Warfarin Blocks Gas6-Mediated Axl Activation Required for ... · 03-09-2015 · Warfarin, a vitamin K "antagonist" used clinically for the prevention of thrombosis for more than

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Warfarin Blocks Gas6-Mediated Axl ActivationRequired for Pancreatic Cancer EpithelialPlasticity and MetastasisAmanda Kirane1,2, Kathleen F. Ludwig2,3, Noah Sorrelle2,4, Gry Haaland5, Tone Sandal5,Renate Ranaweera5, Jason E. Toombs1,2, Miao Wang1,2, Sean P. Dineen1, David Micklem6,Michael T. Dellinger1,2, James B. Lorens5, and Rolf A. Brekken1,2,7

Abstract

Repurposing "old" drugs can facilitate rapid clinical transla-tion but necessitates novel mechanistic insight. Warfarin, avitamin K "antagonist" used clinically for the prevention ofthrombosis for more than 50 years, has been shown to haveanticancer effects. We hypothesized that the molecular mech-anism underlying its antitumor activity is unrelated to its effecton coagulation, but is due to inhibition of the Axl receptortyrosine kinase on tumor cells. Activation of Axl by its ligandGas6, a vitamin K-dependent protein, is inhibited at doses ofwarfarin that do not affect coagulation. Here, we show that

inhibiting Gas6-dependent Axl activation with low-dose war-farin, or with other tumor-specific Axl-targeting agents, blocksthe progression and spread of pancreatic cancer. Warfarin alsoinhibited Axl-dependent tumor cell migration, invasiveness,and proliferation while increasing apoptosis and sensitivity tochemotherapy. We conclude that Gas6-induced Axl signaling isa critical driver of pancreatic cancer progression and its inhi-bition with low-dose warfarin or other Axl-targeting agents mayimprove outcome in patients with Axl-expressing tumors.Cancer Res; 75(18); 1–7. �2015 AACR.

IntroductionVitamin K "antagonists" have been associated anecdotally with

antitumor and anti-metastatic effects in preclinical and clinicalstudies since the 1960s (1–3). Results from dedicated clinicalstudies designed to evaluate the anti-metastatic activity of warfarinhavebeenvariable, inpartdue tocomplicationsassociatedwith fullanticoagulation. The anticancer effects of warfarin are generallyattributed to thromboembolic inhibition, although the molecularmechanism has not been elucidated. The Axl receptor tyrosinekinase is associated with aggressive cancer and poor patient out-come in several malignancies, including pancreatic cancer (4).

Because warfarin blocks vitamin K-dependent g-carboxylation ofglutamic acids (5) and the g-carboxyglutamic acid–rich (GLA)domain of Gas6 is required to induce Axl tyrosine kinase activity(6–8), we hypothesized that the antitumor activity of warfarincould be due to inhibition of Gas6-mediated Axl activation ontumor cells. Warfarin potently inhibits Gas6-dependent Axl acti-vation (9) at an IC50 of �0.6 nmol/L, a concentration well belowthat required to achieve anticoagulation (5, 10).Here, we exploitedthis differential effect to determine whether low-dose (1.5–3.0mmol/L)warfarin treatment impedes pancreatic cancer progressionby inhibiting Axl signaling independent of anticoagulation.

Materials and MethodsCell lines

The human pancreatic cancer cell lines AsPC-1, Panc-1, Capan-1, and Mia PaCa-2 were obtained from the ATCC; the murine cellline Pan02 was obtained from the DCTD tumor repositorymaintained by the NCI at Frederick. C5LM2 is a variant of Panc1developed in our laboratory that was generated through twopassages of growth in vivo and culture of liver metastases and hasbeen characterized previously (11). The C5LM2, AsPC-1, Panc-1,Pan02, andMia PaCa-2 lines were grown in DMEM; Capan-1 wasgrown in IMDM; all cell lines were grown in a humidifiedatmosphere with 5% CO2, at 37�C, and have been DNA finger-printed for provenance using the Power-Plex 1.2 Kit (Promega)and confirmed to be the same as the DNA fingerprint librarymaintained by the ATCC, and were confirmed to be free ofMycoplasma (e-Myco Kit; Boca Scientific).

Animal studiesAll animals were housed in a pathogen-free facility with

24-hour access to food and water. Experiments were approved

1Division of Surgical Oncology, Hamon Center for Therapeutic Oncol-ogy Research, University of Texas Southwestern Medical Center,Dallas, Texas. 2Department of Surgery, Hamon Center for TherapeuticOncologyResearch, Universityof Texas SouthwesternMedical Center,Dallas,Texas. 3DivisionofHematology/Oncology,DepartmentofPedi-atrics, University of Texas Southwestern Medical Center, Dallas, Texas.4Cell Regulation Graduate Program,Universityof Texas SouthwesternMedical Center, Dallas,Texas. 5Department of Biomedicine, Centre forCancer Biomarkers, Norwegian Centre of Excellence, University ofBergen, Bergen, Norway. 6BerGenBio AS, Bergen, Norway. 7Depart-ment of Pharmacology, University of Texas Southwestern MedicalCenter, Dallas, Texas.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

A. Kirane and K.F. Ludwig contributed equally to this article.

Corresponding Author: Rolf A. Brekken, Hamon Center for Therapeutic Oncol-ogy Research, University of Texas Southwestern Medical Center, 6000 HarryHines Boulevard, Dallas, TX 75390-8593. Phone: 214-648-5151; Fax: 214-648-4940; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2887-T

�2015 American Association for Cancer Research.

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by, and conducted in accordance with, an IACUC approvedprotocol at UT Southwestern. LSL-KrasG12D; Cdkn2alox/lox; p48Cre

(KIC) mice were generated as previously described (12). Four- to6-week-old female NOD/SCID and C57/Bl6 mice were obtainedfrom an on campus supplier. A total of 1 � 106 AsPc-1, Panc-1,Mia Paca2, Capan-1, C5LM2,Mia shLuc, andMia shAxl or 5� 105Pan02 cells were injected orthotopically as described previously(12).Mice with established tumors, as determined by sonographywere randomized to receive normal drinking water or watercontaining 1 mg/L (�3.0 mmol/L) warfarin for experiments inimmunocompromised mice and 0.5 mg/L (�1.5 mmol/L) inexperiments in immunocompetent animals with or withoutgemcitabine 25 mg/kg twice weekly depending on experimentaldesign. KIC mice were treated with warfarin 4 weeks starting at 3weeks of age. For all experiments, warfarin containing water wasreplenished every 3 days. For Mia Paca2 tumor–bearing mice,additional conditions of gemcitabine � 10C9 (250 mg i.p. twice/week) were conducted. Mice bearing Panc-1, Capan-1, C5LM2,and Mia Paca2 tumors were sacrificed after 6 weeks of therapy.AsPc-1 tumor–bearing mice received 4 weeks of therapy andPan02 tumor–bearingmice 3 weeks of therapy. ShRNA lines wereallowed to grow for 8 to 10 weeks. For all therapy experimentsprimary tumor burden was established by weighing pancreas andtumor en bloc. Metastatic incidence was determined by visualinspection of the liver and abdominal cavity and confirmed byhematoxylin and eosin (H&E) of liver sections. Tissues were fixedin 10% formalin or snap-frozen in liquid nitrogen for furtherstudies. C5LM2 cells were injected intrasplenically to establishliver metastases, tumors were allowed to grow for 24 weeks andmice were randomized to receive either normal drinking water orwarfarin (1 mg/L) starting 48 hours prior or 48 hours after tumorcell injection. Liver tumor burdenwas determined by liver weight.

Histology and tissue analysisFormalin-fixed tissues were embedded in paraffin and cut in

6-mm sections. Sections were evaluated by H&E and immuno-histochemical analysis using antibodies specific for vimentin(Phosphosolutions), endomucin, E-cadherin (Santa Cruz Bio-technology), phospho-histone H3 (Upstate), cleaved caspase-3(Cell Signaling Technology). Negative controls included omis-sion of primary antibody and immunofluorescence evaluationwas conducted as described previously (12). Necrotic area wasdetermined by quantification of the percentage of viable tumorarea on low magnification of tumor sections by H&E.

Statistical analysisDatawere analyzed usingGraphPad software (GraphPad Prism

version 4.00 for Windows; GraphPad Software; www.graphpad.com). Results are expressed as mean � SEM or SD. Data wereanalyzed by the t test or ANOVA and results are consideredsignificant at P < 0.05.

Additional methods are described in Supplementary Materialsand Methods.

Results and DiscussionWe evaluated the efficacy of low-dose warfarin (0.5–1 mg/L of

drinking water) as a single agent in five murine models ofpancreatic ductal adenocarcinoma (PDA; Fig. 1A and B). Low-dose warfarin therapy was administered when animals had estab-lished intrapancreatic tumors as measured by sonography. Treat-

ment with low-dose warfarin reduced primary tumor growth in asyngenic model (Pan02, Fig. 1A) and in a spontaneous geneticPDA model (KIC, Fig. 1A), but had little effect on the growth ofhuman tumor xenografts (Panc1, AsPC1, Capan-1, Fig. 1A).Importantly, low-dose warfarin consistently and potently inhib-ited metastatic burden (Fig. 1B; Supplementary Table S1) in fourof the five PDA models. Expression analysis revealed that warfa-rin-sensitive tumors expressed detectable levels of Axl, whereasthe nonresponsive Capan-1 tumors did not (Fig. 1C–E). Further-more, Gas6 was expressed at detectable levels in most PDA celllines (data not shown; ref. 4), indicative of autocrine Axl activa-tion. To evaluate the effects of selective Axl inhibition on PDA, weused a stable retroviral shRNA approach. Axl knockdowncompletely suppressed the growth of orthotopic Mia PaCa-2tumors (Fig. 1F). Extended in vivo growth of shAxl Mia Paca-2cells in an independent experiment resulted in 4 of 7 micedeveloping tumors. These tumors were subsequently found toexpress Axl (Supplementary Fig. S1). To validate tumor-selectiveinhibition of Axl activity in the treatment setting, we developed afunction-blocking human-specific anti-Axl monoclonal anti-body, 10C9 (Supplementary Fig. S2). Treatment of establishedorthotopicMia PaCa-2 tumors with 10C9 blunted primary tumorgrowth and potently suppressed metastases (Fig. 1G). Theseresults support the notion that low-dose warfarin inhibits pan-creatic tumor progression in a manner dependent on tumor cellAxl expression.

To determine the effect of warfarin on Gas6-induced Axlsignaling in PDA, we evaluated phosphorylated Axl (pAxl) anddownstream signaling via the PI3K–Akt signaling pathway (13).Warfarin-prevented g-carboxylation of Gas6 in vitro (Fig. 2A) andinhibited basal pAxl levels in Panc-1 cells, an effect that wasrescued by addition of exogenous vitamin K (Fig. 2B). The effectof warfarin on pAxl was validated in Mia PaCa-2 and Panc-1 byimmunocytochemistry (Supplementary Fig. S3). Further warfarinor BGB324, a specific inhibitor of Axl tyrosine kinase activity (14)inhibited phosphorylation of Axl in Panc-1 cells (Fig. 2C). Con-sistent with these results, treatment of Panc-1 cells in vitro with10C9 resulted in decreased Axl and p-Axl levels (SupplementaryFig. S2CandS2D). Furthermore,warfarin inhibitedGas6-inducedactivation of AKT in Panc1 cells in vitro (Fig. 2D). In addition, theeffect of low-dose warfarin treatment on Panc-1 xenografts wasconsistent with the effects on Axl signaling in vitro. Warfarintreatment substantially suppressed the level of pAxl and pAktin Panc-1 tumors (Fig. 2E), decreased expression of phosphory-lated histone H3, a marker of proliferation, and elevated cleavedcaspase-3, and tumor necrosis (Supplementary Fig. S4), andincreased the level of cleaved Parp (Fig. 2E). Low-dose warfarinalso reduced intratumoral microvessel density (SupplementaryFig. S4D) consistent with the reported proangiogenic activity ofAxl (15).

Axl has been associated with enhanced tumor cell migrationand metastatic invasiveness (16). Warfarin reduced basal andGas6-induced cell migration (scratch assay) in an Axl-depen-dent manner (Fig. 2F). Furthermore, tumor cell sphere forma-tion and invasiveness in 3D culture was inhibited by warfarinand shRNA knockdown of Axl in Mia PaCa-2 cells (Fig. 3A–C).Warfarin also inhibited anchorage-independent growth of Axl-expressing cells (Fig. 3D) and inhibited liver colonization ofPanc-1 cells after intrasplenic injection regardless of whetherwarfarin was administered pre- or post (48 hours)-tumor cellinjection (Fig. 3E).

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We also evaluated whether Axl inhibition with warfarin or10C9augmented the efficacyof gemcitabine, the frontline therapyfor pancreatic cancer.Warfarin treatment hadno effect on the IC50value of gemcitabine onAxl-negative cells lines (Capan-1 andMiaPaCa-2 shAxl) in vitro. However, low-dose warfarin potentiatedthe antiproliferative effect of gemcitabine, reducing the IC50 value8.4- and 211-fold in AsPC-1 and Panc-1 cells, respectively. War-farin also lowered the gemcitabine IC50 value in Mia PaCa-2 andPan02 cells (Supplementary Table S2). In vivo blockade of Axlactivation with low-dose warfarin or 10C9-augmented gemcita-bine reduction of primary tumor growth and dramaticallyimproved metastatic control (Fig. 3F and G).

Metastasis and drug resistance are linked to induction ofepithelial-to-mesenchymal transition (EMT) gene programs inpancreatic cancer (17). Axl expression is elevated in tumor cells byEMT and correlated with mesenchymal marker proteins such asvimentin (16). Mia PaCa-2 cells display an EMT-like phenotypeunder basal conditions (18). We found that treatment of MiaPaCa-2 cells withwarfarin for 48hours in vitro reduced pAxl levels,

surface Axl expression, and the mesenchymal markers Zeb1 andvimentin, while elevating the expression of the epithelial markerE-cadherin (Supplementary Fig. S5). Treatment of Panc-1 cells invitro with TGFb and collagen I, conditions that induce EMT,enhancedAxl expression andactivation, an effect thatwas blockedby addition of warfarin (Fig. 4A). Consistent with these results,Zeb1 and nuclear b-catenin levels, another mesenchymal marker,were significantly reduced by warfarin indicative of phenotypicreversal (Fig. 4A). Furthermore, Gas6 addition to Panc1 cells inculture increased the expression of vimentin and Zeb1, an effectthat was blocked by 10C9 (Fig. 4B). In addition, we identified thatexposure to TGFb and collagen inducedAxl expression inCapan-1cells (Capan-EMT), which correlated with increased expression oftranscription factors (Zeb1, Snail, and Twist) that drive EMT. TheEMT-dependent induction of Axl in Capan-1 established auto-crine activation via endogenous Gas6. Correspondingly, theCapan-EMT cells were sensitive to treatment with warfarin, lead-ing to decreased Axl expression, upregulated E-cadherin, andincreased cleaved caspase-3 levels (Supplementary Fig. S6).

Figure 1.Warfarin inhibits tumor progression in Axl-expressing cell lines. A, primary tumor burden after therapy with warfarin. Therapy was initiated when implanted tumorswere visible by ultrasound (�10 mm3) and consisted of control (normal drinking water) or warfarin, administered in the drinking water at 0.5 mg/L[immunocompetent mice: Pan02 (n ¼ 4, control; 3, warfarin); KIC (n ¼ 10, control; 8, warfarin)] or 1 mg/L [Panc-1 (n ¼ 10, control; 8, warfarin); AsPC-1 (n ¼ 8,control; 6, warfarin); Capan-1 (n ¼ 10, control; 7, warfarin)] and continued for 2 to 4 weeks until control mice were moribund. Therapy in KIC mice wasinitiated at 3 weeks of age and continued for 4 weeks. B, metastases were determined grossly upon sacrifice and confirmed by histologic evaluation ofthe liver. Metastatic burden was normalized to mean number of metastases in control-treated animals and is displayed as a fold change. Incidence of metastasisis also indicated. C, murine pancreatic cancer cells express Axl by flow cytometry. D and E, expression of Axl message and protein by human pancreaticcancer cell lines. F, shRNA-mediated knockdown of Axl suppresses growth of orthotopic Mia PaCa-2 tumors (n¼ 8, shLuc; 7, shAxl). Tumor volume determined byserial ultrasound. G, inhibition of Axl with mAb 10C9 reduces tumor growth and suppresses metastasis of MiaPaCa-2 tumors (n ¼ 7, control; 8, 10C9).Therapy with mAb 10C9 (250 mg twice/week) was initiated when tumors were established as above and persisted for 4 weeks. All results were comparedby the unpaired two-tailed t test with Welch's correction; actual P values are shown; error bars, SEM.

Warfarin Inhibits Axl-Mediated Tumor Progression

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Finally, we found that low-dose warfarin treatment of PDA Panc1xenografts reduced expression of vimentin and elevated theexpression of E-cadherin, results consistent with the observedEMT reversal in vitro (Fig. 4C).

Our data show that warfarin exerts its anticancer effects byinhibitingGas6-mediatedAxl activationon tumor cells.We foundthat Gas6-Axl signal transduction is required to maintain epithe-lial–mesenchymal plasticity traits of aggressive pancreatic tumorscomprising tumorigenicity, invasiveness, survival, drug sensitiv-ity, and metastasis. EMT gene-expression patterns are apparentearly in pancreatic cancer development, associated with inflam-matory premalignant lesions, and drive early metastatic spread.Inflammatorymediators such as TGFb that induce EMT transcrip-tion factor–mediated gene reprogramming are prominent inmalignant pancreatic cancer. Consistent with this, Axl expressionis elevated by EMT transcription factors in breast and lungepithelial cells (16, 19, 20). Furthermore, Axl expression is asso-ciated with EMT gene signatures in drug-resistant non–small cell

lung cancer and a requisite effector of EMT-related acquiredresistance to various therapeutics (19). The wide spread expres-sion of Axl in advanced cancer from diverse cellular originssuggests that tumor cell–associated Axl is a fundamental contrib-utor to malignant progression. Inhibition of Axl signaling isassociated with loss of malignant traits, including cell migrationand metastasis (16). Congruently, we show that low-dosewarfarin treatment and tumor-specific Axl–selective targetingpotently block metastasis in several models of PDA. This isassociated with a loss of mesenchymal protein expression andEMT transcription factor expression that result in decreased pro-liferation and increased apoptosis.

Our results demonstrate that low-dose warfarin-mediated Axlinhibition is effective as an anticancer agent without associatedcomplications from anticoagulation. These results strongly sug-gest that the anecdotal antitumor effects observed clinically withcoumarin-based anticoagulants are due in part to specific inhi-bition of Gas6-mediated Axl activation on tumor cells. These

Figure 2.Warfarin inhibits Axl signaling in vitroand in vivo. A, HEK293 cells engineeredto stably express recombinant Gas6were grown in the presence of vitamin K(Vit K) or vitamin K þ warfarin. Gas6levels and g-carboxylation wereassayed by immunoblottingconditioned media. Conditioned mediafrom untransfected HEK293 cells wereused as a negative control. B, Panc1 cellswere grown in the presence of controlmedia, vitamin K,warfarin, orwarfarinþvitamin K. The level of phosphorylatedAxl (pAXL, red) was determined byimmunofluorescence. C, Panc1 cellswere grown overnight in media with1% serum with no additions (control),warfarin (2 mmol/L), or BGB324(2 mmol/L). Lysates were probed fortotal Axl (tAxl) and phosphorylatedAxl (pAxl). D, Panc1 cells were grownovernight in media with 1% serumwith no additions (control), warfarin(1 mmol/L), Gas6 (1.3 nmol/L), or Gas6þwarfarin. Lysates were probed forphosphorylated Akt (pAkt) and actin.E, lysates from Panc1 tumors harvestedfrom mice treated with control orwarfarin were probed for expression oftAxl, pAxl, actin, pAKT, tAKT, andcleaved Parp. F, the effect of warfarin oncell migration was assessed by a"scratch" assay. Monolayers of theindicated cells were wounded with apipet tip. The cells were incubated inmedia containing 2% serum � warfarin(2 mmol/L) or media containing 2%serum þ Gas6 (1.3 nmol/L) � warfarin.Wound closure was monitored at16 hours and is reported as thepercentage of wound closure;� , P < 0.05; �� , P < 0.001 by ANOVA,Bonferroni's MCT. �� , P < .0001.

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Figure 3.Axl inhibition reduces colony formation and enhances chemotherapy. A–C, parental Mia PaCa-2 cells or Mia PaCa-2 cells stably transfectedwith shRNA targeting Axl(Mia shAxl) were grown as spheroids in Matrigel for 7 days in the presence or absence of warfarin (200 ng/mL), n ¼ 4/condition. Mia PaCa-2 cellcolonies form large stellate colonies characteristic of invasive tumor growth. Colonies and cognate cell projections were imaged (A) with a Nikon Phasecontrast microscope using �40 and �200 magnification. Mean total colony number (B) and total colony area � SD (C) reflective of invasive growth werecalculated using ImageJ image analysis; scale bar, 100 mm. �� , P < 0.001 versus Mia PaCa-2 NT; ##, P < 0.01; ###, P < 0.005 by ANOVA with Tukey's MCT.D, soft agar colony formation for AsPC-1, Mia PaCa-2, and Capan-1 cells grown in normal growthmedia in the presence or absence (control) of warfarin (2 mmol/L) for14 days. Mean � SD colonies/hpf are shown. The unpaired two-tailed t test with Welch's correction. E, liver metastases were quantified after intrasplenicinjection of C5LM2 cells. Animals (10/group) were treated with normal drinking water, warfarin (1 mg/L) beginning 48 hours before (preop) or 48 hoursfollowing tumor cell injection (postop), and then continued on warfarin therapy until time of sacrifice. �� , P < 0.005; �� , P < 0.001 versus control; #, P < 0.05versus post-injection treatment group by ANOVA with Tukey's MCT. F–I, mice bearing established orthotopic C5LM2 (F and G) or Mia PaCa-2 (H and I)were treated with saline (control), gemcitabine (Gem), Gemþwarfarin (GemþWar). Mice bearing Mia PaCa-2 tumors were also treated with warfarin alone (War),Gem þ 10C9. Mice were sacrificed when control-treated animals were moribund and primary and metastatic burden was determined. Primary tumor weight � SD(F and H) and fold change in metastases � SD (G and I) are shown. The incidence of metastasis in each group is shown as a percentage. �� , P < 0.01; �� , P < 0.005;�� , P < 0.001 versus control; ##, P < 0.01 versus Gem by ANOVA with Tukey's MCT.






results are consistent with recent studies that show g-carboxyla-tion of Gas6 is required for Gas6-mediated Axl activation (21).Furthermore, Paolino and colleagues (10) demonstrated thatlow-dosewarfarin treatment (0.5mg/L in drinkingwater) inhibitsGas6-mediated activation of TAM receptors, Tyro3, Axl, and Mer(aka Mertk) on natural killer (NK) cells, leading to enhanced NKcell antitumor activity in a murine mammary adenocarcinoma(4T1) model system. We have previously shown that tumor-selective Axl inhibition is sufficient to block metastasis in the4T1model (20). Hence, the effects of systemic Axl inhibitionmayexert antitumor effects through tumor and host–response-depen-dent mechanisms. On the other hand, although each of theanimal models we used has an intact NK compartment, we didnot observe any antitumor activity in Axl-negative Capan-1 cells,suggesting minimal NK cell antitumor activity in these models.Taken together, our results of tumor-selective Axl inhibition inmultiple settings suggest that inhibition of tumor cell Axl tyrosinekinase activity is a critical determinant for the observed efficacy ofwarfarin in cancer patients.

Disclosure of Potential Conflicts of InterestD. Micklem has ownership interest (including patents) in BerGenBio. J.B.

Lorens is a CSO, has ownership interest (including patents), and is a consultant/advisory board member for BerGenBio. R.A. Brekken reports receiving a com-mercial research grant from BerGenBio. No potential conflicts of interest weredisclosed by the other authors.

DisclaimerThe funders had no role in study design, data collection and analysis,

decision to publish, or preparation of the article.

Authors' ContributionsConception and design: A. Kirane, T. Sandal, S.P. Dineen, J.B. Lorens,R.A. BrekkenDevelopment of methodology: A. Kirane, T. Sandal, S.P. Dineen, D. Micklem,J.B. LorensAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Kirane, K.F. Ludwig, N. Sorrelle, G. Haaland,T. Sandal, R. Ranaweera, J.E. Toombs, M. Wang, S.P. Dineen, M.T. Dellinger,J.B. Lorens

Figure 4.Warfarin inhibits Axl-dependent maintenance of EMT. A, the expression level of pAxl, Zeb1, and nuclear b-catenin in Panc1 cells in vitro was measured byimmunofluorescence under normal culture conditions or after growth on collagenmatrix and treatment with TGFb (20 ng/mL) to induce epithelial-to-mesenchymaltransition, with or without warfarin (2 mmol/L). p-Axl was normalized to total Axl area. B, Panc1 cells were treated with either SFM, recombinant Gas 6 (100 ng/mL),or Gas6 following pretreatment with 10C9 (mAb anti-Axl). Transition to a mesenchymal phenotype was characterized by changes in vimentin and nuclearZeb1 expression determined by immunofluoresence. A and B, data are displayed as mean � SEM and represent five images per chamber, with assay performed intriplicate. The percentage of area per image was normalized to cell number. Images were analyzed using Elements software. �, P < 0.05; �� , P < 0.001 byANOVA with Tukey's MCT. C, paraffin-embedded sections of Panc-1 tumors were analyzed by immunofluorescence for markers of EMT. Representativeimages of E-cadherin and vimentin are shown. Total magnification, �200; scale bar, 100 mmol/L. Images were analyzed using Elements software; quantificationof the percentage of area fraction is shown. Data are displayed as mean � SD and represent five images per tumor with 5 animals per group analyzed;�� , P < 0.0001 by the t test.


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Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Kirane, K.F. Ludwig, G. Haaland, T. Sandal,R. Ranaweera, M. Wang, S.P. Dineen, J.B. Lorens, R.A. BrekkenWriting, review, and/or revision of the manuscript: A. Kirane, K.F. Ludwig,N. Sorrelle, G. Haaland, T. Sandal, S.P. Dineen, J.B. Lorens, R.A. BrekkenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):K.F. Ludwig, T. Sandal, J.E. Toombs, S.P.Dineen,D. Micklem, R.A. BrekkenStudy supervision: T. Sandal, S.P. Dineen, R.A. Brekken

AcknowledgmentsThe authors thank Drs. Alan Schroit, Thomas Wilkie, and John Mansour for

critical comments on the text and the members of the Brekken and Lorenslaboratories for advice and helpful discussion.

Grant SupportThe work was supported by the NIH [R21 CA173487 to R.A. Brekken;

T32 CA136515 (PI: J. Schiller) to A Kirane; 5T32GM007062 (PI:D. Mangelsdorf) to N Sorrelle], a sponsored research agreement fromBerGenBio (R.A. Brekken), Effie Marie Cain Scholarship in AngiogenesisResearch (R.A. Brekken), the Children's Cancer Fund (K.F. Ludwig), theDallas VA Research Corporation (DVARC; S. Dineen), Helse Vest (projectno. 911559 to J.B. Lorens), and University of Bergen predoctoral fellowship(G. Haaland).

Received October 1, 2014; revised June 14, 2015; accepted July 1, 2015;published OnlineFirst July 23, 2015.

References1. Brown JM. A study of the mechanism by which anticoagulation with

warfarin inhibits blood-borne metastases. Cancer Res 1973;33:1217–24.2. McCulloch P, George WD. Warfarin inhibition of metastasis: the role of

anticoagulation. Br J Surg 1987;74:879–83.3. Schulman S, Lindmarker P. Incidence of cancer after prophylaxis with

warfarin against recurrent venous thromboembolism. Duration of Antic-oagulation Trial. N Engl J Med 2000;342:1953–8.

4. Song X, Wang H, Logsdon CD, Rashid A, Fleming JB, Abbruzzese JL, et al.Overexpression of receptor tyrosine kinase Axl promotes tumor cell inva-sion and survival in pancreatic ductal adenocarcinoma. Cancer 2011;117:734–43.

5. Nakano T, Kawamoto K, Kishino J, Nomura K, Higashino K, Arita H.Requirement of gamma-carboxyglutamic acid residues for the biologicalactivity of Gas6: contribution of endogenous Gas6 to the proliferation ofvascular smooth muscle cells. Biochem J 1997;323:387–92.

6. Hafizi S, Dahlback B. Gas6 and protein S. Vitamin K-dependent ligands forthe Axl receptor tyrosine kinase subfamily. FEBS J 2006;273:5231–44.

7. Hasanbasic I, Rajotte I, BlosteinM. The role of gamma-carboxylation in theanti-apoptotic function of gas6. J Thromb Haemost 2005;3:2790–7.

8. Varnum BC, Young C, Elliott G, Garcia A, Bartley TD, Fridell YW, et al. Axlreceptor tyrosine kinase stimulated by the vitamin K-dependent proteinencoded by growth-arrest-specific gene 6. Nature 1995;373:623–6.

9. TsouWI, Nguyen KQ, CalareseDA, Garforth SJ, Antes AL, Smirnov SV, et al.Receptor tyrosine kinases, TYRO3, AXL, and MER, demonstrate distinctpatterns and complex regulation of ligand-induced activation. J Biol Chem2014;289:25750–63.

10. PaolinoM, Choidas A,Wallner S, Pranjic B, Uribesalgo I, Loeser S, et al. TheE3 ligase Cbl-b and TAM receptors regulate cancer metastasis via naturalkiller cells. Nature 2014;507:508–12.

11. Melisi D, Ishiyama S, Sclabas GM, Fleming JB, Xia Q, Tortora G, et al.LY2109761, a novel transforming growth factor beta receptor type I andtype II dual inhibitor, as a therapeutic approach to suppressing pancreaticcancer metastasis. Mol Cancer Ther 2008;7:829–40.

12. Ostapoff KT, Cenik BK, Wang M, Ye R, Xu X, Nugent D, et al. Neutralizingmurine TGFbetaR2 promotes a differentiated tumor cell phenotype andinhibits pancreatic cancer metastasis. Cancer Res 2014;74:4996–5007.

13. Sawabu T, SenoH, Kawashima T, Fukuda A, Uenoyama Y, KawadaM, et al.Growth arrest-specific gene 6 and Axl signaling enhances gastric cancer cellsurvival via Akt pathway. Mol Carcinog 2007;46:155–64.

14. Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, et al. R428, a selectivesmall-molecule inhibitor of Axl kinase, blocks tumor spread and prolongssurvival in models of metastatic breast cancer. Cancer Res 2010;70:1544–54.

15. RuanGX, Kazlauskas A. Axl is essential for VEGF-A-dependent activation ofPI3K/Akt. EMBO J 2012;31:1692–703.

16. GjerdrumC, TironC,Hoiby T, Stefansson I,HaugenH, Sandal T, et al. Axl isan essential epithelial-to-mesenchymal transition-induced regulator ofbreast cancer metastasis and patient survival. Proc Natl Acad Sci U S A2010;107:1124–9.

17. RhimAD,Mirek ET, Aiello NM,Maitra A, Bailey JM,McAllister F, et al. EMTand dissemination precede pancreatic tumor formation. Cell 2012;148:349–61.

18. ArumugamT,RamachandranV, Fournier KF,WangH,Marquis L, Abbruzz-ese JL, et al. Epithelial to mesenchymal transition contributes to drugresistance in pancreatic cancer. Cancer Res 2009;69:5820–8.

19. Wilson C, Ye X, Pham TQ, Lin E, Chan SM, McNamara E, et al. AXLinhibition sensitizes mesenchymal cancer cells to anti-mitotic drugs.Cancer Res 2014;74:5878–90.

20. Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. Anepithelial–mesenchymal transition gene signature predicts resistance toEGFR and PI3K inhibitors and identifies Axl as a therapeutic target forovercoming EGFR inhibitor resistance. Clin Cancer Res 2013;19:279–90.

21. Lew ED, Oh J, Burrola PG, Lax I, Zagorska A, Traves PG, et al. DifferentialTAM receptor-ligand-phospholipid interactions delimit differential TAMbioactivities. Elife 2014;3.






Published OnlineFirst July 23, 2015.Cancer Res Amanda Kirane, Kathleen F. Ludwig, Noah Sorrelle, et al. Pancreatic Cancer Epithelial Plasticity and MetastasisWarfarin Blocks Gas6-Mediated Axl Activation Required for

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