The Transmembrane Adaptor Cbp/PAG1 Controls the Malignant ... · ing protein (Cbp), which is also...

Signaling and Regulation

The Transmembrane Adaptor Cbp/PAG1 Controls theMalignant Potential of Human Non–Small Cell LungCancers That Have c-Src Upregulation

Takashi Kanou1,2, Chitose Oneyama1, Kunimitsu Kawahara3, Akira Okimura3, Mitsunori Ohta4,Naoki Ikeda4, Yasushi Shintani2, Meinoshin Okumura2, and Masato Okada1

AbstractThe tyrosine kinase c-Src is upregulated in various human cancers, although the precise regulatory mechanism

underlying this upregulation is unclear. We previously reported that a transmembrane adaptor Csk-bindingprotein (Cbp; PAG1) plays an important role in controlling the cell transformation that is induced by theactivation of c-Src. To elucidate the in vivo role of Cbp, we examined the function of Cbp in lung cancer cell linesand tissues. In this study, we found that Cbp was markedly downregulated in human non–small cell lung cancer(NSCLC) cells. The ectopic expression of Cbp suppressed the anchorage-independent growth of the NSCLC celllines (A549 and Lu99) that had upregulated c-Src, whereas the Cbp expression had little effect on other NSCLCcell lines (PC9 and Lu65) that express normal levels of c-Src. The expression of Cbp suppressed the kinase activityof c-Src in A549 cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (Csk), to lipid rafts. Thetreatment with Src inhibitors, such as PP2, dasatinib, and saracatinib, also suppressed the growth of A549 cells.Furthermore, Cbp expression attenuated the ability of A549 cells to form tumors in nudemice, invade in vitro, andmetastasize in vivo. In addition, we found a significant inverse correlation between the level of Cbp expression andthe extent of lymph node metastasis in human lung cancers. These results indicate that Cbp is required for theCsk-mediated inactivation of c-Src and may control the promotion of malignancy in NSCLC tumors that arecharacterized by c-Src upregulation. Mol Cancer Res; 9(1); 103–14. �2010 AACR.

Introduction

Lung cancer is the leading cause of cancer death amongelderly men and is the second leading cause of cancer deathamong elderly women in Japan. From 2000 to 2007, themortality rate due to lung cancer among the elderly popula-tion was 272.41/100,000 and 62.45/100,000 in men andwomen, respectively (1). Previous reports have demon-strated that lung cancer development involves both envir-onmental and genetic factors (2). Over the past fewdecades, the methods for the diagnosis and treatmentof lung cancer have improved. Recently, targeted mole-cular therapy in the treatment for non–small cell lungcancer (NSCLC) has received significant attention.Although epidermal growth factor receptor (EGFR) inhi-

bitors have been successfully developed, NSCLC patientsstill have unfavorable prognoses (3). Therefore, the devel-opment of novel therapeutic strategies for the treatment ofNSCLC is urgently needed.c-Src was the first identified proto-oncogene product, and

extensive research has shown that c-Src plays crucial roles invarious intracellular signaling pathways that are implicatedin cell growth, adhesion, and migration (4, 5). Severalreports have shown that the overexpression or hyperactivityof c-Src is common in human lung cancers, especially inadenocarcinomas (6–10); however, the C-SRC gene is rarelymutated in human cancers, and the involvement of c-Srcprotein expression or enzymatic activation in the etiology ofhuman cancer remains obscure (11). The kinase activity ofc-Src is tightly regulated by several factors. A critical proteinthat negatively regulates c-Src activation is C-terminal Srckinase (Csk), which phosphorylates the negative regulatorytyrosine residue of c-Src (Tyr529; refs. 12, 13). Csk expres-sion has been reported to be reduced in hepatocellularcarcinoma in comparison with normal tissue, and thisalteration may be correlated with c-Src activation (14).These observations suggest that Csk has a tumor-suppres-sive activity and that its reduced expression may facilitatethe malignant cell behavior that is promoted by c-Src;however, the mechanisms underlying the downregulationof Csk in cancer cells are unclear.

Authors' Affiliations: 1Department of Oncogene Research, ResearchInstitute for Microbial Diseases; 2Department of General Thoracic Surgery,Osaka University Graduate School of Medicine, Osaka University, Suita;Departments of 3Pathology and 4Thoracic Surgery, Osaka PrefecturalMedical Center for Respiratory and Allergic Diseases, Osaka, Japan

Corresponding Author: Masato Okada, Department of OncogeneResearch, Research Institute for Microbial Diseases, Osaka University.3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8297;Fax: 81-6-6879-8298. E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-10-0340

�2010 American Association for Cancer Research.

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Csk is a cytoplasmic protein (15), and, thus, it has beenproposed that Csk requires an appropriate membraneadaptor protein to access the membrane-anchored c-Src.We and another group previously identified the Csk-bind-ing protein (Cbp), which is also known as PAG1 (16), as aspecific membrane adaptor of Csk (17). Cbp exclusivelylocalizes in lipid rafts and is involved in regulating theactivity of Src family kinases (SFK) through the recruitmentof Csk to lipid rafts where SFKs accumulate (17, 18).Several recent studies have implicated Cbp in cancer pro-gression. In colon cancer patients, Cbp expression wassignificantly downregulated in some tumors in comparisonwith adjacent normal tissues (19), suggesting that Cbp has atumor suppressor function for some types of cancer; how-ever, other studies have shown that Cbp is overexpressed inrenal cell carcinoma (RCC) and serves as a positive regulatorof tumor malignancy in RCC patients (20). Thus, it hasbeen suggested that Cbp plays distinct roles in controllingtumor progression depending on the cancer type.In this study, we investigated the function of Cbp in

several human lung cancer cell lines and tissues to addressthe precise in vivo role of Cbp. We show that Cbp expres-sion is significantly downregulated in NSCLC cells and thatthe overexpression of Cbp in NSCLC cells, in which c-Src ishighly activated, results in the suppression of tumor growthby promoting Csk-mediated c-Src inactivation. In addition,we show that Cbp inhibits both the in vitro invasiveness andthe in vivometastasis of a NSCLC cell line, and that there isa significant correlation between the Cbp expression leveland lymph node metastasis. These findings suggest thatCbp can suppress the malignant potentials of NSCLC byattenuating the c-Src–mediated tumorigenic pathway.

Materials and Methods

Cell lines and reagentsThe human NSCLC cell lines (A549, PC9, PC10, Lu65,

Lu99, Lc4a, Lc3, Lc2, Lc23, Lc28, EBC-1, and RERF-LC-MS) were kindly provided by Dr. K. Kodama and Dr. M.Yutsudo. Normal human lung cells (13Lu, 39Lu, and888Lu) were obtained from the European Collection ofCell Cultures (ECACC). A549 cells were cultured inDulbecco's modified Eagle's medium (DMEM) and allother cancer cells were cultured in RPMI1640 (Nacalai).In both cases, the medium was supplemented with 10%FBS. Normal lung cells were maintained in Eagle's mini-mum essential medium (EMEM; DS Pharma Biomedical)that was supplemented with 10% FBS and a 1% nones-sential amino acids solution. PP2 (Calbiochem), Dasatinib(BioVision), and Saracatinib (BioVision) were solubilized inDMSO to obtain 10 to 20mmol/L of stock solutions. Stocksolutions were stored at �20�C and diluted in DMEM foreach experiment.

Cancer specimensNSCLC specimens and paired noncancerous tissues were

snap-frozen in liquid nitrogen immediately after resection.The resected lung tissues were divided upon visual inspec-

tion into tumor (T) and nontumor (N) regions, which werethen histologically confirmed. To compare the Cbp mRNAexpression level in each NSCLC case, we used formalin-fixed, paraffin-embedded (FFPE) samples of human pri-mary lung adenocarcinoma. Sections (10 mm) were cut fromeach block and placed in a 1.5-mL test tube. Total RNA wasthen extracted using the High Pure FFPE RNA Micro Kit(Roche). This study was approved by the ethical reviewboard of the Osaka Prefectural Medical Center for Respira-tory and Allergic Diseases.

Retroviral gene transfer and expressionAll gene transfer and expression protocols were performed

using previously described methods (21). Briefly, thecDNAs that encode wild-type human Cbp and its mutant,which had a substitution of Tyr314 to Phe (Y314F), weresubcloned into the retroviral vectors pCX4puro andpCX4bleo, respectively. All constructs were generated usinga PCR-based procedure. The retroviral vectors were trans-fected into Plat-E, which is an ecotropic murine leukemiavirus-packaging cell line, using Fugene6 (Boehringer)according to the manufacturer's directions. To transferthe gene into human cells, we used pGP þ pE-Ampho(Takara Bio), which is an amphotropic retrovirus-packagingconstruct. Infected cell populations were selected withpuromycin or bleomycin, and the mixed cell populationswere subjected to each assay that is described in this study.

AntibodiesWe generated an anti-human Cbp antibody by immuniz-

ing rabbits with a GST-Cbp (residues 331–430) fusionprotein. We acquired the anti-Src antibody (Ab-1) fromCalbiochem; the anti-Src pY418, anti-Src pY529, and anti-FAK pY397 antibodies from Biosource; the anti-FAK, anti-Csk, and anti-b-tubulin antibodies from Santa Cruz Bio-technology; the anti-Caveolin antibody from BD Transduc-tion Laboratories; the anti-Transferrin receptor fromZymed; and the anti-Akt, anti-Akt pS473, anti-Erk, andanti-Erk pT202/pY204 antibodies from Cell Signaling.Horseradish peroxidase (HRP)-conjugated anti-mouse oranti-rabbit IgG (Zymed) was used as the secondary antibody.

Western blot and immunoprecipitationCells were washed and lysed in ODG buffer (50 mmol/L

of Tris-HCl, pH 7.4, 1 mmol/L of EDTA, 0.25 mol/L ofNaCl, 20 mmol/L of NaF, 1 mmol/L of Na3VO4, 1%Nonidet P-40, 2% Octyl-D-glucoside, 5 mmol/L of b-mer-captoethanol, 0.1% aprotinin, 0.1% leupeptin, 1 mmol/Lof PMSF, and 5% glycerol). Equal amounts of total proteinwere separated using SDS-PAGE and transferred ontonitrocellulose membranes. The membranes were blockedand probed with primary antibodies, followed by incuba-tion with HRP-conjugated secondary antibodies. Forimmunoprecipitation, the precleared lysate (1-mg protein)was incubated with the indicated antibodies for 1 hour andprotein G-Sepharose (GE Healthcare) for 3 hours at 4�C.The immunoprecipitates were washed with ODG bufferand analyzed by Western blotting.

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In vitro proliferation assayIn vitro anchorage-dependent growth was assessed by

using an assay that is based on the cleavage of the tetra-zolium salt WST-1 into formazan by cellular mitochondrialdehydrogenases (Roche). Each cancer cell line was plated in96-well microplates at a density of 1,000 cells per well. Cellswere allowed to grow for 72 hours, and 10 mL of WST-1reagent was applied to each well followed by 1-hour incu-bation. The absorbance at 450 nm was read with a micro-plate reader, and the average of 3 wells was determined.Each experiment was repeated 3 times.

Soft agar assaySingle-cell suspensions of 1 � 104 cells in 1.5 mL of

DMEM that contained 10% FBS and 0.36% agar wereplated onto a layer of 2.5 mL of the same medium thatcontained 0.7% agar in 6-well culture dishes. Viable colo-nies were stained with MTT 10 to 14 days after plating.Colonies larger than approximately 0.1 mm in diameterwere scored.

Invasion assayInvasion assays were conducted using a BioCoat Matri-

gel Invasion Chamber (BD Bioscience) according to themanufacturer's instructions. A cell suspension (5 � 104

cells) in serum-free medium was added to the inserts, andeach insert was placed in the lower chamber, whichcontained a culture medium that was supplemented with10% FBS. After a 24-hour incubation, invasiveness wasevaluated by staining the cells that migrated through theextracellular matrix layer.

Tumor growth and metastasis in nude miceFemale BALB/c athymic nude mice (4 weeks old) were

purchased from Japan CLEA. Cancer cells were grown,harvested, washed with PBS, and resuspended in serum-free DMEM. For the analysis of tumor growth, 1 � 106

cancer cells (0.2 mL) were subcutaneously inoculated intonude mice. Tumor sizes were monitored weekly, andtumor volume was calculated on the basis of the followingformula: volume ¼ 0.5 � length � (width)2. For the lungmetastasis model, 5 � 106 cells were intravenouslyinjected into mice via the tail vein. After 1 month, micewere euthanized using diethyl ether, and the number ofmetastatic nodules was determined in lungs that werefixed in Bouin's solution (22). Mice were handled andmaintained according to the Osaka University guidelinesfor animal experimentation.

Preparation of detergent-resistant membrane domainsThe separation of detergent-resistant membrane

domains (DRM) was performed as previously described(23). Briefly, cells were lysed in a buffer that contained0.25% Triton X-100, and the lysate was placed at thebottom of a discontinuous sucrose gradient (40%/35%/5%). After centrifugation at 40,000 rpm for 6 hours,fractions were collected from the top of the gradient, andaliquots were analyzed by Western blotting. The separa-

tion of DRMs and non-DRMs was confirmed by detect-ing caveolin-1 and transferrin receptor (TFR) as markersfor DRMs and non-DRMs, respectively.

Real-time quantitative PCRTotal RNAwas extracted from cancer cell lines and frozen

tissue samples using Sepasol (Nacalai). The High PureFFPE RNA Micro Kit (Roche) was used to extract totalRNA from the FFPE tissue samples. The Transcriptor FirstStrand cDNA Synthesis Kit (Roche) was used to reversetranscribe all samples for qPCR. The mRNA levels of Cbp(ID: 179693), matrix metalloproteinase (MMP)-2/9 (ID:234422, 957562), and glyceraldehydes-3-phosphate dehy-drogenase (GAPDH; ID: 99999915) were quantified usingTaqMan gene expression assays (Applied Biosystems). PCRwas performed using an Applied Biosystems 7900HT FastReal-time PCR System and Sequence Detection System,Software v2.2.1. The expression of the target genes wasnormalized to that of GAPDH.

ImmunohistochemistryCancer cells were cultured on fibronectin-coated plates

and fixed with 4% PFA (paraformaldehyde) for 15 minutesat room temperature. After blocking with 1% BSA (bovineserum albumin), the cells were incubated with the primaryantibodies overnight at 4�C. The cells were then incubatedwith secondary antibodies for 1 hour at room temperature,and coverslips were mounted on the glass slides. Alexa Fluor488 phalloidin (Invitrogen) was used to stain the F-actin.Images were photographed using a fluorescent confocalmicroscope (Olympus, FV-1000). Immunohistochemistrywas performed on FFPE tumor samples. Sections fromcancerous and corresponding normal tissue were stainedwith an anti-Cbp antibody or a neutralized antibody. Thedetection of immunoreactivity was performed using aniVIEW DAB Detection Kit on Benchmark (Ventana Med-ical Systems).

Wound-healing assayA549 cells were seeded into 6-well plates and incubated in

a medium that was supplemented with 10% FBS until thecells reached confluence. The confluent monolayer cultureswere then scratched with a pipette tip. After incubation for24 hours, the wounded areas were examined with a lightmicroscope and photographed. The wound gap distanceswere then measured. All experiments were performed intriplicate.

Results

Cbp expression is downregulated in NSCLC cellsAs an initial experiment, we compared, by Western

blotting, the expression levels of Cbp and c-Src proteinsin normal lung cells and in a variety of NSCLC cells. Asshown in Figure 1A, the expression of Cbp was markedlydownregulated in NSCLC cells in comparison to those innormal lung cells. In contrast, the expression of c-Src varieddepending on cell types; higher levels of c-Src expression

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were detected in A549 and Lu99 cells. These suggest thatthe expression level of Cbp is not necessarily correlated withthe level of c-Src. However, the majority of NSCLC cellsexhibited a higher ratio of the signal intensity of pY418 (anautoactivation site) to that of total Src protein (Fig. 1A, topright) than normal cells. In addition, the phosphorylationstatus of pY529 (a negative regulatory site that is phos-phorylated by Csk) in several NSCLC cells was lower than

that in normal cells (Fig. 1A, bottom right). These observa-tions demonstrate that c-Src is widely activated in NSCLCcells, although the expression of Csk in NSCLC cells issimilar to that in normal lung cells (Fig. 1A, left).To examine whether Cbp expression was altered in

human cancer tissues, the expression of Cbp mRNA infrozen samples from 4 pairs of NSCLC tissues and theirmatched noncancerous control tissues was analyzed by

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Figure 1.Cbp expression is downregulated in NSCLC cells. A, the protein expressions of Cbp, c-Src, phosphorylated c-Src (pY418 and pY529), and Csk in theindicated cell lines were assessed by Western blotting. Tubulin was used as the loading control (left). The ratio of signal intensity between total Src andpY418 (relative activity of Src; top right graph) or pY529 (relative phosphorylation status of Src pY529; bottom right graph) is shown. B, quantitative real-time(RT) PCR was performed using total RNA that was isolated from fresh frozen tissue samples. The relative levels of Cbp mRNA, which were normalizedto GAPDH mRNA expression, in lung tumors (black bars) and matched normal tissues (white bars) are shown. C, immunohistochemical staining wasperformed for Cbp in lung tissues. Sections of cancerous tissue and normal tissue were stained with anti-Cbp and eosin and observed using lightmicroscopy at 400� magnification.

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quantitative real-time PCR. The expression of CbpmRNA was significantly downregulated in all humanlung cancer samples in comparison to that in theiradjacent normal controls (Fig. 1B). Immunohistochem-istry also indicated that the tumor cells very weaklyexpressed the Cbp protein, although the Cbp proteinwas readily detectable in the normal bronchiolar epithelia(Fig. 1C). These results indicate that Cbp expression isgenerally downregulated in NSCLC cells as well as inNSCLC tissues.

The ectopic expression of Cbp inhibits anchorage-independent growth and tumor growth of NSCLC cellsin which c-Src is upregulatedTo investigate the role of Cbp downregulation in

NSCLC cells, we ectopically expressed Cbp in A549,Lu99, PC9, and Lu65 cells; A549 and Lu99 cells havethe highest levels of c-Src upregulation, whereas PC9 andLu65 cells express normal levels of c-Src. The expressionlevels of Cbp in the transfected cells substantially exceededthose in the normal lung cells (Fig. 2A); however, because

Figure 2. The ectopic expressionof Cbp inhibits the activation of c-Src and the proliferative potentialof lung cancer cells. A, total celllysates from control and Cbp-expressing A549, Lu99, PC9, andLu65 cells and normal lung cellswere analyzed byWestern blottingwith the indicated antibodies. Thedepicted values reflect the foldchanges in signal intensity of thetotal c-Src and pY418 from 2independent experiments, whichare normalized to the signalintensities of A549 mock cells. B,anchorage-dependent growthwas assessed by an in vitroproliferation assay using WST-1.The relative absorbance (mean �SE) in Cbp-expressing cells incomparison to that in mock cellswas obtained from 3 independentexperiments. C, anchorage-independent growth wasexamined by a colony formationassay in soft agar. Colonies werestained with MTT 14 days afterplating. The colony numbers percm2 (mean � SE) that wereobtained from 3 independentexperiments are shown. Student'st tests: *, P < 0.05. D, the ability oftumor formation was examined ina mouse xenograft model. Cells (1� 106) were subcutaneouslyinoculated into nude mice. Thetumor volumes (mm3; mean � SE)that were obtained from 4 mice ineach group are plotted as afunction of days after inoculation.*, P < 0.05.

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the A549 and Lu99 cells exhibited a marked (>10-fold)upregulation of the c-Src protein in comparison to normallung cells (Fig. 1A), the ratio of overexpressed Cbp to c-Srcin these cells appeared to be at a comparable level to that innormal cells. Western blot analysis revealed that thephosphorylation of c-Src Tyr418 in all 4 cell lines wassuppressed by the overexpression of Cbp; although, thelevels of total c-Src and pY418 in PC9 and Lu65 cells weremuch lower than those in A549 cells (Fig. 2A). Theseresults indicate that the kinase activity of c-Src is inhibitedby the expression of Cbp in these cells.We next examined the effects of Cbp expression on the

growth ability of these NSCLC cells. In A549 and Lu99cells, the expression of Cbp significantly suppressed bothanchorage-dependent and anchorage-independent growth,as assessed by a growth assay and a soft agar colonyformation assay (Fig. 2B and C); however, in PC9 andLu65 cells, the expression of Cbp had little effect on bothanchorage-dependent growth and anchorage-independentgrowth. The suppressive effect of Cbp on tumor growth wasfurther examined in a xenograft mouse model. Nude micewere subcutaneously injected with Cbp-overexpressingA549 and PC9 cells or control cells, and tumor formationwas monitored weekly. Similar to anchorage-independentgrowth, the expression of Cbp significantly suppressed thetumor growth of A549 cells but not PC9 cells (Fig. 2C).These data suggest that Cbp can suppress the tumor growthof NSCLC cells, which have upregulated c-Src.

The Cbp-mediated suppression of tumor growthsignaling is associated with a reduction in active c-Src innon–raft compartments.To elucidate the molecular mechanisms by which Cbp

suppresses tumor growth, we investigated the activity statusof several Src downstream molecules, including FAK, Akt,and Erk, in A549 cells. Despite the reduction of total c-Srcactivity in these cells (Fig. 2A), the expression of Cbppreferentially suppressed the activity of FAK (pY379),whereas the activities of Akt and Erk were unchanged(Fig. 3A). To address the mechanism for the inactivationof FAK, we examined the membrane distribution of c-Src,FAK, and Csk by separating DRMs, in which majorcomponents of lipid rafts are concentrated (24). In Cbp-expressing cells, Cbp was enriched in the DRMs (Fig. 3B,right), representing its preferential localization to lipid rafts.In these cells, the activity of c-Src (pY418 signals) wassubstantially reduced in the non-DRMs, whereas the levelof inactive c-Src (pY529) rather increased (Fig. 3B). Quan-titative analysis revealed that the ratio of active c-Src(pY418) in the DRMs to that in the non-DRMs signifi-cantly increased in Cbp-expressing cells (Fig. 3C, left),whereas the levels of inactive c-Src (pY529) in the DRMsdecreased (Fig. 3C, right). These findings demonstrate thatthe expression of Cbp can induce not only the suppressionof c-Src activity but also the accumulation of active c-Src inlipid rafts. Consistent with the reduction of c-Src activity inthe non-DRMs, the activity of FAK (pY397) that is presentin the non-DRMs was appreciably suppressed (Fig. 3B).

This raises the possibility that the Cbp-mediated elimina-tion of active c-Src from the non–raft compartments mayinterfere with the access of active c-Src to FAK, which iscrucial for tumor growth (18).

Cbp-Csk association and inhibition of c-Src activity areinvolved in the growth suppression of A549 cellsTo further elucidate the mechanisms for the suppression

of c-Src activity by the expression of Cbp, we examined theinteraction between c-Src, Csk, and Cbp in A549 cells.Figure 3B demonstrates that Csk was detected in the DRMsfrom Cbp-expressing cells but was absent in the DRMsfrom the mock-treated cells. These data indicate that a partof Csk can be recruited to lipid rafts by the expression ofCbp. The immunoprecipitation assays for Cbp and c-Srcrevealed that Cbp coimmunoprecipitated with both c-Srcand Csk (Fig. 4A), indicating that Cbp, Csk, and c-Src areable to form a ternary complex in lipid rafts. These resultssuggest that active c-Src and Csk are recruited to Cbp,which allows for an efficient inactivation of c-Src by Csk. Toverify the importance of the formation of the Cbp-Cskcomplex in c-Src–induced tumor growth, we expressed aCbp mutant (Cbp Y314F) with a mutation in the Csk-binding site in A549 cells; the mutant was expressed at alevel that was comparable to that of wild-type Cbp(Fig. 4B). The expression of Cbp Y314F failed to suppressanchorage-independent growth in soft agar and tumorformation in a xenograft model (Fig. 4B and C), indicatingthat Cbp-Csk association is crucial for the tumor-suppres-sing effect of Cbp.To examine whether the effects of Cbp expression on cell

growth were mediated through suppressing c-Src activity,we further investigated the effects of Src inhibitors, includ-ing PP2, Dasatinib, and Saracatinib, on the growth of A549cells. As shown in Figure 4D, the addition of each inhibitordecreased the phosphorylation levels of c-Src Tyr418.Consistent with the activity status of c-Src, the Src inhibi-tors dose dependently repressed both anchorage-dependentand anchorage-independent growth of these cells, asassessed by a growth assay and a soft agar colony formationassay (Fig. 4E and F). These data demonstrate that theupregulation of c-Src activity is indeed involved in theenhanced growth ability of A549 cells and suggest thatthe inhibition of c-Src by the Cbp-Csk regulatory circuitcan also contribute to the suppression of tumor growth ofA549 cells.

The expression of Cbp affects the cytoskeletalorganization and motility of lung cancer cellsWe next examined the effects of Cbp expression on the

organization of the actin cytoskeleton because the actincytoskeleton is crucial for regulating the invasion andmetastasis of cancer cells (25). Although the controlA549 cells had prominent actin stress fibers, Cbp expressionsubstantially attenuated stress fiber formations (Fig. 5A). Asdetected by a wound-healing assay, cell motility wassignificantly repressed by Cbp expression (Fig. 5B). In

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contrast, the expression of Cbp Y314F had little effect onboth the actin cytoskeleton and cell motility (Fig. 5A andB), indicating that the Cbp-Csk association and c-Srcactivity are crucial for controlling these cellular functionsas well. The significant reduction in the invasiveness that isassociated with Cbp expression was also observed in aMatrigel invasion assay (Fig. 5C). We further examinedthe expression of invasion-related metalloproteases (MMP-2, MMP-9), which degrade extracellular matrices. Theexpression of MMP-9 was significantly reduced in Cbp-expressing cells compared with control cells, whereas the

expression of MMP-2 was only moderately repressed(Fig. 5D). These results suggest that Cbp can suppressthe invasive potential of lung cancer cells by modulatingcytoskeletal organization, cell motility, and the expression ofMMPs via the inhibition of a c-Src–mediated pathway (5).

The expression of Cbp affects the metastatic ability oflung cancer cellsThe observations that Cbp may control the invasive

potential of lung cancer cells in vitro directed us to examinethe suppressive role of Cbp in tumor metastasis in vivo.

Figure 3. Cbp suppresses Srcsignaling by sequestering activec-Src into DRMs. A, total celllysates from mock and Cbp-expressing A549 cells wereanalyzed by Western blotting withthe indicated antibodies (left). Theactivity status of FAK wasassessed by quantifying therelative intensity of the signal forphosphorylated forms incomparison to that of thenonphosphorylated forms.Relative specific activities (mean� SE) of FAK in Cbp-expressingcells in comparison to those inmock cells were obtained from 3independent experiments. B,DRMs and non-DRMs wereseparated from mock cells andCbp-expressing cells, and eachfraction was analyzed by Westernblotting with the indicatedantibodies. Caveolin 1 and TFRwere detected as markers ofDRMs and non-DRMs,respectively. C, the intensity ofsignals for Src pY418 (left) andpY529 (right) was quantified, andthe ratios of the signals in DRMs tothose in non-DRMs werecompared between mock cellsand Cbp-expressing cells. Therelative values (mean � SE) thatwere obtained from 3 independentexperiments are shown.

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Figure 4. Cbp-Csk association and inhibition of c-Src are involved in the growth suppression of A549 cells. A, cell lysates from mock and Cbp-expressingA549 cells were immunoprecipitated with anti-Cbp antibody, and the immunoprecipitates were analyzed by Western blotting with anti-Src, anti-Csk, oranti-Cbp antibody. To detect Csk in the immunoprecipitates, the sample was not heat denatured so as to avoid an overlap with the IgG heavy chain onthe gel. *, the location of the IgG heavy chain; **, the non-specific bands. B, the anchorage-independent growth of mock cells, Cbp-expressing cells,and Cbp mutant-expressing cells (Cbp Y314F) were examined by a colony formation assay in soft agar. Colonies were stained with MTT 14 days after plating.The colony numbers per cm2 � SE that were obtained from 3 independent experiments are shown (left). The expression levels of Cbp wild typeand Cbp mutants were assessed by Western blotting. C, the ability of tumor formation was compared in mouse xenograft models. The tumor volumes(mean� SE) that were obtained from 4 mice are plotted as a function of days after inoculation. D, A549 cells were incubated with increasing concentrations ofPP2, Dasatinib, Saracatinib or DMSO for 24 hours, and the total cell lysates were subjected to Western blotting with the indicated antibodies. E, in vitroproliferation assays were performed for A549 cells incubated with the indicated Src inhibitor. The relative growth rate (mean � SE) was obtained from 3independent experiments. F, colony formation assays were performed for A549 cells incubated with DMSO, PP2, Dasatinib, or Saracatinib at theconcentrations indicated. Colonies were stained with MTT 10 days after plating. The colony numbers per cm2 (mean� SE) were obtained from 3 independentexperiments. Student's t tests: *, P < 0.05.

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A549 cells, with or without Cbp expression, were intrave-nously injected into nude mice, and the pulmonary meta-static burden was assessed by counting the number of tumornodules on the surface of the lung. Mice in the controlgroups displayed numerous distinguishable pulmonarynodules, whereas mice that were injected with Cbp-expres-sing cells exhibited fewer visible nodules (Fig. 6A).To examine whether the levels of Cbp expression in lung

cancer affected the degree of tumor progression, the expres-sion of Cbp mRNA was examined in primary lung adeno-carcinoma by quantitative PCR using total RNA fromFFPE tissue samples as templates. The lowest score ofCbp mRNA in 12 cases was arbitrarily set as 1.0, and

the relative level of CbpmRNA in each cancer specimen wasevaluated. As shown in Figure 6B, appreciable differences inthe levels of Cbp mRNA were observed among individualsamples, although there was no significant relationshipbetween the Cbp expression levels and the histologic tumorgrade; however, Cbp expression could be significantlycorrelated with lymph node involvement. The mean Cbpscore for tumors without lymph node metastases was 7.71� 4.38, whereas the mean Cbp score for tumors with lymphnode metastases was 3.20 � 2.05 (Fig. 6C). These findingssuggest that the level of Cbp in lung cancer may becorrelated with metastatic potential to the lymph noderather than primary tumor grade.

Figure 5. The impact of Cbpexpression on cytoskeletalorganization and the motility oflung cancer cells. A, actin stressfibers were stained with AlexaFluor 488 phalloidin. B, thewound-healing analysis wasconducted. The wounded areaswere photographed at 0 and 24hours after the monolayer cultureswere scratched (left). The gaps ofthe wound areas were measured,and the relative values (mean �SE) from triplicate experiments areshown (right); *, P < 0.05. C,invasion assays were carried outin Matrigel transwell chambers.After incubation for 24 hours, thecells that had passed through thefilter into the bottom wells werefixed and photographed (left). Thecells in the bottom wells werecounted, and the mean � SE from3 independent experiments isshown (right). D, the expression ofMMP-9 and MMP-2 was analyzedby quantitative real-time PCR.Data represent the relative values(mean � SE); *, P < 0.05.

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Cbp in Lung Cancer





Discussion

In this study, we found that the expression of Cbp issignificantly downregulated in various human NSCLC celllines and tissues. Due to its role as a scaffold for both activec-Src and Csk, Cbp expression was observed to suppress thetumor growth of A549 and Lu99 cells, which have sub-stantially upregulated c-Src. In addition, we have shownthat the expression of Cbp suppresses cell motility, in vitroinvasiveness, and the in vivo metastatic potential of A549cells, and the expression level of Cbp was significantlycorrelated with lymph node involvement in human lungcancers. These findings suggest the important role of Cbp incontrolling the malignant potential of NSCLC, particularlythose that have upregulated c-Src activities.Recently, several studies have implicated a role for Cbp in

tumor malignancy. Cbp may function as a negative reg-ulator of EGF-induced cell transformation (26). In coloncancer cells, Cbp is downregulated and the introduction ofCbp suppresses tumorigenesis (19). In this study, we have

shown that Cbp acts as a suppressor of NSCLC tumorprogression by controlling c-Src signaling. On the otherhand, it has been reported that the expression of Cbp isincreased in a significant majority of diffuse large B-celllymphomas (27). In RCC, Cbp is overexpressed in cancertissues and enhances cell motility and invasiveness, whereinit regulates RhoA activity via its PDZ-binding domain (20).These observations suggest that Cbp has distinct functionsdepending on the cancer type. To elucidate the molecularbasis for the distinct functions of Cbp, a more extensiveanalysis of Cbp function in each case will be necessary.In the immune system, lipid rafts have been thought to

play a positive role in signaling that is initiated through theT-cell, B-cell, and Fc receptors (28, 29). In contrast, wepreviously proposed a role for Cbp in suppressing c-Src–mediated transformation using a model system (19). In thisstudy, we provide firm evidence that Cbp in lipid rafts playsa suppressive role in controlling the c-Src–induced malig-nant potential of human cancer cells. Biochemical analysisdemonstrated that Cbp recruits active c-Src and Csk into

A

B C

Figure 6. The expression of Cbp inhibits metastasis of lung cancer cells. A, mock or Cbp-expressing A549 cells were injected into the tail veins of nudemice asdescribed in the Materials and Methods section. Pulmonary metastatic burden was assessed by counting the number of tumor nodules on the lungsurface (left). The mean values � SE from 5 mice are shown (right). B, quantitative real-time PCR was performed using total RNA that was isolated fromFFPE tissue samples. The relative levels of Cbp mRNA, which were normalized to GAPDH mRNA expression, from the primary cancer specimens of eachcase were compared. Pathologic stages and tumor grades (1: well-differentiated and 2: moderately or poorly differentiated) are indicated at the bottom.C, the levels of Cbp mRNA in cases with lymph node metastases were compared with those of cases without metastases. The bar indicates the mean valueof the Cbp level; ANOVA: *, P < 0.05.

Kanou et al.





lipid rafts by forming a Cbp–Csk–c-Src complex and servesas a scaffold where the efficient inactivation of c-Src by Cskcan be achieved. As a consequence, active c-Src is eliminatedfrom the non–raft compartments, and this eliminationpotentially contributes to the suppression of the non–raftFAK that is crucial for tumor growth. These findingsindicate that the function of c-Src may be regulated notonly on the activity level but also by influencing theintracellular distribution that is determined by the specificscaffold Cbp. In contrast to the overexpression of Cbp, theoverexpression of Csk failed to suppress the tumor growthof A549 cells (data not shown). This intriguing resultfurther supports the requirement of Cbp in Csk-mediatedc-Src regulation as well as in the control of malignancy ofA549 cells.Recent reports have shown that c-Src is altered and highly

activated in NSCLC, especially in adenocarcinomas (7, 9,30). The level of c-Src kinase activity has been reported tocorrelate with tumor size in NSCLC (8). Furthermore, themitogenic effect of both nicotine and asbestos in NSCLCcells is likely to involve the activation of c-Src (31, 32).These observations suggest that c-Src activity is frequentlyassociated with the malignant progression of a variety ofNSCLC cells. Because c-Src is also known to be an impor-tant mediator of upregulated receptor tyrosine kinases,including HER2 and EGFR, therapeutic strategies to inhi-bit c-Src activity are currently being developed (33).Recently, several inhibitors of activated c-Src in cancer cellshave been examined in clinical trials for the treatment of notonly lung but also breast, colon, and prostate cancer (34). Inpreclinical studies, NSCLC cells that were treated withdasatinib (BMS-354825) demonstrated decreased cellgrowth and changes in downstream signaling that resultedin a reduced capability for invasion (35, 36). These reportssuggest that c-Src could be an attractive target for treatmentin a particular subset of patients with NSCLC. Therefore,the c-Src regulatory circuit that consists of Csk, Cbp, andlipid rafts may offer new targets that can prevent theprogression of NSCLC by controlling c-Src function.In addition to tumor growth, the in vivo invasiveness of

A549 cells was also significantly inhibited by the over-expression of Cbp. The attenuation of actin stress fiberformation and the downregulation of MMP-9 expressionmay explain the lower invasiveness of Cbp-expressing cells.

This result was consistent with the previous observation thatc-Src activation promoted tumor invasiveness (25).Recently, it has been demonstrated that Src-FAK signalingthrough JNK (c-jun NH kinase) alters the transcriptionalregulation of MMP-9 (37), and that the pharmacologicblockade of Src activity suppresses VEGF-induced MMPproduction in squamous carcinoma cells (38). These linesof evidence support that the inhibition of c-Src and FAKfunctions through the overexpression of Cbp leads to themodulation of cytoskeletal organization and MMP pro-duction. We have also found that the expression levels ofCbp mRNA that were detected in FFPE NSCLC samples(39) in cases with lymph node involvement were lowerthan those in cases without lymph node involvement. Thisraises the possibility that the level of Cbp in primarytumors is generally relevant to the metastatic potential ofNSCLC; however, the molecular mechanism by which theexpression of Cbp is specifically downregulated in NSCLCremains to be addressed. Because there was no apparentcorrelation between c-Src upregulation and Cbp down-regulation, pathways other than c-Src signaling may alsocontribute to Cbp downregulation mechanisms. Theelucidation of the pathway(s) that lead to Cbp down-regulation may shed light on the mechanisms of tumorprogression and offer potential new opportunities fortherapeutic intervention in NSCLC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

We thank Drs. T. Akagi, K. Kodama, and M. Yutsudo for their generous gifts ofreagents.

Grant Support

TheMinistry of Education, Culture, Sports, Science, and Technology of Japan(grants-in-aid for Scientific Research) and the Uehara Memorial Foundation.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 27, 2010; revised November 29, 2010; accepted December 2, 2010;published OnlineFirst December 14, 2010.

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2011;9:103-114. Published OnlineFirst December 14, 2010.Mol Cancer Res Takashi Kanou, Chitose Oneyama, Kunimitsu Kawahara, et al. That Have c-Src Upregulation

Small Cell Lung Cancers−Malignant Potential of Human Non The Transmembrane Adaptor Cbp/PAG1 Controls the

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