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-PHBY259) 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-PHBY259 interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHBY259 increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the β-catenin—ABCG2 signaling pathway. Moreover, raft-phospho-PHBY259 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-PHBY259 on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and β-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHBY259 axis may provide a new therapeutic strategy for treating patients with ovarian cancer.

中文翻译：

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新型的c-Kit /磷酸化抑制素轴通过Notch3-PBX1和β-catenin-ABCG2信号传导增强卵巢癌的干性和化学抗性。

涉及卵巢癌干性和化学抗性的基本机制仍然未知。在这里，我们探讨了c-Kit和质膜禁止素（PHB）的调节是否影响卵巢癌的干性和化疗耐药性。进行了质谱分析和体外激酶测定，以检查c-Kit在酪氨酸259处PHB的磷酸化。使用基于组织微阵列（TMA）的免疫荧光，蛋白质印迹，免疫沉淀，菌落和球体形成，细胞迁移和细胞生存力测定，确定c-Kit对卵巢癌膜筏-PHB的体外作用。在裸鼠中进行体内肿瘤引发和卡铂治疗。我们发现c-Kit和PHB在筏域共定位，并且在人类卵巢浆液性癌中呈正相关。c-Kit与PHB相互作用并促进膜筏中酪氨酸259（phospho-PHBY259）处PHB的磷酸化，以增强卵巢癌细胞的运动性。异种移植鼠腹水中SKOV3绿色荧光蛋白和萤光素酶（GL）卵巢癌细胞的转移表型SKOV3GL-G4的生成显示了转移潜能与干细胞特征之间的相关性，如c-Kit，Notch3的表达所示，Oct4，Nanog和SOX2。进一步的研究表明，在被c-Kit激活后，筏式磷酸化PHBY259与Notch3相互作用以稳定Notch3并增加下游靶PBX1。筏磷酸-PHBY259的下调通过溶酶体途径增加了Notch3的蛋白质降解，并抑制了β-catenin-ABCG2信号传导途径。此外，筏式磷酸化PHBY259在卵巢癌的干性和致瘤性以及在体外和体内对铂药物治疗的耐药性中起着重要作用。这些发现因此揭示了迄今为止尚未报道的c-Kit与PHB之间的相互关系，以及筏磷酸-PHBY259对Notch3和β-catenin信号通路介导的卵巢癌干性和致瘤性的影响。靶向c-Kit / raft-phospho-PHBY259轴可能为治疗卵巢癌患者提供新的治疗策略。这些发现因此揭示了迄今为止尚未报道的c-Kit与PHB之间的相互关系，以及筏磷酸-PHBY259对Notch3和β-catenin信号通路介导的卵巢癌干性和致瘤性的影响。靶向c-Kit / raft-phospho-PHBY259轴可能为治疗卵巢癌患者提供新的治疗策略。这些发现因此揭示了迄今为止尚未报道的c-Kit与PHB之间的相互关系，以及筏磷酸-PHBY259对Notch3和β-catenin信号通路介导的卵巢癌干性和致瘤性的影响。靶向c-Kit / raft-phospho-PHBY259轴可能为治疗卵巢癌患者提供新的治疗策略。