m-copa, a golgi disruptor, inhibits cell surface ... · 1division of molecular pharmacology, cancer...

Therapeutics, Targets, and Chemical Biology

M-COPA, a Golgi Disruptor, Inhibits Cell SurfaceExpression of MET Protein and Exhibits AntitumorActivity against MET-Addicted Gastric CancersYoshimiOhashi1, MutsumiOkamura1, AsakaHirosawa1, Naomi Tamaki1, AkinobuAkatsuka1,Kuo-Ming Wu2, Hyeong-Wook Choi2, Kentaro Yoshimatsu3, Isamu Shiina4,Takao Yamori1, and Shingo Dan1

Abstract

The Golgi apparatus is responsible for transporting, processing,and sorting numerous proteins in the cell, including cell surface-expressed receptor tyrosine kinases (RTK). The small-moleculecompound M-COPA [2-methylcoprophilinamide (AMF-26)] dis-rupts the Golgi apparatus by inhibiting the activation of Arf1,resulting in suppression of tumor growth. Here, we report anevaluation of M-COPA activity against RTK-addicted cancers,focusing specifically on human gastric cancer (GC) cells with orwithout MET amplification. As expected, the MET-addicted celllineMKN45 exhibited a better response toM-COPA than cell lineswithoutMET amplification. UponM-COPA treatment, cell surface

expression of MET was downregulated with a concurrent accumu-lation of its precursor form. M-COPA also reduced levels of thephosphorylated formofMETalongwith thedownstreamsignalingmolecules Akt and S6. Similar results were obtained in additionalGC cell lineswith amplificationofMETor the FGF receptor FGFR2.MKN45 murine xenograft experiments demonstrated the antitu-mor activity ofM-COPA in vivo. Taken together, our results offer aninitial preclinical proof of concept for the use of M-COPA as acandidate treatment option for MET-addicted GC, with broaderimplications for targeting the Golgi apparatus as a novel cancertherapeutic approach. Cancer Res; 76(13); 3895–903. �2016 AACR.

IntroductionGastric cancer (GC) is the third leading cause of cancer-related

deathworldwide, and its incidence remains high especially in eastAsia, including Japan and Korea (1). GC is often diagnosed at anadvanced stage, and the prognosis of such patients is poor. Owingto its diversity of morphologic forms and considerable heteroge-neity, the classification ofGChas been complicated. Recently, TheCancer Genome Atlas (TCGA) Research Network proposed a newmolecular classification of GC into four subtypes (2). Amongthese, approximately 50% of gastric tumors were categorized asthe chromosomal unstable subtype, containing frequent geneamplification of receptor tyrosine kinases (RTK), such as humanepidermal growth factor receptor 2 (HER2), MET (also called

"hepatocyte growth factor receptor") and fibroblast growth factorreceptor 2 (FGFR2), which have been shown to drive cells tomalignant proliferation and survival (3, 4).

Thanks to recent progress in understanding molecular path-ways underlying carcinogenesis, new targeted treatment optionshave become available for treating cancer patients. In respect tothe development of targeted therapies, monoclonal antibodies(mAb) and small-molecule inhibitors of tyrosine kinase (TKI)activity are ideal candidates that target tumor cells via binding toRTKs. For example, trastuzumab is anmAb developed for treatingHER2-positive breast cancer; gefitinib is a small-molecule TKI-targeting EGFRdeveloped for treating lung cancer. So far, sixmAbsand 23 TKIs have been developed as approved drugs for treatingcancer; however, trastuzumab and ramucirumab are the only twomolecularly targeted drugs approved to date for treating GCpatients. Therefore, the development of new targeted drugsagainst GCs is of keen interest. Because gene amplification ofMET, HER2, and FGFR2 is often observed in GCs, many TKIstargeting these RTKs have been developed and several clinicaltrials in GC patients are in progress.

The Golgi apparatus plays an essential role in the transport,processing, and sorting of numerous proteins (5–8). Most cell-surface and secreted proteins in eukaryotic cells pass through theGolgi apparatus, allowing for posttranslationalmodification suchas processing and glycosylation, and subsequently transport toplasma membrane (9–11). Aberration of Golgi function is asso-ciated with certain forms of inherited diseases, cancer, and dia-betes (12). ADP ribosylation factor 1 (Arf1), a small GTPase and amember of Ras superfamily (13, 14), is required for maintenanceofGolgi structure and function via formationof complex I (COPI)or clathrin-coated vesicles transported among the endoplasmicreticulum (ER), Golgi, and post-Golgi (15–19). We previously

1Division of Molecular Pharmacology, Cancer Chemotherapy Center,Japanese Foundation for Cancer Research, Tokyo, Japan. 2Next Gen-eration Systems, Eisai Inc., Andover, Massachusetts. 3Eisai ProductCreation Systems, Eisai Co., Ltd., Tokyo, Japan. 4Department ofApplied Chemistry, Faculty of Science, Tokyo University of Science,Tokyo, Japan.

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

Current address for K.-M. Wu: Department of Chemical Development, ConcertPharmaceuticals, Inc., Lexington, MA; and current address for T. Yamori: Centerfor Product Evaluation, Pharmaceuticals and Medical Devices Agency, Tokyo,Japan.

Corresponding Author: Shingo Dan, Japanese Foundation for Cancer Research,3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3520-0111; Fax: 81-3-3570-0484; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2220

�2016 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 3895

on May 26, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 12, 2016; DOI: 10.1158/0008-5472.CAN-15-2220

http://cancerres.aacrjournals.org/

demonstrated that M-COPA (2-methylcoprophilinamide, alsocalled "AMF-26") suppressed Arf1-mediated vesicle transport,disrupted the structure of the Golgi apparatus, and exerted anti-tumor activity in vivo against tumors xenografted into mice (20).In fact, M-COPA has been shown to inhibit secretion of intercel-lular adhesion molecule-1 (ICAM-1) and cell surface expressionof VEGFR-2 (21). Therefore, we postulated that M-COPA couldinhibit the processing and transport of RTKs to the cell surface andextracellular space, and thereby exert antitumor activity againstRTK-addicted cancers.

In this study, using GC cell lines with or without MET ampli-fication, we demonstrated that M-COPA inhibited the processingand transport of MET protein onto the cell surface, attenuatedaberrantMET signaling and exerted preferential antitumor activityagainst MET-addicted GCs. Moreover, we obtained similar resultsin FGFR2-amplified signet ring GC cells KATO III. The presentresults suggest that a Golgi-targeted drug could be a novel ther-apeutic modality against MET-addicted GCs, as well as perhapsRTK-addicted cancers from different origins.

Materials and MethodsChemicals

M-COPA (also called "AMF-26," chemical name: (2E,4E)-5-[(1S,2S,4aR,6R,7S,8S,8aS)-7-hydroxy-2,6,8-trimethyl-1,2,4a,5,6,7,8,8a-octahydronaphthalen-1-yl)-2-methyl-N-(pyridin-3-ylmethyl)penta-2,4-dienamide] was totally synthesized byEisai Co., Ltd. according to the methods established previously(22). 5-FU and paclitaxel were purchased from Sigma-AldrichCo. LLC., crizotinib was purchased from Selleck Chemicals.For in vitro studies, these compounds were reconstituted to 20mmol/L in DMSO (Sigma-Aldrich) and stored at �20�C. SN-38 was purchased from Sigma-Aldrich, and cisplatin waspurchased from Nippon Kayaku Co., Ltd. For animal experi-ments, M-COPA was suspended in 0.15N hydrochloride acid(Wako Pure Chemical Industries). Antibodies used for immu-nostaining are listed in Supplementary Table S1.

Cell lines and cell cultureFour GC cell lines (St-4, MKN1, MKN45, and MKN74) are

components of the JFCR39 panel of human cancer cell linesdescribed previously (23, 24). Of these, St-4 was established inour foundation in 1990 (25). MKN1, MKN45, and MKN74 werepurchased from Immuno-Biological Laboratories Co., Ltd. in1991. Other two GC cell lines, Hs-746T, and SNU-5 were pur-chased from the ATCC in 2014. Cell lines were cultured in RPMI-1640 medium (Wako Pure Chemical Industries) supplementedwith 5% (v/v) FBS (Moregate Biotech), penicillin (100 U/mL),streptomycin (100 mg/mL), and kanamycin (1 mg/mL) in ahumidified atmosphere including 5% CO2 at 37�C. Authentica-tion of St-4,MKN1,MKN45, andMKN74 cell lineswas performedby short tandem repeat (STR) analysis usingPowerPlex16 Systems(Promega) according to the manufacturer's instructions by BEXCO., LTD., in 2009 and 2016. Details were described in Supple-mentalMaterials andMethods. Finally, the STR profiles ofMKN1,MKN45 and MKN74 were compared with those in the referencedatabase of the Japanese Collection of Research Bioresources CellBank. Because St-4 was developed in our foundation and noreference data was deposited, we compared the profile deter-mined in 2016 to that in 2009. The KATO III cell line wasoriginally purchased from the ATCC in 2000. Authentication of

the cell linewas performed in 2016 and compared the profile withthat in the ATCC STR database.

Analysis of cell growth inhibitionThe inhibition of cell growthwas assessed bymeasuring changes

in total cellular protein in a culture of each GC cell line after 48hours of drug treatment by use of a sulforhodamine B assay (26).Positive values represent net protein increase before and after drugexposure (% of control growth) and negative values represent celldeath [protein amount after 48 hours-exposure (%) of control cellsat the start of drug exposure]. The drug concentration required for50% reduction in net protein increase (GI50) was calculated asdescribed previously (23, 27, 28).

Flow cytometric analysisCells were incubated withM-COPA for 6 or 24 hours, and then

washed with ice-cold PBS and stained with antibodies againsthuman MET or human FGFR2 conjugated with phycoerythrin(PE). Then cells were washed three times with ice-cold PBS, andstained with propidium iodide (1 mg/mL; Sigma-Aldrich). Thefluorescence intensity of cell surface MET or FGFR2 wasmeasuredby flow cytometric analysis (FACS Calibur or FACS Verse, Becton,Dickinson and Company). The data were analyzed by usingFlowJo (FlowJo LLC.).

Western blot analysisCells were incubated with M-COPA for 1, 6, or 24 hours, and

then lysed as described previously (29). Proteins in cell lysateswere separated in 4% to 15% sodium dodecyl sulfate-polyacryl-amide gel (Bio-Rad Laboratories) electrophoresis, followed byelectroblotting onto a nitrocellulose membrane (Bio-Rad Labo-ratories). Immunoreactive bands were identified with an ODYS-SEY CLx Infrared Imaging System (LI-COR Biosciences).

Animal experimentsThe antitumor effect of M-COPA was tested in vivo against

MKN45-derived human GC xenografts in mice. Animal care andtreatment was performed in accordance with the guidelines of theAnimal Use and Care Committee of the Japanese Foundation forCancer Research, and conformed to theNIHGuide for theCare andUse of Laboratory Animals. Female nude mice of BALB/c geneticbackground were purchased from Charles River LaboratoriesJAPAN, Inc., maintained under specific pathogen-free conditionsand provided with sterile food and water ad libitum. Each nudemouse was subcutaneously inoculated with a generated tumorfragment of size 3mm� 3mm� 3mm.When the tumors reacheda volume of 100 to 300mm3, animals were randomly divided intocontrol and M-COPA groups (each group containing five or sixmice). Then administration of M-COPA was started (day 0). Theexperimental groupofmicewas orally administered a givendoseofM-COPA (50mg/kgofBW)onadaily basis fromday0 to4 (n¼ 6),orweekly (75mg/kgofBW)onday0,7, and14(n¼5). The controlgroup of mice (n ¼ 6) was orally administered with 0.15Nhydrochloride acid solution instead of M-COPA. The tumor vol-ume of tumor-bearing mice was measured as described previously(29). To assess toxicity, the bodyweights of the tumor-bearingmicewere measured.

ImmunohistochemistryFormalin-fixed, paraffin-embedded tissue sections (4-mm-

thick) were deparaffinized in xylene and taken through a series

Ohashi et al.

Cancer Res; 76(13) July 1, 2016 Cancer Research3896




of graded alcohols to water. Then antigens were retrieved throughwet autoclave pretreatment (20 min at 121�C) in 10 mmol/Lcitrate buffer (pH 6.0). Sections were blocked with 3%H2O2 and1% goat serum before incubation with the primary antibody at4�C overnight. By using Dako EnVision Detection System/HRP,Rabbit/Mouse (DABþ; Agilent Technologies Company), sectionswere incubated with horseradish peroxidase (HRP)–conjugatedpolymer secondary antibody, and thereafter peroxidase activitywas visualized by DAB reaction according to the manufacturer'sinstructions. The sections were counterstained with hematoxylin(Dako). The immuno-stained specimens were imaged using amicroscope BX41 (Olympus Corp.) with a 20x, NA 0.50, objec-tive, and DP2-BSW Software (Olympus Corp.).

Statistical analysisThe two-sided Mann–Whitney U test was used to assess the

statistical significanceof theantitumor efficacyofM-COPA in termsof relative tumor growth ratio andbodyweight changeondays2, 4,8, 11, 15 and 18. The number of samples is indicated in thedescription of each experiment. All statistical tests were two-sided.

ResultsOverexpression of MET protein and the Inhibitory effect of M-COPA on its cell surface expression in MET-amplified GC cells

First, we examined the effect of M-COPA on cell surfaceexpression of MET protein in GC cell lines. To this end, we

Figure 1.Inhibitory effect on cell surface expression of MET. M-COPA significantly inhibited cell surface expression of MET protein in cell lines with MET gene amplification.A, baseline expression of MET, EGFR, HER2, HER3, FGFR2, and their phosphorylated forms in the JFCR39 GC cell line panel and MET-addicted cell lines,such as Hs-746T and SNU-5, were examined by immunoblot analysis. Cells were lysed and the proteins in the cell extract were separated by SDS-PAGE andelectroblotted onto a membrane. The membrane was then probed with antibodies against the indicated proteins. Experiments were performed at leasttwice and representative results are indicated. The positive control lysates were as follows: EGFR and p-EGFR from NCI-H3255 that had EGFR-activatingmutation, HER2 and p-HER2 from EGF-stimulated HBC-5, HER3 and p-HER3 from heregulin b1-stimulated MCF-7, and FGFR2 from KATO III. B and C, METexpression on the cell surface was measured by FACS analysis. Cells were treated with M-COPA at the indicated concentrations for 6 or 24 hours and stainedwith a PE-conjugated anti-MET antibody. Lines and areas were used to indicate drug concentrations: Black solid lines with dark gray area, no drug; blackdotted lines, 30 nmol/L; black dashed lines, 100 nmol/L; black long dashed lines, 300 nmol/L; black chain lines with light gray area, 1,000 nmol/L; graysolid lines, stained with isotype-control IgG. Experiments were performed at least twice and representative results are indicated.

Antitumor Effect of a Golgi Disruptor in MET-Addicted Cancer

www.aacrjournals.org Cancer Res; 76(13) July 1, 2016 3897




exploited three GC cell lines with or without MET amplification.As shown in Fig. 1A, the MET-amplified GC cell lines, MKN45,Hs-746T, and SNU-5, overexpressed total MET protein and itsphosphorylated active form, as previously reported (30). The St-4cell line also expressed total and phosphorylated form of MET,even though the intensities of those expression were weaker thanthose of MET-amplified GC cells. We also examined baselineexpression of EGFR, HER2, HER3, and FGFR2, which have beenreported to be amplified in GC cells (2). None of the phosphor-ylated forms of these proteins except HER3 in MKN45 weredetected in any of the GC cell lines examined, although totalEGFR and HER3 were detected in some cell lines.

We then examined cell surface expression ofMET and the effectof M-COPA treatment in these GC cell lines. As shown in Fig. 1B,baseline MET expression on the cell surface was higher in MET-amplified GC cell lines (MKN45, Hs-746T, SNU-5) than thatobserved in other GC cell lines, in parallel with the levels of totalMETprotein determined by immunoblot analysis (Fig. 1A).Upontreatment with M-COPA for 24 hours, cell surface expression ofMET efficiently decreased in a dose-dependent manner; it wassignificantly reduced at concentrations of 100 nmol/L or higher inSNU-5 and MKN45 cells, whereas �300 nmol/L was needed tomediate such reduction in Hs-746T cells. Next, we examined theexpression profile of MET protein over time following treatmentof MKN45 cells with M-COPA. After 6 hours treatment, METprotein expression was reduced to approximately 25% (1/4) atconcentrations of 100 nmol/L or higher and was dramaticallydeclined to negative control levels (IgG-isotype control antibody)within 24 hours at concentrations of 300 nmol/L or higher(Fig. 1C). From these data, we concluded thatM-COPA efficientlyinhibited cell surface expression ofMETprotein inMET-amplifiedGC cells, in a dose- and time-dependent manner.

Growth inhibitory effect of M-COPA against GC cell lines withor without MET amplification

We next evaluated the effect of M-COPA on the growth of GCcell lines with or withoutMET amplification. Expectedly, all threeMET-amplified cell lines were highly sensitive to the MET inhib-itor crizotinib (Fig. 2B), as compared with other GC cell lines,indicating that their growth was addicted to MET, in agreementwith previous reports (31). Interestingly, MET-amplified celllines also exhibited a better drug response to M-COPA thanthose without MET amplification, albeit the selectivity was notas marked as that observed with crizotinib (Fig. 2A). Moreover,the concentrations of M-COPA needed to inhibit cell growthwas comparable with those needed to inhibit cell surfaceexpression of MET in the MET-amplified cell lines; the GI50concentration for the MKN45, Hs-746T, and SNU-5 cell lineswas 29, 40, and 19 nmol/L, respectively. These data indicatedthat M-COPA preferentially inhibited the growth of MET-addicted GC cells and the growth inhibition coincided withdecreased expression of MET protein on the cell surface. Forcomparison, the anticancer effects of chemotherapeutic agentsused in the clinic for GC patients, namely 5-FU, paclitaxel, SN-38, and cisplatin, were examined. However, neither 5-FU,paclitaxel nor cisplatin exhibited significant differences in termsof growth inhibitory activities in the GC cell lines with METamplification with the only exception that SNU-5 tended to behighly sensitive to these agents. On the other hand, SN-38exhibited preferential activity in these MET-amplified cell linesin a similar vein to M-COPA (Fig. 2C–F).

The effect of M-COPA on processing of MET protein and itsdownstream signaling molecules in MET-amplified GC cells

MET is a transmembrane heterodimer that composed of twodisulfide-linked chains of 50 kDa a-subunit and 145 kDa b-sub-unit (32). The molecule is originally synthesized as a single-chain170-kDa precursor (Pr170), which is cotranslationally glycosy-lated. Terminal glycosylation and proteolytic cleavage generatethe mature heterodimer (33). To clarify whether M-COPA coulddisturb processing ofMET, expression levels of the precursor formand amature b-subunit ofMETwere estimated inMKN45 cells byWestern blot analysis. As shown in Fig. 3A, the amount of thePr170 was increased upon treatment with M-COPA in a dose-dependent manner at 6 hours, whereas the active b-subunit andphosphorylated form of MET were decreased. Finally, the activephosphorylated form of MET were dramatically reduced at

Figure 2.Cell growth inhibition of M-COPA against human GC cell lines. M-COPAinhibited the cell growth of MET-addicted cell lines in a more robust mannerthan that observed with non-addicted cell lines. The inhibition of cellproliferation was assessed by measuring changes in total cellular protein.After 48 hours of drug treatment, cells were fixed and stained by use of asulforhodamine B assay. Growth curves under drug treatment with M-COPA(A), crizotinib (B), or typical antitumor agents (C–F) used in GC therapy areshown; 5-FU (C), paclitaxel (D), SN-38 (E), and cisplatin (F). Black circle, St-4;black square, MKN1; black triangle, MKN74; red circle, MKN45; red square, Hs-746T; red triangle, SNU-5. Experiments were performed at least twice andrepresentative results are indicated.

Ohashi et al.





concentrations of 100 nmol/L or higher in 24 hours, in parallel toinhibition of cell surface expression of MET (Fig. 1C) and cellgrowth (Fig. 2A). These results indicated that the processing andthe transport of MET protein onto the cell surface were preventedby M-COPA treatment.

Wenext examined the effect ofM-COPAon the activation statusof the downstream signaling pathway from MET. M-COPAdecreased the levels of phosphorylated Gab2, Akt, and S6 withinthe same drug concentration range and time-course as thoserequired for inhibition of MET processing and transport (Fig.3B). The effectiveness ofM-COPAwas also demonstrated in othertwo MET-addicted GC cell lines, Hs-746T and SNU-5 (Supple-mentary Fig. S1A). M-COPA also inhibited the cell surface expres-sion of MET in St-4 cells (Fig. 1B). Therefore, we investigated theactivation status of MET and its downstream signaling pathwaymolecules. As shown in Supplementary Fig. S1B, M-COPArepressed the MET processing, MET-activation, and phosphory-lation of Akt. However, phosphorylation of S6 ribosomal proteinwas still remained by M-COPA treatment, and concordantly St-4cell line showed lower sensitivity to crizotinib and M-COPA(Fig. 2A). From these data, we concluded that M-COPA inhibitedthe MET-dependent signaling pathway via inhibition of METprocessing and its cell surface expression as mediated by theGolgi apparatus, resulted in attenuating the abnormal cellproliferation in MET-amplified GC cell lines.

The antitumor effect of M-COPA against FGFR2-amplified GCcells

To examine the effect of other RTKs amplified in GCs, weexploited KATO III, which was known as a FGFR2-amplifiedsignet ring cell GC cell line that exhibited a high sensitivity toFGFR2-TKI (34, 35). As we expected, M-COPA caused down-

regulationof cell surface expression, electrophoreticmobility shiftprobably due to inhibition of post-translational modification,and dephosphorylation of FGFR2 protein, and finally decreasedphosphorylated form of downstream signalingmolecules such asAkt and S6 (Fig. 4A and B). These effects were observed in thesimilar concentration range to that required for inhibition of cellgrowth (Fig. 4C). These results suggested that M-COPA sup-pressed cell surface expression of FGFR2 as a result of Golgidysfunction, and thereby exerted antitumor effect in FGFR2-amplified cells, as well as MET-amplified cells.

Antitumor efficacy of M-COPA against MKN45-derived tumorxenografts in vivo

Finally, we tested the antitumor efficacy of M-COPA againsttumor xenografts derived from the MET-amplified GC cell line,

Figure 3.Time-course and dose-dependent effects on processing of MET protein, andphosphorylation status of downstream signaling molecules. M-COPAinhibited cell surface expression, processing, and phosphorylation ofMET andits downstream signaling at the same concentration range and time points asobserved with respect to the inhibitory effect on cell surface expression ofMET in MKN45 cells. A and B, after M-COPA treatment with indicatedconcentrations for 1, 6, or 24 hours, MKN45 cells were lysed. A, processingstatus and phosphorylation status of MET protein. B, phosphorylation statusof signaling molecules, including Gab2, Akt and ribosomal S6 protein (S6),were examined by immunoblot analysis.

Figure 4.The antitumor effect of M-COPA against an FGFR2-amplified KATO III cell linevia inhibition on the cell surface expression of FGFR2. M-COPA repressed cellsurface expression of FGFR2, maturation of FGFR2 protein andphosphorylation status of downstream signaling molecules, and cell growthin vitro in dose–respondmanner. A, FGFR2 expression on the cell surface wasmeasured by FACS analysis. Cells were treated with M-COPA at the indicatedconcentrations for 24 hours and stained with an anti-FGFR2 antibody, inconsequent with a PE-conjugated second antibody. Lines and areas wereused as described in Fig. 1B legend. B, after M-COPA treatment with indicatedconcentrations for 24 hours, KATO III cells were harvested and cell lysateswere prepared. Immunoblot analysis of total and phosphorylated form ofFGFR2 protein and phosphorylation status of downstream signalingmolecules, including Akt and S6 ribosomal protein (S6), were examined.C, the inhibition of cell proliferation was assessed by sulforhodamine B assay.Symbols were used as described in Fig. 2 legend.






MKN45, in vivo. M-COPA was orally administrated daily for thefirst 5 days, or weekly for 3 weeks. In both administration groups,treatment with M-COPA significantly decreased the tumor sizethan that observed in the control group (Fig. 5A, top). Slight butsignificant weight loss was observed in the daily administrationgroupondays 2 and4; however, after stopping administration, nosignificant difference was observed among the three groups onday 8 and later (Fig. 5A, bottom).

To validate the proof of concept that M-COPA administra-tion exerts an antitumor effect via inhibition of cell surfaceexpression of MET, we examined the expression of phosphor-ylated MET in tumor tissue sections by immunohistochemistry.The immunostaining intensities of phosphorylated MET on thecell membrane were markedly decreased in the M-COPA–trea-ted group on day 2 compared with those in the control group(Fig. 5B and C). These data strongly suggested that M-COPAexerted an in vivo antitumor effect on MET-amplified MKN45-derived xenografts via downregulation of cell surface expres-sion of active MET.

DiscussionAs mentioned, MET protein is produced as a 170 kDa single-

chain precursor form, and the precursor is thenposttranslationallyglycosylated, cleaved to yield a mature form consisted of ana-chain (50 kDa) and a b-chain (145 kDa) via theGolgi apparatus(32, 33, 36). In MET-amplified cells, overexpression of METprotein triggers its autophosphorylation and recruitment of its

effectors, resulting in hyperactivation of intracellular downstreamsignaling pathways, including thePI3K–AKTaxis (37). This abnor-mal signal activation is known to drive cells to malignant prolif-eration, thereby entering a "MET-addicted" state (38, 39). In thepresent study, we clearly demonstrated that M-COPA decreasedthe level of the active b-subunit form and increased the precursorform in a dose-dependent manner, consistent with a previousreport showing that a Golgi inhibitor brefeldin A (BFA) abrogatedthe processing of nascent MET protein (36). Moreover, we foundfor the first time that inhibition of MET processing by M-COPAcoincided with downregulation of its cell surface expression andabrogation of its downstream oncogenic signals represented byreduction of phosphorylated forms of Gab, Akt, and S6, andultimately suppressed tumor growth in MET-addicted GC celllines. Downregulation of cell surface expression of activated METprotein in parallel to the observed antitumor effects was alsoconfirmed in the case of MKN45-derived tumor xenografts afteradministrationofM-COPA in vivo. These results strongly suggestedthat inhibition of processing and cell surface expression of METprotein is the main mechanism by which M-COPA exerts antitu-mor effects against MET-addicted GC cells. In addition to MET,phosphorylatedHER3was also detected inMKN45 cells (Fig. 1A),as previously reported (40). MET has been shown to interact withHER3 and activateHER3 signal in gastric and lung cancer cells (40,41). In our study, M-COPA also repressed phosphorylation andcell surface expression ofHER3 (Supplementary Fig. S2), aswell asMET, suggesting that repression of HER3 signal could also beinvolved in antitumor effect of M-COPA in MKN45 cells.

Figure 5.Antitumor efficacy of M-COPA against MET-amplified tumor xenograft in vivo. Tumor fragments derived from human GC cell line MKN45 were subcutaneouslyinoculated into BALB/c nude mice. M-COPA was orally administrated daily for the first 5 days (50 mg/kg BW) or weekly for 3 weeks (75 mg/kg BW).A, the top shows relative tumor growth, whereas the bottom shows body weight change in nude mice. Asterisks represent statistically significant differencesfrom the control group (P < 0.05); error bar, SE. The expression of phosphorylated MET protein in vivo was estimated by immunohistochemistry.B, control tissue section, and M-COPA–treated section on day 2 (C).

Ohashi et al.





Wealso demonstrated that cell surface expression of FGFR2wasalso downregulated by M-COPA treatment in FGFR2-addictedKATO III, a human signet ring cell GC cell line (Fig. 4). FGFR2 isknown as a target for cancer therapy, andnow several FGFR2-TKIs,such as AZD4547, are in clinical study stage. M-COPA alsorepressed the maturation of FGFR2, phosphorylation of its sig-naling pathway molecules, and inhibited cell growth under theconcentration of submicromole order. These data strongly sug-gested that M-COPA could exert antitumor effect in cancer cellsaddicted to not only MET but also FGFR2 and other RTKs,including HER2, mutant EGFR, and so on.

St-4 is a cell line that expresses small amount of MET protein(Fig. 1A). This cell line is not addicted toMETexpression and it didnot respond to crizotinib (Fig. 2B). Our results indicated that M-COPA abolished cell surface expression (Fig. 1B) and processingof MET, but it hardly affected phosphorylation level of S6 ribo-somal protein (Supplementary Fig. S1B) and did not exhibit highsensitivity to M-COPA (Fig. 2A). In contrast, St-4 expresses sub-stantial amount of EGFR at the cell surface, but M-COPA hardlyattenuated cell surface expression of EGFR (Supplementary Fig.S3). The precise mechanism by which these differences in M-COPA response were achieved remains unclear. Interestingly,phosphorylated form of MET was detected but that of EGFR washardly detected in the St-4 cell line before drug exposure (Fig. 1A),suggesting that cell surface expression of RTKs in its activated formcould be selectively abolished by M-COPA treatment. Involve-ment of phosphorylated status of RTKs in M-COPA efficacy isunder investigation.

Besides RTKs, we examined the effect of M-COPA on ABCtransporters such as P-glycoprotein andBCRP. AlthoughM-COPAreduced cell surface expression of BCRP inBCRP-expressing breastcancer HBC-5 cells, it did not affect cell surface expression of P-glycoprotein in adriamycin-resistant AD10 cells derived fromhuman ovarian cancer A2780 cells (Supplementary Fig. S4). Wehave not yet determined the mechanism by which these differ-ences caused, but we supposed that the differences in coatedvesicles (e.g., COPI-coated or clathrin-coated vesicles) may causedifferent M-COPA response. Further studies are needed to clarifythe mechanism by which M-COPA attenuates protein expressionat the cell surface.

There are three approaches to downregulation of HGF/METsignaling in human clinical studies: anti-HGF mAbs, anti-METmAbs, and small-molecule MET TKIs (42). Class I TKIs, such ascrizotinib, bind to the MET ATP–binding pocket and showspecificity againstMET and someother tyrosine kinases, includingALK (43). Crizotinib seemed to exert better antitumor activities toMET-amplified GC xenografts (44), in other words, M-COPAresponse was modest as compared with crizotinib (Fig. 5). How-ever, crizotinib usage has been reported to induce a secondmutation at the gatekeeper position of the ATP-binding pocketof the targeted kinases, resulting in acquired resistance (45, 46).Class II TKIs such as cabozantinib, show broad specificity ascompared with class I inhibitors, binding to a region past thegatekeeper position and occupying a hydrophobic pocket at adeeper location (47). Thepresent results imply thatGolgi-targeteddrugs such as M-COPA could be a novel therapeutic option inaddition to mAbs and TKIs for treating GCs addicted to MET orFGFR2 via inhibition of the processing and the transport of MET/FGFR2onto the cell surface.Moreover, this class of drug could alsobe useful for treating cancers fromdifferent tissues of originwhosegrowth is dependent on RTK expression, especially those harbor-

ing a secondary mutation exhibiting acquired resistance to pre-treated TKIs. In this situation, the Golgi-targeted drugs are alsoexpected to overcome the TKI resistance by inhibiting cell surfaceexpression of the mutated RTKs. The effect of M-COPA on otherRTK-addicted cancer cells (e.g., EGFR-mutated lung cancer) andTKI-resistant cells is under investigation.

We demonstrated that M-COPA exhibited higher sensitivityto MET-addicted cell lines than MET non-addicted cell lines(Fig. 2A); however, the selectivity for MET-addicted cells wasnot as marked as that seen with crizotinib. In other words, M-COPA did partially interfere with the growth of MET non-addicted GC cells. Although we demonstrated that neitherEGFR, HER2, HER3, nor FGFR2 were activated in these celllines, involvement of other RTKs or other cell surface proteinsshould be considered. BFA, another Golgi disruptor, is knownto trigger ER stress and unfolded protein response (48), andsimilar efficacy of M-COPA is expected. Involvement of thispathway in antitumor effect of M-COPA in both MET-addictedand non-addicted cancer cells is under investigation. On theother hand, a selection of chemotherapeutic drugs, apart fromSN-38, did not display any evidence of MET status-specificsensitivity in the panel of MET amplified and unamplified celllines tested (Fig. 2C–F). The reason why SN-38 was moresensitive toward MET-addicted cell lines than nonaddicted celllines remains unclear.

In M-COPA–treated MET-amplified cell lines, processing ofMET protein was suppressed, but the loss of mature MET b-chainwas not accompanied by a corresponding increase of MET pre-cursor, especially in SNU-5 cells. At present, we have not yetelucidated the precisemechanism bywhich this occurred, and thefate of MET protein in M-COPA treated cells remains unclear.Unfolded protein response is known to attenuate translationinitiation via phosphorylation of eIF2 alpha by PERK (48).Therefore, one possibility is that attenuation of total MET proteinmight be occurred as a consequence of general translationsuppression.

We assessed M-COPA–induced toxicity by measuring bodyweight loss. As described before, slight weight loss was observedin the daily administration group, whereas weight loss wasalleviated after stopping administration. Concurrently, loss ofweight was observed in tumor bearing control mice, as well asthoseweekly administered, in accordancewith theprevious reportthat inoculation of MKN45 cells into nude mice caused cachexiaand body weight loss (49), which resulted in no significantdifference among the three groups on day 8 and later.

In conclusion, we demonstrated that M-COPA inhibited theprocessing and the transport of MET protein onto the cell surface,attenuated aberrant MET signaling, and exerted a preferentialantitumor activity of M-COPA against MET-addicted GCs. Thepresent results suggested that a Golgi-targeted drug could be anovel therapeutic modality that has a unique mode of action fortargeting RTK, in addition to mAb and TKI therapies.

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

Authors' ContributionsConception and design: Y. Ohashi, S. DanDevelopment of methodology: Y. Ohashi, M. Okamura, I. ShiinaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Ohashi, M. Okamura, A. Hirosawa, N. Tamaki






Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Ohashi, M. OkamuraWriting, review, and/or revision of the manuscript: Y. Ohashi, H.-W. Choi,I. Shiina, S. DanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Akatsuka, H.-W. Choi, I. ShiinaStudy supervision: K. Yoshimatsu, T. Yamori, S. DanOther (compound synthesis): K.-M. Wu

AcknowledgmentsThe authors thank Kanami Yamazaki and Yumiko Nishimura for their

technical assistance.

Grant SupportThis work is supported by Adaptable & Seamless Technology Transfer

Program through Target-driven R&D (A-STEP; AS2614144Q) in 2014 from theJapan Science and Technology Agency (JST) and in 2015 from the Japan Agencyfor Medical Research and Development (AMED), a grant from the VehicleRacing Commemorative Foundation, and a grant from National Cancer CenterResearch Development Fund (#26-A-5).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 12, 2015; revised February 24, 2016; accepted March 28,2016; published OnlineFirst April 12, 2016.

References1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.

Cancer incidence and mortality worldwide: sources, methods and majorpatterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86.

2. Cancer Genome Atlas Research N. Comprehensive molecular characteri-zation of gastric adenocarcinoma. Nature 2014;513:202–9.

3. Sebolt-Leopold JS, English JM.Mechanisms of drug inhibition of signallingmolecules. Nature 2006;441:457–62.

4. Herbst RS, Fukuoka M, Baselga J. Gefitinib–a novel targeted approach totreating cancer. Nat Rev Cancer 2004;4:956–65.

5. Emr S, Glick BS, Linstedt AD, Lippincott-Schwartz J, Luini A, Malhotra V,et al. Journeys through the Golgi–taking stock in a new era. J Cell Biol2009;187:449–53.

6. Malhotra V, Mayor S. Cell biology: the Golgi grows up. Nature 2006;441:939–40.

7. Patterson GH, Hirschberg K, Polishchuk RS, Gerlich D, Phair RD, Lippin-cott-Schwartz J. Transport through the Golgi apparatus by rapid partition-ing within a two-phase membrane system. Cell 2008;133:1055–67.

8. Xu D, Esko JD. A Golgi-on-a-chip for glycan synthesis. Nat Chem Biol2009;5:612–3.

9. Johannes L, Popoff V. Tracing the retrograde route in protein trafficking.Cell 2008;135:1175–87.

10. Brandizzi F, Barlowe C. Organization of the ER-Golgi interface for mem-brane traffic control. Nat Rev Mol Cell Biol 2013;14:382–92.

11. De Matteis MA, Luini A. Exiting the Golgi complex. Nat Rev Mol Cell Biol2008;9:273–84.

12. UngarD.Golgi linked protein glycosylation and associated diseases. SeminCell Dev Biol 2009;20:762–9.

13. Donaldson JG, Jackson CL. ARF family G proteins and their regulators:roles in membrane transport, development and disease. Nat Rev Mol CellBiol 2011;12:362–75.

14. D'Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane trafficand beyond. Nat Rev Mol Cell Biol 2006;7:347–58.

15. Ooi CE,Dell'Angelica EC, Bonifacino JS. ADP-Ribosylation factor 1 (ARF1)regulates recruitment of the AP-3 adaptor complex to membranes. J CellBiol 1998;142:391–402.

16. Popoff V, Langer JD, Reckmann I, Hellwig A, Kahn RA, Brugger B, et al.Several ADP-ribosylation factor (Arf) isoforms support COPI vesicle for-mation. J Biol Chem 2011;286:35634–42.

17. Puertollano R, Randazzo PA, Presley JF, Hartnell LM, Bonifacino JS. TheGGAs promote ARF-dependent recruitment of clathrin to the TGN. Cell2001;105:93–102.

18. Serafini T, Orci L, Amherdt M, Brunner M, Kahn RA, Rothman JE. ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coatedvesicles: a novel role for a GTP-binding protein. Cell 1991;67:239–53.

19. StamnesMA, Rothman JE. The binding of AP-1 clathrin adaptor particles toGolgi membranes requires ADP-ribosylation factor, a small GTP-bindingprotein. Cell 1993;73:999–1005.

20. Ohashi Y, Iijima H, Yamaotsu N, Yamazaki K, Sato S, Okamura M, et al.AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylationfactor 1 (Arf1) with potential for cancer therapy. J Biol Chem 2012;287:3885–97.

21. Watari K,NakamuraM, FukunagaY, FurunoA, Shibata T, Kawahara A, et al.The antitumor effect of a novel angiogenesis inhibitor (an octahydro-

naphthalene derivative) targeting both VEGF receptor and NF-kappaBpathway. Int J Cancer 2012;131:310–21.

22. Shiina I, Umezaki Y, Ohashi Y, Yamazaki Y, Dan S, Yamori T. Totalsynthesis of AMF-26, an antitumor agent for inhibition of theGolgi system,targeting ADP-ribosylation factor 1. J Med Chem 2013;56:150–9.

23. Yamori T, Sato S, Chikazawa H, Kadota T. Anti-tumor efficacy ofpaclitaxel against human lung cancer xenografts. Jpn J Cancer Res1997;88:1205–10.

24. Yamori T, Matsunaga A, Sato S, Yamazaki K, Komi A, Ishizu K, et al. Potentantitumor activity ofMS-247, a novelDNAminor groove binder, evaluatedby an in vitro and in vivo human cancer cell line panel. Cancer Res1999;59:4042–9.

25. SugimotoY, Tsukahara S,Oh-haraT, Isoe T, TsuruoT.Decreased expressionof DNA topoisomerase I in camptothecin-resistant tumor cell lines asdetermined by a monoclonal antibody. Cancer Res 1990;50:6925–30.

26. SkehanP, StorengR, ScudieroD,MonksA,McMahon J, VisticaD, et al.Newcolorimetric cytotoxicity assay for anticancer-drug screening. J Natl CancerInst 1990;82:1107–12.

27. Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L,et al. Display and analysis of patterns of differential activity of drugs againsthuman tumor cell lines: development of mean graph and COMPAREalgorithm. J Natl Cancer Inst 1989;81:1088–92.

28. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al.Feasibility of a high-flux anticancer drug screen using a diverse panel ofcultured human tumor cell lines. J Natl Cancer Inst 1991;83:757–66.

29. Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, et al.Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinaseinhibitor. J Natl Cancer Inst 2006;98:545–56.

30. KawakamiH,Okamoto I, Arao T,OkamotoW,MatsumotoK, TaniguchiH,et al. MET amplification as a potential therapeutic target in gastric cancer.Oncotarget 2013;4:9–17.

31. Okamoto W, Okamoto I, Arao T, Kuwata K, Hatashita E, Yamaguchi H,et al. Antitumor action of the MET tyrosine kinase inhibitor crizotinib (PF-02341066) in gastric cancer positive for MET amplification. Mol CancerTher 2012;11:1557–64.

32. Giordano S, Ponzetto C, Di RenzoMF, Cooper CS, Comoglio PM. Tyrosinekinase receptor indistinguishable from the c-met protein. Nature1989;339:155–6.

33. Giordano S, Di Renzo MF, Narsimhan RP, Cooper CS, Rosa C, ComoglioPM. Biosynthesis of the protein encoded by the c-met proto-oncogene.Oncogene 1989;4:1383–8.

34. Kunii K, Davis L, Gorenstein J, Hatch H, Yashiro M, Di Bacco A, et al.FGFR2-amplified gastric cancer cell lines require FGFR2 and Erbb3 signal-ing for growth and survival. Cancer Res 2008;68:2340–8.

35. Xie L, Su X, Zhang L, Yin X, Tang L, Zhang X, et al. FGFR2 gene amplificationin gastric cancer predicts sensitivity to the selective FGFR inhibitorAZD4547. Clin Cancer Res 2013;19:2572–83.

36. Crepaldi T, Prat M, Giordano S, Medico E, Comoglio PM. Generation of atruncated hepatocyte growth factor receptor in the endoplasmic reticulum.J Biol Chem 1994;269:1750–5.

37. Eder JP, Vande Woude GF, Boerner SA, LoRusso PM. Novel therapeuticinhibitors of the c-Met signaling pathway in cancer. Clin cancer Res2009;15:2207–14.


Ohashi et al.




38. Comoglio PM, Giordano S, Trusolino L. Drug development of METinhibitors: targeting oncogene addiction and expedience. Nat Rev DrugDiscov 2008;7:504–16.

39. Guo A, Villen J, Kornhauser J, Lee KA, Stokes MP, Rikova K, et al. Signalingnetworks assembled by oncogenic EGFR and c-Met. Proc Natl Acad SciU S A 2008;105:692–7.

40. Yun C, Gang L, Rongmin G, Xu W, Xuezhi M, Huanqiu C. Essential roleof Her3 in two signaling transduction patterns: Her2/Her3 and MET/Her3 in proliferation of human gastric cancer. Mol Carcinog 2015;54:1700–9.

41. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al.MET amplification leads to gefitinib resistance in lung cancer by activatingERBB3 signaling. Science 2007;316:1039–43.

42. Cui JJ.Targeting receptor tyrosine kinase MET in cancer: smallmolecule inhibitors and clinical progress. J Med Chem 2014;57:4427–53.

43. Zou HY, Li Q, Lee JH, Arango ME, McDonnell SR, Yamazaki S, et al. Anorally available small-molecule inhibitor of c-Met, PF-2341066, exhi-bits cytoreductive antitumor efficacy through antiproliferative andantiangiogenic mechanisms. Cancer Res 2007;67:4408–17.

44. Okamoto W, Okamoto I, Arao T, Kuwata K, Hatashita E, Yamaguchi H,et al. Antitumor action of the MET tyrosine kinase inhibitor crizotinib (PF-02341066) in gastric cancer positive for MET amplification. Mol CancerTher 2012;11:1557–64.

45. Qi J, McTigue MA, Rogers A, Lifshits E, Christensen JG, Janne PA, et al.Multiplemutations and bypassmechanisms can contribute to developmentof acquired resistance to MET inhibitors. Cancer Res 2011;71:1081–91.

46. Katayama R, Khan TM, Benes C, Lifshits E, Ebi H, Rivera VM, et al.Therapeutic strategies to overcome crizotinib resistance in non-small celllung cancers harboring the fusion oncogene EML4-ALK. Proc Natl Acad SciU S A 2011;108:7535–40.

47. Grullich C. Cabozantinib: a MET, RET, and VEGFR2 tyrosine kinaseinhibitor. Recent Results Cancer Res 2014;201:207–14.

48. Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential fortranslational regulation and cell survival during the unfolded proteinresponse. Mol Cell 2000;5:897–904.

49. Mori T, Fujiwara Y, Yano M, Tamura S, Yasuda T, Takiguchi S, et al.Prevention of peritoneal metastasis of human gastric cancer cells in nudemice by S-1, a novel oral derivative of 5-Fluorouracil. Oncology 2003;64:176–82.






2016;76:3895-3903. Published OnlineFirst April 12, 2016.Cancer Res Yoshimi Ohashi, Mutsumi Okamura, Asaka Hirosawa, et al. Gastric CancersProtein and Exhibits Antitumor Activity against MET-Addicted M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET

Updated version

10.1158/0008-5472.CAN-15-2220doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2016/04/12/0008-5472.CAN-15-2220.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/76/13/3895.full#ref-list-1

This article cites 49 articles, 17 of which you can access for free 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://cancerres.aacrjournals.org/content/76/13/3895To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-15-2220

http://cancerres.aacrjournals.org/content/suppl/2016/04/12/0008-5472.CAN-15-2220.DC1

http://cancerres.aacrjournals.org/content/76/13/3895.full#ref-list-1

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/76/13/3895


m-copa, a golgi disruptor, inhibits cell surface ... · 1division of molecular pharmacology, cancer...

Documents