探討酪胺酸激酶c-Kit磷酸化prohibitin對於卵巢癌癌幹細胞與抗藥性所扮演的角色

Abstract

如何造成卵巢癌癌幹細胞以及其抗藥性的產生，其致病機轉仍有很多未知的層面存在。在此研究中，我們便是探討酪胺酸激酶c-Kit和細胞膜上的prohibitin (PHB)之間的調控是否會影響卵巢癌幹細胞的表徵以及化療藥物抗性的產生。我們發現c-Kit和PHB蛋白是會共同聚集在細胞膜的脂筏(lipid raft)上，而且與臨床上漿液性卵巢癌的分期是有正相關性的。c-Kit會與PHB連接在一起並促進細胞膜脂筏上的PHB在酪胺酸259的磷酸化，進而增強卵巢癌細胞轉移擴散的能力。我們更進一步將SKOV3帶有GFP和Luciferase螢光蛋白的卵巢癌細胞直接打入小鼠的卵巢，等到其癌細胞擴散到腹腔後再蒐集這些已擴散的癌細胞，重覆幾次後得到擴散轉移能力均較強的SKOV3GL-G4卵巢癌細胞。我們發現SKOV3GL-G4卵巢癌細胞，其c-Kit、Notch3、Oct4、Nanog、SOX2這些幹細胞marker的表現量均有上升的現象。進一步的研究更發現當c-Kit磷酸化細胞膜脂筏上的PHB後，phospho-PHBY259會與Notch3連接在一起，以便穩固Notch3的蛋白不被降解進而增強Notch3的下游PBX1的表現量。降低細胞膜脂筏上PHB蛋白在酪胺酸259的磷酸化，會加速Notch3蛋白被溶酶體分解的降解速率以及去活化β-catenin—ABCG2的訊息傳導途徑。除此之外，我們的實驗也發現細胞膜脂筏上PHB蛋白在酪胺酸259的磷酸化對於卵巢癌的幹細胞特性和對於化療藥物的抗藥性都扮演了很重要的角色。這些包含c-Kit和PHB之間的相互關係，以及細胞膜脂筏上PHB蛋白在酪胺酸259的磷酸化影響Notch3和β-catenin的訊息傳導途徑，進而促進了卵巢癌癌幹細胞和腫瘤的生成都是迄今為止尚未被發現的現象。藉由我們的這項新發現，或許可以透過標靶治療，去抑制c-Kit/raft-phospho-PHBY259的傳導途徑而能有效輔助原本的卵巢癌症療法，減低其復發機率。

How does ovarian cancer regulate its stemness and chemoresistance is largely unclear. Here, we explored the role of c-Kit and plasma membrane prohibitin (PHB) in regulating ovarian cancer stemness and chemotherapy resistance. Our results showed that c-Kit and PHB were colocalized and positively correlated in the lipid raft domain of plasma membrane in human ovarian serous carcinoma. Association of c-Kit with PHB and phosphorylated PHB at tyrosine 259 (phospho-PHBY259) in the membrane raft increased ovarian cancer cell migration. Examination of SKOV3GL-G4, a green fluorescent protein and luciferase (GL) labeling metastatic phenotype of SKOV3 ovarian cancer cells generated from xenograft murine ascites, showed a positive 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 associated with Notch3 to stabilize Notch3 and enhance the downstream target PBX1. Dephosphorylation of raft-PHB at tyrosine 259 degraded Notch3 via a lysosomal pathway and inactivated the β-catenin—ABCG2 signaling. In addition, raft-phospho-PHBY259 played an important role in ovarian cancer stemness and resistance to platinum treatment in vitro and in vivo. These findings disclose a hitherto unreported interrelationship between c-Kit and PHB as well as the involvement of Notch3 and β-catenin signaling pathways in the effects of raft-phospho-PHBY259 on ovarian cancer stemness and tumorigenicity. Targeting the c-Kit/raft-phospho-PHBY259 axis may thus provide a novel therapeutic strategy for treating patients with ovarian cancer.