Src as a Therapeutic Target in Biliary Tract Cancer...Small Molecule Therapeutics Src as a Therapeutic Target in Biliary Tract Cancer Ah-Rong Nam1, Ji-Won Kim2, Ji Eun Park1, Ju-Hee

Small Molecule Therapeutics

Src as a Therapeutic Target in Biliary Tract CancerAh-Rong Nam1, Ji-Won Kim2, Ji Eun Park1, Ju-Hee Bang1, Mei Hua Jin1,Kyung-Hun Lee1,3, Tae-Yong Kim3, Sae-Won Han1,3, Seock-Ah Im1,3,Tae-You Kim1,3, Do-Youn Oh1,3, and Yung-Jue Bang1,3

Abstract

Src, a nonreceptor tyrosine kinase, is involved in a number ofcancer-related signaling pathways and aberrantly activated inbiliary tract cancer (BTC). This study aimed to elucidate thepotential role of Src as a therapeutic target in BTC. We testedbosutinib, an orally active c-Src/Abl kinase inhibitor, alone orin combination with cytotoxic agents using 9 human BTC celllines: SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079,SNU-1196, HuCCT1, TFK-1, and EGI-1. Of these, SNU-308and SNU-478 were relatively sensitive to bosutinib. Bosutinibabrogated phosphorylation of Src and its downstream mole-cules, and significantly increased G1 cell-cycle arrest and apo-ptosis. Bosutinib significantly inhibited cell migration andinvasion and decreased epithelial–mesenchymal transitionmarkers. Bosutinib combined with gemcitabine or cisplatinshowed synergistic antiproliferative and antimigratory effects.

In addition, this combination further inhibited phosphoryla-tion of Src and its downstream molecules and decreased epi-thelial–mesenchymal transition marker expression comparedwith bosutinib alone. We established a SNU-478 xenograftmodel for in vivo experiments, because SNU-478 was moretumorigenic than SNU-308. Bosutinib combined with gemci-tabine or cisplatin showed significantly more potent antitumoreffects than bosutinib alone. Bosutinib combined with gemci-tabine further decreased Ki-67 expression and Src phosphory-lation, and further increased TUNEL expression. Our datasuggest that Src might be a potential therapeutic target in BTC.Bosutinib demonstrated promising antitumor activity alone orin combination with gemcitabine or cisplatin in BTC cells,which supports further clinical development in patients withadvanced BTC. Mol Cancer Ther; 15(7); 1515–24. �2016 AACR.

IntroductionSrc, a nonreceptor tyrosine kinase, is involved in a number of

cancer-related signaling pathways including FAK, PI3K, ERK,and STAT3, and promotes the proliferation, adhesion, migra-tion, invasion, andmetastasis of cancer cells (1–4). Its activity isincreased in a variety of malignancies such as lung, skin,colorectal, breast, ovarian, and head and neck cancer (3–6).In addition, Src activation conferred therapeutic resistance toimatinib treatment for chronic myelogenous leukemia (7, 8),hormone treatment for breast and prostate cancer (9, 10), andtrastuzumab treatment for breast cancer (11, 12). On the basisof these findings, Src-targeted agents have recently been inves-

tigated in preclinical and clinical studies in a variety of malig-nant diseases.

Biliary tract cancer (BTC) is an relatively uncommon malig-nancy with a poor prognosis (13). Most patients are diagnosed atan advanced stage and experience relapse despite radical surgery(14). Although the recent development of targeted therapeuticshas significantly improved the clinical outcome of patientswith advanced solid tumors, still there is no validated therapeutictarget for advanced BTC and the prognosis of patients withadvanced BTC remains still disappointing (15, 16). In recentyears, only gemcitabine plus cisplatin has been established as astandard chemotherapy for BTC patients (17). Therefore, there isstill an urgent unmet need for the development of novel thera-peutic strategies for the treatment of advanced BTC, based onspecific targets on cancer cells.

A recent study demonstrated that Src is also frequently over-expressed in BTC as in other malignant diseases, although itsrelationship with clinicopathologic parameters or histologicorigin was not significant (18). In addition, blockingSrc activity by novel Src inhibitors such as saracatinib(AZD-0530) and AZM555130 reduced the proliferative andinvasive potential of human BTC cell lines (18, 19). However,these studies used only a limited number of BTC cell lines andonly tested Src inhibitor monotherapy, without investigatingcombination strategies with other chemotherapeutic agentsthat are already approved for BTC treatment. Therefore, furtherpreclinical studies are necessary before designing clinical stud-ies using Src inhibitors.

The aim of our study was to investigate Src as a potentialtherapeutic target in BTC. We evaluated the therapeutic potentialof bosutinib (4-anilino-3-quinolinecarbonitrile, also known as

1Cancer Research Institute, Seoul National University College of Med-icine, Seoul, Korea. 2Department of Internal Medicine, Seoul NationalUniversityBundangHospital, Seongnam,Korea. 3Departmentof Inter-nal Medicine, Seoul National University Hospital, Seoul, Korea.

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

A.-R. Nam and J.-W. Kim contributed equally to this article.

Prior presentation: This study was presented in part at the Annual Meeting ofAmerican Association for Cancer Research, held inWashington, DC, in 2013 andin San Diego, CA in 2014.

Corresponding Author: Do-Youn Oh, Department of Internal Medicine, SeoulNational University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republicof Korea (South). Phone: +82-2-2072-0701; Fax: +82-2-762-9662; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0013

�2016 American Association for Cancer Research.

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on December 31, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 22, 2016; DOI: 10.1158/1535-7163.MCT-16-0013

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SKI-606; ref. 20), an orally active small-molecule c-Src/Abl kinaseinhibitor, alone or in combination with cytotoxic agents usingin vitro and in vivo models.

Materials and MethodsHuman BTC cell lines

A total of 9 human BTC cell lines were used in this study.SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079, and SNU-1196 cell lines were purchased from the Korean Cell Line Bankin March 2007 (21). HuCCT1 and TFK-1 cell lines wereobtained from the RIKEN BioResource Center in March2007. EGI-1 cell line was obtained from the Leibniz-InstitutDSMZ (German Collection of Microorganisms and Cell Cul-tures) on March 2007. The origin of each cell line was asfollows: SNU-245, extrahepatic cholangiocarcinoma (commonbile duct); SNU-308, gallbladder adenocarcinoma; SNU-478,ampulla of Vater adenocarcinoma; SNU-869, ampulla of Vateradenocarcinoma; SNU-1079, intrahepatic cholangiocarci-noma; SNU-1196, extrahepatic cholangiocarcinoma (hepaticduct bifurcation); HuCCT1, intrahepatic cholangiocarcinoma;TFK-1, extrahepatic cholangiocarcinoma; and EGI-1, extrahe-patic cholangiocarcinoma (21–24). Most recent authenticationof each cell lines was performed using "AmpFLSTR IdentifilerPCR Amplification Kit (catalog no. 4322288; Applied Biosys-tems)" by the Korean Cell Line Bank on March 8, 2016. The3530xL DNA Analyzer (Applied Biosystems) and the Gene-Mapper v5 (Applied Biosystems) were used for DNA finger-printing analysis. SNU-245, SNU-308, SNU-478, SNU-869,SNU-1079, SNU-1196, HuCCT1, and TFK-1 cell lines weremaintained in RPMI1640 media containing 10% FBS (WelgeneInc.) and 10 mg/mL gentamicin in a humidified atmospherecontaining 5% CO2 at 37�C. EGI-1 cell line was maintained inDMEM supplemented with 10% FBS and 10 mg/mL gentamicinin the same condition.

Tested agentsBosutinib was purchased from Selleck Chemicals LLC for in

vitro experiments and provided by Pfizer Inc. for in vivo experi-ments. The compoundwas dissolved in DMSO. Gemcitabine waspurchased from Lilly Korea Co. Cisplatin was purchased from JWPharmaceutical Co.

Cell growth inhibition assayCells were seeded in 96-well plates and exposed to increasing

concentrations of targeted or cytotoxic agents for 72 hours. Afterdrug treatment, tetrazolium dye (MTT; Sigma-Aldrich) wasadded to each well and incubated for 4 hours at 37�C. Then,the solution was removed carefully and DMSO was added. Cellviability was determined by measuring the absorbance at 540nm with a VersaMax Microplate Reader (Molecular Devices).The half-maximal inhibitory concentration (IC50) of chemo-therapeutic agents was analyzed using SigmaPlot software(Systat Software, Inc.).

Matrigel (Trevigen) was used for three-dimensional cultures.Matrigel was thawed at 4�C and 200 mL was added per well to a 6-well plate. The plate was then incubated at 37�C for 30minutes topromote gelling of the matrix. Next, harvested cells were mixedwith 400 mL of 2% Matrigel, and then added into each wellcontaining gelling matrix. After overnight incubation, each wellwas replaced by 2% Matrigel containing 0.1 or 0.5 mmol/L

bosutinib. Bosutinib-containing Matrigel was replaced every4 days for 12 days.

Western blot analysisCells were treated with bosutinib for 48 hours, and then lysed

in RIPA buffer containing protease inhibitors on ice for 15minutes. Next, protein was obtained by centrifugation at13,000 rpm for 20 minutes. Equal amounts of proteins wereseparated on 10% SDS polyacrylamide gels and transferred ontonitrocellulose membranes. The membranes were probed over-night at 4�C with primary antibodies. Primary antibodiesagainst the following molecules were purchased from CellSignaling Technology: Src, phosphorylated Src (Tyr416); FAK,phosphorylated FAK (Tyr397 and Tyr925); AKT, phosphorylat-ed AKT (Ser473); ERK, phosphorylated ERK (Thr202/Tyr204),STAT3, and phosphorylated STAT3 (Tyr705). Anti-cyclin D,cyclin E, cyclin A, cyclin B, p27, b-catenin, Lamin B, Vimentin,and Snail antibodies were purchased from Santa Cruz Bio-technology. Anti-b-actin antibody was purchased from Sigma-Aldrich. Antibody binding was detected using an enhancedchemiluminescence system according to the manufacturer'sprotocol (Amersham Biosciences). Anti-mouse and -rabbit sec-ondary antibodies were purchased from Thermo Fisher Scien-tific Inc. The data were normalized and quantified by ImageJsoftware (NIH).

Cell-cycle analysisCells treated with bosutinib at various concentrations for 24

hours were harvested, fixed with cold 70% ethanol, and stored at�20�C. The fixed cells were harvested by centrifugation, dissolvedin 20 mg/mL RNase A (Invitrogen), and incubated at 37�C for 10minutes. Next, the cells were stained with 20 mg/mL propidiumiodide (PI; Sigma-Aldrich). The DNA content of 10,000 cells pereach experimental groupwas analyzed using a FACSCalibur FlowCytometer (BD Biosciences). Three independent experimentswere performed for each condition.

Apoptosis assaysAfter the cells were treated with 1.0 mmol/L bosutinib, the

degree of apoptosis was measured using Annexin V-FITC and PIdouble staining according to the protocols of the manufacturer(BD Biosciences). The cells were analyzed using a FACS Caliburflow cytometer (BD Biosciences). Early apoptosis was defined asAnnexin V–FITC-positive and PI-negative, whereas late apoptosiswas Annexin V–FITC- and PI-positive. Results are expressed as themean value of three independent experiments.

Migration and invasion assaysFor the migration assay, cells were grown as monolayers in 6-

well culture plates. After 24 hours, confluent monolayers weregently scratched with a sterile 200-mL pipette tip. The plates werewashed with PBS, and then DMSO or 0.1 mmol/L or 1.0 mmol/Lbosutinib was added to the wells in medium. After 72 hours forSNU-308 cells and 24 hours for SNU-478 cells, cell movementback into the area of the scratchwas recorded by lightmicroscopy.Cell migration was measured in 10 randomly selected micro-scopic fields for each experiment. Three independent experi-ments were performed for each condition.

The cell invasion assay was performed using a Cytoselect24-well Cell Invasion Assay Kit (Cell Biolabs, Inc.). The kitincluded polycarbonate membrane inserts (8-mm pore size). The

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Mol Cancer Ther; 15(7) July 2016 Molecular Cancer Therapeutics1516




top surface of the insert membrane was coated with a uniformlayer of dried murine laminin I matrix. Cells were serum starvedfor 24 hours then a cell suspension containing 1� 106 cells/mL inserum-free media alone or serum-free media with 1.0 mmol/L ofbosutinib was added to the inside of each insert. Each insert wasthen transferred to a bottom well of the plate filled with mediacontaining 20% FBS. After incubation for 24 hours, the invadingcellswere stained and extracted, and thenquantifiedbymeasuringthe absorbance at 560 nm with a VersaMax microplate reader(Molecular Devices). The data presented are representative of twoindependent experiments.

In vivo studyAnimal experiments were performed at the Biomedical Cen-

ter for Animal Resource Development of Seoul National Uni-versity (Seoul, Korea) according to the institutional guidelineswith prior approval from the Institutional Animal Care and UseCommittee. Of SNU-308 and SNU-478 cell lines that weresensitive to bosutinib in this study, we used the SNU-478

xenograft model for in vivo experiments, as the SNU-478 cellline was more tumorigenic in female Balb/c athymic nude micethan the SNU-308 cell line. A total of 30 female Balb/c athymicnude mice ages 4 to 6 weeks were supplied from Central LabAnimal, Inc. The mice were adapted to local conditions for1 week, and then injected subcutaneously in the right flank with1 � 107 SNU-478 cells in 100 mL of PBS. After implantation ofthe tumor cells, the tumor volume was measured every weekusing calipers and calculated using the following formula:(width2 � height)/2. When the tumor volume reached 200mm3, the mice were randomly divided into 6 treatment groups:control, bosutinib, gemcitabine, cisplatin, bosutinib plus gem-citabine, and bosutinib plus cisplatin. The control group wastreated with 0.5% methanol and 0.4% Tween 80 in deionizedwater via oral gavage. Bosutinib at a dose of 150 mg/kg wasadministered via oral gavage once daily for 28 days. Gemcitabine(100 mg/kg) and cisplatin (4.5 mg/kg) were injected intraperito-neally twice a week for 28 days. When the tumor volume reached1,500mm3, themice were euthanizedwith CO2. The tumors were

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Figure 1.Bosutinib inhibits the proliferation of BTC cells via the inhibition of Src phosphorylation. A, the effects of bosutinib on BTC cell proliferation were evaluated byMTT assays. Cells were treated with increasing doses of bosutinib for 72 hours. B, a Matrigel-embedded three-dimensional culture system was used toinvestigate the antiproliferative activity of bosutinib in SNU-308 and SNU-478 cells. Cells were treated with 0 (DMSO), 0.1, and 0.5 mmol/L of bosutinibevery 3 days for 12 days, and then examined under light microscopy. C, Western blot analysis was performed to evaluate the effect of bosutinib onSrc signaling pathways in BTC cells. SNU-308, SNU-478, and TFK-1 cells were treated with increasing concentrations of bosutinib [0 (DMSO), 0.1, 0.5, and1.0 mmol/L] for 48 hours, after which protein extracts were immunoblotted with the indicated antibodies. The molecular weight of each protein is asfollows: Src, 60 kDa; FAK, 125 kDa; AKT, 60 kDa; ERK, 42/44 kDa; STAT3, 79/86 kDa; and b-actin, 42 kDa.

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excised and stored in liquid nitrogen for further Western blotanalysis or immunohistochemical staining.

IHCFour-micron thick sections from paraffin-embedded xeno-

graft tumor tissues were deparaffinized and dehydrated. IHCdetection of proliferating cells was conducted using ananti-Ki-67 antibody (GeneTex, Inc.) at a dilution of 1:100.Terminal deoxynucleotidyl transferase–mediated dUTP nickend labeling (TUNEL) assays were conducted for the IHCdetection of apoptosis using an ApopTag In situ ApoptosisDetection Kit (EMD Millipore), in accordance with the man-ufacturer's protocol. A phosphorylated Src antibody was used ata dilution of 1:200 and was purchased from Cell SignalingTechnology.

Statistical analysisExperimental data were expressed as the mean � SE and com-

pared using the Student t test. Data were analyzed and displayedusingSigmaPlot software (Systat Software, Inc.). All statistical testswere two-sided, with significance defined as P < 0.05.

ResultsBosutinib inhibits BTC cell proliferation by inhibition of Srcphosphorylation and abrogation of its downstream signalingpathways

A total of nine BTC cell lines were treated with bosutinib.Among them, SNU-308 and SNU-478 cells were sensitive tobosutinib with IC50 values of 0.65 � 0.06 and 0.63 � 0.03mmol/L, respectively, compared with other cells (Fig. 1A; Supple-mentary Table S1). In contrast, TFK-1 cells were relatively resistantto bosutinib with an IC50 value of 4.45 mmol/L. In a three-dimensional culture system, bosutinib also showed an antipro-liferative effect that was dose dependent in both SNU-308 andSNU-478 cells (Fig. 1B).

Next,Western blot analysis was performed to evaluate the effectof bosutinib on the downstream signaling pathways of Src (Fig.1C). In SNU-308 and SNU-478 cells, bosutinib abrogated SrcTyr416 and FAK Tyr397 phosphorylation in a dose-dependentmanner. In contrast, FAK Tyr925 phosphorylation was slightlyinhibited by high-dose bosutinib in the SNU-308 cell line only,whereas it was upregulated in SNU-478 and TFK-1 cell lines. Inaddition, the phosphorylation of AKT and ERK in SNU-308 andSNU-478 cells was decreased with increasing concentrations ofbosutinib. Bosutinib treatment decreased STAT3 phosphoryla-tion in SNU-478 cells but it was increased in SNU-308 and TFK-1cells. In TFK-1 cells, bosutinib did not significantly influence thephosphorylation of Src, AKT, and ERKwhen comparedwith SNU-308 and SNU-478 cells.

Inhibition of Src by bosutinib induces G1 cell-cycle arrest insensitive BTC cell lines

The effects of bosutinib on the cell cycle of SNU-308, SNU-478, and TFK-1 cells were evaluated using flow cytometry.Bosutinib significantly increased G1 cell-cycle arrest in adose-dependent manner in both SNU-308 and SNU-478 cells(Fig. 2A). The sub-G1 fraction was significantly increased bybosutinib treatment in SNU-478 cells (P ¼ 0.008), and showeda tendency to be increased in SNU-308 cells with borderlinesignificance (P ¼ 0.062). In TFK-1 cells, G1 cell-cycle arrest wassignificantly increased after 1 mmol/L bosutinib treatment butthis was not observed at lower concentrations. The sub-G1

fraction in TFK-1 cells was not significantly increased by bosu-tinib treatment (P ¼ 0.572). After 1 mmol/L bosutinib treat-ment for 48 hours, the Annexin V-FITC/PI stain revealed thatbosutinib significantly induced both early and late apoptosis inSNU-308 and SNU-478 cells (Fig. 2B). However, the tendencywas not apparent in TFK-1 cells. Bosutinib decreased cyclin D,cyclin E, cyclin A, and cyclin B expression, and increased p27expression in both SNU-308 and SNU-478 cells, but not in TFK-1 cells (Fig. 2C). Of note, the nuclear expression of p27 wassignificantly increased in both SNU-308 and SNU-478 cells, butnot in TFK-1 cells (Fig. 2D).

Bosutinib inhibits the migration and invasion ofBTC cells

Migration and invasion assays were performed to determinethe effect of bosutinib on the migration and invasion activityof BTC cells. The migration assays indicated that bosutinibsignificantly inhibited the migratory activity of both SNU-308and SNU-478 cells (Fig. 3A). Furthermore, bosutinibsignificantly inhibited the invasive activity of SNU-478 cells(Fig. 3B).

Next, we performed Western blot analysis to investigatethe effect of bosutinib on the expression levels of proteinsrelated to epithelial–mesenchymal transition (EMT) in SNU-308 and SNU-478 cells. Bosutinib did not influence the expres-sion levels of E-cadherin and b-catenin in both cell lines.However, Vimentin levels were slightly decreased and Snailexpression was potently abrogated by bosutinib in a dose-dependent manner (Fig. 3C).

Src inhibition enhances the antiproliferative and antimigratoryeffects of cytotoxic agents in BTC

Because gemcitabine and cisplatin are standard chemother-apeutic agents in patients with advanced BTC, we investigatedthe combined effects of bosutinib with either gemcitabine orcisplatin in BTC cells. Bosutinib demonstrated synergistic anti-proliferative effects in combination with gemcitabine or cis-platin in SNU-308 and SNU-478 cells (Fig. 4A). The migratory

Figure 2.Bosutinib induces the G1 cell-cycle arrest of sensitive BTC cells. A, cell-cycle analysis of SNU-308, SNU-478, and TFK-1 cells was performed using flowcytometry after increasing concentrations of bosutinib treatment [0 (DMSO), 0.1, 0.5, and 1.0 mmol/L] for 24 hours. The percentage of cells in the sub-G1,G1, S, and G2–M phases are shown in the bar graph. The columns represent the mean of three independent experiments and error bars are shown(�SE). � , P < 0.01. B, after the cells were treated with 1.0 mmol/L bosutinib, apoptosis was quantified using Annexin V-FITC and PI staining and flowcytometry. Early apoptosis was defined as Annexin V-FITC–positive and PI-negative, whereas late apoptosis was Annexin V-FITC– and PI-positive. Resultsare expressed as the mean value of three independent experiments. � , P < 0.05. C, Western blot analysis demonstrated the effect of bosutinib on theexpression levels of cell-cycle checkpoint molecules including cyclin D, cyclin E, cyclin A, cyclin B, and p27 in SNU-308, SNU-478, and TFK-1 cells. Thecells were treated with increasing doses of bosutinib [0 (DMSO), 0.1, 0.5, and 1.0 mmol/L] for 48 hours. D, nuclear or cytosolic expression levels of p27 weremeasured after bosutinib treatment. Western blot data were normalized to Lamin B levels using ImageJ software (NIH). � , P < 0.01.






effects of cytotoxic agents were potentiated by the addition ofbosutinib (Fig. 4B). In addition, combination treatment ofbosutinib with gemcitabine or cisplatin for 72 hours signifi-cantly inhibited the migration of SNU-478 cells when com-pared with bosutinib monotherapy. In SNU-478 cells, combi-

nation treatment of bosutinib with gemcitabine or cisplatinfurther abrogated Src phosphorylation compared with bosuti-nib alone, and further downregulated Vimentin and Snailexpression compared with bosutinib, gemcitabine, or cisplatinalone (Fig. 4C).

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Figure 3.Bosutinib inhibits the migration and invasion of BTC cells. A, migration assays were performed to visualize the effects of bosutinib on migration activity inBTC cells. SNU-308 and SNU-478 cells were seeded in a 6-well plate. After 24 hours, cell monolayers were scratched with a sterile 200-mL pipette tipand incubated in culture medium alone or with bosutinib. After 72 hours for SNU-308 cells and 24 hours for SNU-478 cells, the cell migration activitywas observed under light microscopy. �� , P < 0.005. B, invasion activity was examined using a chamber system coated with laminin I matrix. Theinvaded cells were stained and quantified at 560 nm. ��, P < 0.005. C, Western blot analysis of proteins associated with EMT was performed aftertreatment with increasing concentrations of bosutinib [0 (DMSO), 0.1, 0.5, and 1.0 mmol/L].

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Src inhibition has potent antitumor effects in a xenograftmodel

A SNU-478 xenograft model using female Balb/c athymic nudemice demonstrated that bosutinib alone significantly delayedtumor growth compared with controls (Fig. 5A). Bosutinib com-bined with gemcitabine or cisplatin showed significantly morepotent antitumor effects when compared with bosutinib, gemci-tabine, or cisplatin monotherapy. Moreover, the combination

treatment did not significantly influence the body weight of mice(data not shown).

Tumors treated with bosutinib alone exhibited an apparentdecrease in cell proliferation by Ki-67 assays and an apparentincrease in apoptosis by TUNEL assays (Fig. 5B). In addition,bosutinib treatment apparently decreased Src phosphorylation.Combination treatment of bosutinib and gemcitabine furtherdecreased Ki-67 expression and Src phosphorylation, and further

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Figure 4.Bosutinib enhances the antiproliferative and antimigratory effects of cytotoxic agents. A, antiproliferative activity of 0.5 mmol/L bosutinib (Bos) incombination with 0.01 mmol/L gemcitabine (Gem) or 1.0 mmol/L cisplatin (Cis) was evaluated by MTT assays. � , P < 0.01; �� , P < 0.005; N.S., not significant.B, for migration assays, SNU-478 cell monolayers were scratched with a sterile 200-mL pipette tip, and then treated with 0.1 mmol/L bosutinib, 0.1 mmol/Lgemcitabine, or 2.0 mmol/L cisplatin alone or in combination. The distance between the cells was measured. �� , P < 0.005. C, in SNU-478 cells, theWestern blot analysis of molecules associated with Src signaling pathways and EMT was performed after treatment with 1.0 mmol/L bosutinib, 0.1 mmol/Lgemcitabine, or 2.0 mmol/L cisplatin alone or in combination for 48 hours. Bos þ Cis, bosutinib plus cisplatin; Bos þ Gem, bosutinib plusgemcitabine; C and Con, control.






increased TUNEL expression. In tumor lysates, the phosphoryla-tion of Src and STAT3 was downregulated by bosutinib mono-therapy. Moreover, when bosutinib was combined with gemci-tabine, a greater decrease in Src and STAT3 phosphorylation wasobserved (Fig. 5C).

DiscussionOur data suggested that Src might be a potential thera-

peutic target in BTC. A previous study indicated that about80% of BTC specimens expressed an activated Src proteinsimilar to other malignancies (3–6, 18). In our study, a totalof nine BTC cell lines were tested, and the IC50 values rangedfrom 0.63 to 4.45 mmol/L in 3-day MTT assays. Our datasupport that a subset of BTC may be sensitive to the Srcinhibitor, bosutinib.

Bosutinib monotherapy inhibited the phosphorylation of SrcTyr416 and FAK Tyr397, which is an autophosphorylation sitefor integrins (25–27). In contrast, bosutinib did not inhibit thephosphorylation of FAK Tyr925 residue, which is a Src-specificphosphorylation site. Previous preclinical studies of bosutinibshowed conflicting results in terms of FAK phosphorylation. Incolon cancer and breast cancer, bosutinib decreased FAKTyr925 phosphorylation, whereas phosphorylation of the

Tyr397 residue was unchanged (28, 29). In contrast, FAKTyr397 phosphorylation was significantly decreased by bosu-tinib in thyroid cancer (30). Another Src/Abl inhibitor dasati-nib also inhibited FAK Tyr397 phosphorylation in colon cancerand pancreatic cancer (31, 32). The precise molecular mechan-isms for these conflicting results need to be investigated furtherto determine how to abrogate the downstream signaling path-ways of Src using Src inhibitors more effectively. In addition,bosutinib decreased AKT and ERK phosphorylation in a dose-dependent manner in SNU-308 and SNU-478 cells, which wererelatively sensitive to bosutinib. In contrast, conflicting resultswere shown for STAT3 phosphorylation. In SNU-478 cells,STAT3 phosphorylation was downregulated by bosutinib.However, in SNU-308 and TFK-1 cells, it was upregulated. Thisfinding is in line with a previous study that demonstratedsustained Src inhibition induced altered JAK–STAT3 binding,leading to aberrant STAT3 activation (33). These results suggestthat a STAT3 targeting strategy might be an option to increasethe therapeutic efficacy or to overcome the resistance of Srcinhibitors.

Cell-cycle analysis demonstrated that bosutinib induced G1

arrest and increased the sub-G1 fraction in a dose-dependentmanner in SNU-308 and SNU-478 cells. Bosutinib decreasedcyclin D, cyclin E, cyclin A, and cyclin B expression, and

A SNU-478 Xenograft

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Figure 5.Bosutinib (Bos) has potentantitumor effects in combinationwith gemcitabine (Gem) or cisplatin(Cis) in a SNU-478 xenograft model.A, mice were treated with vehiclealone, bosutinib (150 mg/kg),gemcitabine (100 mg/kg), cisplatin(4.5 mg/kg), bosutinib plusgemcitabine (Bos þ Gem), orbosutinib plus cisplatin (Bos þ Cis)for 28 days. � , P < 0.01. B, the tumorswere harvested and analyzed byIHC. Ki-67 and TUNEL expressionand Src phosphorylation wereevaluated in a SNU-478 xenograftmodel. C, proteins were extractedfrom excised tumors forimmunoblotting with the indicatedantibodies. Results of representativesamples were shown. H&E, eosin.

Nam et al.





increased p27 expression in both sensitive BTC cell lines. Inparticular, the nuclear stabilization of p27 was also observedafter bosutinib treatment, which has been suggested to be amechanism of apoptosis induced by the Src inhibitor (34).Moreover, bosutinib monotherapy demonstrated potent anti-migratory and anti-invasive activity against BTC cell lines, anddownregulated the expression of proteins related to EMT,which is associated with cancer progression and metastasis andoften mediated by the MAPK and PI3K/AKT pathways (35, 36).These findings are also consistent with previous findings ofbosutinib in other type of cancers (29, 30, 37, 38). The resultsof our preclinical studies support bosutinib as a single agentwith promising antitumor activity in BTC, which warrantsfurther clinical studies.

Our in vitro and in vivomodels alsodemonstrated that bosutinibhas synergistic antiproliferative activity in combination withcytotoxic agents including gemcitabine and cisplatin, which arecurrently the standard treatment in patients with advanced BTC.Importantly, there was no significant effect on body weight inthe in vivo models (15). The combination treatment furtherinhibited the migratory activity of BTC cells, decreased the phos-phorylation of Src and its downstream molecules, decreasedEMT marker expression levels such as Vimentin and Snail, andincreased apoptosis in BTC cells. These results indicate that thisnovel combination strategy is worthy of further clinical studies inpatients with advanced BTC, because there is currently no vali-dated therapeutic target for advanced BTC despite the poorprognosis of these patients.

In spite of these promising results, alternative cancer survivaland growth machineries are serious issues in anticancer drugdiscovery as it may eventually induce acquired resistance to Srcinhibitors. Similarly, a previous study using a murine melano-ma model indicated that adoptive cell transfer therapy withcytotoxic T cells unexpectedly induced treatment resistancethrough an inflammation-induced reversible loss of melano-cytic antigens (39). More recently, Obenauf, and colleaguesdemonstrated that targeted therapy induces secretome changesin drug-sensitive cancer cells, paradoxically generating a tumormicroenvironment that promotes the growth of drug-resistantclones (40). In case of Src inhibition, only few have beenknown about acquired resistance mechanisms. Aforemen-tioned aberrant STAT3 activation by bosutinib could be aresistance mechanism of Src inhibitors (33). Recently, Lu andcolleagues demonstrated that IGFBP2/FAK pathway may beassociated with dasatinib resistance in non–small cell lungcancer cells (41). Aberrant deregulation of FAK or its down-stream molecules by alternative pathway activation may con-tribute to Src inhibitor resistance. Therefore, further preclinicalstudies should focus on the identification and overcome of thealternative cancer survival and growth machineries of Src inhi-bitors in BTC.

A recent study identified that a bosutinib isomer (Bos-I) wasmore potent to inhibit Chk1 and Wee1 than "authentic"bosutinib and synergized with gemcitabine in a pancreaticcancer cell line (42). However, the ability of Bos-I to inhibitSrc and Abl was relatively less potent compared with "authen-tic" bosutinib. Nevertheless, as Bos-I showed greater antitumoractivity than "authentic" bosutinib toward pancreatic cancercells when combined with gemcitabine, further preclinical orclinical studies are needed to focus on the antiproliferativeactivity of Bos-I combined with gemcitabine or cisplatin in BTCcells.

In summary, our data suggest that Src might be a potentialtherapeutic target in BTC. Bosutinib, an orally active smallmolecule c-Src/Abl kinase inhibitor, demonstrated promisingantitumor activity alone or in combination with gemcitabine orcisplatin by significantly inhibiting the phosphorylation of Srcand its downstream molecules, inducing G1 cell-cycle arrest viathe nuclear stabilization of p27 in part, decreasing migration,invasion, and EMT, and increasing apoptosis in BTC in vitro andin vivo models. The results of our preclinical study support thefurther clinical development of bosutinib monotherapy orcombination therapy with other cytotoxic agents in patientswith advanced BTC.

Disclosure of Potential Conflicts of InterestS.-A. Im is a consultant/advisory board member for AstraZeneca, Hanmi,

Novartis, and Roche. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: A.-R. Nam, D.-Y. Oh, Y.-J. BangDevelopment of methodology: A.-R. Nam, Y.-J. BangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.-R. Nam, K.-H. Lee, T.-Y. Kim, S.-A. Im, T.-Y. Kim,D.-Y. Oh, Y.-J. BangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.-R. Nam, J.-W. Kim, S.-A. Im, D.-Y. Oh, Y.-J. BangWriting, review, and/or revision of the manuscript: A.-R. Nam, J.-W. Kim,K.-H. Lee, S.-A. Im, D.-Y. Oh, Y.-J. BangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.E. Park, J.-H. Bang, M.H. Jin, K.-H. Lee, S.-W.Han, S.-A. Im, D.-Y. OhStudy supervision: D.-Y. Oh

Grant SupportThis study was supported by a grant from the National R&D Program for

CancerControl (grant no. 1320090),Ministry ofHealth andWelfare, Korea, anda grant from SK Telecom (grant no. 3420130140), Seoul, Korea (to D.-Y. Oh).

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 January 11, 2016; revised March 31, 2016; accepted April 13, 2016;published OnlineFirst April 22, 2016.

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2016;15:1515-1524. Published OnlineFirst April 22, 2016.Mol Cancer Ther Ah-Rong Nam, Ji-Won Kim, Ji Eun Park, et al. Src as a Therapeutic Target in Biliary Tract Cancer

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Src as a Therapeutic Target in Biliary Tract Cancer...Small Molecule Therapeutics Src as a Therapeutic Target in Biliary Tract Cancer Ah-Rong Nam1, Ji-Won Kim2, Ji Eun Park1, Ju-Hee