Fer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma...

Signal Transduction

Fer Protein-Tyrosine Kinase Promotes LungAdenocarcinoma Cell Invasion and Tumor Metastasis

Joseph Ahn1,3, Peter Truesdell1,3, Jalna Meens1,3, Carli Kadish1, Xiaolong Yang2, Alexander H. Boag2, andAndrew W.B. Craig1,3

AbstractEpidermal growth factor receptor (EGFR) is frequently amplified or mutated in non–small cell lung cancer

(NSCLC). Although Fer protein-tyrosine kinase signals downstream of EGFR, its role in NSCLC tumorprogression has not been reported. Here, Fer kinase was elevated in NSCLC tumors compared to normal lungepithelium. EGFR signaling in NSCLC cells fosters rapid Fer activation and increased localization to lamellipodia.Stable silencing of Fer inH1299 lung adenocarcinoma cells (Fer KD) caused impaired EGFR-induced lamellipodiaformation compared to control cells. Fer KD NSCLC cells showed reduced Vav2 tyrosine phosphorylation thatwas correlated with direct Fer-mediated phosphorylation of Vav2 on tyrosine-172, which was previously reportedto increase the guanine nucleotide exchange factor activity of Vav2. Indeed, Fer KD cells displayed defects in Rac-GTP localization to lamellipodia, cell migration, and cell invasion in vitro. To test the role of Fer in NSCLCprogression andmetastasis, control and Fer KD cells were grown as subcutaneous tumors inmice. Although Fer wasnot required for tumor growth, Fer KD tumor-bearing mice had significantly fewer numbers of spontaneousmetastases. Combined, these data demonstrate that Fer kinase is elevated in NSCLC tumors and is important forcellular invasion and metastasis.Implications: Fer protein-tyrosine kinase is a potential therapeutic target in metastatic lung cancer.Mol Cancer

Res; 11(8); 952–63. �2013 AACR.

IntroductionNon–small cell lung carcinoma (NSCLC) is the most

common form of lung cancer and the leading cause ofcancer-related deaths worldwide. NSCLC has a 5-yearsurvival rate of only 15% due in part to advanced diseaseat diagnosis and high rates of metastases. A subset ofNSCLC's acquire increased copy number or gain-of-func-tion mutations in EGF receptor (EGFR) leading to consti-tutive EGFR signaling to pathways controlling cell growth,survival, and motility (1). EGFR is a known driver ofNSCLC as transgenic mice with inducible expression ofmutant alleles of EGFR (e.g.,D747-752 or L858R) in type IIlung pneumocytes develop lung adenocarcinomas thatregressed upon treatment with an EGFR inhibitor (2).Indeed, patients with lung cancer with activating EGFRmutations who are treated with EGFR inhibitors frequentlyshow a positive initial response but eventually relapse due to

development of resistance (3). Therefore, identifying newtargets for molecular-targeted therapies and development ofcombinational therapies may lead to improved NSCLCpatient outcomes.Fer (Fes-related) is a widely expressed nonreceptor tyro-

sine kinase that is activated downstream of EGFR (4), andwas recently reported to promote resistance to quinacrine(5), an antimalarial drug with potential as a cancer thera-peutic (6). Fer is composed of 4 domains: F-BAR (Fer/CIP4homology-Bin/amphiphysin/RVS), FX (extended F-BAR),SH2 (Src homology 2), and a protein-tyrosine kinasedomain. F-BAR domains form crescent-shaped dimers thatbind phosphoinositides that are enriched in membranemicrodomains at sites of endocytosis and filamentous actin(F-actin) polymerization (7). The F-BARdomains of Fer andFes, a more tissue-restricted family member (8), were ini-tially shown to bind PI(4,5)P2 (9, 10).More recently, the FXdomain in Fer was shown to bind phosphatidic acid (PA),leading to enhanced Fer kinase activity and localization tolamellipodia (11). Itoh and colleagues also show that Ferpromotes cell motility in a phospholipase D (PLD)/PA–dependent pathway (11). Recently, Fes was also shown tobind phosphatidic acid and participate in a PLD-inducedpathway of myeloid differentiation (12). The SH2 domainof Fer confers binding to tyrosine-phosphorylated proteins,including receptor tyrosine kinases [e.g., EGFR, platelet-derived growth factor receptor (PDGFR), etc.], adaptorproteins (e.g., cortactin, Gab1, etc.), and immune receptors(8). Recently, the structure of the SH2-kinase domain

Authors' Affiliations: Departments of 1Biomedical and MolecularSciences and 2Pathology & Molecular Medicine, 3Division of CancerBiology & Genetics, Queen's University, Kingston, Ontario, Canada

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

Corresponding Author: Andrew W.B. Craig, Division of Cancer Biology &Genetics, Queen's University, Botterell Hall, 3rd Fl, CRI315, 18 Stuart St.,Kingston, ON K7L 3N6, Canada. Phone: 613-533-2496; Fax: 613-533-6830; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-13-0003-T

�2013 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 11(8) August 2013952

on August 15, 2019. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst May 22, 2013; DOI: 10.1158/1541-7786.MCR-13-0003-T

http://mcr.aacrjournals.org/

fragment of Fes was solved, and this revealed a criticalinterface allowing allosteric regulation between the SH2and kinase domains (13). The implications of this studyare that Fes kinase activation occurs only upon ligandbinding to its SH2 domain. Because of conservation ofthese critical interface residues between Fer and Fes, it islikely that Fer kinase activation also requires the SH2domain to be bound to ligands such as EGFR.A number of studies have identified Fer activation or

upregulation in a number of different cancers, includinglung (14), hepatic (15), prostate (16), and triple-negativebreast cancer (17). Overexpression of Fer was also linkedto quinacrine resistance and enhanced activation of NF-kB pathway (5). Other Fer substrates are linked to tumorprogression and metastasis. The transcription factorSTAT3 is a known substrate of Fer, and has been linkedto prostate tumor progression (16). The actin regulatoryprotein cortactin is a substrate of Fer downstream ofgrowth factor receptors and integrins (18–20). Recentstudies also implicate Fer in signaling via Rac GTPase topromote lamellipodia formation and cell motility (11).Overexpression of Fer also correlated with enhancedtyrosine phosphorylation of Vav2 (11), a Rac guanineexchange factor (GEF) that is activated via tyrosine phos-phorylation downstream of EGFR (21). Fer may alsopromote Rac activation by phosphorylating RhoGDIa tolimit Rac1 binding (22), and prevent sequestration byRhoGDIa (23).In this report, we show that Fer is expressed in normal

lung tissue, primarily in type II pneumocytes, and atelevated levels in NSCLC (adenocarcinoma and squamoussubtypes). EGF treatment of H1299 lung adenocarcino-ma cells led to rapid Fer activation and localization toleading edge membrane projections. Fer silencing reducedlamellipodia formation, motility, and cell invasion viadefects in the Vav2/Rac signaling axis. Stable silencingof Fer also reduced spontaneous metastases in subcuta-neous tumor xenograft assays in mice. Together, thesefindings identify Fer as a positive regulator of EGFR-induced lamellipodia formation, cell motility, invasion,and tumor metastasis.

Materials and MethodsCell culture, transfections, and EGF treatmentNCI-H1299 were purchased from American Type Cul-

ture Collection andweremaintained inDulbecco'smodifiedEagle medium (DMEM) supplemented with 10% FBS(Sigma-Aldrich), 1% glutamine (Invitrogen), and 1% anti-mycotic/antibiotics (Invitrogen) at 37�C in a 5% CO2atmosphere. A549 cells were acquired from Dr. Susan Cole(Queen's University, Kingston, ON, Canada) and grown in5%FBS as earlier. Immortalized wild-type (WT; ferþ/þ) andfer-deficient (ferDR/DR) mouse embryonic fibroblasts (MEF)were described previously (19, 20). Transient transfectionsof cells with plasmid DNA were conducted using FuGENEHD (Roche Diagnostics Inc.) or X-tremeGENE (RocheApplied Science) according to the manufacturer's directions.EGF stimulation of H1299 cells were conducted by starving

overnight in serum-free media (16 hours) before treatmentwith human EGF (50 or 100 ng/mL; Peprotech) for indi-cated times in figure legends. EGF stimulation of MEF cellswas conducted by starving in serum-free media for 48 hoursbefore treatment with human EGF (100 ng/mL for 5minutes).

Generation of stable Fer knockdown cell linespGIPZ-based lentiviral transfection encoding a nontar-

geting short hairpin RNA (shRNA) and Fer-specificshRNA (FerKD1: 50-CTATCCAAATTTGAATCTA-30,FerKD2: 50–AGGCTCACCATGATGATTA-30) wereobtained from Open Biosystems. Lentiviral plasmids werecotransfected with packaging and envelope plasmids(CMVDR8.91 and MD.2G) in HEK 293T humanembryonic kidney cells and viral supernatants collectedat 48 hours. H1299 and A549 cells were transduced withthe above viruses and stable cell pools were selected after48 hours in media with puromycin (2 mg/mL). Cell poolswere passaged several times and lysates prepared to test theefficiency of Fer silencing by immunoblotting.

Immunoprecipitation and immunoblottingFor immunoprecipitation assays, cells were lysed in

Mg2þ lysis buffer [50 mmol/L Tris–HCL (pH 7.4),150 mmol/L NaCL, 1% Triton X-100, 0.25% sodiumdeoxycholate, 10% glycerol, 25 nmol/L NaF, 10 mmol/LMgCl2, 1 mmol/L EDTA, 10 mg/mL aprotinin, 10 mg/mLleupeptin, 1 mmol/L Na3VO4, and 100 mmol/L phenyl-methylsulfonyl fluoride] and soluble cell lysates (SCL)were subjected to immunoprecipitation with rabbit anti-Fer (24) antibodies or mouse anti-Vav2 antibodies (SantaCruz Biotechnology; H-200), and recovered with 25 mL ofGammaBind Sepharose (50% v/v slurry in PBS), per 1 mLSCL. After extensive washing, the immune complex wassubjected to SDS-PAGE as described later.For immunoblot assays, cells were lysed using kinase

lysis buffer [20 mmol/L Tris–HCL (pH 7.5), 150 nmol/LNaCl, 1 mmol/L EDTA, 1%Nonidet P-40, 0.5% sodiumdeoxycholate, 10 mg/mL aprotinin, 10 mg/mL leupeptin,1 mmol/L Na3VO4, 100 mmol/L phenylmethylsulfonylfluoride]. Cell lysates were separated by SDS-PAGE underreducing conditions and transferred to Immobilon Pmembranes (Millipore) using a semi-dry transfer appara-tus (Bio-Rad). Membranes were blocked in either 5%bovine serum albumin (BSA) for antiphosphotyrosineantibodies (pY99), or 5% nonfat milk for all other anti-bodies and then probed with antibodies directed against:Fer (1:1,000; ref. 24), p-Tyr (1:1,000; Santa Cruz Bio-technology; PY99), tubulin (1:1,000; Sigma; T6074),actin (1:1,000; Santa Cruz Biotechnology; C4), Vav2(1:1,000; Santa Cruz Biotechnology; H-200), phospho-172 Vav2 (1:1,000; ECM Biosciences), Rac1 (1:1,000;Millipore; 23A8), active Rac1-GTP (1:1,000; NewEastBiosciences), and transferrin (1:1,000; Invitrogen; H68.4)overnight at 4�C. Antibody–antigen complexes weredetected using either horseradish peroxidase (HRP)–con-jugated goat anti-mouse immunoglobulin (1:10,000; GE

Fer Promotes Lung Cancer Metastasis

www.aacrjournals.org Mol Cancer Res; 11(8) August 2013 953




Healthcare) or HRP-conjugated goat anti-rabbit immu-noglobulin (1:10,000; GE Healthcare) and followed byenhanced chemiluminescence (ECL; Applied BiologicalMaterials).

Immunofluorescence staining and confocal microscopyImmunofluorescence staining was conducted on serum-

starved H1299 cells plated on fibronectin (20 ng/mL)–coated coverslips, and treated with EGF (50 ng/mL) forthe times indicated in figure legends. Cells were incubatedwith mouse anti-Fer antibodies (1:50; Selleck; 5D2C4) ormouse anti-active Rac1 (RacGTP-specific) antibody (1:50;NewEast Biosciences), detected with either Alexa Fluor 568-or 633–conjugated goat anti-rabbit immunoglobulin G(IgG; 1:200; Invitrogen) or Alexa Fluor 568- or 633–conjugated goat anti-mouse IgG (1:200; Invitrogen) andcounterstained with Tritc-phalloidin (1:400; Invitrogen)and 40,6-diamidino-2-phenylindole (DAPI; 1:400; Invitro-gen) and analyzed by confocal microscopy (HCX PL APODIC 63X/132 Oil CS objective; WaveFX-X1 spinning discconfocal microscope; Quorum Technologies Inc.).

Analysis of Fer expression inNSCLC cell lines and tumorhomogenatesFer expression was analyzed in a panel of normal lung

epithelial (NL20) and NSCLC cell lines (NCI-H1299,A549, NCI-H1975, HCC-827, SK-LuCi-6, QU-DB, andLC-T; described in ref. 25). Tumor and adjacent normallung tissues were acquired from the Ontario Tumor Bank(Ontario Institute for Cancer Research, Toronto, ON,Canada). These biopsies were collected at initial diagnosisof 12 patients with NSCLC (ages, 48–75 years; grade 2–3).Homogenates were prepared and along with cell lysatesabove, were subjected to immunoblot with Fer and actinantibodies.

Cell growth assaysCell growth assays were conducted on H1299 control

and Fer knockdown (KD) cells grown to 80% confluence.Cells were trypsinized, fixed in 70% ethanol overnight at4�C, and treated with 0.1 mg/mL RNaseA in PBS for 30minutes at room temperature. Cells were permeabilizedwith PBS with 0.5% Triton X-100 and resuspended inpropidium iodide (PI; 10 mg/mL)/RNase A (0.1 mg/mL)at 37�C for 20 minutes. Samples were analyzed by flowcytometry.

Cell migration assaysWound-healing assays were conducted on confluent

monolayers of H1299 and A549 cells (nontargeting andFer knockdown) that were serum-starved overnight beforeforming a wound using a pipette tip and removal-sus-pended cells. Cells were stimulated with media supple-mented with EGF (50 ng/mL) and phase contrast micro-graphs acquired (0 hours). After 18 to 24 hours ofincubation, cells were fixed in 4% paraformaldehyde andstained with crystal violet and images were taken at thesame positions taken at 0 hours. Cell migration was

quantified as the difference between cell-free areas at time0 and 18 hours for the same field of view. Agarose dropchemotaxis assays were conducted as previously describedwith minor alterations (26). Coverslips were coated withfibronectin (20 ng/mL) for 24 hours at room temperaturebefore adding an agarose drop (0.1% low-melting tem-perature agarose) supplemented with EGF (final concen-tration of 20 ng/mL). H1299 cells (20,000) were stainedwith either CellTracker orange or CellTracker green(Invitrogen; 10 mmol/L) for 1 hour before plating oncoverslips and allowed to adhere for 6 hours at 37�C.Postadherence images from the edge of the agarose dropwere acquired by the Quorum Wave FX microscope (�10objective) every 10 minutes for 18 hours in a chamberheated to 37�C in a 5% CO2 atmosphere. Migrationanalysis was conducted using the manual tracking suite inImageJ (NIH, Bethesda, MD, USA).

Invasion assaysBoyden chamber invasion assays were conducted using

Transwell inserts (8 mm; BDBiosciences) coated with 17 mLof growth factor–reduced Matrigel (BD Biosciences) thatwere allowed to solidify at 37�C for 30 minutes. The insertswere placed in a 24-well plate containing DMEM supple-mented with 10% FBS and 50,000 H1299 cells in DMEMwith 0.5% FBS were plated into the upper chamber. Forassays with A549 cells, TGF-b1 (2 ng/mL) was included toincrease their invasive potential. After 48 hours for A549 and60 hours for H1299 cells, the filters were fixed in 4%paraformaldehyde, Matrigel from the upper chamber wasremoved andfilters were stainedwithDAPI. Epifluorescencewas used to detect nuclei of cells that had invaded and imagefields were scored using Image Pro Plus software (MediaCybernetics).

Vav2 constructs and in vitro kinase assaysPlasmids encoding Vav2 glutathione S-transferase (GST)

fusions with different point mutations were obtained fromDr. Buday Laszlo (Hungarian Academy of Sciences, Buda-pest, Hungary; ref. 21). All plasmids were transformed intoBL21 Escherichia coli, grown to mid log phase in Luria Broth(LB) and induced with 1 mmol/L isopropyl-l-thio-B-[scap)d[r]-galactopyranoside (IPTG). Cell pellets were resus-pended in A1000 buffer [1 mol/L NaCl, 20 mmol/LTris–HCL (pH 8), 0.2 mmol/L EDTA, 0.5 mmol/L dithio-threitol (DTT), 0.1% Triton-X100, 10 mg/mL aprotinin,10 mg/mL leupeptin, and 100 mmol/L phenylmethylsulfo-nyl fluoride], treated with lysozyme (166 ng/mL) for 30minutes, sonicated, and cleared by centrifugation. GST-fusion proteins were purified on prepared glutathione–sepharose beads and subsequently eluted using glutathioneelution buffer [50 mmol/L Tris–HCL (pH 8) and 3.1 mg/mL reduced glutathione]. Purified GST-fusion proteinswere treated with thrombin (10 U/mg) to remove the GSTtag. For kinase reactions, cleaved Vav2 constructs (1 mg)were incubated with or without 250 ng of active Fer (GST-fusion to SH2-kinase; Signal Chem) in kinase reaction buffer[0.2 mol/L Tris–HCL (pH 7.4), 0.1 mmol/L Na3VO4, 10

Ahn et al.

Mol Cancer Res; 11(8) August 2013 Molecular Cancer Research954




mmol/L MgCl2, and 0.5 mmol/L ATP] for 20 minutes atroom temperature. The reaction was stopped with 6� SDSand subjected to SDS-PAGE and anti-pY immunoblot.

Tumor xenograft assaysRag2�/�:gc�/� mice (BALB/c background) are deficient

inNK/B/T cells and recently shown to be an excellentmodelfor detecting lung metastases (27). All experiments wereapproved by the Queen's University Animal Care Commit-tee. H1299 nontargeting and Fer knockdown cells weregrown to 80% to 90% confluence, trypsinized and counted.For xenografting assays, cells were injected subcutaneouslyinto the flank area of the mice ((2.5 � 106 cells in 100 mL).After 5 weeks, mice were sacrificed and tumor masses wererecorded. The tumor and lung tissues were resected, fixed informalin, and embedded in paraffin. Paraffin blocks wereserially sectioned and stained with hematoxylin and eosin forhistologic examination. Three sections, spaced 25 mm apart,from each mouse were digitally scanned and metastaticnodules were manually scored in a blinded fashion.

Tumor tissue microrrays and immunohistochemistryTissue microarrays covering a range of lung cancer types

(LC1005; US Biomax) were stained using the VMSI Dis-covery XT Automated Immunostainer (Ventana MedicalSystems, Inc.). Antigens were retrieved with EDTA (pH 8)solution and incubated with mouse anti-Fer (Selleck;5D2C4; diluted 1:100 in TBS/1% BSA). As a control forspecificity, an aliquot of mouse anti-Fer that was preclearedwith beads containing GST-Fer-FX domain that overlapsthe antigenic region (amino acids 280–474) was also used.Immunohistochemistry (IHC) signals were developed usingbiotinylated goat anti-mouse secondary antibody followedby 3,30-diaminobenzidine (DAB) and hematoxylin coun-terstained. Tissue microarrays were digitally scanned andwere scored using ImageScope software (Aperio) to calculateH scores according to the formulaH¼ (% negative� 0)þ(%weak positive� 100)þ (% positive� 200)þ (% strongpositive � 300), as described in ref. (28).

Statistical and image analysisStatistical significance (�, P� 0.05) was determined using

paired Students t test in Microsoft Excel. Graphs weregenerated using GraphPad Prism. ImageJ image-analysissoftware with the manual tracking package was used forquantification analysis of cell migration.

ResultsExpression of Fer kinase in normal human lung tissueand NSCLC tumorsTo evaluate Fer expression in normal lung and NSCLC

tumors, we surveyed Fer levels in NSCLC tumors andmatched normal lung tissue homogenates by immunoblot.Although Fer was expressed in all samples, we observedincreased Fer protein levels in most tumors (T) comparedwith their normal (N) lung tissue controls (Fig. 1A; 2.2 �0.4 (mean � SEM) for T:N ratio; actin served as a loadingcontrol). To visualize Fer expression in these tissues, we first

established optimal immunohistochemical staining condi-tions (Supplementary Fig. S1). Importantly, antisera deple-tion using a recombinant Fer construct (FX domain region)abrogated the immunohistochemical signal (SupplementaryFig. S1). Next, we conducted Fer immunohistochemicalstaining in NSCLC tumor tissue microarrays with matchingnormal adjacent lung tissue. Fer expression was detectable innormal lung tissues, with highest expression in type IIpneumocytes that secrete surfactant (Fig. 1B; see arrowswithin insets). Fer was highly expressed in NSCLC tumors,including most adenocarcinomas and squamous carcinomas(Fig. 1B). Quantification of the immunohistochemicalstaining (H score) revealed a significant increase in Fer levelsin adenocarcinomas or squamous carcinomas comparedwith normal adjacent lung tissue (Fig. 1C). Together, theseresults are consistent with high Fer expression in human lungtumors.

N T1

5.12 1.68 3.22 0.62 1.17 2.56 3.34 1.59 1.14 4.49 1.46 0.59

95

43

IB: Fer

IB: Actin

T:N ratio

Normal

A

B

C 300

200

100

0

Squam

ous

Adeno

Nor

mal A

dj

Nor

mal A

dj

H s

co

re

Squamous Adenocarcinoma

2 3 4 5 6 7 8 9 10 11 12N T N T N T N T N T N T N T N T N T N T N T

Figure 1. Fer kinase expression in normal human lung andNSCLC tumors.A, homogenates from human lungNSCLC adenocarcinoma tumor tissue(T) with matching adjacent normal lung tissue (N) from 12 patients wereanalyzed for Fer expression by immunoblot (IB) with Fer antisera. Foldchange between tumor and normal homogenate were quantified bydensitometry and normalized to actin. B, human NSCLC tumors andmatched normal adjacent lung tissue microarrays were screened for Ferexpression using IHC. Representativemicrographs are shownwith insetscorresponding to higher magnification of the boxed region. Arrowsindicate type II pneumocytes positive for Fer. C, Fer expression wasquantified using imaging software to generate H scores (as described inMaterials and Methods) for matched samples of squamous oradenocarcinoma and adjacent normal tissue [N ¼ 11/subtype; �, asignificant difference between normal and tumor based on pairedStudents t test (P < 0.05)].






EGFR-induced Fer activation and localization tolamellipodia in NSCLC cellsTo identify potential cell models to test Fer signaling and

function, a panel of human lung epithelium-derived celllines was screened for Fer expression. This panel included animmortalized, normal lung epithelial cell lines (HBE154,HBE158, and NL20), and 6 NSCLC cell lines. Fer immu-noblot analysis revealed expression of Fer in normal lungepithelial cell lines, and all NSCLC cell lines tested (Fig. 2A).Because previous studies have implicated Fer in signalingdownstream of EGFR (4, 5), we selected H1299 lungadenocarcinoma cells that express WT EGFR, and effec-tively model EGFR-driven metastasis in mice (29, 30).To test for EGF-induced activation of Fer in H1299 cells,

serum-starved cells were treated with or without EGF for 5

minutes. Following immunoprecipitation of Fer from SCLs,tyrosine phosphorylation (pY) of Fer was assessed by immu-noblot. We observed increased Fer pY in EGF-treated celllysates, consistentwith increased EGFRpY in SCLs (Fig. 2B;see arrow at 170 kDa). Because we observed equal recoveryof Fer in the immunoprecipitations (Fig. 2B, bottom), weconclude that Fer signals downstream of EGFR in H1299NSCLC cells.Fer was recently shown to localize to lamellipodia via N-

terminal F-BAR/FX domains that bind the PLD productphosphatidic acid and enhance Fer kinase activation (31). Totest whether EGFR signaling alters Fer localization inNSCLC, H1299 cells were plated on poly-L-lysine/fibro-nectin–coated coverslips, serum-starved, and treated with orwithout EGF for 5 minutes. Immunofluorescence stainingfor Fer along with staining of filamentous (F)-actin andnuclei was conducted before confocal microscopy. In serum-starvedH1299 cells, Fer localized within the cytosol (green),and displayed no localization to cortical F-actin (red) or thecell nucleus (Fig. 2C, top). Upon EGF treatment, H1299cells acquired amigratory phenotype featuring a leading edgewith broad lamellipodial protrusions (Fig. 2C, bottom).Interestingly, Fer was enriched within F-actin–rich regionsof the lamellipodia (Fig. 2C, see arrows). Together, theseresults are consistent with EGF-induced Fer activation andlocalization to the leading edge of NSCLC cells undergoingdynamic F-actin polymerization andmembrane protrusions.

Stable silencing of Fer impairs EGF-inducedlamellipodia formation in H1299 NSCLCTo probe the function of Fer in NSCLC cells, we selected

H1299 cell pools transduced with lentiviruses encodingeither a nontargeting shRNA control or shRNAs specificfor Fer. Fer expression was analyzed following several cellpassages, and this revealed efficient Fer knockdown withshRNA1 (KD1) and shRNA2 (KD2) compared with non-targeting control (Fig. 3A). Densitometry analysis for severalcell passages revealed a stable reduction in Fer levels (relativeto tubulin) by approximately 80% and 60% in Fer KD1,KD2 cell lysates, respectively.To test whether Fer silencing affects growth of H1299

cells, the cell-cycle profiles of nontargeting and Fer knock-down cells were compared. Following PI staining of sub-confluent H1299 nontargeting and Fer knockdown cellsgrown in presence of serum, the DNA content of cells wasanalyzed by flow cytometry. No significant differences wereobserved for the percentage of cells residing in G1, S, orG2–M phases of the cell cycle between nontargeting, FerKD1, and Fer KD2 (Fig. 3B). Thus, Fer silencing does notregulate growth or survival of H1299 cells.Because we observed Fer localizationwithin EGF-induced

lamellipodia in H1299 cells, we compared the proportion ofnontargeting and Fer knockdown cells with prominentleading edge lamellipodia marked by F-actin staining. Fol-lowing EGF treatment for 5 minutes, Fer knockdown cellsappeared less polarized, and had less extensive F-actin–richmembrane ruffles compared with nontargeting control cells(Fig. 3C). Next, we scored the percentage of cells in these

NormalA

95

43

170130

95

72

55

95

95

NSCLC

IB: Fer

IB: pY

– + EGFB

C

IP: Fer

IP: Fer

IB: pY

IB: Fer

Actin Fer DAPI Merge

Seru

m s

tarv

ed

EG

F (

50 n

g/m

L)

:

5 m

in

IB: Actin

Figure 2. EGF-induced Fer activation and localization to lamellipodia inNSCLC cells. A, expression of Fer was surveyed by immunoblot (IB) oflysates from a panel of immortalized and NSCLC cell lines using Ferantisera. B, starvedH1299 cells were stimulatedwith EGF (100 ng/mL) for5 minutes before being harvested and subjected to immunoprecipitation(IP) with mouse Fer antisera. Lysates and immunoprecipitation's weresubjected to immunoblot analysis with the indicated antibodies. C,representative micrographs for serum-starved H1299 cells stimulatedwith EGF (50 ng/mL) for 5 minutes or left unstimulated before beingprocessed for immunofluorescence staining of endogenous Fer (green)and counterstained for F-Actin (red) and nuclear DNA (blue). Scale bars,25 mm.

Ahn et al.





assays with extensive lamellipodia (>50% of cell periphery),and observed significantly fewer Fer knockdown cells withthis migratory phenotype compared with nontargeting con-trol (Fig. 3D; P < 0.05). Taken together, these resultsimplicate Fer kinase in enhancing EGFR signaling to path-ways promoting lamellipodia formation.

Fer phosphorylates Vav2 downstream of EGFRA recent study reported that overexpression of Fer

enhanced tyrosine phosphorylation of the GEF Vav2 andformation of lamellipodia (11). Because EGF-induced Vav2pY was shown to enhance its GEF activity toward RacGTPase (21), we hypothesized that Fer silencing impairsVav2 pY leading to defects in Rac activation, a key inducer oflamellipodia formation (32). To test this, serum-starvedH1299 nontargeting and Fer knockdown cells were treatedwith or without EGF for 5 minutes and then harvested. Noovert defect in global pY levels was observed in SCLs fromFer knockdown cells compared with nontargeting control(Fig. 4A, top). EGF treatment led to increased Vav2 pY inboth nontargeting and Fer knockdown cells, however, thelevels of Vav2 pY were reduced in Fer knockdown cells (Fig.4A, third panel). This difference was not due to altered levelsor recovery of Vav2 in Fer knockdown cells (Fig. 4A,bottom).To complement these findings, and rule out the possibility

of off target effects of our shRNAs, we tested Vav2 pYphosphorylation status in MEF from WT (ferþ/þ) andhomozygous D743R knockin mice (ferDR/DR) devoid ofFer kinase activity (19), (20). MEFs were serum-starved andtreated with or without EGF for 5 minutes. As expected,EGF treatment led to increased pY of many prominentproteins in lysates for both genotypes (Fig. 4B, top). Aspreviously reported (19), Fer levels were greatly reduced inferDR/DRMEFs due to instability of FerDR protein. Althoughno differences were observed in levels of Vav2 betweengenotypes, ferDR/DR MEFs showed reduced Vav2 pY uponEGF treatment comparedwith that observed in ferþ/þMEFs(Fig. 4B, second and fourth panels). Taken together, theseresults indicate that EGFR-induced Vav2 pY is partiallydependent on Fer kinase in NSCLC and MEFs.Vav2 has 3 tyrosines within an acidic domain, and

phosphorylation of these residues leads to enhanced RacGEF activity (Supplementary Fig. S2A and S2B; ref. 33).Wepredicted that Fer may phosphorylate at least one of thesesites, due to the observed partial reduction inVav2 pY in Fer-deficient cells. To address this, we compared Vav2 pY siteswith an autophosphorylation site in Fer (Y402), and that of aknown Fer substrate (cortactin Y421; refs. 13, 20). Of the 3sites in Vav2 acidic domain, Y172 showed some conserva-tion of acidic residues inþ1 andþ2 positions with Fer Y402and cortactin Y421 (Supplementary Fig. S2C). To testwhether Vav2-Y172 is a target of Fer kinase in NSCLCcells, we subjected lysates from nontargeting, Fer KD1, andKD2 cell treated with EGF for 0 to 15 minutes to immu-noblot analyses with pY172-Vav2 antisera. We observedincreased Vav2 pY172 within 5 minutes of EGF treatmentin nontargeting cells (Fig. 4C). In contrast, EGF treatment

25 µm 25 µm 25 µm

H1299 cellsA

B

C

D

NT

95

55

KD1 KD2

IB: Fer

IB: Tubulin

NTKD1

70

60

50

40

30

20

10

0

70

60

50

40

30

20

10

0

G1 G2–MS

NT KD1 KD2

NT KD1 KD2

% C

ell

po

pu

latio

n

% o

f ce

lls fo

rmin

g la

me

llip

od

ia

KD2

Figure 3. Fer knockdown impairs lamellipodia formation but not cellproliferation. A, two separate shRNA constructs for Fer (KD1 andKD2) and one nontargeting control (NT) were transduced into H1299cells. Selection was achieved with puromycin (2 mg/mL) for 1 weekand knockdown of Fer was measured after selection with puromycinby immunoblot (IB) with Fer antisera. B, H1299 nontargeting,Fer KD1, and Fer KD2 cells were stained with PI for DNA contentand quantified by flow cytometry analysis to measure cell-cyclefractions. C, representative micrographs for serum-starved H1299nontargeting, Fer KD1, and Fer KD2 cells stimulated with EGF(50 ng/mL) for 5 minutes before being processed forimmunofluorescence staining of endogenous Fer (green) andcounterstained for F-Actin (red) and Nuclei (blue). D, thequantification of the percentage of the cell population forminglamellipodia for H1299 nontargeting, Fer KD1, and Fer KD2 cellsstimulated with EGF [mean � SEM; N ¼ 3 replicates; �, a significantdifference between nontargeting and knockdown based on pairedStudents t test (P < 0.05)].






failed to induce detectable Vav2 pY172 in Fer knockdowncells (Fig. 4C). These results are consistent with Fer pro-moting Vav2 pY downstream of EGFR, but do not addresswhether Vav2 is a direct substrate of Fer. To test this, weconducted in vitro kinase assays with purified, active Fer andpurified Vav2 N-terminal domain constructs (GST-Vav2nontargeting) with individual or combined mutations of pYsites to phenylalanine (F) residues (Fig. 4D). Interestingly,Fer induced Vav2 pY in the WT construct and this was lostin the Y172F-mutant construct (Fig. 4D). Additional muta-tions to Y142 and Y159 did not have an effect in this assay(Fig. 4D), suggesting that Fer specifically phosphorylatesVav2 Y172 in vitro and in NSCLC cells.

Fer promotes EGFR signaling to Rac1 GTPaseBecause phosphorylation of Vav2 Y172 enhances GEF

activity toward Rac (34), and Vav2 was hypophosphorylatedin Fer knockdown NSCLC cells, we tested for potentialdifferences in RacGTP levels. H1299 cells (nontargeting,KD1, KD2) were serum-starved and treated with EGF for5 minutes, and processed for immunofluorescence stainingwith a Rac1GTP-specific antibody, along with counterstain-ing of F-actin and cell nuclei. Subsequent confocal micros-copy analysis revealed the expected accumulation ofRac1GTP (green) within the F-actin (red)–rich lamellipodiain H1299 nontargeting cells treated with EGF (Fig. 5,see arrows). In contrast, Fer knockdown cells contained acytosolic pool of Rac1GTP, which may reflect the endosomalpool of this protein (35), and only limitedRac1GTP at the cellperiphery was observed (Fig. 5). These results are consistentwith EGFR signaling leading to activation of a membrane-targeted pool of Fer that phosphorylates Vav2 and enhancesGTP loading of Rac at leading edge membrane protrusions.

Fer silencing impairs NSCLC cell motility and invasionBecause Rac1 promotes lamellipodia formation and cell

motility (36), and Fer silencing impaired EGFR and Vav2signaling to Rac1, we tested whether Fer regulates NSCLCcell motility. Initial experiments using a scrape wound–induced cell motility assay, we observed a significant reduc-tion in wound area closure in Fer knockdown cells (55% and74% reductions in KD1 and KD2, respectively) comparedwith nontargeting control cells (Supplementary Fig. S3).Wefurther tested the effect of Fer knockdown on cell motilityusing an adapted EGF-embedded agarose drop chemotaxisassay (26) to allow simultaneous analysis of H1299 non-targeting and Fer knockdown cells under identical condi-tions. Cell lines were cultured with cell permeable fluores-cent dyes (nontargeting, red channel; Fer knockdown, greenchannel) before plating of an equalmixture on poly-L-lysine/fibronectin–coated coverslips containing an agarose dropenriched with EGF. Time-lapse confocal microscopy wasconducted on live cells over 18 hours, and we observed cellsaround the margin of the agarose drop begin to invadeunderneath the drop (Fig. 6A and Supplementary Video S1).Compared with nontargeting control cells, we observedfewer Fer knockdown cells migrating under the agarose dropin multiple separate fields of view (Fig. 6B; 50% reductioncompared with control; P < 0.05). Analysis of cells migratingunder the agarose drop revealed a significant reduction in cellmigration velocity with Fer silencing (Fig. 6C; 41% reduc-tion; P < 0.05). To extend on these results, we compared theinvasive properties of H1299 nontargeting and Fer knock-down cells after culturing them in Transwell chambersoverlayed with Matrigel. We observed a 75% reduction incell invasion in Fer knockdown cells compared with non-targeting control (Fig. 6D; P < 0.05). To test the role of Ferin another NSCLC cell line, we achieved stable Fer silencingin A549 cells and observed similar defects in wound healingmigration and cell invasion in Fer knockdown cells com-pared with nontargeting control (Supplementary Fig. S4).Taken together, these results are consistent with an EGFR/

NTA

C

D

BKD

NT KD1 KD2

–

170130

95

72

55

95

95

95

170130

95

72

55

95

95

95

95

95

95

95

55

+ – + EGF – + – + EGF

EGF

IB: pY

IB: Vav2

IB: Vav2

0 5 15 0 5 15 0 5 15

IB: pYIP: Vav2

IP: Vav2

IB: pY

IB: pY

34

26

34

26

55

IB: pY172-Vav2

IB: Vav2

IB: Vav2

IB: Tubulin

GST-Vav2 NT

WTY172FY142F/Y172FY142F/Y159F/Y172F

IB: Vav2

IB: pY

IB: Fer

IB: Fer

IB: Vav2

IB: Fer

− Fer + Fer

fer +/+ fer DR/DR

IP: Vav2

IP: Vav2

Figure 4. Fer promotes Vav2 phosphorylation downstream of the EGFR.A, serum-starvedH1299 nontargeting (NT) andKD1were stimulatedwithEGF (100 ng/mL) for 5 minutes before being harvested and beingsubjected to immunoprecipitation (IP) with mouse anti-Vav2 antisera.Immunoprecipitation and SCL fractions were probed with indicatedantibodies. B, serum-starved MEF cells [ferþ/þ (WT) and ferDR/DR] werestimulated with EGF (100 ng/mL) for 5 minutes before being harvestedand subjected to immunoprecipitation with mouse anti-Vav2 antisera.Immunoprecipitation and SCL fractions were probed with indicatedantibodies. C, starvedH1299 cells were stimulatedwith EGF (100 ng/mL)at multiple time points, harvested and probed with phosphotyrosine172 Vav2 antisera. D, GST cleaved N-terminal fragments of Vav2 withtyrosine to phenylalanine (YF) mutations were subjected to an in vitrokinase assay with ATP and with or without active Fer. To assessphosphorylation, sampleswere subjected to immunoblot (IB) withmouseantiphosphotyrosine antisera.

Ahn et al.





Fer/Vav2/Rac pathway that promotes directional migrationand invasion in NSCLC cells.

Fer kinase promotes NSCLC metastasis in vivoTo test the in vivo effects of Fer silencing on NSCLC

progression and metastasis, we generated subcutaneoustumor xenografts for H1299 nontargeting or Fer knock-down cells in mice lacking NK, B, and T cells (Rag2�/�:gc�/�), that were effective to model metastatic disease(37). At 5 weeks postinjection, mice were sacrificed andprimary tumors and several secondary organs were har-vested. We observed no significant differences in primarytumor mass in mice injected with nontargeting controlcells or Fer knockdown cells (Fig. 7A; N ¼ 17 mice/groupfrom 3 separate experiments). To test whether Fer silenc-ing was maintained in vivo, tumor homogenates wereprepared and analyzed for Fer expression by immunoblot.We observed reduced levels of Fer in 3 separate tumorsfrom Fer KD1 cells compared with nontargeting controltumors (Fig. 7B). Because H1299 cells were previouslyshown to form spontaneous metastases in mice (29, 30),we screened other organs (e.g., lung, liver, etc.) for GFP-expressing tumor cells. The only tissue in which weconsistently observed spontaneous GFPþ metastases waswithin the lungs (data not shown). Indeed, metastaticnodules were present within lung tissue sections fromthese mice harboring subcutaneous H1299 tumors (Fig.7C, see arrows). Upon analysis of serial lung sections fromeach mouse, we observed significantly reduced numbers oflung metastases for mice harboring Fer knockdown sub-cutaneous tumors than those with nontargeting control

tumors (Fig. 7D; P < 0.05). Together, these resultsidentify an EGFR/Fer/Vav2/Rac signaling axis that pro-motes metastasis of NSCLC, and suggests that Fer is apotential therapeutic target for metastatic NSCLC.

DiscussionAberrant EGFR signaling is known to drive a number of

NSCLC-promoting tumor growth and invasiveness (1). Inthis study, we provide evidence that Fer propagates EGFRsignals that enhance cell migration, invasion, and tumormetastasis. EGFR signaling via Fer is facilitated by high levelFer expression in human NSCLC tumors and derived celllines. In H1299 adenocarcinoma cells, EGFR signaling ledto rapid Fer activation and localization to leading edgemembrane protrusions. This membrane pool of Fer isfunctional, as stable silencing of Fer caused defects inEGF-induced lamellipodia formation, cell migration, andinvasion. We went on to define Fer substrates and pathwaysto explain these defects. Because Fer overexpression wasshown to enhance Vav2 pY and Rac-dependent lamellipodiaformation (11), we tested whether Fer knockdown cellsdisplayed defects in this signaling axis. Indeed, Vav2 wasidentified as a direct substrate of Fer, with Y172 identified asthe preferred site. Because EGF-induced Vav2 pY waspreviously shown to enhance GEF activity toward Rac(21), our study implicates Fer in participating in this keypathway at leading edge membrane protrusions. ReducedRac1 signaling to actin nucleation promoting factors (e.g.,WAVE) at these sites may explain the defects in H1299 cellmigration (reduced velocity and persistence) observed by livecell imaging. Similar results were observed with Fer silencing

Actin Rac1GTP DAPI Merge

NT

KD

1K

D2

Figure 5. Fer promotes Rac1membrane localizationdownstream of the EGFR.Representative micrographsfor serum-starved H1299nontargeting, Fer KD1, andFer KD2 cells stimulatedwith EGF (50 ng/mL) for 5 minutesbefore being processed forimmunofluorescence staining ofactive GTP-bound Rac1 (green)and counterstained for F-Actin(red) and nuclear DNA (blue).Scale bars, 20 mm.






in A549NSCLC cells, which displayed reducedmotility andcell invasion. The significance of these defects observed usingin vitro assays was tested by conducting tumor xenograftassays in mice using control and Fer knockdown H1299cells. We observed no effects of Fer silencing on growth ofsubcutaneous NSCLC tumors, but a significant defect innumbers of spontaneous lung metastases. Taken together,these findings identify Fer as a key mediator of EGFRsignaling in NSCLC that enhances tissue invasion andmetastasis pathways.EGFR-induced Fer activation and downstream signaling

is likely to involve more than the direct interaction of theseproteins, such as production of second messengers thatattract Fer and its substrates to the plasmamembrane. EGFRactivation is known to activate PLD and enhance phospha-tidic acid levels in membranemicrodomains (31). It is worthnoting that both the F-BAR/FX of Fer and the poly-basic Cdomain of Rac1 bind to phosphatidic acid, leading to Feractivation and reduced Rac1 sequestration by RhoGDIa(11), (38), (39). Fer can also phosphorylate RhoGDIadirectly to limit Rac1 binding (22). Along with enhancedVav2 pY, this may also contribute to the ability of Fer to

enhance Rac1GTP at the leading edge protrusions in NSCLCcells (this article).Fer phosphorylates multiple substrates linked to the

regulation of actin polymerization. One of these proteinsis the actin branching protein cortactin, which is phosphor-ylated by Fer in response to integrin-induced reactive oxygenspecies and F-actin depolymerization (20), (40). Defects incortactin pYmay also contribute to the defects in actin-basedmotility in Fer knockdown NSCLC cells, although noconsistent defects in cortactin pY were observed in Ferknockdown H1299 cells (data not shown). However, wedo provide evidence for a Fer-mediated Vav2/Rac pathwaythat enhances F-actin branching and cell motility via acti-vation of WAVE-Arp2/3 complex and inhibition of F-actinsevering by cofilin (41), (42). These findings suggest that Fersignals through multiple actin polymerization pathways toultimately promote cell migration and invasion.Although Fer pY was identified in proteomic studies of

lung cancer (14), there have been limited studies of Ferfunction in lung cancer. In addition to our findingslinking Fer to NSCLC cell invasion and tumor metastasis,it will be important to further characterize potential roles

16

14

12

10

8

6

4

2

0

30

25

20

15

10

5

0

T~11 h T~14.5 h T~18 h

T~7.5 hT~3.5 hT = 0

WT

Fer KD

60

50

40

30

20

10

0

A

B C D

Mig

rating c

ells

/fie

ld

Inva

ded c

ells

/are

a

Velo

city,

µm

/h

NT KD1 NT KD1 NT KD1

Figure 6. Stable silencing of Ferimpairs motility and invasivenessof H1299 cells. A, representativetime-lapse images of H1299nontargeting (NT; red) and KD1(green) cells migrating toward andunder an agarose drop enrichedwith EGF (20 mg/mL) over thecourse of 18 hours. B and C, thequantification of the percentage ofthe cell population migrating (B)and the migratory velocity (C) forH1299 nontargeting and Ferknockdown cells under an EGFenriched agarose drop [mean �SD; N ¼ 10; �, a significantdifference between nontargetingand knockdown based on pairedStudents t test (P < 0.05)]. D,invasion assays were conductedfor H1299 nontargeting and Ferknockdown cells as described inMaterials and Methods. Graphdepicts average numbers ofinvading cells per field [mean�SD;N ¼ 3 experimental replicatesconducted in triplicate; �, asignificant difference betweennontargeting and knockdownbased on paired Students t test(P < 0.05)].

Ahn et al.





of Fer in regulating key transcription factors linked tocancer progression and resistance to potential cancertherapies. Recently, an insertional mutagenesis screen ledto the discovery that Fer overexpression confers resistanceto quinacrine-induced cell death via enhancing EGFR

signaling to NF-kB (5). Thus, it will be important totest whether Fer knockdown NSCLC cells display defectsin NF-kB target genes that may contribute to NSCLCmetastasis. These include NF-kB–induced cell adhesionproteins linked to chemotherapy resistance and lungcancer metastasis (43, 44). Another recent study identifieda Fer/STAT3 pathway that represses expression of lamininbinding glycans and glycosyltransferases (e.g., LARGE)that inhibit the motility of carcinomas (45). These glycansregulate airway wound-healing responses (46). Loss oflaminin binding in lung cancers was recently shown toinvolve downregulation of LARGE, and migration wasinhibited following ectopic expression of LARGE (47). Infuture studies, it will be worth testing whether Fer reg-ulates airway wound-healing responses or NSCLC metas-tasis via the STAT3 pathway. Although this study wasunder review, another group reported that Fer overexpres-sion is associated with poor prognosis in patients withNSCLC that had undergone surgery, and this associationwas independent of tumor stage, treatment regimen, orEGFR status (48). Together with our results implicatingFer in promoting NSCLC metastasis, this lends furthersupport to pursuing Fer as a therapeutic target in NSCLC.Fer is likely to promote the progression or metastasis in

other cancer types as well as lung cancer. Recently, Fer wasshown to promote prostate cancer progression via an IL-6/Fer/STAT3 pathway (16). Increased Fer phosphorylationhas also been observed in metastatic hepatocellular carcino-mas (15) and highly metastatic breast cancers (17). BecauseFer-deficient mice have no overt phenotypes (19), it is likelyto be a drugable target. It is also promising that severalinhibitors of Fes kinase were recently identified and used toinhibit osteoclast differentiation (49). On the basis of theirhigh degree of homology, it is likely that at least a subset ofFes inhibitors are also Fer inhibitors, and this will be worthtesting in future using preclinical models of metastatic lungcancer and other relevant cancer models. In conclusion, Feris an emerging therapeutic target to develop additionalantimetastasis treatment regimens.

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

Authors' ContributionsConception and design: P. Truesdell, C. Kadish, A.H. Boag, A.W.B. CraigDevelopment of methodology: P. Truesdell, C. Kadish, A.H. BoagAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J. Ahn, P. Truesdell, J. Meens, C. Kadish, X. YangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): J. Ahn, C. Kadish, A.H. Boag, A.W.B. CraigWriting, review, and/or revision of themanuscript: J. Ahn, P. Truesdell, C. Kadish,X. Yang, A.H. Boag, A.W.B. CraigAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J. MeensStudy supervision: A.W.B. Craig

AcknowledgmentsThe authors thank Buday Laszlo for providing Vav2 constructs and Stephanie

Everingham for technical assistance. The authors also thank Jeff Mewburn and MattGordon (Queen's flow cytometry and confocal imaging facility) for help with confocalimaging, and Lee Boudreau (Queen's Laboratory for Molecular Pathology) for helpoptimizing immunohistochemical staining.

A

B

C

D

95

55

IB: Fer

IB: Actin

1.2

1.0

0.8

0.6

0.4

0.2

0.0

60

50

40

30

20

10

0

NT

NT

T1 T2 T3 T1 T2 T3

KD1

KD1

KD2

NT KD1 KD2

NT KD1 KD2

Tum

or

mass (

g)

Lung m

ets

per

mouse

Figure 7. Fer promotes NSCLC metastasis in mice. A, the graph depictsprimary tumor mass (mean � SEM) from subcutaneous xenograftedH1299 nontargeting and Fer knockdown cells in male Rag2�/�:gc�/�

mice (N¼ 17 mice/group from 3 experimental replicates). B, persistenceof shRNA silencing in H1299 cells over the course of xenograftexperiments was measured by immunoblot with Fer antisera. C,representative micrograph of hematoxylin and eosin–stained lungsections from mice with subcutaneous H1299 nontargeting and KD1tumors (arrows indicate positions of metastases). D, the graph depictsquantification of spontaneous lung metastasis per mouse in lungsections from mice xenografted with H1299 nontargeting and Ferknockdown cells in male Rag2�/�:gc �/� mice [mean � SEM; N ¼ 3replicates; �, a significant difference between nontargeting andknockdown based on paired Students t test (P < 0.05)].






Grant SupportThis research was supported by operating grants from Canadian Cancer Society

Research Institute (#020217) and Canadian Institutes for Health Research(MOP119562) to A.W.B. Craig. J. Ahn was supported by the Terry FoxFoundation Multidisciplinary Cancer Research training program at Queen'sUniversity.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received January 7, 2013; revised April 19, 2013; accepted April 22, 2013;published OnlineFirst May 22, 2013.

References1. daCunhaSantosG, Shepherd FA, TsaoMS. EGFRmutations and lung

cancer. Ann Rev Pathol 2011;6:49–69.2. Politi K, ZakowskiMF, FanPD,Schonfeld EA, PaoW,VarmusHE. Lung

adenocarcinomas induced in mice by mutant EGF receptors found inhuman lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev 2006;20:1496–510.

3. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al.Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib isassociated with a second mutation in the EGFR kinase domain. PLoSMed 2005;2:e73.

4. Kim L, Wong TW. The cytoplasmic tyrosine kinase FER is associatedwith the catenin-like substrate pp120 and is activated by growthfactors. Mol Cell Biol 1995;15:4553–61.

5. Guo C, Stark GR. FER tyrosine kinase (FER) overexpression mediatesresistance to quinacrine through EGF-dependent activation of NF-kappaB. Proc Natl Acad Sci U S A 2011;108:7968–73.

6. ChauCH, FiggWD. New tricks from an old drug: a role for quinacrine inanti-cancer therapy? Cell Cycle 2009;8:4024–5.

7. Suetsugu S, Gautreau A. Synergistic BAR-NPF interactions in actin-driven membrane remodeling. Trends Cell Biol 2012;22:141–50.

8. Craig AW. FES/FER kinase signaling in hematopoietic cells and leu-kemias. Front Biosci 2012;17:861–75.

9. McPherson VA, Everingham S, Karisch R, Smith JA, Udell CM, ZhengJ, et al. Contributions of F-BAR and SH2 domains of Fes proteintyrosine kinase for coupling to the FcepsilonRI pathway in mast cells.Mol Cell Biol 2009;29:389–401.

10. Tsujita K, Suetsugu S, Sasaki N, Furutani M, Oikawa T, Takenawa T.Coordination between the actin cytoskeleton and membrane defor-mation by a novel membrane tubulation domain of PCH proteins isinvolved in endocytosis. J Cell Biol 2006;172:269–79.

11. Itoh T, Hasegawa J, Tsujita K, Kanaho Y, Takenawa T. The tyrosinekinase Fer is a downstream target of the PLD-PA pathway thatregulates cell migration. Sci Signal 2009;2:ra52.

12. Di FulvioM, Frondorf K,HenkelsKM,GrunwaldWCJr, Cool D,Gomez-Cambronero J. Phospholipase D2 (PLD2) shortens the time requiredfor myeloid leukemic cell differentiation: mechanism of action. J BiolChem 2012;287:393–407.

13. FilippakopoulosP, KoflerM,HantschelO,GishGD,Grebien F, SalahE,et al. Structural coupling of SH2-kinase domains links Fes and Ablsubstrate recognition and kinase activation. Cell 2008;134:793–803.

14. Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, et al. Globalsurvey of phosphotyrosine signaling identifies oncogenic kinases inlung cancer. Cell 2007;131:1190–203.

15. Li H, Ren Z, Kang X, Possemato A, Yu J, HaackH, et al. Identification oftyrosine-phosphorylated proteins associated with metastasis andfunctional analysis of FER in human hepatocellular carcinoma cells.BMC Cancer 2009;9:366.

16. Zoubeidi A, Rocha J, Zouanat FZ, Hamel L, Scarlata E, Aprikian AG,et al. The Fer tyrosine kinase cooperates with interleukin-6 to activatesignal transducer and activator of transcription 3 and promote humanprostate cancer cell growth. Mol Cancer Res 2009;7:142–55.

17. Albeck JG, Brugge JS. Uncovering a tumor suppressor for triple-negative breast cancers. Cell 2011;144:638–40.

18. Kim L, Wong TW. Growth factor-dependent phosphorylation of theactin-binding protein cortactin ismediated by the cytoplasmic tyrosinekinase FER. J Biol Chem 1998;273:23542–8.

19. Craig AW, Zirngibl R, Williams K, Cole LA, Greer PA. Mice devoidof fer protein-tyrosine kinase activity are viable and fertile butdisplay reduced cortactin phosphorylation. Mol Cell Biol 2001;21:603–13.

20. Sangrar W, Gao Y, Scott M, Truesdell P, Greer PA. Fer-mediatedcortactin phosphorylation is associated with efficient fibroblast migra-tion and is dependent on reactive oxygen species generation duringintegrin-mediated cell adhesion. Mol Cell Biol 2007;27:6140–52.

21. TamasP,Solti Z, BauerP, Ill�esA, Sipeki S, Bauer A, et al.Mechanismofepidermal growth factor regulation of Vav2, a guanine nucleotideexchange factor for Rac. J Biol Chem 2003;278:5163–71.

22. Fei F, Kweon SM, Haataja L, De Sepulveda P, Groffen J, HeisterkampN. The Fer tyrosine kinase regulates interactions of Rho GDP-Disso-ciation Inhibitor alpha with the small GTPase Rac. BMC Biochem2010;11:48.

23. Boulter E,Garcia-MataR,GuilluyC, DubashA, RossiG, BrennwaldPJ,et al. Regulation of Rho GTPase crosstalk, degradation and activity byRhoGDI1. Nat Cell Biol 2010;12:477–83.

24. Haigh J, McVeigh J, Greer P. The fps/fes tyrosine kinase is expressedin myeloid, vascular endothelial, epithelial, and neuronal cells and islocalized in the trans-golgi network. Cell Growth Differ 1996;7:931–44.

25. Zhou Z, Hao Y, Liu N, Raptis L, Tsao MS, Yang X. TAZ is a noveloncogene in non–small cell lung cancer. Oncogene 2011;30:2181–6.

26. Wiggins H, Rappoport J. An agarose spot assay for chemotacticinvasion. BioTechniques 2010;48:121–4.

27. Le Devedec SE, van Roosmalen W, Maria N, Grimbergen M, Pont C,Lalai R, et al. An improved model to study tumor cell autonomousmetastasis programs using MTLn3 cells and the Rag2(�/�) gammac(�/�) mouse. Clin Exp Met 2009;26:673–84.

28. Finn RS, Press MF, Dering J, Arbushites M, Koehler M, Oliva C, et al.Estrogen receptor, progesterone receptor, human epidermal growthfactor receptor 2 (HER2), andepidermal growth factor receptor expres-sion and benefit from lapatinib in a randomized trial of paclitaxel withlapatinib or placebo as first-line treatment in HER2-negative orunknown metastatic breast cancer. J Clin Oncol 2009;27:3908–15.

29. Huang MS, Wang TJ, Liang CL, Huang HM, Yang IC, Yi-Jan H, et al.Establishment of fluorescent lung carcinomametastasis model and itsreal-time microscopic detection in SCID mice. Clin Exp Met 2002;19:359–68.

30. Onn A, Isobe T, Itasaka S, Wu W, O'Reilly MS, Ki Hong W, et al.Development of an orthotopic model to study the biology and therapyof primary human lung cancer in nude mice. Clin Cancer Res 2003;9:5532–9.

31. Mazie AR, Spix JK, Block ER, Achebe HB, Klarlund JK. Epithelial cellmotility is triggered by activation of the EGF receptor through phos-phatidic acid signaling. J Cell Sci 2006;119(Pt 8):1645–54.

32. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The smallGTP-binding protein rac regulates growth factor-induced membraneruffling. Cell 1992;70:401–10.

33. Tybulewicz VL. Vav-family proteins in T-cell signalling. Curr OpinImmunol 2005;17:267–74.

34. Aghazadeh B, Lowry WE, Huang XY, Rosen MK. Structural basis forrelief of autoinhibition of the Dbl homology domain of proto-oncogeneVav by tyrosine phosphorylation. Cell 2000;102:625–33.

35. Palamidessi A, Frittoli E, Garre M, Faretta M, Mione M, Testa I, et al.Endocytic trafficking of Rac is required for the spatial restriction ofsignaling in cell migration. Cell 2008;134:135–47.

36. Heasman SJ, Ridley AJ. Mammalian Rho GTPases: new insights intotheir functions from in vivo studies. Nat Rev 2008;9:690–701.

37. Chander H, Truesdell P, Meens J, Craig AW. Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion andmetastasis. Oncogene. 2012 Jul 23:[Epub ahead of print].

38. Chae YC, Kim JH, Kim KL, Kim HW, Lee HY, Heo WD, et al. Phos-pholipase D activity regulates integrin-mediated cell spreading and

Ahn et al.





migration by inducing GTP-Rac translocation to the plasma mem-brane. Mol Biol Cell 2008;19:3111–23.

39. AbramoviciH,MojtabaieP,ParksRJ, ZhongXP,KoretzkyGA, TophamMK, et al. Diacylglycerol kinase zeta regulates actin cytoskeletonreorganization through dissociation of Rac1 from RhoGDI. Mol. Biol.Cell 2009;20:2049–59.

40. Fan L, Di Ciano-Oliveira C, Weed SA, Craig AW, Greer PA, RotsteinOD, et al. Actin depolymerization-induced tyrosine phosphorylationof cortactin: the role of Fer kinase. Biochem J 2004;380(Pt 2):581–91.

41. Chan AY, Coniglio SJ, Chuang YY, Michaelson D, Knaus UG, PhilipsMR, et al. Roles of the Rac1 and Rac3 GTPases in human tumor cellinvasion. Oncogene 2005;24:7821–9.

42. Suetsugu S, Kurisu S, Oikawa T, Yamazaki D, Oda A, Takenawa T.Optimization of WAVE2 complex-induced actin polymerization bymembrane-bound IRSp53, PIP(3), and Rac. J Cell Biol 2006;173:571–85.

43. Schmidmaier R, Baumann P. ANTI-ADHESION evolves to a prom-ising therapeutic concept in oncology. Curr Med Chem 2008;15:978–90.

44. Liang CH, Chiu SY, Hsu IL, Wu YY, Tsai YT, Ke JY, et al. a-Catulindrives metastasis by activating ILK and driving an avb3 integrinsignaling axis. Cancer Res 2013;73:428–38.

45. Yoneyama T, Angata K, Bao X, Courtneidge S, Chanda SK, FukudaM.Fer kinase regulates cell migration through alpha-dystroglycan glyco-sylation. Mol Biol Cell 2012;23:771–80.

46. White SR, Wojcik KR, Gruenert D, Sun S, Dorscheid DR. Airwayepithelial cell wound repair mediated by alpha-dystroglycan. Am JResp Cell Mol Biol 2001;24:179–86.

47. de Bernabe DB, Inamori K, Yoshida-Moriguchi T, Weydert CJ, HarperHA, Willer T, et al. Loss of alpha-dystroglycan laminin binding inepithelium-derived cancers is caused by silencing of LARGE. J BiolChem 2009;284:11279–84.

48. Kawakami M, Morita S, Sunohara M, Amano Y, Ishikawa R, WatanabeK, et al. FER overexpression is associated with poor postoperativeprognosis and cancer-cell survival in non–small cell lung cancer. Int JClin Exp Pathol 2013;6:598–612.

49. Hellwig S,Miduturu CV, Kanda S, Zhang J, Filippakopoulos P, Salah E,et al. Small-molecule inhibitors of the c-Fes protein-tyrosine kinase.Chem Biol 2012;19:529–40.






2013;11:952-963. Published OnlineFirst May 22, 2013.Mol Cancer Res Joseph Ahn, Peter Truesdell, Jalna Meens, et al. Invasion and Tumor MetastasisFer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma Cell

Updated version

10.1158/1541-7786.MCR-13-0003-Tdoi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2013/05/22/1541-7786.MCR-13-0003-T.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/11/8/952.full#ref-list-1

This article cites 48 articles, 21 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/11/8/952.full#related-urls

This article has been cited by 5 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/11/8/952To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-13-0003-T

http://mcr.aacrjournals.org/content/suppl/2013/05/22/1541-7786.MCR-13-0003-T.DC1

http://mcr.aacrjournals.org/content/11/8/952.full#ref-list-1

http://mcr.aacrjournals.org/content/11/8/952.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/11/8/952


Fer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma...

Documents

Transcript of Fer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma...