL-6are mediated via IL-6 induction of AR signaling (23–25).

Authors' Affiliations: Departments of 1Cancer Biology, 2Urology, and3Medical Oncology, Kimmel Cancer Center; 4Department of Pharma-cology and Experimental Therapeutics, Thomas Jefferson University,Philadelphia, Pennsylvania; 5Oncology iMED, AstraZeneca R&D Boston,Waltham, Massachusetts; 6Department of Pathology, Haartman Insti-tute; 7Institute of Molecular Medicine, University of Helsinki, Helsinki;and 8Department of Pathology, Institute of Biomedicine, University ofTurku, Turku, Finland

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

Current address for P. Vogiatzi: University of Michigan; current address forA.L. Romero-Weaver, University of Pennsylvania, 195 John Morgan Build-ing, 3620 Hamilton Walk, Philadelphia, PA 19104-6072; and currentaddress for J. Abdulghani, Penn State Hershey Cancer Institute.

Corresponding Author: Marja T. Nevalainen, Thomas Jefferson Univer-sity, 233 S. 10th Street, Philadelphia, PA 19107. Phone: 215-503-9250;Fax: 215-503-9245; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0605

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(5) May 20141246

on September 27, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 27, 2014; DOI: 10.1158/1535-7163.MCT-13-0605

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The existing literature on IL-6 regulation of prostatecancer cell growth and viability has been controversial,including reports documenting both growth-stimulatoryand -inhibitory effects (26–32). Our previous work dem-onstrated that Stat3 activation predominantly affectsprostate cancer progression by inducing metastaticbehavior of prostate cancer cells in vitro and massivemetastasis formation in vivo, rather than promoting pros-tate cancer cell growth (33, 34). Given that Stat3 is one ofthe key IL-6–activated signaling molecules in prostatecancer, we hypothesized that IL-6 promotes metastaticprogression of prostate cancer.Here, we show that the IL-6 protein is expressed in the

majority of distant metastases of clinical prostate cancers.IL-6 induced a robust increase in prostate cancer metas-tasis formation in nude mice in an experimental metas-tasis assay, which was accompanied by IL-6–driven pros-tate cancer cell migration and heterotypic adhesionin vitro. We further demonstrate that IL-6 stimulationleads to activation of Janus kinase (Jak1/2) and predom-inantly Stat3 signaling pathway in AR-negative andAR-positive prostate cancer cells. Most important froma therapeutic perspective, pharmacologic targeting ofJak1/2 by a potent ATP-competitive small-moleculeinhibitor of Jak1/2 kinase, AZD1480 (35) prevented IL-6–driven development of a migratory prostate cancer cellphenotype in vitro and suppressed IL-6–drivenmetastaticdissemination of prostate cancer in nude mice. In sum-mary, Jak1/2 may represent a therapeutic target proteinfor prostate cancer, and prevention of metastatic progres-sion of primary prostate cancer in patients could poten-tially be achieved by small-molecule Jak1/2 inhibitorscurrently in clinical development (36, 37).

Materials and MethodsClinical samples of distant metastases of prostatecancersArchival and deidentified, formalin-fixed, paraffin-

embedded specimens of distant metastases of clinicalprostate cancers (n ¼ 87; regional lymph nodes, n ¼ 36;bone, n ¼ 3; other organs, n ¼ 48; Table 1) were obtainedfrom University of Turku in Finland. The immunohisto-chemical analysis of the deidentified archival tissues wasgranted an exemption from full Institutional ReviewBoard review by Thomas Jefferson University (Philadel-phia, PA).

Cell culture and reagentsHuman prostate cancer cell lines CWR22Rv1, DU145,

and LNCaP (American Type Culture Collection, ATCC)were cultured in RPMI 1640 (Mediatech; #15-041-CV)containing 10% FBS(Quality Biological; #100106) andpenicillin/streptomycin (Mediatech, Inc.; 50 IU/mLand 50 mg/mL, respectively; #30-002-CL). LNCaP cellswere cultured in the presence of 0.5 nmol/L dihydro-testosterone (Sigma; #512-18-6). CWR22Rv1 cells wereobtained in 2005 from Dr. Thomas Pretlow (Case West-ern Reserve University, Cleveland, OH) and LNCaP

and DU145 cells in 2009 from ATCC. AZD1480 (35) wasprovided by AstraZeneca. IL-6–producing lentivirus(LV-II-human-IL-6 virus, 3.8 � 108 IU/mL) was pur-chased from Capital Biosciences and recombinant IL-6was purchased from ProSpec Protein Specialists (#CYT-213).

Immunostaining of paraffin-embedded tissuesections

Immunohistochemistry of distant prostate cancermetas-taseswasperformedasdescribedpreviously (33, 34, 38–40).The primary monoclonal antibody (mAB) IL-6 (Santa CruzBiotechnology; sc-130326) was used at a concentration of5 mg/mL.

ELISADU145 were mock-infected or infected with lentivirus

expressing the IL-6 gene (multiplicity of infection, MOI¼5), followed by collection of cell culturemedia at 4, 10, and20 days and analysis for IL-6 protein levels by ELISAaccording to the manufacturer’s instructions (R&D Sys-tems; #Q6000B).

Homotypic cell adhesion assayDU145 and CWR22Rv1 cells were seeded on Matrigel

(BD Biosciences; #354234), serum starved for 18 hours,and treated with recombinant IL-6 (5 nmol/L) for 24, 48,72, or 96 hours.

Heterotypic cell adhesion assayDU145 cells, cultured in the presence of 1% charcoal-

stripped FBS (CS-FBS), were infected with IL-6 and Stat3-shRNA and/or ERK1/2-shRNA lentiviruses (atMOI¼ 5)and/or treated with recombinant IL-6 (5 nmol/L) with or

Table 1. IL-6 expression in distant metastasesof clinical prostate cancers

Number of patients %

Prostate cancer metastases (lymph node metastases, bonemetastases, and metastases to other organs)IL-6 expression status 87 100Negative 19 22Positive 68 78

Prostate cancer metastases to regional lymph nodesIL-6 expression status 36 100Negative 4 11Positive 32 89

Prostate cancer metastases to boneIL-6 expression status 3 100Negative 1 33Positive 2 67

Prostate cancer metastases to other organsIL-6 expression status 48 100Negative 14 29Positive 34 71

JAK1/JAK2 Inhibition Suppresses Prostate Cancer Metastases

www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1247




without AZD1480 (800 nmol/L). DU145 and CWR22Rv1cell adhesion to the endothelial cells was determined asdescribedpreviously (41) and in the SupplementaryMate-rials and Methods.

Immunofluorescence cytochemistry of tubulinDU145 cells grown on cover glasses were serum-

starved for 18 hours, treated with recombinant IL-6 (5nmol/L), and fixed 48 hours after IL-6 treatment with 4%paraformaldehyde. Cells were incubated with anti-tubu-lin mAb (Invitrogen; 0.5 mg/mL; #A11126), followed byincubation with fluorescein isothiocyanate (FITC)-conju-gated secondary antibodies (Invitrogen; #A11001) and thenuclei were stained with mounting medium containing40,6-diamino-2-phenylindole (Vector Labs; #H1200). Theimmunostaining was examined under Zeiss LSM 510META Confocal Laser Scanning Microscope with anX63 objective (Zeiss) and photographed.

Boyden chamber migration assayDU145 and CWR22Rv1 cells were cultured in 1% CS-

FBS, phenol-red free RPMI medium in the presence orabsence of IL-6 (5 nmol/L) for 72 hours. In the indicatedtreatment groups, the cells were infected with lenti-viruses expressing control-shRNA, Stat3-shRNA, and/orERK1/2-shRNA 24 hours before the start of the IL-6 treat-ment. Also, in the indicated treatment groups, AZD1480(800 nmol/L) was added to the culture medium 1 hourbefore the start of the IL-6 treatment. The cells werecounted and suspended to the upper chambers (2.5 �104 cells/chamber) of themotility chamber system (8.0mmpore size; BDBioSciences; #353097) using FBS (10%) as thechemoattractant, as described previously (33, 41). After 16hours, the cells that had traversed the membrane poreswere fixed, stained, and counted. Each experiment wasrepeated four times.

Protein solubilization, immunoprecipitation, andimmunoblotting

CWR22Rv1, DU145, and LNCaP cells were solubilizedand immunoprecipitations and immunoblottings wereperformed as described previously (33, 34, 41–44). Forimmunoprecipitation, antibodies usedwere anti-Stat5a orStat5b polyclonal antibody (pAb ; 1:1,000; Advantex Bio-Reagents; 5a: #AX551, 5b: #AX554), anti-Jak1 or anti-Jak2pAbs (Upstate; #06-255) at 5 mL/immunoprecipitation(IP). Primary antibodies used for immunoblotting wereanti–phosphotyrosine-Stat5a/b (Y694)mAb (0.25 mg/mL;BD Biosciences; #611965), anti-Stat5a/b mAb (0.25 mg/mL; BD Biosciences; #611965), anti-Stat3 Ab (0.2 mg/mL;Santa Cruz Biotechnology; #SC-483), anti–phosphotyro-sine-Stat3 (Y705) Ab (1:1,000; Cell Signaling Technology;#9145S), anti-Akt (1:1,000; Cell Signaling Technology;#9272), anti–phospho-Akt (1:1,000; Cell Signaling Tech-nology; #9275S), anti-Jak1 mAb (0.25 mg/mL; BD Bios-ciences; #610231), anti-Jak2 mAb (1:1,000; Biosource; #44-426G), anti-phosphotyrosine 4G10 (0.1 mg/mL; Upstate;#05-321), and anti-actin pAb (0.2 mg/mL; Sigma; #A2066).

Prostate cancer cellswere lysed in lysis buffer [10mmol/LTris-HCl (pH 7.6), 5 mmol/L EDTA, 50 mmol/LNaCl, 30mmol/L sodium pyrophosphate, 50 mmol/L sodiumfluoride, 1 mmol/L sodium orthovanadate, 1% TritonX-100, 1 mmol/L phenylmethlysulphonylfluoride, 5mg/mL aprotinin, 1 mg/mL pepstatin A, and 2 mg/mLleupeptin]. The cell lysates were separated on 4% to 12%SDS-PAGE (Invitrogen; #NP0321BOX) and transferredelectrophoretically to polyvinylidene fluoride membrane(Millipore; #IPVH00010). For immunoblotting, blockingbuffer used was Tris Buffered Saline with Tween 20(TBST; 0.15mol/LNaCl; 0.1% Tween 20; 50mmol/L Tris,pH 8.0) with 3% bovine serum albumin (BSA).

Cell viability assayDU145 cells were treated with AZD1480 or dimethyl

sulfoxide (as vehicle control) or infectedwith IL-6–expres-sing lentivirus (MOI ¼ 5) for 72 hours. Cell viability wasanalyzed by the CellTiter 96 AQueous Assay Kit (Pro-mega; #G3580) according to the manufacturer’s protocol.

Tail vein injections of human prostate cancer cellsMale athymic nude mice (for DU145 cells) were pur-

chased from Taconic and severe combined immunode-ficient mice (for CWR22Rv1 cells) were purchased fromThe Jackson Laboratory, and cared for according to insti-tutional guidelines. DU145 and CWR22Rv1 cells wereinfected with IL-6 lentivirus (MOI ¼ 5) alone or in com-bination with Stat3-shRNA lentivirus (MOI ¼ 5) and/orERK1/2-shRNA (MOI¼ 5) lentivirus. After 24 hours, 1�106 cells were suspended in 0.2 mL of phosphate bufferedsaline (PBS) and injected into the lateral tail veins of nudemice using a 27-gauge needle, as described previously(33, 34, 41). In some of the experiments, the mice weretreated daily by oral gavage of AZD1480 at 50 mg/kgbody weight or vehicle [0.5% hydroxypropyl methylcellulose (HPMC)/0.1% Tween-80] starting on day 3 untilthe mice were sacrificed (8 weeks after cell inoculation).The lungswere perfusedwith 1.5mLof 15% India Ink dyein 3.7% formalin, removed and bleached in Fekete’s solu-tion (70% ethanol, 3.7% formaldehyde, and 0.75 mol/Lglacial acetic acid). The livers were fixed in 10% formalin.Lung surfaces were photographed and number of surfacelung metastases was scored.

Statistical analysisStatistical analyses are provided in the Supplementary

Materials and Methods.

ResultsIL-6 is expressed in the majority of distantmetastases of clinical prostate cancers

To test the hypothesis that IL-6 promotes metastaticbehavior of human prostate cancer cells, we first deter-mined the frequency of IL-6 expression in clinical prostatecancer metastases (n ¼ 87) using immunohistochemicaldetection of IL-6 in paraffin-embedded tissue sections.Representative prostate cancer metastases positive or

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Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics1248




negative for IL-6 are presented in Fig. 1A. A positiveimmunoreaction for IL-6 was detected in 78% (68/87) ofprostate cancer metastases (Table 1). In prostate cancermetastases to regional lymph nodes, an intense immunor-eaction for IL-6 was detected in 89% (32/36) of the speci-mens, whereas IL-6 was expressed in two of three bonemetastases. In addition, IL-6was expressed in 71% (34/48)of prostate cancer metastases to distant organs other thanbone. In summary, our results indicate that themajority ofprostate cancer metastases robustly express IL-6 protein.

IL-6 induces experimental metastases formation innude miceTo determine if IL-6 affects metastatic dissemination of

human prostate cancer cells in vivo, we performed in vivoexperimental metastases assays in nude mice. We gener-

ated lentivirus expressing the IL-6 gene, and evaluated theefficacy of this lentiviral construct in producing IL-6 inprostate cancer cells by enzyme-linked immunosorbentassay. Lentiviral expression of the IL-6 gene in DU145 cellsresulted in a 3-fold increase in IL-6 production into theculture medium (2.2 ng/mL; 0.11 nmol/L) at 20 dayscompared with mock-infected cells (0.7 ng/mL; 0.04nmol/L; Fig. 1B). Next, we infected DU145 cells with theIL-6 lentivirusand24hoursafter the lentiviral genedelivery(MOI ¼ 5), DU145 cells were inoculated in athymic nudemice through tail vein injection (1 � 106 cells per mouse).After 8weeks, the lungswere harvested, stainedwith IndiaInk, bleachedwith Fekete’s solution, and scored for surfacelungmetastases.As demonstrated in Fig. 1C, the number ofmetastases in mice injected with DU145 cells infected withIL-6 lentivirus was robustly increased when compared

Figure 1. IL-6 is produced in distantmetastases of clinical prostatecancers, and autocrine IL-6induces metastatic colonization ofhuman prostate cancer cells in thelungs of nude mice in anexperimental metastases assay. A,IL-6 protein is expressed in distantmetastases of human prostatecancers. IL-6 in prostate cancermetastases was analyzed byimmunohistochemical staining.Distant prostate cancermetastasesnegative (left) or positive (right) forimmunostaining of autocrine IL-6are shown. B, ELISA demonstratingincreased IL-6protein expression inculture media of DU145 cellsinfected with lentivirus (MOI ¼ 5)expressing the IL-6 gene. C,IL-6 increases metastasesformation of prostate cancer cellsto the lungs of athymic nude mice.Athymic nude mice were injectedwith DU145 cells infected withlentivirus expressing IL-6 gene atMOI ¼ 5 through the tail veins.Increased autocrine IL-6expression in DU145 cells resultedin a significant increase (71-fold;mean, 212; SEM, 102) comparedwith control cells (mean, 3; SEM, 2;top). Representative photographsof India Ink–stained lungs derivedfrom athymic nude mice (bottom).






with control mice. Quantitatively, injection of DU145cells infected with IL-6 lentivirus resulted in an averageof 212 (SEM¼ 102) metastasesper lung, as comparedwith3 metastases per lung using mock-infected DU145 cells(SEM¼ 2.0). As depicted visually in Fig. 1C (bottom), IL-6robustly promoted development of metastases in thelungs of nude mice (P¼ 0.0053), evidenced by the densityand large number of white metastatic nodules presentthroughout the lungs. To verify the concept that IL-6 pro-motes prostate cancer metastases formation in vivo, asecond cell line CWR22Rv1, which is AR-positive, wasinfectedwith lentivirus expressing the IL-6 gene (MOI¼ 5)and inoculated inmice through tail vein injections (1� 106

cells permouse). IL-6 increased livermetastases formationof CWR22Rv1 cells significantly (Supplementary Fig. S1A)as evidenced by increased liver weight (P ¼ 0.0079; Sup-plementary Fig. S1B) and decreased overall survival (Sup-plementary Fig. S1C). Collectively, these data are the firstdemonstration that IL-6 increases the intrinsic ability ofprostate cancer cells to metastasize in vivo.

IL-6 promotes prostate cancer cell migration,decreases homotypic adhesion of prostate cancercells, and increases heterotypic adhesion of prostatecancer cells to endothelial cells

Given that IL-6 was capable of inducing extensivemetastases formation in vivo, we next investigated if IL-6 is involved in the regulation of metastatic behavior ofprostate cancer cells in vitro. Development of metastasesin the lungs and the livers of mice following tail veininjection of prostate cancer cells requires a cascade ofwell-characterized biologic events, including decreased homo-typic adhesion, increased heterotypic adhesion, extrava-sation, increased migration, increased invasion of cellsinto the extracellular matrix, and increased cell viabilityduring migration (45). In Boyden chamber assays, migra-tion of DU145 and CWR22Rv1 cells was increased by 46%(P ¼ 0.045) and 200% (P < 0.0001), respectively, by IL-6 (5nmol/L) after 16-hour incubation using 10% FBS as thechemoattractant (Fig. 2A and Supplementary Fig. S2A).Furthermore, IL-6 induced morphologic changes charac-teristic of motile cells (Fig. 2B). Specifically, microtubulesare known to be important for intrinsic cell polarizationand directional cell migration, and can be linked to actinpolymers directly or indirectly through intermediate pro-teins or signaling molecules (46–48). DU145 cells werestained for the presence of a-tubulin using FITC-conju-gated secondary antibodies (Fig. 2B). IL-6 (5 nmol/L)induced outward polarization of microtubules from thecentrosomes, forming a dense meshwork facing the plas-ma membrane. In contrast, the microtubule network incontrol DU145 cells remained disrupted, which is char-acteristic of nonmigratory cells (Fig. 2B).

To investigate if IL-6 disrupts homotypic adhesion ofprostate cancer cells, we examined the effects of IL-6 onDU145 and CWR22Rv1 cells cultured on Matrigel, acollagen-rich extracellular matrix that provides a morephysiologic growth environment thanplastic.DU145 cells

(Fig. 2C) and CWR22Rv1 cells (Supplementary Fig. S2B)were treated with recombinant IL-6 (5 nmol/L) and cul-tured on Matrigel for 4 days. In the control group, DU145cells grew as adherent sheets of cells attached toMatrigel.In contrast, cells treated with IL-6 (5 nmol/L) were partlydispersed as single cells or small, scattered cell clusters(Fig. 2C and Supplementary Fig. S2B). These data provid-ed evidence that IL-6 disrupts homotypic adhesion ofDU145 and CWR22Rv1 cells, reducing epithelial cell-to-cell contact and suggesting increasedmigratory and inva-sive potential.

Finally, the initial arrest and attachment of cancer cells tovascular endothelium precedes extravasation from thebloodstream, and is a crucial step in themetastatic cascade.To determine if IL-6 promotes adhesion of prostate cancercells to vascular endothelial cells, IL-6 was expressedin DU145 and CWR22Rv1 cells using IL-6 lentivirus for72 hours and tested for adhesion to two types of humanendothelial cells: human umbilical vein endothelial cells(HUVEC) and human bone marrow endothelial cells(HBMEC). Notably, IL-6 increased binding of DU145 cellstoHUVECcells by 67% (P¼ 0.0022) and toHBMECcells by125% (P ¼ 0.0079; Fig. 2D). In addition, IL-6 increasedbinding of CWR22Rv1 cells to HBMEC cells by 51% (P <0.001; Supplementary Fig. S2C). Collectively, the resultspresented here indicate that IL-6 induces a migratory phe-notype in prostate cancer cells and increases heterotypicadhesion of prostate cancer cells to endothelial cells.

Stat3 is the keymediator of IL-6–driven developmentof amigratory prostate cancer cell phenotype in vitro

To identify signaling pathways that mediate the bio-logic effects of IL-6 in DU145 cells, cells were starvedovernight, stimulatedwith IL-6 (5nmol/L) for 30minutes,and immunoblotted for active phosphorylated forms ofStat3, ERK1/2, and Stat5a/b. As shown in Fig. 3A, i and ii,both Stat3 and ERK1/2, but not AKT or Stat5a/b, wereactivated by IL-6 stimulation in the absence of serum inthe culture medium in DU145 cells. Moreover, IL-6 acti-vated Jak2 in CWR22Rv1, DU145, and LNCaP cells, andJak1 in DU145 cells, whereas Jak1 was not expressed inLNCaP and CWR22Rv1 cells (Fig. 3A, iii). In CWR22Rv1cells, IL-6 activated only Stat3 but not ERK1/2 or Akt(Supplementary Figs. S3A and S3B). Next, we generatedlentiviruses expressing shRNAs targeting Stat3, ERK1,and ERK2, and verified protein knockdown efficiency inDU145 cells byWestern blotting (Fig. 3B). To evaluate thecontributions of Stat3 and ERK1/2 signaling to IL-6–induced prostate cancer cell migration, DU145 cells wereinfectedwith lentiviruses expressing Stat3 shRNAand/orERK1/2-shRNA and treated with IL-6 (5 nmol/L) for 72hours, after which the cells were tested for migratorypotential in Boyden chamber assays for 16 hours. IL-6significantly increased migration of DU145 cells (P ¼0.0017; Fig. 3C). Genetic knockdown of Stat3 (P ¼ 0.029)as well as ERK1/2 (P ¼ 0.057) inhibited IL-6–inducedmigration of DU145 cells cultured in the presence of FBS(Fig. 3C).

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To evaluate if Stat3 and ERK1/2 pathways mediateIL-6–induced heterotypic adhesion of prostate cancercells to endothelial cells, DU145 cells were infected withlentiviruses expressing IL-6, Stat3 shRNA, and/orERK1/2-shRNA for 72 hours and tested for adhesionto HBMEC cells. IL-6 significantly increased binding ofDU145 cells to HBMEC cells (P ¼ 0.0001). Geneticknockdown of either Stat3 (P ¼ 0.0006) or ERK1/2 (P¼ 0.0006) reduced IL-6–induced prostate cancer celladhesion to HBMEC endothelial cells (Fig. 3D). In con-clusion, the data presented here demonstrate that Stat3signaling pathway predominantly mediates IL-6–

induced prostate cancer cell migration and heterotypicadhesion in DU145 and CWR22Rv1 cells, whereasERK1/2 may also contribute to mediation of IL-6 effectsin DU145 cells to a lesser extent.

Pharmacologic targeting of Jak2 by AZD1480suppresses IL-6–inducedStat3 andERK1/2 signalingand development of a migratory prostate cancer cellphenotype

Because Jak1 and Jak2 are the key tyrosine kinasesactivated by IL-6 in prostate cancer cells, we wanted todetermine if the small-molecule Jak1/2 inhibitor

Figure 2. IL-6 induces a migratory phenotype in prostate cancer cells. A, IL-6 induces migration of prostate cancer cells in Boyden chamber assays. DU145cells were cultured in the presence or absence of IL-6 (5 nmol/L) for 72 hours, counted, and equal numbers of cells in different treatment groups weresuspended within the motility chamber system using 10% FBS as a chemoattractant. The cells that traversed the membrane pores were counted after16hours. B, IL-6 induces polarization of themicrotubule network in prostate cancer cells. DU145 cells cultured in the presenceor absence of IL-6 (5 nmol/L) for48 hours were fixed and incubated with anti-tubulin mAb followed by FITC-conjugated secondary antibody. Representative images from one of threeindependent experiments are shown. C, IL-6 disrupts homotypic adhesion of prostate cancer cells. DU145 cells were seeded on Matrigel, treated withIL-6 (5 nmol/L) for the indicated periods of time, andmorphologic alterations were photographed. Note the dispersed and scattered cells in the IL-6 treatmentgroup. D, IL-6 induces heterotypic adhesion of prostate cancer cells to endothelial cells. DU145 cells were cultured in the presence or absence ofIL-6 (5 nmol/L) for 72 hours, stained with a fluorescent dye, and allowed to adhere to HUVECs (left) or HBMECs (right) for 60minutes, after which the adherentcells were quantitated. Averages of three independent experiments are shown.






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AZD1480 (35) is capable of suppressing IL-6–inducedmetastatic behavior of humanprostate cancer cells in vitro.We first tested the efficacy of AZD1480 in blocking IL-6–activated cell signaling in prostate cancer cells. AZD1480inhibited IL-6–induced phosphorylation of Stat3 byapproximately 50% (IC50) at a concentration of 9.4 nmol/L in DU145 cells, while the IC50 values for IL-6–inducedERK1 and ERK2 phosphorylation were 26 and 21 nmol/L,respectively (Fig. 4A). Next, we evaluated if AZD1480suppresses IL-6–driven migration of DU145 cells (Fig.4B). DU145 cells were treated with or without recombin-ant IL-6 (5 nmol/L) for 72 hours in the presence or absenceof AZD1480 (800 nmol/L), after which equal numbers ofcells per group were assayed for migratory ability in Boy-den chambers for 16 hours. IL-6 increased migration ofDU145 cells by 48% (P ¼ 0.057), which was potentlysuppressed by AZD1480 (P ¼ 0.029; Fig. 4B). Westernblotting of cell lysates from parallel wells indicated thatAZD1480 primarily inhibited Stat3 and ERK1 phosphory-lation, with less inhibition of ERK2 phosphorylation, dur-ing long-term IL-6 treatment of DU145 cells (Fig. 4B).Similarly, AZD1480 inhibited (P < 0.0001) effectivelyIL-6–induced (P ¼ 0.002) migration of CWR22Rv1 cells(Supplementary Fig. S3C).To investigate if AZD1480 reduces heterotypic adhe-

sion of prostate cancer cells to endothelial cells, DU145prostate cancer cells were treated with recombinant IL-6(5 nmol/L) or vehicle with or without AZD1480 (800nmol/L) for 72 hours and assayed for binding toHUVEC and HBMEC cells (Fig. 4C). Increased hetero-typic adhesion of DU145 cells to HUVEC (43%; P <0.0001) and HBMEC cells (63%; P < 0.0001) induced byIL-6 was effectively blocked by AZD1480 (HUVEC, P ¼0.0022; HBMEC, P ¼ 0.0011; Fig. 4C). Similarly,increased heterotypic adhesion of CWR22Rv1 cells toHBMEC cells by IL-6 (P ¼ 0.04) was inhibited byAZD1480 (P < 0.0001).Finally, we evaluated if the ability of AZD1480 to block

IL-6–driven metastatic behavior of prostate cancer cellswas due to induction of apoptosis in prostate cancer cells.Cell viability of DU145 cells cultured with or without IL-6and/orAZD1480wasdeterminedbyMTTassay (Fig. 4D).

IL-6 showed no effect on viability or growth of DU145cells, indicating that IL-6–driven metastasis formationwas not due to IL-6–stimulated prostate cancer cell pro-liferation. At the same time, AZD1480 failed to decreasethe number of viable DU145 cells, indicating that sup-pression of prostate cancer cell migration and heterotypicadhesion by AZD1480 was not caused by AZD1480-induced prostate cancer cell death (Fig. 4D). In summary,the data presented here demonstrate that AZD1480 spe-cifically blocked the stimulatory effects of IL-6 on prostatecancer cell migration and heterotypic adhesion, ratherthan affecting prostate cancer cell viability.

Stat3 and ERK1/2 mediate IL-6–driven metastasesformation in nude mice, which can be effectivelytargeted by pharmacologic inhibition of Jak1/2 usingAZD1480

Given that IL-6 promoted metastatic behavior of pros-tate cancer cells in vitro, which was potently inhibited bygenetic knockdown of both Stat3 and ERK1/2, we inves-tigated if Stat3 andERK1/2 signalingmediate IL-6–drivenmetastatic colonization in vivo. Most importantly, wewanted to determine if IL-6–induced migratory cell phe-notype in vitro andmetastatic dissemination in vivo can besuppressed pharmacologically using the Jak1/2 inhibitorAZD1480.

DU145 cells were infected with IL-6 lentivirus (MOI ¼5) alone or in combination with Stat3-shRNA lentivirus(MOI ¼ 5) and/or ERK1/2-shRNA lentivirus (MOI ¼ 5),after which the cells were injected into the tail veins ofathymic nude mice (Fig. 5). IL-6 induced a 7-fold increasein metastases formation (mean, 787; SEM, 92) comparedwith the control group (mean, 117; SEM, 28; P < 0.0001),whichwas substantially blockedbygenetic knockdownofStat3 alone (mean, 146; SEM, 48;P < 0.0001), ERK1/2 alone(mean, 229; SEM, 62; P < 0.0001), or Stat3 and ERK1/2combined (mean, 122; SEM, 44; P < 0.0001; Fig. 5). Toevaluate if AZD1480 was able to suppress IL-6–drivenmetastatic progression of prostate cancer, mice were trea-ted daily by oral gavage of AZD1480, starting on day 3until sacrifice. AZD1480 significantly suppressed IL-6–driven metastases formation in these mice (mean, 232;

Figure 3. Stat3 and ERK1/2 signaling mediate the biologic effects of IL-6 in induction of migratory prostate cancer cell phenotype. A, IL-6 activatesphosphorylation of Stat3, ERK1/2, and Jak1/2 but not Stat5 or Akt. (i) DU145 cells were serum starved (0% FBS) overnight followed by stimulation of thecells with IL-6 (5 nmol/L) for 20 minutes and immunoblotted for p(Tyr705)Stat3, Stat3, p(Thr202/Tyr204)ERK, ERK1/2 or p(Ser473)Akt or Akt. (ii) Stat5aand Stat5b were immunoprecipitated (IP) and immunoblotted for pYStat5a/b and total Stat5a/b. Whole-cell lysates of parallel samples wereimmunoblotted for actin. (iii) CWR22Rv1, DU145, and LNCaP cells were serum starved overnight followed by stimulation with IL-6 (5 nmol/L) for 20minutes. Jak1 and Jak2 were immunoprecipitated and blotted with anti-phosphotyrosine (4G10), anti-Jak1, or anti-Jak2 mAbs. B, lentivirusexpressing shRNAs targeting Stat3, ERK1, and ERK2 expression in DU145 cells. DU145 cells infected with each lentiviral construct wereimmunoblotted with anti-Stat3, anti-actin, anti-ERK1, or anti-ERK2 antibodies. C, inhibition of Stat3 and/or ERK1/2 by RNA interference blocksIL-6–induced prostate cancer cell migration. DU145 cells were cultured in the presence of 1% CS-FBS and infected with lentiviruses expressingcontrol-shRNA, Stat3-shRNA, and/or ERK1/2-shRNA (MOI ¼ 5) and treated with or without IL-6 (5 nmol/L) for 72 hours. Cells were counted and equalnumbers of cells in each treatment group were assayed for migration in Boyden chambers for 16 hours. D, inhibition of Stat3 and/or ERK1/2signaling blocks IL-6–induced increase in adhesion of prostate cancer cells to endothelial cells. DU145 cells were cultured in the presence of 1% CS-FBS and infected with lentiviruses expressing control-shRNA, Stat3-shRNA, and/or ERK1/2-shRNA (MOI ¼ 5) and treated with or without IL-6 (5 nmol/L)for 72 hours. Cells were stained with a fluorescent dye and allowed to adhere to HBMEC cells for 60 minutes followed by quantitation of adhered cells byfluorescent reader. Averages of three independent experiments are shown.






SEM, 31; P < 0.0001), compared with the IL-6–expressinggroup without AZD1480 treatment (mean, 787; SEM,92; Fig. 5). Collectively, these data demonstrate that phar-

macologic inhibition of Jak1/2 effectively prevents IL-6–driven metastatic colonization of human prostate cancercells in nude mice.

Figure 4. Pharmacologic Jak1/2 inhibitor AZD1480 suppresses IL-6–activated Stat3 and ERK1/2 signaling in prostate cancer cells and IL-6–induced migratoryprostate cancer cell phenotype. A, AZD1480disrupts IL-6–induced phosphorylation of Stat3 andERK1/2 in prostate cancer cells. DU145cellswere serumstarved(0% FBS) overnight, pretreated with AZD1480 at indicated concentrations for 1 hour followed by stimulation of the cells with IL-6 (50 nmol/L) for 20 minutes,and immunoblotted for anti-p(Tyr705)Stat3, anti-p(Thr202/Tyr204)ERK, anti-Stat3, anti-ERK1/2, or anti-Actin, as indicated. B, AZD1480 inhibits IL-6–inducedmigration of DU145 cells. DU145 cells were cultured in 1% CS-FBS in the presence or absence of IL-6 (5 nmol/L) with or without pretreatment of thecells with AZD1480 (800 nmol/L). Equal numbers of cells in each treatment group were analyzed for migration in Boyden chambers for 16 hours (top). Cell lysatesfrom parallel wells were immunoblotted for anti-p(Tyr705)Stat3, anti-p(Thr202/Tyr204)ERK, Stat3, or anti-actin (bottom). C, AZD1480 inhibits IL-6–inducedheterotypic adhesion of prostate cancer cells to endothelial cells. DU145 cells were cultured in 1%CS-FBS in the presence or absence of IL-6 (5 nmol/L) with orwithoutpretreatmentof thecellswithAZD1480 (800nmol/L).Averagesof three independentexperimentsareshown.D, IL-6orAZD1480didnotaffect theviabilityofDU145 cells. (i) DU145 cells were cultured in 1% CS-FBS for 7 days in the presence or absence of IL-6 (5 nmol/L). The bars represent the numbers of viableDU145cells in both treatmentgroupsat indicated times. (ii) Thenumber ofDU145cells cultured in1%CS-FBS for 7days in thepresenceorabsenceofAZD1480atindicated concentration.

Gu et al.





DiscussionA significant fraction of patients with organ-confined

prostate cancers treated by radical prostatectomy devel-op recurrent metastatic disease (39, 40), which suggeststhat tumor cells with metastatic potential may havealready disseminated at the time of the primary treat-ment. Current therapeutic strategies for metastatic pro-state cancer are directed against AR signaling andeventually fail because of progression to the CR stageof prostate cancer (2–6). CRPC is treated with nontar-geted cytotoxic therapeutic regimens that provideonly limited additional survival benefit (2–6). Therefore,improved therapeutic strategies are needed both toprevent metastatic dissemination of organ-confinedprostate cancer and to effectively treat existing meta-static disease. In this work, we show for the first timethat IL-6–activated Jak1/2 signaling pathways inducemetastatic behavior of AR-positive (CWR22Rv1) andAR-negative (DU145) prostate cancer cells in vitroand excessive metastasis formation in vivo, using exper-imental metastasis assays in mice. These biologic pro-metastatic effects were predominantly mediated by Jak–Stat3 signaling pathway and to a lesser extent by Jak1/2–ERK1/2. Most important from a therapeutic perspec-tive, IL-6–driven metastatic progression of prostate can-cer in vivowas potently suppressed by a small-moleculepharmacologic Jak1/2 inhibitor, AZD1480.To our knowledge, this is the first evidence of IL-6–

activated Jak1/2 signaling pathways promotingmetastat-

ic processes in prostate cancer. IL-6 increased prostatecancer cell migration, microtubule reorganization, andheterotypic adhesion of prostate cancer cells to vascularendothelial cells, while reducing homotypic prostate can-cer cell adhesion. IL-6 acts to directly upregulate meta-static processes in prostate cancer cells, because IL-6treatment did not increase prostate cancer cell viability.Intriguingly, IL-6–activated Jak1/2 signaling resulted in arobust 7-fold increase in metastatic colonization of pros-tate cancer cells in the lungs of nudemice. In prior studies,IL-6 has been primarily investigated for its growth-pro-moting ability in prostate cancer using various modelsystems such as human prostate cancer cell lines, long-term exposure of prostate cancer cells to elevated IL-6concentrations, or stable prostate cancer cell clones over-expressing IL-6 (26–32). Results have been contradictory,with some studies indicating that IL-6 stimulation con-ferred agrowth advantageonprostate cancer cells,where-as other studies demonstrated growth-inhibitory effectsof IL-6 on prostate cancer cells (26–32). Also, IL-6 has beensuggested to induce neuroendocrine differentiation ofprostate cancer cells (49, 50). The current work tested thehypothesis that autocrine IL-6 activation of Jak1/2 sig-naling promotes metastatic progression of prostate can-cer. The results presented here provide a new perspectivein understanding the association of autocrine/circulatingIL-6 with surrogate markers of clinical prostate cancerprogression in patients, which has been previously dem-onstrated in numerous studies (10–16).

Figure 5. Pharmacologic Jak1/2inhibitor AZD1480 blocksIL-6–Jak–Stat3/ERK1/2–drivenmetastases formation in nudemice. Genetic knockdown of Stat3and/or ERK1/2, as well aspharmacologic inhibition of Jak1/2by AZD1480, suppresses prostatecancer metastases formation innude mice. DU145 cells infectedwith lentivirus expressing IL-6 (MOI¼ 5) with or without simultaneousinfection of lentiviruses expressingshRNA-Stat3 (MOI ¼ 5) and/orshRNA-ERK1/2 (MOI ¼ 5) wereinjected in athymic nude micethrough tail veins. AZD1480 wasadministered to indicated groupsof mice daily by oral gavage(50 mg/kg). The lungs were stainedwith India Ink and scored forsurface metastases.Representative photographs ofIndia Ink–stained lungs (bottom).






The biologic prometastatic effects of IL-6 in DU145 andCWR22Rv1 prostate cancer cells were predominantlymediated by the Jak1/2–Stat3 pathway, while ERK1/2may alsoplay some role inDU145 cells. Thefindings of thecurrent study are in line with our previous work, whichput forth a key role for Stat3 in promotion of metastaticdissemination of prostate cancer cells in vivo, induction ofmigratory phenotype in vitro, and induction of geneexpression profiles associated with metastatic processesin prostate cancer (33, 34). Although IL-6 can promote ARsignaling in some prostate cancer cell models (23–25), arecent study demonstrated that IL-6 secreted by endothe-lial cells promoted metastatic behavior of prostate cancercells via downregulation of AR (51). Loss of AR signaling,in turn, resulted in increased TGF-b production, followedby induction of epithelial-to-mesenchymal transition(EMT) and acquisition of an invasive prostate cancer cellphenotype (51). Our data presented here, however, showthat IL-6 directly promoted metastatic processes throughJak1/2 signaling independently of AR, because DU145cells are negative for expression of functional AR. In otherwords, IL-6 is capable of promotingmetastatic behavior ofprostate cancer cells independently of theAR–TGF-b axis.Future work will need to determine if IL-6 is capable ofpromoting metastatic processes simultaneously throughmultiple signaling pathways, including Jak1/2–Stat3,Jak1/2–ERK1/2, and AR–TGF-b in prostate cancer cellspositive for AR expression.

The finding of key clinical importance in the presentwork is that pharmacologic targeting of IL-6–Jak1/2–Stat3signaling by the Jak1/2 inhibitor AZD1480 effectivelysuppressed IL-6–induced migratory prostate cancer cellphenotype and metastatic dissemination in nude mice.This is critically important as numerous Jak1/2 inhibitorsare currently in active clinical development for hemato-poietic proliferative disorders and malignancies (36, 37).Jak1/2 inhibitors could potentially provide an adjuvanttherapy for organ-confined or locally-advanced prostatecancer as a strategy to prevent metastases developmentafter primary treatment. In addition, Jak1/2 inhibitorsmightprovide therapeutic benefit for treatment of existingmetastases in both hormone-responsive and CR prostatecancer. In the presentwork,we also demonstrated that IL-6 is expressed in the majority of distant metastases ofclinical prostate cancers, similar to what we have previ-ously shown for active Stat3 expression in clinical prostatecancer metastases (33). Future work should establish ifautocrine IL-6 production inprostate cancer predicts earlydevelopment of recurrent metastatic prostate cancer.Moreover, it will be important to evaluate if IL-6 expres-sion in prostate cancer metastases predicts early prostate

cancer–specific death. Finally, IL-6 production/positivityof clinical prostate cancers may serve as a biomarker toidentify patients who would be most responsive to Jak2inhibitor–based therapies. Siltuximab (CNTO328), amonoclonal antibody against IL-6, has been evaluated inseveral phase I and II trials in CRPC in combination withchemotherapy or in the postchemotherapy setting (52–54). The efficacy of siltuximabwas limited in these studies(52–54). However, this may be due to the fact that nopatient selection based on IL-6 positivity or Stat3 activa-tion of CRPC was conducted.

In summary, the findings presented in this work are thefirst to demonstrate induction of metastatic processes inprostate cancer by IL-6–Jak1/2–Stat3/ERK1/2 pathways,and the therapeutic potential of pharmacologic Jak1/2inhibition as a novel treatment strategy for advanced,metastatic prostate cancer.

Disclosure of Potential Conflicts of InterestM. Zinda and D. Huszar have ownership interest in AstraZeneca. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Gu, A.L. Romero-Weaver, J. Abdulghani,K. Alanen, M. Zinda, D. Huszar, M.T. NevalainenDevelopment of methodology: L. Gu, P. Vogiatzi, J. Abdulghani, M.T.NevalainenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Gu, P. Talati, A.L. Romero-Weaver, Z. Liao,D.T. Hoang, T. Mirtti, M.T. NevalainenAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): L. Gu, P. Talati, P. Vogiatzi, A.L. Romero-Weaver, J. Abdulghani, B. Leiby, T. Mirtti, M.T. NevalainenWriting, review, and/or revision of the manuscript: L. Gu, P. Talati,P. Vogiatzi, A.L. Romero-Weaver, J. Abdulghani, D.T. Hoang, T. Mirtti,M. Zinda, D. Huszar, M.T. NevalainenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Gu, P. Vogiatzi, T. Mirtti,K. Alanen, M.T. NevalainenStudy supervision: L. Gu, M.T. Nevalainen

AcknowledgmentsThe authors thank Geraldine Bebernitz, Shenghua Wen, Denise

Hughes, and Corinne Reimer at AstraZeneca for their contributions.

Grant SupportThis work was supported by grants from National Cancer Institute

(2RO1CA11358-06, to M.T. Nevalainen), AstraZeneca (to M.T. Nevalai-nen), and Pennsylvania Department of Health (to M.T. Nevalainen).Shared Resources of Kimmel Cancer Center at Thomas Jefferson Univer-sity are partially supported by NIH grant CA56036-08 (Cancer CenterSupport Grant to Kimmel Cancer Center, Thomas Jefferson University).

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 July 29, 2013; revised January 13, 2014; accepted January 29,2014; published OnlineFirst February 27, 2014.

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Gu et al.




2014;13:1246-1258. Published OnlineFirst February 27, 2014.Mol Cancer Ther Lei Gu, Pooja Talati, Paraskevi Vogiatzi, et al. Cancer Metastases Formation

Induced Experimental Prostate−AZD1480 Potently Inhibits IL-6 Pharmacologic Suppression of JAK1/2 by JAK1/2 Inhibitor

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