Agricultural Biotechnology Research Center, Academia Sinica, 128 Academia Rd, Sec. 2, Taipei, 11529, Taiwan.
Institute of Biotechnology, National Taiwan University, 4F, No. 81, Chang-Xing St, Taipei, 10672, Taiwan.
J Biomed Sci. 2020 Mar 13;27(1):42. doi: 10.1186/s12929-020-00638-x.
The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown. Here, we explored whether the regulation of c-Kit and plasma membrane prohibitin (PHB) affects ovarian cancer stemness and chemotherapy resistance.
Mass spectrum analysis and an in vitro kinase assay were conducted to examine the phosphorylation of PHB at tyrosine 259 by c-Kit. The in vitro effects of c-Kit on membrane raft-PHB in ovarian cancer were determined using tissue microarray (TMA)-based immunofluorescence, western blotting, immunoprecipitation, colony and spheroid formation, cell migration and cell viability assays. In vivo tumor initiation and carboplatin treatment were conducted in nude mice.
We found that c-Kit and PHB colocalized in the raft domain and were positively correlated in human ovarian serous carcinoma. c-Kit interacted with PHB and facilitated the phosphorylation of PHB at tyrosine 259 (phospho-PHB) in the membrane raft to enhance ovarian cancer cell motility. The generation of SKOV3GL-G4, a metastatic phenotype of SKOV3 green fluorescent protein and luciferase (GL) ovarian cancer cells, in xenograft murine ascites showed a correlation between metastatic potential and stem cell characteristics, as indicated by the expression of c-Kit, Notch3, Oct4, Nanog and SOX2. Further study revealed that after activation by c-Kit, raft-phospho-PHB interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHB increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the β-catenin-ABCG2 signaling pathway. Moreover, raft-phospho-PHB played an important role in ovarian cancer stemness and tumorigenicity as well as resistance to platinum drug treatment in vitro and in vivo.
These findings thus reveal a hitherto unreported interrelationship between c-Kit and PHB as well as the effects of raft-phospho-PHB on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and β-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHB axis may provide a new therapeutic strategy for treating patients with ovarian cancer.
卵巢癌干性和化疗耐药的潜在机制在很大程度上仍不清楚。在这里，我们探讨了 c-Kit 和质膜抑素（PHB）的调节是否影响卵巢癌干性和化疗耐药性。
采用质谱分析和体外激酶测定法检测 c-Kit 对 PHB 酪氨酸 259 的磷酸化。采用组织微阵列（TMA）免疫荧光、Western blot、免疫沉淀、集落和球体形成、细胞迁移和细胞活力测定，研究 c-Kit 对卵巢癌细胞质膜筏 PHB 的体外作用。在裸鼠中进行体内肿瘤起始和卡铂治疗。
我们发现 c-Kit 和 PHB 在筏域中共定位，并且在人卵巢浆液性癌中呈正相关。c-Kit 与 PHB 相互作用，并促进 PHB 在质膜筏中的酪氨酸 259 磷酸化（磷酸化 PHB），从而增强卵巢癌细胞的迁移能力。SKOV3GL-G4 的产生，一种 SKOV3 绿色荧光蛋白和荧光素（GL）卵巢癌细胞的转移表型，在异种移植鼠腹水的转移潜能和干细胞特征之间存在相关性，这表明 c-Kit、Notch3、Oct4、Nanog 和 SOX2 的表达。进一步的研究表明，c-Kit 激活后，筏 PHB 与 Notch3 相互作用，稳定 Notch3 并增加下游靶标 PBX1。通过溶酶体途径下调筏 PHB 增加 Notch3 的蛋白降解，并抑制 β-catenin-ABCG2 信号通路。此外，筏 PHB 在体外和体内对卵巢癌干性和致瘤性以及对铂类药物治疗的耐药性起着重要作用。
这些发现揭示了 c-Kit 和 PHB 之间以前未报道的相互关系，以及筏 PHB 通过 Notch3 和 β-catenin 信号通路对卵巢癌干性和致瘤性的影响。靶向 c-Kit/筏 PHB 轴可能为治疗卵巢癌患者提供新的治疗策略。
