M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET

Published OnlineFirst April 12, 2016; DOI: 10.1158/0008-5472.CAN-15-2220 Cancer Therapeutics, Targets, and Chemical Biology Research

M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET Protein and Exhibits Antitumor Activity against MET-Addicted Gastric Cancers Yoshimi Ohashi1, Mutsumi Okamura1, Asaka Hirosawa1, Naomi Tamaki1, Akinobu Akatsuka1, Kuo-Ming Wu2, Hyeong-Wook Choi2, Kentaro Yoshimatsu3, Isamu Shiina4, Takao Yamori1, and Shingo Dan1

Abstract

The Golgi apparatus is responsible for transporting, processing, expression of MET was downregulated with a concurrent accumu- and sorting numerous proteins in the cell, including cell surface- lation of its precursor form. M-COPA also reduced levels of the expressed receptor tyrosine kinases (RTK). The small-molecule phosphorylated form of MET along with the downstream signaling compound M-COPA [2-methylcoprophilinamide (AMF-26)] dis- molecules Akt and S6. Similar results were obtained in additional rupts the Golgi apparatus by inhibiting the activation of Arf1, GC cell lines with amplification of MET or the FGF receptor FGFR2. resulting in suppression of tumor growth. Here, we report an MKN45 murine xenograft experiments demonstrated the antitu- evaluation of M-COPA activity against RTK-addicted cancers, mor activity of M-COPA in vivo. Taken together, our results offer an focusing specifically on human gastric cancer (GC) cells with or initial preclinical proof of concept for the use of M-COPA as a without MET amplification. As expected, the MET-addicted cell candidate treatment option for MET-addicted GC, with broader line MKN45 exhibited a better response to M-COPA than cell lines implications for targeting the Golgi apparatus as a novel cancer without MET amplification. Upon M-COPA treatment, cell surface therapeutic approach. Cancer Res; 76(13); 1–9. 2016 AACR.

Introduction "hepatocyte growth factor receptor") and fibroblast growth factor receptor 2 (FGFR2), which have been shown to drive cells to Gastric cancer (GC) is the third leading cause of cancer-related malignant proliferation and survival (3, 4). death worldwide, and its incidence remains high especially in east Thanks to recent progress in understanding molecular path- Asia, including Japan and Korea (1). GC is often diagnosed at an ways underlying carcinogenesis, new targeted treatment options advanced stage, and the prognosis of such patients is poor. Owing have become available for treating cancer patients. In respect to to its diversity of morphologic forms and considerable heteroge- the development of targeted therapies, monoclonal antibodies neity, the classification of GC has been complicated. Recently, The (mAb) and small-molecule inhibitors of tyrosine kinase (TKI) Cancer Genome Atlas (TCGA) Research Network proposed a new activity are ideal candidates that target tumor cells via binding to molecular classification of GC into four subtypes (2). Among RTKs. For example, trastuzumab is an mAb developed for treating these, approximately 50% of gastric tumors were categorized as HER2-positive breast cancer; gefitinib is a small-molecule TKI- the chromosomal unstable subtype, containing frequent gene targeting EGFR developed for treating lung cancer. So far, six mAbs amplification of receptor tyrosine kinases (RTK), such as human and 23 TKIs have been developed as approved drugs for treating epidermal growth factor receptor 2 (HER2), MET (also called cancer; however, trastuzumab and ramucirumab are the only two molecularly targeted drugs approved to date for treating GC patients. Therefore, the development of new targeted drugs 1Division of Molecular Pharmacology, Cancer Chemotherapy Center, fi 2 against GCs is of keen interest. Because gene ampli cation of Japanese Foundation for Cancer Research, Tokyo, Japan. Next Gen- MET HER2 FGFR2 eration Systems, Eisai Inc., Andover, Massachusetts. 3Eisai Product , , and is often observed in GCs, many TKIs Creation Systems, Eisai Co., Ltd., Tokyo, Japan. 4Department of targeting these RTKs have been developed and several clinical Applied Chemistry, Faculty of Science, Tokyo University of Science, trials in GC patients are in progress. Tokyo, Japan. The Golgi apparatus plays an essential role in the transport, Note: Supplementary data for this article are available at Cancer Research processing, and sorting of numerous proteins (5–8). Most cell- Online (http://cancerres.aacrjournals.org/). surface and secreted proteins in eukaryotic cells pass through the Current address for K.-M. Wu: Department of Chemical Development, Concert Golgi apparatus, allowing for posttranslational modification such Pharmaceuticals, Inc., Lexington, MA; and current address for T. Yamori: Center as processing and glycosylation, and subsequently transport to for Product Evaluation, Pharmaceuticals and Medical Devices Agency, Tokyo, plasma membrane (9–11). Aberration of Golgi function is asso- Japan. ciated with certain forms of inherited diseases, cancer, and dia- Corresponding Author: Shingo Dan, Japanese Foundation for Cancer Research, betes (12). ADP ribosylation factor 1 (Arf1), a small GTPase and a 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan. Phone: 81-3-3520-0111; Fax: 81-3- member of Ras superfamily (13, 14), is required for maintenance 3570-0484; E-mail: [email protected] of Golgi structure and function via formation of complex I (COPI) doi: 10.1158/0008-5472.CAN-15-2220 or clathrin-coated vesicles transported among the endoplasmic 2016 American Association for Cancer Research. reticulum (ER), Golgi, and post-Golgi (15–19). We previously

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demonstrated that M-COPA (2-methylcoprophilinamide, also the cell line was performed in 2016 and compared the profile with called "AMF-26") suppressed Arf1-mediated vesicle transport, that in the ATCC STR database. disrupted the structure of the Golgi apparatus, and exerted antitumor activity in vivo against tumors xenografted into mice (20). Analysis of cell growth inhibition In fact, M-COPA has been shown to inhibit secretion of intercel- The inhibition of cell growth was assessed by measuring changes lular adhesion molecule-1 (ICAM-1) and cell surface expression in total cellular protein in a culture of each GC cell line after 48 of VEGFR-2 (21). Therefore, we postulated that M-COPA could hours of drug treatment by use of a sulforhodamine B assay (26). inhibit the processing and transport of RTKs to the cell surface and Positive values represent net protein increase before and after drug extracellular space, and thereby exert antitumor activity against exposure (% of control growth) and negative values represent cell RTK-addicted cancers. death [protein amount after 48 hours-exposure (%) of control cells In this study, using GC cell lines with or without MET ampli- at the start of drug exposure]. The drug concentration required for fication, we demonstrated that M-COPA inhibited the processing 50% reduction in net protein increase (GI50) was calculated as and transport of MET protein onto the cell surface, attenuated described previously (23, 27, 28). aberrant MET signaling and exerted preferential antitumor activity against MET-addicted GCs. Moreover, we obtained similar results Flow cytometric analysis in FGFR2-amplified signet ring GC cells KATO III. The present Cells were incubated with M-COPA for 6 or 24 hours, and then results suggest that a Golgi-targeted drug could be a novel ther- washed with ice-cold PBS and stained with antibodies against apeutic modality against MET-addicted GCs, as well as perhaps human MET or human FGFR2 conjugated with phycoerythrin RTK-addicted cancers from different origins. (PE). Then cells were washed three times with ice-cold PBS, and stained with propidium iodide (1 mg/mL; Sigma-Aldrich). The Materials and Methods fluorescence intensity of cell surface MET or FGFR2 was measured by flow cytometric analysis (FACS Calibur or FACS Verse, Becton, Chemicals Dickinson and Company). The data were analyzed by using M-COPA (also called "AMF-26," chemical name: (2E,4E)-5- FlowJo (FlowJo LLC.). [(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- Western blot analysis ylmethyl)penta-2,4-dienamide] was totally synthesized by Cells were incubated with M-COPA for 1, 6, or 24 hours, and Eisai Co., Ltd. according to the methods established previously then lysed as described previously (29). Proteins in cell lysates (22). 5-FU and paclitaxel were purchased from Sigma-Aldrich were separated in 4% to 15% sodium dodecyl sulfate-polyacryl- Co. LLC., crizotinib was purchased from Selleck Chemicals. amide gel (Bio-Rad Laboratories) electrophoresis, followed by For in vitro studies, these compounds were reconstituted to 20 electroblotting onto a nitrocellulose membrane (Bio-Rad Labo- mmol/L in DMSO (Sigma-Aldrich) and stored at 20C. SN- ratories). Immunoreactive bands were identified with an ODYS- 38 was purchased from Sigma-Aldrich, and cisplatin was SEY CLx Infrared Imaging System (LI-COR Biosciences). purchased from Nippon Kayaku Co., Ltd. For animal experiments, M-COPA was suspended in 0.15N hydrochloride acid Animal experiments (Wako Pure Chemical Industries). Antibodies used for immu- The antitumor effect of M-COPA was tested in vivo against nostaining are listed in Supplementary Table S1. MKN45-derived human GC xenografts in mice. Animal care and treatment was performed in accordance with the guidelines of the Cell lines and cell culture Animal Use and Care Committee of the Japanese Foundation for Four GC cell lines (St-4, MKN1, MKN45, and MKN74) are Cancer Research, and conformed to the NIH Guide for the Care and components of the JFCR39 panel of human cancer cell lines Use of Laboratory Animals. Female nude mice of BALB/c genetic described previously (23, 24). Of these, St-4 was established in background were purchased from Charles River Laboratories our foundation in 1990 (25). MKN1, MKN45, and MKN74 were JAPAN, Inc., maintained under specific pathogen-free conditions purchased from Immuno-Biological Laboratories Co., Ltd. in and provided with sterile food and water ad libitum. Each nude 1991. Other two GC cell lines, Hs-746T, and SNU-5 were pur- mouse was subcutaneously inoculated with a generated tumor chased from the ATCC in 2014. Cell lines were cultured in RPMI- fragment of size 3 mm 3mm 3 mm. When the tumors reached 1640 medium (Wako Pure Chemical Industries) supplemented a volume of 100 to 300 mm3, animals were randomly divided into with 5% (v/v) FBS (Moregate Biotech), penicillin (100 U/mL), control and M-COPA groups (each group containing five or six streptomycin (100 mg/mL), and kanamycin (1 mg/mL) in a mice). Then administration of M-COPA was started (day 0). The humidified atmosphere including 5% CO2 at 37 C. Authentica- experimental group of mice was orally administered a given dose of tion of St-4, MKN1, MKN45, and MKN74 cell lines was performed M-COPA (50 mg/kg of BW) on a daily basis from day 0 to 4 (n ¼ 6), by short tandem repeat (STR) analysis using PowerPlex16 Systems or weekly (75 mg/kg of BW) on day 0, 7, and 14 (n ¼ 5). The control (Promega) according to the manufacturer's instructions by BEX group of mice (n ¼ 6) was orally administered with 0.15N CO., LTD., in 2009 and 2016. Details were described in Supple- hydrochloride acid solution instead of M-COPA. The tumor vol- mental Materials and Methods. Finally, the STR profiles of MKN1, ume of tumor-bearing mice was measured as described previously MKN45 and MKN74 were compared with those in the reference (29). To assess toxicity, the body weights of the tumor-bearing mice database of the Japanese Collection of Research Bioresources Cell were measured. Bank. Because St-4 was developed in our foundation and no reference data was deposited, we compared the profile deter- Immunohistochemistry mined in 2016 to that in 2009. The KATO III cell line was Formalin-fixed, paraffin-embedded tissue sections (4-mm- originally purchased from the ATCC in 2000. Authentication of thick) were deparaffinized in xylene and taken through a series

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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 and electroblotted onto a membrane. The membrane was then probed with antibodies against the indicated proteins. Experiments were performed at least twice and representative results are indicated. The positive control lysates were as follows: EGFR and p-EGFR from NCI-H3255 that had EGFR-activating mutation, 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, MET expression 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 stained with a PE-conjugated anti-MET antibody. Lines and areas were used to indicate drug concentrations: Black solid lines with dark gray area, no drug; black dotted 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; gray solid lines, stained with isotype-control IgG. Experiments were performed at least twice and representative results are indicated.

of graded alcohols to water. Then antigens were retrieved through Statistical analysis wet autoclave pretreatment (20 min at 121C) in 10 mmol/L The two-sided Mann–Whitney U test was used to assess the citrate buffer (pH 6.0). Sections were blocked with 3% H2O2 and statistical significance of the antitumor efficacy of M-COPA in terms 1% goat serum before incubation with the primary antibody at of relative tumor growth ratio and body weight change on days 2, 4, 4C overnight. By using Dako EnVision Detection System/HRP, 8, 11, 15 and 18. The number of samples is indicated in the Rabbit/Mouse (DABþ; Agilent Technologies Company), sections description of each experiment. All statistical tests were two-sided. were incubated with horseradish peroxidase (HRP)–conjugated polymer secondary antibody, and thereafter peroxidase activity was visualized by DAB reaction according to the manufacturer's Results instructions. The sections were counterstained with hematoxylin Overexpression of MET protein and the Inhibitory effect of M- (Dako). The immuno-stained specimens were imaged using a COPA on its cell surface expression in MET-amplified GC cells microscope BX41 (Olympus Corp.) with a 20x, NA 0.50, objec- First, we examined the effect of M-COPA on cell surface tive, and DP2-BSW Software (Olympus Corp.). expression of MET protein in GC cell lines. To this end, we

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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 its phosphorylated active form, as previously reported (30). The St-4 cell line also expressed total and phosphorylated form of MET, even though the intensities of those expression were weaker than those of MET-amplified GC cells. We also examined baseline expression of EGFR, HER2, HER3, and FGFR2, which have been reported to be amplified in GC cells (2). None of the phosphorylated forms of these proteins except HER3 in MKN45 were detected in any of the GC cell lines examined, although total EGFR and HER3 were detected in some cell lines. We then examined cell surface expression of MET and the effect of 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 that observed in other GC cell lines, in parallel with the levels of total MET protein determined by immunoblot analysis (Fig. 1A). Upon treatment with M-COPA for 24 hours, cell surface expression of MET efficiently decreased in a dose-dependent manner; it was significantly reduced at concentrations of 100 nmol/L or higher in SNU-5 and MKN45 cells, whereas 300 nmol/L was needed to mediate such reduction in Hs-746T cells. Next, we examined the expression profile of MET protein over time following treatment of MKN45 cells with M-COPA. After 6 hours treatment, MET protein expression was reduced to approximately 25% (1/4) at concentrations of 100 nmol/L or higher and was dramatically declined 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 that M-COPA efficiently inhibited cell surface expression of MET protein in MET-amplified GC cells, in a dose- and time-dependent manner.

Growth inhibitory effect of M-COPA against GC cell lines with Figure 2. or without MET amplification Cell growth inhibition of M-COPA against human GC cell lines. M-COPA We next evaluated the effect of M-COPA on the growth of GC inhibited the cell growth of MET-addicted cell lines in a more robust manner MET fi than that observed with non-addicted cell lines. The inhibition of cell cell lines with or without ampli cation. Expectedly, all three proliferation was assessed by measuring changes in total cellular protein. MET fi -ampli ed cell lines were highly sensitive to the MET inhib- After 48 hours of drug treatment, cells were fixed and stained by use of a itor crizotinib (Fig. 2B), as compared with other GC cell lines, sulforhodamine B assay. Growth curves under drug treatment with M-COPA indicating that their growth was addicted to MET, in agreement (A), crizotinib (B), or typical antitumor agents (C–F) used in GC therapy are with previous reports (31). Interestingly, MET-amplified cell shown; 5-FU (C), paclitaxel (D), SN-38 (E), and cisplatin (F). Black circle, St-4; lines also exhibited a better drug response to M-COPA than black square, MKN1; black triangle, MKN74; red circle, MKN45; red square, Hs- MET fi 746T; red triangle, SNU-5. Experiments were performed at least twice and those without ampli cation, albeit the selectivity was not representative results are indicated. as marked as that observed with crizotinib (Fig. 2A). Moreover, the concentrations of M-COPA needed to inhibit cell growth was comparable with those needed to inhibit cell surface expression of MET in the MET-amplified cell lines; the GI50 The effect of M-COPA on processing of MET protein and its concentration for the MKN45, Hs-746T, and SNU-5 cell lines downstream signaling molecules in MET-amplified GC cells was 29, 40, and 19 nmol/L, respectively. These data indicated MET is a transmembrane heterodimer that composed of two that M-COPA preferentially inhibited the growth of MET- disulfide-linked chains of 50 kDa a-subunit and 145 kDa b-sub- addicted GC cells and the growth inhibition coincided with unit (32). The molecule is originally synthesized as a single-chain decreased expression of MET protein on the cell surface. For 170-kDa precursor (Pr170), which is cotranslationally glycosy- comparison, the anticancer effects of chemotherapeutic agents lated. Terminal glycosylation and proteolytic cleavage generate used in the clinic for GC patients, namely 5-FU, paclitaxel, SN- the mature heterodimer (33). To clarify whether M-COPA could 38, and cisplatin, were examined. However, neither 5-FU, disturb processing of MET, expression levels of the precursor form paclitaxel nor cisplatin exhibited significant differences in terms and a mature b-subunit of MET were estimated in MKN45 cells by of growth inhibitory activities in the GC cell lines with MET Western blot analysis. As shown in Fig. 3A, the amount of the amplification with the only exception that SNU-5 tended to be Pr170 was increased upon treatment with M-COPA in a dose- highly sensitive to these agents. On the other hand, SN-38 dependent manner at 6 hours, whereas the active b-subunit and exhibited preferential activity in these MET-amplified cell lines phosphorylated form of MET were decreased. Finally, the active in a similar vein to M-COPA (Fig. 2C–F). phosphorylated form of MET were dramatically reduced at

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regulation of cell surface expression, electrophoretic mobility shift probably due to inhibition of post-translational modification, and dephosphorylation of FGFR2 protein, and finally decreased phosphorylated form of downstream signaling molecules such as Akt and S6 (Fig. 4A and B). These effects were observed in the similar concentration range to that required for inhibition of cell growth (Fig. 4C). These results suggested that M-COPA suppressed cell surface expression of FGFR2 as a result of Golgi dysfunction, and thereby exerted antitumor effect in FGFR2- amplified cells, as well as MET-amplified cells.

Antitumor efficacy of M-COPA against MKN45-derived tumor xenografts in vivo Finally, we tested the antitumor efficacy of M-COPA against tumor xenografts derived from the MET-amplified GC cell line,

Figure 3. Time-course and dose-dependent effects on processing of MET protein, and phosphorylation status of downstream signaling molecules. M-COPA inhibited cell surface expression, processing, and phosphorylation of MET and its downstream signaling at the same concentration range and time points as observed with respect to the inhibitory effect on cell surface expression of MET in MKN45 cells. A and B, after M-COPA treatment with indicated concentrations for 1, 6, or 24 hours, MKN45 cells were lysed. A, processing status and phosphorylation status of MET protein. B, phosphorylation status of signaling molecules, including Gab2, Akt and ribosomal S6 protein (S6), were examined by immunoblot analysis. concentrations of 100 nmol/L or higher in 24 hours, in parallel to inhibition of cell surface expression of MET (Fig. 1C) and cell growth (Fig. 2A). These results indicated that the processing and the transport of MET protein onto the cell surface were prevented by M-COPA treatment. We next examined the effect of M-COPA on the activation status of the downstream signaling pathway from MET. M-COPA decreased the levels of phosphorylated Gab2, Akt, and S6 within the same drug concentration range and time-course as those required for inhibition of MET processing and transport (Fig. 3B). The effectiveness of M-COPA was also demonstrated in other two MET-addicted GC cell lines, Hs-746T and SNU-5 (Supple- mentary Fig. S1A). M-COPA also inhibited the cell surface expression of MET in St-4 cells (Fig. 1B). Therefore, we investigated the activation status of MET and its downstream signaling pathway molecules. As shown in Supplementary Fig. S1B, M-COPA repressed the MET processing, MET-activation, and phosphorylation of Akt. However, phosphorylation of S6 ribosomal protein was still remained by M-COPA treatment, and concordantly St-4 Figure 4. cell line showed lower sensitivity to crizotinib and M-COPA The antitumor effect of M-COPA against an FGFR2-amplified KATO III cell line (Fig. 2A). From these data, we concluded that M-COPA inhibited via inhibition on the cell surface expression of FGFR2. M-COPA repressed cell the MET-dependent signaling pathway via inhibition of MET surface expression of FGFR2, maturation of FGFR2 protein and phosphorylation status of downstream signaling molecules, and cell growth processing and its cell surface expression as mediated by the in vitro in dose–respond manner. A, FGFR2 expression on the cell surface was Golgi apparatus, resulted in attenuating the abnormal cell measured by FACS analysis. Cells were treated with M-COPA at the indicated proliferation in MET-amplified GC cell lines. concentrations for 24 hours and stained with an anti-FGFR2 antibody, in consequent with a PE-conjugated second antibody. Lines and areas were The antitumor effect of M-COPA against FGFR2-amplified GC used as described in Fig. 1B legend. B, after M-COPA treatment with indicated cells concentrations for 24 hours, KATO III cells were harvested and cell lysates were prepared. Immunoblot analysis of total and phosphorylated form of To examine the effect of other RTKs amplified in GCs, we FGFR2 fi FGFR2 protein and phosphorylation status of downstream signaling exploited KATO III, which was known as a -ampli ed molecules, including Akt and S6 ribosomal protein (S6), were examined. signet ring cell GC cell line that exhibited a high sensitivity to C, the inhibition of cell proliferation was assessed by sulforhodamine B assay. FGFR2-TKI (34, 35). As we expected, M-COPA caused down- Symbols were used as described in Fig. 2 legend.

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Figure 5. Antitumor efficacy of M-COPA against MET-amplified tumor xenograft in vivo. Tumor fragments derived from human GC cell line MKN45 were subcutaneously inoculated 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 differences from 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).

MKN45, in vivo. M-COPA was orally administrated daily for the effectors, resulting in hyperactivation of intracellular downstream first 5 days, or weekly for 3 weeks. In both administration groups, signaling pathways, including the PI3K–AKT axis (37). This abnor- treatment with M-COPA significantly decreased the tumor size mal signal activation is known to drive cells to malignant prolif- than that observed in the control group (Fig. 5A, top). Slight but eration, thereby entering a "MET-addicted" state (38, 39). In the significant weight loss was observed in the daily administration present study, we clearly demonstrated that M-COPA decreased group on days 2 and 4; however, after stopping administration, no the level of the active b-subunit form and increased the precursor significant difference was observed among the three groups on form in a dose-dependent manner, consistent with a previous day 8 and later (Fig. 5A, bottom). report showing that a Golgi inhibitor brefeldin A (BFA) abrogated To validate the proof of concept that M-COPA administra- the processing of nascent MET protein (36). Moreover, we found tion exerts an antitumor effect via inhibition of cell surface for the first time that inhibition of MET processing by M-COPA expression of MET, we examined the expression of phosphor- coincided with downregulation of its cell surface expression and ylated MET in tumor tissue sections by immunohistochemistry. abrogation of its downstream oncogenic signals represented by The immunostaining intensities of phosphorylated MET on the reduction of phosphorylated forms of Gab, Akt, and S6, and cell membrane were markedly decreased in the M-COPA–trea- ultimately suppressed tumor growth in MET-addicted GC cell ted group on day 2 compared with those in the control group lines. Downregulation of cell surface expression of activated MET (Fig. 5B and C). These data strongly suggested that M-COPA protein in parallel to the observed antitumor effects was also exerted an in vivo antitumor effect on MET-amplified MKN45- confirmed in the case of MKN45-derived tumor xenografts after derived xenografts via downregulation of cell surface expres- administration of M-COPA in vivo. These results strongly suggested sion of active MET. that inhibition of processing and cell surface expression of MET protein is the main mechanism by which M-COPA exerts antitumor effects against MET-addicted GC cells. In addition to MET, Discussion phosphorylated HER3 was also detected in MKN45 cells (Fig. 1A), As mentioned, MET protein is produced as a 170 kDa single- as previously reported (40). MET has been shown to interact with chain precursor form, and the precursor is then posttranslationally HER3 and activate HER3 signal in gastric and lung cancer cells (40, glycosylated, cleaved to yield a mature form consisted of an 41). In our study, M-COPA also repressed phosphorylation and a-chain (50 kDa) and a b-chain (145 kDa) via the Golgi apparatus cell surface expression of HER3 (Supplementary Fig. S2), as well as (32, 33, 36). In MET-amplified cells, overexpression of MET MET, suggesting that repression of HER3 signal could also be protein triggers its autophosphorylation and recruitment of its involved in antitumor effect of M-COPA in MKN45 cells.

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We also demonstrated that cell surface expression of FGFR2 was ing a secondary mutation exhibiting acquired resistance to pre- also downregulated by M-COPA treatment in FGFR2-addicted treated TKIs. In this situation, the Golgi-targeted drugs are also KATO III, a human signet ring cell GC cell line (Fig. 4). FGFR2 is expected to overcome the TKI resistance by inhibiting cell surface known as a target for cancer therapy, and now several FGFR2-TKIs, expression of the mutated RTKs. The effect of M-COPA on other such as AZD4547, are in clinical study stage. M-COPA also RTK-addicted cancer cells (e.g., EGFR-mutated lung cancer) and repressed the maturation of FGFR2, phosphorylation of its sig- TKI-resistant cells is under investigation. naling pathway molecules, and inhibited cell growth under the We demonstrated that M-COPA exhibited higher sensitivity concentration of submicromole order. These data strongly sug- to MET-addicted cell lines than MET non-addicted cell lines gested that M-COPA could exert antitumor effect in cancer cells (Fig. 2A); however, the selectivity for MET-addicted cells was addicted to not only MET but also FGFR2 and other RTKs, not as marked as that seen with crizotinib. In other words, M- including HER2, mutant EGFR, and so on. COPA did partially interfere with the growth of MET non- St-4 is a cell line that expresses small amount of MET protein addicted GC cells. Although we demonstrated that neither (Fig. 1A). This cell line is not addicted to MET expression and it did EGFR, HER2, HER3, nor FGFR2 were activated in these cell not respond to crizotinib (Fig. 2B). Our results indicated that M- lines, involvement of other RTKs or other cell surface proteins COPA abolished cell surface expression (Fig. 1B) and processing should be considered. BFA, another Golgi disruptor, is known of MET, but it hardly affected phosphorylation level of S6 ribo- to trigger ER stress and unfolded protein response (48), and somal protein (Supplementary Fig. S1B) and did not exhibit high similar efficacy of M-COPA is expected. Involvement of this sensitivity to M-COPA (Fig. 2A). In contrast, St-4 expresses sub- pathway in antitumor effect of M-COPA in both MET-addicted stantial amount of EGFR at the cell surface, but M-COPA hardly and non-addicted cancer cells is under investigation. On the attenuated cell surface expression of EGFR (Supplementary Fig. other hand, a selection of chemotherapeutic drugs, apart from S3). The precise mechanism by which these differences in M- SN-38, did not display any evidence of MET status-specific COPA response were achieved remains unclear. Interestingly, sensitivity in the panel of MET amplified and unamplified cell phosphorylated form of MET was detected but that of EGFR was lines tested (Fig. 2C –F). The reason why SN-38 was more hardly detected in the St-4 cell line before drug exposure (Fig. 1A), sensitive toward MET-addicted cell lines than nonaddicted cell suggesting that cell surface expression of RTKs in its activated form lines remains unclear. could be selectively abolished by M-COPA treatment. Involve- In M-COPA–treated MET-amplified cell lines, processing of ment of phosphorylated status of RTKs in M-COPA efficacy is MET protein was suppressed, but the loss of mature MET b-chain under investigation. was not accompanied by a corresponding increase of MET pre- Besides RTKs, we examined the effect of M-COPA on ABC cursor, especially in SNU-5 cells. At present, we have not yet transporters such as P-glycoprotein and BCRP. Although M-COPA elucidated the precise mechanism by which this occurred, and the reduced cell surface expression of BCRP in BCRP-expressing breast fate of MET protein in M-COPA treated cells remains unclear. cancer HBC-5 cells, it did not affect cell surface expression of P- Unfolded protein response is known to attenuate translation glycoprotein in adriamycin-resistant AD10 cells derived from initiation via phosphorylation of eIF2 alpha by PERK (48). human ovarian cancer A2780 cells (Supplementary Fig. S4). We Therefore, one possibility is that attenuation of total MET protein have not yet determined the mechanism by which these differ- might be occurred as a consequence of general translation ences caused, but we supposed that the differences in coated suppression. vesicles (e.g., COPI-coated or clathrin-coated vesicles) may cause We assessed M-COPA–induced toxicity by measuring body different M-COPA response. Further studies are needed to clarify weight loss. As described before, slight weight loss was observed the mechanism by which M-COPA attenuates protein expression in the daily administration group, whereas weight loss was at the cell surface. alleviated after stopping administration. Concurrently, loss of There are three approaches to downregulation of HGF/MET weight was observed in tumor bearing control mice, as well as signaling in human clinical studies: anti-HGF mAbs, anti-MET those weekly administered, in accordance with the previous report mAbs, and small-molecule MET TKIs (42). Class I TKIs, such as that inoculation of MKN45 cells into nude mice caused cachexia crizotinib, bind to the MET ATP–binding pocket and show and body weight loss (49), which resulted in no significant specificity against MET and some other tyrosine kinases, including difference among the three groups on day 8 and later. ALK (43). Crizotinib seemed to exert better antitumor activities to In conclusion, we demonstrated that M-COPA inhibited the MET-amplified GC xenografts (44), in other words, M-COPA processing and the transport of MET protein onto the cell surface, response was modest as compared with crizotinib (Fig. 5). How- attenuated aberrant MET signaling, and exerted a preferential ever, crizotinib usage has been reported to induce a second antitumor activity of M-COPA against MET-addicted GCs. The mutation at the gatekeeper position of the ATP-binding pocket present results suggested that a Golgi-targeted drug could be a of the targeted kinases, resulting in acquired resistance (45, 46). novel therapeutic modality that has a unique mode of action for Class II TKIs such as cabozantinib, show broad specificity as targeting RTK, in addition to mAb and TKI therapies. compared with class I inhibitors, binding to a region past the gatekeeper position and occupying a hydrophobic pocket at a Disclosure of Potential Conflicts of Interest deeper location (47). The present results imply that Golgi-targeted No potential conflicts of interest were disclosed. drugs such as M-COPA could be a novel therapeutic option in addition to mAbs and TKIs for treating GCs addicted to MET or Authors' Contributions FGFR2 via inhibition of the processing and the transport of MET/ Conception and design: Y. Ohashi, S. Dan FGFR2 onto the cell surface. Moreover, this class of drug could also Development of methodology: Y. Ohashi, M. Okamura, I. Shiina be useful for treating cancers from different tissues of origin whose Acquisition of data (provided animals, acquired and managed patients, growth is dependent on RTK expression, especially those harbor- provided facilities, etc.): Y. Ohashi, M. Okamura, A. Hirosawa, N. Tamaki

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Ohashi et al.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Grant Support computational analysis): Y. Ohashi, M. Okamura This work is supported by Adaptable & Seamless Technology Transfer Writing, review, and/or revision of the manuscript: Y. Ohashi, H.-W. Choi, Program through Target-driven R&D (A-STEP; AS2614144Q) in 2014 from the I. Shiina, S. Dan Japan Science and Technology Agency (JST) and in 2015 from the Japan Agency Administrative, technical, or material support (i.e., reporting or organizing for Medical Research and Development (AMED), a grant from the Vehicle data, constructing databases): A. Akatsuka, H.-W. Choi, I. Shiina Racing Commemorative Foundation, and a grant from National Cancer Center Study supervision: K. Yoshimatsu, T. Yamori, S. Dan Research Development Fund (#26-A-5). Other (compound synthesis): K.-M. Wu The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments The authors thank Kanami Yamazaki and Yumiko Nishimura for their Received August 12, 2015; revised February 24, 2016; accepted March 28, technical assistance. 2016; published OnlineFirst April 12, 2016.

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M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET Protein and Exhibits Antitumor Activity against MET-Addicted Gastric Cancers

Yoshimi Ohashi, Mutsumi Okamura, Asaka Hirosawa, et al.

Cancer Res Published OnlineFirst April 12, 2016.

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