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Priority Report
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.
CancerResearch
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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).
Kirane et al.
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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.
Kirane et al.
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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.
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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.
Cancer Res; 75(18) September 15, 2015 Cancer ResearchOF6
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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.
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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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