Antitumor Activity of Src Inhibitor Saracatinib (AZD-0530) in … · Preclinical Development...

Preclinical Development

Antitumor Activity of Src Inhibitor Saracatinib (AZD-0530)in Preclinical Models of Biliary Tract Carcinomas

Giuliana Cavalloni1,3, Caterina Peraldo-Neia1, Ivana Sarotto3, Loretta Gammaitoni1,3, Giorgia Migliardi2,Marco Soster4, Serena Marchi�o4, Massimo Aglietta1,3, and Francesco Leone1,3

AbstractBiliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an

urgent need to identify new targets and to design innovative therapeutic approaches. Among potential

candidatemolecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects

of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was

investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib

treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines

TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation andmigration was analyzed

in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-

modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was

activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counter-

acted the activation of Src and of its downstream effectors, increased the fraction of cells in G0–G1 phase, and

inhibited cell migration. At high concentrations (median dose from 2.26–6.99 mmol/L), saracatinib was also

capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth,

associatedwith an impairedvascular network.Here,weprovide ademonstration that the targeted inhibition of

Src kinase by saracatinib is of therapeutic benefit in preclinicalmodels of BTC.Wepropose our results as a basis

for the design of saracatinib-based clinical applications. Mol Cancer Ther; 11(7); 1528–38. �2012 AACR.

IntroductionBiliary tract carcinoma (BTC) is a highly malignant

epithelial cancer with a poor prognosis, due to its aggres-siveness and the availability of limited therapeuticoptions. At present, only surgical resection is associatedwith an improvement in patients’ outcome, with 5-yearsurvival rates of 20% to 40% (1). However, the presence ofdistant metastases, extensive regional lymph nodemetas-tases, and vascular encasement or invasion precludesresection. Consequently, patients with unresectable dis-ease have a survival of less than 12 months from thediagnosis.

Chemotherapy has been commonly used to managepatients with BTC, with the dual intention of improvingtheir outcome and controlling tumor progression (2).Among the chemotherapeutic agents used in BTC, gem-citabine has shown efficacy both as a single agent and incombination with other cytotoxic drugs. The responserates of single-agent gemcitabine range from 8% to 60%,depending on the cohort reported (3). The best result ofcombination chemotherapy in a phase II study wasobtained for a regimen based on gemcitabine plusoxaliplatin (GEMOX), for which a 36% of response rateand 15.4 months of median survival have been achieved(4). Recently, the U.K. ABC-02 randomized phase IIItrial showed that the association of gemcitabine withcisplatin increased both progression-free survival (PFS)and overall survival (OS), compared with gemcitabinealone (median, 8 vs. 5 months and 11.7 vs. 8.1 months,respectively; ref. 5). Although significant, the resultsachieved in these clinical trials witness a modestincrease in both PFS and OS rates, evidencing thatcurrent therapies have a limited potential of improvinga patient’s outcome. Thus, there is an urgent need todevelop novel therapeutic strategies for the treatment ofBTC, based on specific molecules on cancer cells astargets for innovative therapies.

Src is a nonreceptor tyrosine kinase associated withgrowth factor/cytokine receptors, and plays a key rolein the regulation of multiple cellular mechanisms in both

Authors' Affiliations: 1Department of Medical Oncology, University ofTurin Medical School; 2Unit of Molecular Pharmacology, Department ofOncological Sciences, University of Turin; 3Unit of Pathology, Fondazionedel Piemonte per l'Oncologia; and 4Laboratory of Tumor Microenviron-ment, Fondazione Piemontese per la Ricerca sul Cancro, Institute forCancer Research and Treatment (IRCC), Candiolo, Turin, Italy

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

G. Cavalloni and C. Peraldo-Neia contributed equally to this work.

Corresponding Author:Giuliana Cavalloni, Department ofMedical Oncol-ogy, University of Turin Medical School, Str. Provinciale 142, Candiolo,Torino 10060, Italy. Phone: 390119933503; Fax: 390119933522; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-1020

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(7) July 20121528

on April 21, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 27, 2012; DOI: 10.1158/1535-7163.MCT-11-1020

http://mct.aacrjournals.org/

normal and cancer cells, including migration, adhesion,invasion, survival, proliferation, angiogenesis, andinflammation (6). Src regulates important signaling cas-cades, including the focal adhesion kinase (FAK), thephosphoinositide 3-kinase (PI3K), and the signal trans-ducer and activator of transcription 3 (STAT3) pathways(7–10). An increase in Src expression has been reported inseveral human cancers including colorectal, pancreatic,and breast carcinomas (11–14). Recently, the small-mole-cule Src inhibitor AZM555130 has been shown to signif-icantly reduce the proliferative and invasive potential ofthe HuCCA-1 human cholangiocarcinoma cell line (15),suggesting that targeting Src tyrosine kinase mightprovide a therapeutic benefit for the management ofcholangiocarcinoma.A growing number of pharmacologic Src inhibitors are

currently being tested in clinical trials (16, 17). The dualSrc/Abl inhibitor dasatinib (BMS-354825) has shown pre-clinical efficacy in solid tumors and clinical activity inleukemia (18). In vitro, the Src inhibitor saracatinibreduces migration and, in combination with tamoxifen,blocks proliferation of breast cancer cells (14). In ortho-topic prostate cancer models, saracatinib inhibits bonemetastasis formation (19). Thismolecule is currentlybeingtested in phase I and phase II clinical trials for varioussolid tumors (20, 21).Here, we investigate the effects of targeting Src with

saracatinib in preclinical models of BTC, and we reportthat this inhibitor is capable of reducing cellmigration andproliferation in vitro, and of delaying tumor growth invivo, in human BTC xenografts.

Materials and MethodsTissue specimensThe study was conducted on formalin-fixed tissue

derived from 19 patients with BTC, including 4 intrahe-patic cholangiocarcinomas (ICC), 10 extrahepatic cholan-giocarcinomas (ECC), and 5 gallbladder carcinomas(GBC). All patients were of Italian origin and were 14males and 5 females aged from 46 to 75 years (median, 63years). Surgical samples were obtained from the patientsbefore any systemic treatment was administrated andcollected according to conventional histopathologicdiagnostic protocols. Histologic diagnosis and gradingwere conducted according to the World Health Organi-zation Classification of Tumors (2002), and staging wasdetermined according to the tumor–node–metastasissystem (22). The clinical profiles of patients as well astheir pathologic features are reported in SupplementaryTable S1.

Immunohistochemistry, immunocytochemistry, andimmunofluorescenceFor patients with tumor, histologic sections of 4 mm

were mounted on glass slides. Sections were decoratedwith primary mouse anti-p-Src polyclonal antibody(Tyr419, Cell Signaling, Inc.) and then incubated with a

dextran polymer conjugated to horseradish peroxidase.The final reaction was visualized using 3,30-diamino-benzidine. Finally, sections were counterstained withHarris hematoxylin, dehydrated, and mounted in DPX.Staining scores were established semiquantitativelyfrom the percentage of p-Srcþ cells and the stainingintensity. Tumors were graded as negative (less than 1%positive cells), þ (low intensity, 1–10% positive cells),þþ (moderate intensity, >25%–<50% positive cells), andþþþ (high intensity, >50% positive cells). For ornithinecarbamyl transferase–frozen tissues, sections of 10 mmwere mounted on SuperFrost Plus glass slide (Menzel-Gl€aser). Sections were decorated with rabbit polyclonalanti-HIF-1a (Upstate Millipore) and Dako EnVisionþSystem-HRP Labeled Polymer Anti-Rabbit; the DakoAECþ High Sensitivity Substrate Chromogen was usedto visualize the reaction. For immunofluorescence, sec-tions were decorated with the purified rat anti-mouseCD31 clone MEC 13.3 primary antibody (BD Pharmin-gen) and with goat anti-rat secondary antibody (Invi-trogen). Apoptosis was evaluated by terminal deoxy-nucleotidyl transferase–mediated dUTP nick end label-ing (TUNEL) assay using In Situ Cell Death DetectionKit, TMR red (Roche), using manufacturer’s instruc-tions. To evaluate proliferation, slides were decoratedwith primary antibody anti-Ki-67 clone MIB1 (Dako)and with donkey anti-mouse secondary antibody (Invi-trogen). For immunocytochemistry, 100,000 cells werespotted per glass slide (Menzel-Gl€aser) and formalinfixed. Apoptosis was conducted by TUNEL assay aspreviously described. Fluorescent images wereacquired with a DMIRE2 confocal microscope fromLeica TCS SP5 II confocal microscope. The images wereanalyzed with the Image Processing and Analysis soft-ware in Java (ImageJ), version 1.44h.

Cell lines and drug preparationThe ECC cell lines EGI-1 and TFK-1 were kindly pro-

vided by Scherubl from the Institute of Physiology,Charit�e-Universit€atsmedizin (Berlin, Germany). The ICCcell line HuH28 and the GBC cell line TGBC1-TKB wereobtained from Cell Bank, RIKEN BioResource Center. Allthe cell lines were cultured in RPMI-1640 medium con-taining 10% FBS, 100 U/mL penicillin, and 100 mg/mLstreptomycin. The murine endothelial cell line b-ENDwas cultured in complete Dulbecco’s Modified Eagle’sMedium (23).

Saracatinib was from Sequoia (Sequoia Research Pro-ducts), dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at �20� C. For in vivo experiments,saracatinib was dissolved in Cremophor EL (Sigma-Aldrich) with 95% ethanol (50:50).

Cell growth and colony formation assayCells (3,000 cells per well) were seeded onto 96-well

tissue culture plates and treated 24 hours later withdifferent drug doses (0.625–10 mmol/L) for 72 hours.Proliferation was evaluated with Cell Titer-Glo Cell

Antitumor Activity of Saracatinib in BTC Preclinical Models

www.aacrjournals.org Mol Cancer Ther; 11(7) July 2012 1529




Viability Assay (Promega, Corporation), following themanufacturer’s protocol. The luminescent signal wasmeasured by GloMax (GloMax-Multi Detection System,Promega). All tests were conducted in quadruplicate andrepeated in 3 independent experiments. Themedian dose(Dm) value (concentration inhibiting 50% of cell growthcompared with 0.001% of DMSO control) was calculatedfor each cell line after 72 hours of treatment using Calcu-Syn software (Biosoft) based on Chou-Talalay method.Colonies derived from single cells were obtained byplating 300 cells per well on 24-well tissue culture platesin RPMI plus 10% FBS, and treatedwith different doses ofsaracatinib (from 80 nmol/L to 5 mmol/L). After 3 days,colonies were stained with 0.1% crystal violet (Sigma-Aldrich).

Cell-cycle measurements and flow cytometryCell-cycle distribution was determined by flow cyto-

metry. Briefly, 1 � 106 harvested cells were fixed with70% cold ethanol at �20�C for 16 hours. After washingwith PBS, cells were resuspended in staining solution(50 mg/mL propidium iodide þ 100 mg/mL RNaseA inPBS, all from Sigma-Aldrich) and incubated at 4�Covernight. The analysis was conducted by fluorescence-activated cell sorting using Summit Research Software(Becton Dickinson).

Wound-healing assayCells were seeded in triplicate in 6-well tissue culture

plates andallowed to growuntil 100%confluence. The celllayer was gently "wounded" through the central axis ofthe plate using a pipette tip, then treated with differentdoses of saracatinib in RPMI plus 10% fetal calf serum.Cell migration toward the scraped area was observed in9 randomly selected microscopic fields for each condi-tion and timepoint (up to 24hours). Imageswere acquiredwith a Leica DM13000B Inverted Microscope (Leica).

Western blot analysisCells were lysed with lysis buffer (10% SDS, 0.5 mol/L

Tris-HCl, pH 6.8) at 100�C; 20 mg of proteins were electro-phoresed on 7.5% SDS-PAGE, and transferred to 0.45-mmnitrocellulose membranes (Hybond�-C Extra). Lumines-cence was revealed with a chemiluminescence reagent(Euroclone). Anti-mouse and anti-rabbit antibodieslinked with horseradish peroxidase, anti-p-Src (Tyr419), Src, p-paxillin (Tyr 118), p-p38 (Tyr180–182), p38,p-MAPK (Tyr 202–204), mitogen-activated protein kinase(MAPK), p-Akt (Ser 473), Akt, and cyclin D1 were fromCell Signaling Technology; antibodies anti-p-FAK (Tyr861), FAK, and paxillin were from Millipore. For blotprotein quantification, densitometric analysis of thedetected bands was conducted with the Quantity OneSoftware (Bio-Rad). Band intensities of total proteinswerenormalized to the intensity of corresponding actin bands,and band intensities of phosphorylated proteins werenormalized to the intensity of corresponding normalizedtotal proteins values.

VEGF ELISA assayCells were plated at 600,000 cells per well on 24-well

tissue culture plates in appropriate medium for 24 hours,then cultured in serum-free medium for 24 hours. Freshmedium supplemented with 5% FBS and containing var-ious doses of saracatinib (from 10–0.625 mmol/L), wasadded for additional 24 hours. The supernatant washarvested, and secreted VEGF was measured by ELISAusing theHumanVEGFQuantikine ELISA ImmunoassayKit (R&D Systems) according to the manufacturer’sprotocol.

Mice xenograft modelsNOD (nonobese diabetic)/Shi-scid (severe combined

immunodeficient) IL2rgnull female mice (5–6 weeks old)were used for in vivo experiments. Animals were main-tained at the animal facilities of our institution (IRCC,Candiolo, Torino, Italy) and handled according to insti-tutional regulations. EGI-1 cells were used for theseexperiments because of their tumorigenicity.

In 3 different experiments, mice were subcutaneouslyinjected into the right flankwith 5� 106 EGI-1 cells in 50%growth factor–reduced BDMatrigel basement membranematrix.When tumors reached a volume of about 200mm3

(about 2 weeks after injection), animals were treateddaily with either saracatinib (25 mg/kg/die) or vehicleby oral gavage for 21 days. Subcutaneous xenograftdiameters were measured every 7 days. Ten mice wereused for each treatment group. At the end of the treat-ment, mice were euthanized, tumor diameters mea-sured, and volumes calculated using the followingformula: V ¼ A � B2/2 (V ¼ tumor volume, A ¼ largestdiameter, B ¼ smallest diameter). Mean volumes oftreated and untreated xenografts were compared usingan unpaired t test (Student t test) considering as statis-tically significant a P value less than 0.05 (confidenceinterval 95%).

Microarray analysisTotal RNAwas isolated fromEGI-1 cells, either untreat-

ed or treated with 10 mmol/L saracatinib for 24 hours,using TRIzol (Life Technologies). For frozen EGI-1 xeno-graft tissues, RNA was extracted with TRI Reagent (Sig-ma). RNAquantificationwas conductedwith Bioanalyzer2100 (Agilent Technologies), and 1 mg of mRNA wasamplified using the MessageAmp II aRNAAmplificationKit (Ambion Inc.). Amino-allyl–modified nucleotideswere incorporated according to the manufacturer’s pro-tocol. Labeling was conducted using NHS (N-hydroxy-succinimidyl) ester Cy3 or Cy5 dyes (GEHealthcare). Thedye-swap replication procedure was applied. Sampleswere hybridized on 8 � 60 K glass arrays (Agilent Tech-nologies). Arrays were scanned and images analyzed bythe Feature Extraction Software from Agilent Technolo-gies (version 9.5), and the text files were then processedusing the Bioconductor package Limma (Linear modelsfor microarray analysis). Microarray data were depositedin Gene Expression Omnibus (GSE36622).

Cavalloni et al.

Mol Cancer Ther; 11(7) July 2012 Molecular Cancer Therapeutics1530




Real-time quantitative PCRRNAwas extracted and retro-transcribed in cDNAwith

the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems). The cDNA was used for amplifi-cation of CXCL10, CAV1, SAA4, RRM2, CFB genes, andPGK housekeeping gene with specific primers (Supple-mentary Table S2). Real-time PCR (RT-PCR) was carriedout in triplicate. Quantitative analysis was conducted bythe measurement of Ct values (24).

ResultsSrc protein is phosphorylated in human BTC tissuesand cell linesThe presence of an activated Src protein was deter-

mined by immunohistochemistry in a panel of 19 BTCspecimens, revealing amoderate to strongpositivity for p-Src expression in 15 samples (5were classified 1þ, five 2þ,and five 3þ). Only 4 samples were negative. Supplemen-tary Fig. S1 shows representative immunostaining of p-Src in BTC samples. We did not find any statisticallysignificant association between p-Src amounts and anyclinicopathologic parameter or histologic origin (data notshown).We also evaluated the presence of p-Src in 4 human

BTC lines of different origin (i.e., TFK-1, EGI-1, HuH28,and TGBC1-TKB), observing variable basal levels of Srcactivation in these cultured cells (Supplementary Fig. S2).

Saracatinib inhibits theproliferationofBTCcell linesTo assess the capability of saracatinib (Fig. 1A) to

interfere with cell proliferation, BTC cell lines were trea-ted with different concentrations of this Src inhibitor. TheICC-derivedHuH28 cell linewas themost responsive (Dm

¼ 2.26 mmol/L). The ECC cell lines TFK-1 and EGI-1showed a similar behavior, with Dm of 4.02 mmol/L and3.82 mmol/L, respectively. The GBC cell line TGBC1-TKBwas less sensitive (Dm ¼ 6.99 mmol/L; Table 1).Figure 1B showed representative dose–effect curves of

each cell line. A statistical significant decrease in cellproliferation was seen in all the cell lines up to 1.25mmol/L, except for TGBC1-TKB, in which no significanteffect was revealed (Supplementary Fig. S3).By conducting the colony formation assay, we showed

that saracatinib had the ability to reduce the formation ofcellular foci in all the cell lines, starting from 1 mmol/L(Supplementary Fig. S4). We further evaluated the effectof a proliferation inhibitory dose (5 mmol/L) of saraca-tinib on cell-cycle progression. After 24 hours of drugtreatment, an increase in the fraction of cells in G0–G1

was revealed in TFK-1, EGI-1, and HuH28 (Fig. 1C). Thiseffect was not improved by longer treatments (data notshown). In HuH28 cells, which proved to be the mostsensitive to Saracatinib in terms of growth inhibition,this drug also increased the sub-G1 fraction, whereas inthe less responsive TGBC1-TKB cells, the effect on G1

checkpoint arrest was weak (Fig. 1C). Moreover, weinvestigated the effect of saracatinib on a key cell-cycle

regulator, cyclin D1. In agreement with the weakly effecton BTC growth, a slight inhibition of cyclin D1 expres-sion was detected only in EGI-1 cells, whereas in theother models, saracatinib was not able to inhibit thisprotein (data not shown). We have also evaluated theeffect of saracatinib on apoptosis by TUNEL assay. After24 hours of treatment, a significant increase in thenumber of apoptotic cells was revealed in all cell lines,but the effect was prominent only in TGBC1-TKB cells(P value < 0.05; Supplementary Fig. S5).

Saracatinib inhibits BTC cell migrationWe investigated the impact of saracatinib on themigra-

tion potential of the cited BTC cell lines using the wound-healing assay. A wound was produced on a layer of

A

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Saracatinib structure (www.lclab.com)

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TFK11.00

0.80

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Figure 1. Impact of saracatinib on BTC cell growth. A, molecular structureof saracatinib. B, representative dose–effect curves in BTC cell lines. C,effect of saracatinib on BTC cell-cycle progression. Each barrepresents the average percent cells in each phase. NT, not treated; Sar,saracatinib treated.






confluent cells, followedby treatmentwithdifferent dosesof saracatinib (5–1–0.5–0.1 mmol/L) for 24 hours. Thisassay revealed a significant dose-dependent inhibition ofmigration by saracatinib, whichwas induced in all the celllines (data not shown). Figure 2 shows a representativeassay conducted with HuH28.

Saracatinib inhibits the activation of Src and of itsdownstream effectors

To evaluate the target effect of saracatinib on Src and onits main transducers, all the cell lines were treated with 5mmol/L of inhibitor for 2 hours, followed byWestern blotanalysis for the presence of the cognate phosphoproteins.This assay revealed that a switch-off of the pathwayinvolving Src, FAK, and paxillin was induced in allthe cell lines, as a consequence of saracatinib treatment(Fig. 3A and B).

We further evaluated whether saracatinib was capableof interfering with the MAPK and PI3K/Akt signalingcascades. In this case, we observed that only the down-streameffector ofMAPK,p-p38was switched-off in all thecell lines examined (Fig. 3C). A weak downregulation ofp-MAPK was revealed in EGI-1 and HuH28 only. Con-versely, no downregulation of p-Akt was seen in any cellline (data not shown).

Saracatinib delays tumor growth of EGI-1 xenograftsHaving observed a targeted antitumor effect of saraca-

tinib in vitro in BTC cell models, we evaluated an in vivoantitumor activity on the xenograft implantation of thehighly tumorigenic EGI-1 cell line. In 3 independentexperiments, NOD/SCID IL2rgnull mice were injectedsubcutanoeusly with EGI-1 cells, and 10 mice per groupwere orally treated daily for 21 days, with saracatinib orwith drug vehicle. Saracatinib was able to significantlydelay the growth of EGI-1 xenografts in comparison withcontrol mice. The median fold-increase in tumor volumewas 4.9 and 13.6, respectively (Fig. 4A; P value ¼ 0.06).Values were obtained from the ratio of volume at day 21and volume at day 0 from each cohort of mice. In Fig. 4B,representative tumors harvested from control and sara-catinib-treated EGI-1 xenografts are shown. On the sametumors, to evaluate the effect of saracatinib on cell pro-liferation, theKi-67 stainingwas conducted.A statisticallysignificant decrease of cell proliferation was revealedin treated mice (P value ¼ 0.0003 Supplementary Fig.S6A–S6C).

Table 1. Median proliferation-inhibitory doses(Dm) of saracatinib inBTCcell lines after 72 hoursof incubation with different doses of drug.

Cell lineSaracatinib Dm

(mmol/L)

TFK-1 4.02EGI-1 3.82HuH28 2.26TGBC1-TKB 6.99

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5 μmol/L 0.35

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Figure 2. Representative wound-healing assays on HuH28 cellstreated with the indicated doses ofsaracatinib for 24 hours. Time 0,time of the wound; NT, not treatedcells.

Cavalloni et al.





Saracatinib inhibits in vivo and in vitro tumorangiogenesisWe noticed that saracatinib-treated tumors seemed

macroscopically less vascularized compared with thecontrols (see Fig. 4B for examples). This prompted us toverify the status of tumor vasculature at the microscopiclevel. Sections of tumor tissues from control and saraca-tinib-treated EGI-1 xenografts were stained for the endo-thelial marker CD31. A significant reduction (P < 0.0001)in the number of tumor blood vessels was observed insaracatinib-treated mice (Fig. 5A–C). Confirming thereduced blood support in these tumors, a significantincrease of the hypoxia marker HIF-1a was revealed intreated mice (P¼ 0.008; Fig. 5D–F). As a consequence, thenumber of apoptotic cells was significantly enhancedupon saracatinib treatment (P ¼ 0.04; Fig. 5G–I). Havingobserved a potent antiangiogenic effect of saracatinib ontumor xenografts, we further evaluated the in vitro effectof Src inhibitor on proliferation and migration of themurine endothelial cell line b-END, and on VEGF secre-tion by BTC cell lines treated with different doses of thedrug. The Dm for b-END cells was 0.97 mmol/L, lower

than those found in BTC cells, whereas the effect onmigration is less marked compared with BTC cells (Fig.6). We did not find any statistically significant reductionof VEGF secretion in BTC cells upon saracatinib treatment(P > 0.05; data not shown).

Saracatinib induces a modulation of gene expressionin BTC preclinical models

The physiopathologic responses observed both in vitroand in vivo in the described preclinical models of humanBTC were suggestive of a transcriptional modulationdriven by saracatinib treatment. We therefore evaluatedsaracatinib-induced gene expression profiles by micro-array analysis of samples deriving from cultured EGI-1cells and corresponding xenografts.

For the in vitro experiments, to maximize the effect ofdrug, EGI-1 cells were treated for 24 hours either withvehicle or with high dose (10 mmol/L) of saracatinib,followed by quantification of transcript genes. Raw datawere filtered by using a jLogFCj >1 and an adjusted Pvalue less than 0.01. The comparison between saracatinib-and vehicle-treated EGI-1 cells revealed 647 modulated

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Figure 3. Effect of 5mmol/L of saracatinib after 2 hours of treatment onBTCcell lines. A, an evident switching off of p-Src, and p-FAKwas observed in all the celllines. B, a switching off of p-Paxillin was revealed in all the cell lines, more moderate in TFK-1. C, a slight inhibition effect was seen on p-p38 in BTC cell lines.






probes, of which 303 upregulated and 344 downregulated(Supplementary Table S2). We found that the expressionof 20 genes was commonly regulated both in vitro and invivo by saracatinib treatment (Supplementary Table S2).

A gene ontology analysis with the Database for Anno-tation, Visualization, and Integrated Discovery (DAVID)tool (david.abtcc.ncifcrf.gov) revealed that the biologicprocesses, significantly perturbed by saracatinib in cul-tured cells, are related to proliferation, cell cycle, neovas-cularization, and inflammation/chemotaxis. We furthercontextualized the deregulated genes in cellular signalingby the use of the PathwayMiner tool (www.biorag.org),finding a significant involvement in the G1–S check point,cyclin/cell cycle, p38 MAPK, and p53 pathways.

For the evaluation of saracatinib-induced transcrip-tome in vivo, we harvested tumor xenografts after 21 daysof treatment. A comparison between tumors from sara-catinib-treated and control mice revealed amodulation of83 probes only, 46 upregulated and 37 downregulated(Supplementary Table S2). The gene ontology analysisshowed that upregulated genes are involved in inflam-mation and immune response, whereas downregulatedgenes are generically related to tissue development. Inter-estingly, the genes modulated by saracatinib in vivo areinvolved in the metalloproteinase- and the chemokine-related pathways, as revealed by the PathwayMineranalysis.

To confirmresults obtainedbymicroarray experiments,we selected 5 deregulated genes for further validation byquantitative RT-PCR, that is, the upregulated chemokine(C-X-C motif) ligand 10 (CXCL10), complement factor B(CFB), constitutive serum amyloid A4 (SAA4) transcripts,

and the downregulated ribonucleotide reductase M2(RRM2) and the caveolin 1 (CAV1). From the 2 techniques,we obtained the same trend of selected genes (data notshown).

DiscussionGrowing evidence indicates that a deregulation of Src, a

protein implicated in a variety of mechanisms includingcell proliferation, survivals and trafficking, is involved inthe development and progression of solid tumors.Accordingly, Src inhibition is being actively pursued asa potential therapeutic strategy formany common cancers(10). We here provide a first report of the efficacy ofSrc-targeted therapeutic approach in preclinical modelsof human BTC.

Weanalyzedapanel of humanBTC specimens showingthat in about 80% of the samples, an activated Src proteinis expressed. Similar results have been previouslyobtained in other cancer types, particularly in breast,colon, and hepatocellular carcinomas (25–27).

We therefore evaluated the in vitro activity of the small-molecule Src inhibitor saracatinib on a panel of BTC celllines. Although all these cell lines expressed activated Src,the antiproliferative effect of saracatinib in culture wasmoderate and resulted in a broad range of responses, fromvery low to moderate. However, these results are consis-tent with data present in the literature. Chang and col-leagues (28) reported median doses ranging from 1 to 16mmol/L for saracatinib-mediated growth inhibition in apanel of prostate cancer cell lines. In another study (29),the antiproliferative effect of saracatinib on cell lines fromdifferent tumor types was achieved with very variablemedian doses (from 0.2–14 mmol/L). Our analysis on BTCcell-cycle progression showed that saracatinib is veryspecific in blocking cells in the G0–G1 phase, but not inapoptosis. Similar results were observed by Ammer andcolleagues in head andneck squamous cell carcinoma andin colorectal cancer (30, 31). The saracatinib-modulatedgene expression model supported these observations byshowing a downregulation of genes involved in mitosisand in cell-cycle checkpoint.

In the evaluation of saracatinib-modulated pathways inBTC cell lines, we observed only a partial inhibition of theMAPKpathway inHuH28 and EGI-1 cells (essentially, aninactivation of p38MAPK), and no effect on Akt (data notshown). These findings indicate that the inhibition of Srcby saracatinib is probably counteracted by compensatorymechanisms involved in cell proliferation and survival.We have previously showed, in BTC preclinical models,that the EGFR/HER2 pathways are inhibited by smallmolecules targeted to these tyrosine kinase receptors(erlotinib, gefitinib, and lapatinib) or to their downstreameffectors (everolimus and sorafenib), leading to an inhi-bition of the proliferation mediated by MAPK and/orPI3K/AKT pathways (32). Together, these data suggestthat an association of saracatinibwith one ormore of theseinhibitors might be a future strategy for combined

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inhibition of cell migration and proliferation in BTCtreatment.Despite having a poor antiproliferative effect, the

antitumor activity of saracatinib might be explained byother mechanisms. In fact, we showed that saracatinib isan efficient inhibitor of BTC cell migration. Accordingly,we evidenced a saracatinib-mediated switch-off of theSrc/FAK/paxillin signaling pathway, which is a mainplayer in the regulation of cell adhesion and migration.The capability of a cell to detach from the primarytumor mass and to move into the surrounding stromaand vasculature is a necessary step towards cancerinvasion and progression to distant metastasis. Indeed,very recent works proposed that saracatinib couldreduce the metastatic potential of cancer cells, at leastin preclinical models of fibrosarcoma (33) and of non–small cell lung carcinoma (34). Our data support thehypothesis that such a role might be exerted by Src inBTC models as well.A further step in the evaluation of Src inhibition in BTC

preclinical models was the assessment of saracatinibeffects in an in vivo BTCmodel obtained by subcutaneousimplant of EGI-1 cells. In mice treated with saracatinib, asignificant delay in xenograft tumor growth was measur-able after 21 days of drug administration. These dataconfirm the efficacy of an Src-targeted approach as anefficient strategy to counteract tumor progression, and are

consistent with previous works conducted on gastric (35)and pancreatic (8) carcinoma xenograft models.

A series of scientific evidences showed that, besideshaving a role in tumorprogression andmetastasis (11), Srcis also implicated in the regulation of tumor angiogenesisand inflammatory stromal reaction (17). Consistently, weobserved that saracatinib-treated EGI-1 xenograft tumorswere macroscopically less angiogenic compared withvehicle-treated tumor tissues. These considerations arefurther supported by our analysis of saracatinib-modu-lated transcriptome in the BTC xenograft model, whichrevealed a significant deregulation of genes involved invasculature/blood vessel development and morphogen-esis. We therefore evaluated the presence and number ofblood vessels in these tissues by specific staining followedby confocal microscope analysis, confirming a significantreduction of vessel areas in tumor specimens fromsaracatinib-treated mice compared with untreated mice(P < 0.0001). Such reduced neovascularization was asso-ciated to increased hypoxia of the tumor tissues, withconsequent upregulation of the HIF-1a transcription fac-tor (P ¼ 0.0008). To support these in vivo data, we inves-tigated the effect of saracatinib on murine endothelial cellgrowth and migration in vitro. We found that the Srcinhibition affected the endothelial cell proliferation at lowdoses of the drug, whereas the effect on migration is lessmarked.

Figure 5. CD31 staining of sectionsderived from EGI-1 xenograft tumorsgrown in vehicle- (A) and saracatinib-treated (B) mice. A statisticalsignificant reduction of vesselformation (CD31 staining) waspresent in the saracatinib-treated (C)group. HIF-1a staining of tumorsections derived from vehicle- (D)and saracatinib-treated (E) EGI-1xenografts. A statistical significantincrease in the expression of HIF-1awas seen in tumor tissues fromsaracatinib-treated mice (F). TUNELstaining of tumor sections derivedfrom vehicle- (G) and saracatinib-treated (H) group. A statisticalsignificant increase of apoptosis wasseen in saracatinib-treated mice (I).NT, vehicle-treated mice.

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Because of the strict relation of Src with VEGF, we havefurther evaluated the effect of saracatinib on its produc-tion in BTC in vitro models; no statistical significantinhibition of secreted VEGF was found, as shown by Baiand colleagues (36). This could, at least in part, explainwhy saracatinib treatment, albeit inducing a significantreduction of the tumor mass and an increase in thenumber of tumor apoptotic cells, did not prove to be along-term curative approach by itself. Similar results arealso being observed in studies involving human subjects.Several phase II clinical trials on patients with headand neck, gastric, and pancreatic cancer revealed the poorefficacy of saracatinib, when used as single agent (37–39).Further studies are currently reassessing the role of thisdrug in combination with chemotherapeutic agents. Inparticular, it has been shown that the inhibition of Srcobtained by a Src family kinase inhibitor, alone or incombination with gemcitabine, results in delayed growthand metastasis of orthotopically implanted human pan-creatic carcinoma cells, as a consequence of a reduction in

tumor cell proliferation and microvessel density, and byan increase in the apoptotic index (40). In our in vitropreclinical models, we showed that there is a synergisticeffect of saracatinib and gemcitabine in 2 of the 4 cell lines(41). In this connection and on the basis of our preclinicalresults, we can hypothesize to design a clinical study ofsaracatinib given as a maintenance therapy to verify thecapability of the drug to improve the PFS status in thosepatients in which a disease control was reached bychemotherapy.

In conclusion, we showed that activated Src is widelypresent in biopsies from patients with BTC and that itssmall-molecule inhibitor, saracatinib has a potent inhib-itory effect on cell BTC migration in vitro, and on tumorgrowth and neovascularization in vivo. Taken together,our findings encourage further studies in which the com-bination of saracatinib with chemotherapeutic agent or itsadministration upon the obtaining of a stable disease willbe investigated to improve the management of patientswith BTC.

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Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed by the authors.

Authors' ContributionsConception and design: G. Cavalloni, C.P. Neia, M. Aglietta, F. LeoneDevelopment of methodology: G. Cavalloni, C. Peraldo-Neia, I. Sarotto,G. Migliardi, F. LeoneAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Cavalloni, C. Peraldo-Neia, I. Sarotto,L. Gammaitoni, F. LeoneAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): G. Cavalloni, C. Peraldo-Neia, L. Gammai-toni, M. Soster, F. LeoneWriting, review, and/or revision of the manuscript: G. Cavalloni,C. Peraldo-Neia, S. Marchio, M. Aglietta, F. LeoneAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): I. Sarotto, F. LeoneStudy supervision: G. Cavalloni, M. Aglietta, F. Leone

Grant SupportThis work was supported by grants from "Progetti di Ricerca Rete

Oncologia Piemonte-Valle d’Aosta" and "Associazione Italiana Ricercasul Cancro –AIRC 5 � 1000" and by Fondazione Piemontese per laRicerca sul Cancro (FPRC) Intramural Grant, AIRC-MFAG. G. Caval-loni is supported by (A.D.I.S.C.O.) and Fondazione del Piemonte perl’Oncologia (FPO). C. Peraldo-Neia has a Ph.D. Fellowship sponsoredby MIUR (University of Turin). I. Sarotto and L. Gammaitoni weresupported by FPO. G. Migliardi is supported by FIRB, University ofTurin. M. Soster has a PhD fellowship sponsored by AIRC-MFAG.S. Marchio is supported by Banca d’Alba. M. Aglietta and F. Leone aresupported by the University of Turin.

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.

ReceivedDecember 15, 2011; revisedMarch 21, 2012; acceptedMarch 21,2012; published OnlineFirst March 27, 2012.

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2012;11:1528-1538. Published OnlineFirst March 27, 2012.Mol Cancer Ther Giuliana Cavalloni, Caterina Peraldo-Neia, Ivana Sarotto, et al. Preclinical Models of Biliary Tract CarcinomasAntitumor Activity of Src Inhibitor Saracatinib (AZD-0530) in

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