Hara Yasushi, Obata Yuuki, Horikawa Keita, Tasaki Yasutaka, Suzuki Kyohei, Murata Takatsugu, Shiina Isamu, Abe Ryo
Division of Immunobiology, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba, Japan.
Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan.
PLoS One. 2017 Apr 12;12(4):e0175514. doi: 10.1371/journal.pone.0175514. eCollection 2017.
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.
Kit受体酪氨酸激酶的功能获得性突变会导致多种癌症的发生，如肥大细胞瘤、胃肠道间质瘤（GISTs）、急性髓系白血病和黑色素瘤。药物伊马替尼是Kit的选择性抑制剂，用于治疗Kit突变阳性的癌症。然而，在肿瘤性肥大细胞中经常发现的Kit激酶结构域突变会导致对伊马替尼产生耐药性，表达这些突变体的癌症在伊马替尼存在的情况下仍能增殖。最近，我们发现，在内源性表达伊马替尼耐药Kit突变体的肿瘤性肥大细胞中，Kit分别仅在内溶酶体和内质网（ER）上导致磷脂酰肌醇3-激酶-蛋白激酶B（PI3K-Akt）途径和信号转导及转录激活因子5（STAT5）的致癌激活。在此，我们展示了一种通过该分泌途径的抑制剂M-COPA（2-甲基粪卟啉酰胺）抑制肿瘤性肥大细胞中Kit-PI3K-Akt途径的策略。在经M-COPA处理的细胞中，Kit在内质网中的定位显著增加，而内溶酶体中的Kit消失，这表明M-COPA阻断了Kit从内质网的生物合成运输。该药物极大地抑制了致癌性Akt激活，而不影响Kit与PI3K的结合，表明内质网定位的Kit-PI3K复合物无法激活Akt。重要的是，M-COPA而非伊马替尼通过抑制抗凋亡性Akt激活来抑制肿瘤性肥大细胞增殖。我们的M-COPA处理试验结果表明，Kit不仅能在内质网上激活细胞外信号调节激酶（Erk），还能在其他区室激活。此外，Kit中的酪氨酸568/570、酪氨酸703、酪氨酸721和酪氨酸936在内质网上被磷酸化，这表明这五个酪氨酸残基在突变型Kit到达质膜（PM）之前均已被磷酸化。我们的研究提供了证据表明Kit在内质网上合成后不久就被酪氨酸磷酸化，但无法激活Akt，并且还证明了M-COPA对抑制肿瘤性肥大细胞生长有效。
