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Small Molecule Therapeutics

Bazedoxifene as a Novel GP130 Inhibitor forPancreatic Cancer TherapyXiaojuan Wu1,2, Yang Cao2,3, Hui Xiao2, Chenglong Li4, and Jiayuh Lin5

Abstract

The IL6/GP130/STAT3 pathway is crucial for tumorigenesis inmultiple cancer types, including pancreatic cancer, and presents asa viable target for cancer therapy. We reported Bazedoxifene,which is approved as a selective estrogen modulator by FDA, asa novel inhibitor of IL6/GP130 protein–protein interactionsusingmultiple ligand simultaneous docking and drug reposition-ing approaches. STAT3 is one of the major downstream effectorsof IL6/GP130. Here, we observed Bazedoxifene inhibited STAT3phosphorylation and STAT3 DNA binding, induced apoptosis,and suppressed tumor growth in pancreatic cancer cells withpersistent IL6/GP130/STAT3 signaling in vitro and in vivo. Inaddition, IL6, but not INFg , rescued Bazedoxifene-mediatedreduction of cell viability. Bazedoxifene also inhibited STAT3

phosphorylation induced by IL6 and IL11, but not by OSM orSTAT1 phosphorylation induced by INFg in pancreatic cancercells, suggesting that Bazedoxifene inhibits the GP130/STAT3pathway mediated by IL6 and IL11. Furthermore, Bazedoxifenecombinedwith paclitaxel or gemcitabine synergistically inhibitedcell viability and cell migration in pancreatic cancer cells. Theseresults indicate that Bazedoxifene is a potential agent and cangenerate synergism when combined with conventional chemo-therapy in human pancreatic cancer cells and tumor xenograftin mice. Therefore, our results support that Bazedoxifene as anovel inhibitor of GP130 signaling and may be a potentialand safe therapeutic agent for human pancreatic cancer therapy.Mol Cancer Ther; 15(11); 2609–19. �2016 AACR.

IntroductionHuman pancreatic cancer is one of the deadly malignant

diseases with very poor clinical outcome. The median overallsurvival is approximately 6 months after surgical and chemor-adiotherapies for locally advanced and metastatic stages ofpancreatic cancer and the 5-years overall survival rate is lessthan 5%. Because of the absence of specific symptoms, the lackof early detection techniques, pancreatic cancer is usuallydiagnosed at advanced and metastatic stages and is not resectedby surgery (1, 2). To date, chemo- and radiotherapy have onlylimited success because of high resistance (3). Unfortunately,only less than 15% of all pancreatic cancer patients have achance for surgical resection, after which 5-year survival rarelysucceed to 20% to 25% (4, 5).

The IL6/GP130/STAT3 signaling pathway is frequently acti-vated in many human cancer and contributes to oncogenesisand cancer progression (6, 7). The present review is to highlightthe role of IL6 in pancreatic cancer development and progres-sion (8). It is well-established that IL6 is elevated in the serum

of pancreatic cancer patients compared with healthy controlsand those with chronic pancreatitis (9–13). Several studiesraised strong evidence that elevated levels of IL6 protein andmRNA in serum and tumor samples of patients with pancreaticcancer is associated with increased tumor size and poor prog-nosis (14). IL6 binds a nonsignaling a-receptor IL6R to form abinary complex (IL6/IL6Ra), which, after dimerization withGP130, leads to activation of receptor-associated JAKs. In turn,these several phosphorylate downstream targets, includingcytoplasmic STAT3, which after dimerization rapidly translo-cate to the nucleus and promotes pancreatic cancer progressionthrough transcriptional regulation of antiapoptotic and pro-proliferative genes (14). STAT3 has been identified as a keyoncogenic factor in a number of human cancers and is requiredfor oncogenesis in mouse model of cancers (15, 16). In pan-creatic cancers, constitutive activation of STAT3 by phosphor-ylation of Tyr705 has been reported in 30% to 100% of humantumor specimens, as well as in many pancreatic cancer cell lines(17, 18). In contrast, this pathway is inactive in normal pan-creas, and correspondingly STAT3 is not required for pancreaticdevelopment or homeostasis (19). These studies suggest thatSTAT3 activation by IL6/GP130 signaling pathway plays animportant role in human pancreatic cancer development andprogression.

Inhibiting IL6/GP130 signaling might be a new therapeuticoption for pancreatic cancer. One possibility would be the treat-ment with the humanized monoclonal anti–IL6R antibodies,which is already approved for the treatment of some inflamma-tion disease (20). However, its potential therapeutic effect onpancreatic cancer has not yet examined. Selective inhibitors ofIL6/GP130/STAT3 are more effective options for treatment ofpancreatic cancer. Our previous study explored that a small-molecular inhibitor of STAT3, LLL12, was proposed selectivelyblocking exogenous IL6-induced STAT3 phosphorylation andnuclear translocation in two human pancreatic cancer cell lines

1Department of Pediatric Surgery, Tongji Hospital, Huazhong Univer-sity of Science and Technology,Wuhan, China. 2Department of Pedi-atrics, Center for Childhood Cancer and Blood Diseases, the ResearchInstitute at Nationwide Children's Hospital, College of Medicine, TheOhio State University, Columbus, Ohio. 3Department of Hematology,Tongji Hospital, Huazhong University of Science and Technology,Wuhan, China. 4Division of Medicinal Chemistry and Pharmacognosy,College of Pharmacy, The Ohio State University, Columbus, Ohio.5Department of Biochemistry and Molecular Biology, University ofMaryland School of Medicine, Baltimore, Maryland.

Corresponding Author: Jiayuh Lin, University of Maryland School of Medicine,655 W Baltimore S, Baltimore, MD 21201. Phone: 410-706-7469; Fax: 410-706-8297; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0921

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 2609

on June 5, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 17, 2016; DOI: 10.1158/1535-7163.MCT-15-0921

http://mct.aacrjournals.org/

(21). Because of the importance of small molecules in drugdiscovery, and as there are no any examples of small moleculesinhibiting the IL6/GP130 pathway associated in pancreatic cancertreatment, it would be a good strategy to use small molecularinhibitors for the same.

Bazedoxifene is known as a selective estrogen modulator andcommonly used for the prevention for osteoporosis. Recently,we have discovered Bazedoxifene as a novel small molecularGP130 inhibitor, which binds to GP130 D1 domain (22). Itmay be expected to speed up the development of clinicaltherapies for the IL6/GP130/STAT3–dependent cancers. In thisstudy, we report the new role of Bazedoxifene as a GP130inhibitor to inhibit the GP130/STAT3 signaling pathway medi-ated by IL6 and IL11, induce apoptosis in pancreatic cancercells, and suppress the tumor growth in human pancreaticcancer xenograft, suggesting that Bazedoxifene may serve as anovel therapeutic drug for pancreatic cancer by targeting theGP130/STAT3 signaling pathway.

Materials and MethodsCell lines and reagents

Human pancreatic cancer cell lines (AsPC-1, PANC-1, HPAF-II,BxPC-3, HPAC, Capan-1) were purchased from the ATCC.HPAF-II cells were cultured in Eagle's Minimum EssentialMedium (DMEM), Capan-1 cells were maintained in Iscove'sModified Dubecco's Medium (IMDM) supplemented with 20%FBS and the others in DMEM supplemented 10% FBS and 1%penicillin/streptomycin. HPAF-II was purchased within 2months before the experiments about HPAF-II was performed.AsPC-1, PANC-1, BxPC-3, Capan-1, and HPAC cell lines werefrozen within 2 months of receipt and were resuscitated fromearly passage liquid nitrogen stocks as needed. Cells werecultured for less than 3 months before reinitiating cultures andwere routinely inspected microscopically for stable phenotype.All cell lines were cultured in a humidified 37�C incubator with5% CO2.

IL6, IL11, OSM, and IFNg were purchased from Cell SignalingTechnology. The powder was dissolved in sterile PBS to make a100 ng/mL stock solution. Bazedoxifene was purchased fromAcesys Pharmatech, and paclitaxel and gemcitabine were boughtfrom LC Laboratories. These three drugs were dissolved in sterileDMSO tomake a 20mmol/L stock solution. Aliquots of the stocksolution were stored at �20�C.

Western blotting assayHuman pancreatic cancer cell lines (HPAF-II, BxPC-3, Capan-1,

and HPAC) were harvested after treatment with Bazedoxifeneor DMSO at 50% to 60% confluence overnight, then lysed incold RIPA lysis buffer containing protease inhibitors cocktailand phosphatase inhibitor cocktail. The lysates were subjectedto 10% or 12% SDS-PAGE gel and transferred to a PVDFmembrane. Membranes were probed with a 1:1,000 dilutionof specific primary antibody and 1:10,000 horseradish perox-idase–conjugated secondary antibody. Primary antibodiesagainst phosphorylated STAT3 (Tyr705), STAT3, phosphorylat-ed STAT1 (Tyr701), STAT1, cleaved caspase-3, phospho-specificextracellular signal-regulated kinase (ERK) 1/2 (Threonine 202/Tyrosine 204), P-AKT (Ser473), GAPDH and secondary anti-body are all from Cell Signaling Technology. Membranes wereanalyzed using enhanced chemiluminescence plus reagents and

scanned with the Storm Scanner (Amersham Pharmacia Bio-tech Inc.).

STATs phosphorylation induced by cytokines or growth factorsPANC-1, AsPC-1, and HPAF-II pancreatic cancer cells were

seeded in 10-cm plates and allowed to adhere overnight. Thefollowing night, the cells were serum starved. The cells were thenleft untreated orwere treatedwith Bazedoxifene (5–20mmol/L) orDMSO. After 2 hours, the untreated and Bazedoxifene-treatedcells were stimulated by IL6 (50 ng/mL), IL11 (50 ng/mL), OSM(50 ng/mL), or INFg (50 ng/mL) for 30 minutes. The cells wereharvested and analyzed by Western blot analysis for p-STAT3Y705

or p-STAT1Y701.

Reverse transcriptase-PCRCells were treated with Bazedoxifene (5–20 mmol/L) or DMSO

at 50% to 60% confluence in the presence of 10% FBS for 24hours. RNA from the cells was then extracted using RNeasy Kits(Qiagen) according to the manufacturer's instruction. Reversetranscription was done using an Omniscript reverse transcriptionkit (Qiagen). PCR amplification was performed under the fol-lowing conditions: 5 minutes at 94�C followed by 30 cycles of 30seconds at 94�C, 30 seconds at 48–55�C, and 60 seconds at 72�Cwith a final extension of 10 minutes at 72�C. The followingprimers were used: Cyclin D1, annealing at 52�C (For): 50-GCTGGAGCCCGTGAAAAAGA-30 (Rev): 50-CTCCGCCTCTGG-CATTTTG-30; Bcl-Xl, annealing at 48�C (For): 50-TTGGACAATG-GACTGGTTGA-30 (Rev): 50-GTAGAGTGGATGGTCAGTG-30; Sur-vivin, annealing at 52�C (For): 50-ACCAGGTGAGAAGTGAGG-GA-30 (Rev): 50-AACAGTAGAGGAGCCAGGGA-30; GAPDH,annealing at 52�C (For): 50-TGATGACATCAAGAAGGTGGT-GAAG-30 (Rev): 50-TCCTTGGAGGCCATGTGGGCAT-30 (integrat-ed DNA Technologies).

MTT cell viability assayHuman pancreatic cancer cell lines (HPAC, PANC-1, HPAF-II,

BxPC-3, and Capan-1), were seeded in 96-well plates at a densityof 3,000 cells per well. The next day, IL6 (50 ng/mL), INFg (50 ng/mL), or Bazedoxifene (10 mmol/L) alone, or combination of IL6or INFg with Bazedoxifene were added in triplicate to the plates inthe presence of 0% FBS in HPAF-II cells for 24 hours. Differentconcentrations of Bazedoxifene (5–10 mmol/L), paclitaxel (1–2.5mmol/L), or gemcitabine (5 mmol/L) alone, or Bazedoxifene pluspaclitaxel or gemcitabine were add in triplicate to the plates in thepresence of 10% FBS in BxPC-3 or Capan-1 cells or paclitaxelplus gemcitabine were add in the plates in PANC-1, HPAC,BxPC-3 and Capan-1 cells. The cells were incubated at 37�Cfor a period of 24 to 48 hours. BxPC-3 and Capan-1 cells wereseeded in 96-well plates at a density of 3,000 cells per welland cultured at 37�C. The next day, GP130 siRNA (100 nmol/L) or negative control siRNA was transfected into cells intriplicate using lipofectamine 2000 for 72 hours. Twenty-fivemL of 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bro-mide (MTT, Sigma) was added to each sample in a volumeof 100 mL and incubated for 4 hours. Then 150 mL of N, N-dimethylformamide (Sigma) solubilization solution wasadded to each well. The absorbance was read at 595 nm.Combination index (CI) was performed using data obtainedfrom MTT assay with CompuSyn software. The CI valuesindicate a synergistic effect when 1, and an additive effect when equal to 1 (23).

Wu et al.

Mol Cancer Ther; 15(11) November 2016 Molecular Cancer Therapeutics2610




STAT3 DNA binding assayBxPC-3 cells were seeded in a 10-cm plate and treated with

Bazedoxifene (5–10 mmol/L) or DMSO for 24 hours. The NuclearExtract Kit (Clontech Inc.) was used to prepare cell nuclear extractsfollowing the manufacturer's protocol. Nuclear extracts wereanalyzed for STAT3 DNA–binding activity using a STAT3 DNAbinding ELISA kit (Active Motif) with an ELISA-based method.Absorbance was read at 450 nm.

Wound-healing/cell migration assayWhen HPAC cells were 100% confluent, the monolayer was

scratched in same width using a pipette tip. After washing, HPACcells were then treated with different concentrations of Bazedox-ifene or DMSO. In addition, we treated HPAC cells with Bazedox-ifene, paclitaxel alone, or combination of them. After 24 hoursculture, when thewound in theDMSO control was closed, imageswere captured by Leica Microsystems.

ImmunofluorescenceHPAF-II cells were seeded on glass coverslips in 6-well plate.

The next day, the cells were cultured in serum free medium for24 hours and pretreated with Bazedoxifene (20 mmol/L) for2 hours, followed by induction with 50 ng/mL IL6 for 30 min-utes. Cells were fixed with cold methanol for 15 minutes andblocked with 5% normal goat serum and 0.3% Triton X-100 inPBS for 1 hour. The cells were incubated with primary anti-bodies of p-STAT3Y705 (Cell Signaling Technology, 1:100)overnight at 4�C. After incubation with anti-rabbit FIFC-con-jugated secondary antibody (Invitrogen, 1: 200), the cells weremounted with Vectashield Hardset mounting medium withDAPI (Vector Laboratories). Photomicrographs were capturedby Leica Microsystems.

Mouse xenograft tumor modelAll animal studies were conducted in accordance with the

principles and standard procedures approved by IACUC of theResearch Institute at Nationwide Children's Hospital. Capan-1(3�106) andHPAF-II (3�106) cells inMatrigel (BDBiosciences)were injected subcutaneously into the both side of flank area of6-week-old female athymic nude mice which were purchasedfrom Harlan. After Capan-1 tumor development, which was 1week after initial implantation, mice were divided into twotreatment groups consisting of four mice (tumors: n¼ 8): DMSOvehicle control and gavage injection of Bazedoxifene (5mg/kg/d).Mice bearing HPAF-II tumor were irrigated with Bazedoxifene(5 mg/kg/d) and/or injected via abdomen with paclitaxel (15mg/kg, 2/w). Tumor growth was determined by measured thelength (L) and width (W) of the tumor every other day with acaliper, and tumor volume was calculated on the basis of thefollowing formula: volume ¼ 0.52 � LW2. After 21 days oftreatment, tumorswereharvested, snap-frozen indry ice, andstoredat�80�C. Tumors tissue homogenates were lysed and separated bySDS-PAGE to examine the expression of STAT3 phosphorylation,P-ERK1/2, P-AKT (Ser473), and cleaved caspase-3.

Statistical analysisSignificance of correlations was done using GraphPad Prism

software. Unpaired t tests were used for analyses assuming Gauss-ian populations with a 95% confidence interval. Data are pre-sented asmean� SE.Differences were analyzedwith the Student t

test, and significance was set at P < 0.05; �, P < 0.05; ��, P < 0.01;and ��, P < 0.001, respectively.

ResultsBazedoxifene, a novel small molecule inhibitor that targetsGP130

IL6/IL6Ra or IL11/IL11R binds to the GP130 D1 domainthrough a few hot residues to form the IL6/IL6Ra/GP130 orIL11/IL11R/GP130 heterotrimers and dimerization of the trimersactivates IL6/GP130/STAT3 or IL11/GP130/STAT3 signalingpathway, which is crucial for the progression in multiple humancancers. We discovered Bazedoxifene as a novel molecule inhib-itor of IL6/GP130 or IL11/GP130 protein–protein interactions(PPI) using multiple ligand simultaneous docking (MLSD).We identified that Bazedoxifene disables the dimerization of theIL6/IL6Ra/GP130 or IL11/IL11R/GP130 heterotrimers usingMLSD method (Fig. 1; ref. 22). Bazedoxifene targets the humanestrogen receptor (ER) and is approved by FDA as a drug for theprevention of osteoporosis. In our study, we confirmed thatBazedoxifene inhibits STAT3 phosphorylation induced by IL6and IL11 in GP130/STAT3 pathway signaling.

Bazedoxifene inhibits STAT3 phosphorylation induced bycytokines in human pancreatic cancer cells

IL6 family cytokines such as IL6, IL11, and OSM can induceSTAT3 phosphorylation. AsPC-1, HPAF-II, and PANC-1 pancre-atic cancer cells, which do not express phosphorylated STAT3 inserum-free medium for 24 hours, were used to examine if Baze-doxifene can inhibit IL6, IL11, or OSM induced STAT3 phosphor-ylation. In this study, we found IL6 as well as IL11 could stimulatephosphorylation of STAT3 and Bazedoxifene could decrease thephosphorylation in a dose-dependent manner. However, Baze-doxifene could not suppress p-STAT3 induced by OSM (Fig. 2A–C) or phosphorylation of STAT1 stimulated by INFg in AsPC-1andHPAF-II cells (Fig. 2D). These results indicate Bazedoxifene isan inhibitor of GP130/STAT3 signaling pathway mediated by IL6and IL11 in pancreatic cancer cells.

Figure 1.

Bazedoxifene (ball-and-stick) binds to GP130 D1 domain (ribbon model). Ile83and Phe36 offer important hydrophobic interaction with bazedoxifene'sazepanylring; and Tyr94 and Asn92 form aromatic and hydrogen-bondinginteractions with bazedoxifene, respectively.

Bazedoxifene as a Novel GP130 Signaling Inhibitor

www.aacrjournals.org Mol Cancer Ther; 15(11) November 2016 2611




Bazedoxifene inhibits phosphorylation of STAT3, inducesapoptosis, and suppresses transcription of STAT3 downstreamtargets in human pancreatic cancer cells

Bazedoxifene was assessed for its inhibitory effect of GP130/STAT3 signaling on Capan-1, BxPC-3, HPAF-II, and HPAChuman pancreatic cancer cells which expressed persistentSTAT3 phosphorylation. The results showed that Bazedoxifenedecreased expression of p-STAT3Y705 in dose-dependent man-ner in all four cell lines (Fig. 2F). The inhibition of phosphor-ylated STAT3 by Bazedoxifene was consistent with the induc-tion apoptosis evidenced by the increasing caspase-3 in all celllines (Fig. 2F). However, Bazedoxifene could impact differentdownstream targets of GP130 in different pancreatic cell lines.

As shown in Fig. 2C, p-AKT (S473) was downregulated byBazedoxifene in BxPC-3, HPAF-II, and HPAC except Capan-1cells, but only in BxPC-3, Capan-1, and HPAC cells suppressionof p-ERK1/2 (T202/Y204) was seen. In addition, to examine theinfluence of Bazedoxifene on the inhibition of STAT3 pathway,we detected the RNA expression of its downstream targets byreverse transcription (RT)-PCR, including CyclineD1, Bxl-cL,and Survivin. We found these STAT3 targets were decreased inCapan-1, BxPC-3, and HPAF-II cells when they were treatedwith different concentration of Bazedoxifene (5–20 mmol/L;Fig. 2E). This indicates that Bazedoxifene is an effective GP130inhibitor through donwregulation of its downstream targets inpancreatic cancer cells.

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Figure 2.

Bazedoxifene inhibits STAT3 phosphorylation induced by IL6 and IL11, downregulates expression of STAT3 downstream target genes, and decreasesthe STAT3 DNA–binding activity in pancreatic cancer cells. P-STAT3Y705, P-AKT, and P-ERK were analyzed by Western blot analysis in AsPC-1 (A), PANC-1 (B),and HPAF-II (C) cells after induction by IL6, IL11, and OSM (50 ng/mL). D, AsPC-1 and HPAF-II cells were stimulated by INFg (50 ng/mL) and thecells were harvested and analyzed for p- STAT1Y701. E, the mRNA expression of CyclinD1, Bcl-xL, and Survivin was detected at by RT-PCR in Capan-1,BxPC-3, and HPAF-II after treatment with Bazedoxifene. F, Western blot analysis of p-STAT3Y705, p-AKT (S473), p-ERK (T202/Y204), and cleaved caspase-3were performed after treatments with Bazedoxifene at the indicated concentrations in Capan-1, BxPC-3, HPAF-II, and HPAC cells. G, STAT3 DNA–bindingactivity in BxPC-3 or Capan-1 cells treated with Bazedoxifene for 24 hours was detected by DNA-binding assay (� , P < 0.05; �� , P < 0.01). H, HPAF-IIcells were treated with cytokines, including IL6 and INFg (50 ng/mL), with or without Bazedoxifene (10 mmol/L) in serum-free condition for 24 hours(� , P < 0.05). Cell viability was detected by MTT assay.

Wu et al.





Bazedoxifene inhibits STAT3 DNA binding and STAT3 nucleartranslocation induced by IL6; IL6 partially rescuesbazedoxifene-mediated decrease of cell viability in humanpancreatic cancer cells

IL6 activates GP130/STAT3 signaling pathway throughSTAT3 nuclear translocation to bind DNA. To evaluate theinhibition of STAT3 DNA binding activity, BxPC-3 and Capan-1 cells were treated with Bazedoxifene (5–20 mmol/L) over-night and were harvested and performed STAT3 DNA-bindingactivity assay as described in Materials and Methods. We foundthat Bazedoxifene caused a significant different inhibitionof STAT3 DNA-binding activity in BxPC-3 and Capan-1 cells(Fig. 2G). Furthermore, HPAF-II cells were starved in serum-free medium for 24 hours and pretreated with Bazedoxifene(20 mmol/L) for 2 hours followed by IL6 stimulation. Immu-nofluorescence results showed STAT3 nuclear translocationinduced by IL6 was blocked by Bazedoxifene (Fig. 3A). How-ever, Bazedoxifene could not inhibit STAT1 nuclear translo-cation induced by INFg (Fig. 3B). On the other hand, toexplore whether exogenous IL6 could rescue the decreased cellviability by Bazedoxifene, we treated HPAF-II cells with 10mmol/L Bazedoxifene with or without IL6 or INFg (50 ng/mL)in serum-free medium for 24 hours. The data in Fig. 2Hshowed IL6 could partially rescue Bazedoxifene-mediatedinhibition in HPAF-II cells.

Inhibition of GP130/STAT3 using Bazedoxifene andSiRNA-sensitized pancreatic cancer cells to anticancer drugs,gemcitabine, or paclitaxel

To evaluate the synergistic effect of suppression of GP130/STAT3 with other anticancer drugs, we first treated pancreaticcancer cells in combination of Bazedoxifene with gemcitabineor paclitaxel, which are the standards of care for humanpancreatic cancer at present. As shown in Fig. 4A, the combi-nation effect of Bazedoxifene with gemcitabin or paclitaxel for48 hours in BxPC-3 and Capan-1 cells showed cell viability wasmore significantly decreased in the combination treatmentgroup than single drug group. The CI values of all the combi-nation treatments were less than 1, suggesting there was syn-ergism in the combination treatments of Bazedoxifene withgemcitabin. To further determine the role of inhibition of theIL6/GP130/STAT3 pathway in combination treatment withother anticancer drugs, BxPC-3 and Capan-1 cells were trans-fected with GP130 or IL6 SiRNA for 48 hours and treated withgemcitabine or paclitaxel for more 24 hours. Knockdown ofGP130 or IL6 was confirmed by western blot, as shownin Fig. 4B, p-SATA3Y705, GP130, or IL6 was decreased. Cellviability was further reduced in GP130-knockdown-cells andIL6-knockdown-cells treated with gemcitabine or paclitaxelthan control groups (Fig. 4C). Furthermore, our results showedthe greater inhibition was seen in the combination treatment of

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Bazedoxifene inhibits STAT3 nucleartranslocation induced by IL6, but notp-STAT1 by INFg . HPAF-II cells withlower expression of p-STAT3Y705 werepretreated with bazedoxifene(20 mmol/L) for 2 hours. After that, thecells were stimulated by IL6 or INFg (50ng/mL) for half an hour, and STAT3 (A)or STAT1 (B) nuclear translocation wasdetected by immunofluorescence.






Bazedoxifene with paclitaxel or gemcitabine than paclitaxelplus gemcitabine in HPAC, Capan-1, HPAF-II and BxPC-3 cells(Fig. 4D). These results revealed that suppression of GP130/STAT3 signaling pathway could sensitize pancreatic cancer cellsto the first-line chemotherapeutic agents, gemcitabine, andpaclitaxel.

Bazedoxifene inhibits cell migrationGP130/STAT3 activation is involved in cell migration, so sup-

pression ofGP130might block cellmigration in pancreatic cancercells. Therefore, we next evaluated whether Bazedoxifene couldblock cell migration which is an important process in tumorinvasion and metastasis. As shown in Fig. 5A, Bazedoxifenetreatment reduced migration ability in a dose-dependent mannerinHPAC cells.Moreover, combination treatment blocked the cellsmigration more significantly than single group (Fig. 5B). Theseresults suggest that inhibition of GP130 by Bazedoxifene blocksthe cellsmigration in pancreatic cancer cells and the cellmigration

was more significantly inhibited in the combination treatmentgroup than single-agent groups.

Bazedoxifene inhibits capan-1 tumor growth in mouse modelin vivo

We further verified whether Bazedoxifene suppressed thetumor growth in vivo as in vitro. Capan-1 cells (3 � 106)injection was performed as previously described in Materialsand Methods. One week after initial implantation, when thetumors reached a size of 0.05 to 0.1cm3, the mice were given5 mg/kg Bazedoxifene in the treated group or DMSO in vehiclegroup daily for 18 days. As shown in Fig. 6A, Bazedoxifenesignificantly suppressed tumor growth compared with thevehicle group. P-STAT3Y705 of tumor tissue sample in Bazedox-ifene-treated group was reduced, and caspase-3 was induced(Fig. 6A), suggesting that Bazedoxifene could suppress pancre-atic cancer xenograft tumor growth and induce apoptosis intumor cells.

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Figure 4.

Blocking GP130 signaling using Bazedoxifene, GP130 SiRNA, or IL6 SiRNA enhances the effect of treatment with paclitaxel and gemcitabine in pancreaticcancer cells. A, cell viability was performed by MTT after treatment with bazedoxifene, paclitaxel, gemcitabine alone or combination in BxPC-3 and Capan-1 cells(� , P < 0.05; �� , P < 0.01; �� , P < 0.001). B, BxPC-3 and Capan-1 cells were transfected with GP130 SiRNA or IL6 SiRNA for 72 hours. IL6, GP130, p-STAT3Y705,p-AKT (S473), and p-ERK (T202/Y204) were assessed by Western blot analysis. C, BxPC-3 and Capan-1 cells were transfected with GP130 SiRNA or IL6SiRNA for 48hours and treatedwith paclitaxel for another 24hours. Cell viabilitywasdetected (�,P

Finally, we tested whether the combined Bazedoxifene andpaclitaxel had stronger inhibitory effects than single drug treat-ment in HPAF-II xenograft tumor growth. HPAF-II tumor-bearingmice were treated with Bazedoxifene and paclitaxel either indi-vidually or in combination as described in Materials and Meth-ods. Compared with vehicle-treated mice, tumor volumewas significantly decreased in mice treated with Bazedoxifene(P < 0.05) or paclitaxel alone (P < 0.05). Furthermore, combi-nation of Bazedoxifene and paclitaxel dramatically decreasedtumor growth compared with both vehicle and monotherapy(P < 0.05, Fig. 6B). Correspondingly, the combination therapywas well tolerated and did not result in any significant in vivotoxicity (Fig. 6B). As Fig. 6B shown, P-STAT3Y705 of tumor tissuesample in Bazedoxifene- and combination-treated groups wasreduced, and caspase-3 was more induced in the combination-treated group than the Bazedoxifene-treated group.

DiscussionPancreatic cancer is an extremely aggressive malignant tumor

characterized by extensive invasion and early metastasis (24).Pancreatitis is known as the most common precursor lesions ofpancreatic cancer. Recent evidences indicate several inflamma-tory cytokines, including IL6, express abnormally highly inchronic pancreatitis, at all stages of human pancreatic carcino-genesis in mouse models of this disease (7, 25–28). Given bythe fact that IL6 plays important role during the initiation,maintenance, and progression of pancreatic cancer (14, 26).

Several studies have revealed that the inhibition of IL6 provedanti–IL6–blocking antibodies or selective molecule sgp130Fcto inhibit IL6 signaling and induce cell apoptosis in pancreaticcancer cells and animal models (20, 29). However, antibodytreatment led to massive systemic elevations in IL6 (30). Toovercome such difficulties, inhibitors of GP130, as an impor-tant part of receptor signaling complexes of IL6/IL6R/GP130,are required. SC144, a GP130 inhibitor, was reported that itinhibits the GP130/STAT3 pathway through decrease constitu-tive STAT3 phosphorylation and its downstream genes expres-sion in ovarian cancer (31, 32). Though their study showed thatGP130 is directly inhibited by SC144, the domain that binds toGP130 was not examined and still unclear. Existing drug,Bazedoxifene, which is approved by FDA as an estrogen recep-tor modulator and commonly used as treatment for osteopo-rosis (33, 34). As shown in Fig. 1, Bazedoxifene binds to GP130D1 domain through spots Ile83, Phe36, Tyr94 and Asn92,which suggesting that Bazedoxifene could be a novel inhibitorof IL6/GP130 signaling (22). Because the IL6/GP130/STAT3signaling pathway is involved in cancer growth, progression,and drug resistance in a variety of human cancers, includingpancreatic cancer (26, 35, 36), targeting this signaling pathwaywould be a promising therapy for the treatment of pancreaticcancer (37). The in vitro and in vivo results obtained in thisstudy, confirmed that the inhibition of persistent STAT3 acti-vation by Bazedoxifene, including suppressing the STAT3 phos-phorylation induced by IL6, reducing the downstream genesexpression, inhibiting cell migration in pancreatic cancer cells,

(μmol/L)B: Bazedoxifene

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Wound-healing assay was done in HPAC cells. A, HPAC cells were treated with different concentrations of bazedoxifene or DMSO (�� , P < 0.01). B, HPAC cellswere treated by bazedoxifene and paclitaxel alone or combination of both drugs (� , P < 0.05; �� , P < 0.01).






as well as suppress the pancreatic tumor growth in mousemodel in vivo.

Pancreatic cancer cell lines, BxPC-3,Capan-1,HPACandHPAF-IIcells were reported to secrete IL6 (38, 39). Therefore, BxPC-3,Capan-1, HPAC and HPAF-II cells expressing persistent IL6/GP130/STAT3 signaling were used to explore the inhibitory effecton the IL6/GP130/STAT3 signaling pathway by Bazedoxifene. Asthe major downstream effector of IL6/GP130 pathway, phosphor-ylation of STAT3 was downregulated by Bazedoxifene in all fourpancreatic cancer cell lines and the downstream target genes ofSTAT3, including cyclinD1, Bcl-xL, and survivin, were decreasedusing RT-PCR in Capan-1, BxPC-3 and HPAF-II cells, which con-firmed that Bazedoxifene is an effective inhibitor of IL6/GP130signaling. Our results also showed that Bazedoxifene could inhibitP-AKTandP-ERK1/2 in threeout of fourpancreatic cancer cell lines.We found P-AKT (S473) was much lower in Capan-1 cells than theother three cancer cell lines, and Bazedoxifene, IL6, and GP130siRNA all induced P-AKT insteadof inhibited P-AKT in this cell line.The observation that Bazedoxifene could inhibit P-AKT in BXPC-3cells and P-ERK1/2 in BXPC-3 and Capan-1 cells but IL6 or GP130siRNAcouldnot, suggestingapossiblemechanism:P-ERK inBXPC-3 and Capan-1 cells and P-AKT in BXPC-3 cells are not mainlydependent on IL6 or GP130 signaling. In addition, to investigate

whether suppression of IL6/GP130 signaling could induce cellapoptosis as reported in vitro and in vivo (40–43), apoptoticmarker cleaved caspase-3 was examined in Bazedoxifene-treatedpancreatic cancer cells. The results showed that Bazedoxifenetreatment induced cell apoptosis in pancreatic cancer cells. Onthe other hand, IL6 partially rescued Bazedoxifene-mediatedinhibition of cell viability inHPAF-II cells. Our results, therefore,support the idea that Bazedoxifene is a potent inhibitor ofGP130, which is consistent with suppression of GP130 inhibitsSTAT3 activity and induces cell apoptosis (42, 44, 45). Bazedox-ifene also inhibits pancreatic cancer cell migration. Pancreaticcancer cell lines we tested here secrete IL6. IL6 can induceP-STAT3 and other downstream target (such as AKT or ERK)through the autocrine pathway. Therefore, the ability of Baze-doxifene to inhibit cell viability and migration is likely due to itsability to inhibit one of the pathways ormore than one pathwayscombined: (i) autocrine IL6 induction of P-STAT3; (ii) autocrineIL6 and non-GP130 pathway(s) induction of P-ERK and P-AKT;(iii) other pathway(s) in addition to STAT3, ERK, and AKT.Furthermore, Bazedoxifene suppresses human pancreatic tumorgrowth in a mouse xenograft model, which is showing Bazedox-ifene as a potent inhibitor of pancreatic cancer cells expressingpersistent GP130/STAT3 signaling.

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Inhibitory efficacy of Bazedoxifene on tumor growth in the Capan-1 tumor xenograft mouse model. Combined Bazedoxifene and paclitaxel treatment results inrobust efficacy in HPAF-II tumor xenograft model. A, growth rate of the Capan-1 tumor xenograft in mouse model treated with vehicle and Bazedoxifene (�� , P < 0.01;�� , P < 0.001). B, growth rate of HPAF-II tumor xenograft in mouse model treated with vehicle, Bazedoxifene, paclitaxel, or Bazedoxifene/paclitaxel treatment(� , P < 0.05; �� , P < 0.01; ��, P < 0.001). P-STAT3Y705 and caspase-3 of Capan-1 tumor tissue samples from these mice were detected by Western blot analysis.

Wu et al.





IL6 family cytokines, IL6 and IL11, act on the cells usingreceptor GP130 by similar molecular interactions, which leadsto the intracellular signal (46, 47). In this study, Bazedoxifeneinhibited STAT3 phosphorylation induced by IL6 and IL11, butnot STAT1 phosphorylation induced by INFg was not inhibitedfurther indicating its selectivity on STAT3 over STAT1. IL6 binds toIL6Ra to form a binary complex and then recruits GP130 to formthe IL6/IL6Ra/GP130 heterotrimer. In addition, homodimeriza-tion of the IL6/IL6Ra/GP130 heteotrimers occurs by interactionsbetween IL6 of one trimer and the D1 domain of GP130 of theother trimer, forming a hexamer (48, 49). The reciprocal homo-dimerization of the IL6/IL6Ra/GP130 trimers triggers a signalingcascade downstream such as JAK/STAT3. Interestingly, from ourcomputational modeling, we also found that IL11 exhibits verysimilar hexamer formation as IL6. IL11 also binds to IL11Ra toform a binary complex and then recruits GP130 to form theIL11/IL11Ra/GP130 heterotrimer. Homodimerization of theIL11/IL11Ra/GP130 heteotrimers also occurs by interactionsbetween IL11 of one trimer and the D1 domain of GP130 of theother trimer to form a hexamer. Bazedoxifene specifically bindsto D1 domain of GP130 (but not D2 and D3 domains) andblocking IL6 or IL11 of one trimer to bind to the D1 domain ofGP130 on the other trimer, and thus further blocking hexamerformation and the signaling cascade downstream to STAT3.Our computational modeling is further supported by Bazedox-ifene can inhibit P-STAT3 induced by IL6 and IL11. IL11 isreported to be involved in tumorigenesis in gastric and breastcancers and is also a potential cancer therapeutic target (50–52). The expression of IL11 is also elevated in pancreatic cancer,suggesting that it may play a role in the oncogenesis of pan-creatic cancer (53). Furthermore, from our computationalmodeling, LIF and OSM, however, only form trimer to signal-ing downstream and do not form hexamerization through D1domain of GP130 protein. Therefore, Bazedoxifene did notinhibit P-STAT3 induced by OSM (this study) or LIF (22). Theseresults support the selectivity of Bazedoxifene for inhibitingGP130/STAT3 signaling mediated by IL6 and IL11 but not OSMand LIF. These results also suggest that Bazedoxifene could be adual inhibitor of IL6 and IL11 for cancer therapy.

Majority of the pancreatic cancer patients are relying on theneoadjuvant chemotherapy or chemoradiotherapy may beused in cases that are considered to be borderline resectableto reduce the cancer to such a level where surgery is could bedelayed and this in turn, given the fact that operation is notpreferable in many cases. However, this strong limitation ofconventional treatment is mainly due to the drug resistanceon the current standard-of-care treatment (54). The IL6/GP130/STAT3 pathway is involved in drug resistance in avariety of human cancers, including pancreatic cancer (55,56). In this study, we show that Bazedoxifene or knockdownof GP130 works synergistically with gemcitabine or paclitaxel

in BxPC-3 and Capan-1 pancreatic cancer cells, respectively.Furthermore, the experiment of cell migration with the com-bination treatment of Bazedoxifene and paclitaxel in HPACcells provides more evidences that Bazedoxifene sensitizespancreatic cancer to other anticancer drugs. In vivo, combina-tion of Bazedoxifene and paclitaxel was superior to bothvehicle and monotherapy in HPAF-II tumor xenograft mice.The ability of Bazedoxifene to generate stronger inhibition ofcell viability when combined with gemcitabine or paclitaxel,and is likely due to its ability to inhibit one of the pathway ormore than one pathways combined: (i) autocrine IL6 induc-tion of P-STAT3; (ii) autocrine IL6 and non-GP130 pathway(s) induction of P-ERK in BXPC-3 and Capan-1 cells andinduction of P-AKT in BXPC-3 cells; (iii) other pathway(s) inaddition to STAT3, ERK, and AKT. These results indicate thatIL6/GP130 signaling contributes to the drug resistance andBazedoxifene has stronger inhibition effect in combinationwith paclitaxel or gemcitabine than as a single agent.

On thebasis of ourfindings,we suggested that Bazedoxifene is apotent inhibitor of GP130/STAT3 signaling mediated by IL6 andIL11. Not only effectively it blocks activation of STAT3, but alsosuppresses pancreatic cancer growth in vitro and in vivo andsensitizes pancreatic cancer cells to paclitaxel and gemcitabine.Thus, Bazedoxifene is a potential therapeutic small molecularagent that could be useful for the treatment of human pancreaticcancer when combined with paclitaxel and gemcitabine.

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

Authors' ContributionsConception and design: X. Wu, C. Li, J. LinDevelopment of methodology: X. Wu, C. Li, J. LinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Wu, Y. Cao, C. Li, J. LinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Wu, J. LinWriting, review, and/or revision of the manuscript: X. Wu, H. Xiao, J. LinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Wu, J. LinStudy supervision: X. Wu, J. Lin

Grant SupportThis work was supported by the grants NIH/NCI/R21, CA173473-01 (PI: J.

Lin). NIH/R01, NS087213-01A1 (PI: J. Lin). AACR-Pancreatic Cancer Networkresearch grants (PI: J. Lin). National Natural Science Funding of China,81500395 (PI: X. Wu).

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

Received November 17, 2015; revised August 2, 2016; accepted August 4,2016; published OnlineFirst August 17, 2016.

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2016;15:2609-2619. Published OnlineFirst August 17, 2016.Mol Cancer Ther Xiaojuan Wu, Yang Cao, Hui Xiao, et al. TherapyBazedoxifene as a Novel GP130 Inhibitor for Pancreatic Cancer

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