Original Articles Bortezomib enhances the therapeutic efficacy of dasatinib by promoting c-KIT internalization-induced apoptosis in gastrointestinal stromal tumor cells Ying Dong a,1 , Chao Liang b,1 , Bo Zhang c , Jianjuan Ma d , Xuexin He a , Siyu Chen e , Xianning Zhang f , Wei Chen b, * a Department of Oncology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China b Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China c National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China d Department of Internal Medicine, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China e Department of Oncology, Xinhua Hospital Affiliated to Medical School of Shanghai Jiaotong University, Shanghai 200092, China f Department of Cell Biology and Medical Genetics, Research Center of Molecular Medicine, Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou 310058, China ARTICLE INFO Article history: Received 6 January 2015 Received in revised form 17 February 2015 Accepted 26 February 2015 Keywords: GISTs Dasatinib Bortezomib Treatment Efficacy c-KIT A B ST R AC T Dasatinib-based therapy is often used as a second-line therapeutic strategy for imatinib-resistance gas- trointestinal stromal tumors (GISTs); however, acquired aberrant activation of dasatinib target proteins, such as c-KIT and PDGFRβ, attenuates the therapeutic efficiency of dasatinib. Combination therapy which inhibits the activation of dasatinib target proteins may enhance the cytotoxicity of dasatinib in GISTs. Bortezomib, a proteasome inhibitor, significantly inhibited cell viability and promoted apoptosis of dasatinib-treated GIST-T1 cells, whereas GIST-T1 cells showed little dasatinib cytotoxicity when treated with dasatinib alone, as the upregulation of c-KIT caused by dasatinib itself interfered with the inhibi- tion of c-KIT and PDGFRβ phosphorylation by dasatinib. Bortezomib induced internalization and degradation of c-KIT by binding to Cbl, an E3 ubiquitin-protein ligase, and the subsequent release of Apaf-1, which was originally bound to the c-KIT-Hsp90β-Apaf-1 complex, induced primary apoptosis in GIST-T1 cells. Combined treatment with bortezomib plus dasatinib caused cell cycle arrest in the G1 phase through inactivation of PDGFRβ and promoted bortezomib-induced apoptosis in GIST-T1 cells. Our data suggest that combination therapy exerts better efficiency for eradicating GIST cells and may be a promising strat- egy for the future treatment of GISTs. © 2015 Published by Elsevier Ireland Ltd. Introduction Gastrointestinal stromal tumors (GISTs) are the largest subset of mesenchymal tumors of the digestive tract [1,2]. GISTs, derived from interstitial cell of Cajal (ICC) or gastrointestinal mesenchymal stem cells, are distinguished from other mesenchymal tumors occur- ring in the digestive tract, such as leiomyoma, leiomyosarcoma, and schwannoma, by their aberrant expression of c-KIT (CD117), a class III receptor tyrosine kinase (RTK) [3–5]. Over the past 10 years, based on our understanding of the role of RTKs in the potential oncogen- esis of GISTs, the implementation of tyrosine kinase inhibitors (TKIs) has revolutionized the therapeutic strategy for GISTs [6,7]. Imatinib, the first-line TKI in treatment of chronic myelogenous leukemia (CML), has been approved and widely applied for GIST therapy [8]. c-KIT (mammalian cellular homolog of v-KIT in Hardy– Zuckerman-feline sarcoma virus) proto-oncogene is a 145-kD transmembrane RTK, consisting of a extracellular ligand-binding domain, a single transmembrane domain, an intracellular juxtamembrane region, and a split intracellular kinase domain [9]. When the c-KIT ligand is bound, the stem cell factor (SCF) and two neighboring c-KIT proteins undergo dimerization followed by phos- phorylation of tyrosine residues in the juxtamembrane region and kinase domains, which formatted as the dock sits of Src homology 2 (SH2) domains of a variety of signal molecules, including PI3K, AKT, ERK, and so on [9,10]. The activation of factors downstream of the c-KIT signaling cascade is extremely important for pro-oncogenic cellular progress, and specifically for anti-apoptosis Abbreviations: Apaf-1, apoptotic protease activating factor 1; BOR, bortezomib; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehy- drogenase; GISTs, gastrointestinal stromal tumors; Hsp90β, heat shock protein 90 β; PDGFRβ, platelet-derived growth factor receptor β; PI, propidium iodide; RTK, re- ceptor tyrosine kinase; SCF, stem cell factor; TKIs, tyrosine kinase inhibitors. * Corresponding author. Tel.: +86 88981599; fax: +86 88981599. E-mail address: wei_chen@zju.edu.cn (W. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2015.02.044 0304-3835/© 2015 Published by Elsevier Ireland Ltd. Cancer Letters ■■ (2015) ■■–■■ ARTICLE IN PRESS Please cite this article in press as: Ying Dong, et al., Bortezomib enhances the therapeutic efficacy of dasatinib by promoting c-KIT internalization-induced apoptosis in gastrointes- tinal stromal tumor cells, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.044 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Q1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84