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Open Access

Peer-reviewed

Research Article

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

  • Yasushi Hara,

    Affiliation Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan

    ⨯
  • Yuuki Obata ,

    yobata@rs.tus.ac.jp

    Affiliation Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan

    ⨯
  • Keita Horikawa,

    Affiliation Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan

    ⨯
  • Yasutaka Tasaki,

    Affiliation Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan

    ⨯
  • Kyohei Suzuki,

    Affiliation Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan

    ⨯
  • Takatsugu Murata,

    Affiliation Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan

    ⨯
  • Isamu Shiina,

    Affiliation Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan

    ⨯
  • Ryo Abe

    Affiliation Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan

    ⨯

Abstract

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.

Citation: Hara Y, Obata Y, Horikawa K, Tasaki Y, Suzuki K, Murata T, et al. (2017) M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells. PLoS ONE 12(4): e0175514. https://doi.org/10.1371/journal.pone.0175514

Editor: Kevin D. Bunting, Emory University, UNITED STATES

Received: December 13, 2016; Accepted: March 27, 2017; Published: April 12, 2017

Copyright: © 2017 Hara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (to Y. Obata 16K18427, and R. Abe 15K06843, http://www.jsps.go.jp) and the Uehara Memorial Foundation (to Y. Obata K15-148, http://www.ueharazaidan.or.jp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Kit, a cell-surface receptor for stem cell factor (SCF), belongs to the type III receptor tyrosine kinase family that includes platelet-derived growth factor receptor α/β (PDGFRα/β), Flt3, and Fms [1–4]. It plays a pivotal role in the development of mast cells, interstitial cells of Cajal (ICC), hematopoietic cells, germ cells, and melanocytes [3–5].

Kit is composed of five N-glycosylated immunoglobulin domains in the N-terminal extracellular portion that bind SCF, a transmembrane domain, and the carboxy-terminal intracellular tyrosine kinase domain [4–6]. The binding of SCF autophosphorylates Kit on specific tyrosine residues (Tyr), such as Tyr568/570, Tyr703, Tyr721, and Tyr936 [5–8]. Kit then binds to other cytoplasmic enzymes, such as PI3K and Src kinases, and this complex activates downstream molecules [5–9]. This activates the Akt-Bad pathway and the Ras-Mek-Erk cascade, which regulate gene expression and cytoskeletal structures, resulting in cell proliferation and survival [7–11].

In many mast cell neoplasms and GISTs, Kit develops gain-of-function mutations, causing permanent, ligand-independent activation of the receptor [12–15]. Mutant Kit transforms mast cells and ICC through permanent activation of the PI3K-Akt pathway and STATs resulting in the development of mast cell tumours and GISTs [16–20]. Thus, the drug imatinib, a selective inhibitor of Kit, improved the prognosis of GIST patients [15,21,22]. However, imatinib treatment is ineffective in most cases of mast cell tumours because they express imatinib-resistant Kit that has a mutation in the kinase domain [12,13,23,24]. Furthermore, GISTs frequently acquire a secondary mutation in the Kit kinase domain, resulting in imatinib resistance [15,25]. Considering that other cancer-causing receptors develop drug resistance in a manner similar to imatinib-resistant Kit [26], a new strategy for inhibition of receptors bearing a mutation in the kinase domain is desirable.

In human neoplastic mast cell disorders such as mastocytosis and mast cell leukemia, Kit often has an Asp816Val substitution in the kinase domain (D816V) [12,13,27] (see Fig 1A). Similar mutations are also found in mouse mastocytoma (eg, D814Y and D814V) [12,13,28]. We recently reported that KitD814Y activates the PI3K-Akt pathway and STAT5 on endolysosomes and the ER, respectively [29]. Furthermore, a recent study described a new inhibition strategy for Flt3 receptor kinase bearing a mutation in the kinase domain through blocking the receptor trafficking with fluvastatin [30]. These findings provided evidence that intracellular trafficking of mutant receptors is a promising target for the treatment of cancers.

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Fig 1. Mutations in the Kit kinase domain confer imatinib resistance to neoplastic mast cells.

(A) Schematic representations of wild-type Kit (KitWT) and mutant Kit showing the extracellular domain (ECD), the transmembrane domain (TM), and the kinase domain. Asp814 and Asp816, D in black; Tyr814 and Val816, Y and V in red. In addition to D816V, V560G is found in HMC-1.2’s Kit, but we did not find a difference between RCM and HMC-1.2 in Kit localization and oncogenic signalling. (B) RCM (left) and HMC-1.2 (right) were treated with vehicle (0), imatinib (Kit kinase inhibitor, closed circles), or PKC412 (inhibits type III tyrosine kinase, open circles) for 24 hours. Proliferation was assessed by [3H]-thymidine incorporation. Results (c.p.m.) are means ± SD (n = 3). (C) RCM and HMC-1.2 were treated with vehicle, 1 μM PKC412 or 5 μM imatinib (IMA) for 16 hours. Cell lysates were immunoblotted with anti-Kit, anti-phospho-KitTyr721 (anti-pKitTyr721), anti-Akt, anti-pAkt, anti-STAT5, anti-pSTAT5, and anti-cleaved caspase-3. Note that imatinib did not affect the phosphorylation of Kit or cell proliferation.

https://doi.org/10.1371/journal.pone.0175514.g001

M-COPA is a novel inhibitor of ADP-ribosylation factor 1 (ARF1), which plays a role in vesicular trafficking [31,32]. Recent studies showed that M-COPA blocks the trafficking of Met receptor kinase along the secretory pathway to the PM, resulting in suppression of receptor-ligand binding [33,34]. Thus, M-COPA has an anti-tumour effect in vivo. However, whether M-COPA inhibits oncogenic signalling of mutant receptors that are localized on organelles such as endolysosomes is unknown.

Here, we show that in neoplastic mast cells from mice and humans, M-COPA significantly inhibits the biosynthetic trafficking of imatinib-resistant Kit mutant (hereafter, referred to as Kitmut) from the ER. In M-COPA-treated cells, Kitmut is unable to activate Akt because endolysosomal Kitmut disappears. Since Kitmut activates STAT5 selectively on the ER, M-COPA enhances STAT5 activation. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting the anti-apoptotic Akt activation that occurs only on endolysosomes. Results of our M-COPA treatment assay show that Kitmut can activate Erk not only on the ER but also on other organelles. Furthermore, Kit Tyr568/570, Tyr703, Tyr721, and Tyr936 are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated on the ER before Kitmut reaches the PM. Our study provides evidence that Kitmut is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.

Materials and methods

Cell culture

RCM (R cell mutant Kit) cells [29] were established from splenocytes of DO11.10 mice by repeated stimulation with ovalbumin peptides in vitro. This cell line exhibits a mast cell-like surface phenotype, Kit+ FcεRI+, and mast cell-like expression profiles of proteases. Moreover, RCM cells can secrete biologically active products, such as histamine and β-hexosaminidase, upon stimulation. They homozygously express KitD814Y and proliferate autonomously. HMC-1.2 (human mast cell line-1.2) cells [27] were established from a patient with mast cell leukemia. The cell line heterozygously expresses KitV560G,D816V and shows autonomous proliferation. Both cell lines were cultured at 37°C in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin, glutamine (Pen/Strep/Gln), and 50 μM 2-mercaptoethanol. A lung adenocarcinoma cell line A549 was purchased from American Type Culture Collection (Manassas, VA, USA), and the cells were cultured at 37°C in RPMI1640 medium supplemented with 10% FCS, and Pen/Strep/Gln.

Cell proliferation assay

RCM and HMC-1.2 cells were cultured with inhibitors for 16 hours, and then treated with [3H]-thymidine deoxyribonucleotide (TdR) for 8 hours. Cell proliferation was evaluated by the incorporation of [3H]-TdR.

Antibodies

The following antibodies were purchased: Kit (M-14), STAT5 (C-17), Erk2 (K-23) and cathepsin D (H-75) from Santa Cruz Biotechnology (Dallas, TX); Kit[pTyr719], Kit[pTyr703], Akt (40D4), Akt[pT308] (C31E5E), STAT5[pTyr694] (D47E7), Erk[pThr202/pTyr204] (E10), and cleaved caspase-3 from Cell Signaling Technology (Danvers, MA); p85, phospho-tyrosine (4G10), and Kit[pTyr568/570] from Millipore (Billerica, MA); LAMP1 from Sigma (St. Louis, MO); Kit[pTyr936] from Thermo Scientific Pierce (Rockford, IL) and calnexin from Enzo Life Sciences (Farmingdale, NY). HRP-labeled anti-mouse Ig, anti-rabbit Ig, and anti-goat Ig secondary antibodies were purchased from the Jackson Laboratory (Bar Harbor, MA). Alexa-Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).

Chemicals

Imatinib (Cayman Chemical, Ann Arbor, MI), PKC412 (Santa Cruz Biotechnology), and Akt inhibitor VIII (Millipore) were dissolved in dimethylsulfoxide (DMSO). Monensin (Biomol, Exeter, UK), brefeldin A (Wako, Osaka, Japan), and bafilomycin A1 (Sigma) were dissolved in ethanol. M-COPA was synthesized as previously described [31] and dissolved in DMSO.

Immunofluorescence confocal microscopy

Cells were fixed with methanol for 10 minutes at -20°C, then cyto-centrifuged onto coverslips. Fixed cells were permeabilized and blocked for 30 minutes in PBS supplemented with 0.1% saponin and 3% BSA, and then incubated with a primary and secondary antibody for 1 hour each. After washing with PBS, cells were mounted with Fluoromount (DiagnosticBioSystems, Pleasanton, CA). Confocal images were obtained with a Fluoview FV10i laser scanning microscope with an x60 1.20 N.A. water-immersion objective (Olympus, Tokyo, Japan). Composite figures were prepared with Photoshop Elements 10 and Illustrator CS6 software (Adobe, San Jose, CA). Pearson’s R correlation coefficients were calculated with NIH ImageJ 1.48v software.

Immunoprecipitation and western blotting

Lysates from 1~2 x 106 cells were prepared in SDS-PAGE sample buffer or NP-40 lysis buffer (50 mM HEPES, pH 7.4, 10% glycerol, 1% NP-40, 4 mM EDTA, 100 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM PMSF, and 1 mM Na3VO4). Immunoprecipitation was performed at 4°C for 5 hours using protein G pre-coated with antibody. Immunoprecipitates were dissolved in SDS-PAGE sample buffer, subjected to SDS-PAGE, and electro-transferred onto PVDF membranes. Immunodetection was performed by ECL (PerkinElmer, Waltham, MA). Sequential re-probing of membranes was performed after the complete removal of primary and secondary antibodies in stripping buffer, or by inactivation with peroxidase in 0.1% NaN3. Results were analyzed with an LAS-3000 image analyzer with Science Lab software (Fujifilm, Tokyo, Japan) or with a c-Digit imaging system with Image Studio Digit software (Licor Biosciences, Lincoln, NE).

Analysis of protein glycosylation

Following the manufacturer’s instructions (New England Biolabs, Ipswich, MA), NP-40 cell lysates were treated with endoglycosidase H (endo H) or peptide-N-glycosidase F (PNGase F) for 1 hour at 37°C. The reactions were stopped with SDS-PAGE sample buffer, and products were resolved by SDS-PAGE and immunoblotted.

Gene silencing of KitD814Y with small interfering RNAs (siRNAs) and electroporation

For silencing KitD814Y, siRNA duplexes were purchased from Sigma (Kit1: GAAGGAUUAUGUCAAAUCUTT, Kit2: GACAUGAAGCCUGGCGUUUTT). The control siRNA duplex was also purchased from Sigma (Mission negative control SIC-001). For knockdown of KitD814Y, cells were transfected using a Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA) and cultured for 20 hours.

Statistical analyses

For statistical analysis, experiments were repeated as three biological replicates. Differences between two or more groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post-hoc test. All significant differences showed a 5% level of probability.

Results

RCM and HMC-1.2 can proliferate in the presence of imatinib due to mutations in the Kit kinase domain

We recently established a mast cell line from mouse splenocytes, made up of RCM cells bearing Kit and FcεRI [29]. These cells grow without cytokines and develop tumours in vivo. RCM cells homozygously express KitD814Y, an imatinib-resistant mutant [29] (Fig 1A). First, to confirm whether the proliferation of RCM was imatinib-resistant, we performed an [3H]-thymidine deoxyribonucleotide ([3H]-TdR) uptake assay. Fig 1B shows that RCM cells proliferated in the presence of imatinib at any of the concentrations used (closed circles). Similarly, the proliferation of HMC-1.2 (a human mast cell line established from a mast cell leukemia patient), which heterozygously express KitV560G,D816V [12,13,27] (Fig 1A), was unaffected by imatinib treatment (Fig 1B, right, closed circles). These results indicate that imatinib does not have an inhibitory effect on growth of these neoplastic mast cells. In support of this, Western blotting analysis showed that the phosphorylation of Kit as well as of Akt and STAT5 was unaffected by imatinib treatment (Fig 1C). As previously reported [16,18,29,35], Kitmut knockdown by siRNAs and Kit inhibition by PKC412 (multi-tyrosine kinase inhibitor) greatly inhibited the growth of RCM and HMC-1.2 (Fig 1B, open circles and S1 Fig), confirming Kit-dependent growth of these cells. In addition, PKC412 markedly decreased the activation of Kitmut, resulting in inhibition of Akt and STAT5 (Fig 1C). Inhibition of Kitmut by PKC412 can induce cleavage of caspase-3, a sign of apoptosis [16,19], but imatinib did not have the same effect (Fig 1C, lower panels). Taken together, the results suggest that these cells proliferate in the presence of imatinib in a manner dependent on their Kit kinase domain mutant.

M-COPA, a novel inhibitor of the secretory pathway, blocks biosynthetic transport of Kitmut from the ER

We previously reported that in neoplastic mast cells, Kit-dependent activation of Akt and STAT5 occurs on endolysosomes and the ER, respectively, and that inhibition of Akt is sufficient for suppression of cell proliferation [29]. Recent studies showed that M-COPA has an anti-cancer effect in Met tyrosine kinase-addicted cancers in vivo through inhibition of the trafficking of the receptor to the PM [31–33]. Therefore, we investigated the effect of M-COPA on oncogenic Kit signalling in neoplastic mast cells. Immunofluorescence confocal microscopic analysis showed that in RCM and HMC-1.2, Kit co-localized with the endolysosome marker cathepsin D (Fig 2A and S2A Fig), as previously described [29]. M-COPA decreased the endolysosomal localization of Kit in a dose-dependent manner (Fig 2B and S2B Fig). With Pearson’s R correlation coefficient intensity analysis, we found that M-COPA significantly increased co-localization of Kit with the ER marker calnexin (Fig 2C and S2B Fig), indicating that the trafficking of Kit from the ER is inhibited by M-COPA treatment. Taken together with the fact that Kitmut localized to endolysosomes through endocytosis from the PM after moving along the secretory pathway [29], these results suggest that M-COPA decreases endolysosomal Kit through blocking the biosynthetic transport of Kit from the ER.

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Fig 2. In neoplastic mast cells, M-COPA inhibits Kit trafficking from the ER, resulting in a decrease in endolysosomal Kit.

(A) RCM cells were immunostained with anti-Kit (green) and anti-cathepsin D (endolysosome marker, red). Insets indicate magnified images of the boxed area. Bar, 10 μm. (B) RCM cells were treated with vehicle or 0.1~5 μM M-COPA for 16 hours, then stained for Kit (green) and calnexin (ER marker, red). Insets indicate magnified images of the boxed area. Bars, 10 μm. (C) Pearson’s R correlation coefficients were calculated by intensity analysis of Kit vs. calnexin. Results are means ± SD (n = 11~36). Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. ***P < 0.001; NS, not significant. Note that in RCM cells, co-localization of Kit with calnexin was significantly increased by M-COPA treatment.

https://doi.org/10.1371/journal.pone.0175514.g002

M-COPA can inhibit Akt activation through blocking the localization of Kitmut to endolysosomes in naoplastic mast cells

Next, we tested whether M-COPA suppressed oncogenic activation of Akt through blocking Kit trafficking in RCM and HMC-1.2. As shown in Fig 3A and 3B, Kit shifted to a lower molecular weight form on M-COPA treatment. Since partially glycosylated receptors in the ER subsequently move to the Golgi apparatus for further glycosylation [24,29,36,37], these results support our observation that M-COPA blocks Kit trafficking from the ER. Phosphorylation of Kit at Tyr721, which is critical for activation of the PI3K-Akt pathway [5,10,38], was unaffected by M-COPA treatment (Fig 3A and 3B), indicating that phosphorylation of the tyrosine residue occurs on the ER. Furthermore, using a co-immunoprecipitation assay, we tested whether M-COPA decreased the association of Kit with PI3K. Fig 3C shows that the PI3K p85 subunit was co-immunoprecipitated with Kit, and that ER accumulation of Kit by M-COPA did not affect the association of Kit with p85. Interestingly, M-COPA markedly decreased the activation of Akt, and the effect was correlated with a decreased ER export of Kit (Fig 3A and 3B). These results indicate that in the ER, the Kit-PI3K complex is unable to activate Akt. On the other hand, STAT5 phosphorylation was enhanced by M-COPA treatment (Fig 3A and 3B), supporting our previous finding that Kitmut activates STAT5 on the ER in neoplastic mast cells [29]. Similar results were obtained with RCM cells treated with a well-known ER-to-Golgi trafficking inhibitor brefeldin A (BFA) (S3A–S3C Fig), indicating that through blocking ER export of Kitmut, M-COPA inhibits and enhances the activation of Akt and of STAT5, respectively.

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Fig 3. In neoplastic mast cells, M-COPA inhibits Kit-dependent Akt activation through blocking the biosynthetic transport of Kit from the ER.

(A-C) RCM (A) and HMC-1.2 (B) were treated for 16 hours with vehicle (0) or 0.1~5 μM M-COPA. Cell lysates were immunoblotted with anti-Kit, anti-phospho-KitTyr721 (anti-pKitTyr721), anti-Akt, anti-pAkt, anti-STAT5, anti-pSTAT5, and anti-cleaved caspase-3. The graphs show the levels of pSTAT5 (open circles) or pAkt (closed circles) expressed relative to lysate from vehicle-treated cells. CG, complex-glycosylated form; HM, high mannose form. (C) RCM and HMC-1.2 were treated with 5 μM M-COPA for 16 hours. Anti-Kit immunoprecipitates were immunoblotted with anti-Kit and anti-PI3K p85. Note that M-COPA inhibited Akt activation without affecting the phosphorylation of Kit at Tyr721 or the association of Kit with PI3K p85. (D) RCM (left) and HMC-1.2 (right) were treated with vehicle (0), M-COPA (open circles) or imatinib (closed circles) for 24 hours. Proliferation was assessed by [3H]-thymidine incorporation. Results (c.p.m.) are means ± SD (n = 3).

https://doi.org/10.1371/journal.pone.0175514.g003

Next, we determined whether M-COPA inhibited Akt activation through blockade of Kit glycosylation or Kit trafficking. Previously, we reported that bafilomycin A1 (bafA1) inhibits Kit-dependent Akt activation through endosome-endolysosome trafficking [29] (S4A Fig). Thus, we treated Kit from bafA1-treated cells with endoglycosidase H (endo H), which digests immature glycan but not mature glycan. In bafA1-treated cells, a majority of Kit was not digested by endo H, indicating that Kitmut cannot activate Akt before reaching endolysosomes even if Kit glycosylation is normal (S4B Fig). These results suggest that M-COPA inhibits the Kit-Akt pathway through blocking Kit localization to endolysosomes, probably rather than through inhibiting Kit glycosylation.

In neoplastic mast cells, M-COPA induces apoptosis through inhibiting Kit-dependent Akt activation that occurs only on endolysosomes

S5A and S5B Fig show that blockade of Akt with Akt inhibitor VIII (Akti VIII) suppressed RCM proliferation and induced apoptosis. Furthermore, induction of apoptosis by M-COPA was correlated with the decrease in Akt activation (Fig 3A and 3B, bottom panels). To confirm whether M-COPA induced apoptosis through inhibiting Akt phosphorylation, we treated a Kitmut-negative lung adenocarcinoma cell line A549 with M-COPA. As shown in S5C Fig, blockade of ER export with M-COPA did not result in Akt inhibition and caspase-3 cleavage in A549 cells, supporting our finding that the drug can cause apoptosis through inhibition of the Kit-PI3K-Akt pathway. As shown in Fig 3D, RCM and HMC-1.2 were unable to proliferate in the presence of M-COPA because they underwent apoptosis. These results suggest that M-COPA inhibits the growth of neoplastic mast cells through blocking the anti-apoptotic activation of the Kit-Akt pathway that occurs only on endolysosomes.

In neoplastic mast cells, Kitmut can activate Erk not only on the ER but also on other compartments

Previous studies showed that Kit activates Erk in various cells [4,5,11]. Indeed, Kitmut knockdown with siRNA and Kit inhibition with PKC412 decreased Erk phosphorylation in RCM cells (Fig 4A and S6A Fig), confirming that Kitmut activates Erk in RCM cells. We thus investigated the effect of M-COPA on Kit-dependent Erk activation. In RCM cells, Erk phosphorylation was decreased but remained in M-COPA-treated cells (Fig 4B). Similar results were obtained with BFA-treated RCM cells (S6B Fig). Next, we tested the effect of bafA1 (inhibitor of endosome trafficking) or monensin (inhibitor of Golgi export) on Erk activation. As shown in S6C Fig, similar to M-COPA, these inhibitors had only a partial inhibitory effect on Erk phosphorylation. Although we could not determine the signalling platform for the Kit-Erk pathway in this study, these results indicate that Kitmut can activate Erk not only on the ER but also on other organelles. We previously reported that Erk inhibition by U0126 does not affect proliferation of RCM cells [29]. However, it is possible that the Kit-Erk pathway in neoplastic mast cells plays a role in other cellular events, such as migration and degranulation [39]. Thus, further study will be required for understanding the role of the Kit-Mek-Erk pathway in the pathogenesis of mast cell tumours.

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Fig 4. Effect of M-COPA treatment on Kit-dependent Erk activation in RCM cells.

(A and B) RCM cells were treated with vehicle (0), (A) 1 μM PKC412 for 24 hours, or (B) 0.2~5 μM M-COPA for 16 hours. Cell lysates were immunoblotted with anti-Erk and anti-phospho-Erk (anti-pErk).

https://doi.org/10.1371/journal.pone.0175514.g004

In neoplastic mast cells, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kitmut are phosphorylated on the ER

The critical tyrosine phosphorylation sites in Kit responsible for its downstream activation are in the juxtamembrane region, the kinase domain, and the carboxy-terminal region (referred to henceforth as pTyr568/570, pTyr703, pTyr721, and pTyr936, see Fig 5A) [5–8,10,40]. Previously, we reported that in GIST cell lines, Tyr568/570 and Tyr703 in mutant Kit are dephosphorylated on the ER [24]. Thus we examined whether these five tyrosine residues were phosphorylated on the ER in neoplastic mast cells. Interestingly, pTyr568/570, pTyr703, and pTyr936 remained both in M-COPA-treated and BFA-treated cells, similar to pTyr721 (Fig 5B–5D, and S3D and S3E Fig; see also Fig 3A and 3B). In addition, phosphorylation of these tyrosine residues was unaffected by treatment with monensin, an inhibitor of Golgi export (Fig 5E and 5F). These results suggest that in neoplastic mast cells, the five tyrosine residues in Kitmut are phosphorylated on the ER, not after reaching the PM.

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Fig 5. In neoplastic mast cells, Kit phosphorylation at Tyr568/570, Tyr703, Tyr721, and Tyr936 occurs on the ER.

(A) Tyrosine phosphorylation sites in human Kit. (B-F) Cells were treated with vehicle or (B-D) 5 μM M-COPA for 16 hours or (E and F) 250 nM monensin for 24 hours. Anti-Kit immunoprecipitates (B and E) and cell lysates (C, D, and F) were immunoblotted with anti-Kit, anti-phospho-KitTyr568/570 (anti-pKitTyr568/570), anti-pKitTyr703, anti-pKitTyr721, and anti-pKitTyr936. Note that ER-localized Kit was phosphorylated at Tyr568/570, Tyr703, Tyr721 and Tyr936 in neoplastic mast cells.

https://doi.org/10.1371/journal.pone.0175514.g005

Discussion

In this study, we demonstrate that M-COPA inhibits autonomous proliferation of neoplastic mast cells through blocking the trafficking of Kitmut from the ER since oncogenic activation of the Kit-PI3K-Akt pathway occurs only on endolysosomes (Fig 6A). Furthermore, results of our M-COPA treatment assay show that Kit Tyr568/570, Tyr703, Tyr721, and Tyr936 are phosphorylated on the ER, not after reaching the PM (Fig 6B). In addition to previous reports of a leukemia therapy using inhibition of Flt3 trafficking [30,37], our study shows that inhibition of mutant receptor trafficking represents a promising strategy for the treatment of cancers.

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Fig 6. Model of the effect of M-COPA on Kit signalling and on autophosphorylation.

(A) In neoplastic mast cells, mutant Kit is trafficked to the PM along the secretory pathway and then moves to endolysosomes through endocytosis. Kit activates Akt only on endolysosomes as previously described (Obata et al., 2014). Note that M-COPA inhibits the activation of Akt through blocking ER export of Kit. STAT5 activation is enhanced by M-COPA because it is activated by ER-localized Kit. HM, high mannose; CG, complex glycosylation. (B) Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER in neoplastic mast cells.

https://doi.org/10.1371/journal.pone.0175514.g006

Previous studies showed that cancer-causing receptors accumulate and initiate oncogenic signals on intracellular compartments [41,42]. PDGFRαV561D expressed in HEK293 and FGFR3K650E in multiple myeloma cells accumulate on the Golgi apparatus where they activate downstream molecules such as Akt, STATs, and Erk [41,43–45]. EGFRL858R in lung adenocarcinoma and gp130(ΔYY) and Met in hepatoma cause oncogenic signalling on endosomes [42,46–50]. In addition, the localization of receptors bearing secondary mutations that confer a drug resistance is similar to that of receptors bearing primary mutations [24,46,47]. Considering that all mutant receptors are biosynthetically trafficked from the ER and that M-COPA can block the localization of receptors to the signalling platforms, the drug may inhibit their signalling. Our study will help to develop a strategy for the treatment of cancers that have receptors with secondary mutations.

Similar to mast cell tumours, GISTs frequently have constitutively active mutations. Mutant Kit in GISTs is localized and activated on the perinuclear compartment characterized as the Golgi apparatus [24,51,52]. Unlike mast cell tumours, Kit activates Akt, STAT5, and Erk only on the Golgi apparatus in GISTs [24]. Although its sub-cellular localization in GISTs is different from that in mast cell tumours, M-COPA may inhibit oncogenic signalling through blocking the localization of Kit to the signalling platform. Investigation of the effect of M-COPA on GIST growth is now under way.

In neoplastic mast cells, Kit Tyr568/570 and Tyr703 are phosphorylated, whereas in GISTs, the tyrosine residues are dephosphorylated [24]. This may explain why Kitmut in the ER can activate STAT5 and Erk only in mast cell tumours. Since in GIST cells, mast cell tumour-type Kit mutant (KitD814Y) is localized and autophosphorylated on the Golgi apparatus [24], the host environment may determine the Kit signaling platform. Further study will be required for understanding the differences between mast cell tumours and GISTs in the role of ER-localized Kit.

Several inhibitors of protein trafficking, such as brefeldin A (BFA), Exo1, Exo2, and AG1478, have been reported [53–56]. However, BFA has less stability in vivo while the others exhibit weaker inhibition of cell growth, and their development as drugs has not progressed. On the other hand, M-COPA had an anti-cancer effect in vivo experiments using breast cancer or gastric cancer xenograft models [32,33], and it is now considered a novel anticancer drug candidate. Together with the fact that M-COPA suppressed imatinib-resistant Kit signalling, inhibition of receptor trafficking is a promising approach for the treatment of cancers bearing a resistant mutation to targeting therapy.

Conclusion

We demonstrate that M-COPA can suppress the proliferation of neoplastic mast cells through blockade of Kitmut trafficking to the signalling platform such as at endolysosomes. Furthermore, this study shows the phosphorylation states of Kitmut on the ER. Our observations will open new fields for developing therapies for cancers that express mutant Kit, such as mast cell tumours, GISTs, acute myeloid leukemia, and melanomas and will be helpful for understanding the spatiotemporal regulation of tyrosine phosphorylation signalling.

Supporting information

S1 Fig. KitD814Y is essential for autonomous proliferation of RCM cells.

(A and B) RCM cells were transfected with control siRNA or Kit siRNAs (Kit1 or Kit2) and cultured for 20 hours. (A) Cell lysates were immunoblotted with anti-Kit and anti-phospho-tyrosine (anti-pTyr). Total protein levels were confirmed by Coomassie staining. (B) The graph shows the levels of [3H]-thymidine incorporation into RCM cells. Results (c.p.m.) are means ± s.d. (n = 3). Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. ***P < 0.001.

https://doi.org/10.1371/journal.pone.0175514.s001

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S2 Fig. M-COPA inhibits Kit trafficking from the ER in HMC-1.2 cells.

(A) HMC-1.2 cells were immunostained with anti-Kit (green) and anti-cathepsin D (endolysosome marker, red) Insets indicate magnified images of the boxed area. Bar, 10 μm. (B) HMC-1.2 cells were treated with vehicle or 0.5~5 μM M-COPA for 16 hours, then stained for Kit (green) and calnexin (ER marker, red). Insets indicate magnified images of the boxed area. Bars, 10 μm. The graph shows Pearson’s R correlation coefficients calculated between Kit and calnexin. Results are means ± SD (n = 14~30). Data were subjected to one-way ANOVA with Dunnett’s multiple comparison post-hoc test. ***P < 0.001. Note that in HMC-1.2 cells, co-localization of Kit with calnexin was significantly increased by M-COPA treatment.

https://doi.org/10.1371/journal.pone.0175514.s002

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S3 Fig. Effect of BFA on Kit trafficking and oncogenic signalling.

(A) RCM cells were treated with vehicle or 5 μM BFA for 16 hours, then immunostained with anti-Kit (green) and anti-calnexin (ER marker, red). Bars, 10 μm. (B-E) RCM cells were treated for 16 hours with vehicle (0) or 1~5 μM BFA. (B) Cell lysates were immunoblotted with anti-Kit, anti-phospho-KitTyr721 (anti-pKitTyr721), anti-Akt, anti-pAkt, anti-STAT5, anti-pSTAT5, and anti-cleaved caspase-3. The graph shows the levels of pSTAT5 (open circles) or pAkt (closed circles) expressed relative to lysate from vehicle-treated cells. (C-E) RCM cells were treated with 5 μM BFA for 16 hours. Anti-Kit immunoprecipitates (C and D) or lysates (E) were immunoblotted with the indicated antibody.

https://doi.org/10.1371/journal.pone.0175514.s003

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S4 Fig. Blockade of Kit trafficking to endolysosomes inhibits Akt activation.

(A and B) RCM cells were treated with vehicle or 100 nM BafA1 for 24 hours. (A) Lysates were immunoblotted with the indicated antibody. (B) Lysates were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted. CG, complex-glycosylated form; HM, high mannose form; DG, deglycosylated form.

https://doi.org/10.1371/journal.pone.0175514.s004

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S5 Fig. Inhibition of Akt induces apoptosis in RCM cells.

(A) RCM cells were treated with vehicle (0), or Akt inhibitor VIII (Akti VIII) for 24 hours. Proliferation was assessed by [3H]-thymidine incorporation. Results (c.p.m.) are means ± SD (n = 3). (B) Immunoblots, lysates from RCM cells treated with vehicle or 10 μM Akti VIII for 24 hours. Note that Akt inhibition induced apoptosis in RCM cells. (C) A549 or HMC-1.2 were treated with vehicle (0) or 1~5 μM M-COPA for 16 hours. Lysates were immunoblotted. Total protein levels were confirmed by Coomassie staining. Note that M-COPA did not affect the Akt activation and cleavage of caspase-3.

https://doi.org/10.1371/journal.pone.0175514.s005

(EPS)

S6 Fig. Effect of inhibition of Kit trafficking on Erk activation.

(A) RCM cells were transfected with control siRNA or Kit siRNAs (Kit1 or Kit2) and cultured for 20 hours. Cell lysates were immunoblotted with anti-Erk and anti-phospho-Erk (anti-pErk). (B and C) RCM cells were treated with (B) vehicle (0), 1~5 μM BFA for 16 hours, (C) 250 nM monensin or 100 nM BafA1 for 24 hours. Cell lysates were immunoblotted.

https://doi.org/10.1371/journal.pone.0175514.s006

(EPS)

Acknowledgments

The authors thank Dr. Yoshimi Ohashi, Dr. Akinori Akatsuka, and Dr. Shingo Dan (Japanese Foundation for Cancer Research), Dr. Kentaro Yoshimatsu (Eisai Inc.) and Dr. Takachika Azuma (Tokyo University of Science) for their helpful advice and sharing of materials throughout this study. This work was supported by a grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (16K18427 to Y.O., 15K06843 to R.A.) and from the Uehara Memorial Foundation (to Y.O.).

Author Contributions

  1. Conceptualization: YO RA.
  2. Formal analysis: YH YO KH.
  3. Funding acquisition: YO RA.
  4. Investigation: YH YO KH.
  5. Methodology: YO.
  6. Project administration: YO RA.
  7. Resources: YT KS TM IS.
  8. Supervision: YO RA.
  9. Validation: YH YO KH.
  10. Visualization: YH YO.
  11. Writing – original draft: YH YO.
  12. Writing – review & editing: YO RA.

References

  1. 1. Besmer P, Murphy JE, George PC, Qiu FH, Bergold PJ, Lederman L, et al. A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. Nature. 1986; 320: 415–421. pmid:3007997
  2. 2. Yarden Y, Kuang WJ, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, et al. Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987; 6: 3341–3351. pmid:2448137
  3. 3. Broudy VC. Stem cell factor and Hematopoiesis. Blood. 1997; 90: 1345–1364. pmid:9269751
  4. 4. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature. 2001; 411: 355–365. pmid:11357143
  5. 5. Lennartsson R, Rönnstrand R. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol Rev. 2012; 92: 1619–1649. pmid:23073628
  6. 6. Roskoski R. Structure and regulation of Kit protein-tyrosine kinase—the stem cell factor receptor. Biochem Biophys Res Commun. 2005; 338: 1307–1315. pmid:16226710
  7. 7. Masson K, Heiss E, Band H, Rönnstrand L. Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation. Biochem J. 2006; 399: 59–67. pmid:16780420
  8. 8. Timokhina I, Kissel H, Stella G, Besmer P. Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation. EMBO J. 1998; 17: 6250–6262. pmid:9799234
  9. 9. Broudy VC, Lin NL, Liles WC, Corey SJ, O'Laughlin B, Mou S, et al. Signaling via Src family kinases is required for normal internalization of the receptor c-Kit. Blood. 1999; 94: 1979–1986. pmid:10477727
  10. 10. Blume-Jensen P, Janknecht R, Hunter T. The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136. Curr Biol. 8: 1998; 779–782. pmid:9651683
  11. 11. Kon S, Minegishi N, Tanabe K, Watanabe T, Funaki T, Wong WF, et al. Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia. J Clin Invest. 2013; 123: 1123–1137. pmid:23434593
  12. 12. Boissan M, Feger F, Guillosson JJ, Arock M. c-Kit and c-kit mutations in mastocytosis and other hematological diseases. J Leukoc Biol. 2000; 67: 135–148. pmid:10670573
  13. 13. Bibi S, Arslanhan MD, Langenfeld F, Jeanningros S, Cerny-Reiterer S, Hadzijusufovic E, et al. Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy. Haematologica. 2014; 99: 417–429 pmid:24598853
  14. 14. Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science. 1998; 279: 577–580. pmid:9438854
  15. 15. Lasota J, Miettinen M. Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours. Histopathology. 2008; 53: 245–266. pmid:18312355
  16. 16. Baumgartner C, Cerny-Reiterer S, Sonneck K, Mayerhofer M, Gleixner KV, Fritz R, et al. Expression of activated STAT5 in neoplastic mast cells in systemic mastocytosis: subcellular distribution and role of the transforming oncoprotein KIT D816V. Am J Pathol. 2009; 175, 2416–2429. pmid:19893034
  17. 17. Chaix A, Lopez S, Voisset E, Gros L, Dubreuil P, De Sepulveda P. Mechanisms of STAT protein activation by oncogenic KIT mutants in neoplastic mast cells. J Biol Chem. 2011; 286: 5956–5966. pmid:21135090
  18. 18. Aichberger KJ, Gleixner KV, Mirkina I, Cerny-Reiterer S, Peter B, Ferenc V, et al. Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs. Blood. 2009; 114: 5342–5351. pmid:19850739
  19. 19. Bauer S, Duensing A, Demetri GD, Fletcher JA. KIT oncogenic signaling mechanisms in imatinib-resistant gastrointestinal stromal tumor: PI3-kinase/AKT is a crucial survival pathway. Oncogene. 2007; 26: 7560–7568. pmid:17546049
  20. 20. Gordon PM, Fisher DE. Role for the proapoptotic factor BIM in mediating imatinib-induced apoptosis in a c-KIT-dependent gastrointestinal stromal tumor cell line. J Biol Chem. 2010; 285: 14109–14114. pmid:20231287
  21. 21. Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med. 2001; 344: 1052–1056. pmid:11287975
  22. 22. Tuveson DA, Willis NA, Jacks T, Griffin JD, Singer S, Fletcher CD, et al. STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: biological and clinical implications. Oncogene. 2001; 20: 5054–5058. pmid:11526490
  23. 23. Zermati Y, De Sepulveda P, Féger F, Létard S, Kersual J, Castéran N, et al. Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms. Oncogene. 2003; 22: 660–664. pmid:12569358
  24. 24. Obata Y, Horikawa K, Takahashi T, Akieda Y, Tsujimoto M, Fletcher JA, et al. Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors. Oncogene. 2017.
  25. 25. Rubin BP, Duensing A. Mechanisms of resistance to small molecule kinase inhibition in the treatment of solid tumors. Lab Invest. 2006; 86: 981–986. pmid:16924245
  26. 26. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005; 2: e73. pmid:15737014
  27. 27. Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest. 1993; 92: 1736–1744. pmid:7691885
  28. 28. Tsujimura T, Furitsu T, Morimoto M, Isozaki K, Nomura S, Matsuzawa Y, et al. Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation. Blood. 1994; 83: 2619–2626. pmid:7513208
  29. 29. Obata Y, Toyoshima S, Wakamatsu E, Suzuki S, Ogawa S, Esumi H, et al. Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation. Nat Commun. 2014; 5: 5715. pmid:25493654
  30. 30. Williams AB, Li L, Nguyen B, Brown P, Levis M, Small D. Fluvastatin inhibits FLT3 glycosylation in human and murine cells and prolongs survival of mice with FLT3/ITD leukemia. Blood. 2012; 120: 3069–3079. pmid:22927251
  31. 31. Shiina I, Umezaki Y, Ohashi Y, Yamazaki Y, Dan S, Yamori T. Total synthesis of AMF-26, an antitumor agent for inhibition of the Golgi system, targeting ADP-ribosylation factor 1. J Med Chem. 2013; 56: 150–159. pmid:23214926
  32. 32. 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-ribosylation factor 1 (Arf1) with potential for cancer therapy. J Biol Chem. 2012; 287: 3885–3897. pmid:22158626
  33. 33. Ohashi Y, Okamura M, Hirosawa A, Tamaki N, Akatsuka A, Wu KM, et al. M-COPA, a Golgi disruptor, inhibits cell surface expression of MET protein and exhibits antitumor activity against MET-addicted gastric cancers. Cancer Res. 2016; 76: 3895–3903. pmid:27197184
  34. 34. Watari K, Nakamura M, Fukunaga Y, Furuno A, Shibata T, Kawahara A, et al. The antitumor effect of a novel angiogenesis inhibitor (an octahydronaphthalene derivative) targeting both VEGF receptor and NF-κB pathway. Int J Cancer. 2012; 131: 310–321. pmid:21826646
  35. 35. Growney JD, Clark JJ, Adelsperger J, Stone R, Fabbro D, Griffin JD, et al. Activation mutations of human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine kinase inhibitor PKC412. Blood. 2005; 106: 721–724. pmid:15790786
  36. 36. Helenius A, Aebi M. Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem. 2004; 73: 1019–1049. pmid:15189166
  37. 37. Choudhary C, Olsen JV, Brandts C, Cox J, Reddy PN, Böhmer FD, et al. Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes. Mol Cell. 2009; 36: 326–339. pmid:19854140
  38. 38. Kim MS, Rådinger M, Gilfillan AM. The multiple roles of phosphoinositide 3-kinase in mast cell biology. Trends Immunol. 2008; 29, 493–501. pmid:18775670
  39. 39. Sivalenka RR, Jessberger R. SWAP-70 regulates c-kit-induced mast cell activation, cell-cell adhesion, and migration. Mol Cell Biol. 2004; 24:10277–10288. pmid:15542837
  40. 40. Chaix A, Arcangeli ML, Lopez S, Voisset E, Yang Y, Vita M, et al. KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570. Oncogene. 2003; 33: 872–881.
  41. 41. Toffalini F, Demoulin JB. New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases. Blood. 2010; 116: 2429–2437. pmid:20581310
  42. 42. Kon S, Kobayashi N, Satake M. Altered trafficking of mutated growth factor receptors and their associated molecules: implication for human cancers. Cell Logist. 2014; 18; 4: e28461.
  43. 43. Bahlawane C, Eulenfeld R, Wiesinger MY, Wang J, Muller A, Girod A, et al. Constitutive activation of oncogenic PDGFRα-mutant proteins occurring in GIST patients induces receptor mislocalisation and alters PDGFRα signalling characteristics. Cell Commun Signal. 2015; 13: 21. pmid:25880691
  44. 44. Ronchetti D, Greco A, Compasso S, Colombo G, Dell'Era P, Otsuki T, et al. Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations. Oncogene. 2003; 20: 3553–3562.
  45. 45. Gibbs L, Legeai-Maller L. FGFR3 intracellular mutations induce tyrosine phosphorylation in the Golgi and defective glycosylation. Biochim Biophys Acta. 2007; 1773: 502–512. pmid:17320202
  46. 46. Chung BM, Raja SM, Clubb RJ, Tu C, George M, Band V, et al. Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src. BMC Cell Biol. 2009; 10: 84. pmid:19948031
  47. 47. Watanuki Z, Kosai H, Osanai N, Ogama N, Mochizuki M, Tamai K, et al. Synergistic cytotoxicity of afatinib and cetuximab against EGFR T790M involves Rab11-dependent EGFR recycling. Biochem Biophys Res Commun. 2014; 455: 269–276. pmid:25446083
  48. 48. Schmidt-Arras D, Müller M, Stevanovic M, Horn S, Schütt A, Bergmann J, et al. Oncogenic deletion mutants of gp130 signal from intracellular compartments. J Cell Sci. 2014; 127: 341–353. pmid:24213527
  49. 49. Rinis N, Küster A, Schmitz-Van de Leur H, Mohr A, Müller-Newen G. Intracellular signaling prevents effective blockade of oncogenic gp130 mutants by neutralizing antibodies. Cell Commun Signal. 2014; 12:14. pmid:24612692
  50. 50. Joffre C, Barrow R, Ménard L, Calleja V, Hart IR, Kermorgant S. A direct role for Met endocytosis in tumorigenesis. Nat Cell Biol. 2010; 13, 827–837.
  51. 51. Tabone-Eglinger S, Subra F, El Sayadi H, Alberti L, Tabone E, Michot JP, et al. KIT mutations induce intracellular retention and activation of an immature form of the KIT protein in gastrointestinal stromal tumors. Clin Cancer Res. 2008; 14: 12285–12294.
  52. 52. Jaramillo S, Ríos-Moreno MJ, Hernández A, Amérigo J, Trigo-Sánchez I, González-Cámpora R. Gastrointestinal stromal tumors (GISTs): role of CD 117 and PDGFRA Golgi-like staining pattern in the recognition of mutational status. Rev Esp Enferm Dig. 2012; 104: 128–133. pmid:22449154
  53. 53. Klausner RD, Donaldson JG, Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992; 116: 1071–1080. pmid:1740466
  54. 54. Feng Y, Yu S, Lasell TK, Jadhav AP, Macia E, Chardin P, et al. Exo1: a new chemical inhibitor of the exocytic pathway. Proc Natl Acad Sci U S A. 2003; 100: 6469–6474 pmid:12738886
  55. 55. Feng Y, Jadhav AP, Rodighiero C, Fujinaga Y, Kirchhausen T, Lencer WI. Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans-Golgi network but not the Golgi apparatus in Exo2-treated cells. EMBO Rep. 2004; 5: 596–601. pmid:15153932
  56. 56. Pan H, Yu J, Zhang L, Carpenter A, Zhu H, Li L, et al. A novel small molecule regulator of guanine nucleotide exchange activity of the ADP-ribosylation factor and golgi membrane trafficking. J Biol Chem. 2008; 283: 31087–31096. pmid:18799457