ATP11B调控卵巢癌肿瘤干细胞特性介导铂类耐药的机制研究

Epithelial ovarian cancer (EOC) is the leading cause of death among gynecological malignancies. The primary cause of recurrence and death is chemotherapy resistance. We employed the proteomic technique Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC) to compare proteins expressed in side population (SP) ovarian cancer cells with those in non-SP ones. CDC50A was identified and proved to be a new potential marker for ovarian cancer stem cells (OCSCs). CDC50A is a subunit of ATP11B proteins. In our previous study, we found that EOC cells with CDC50A overexpression could not play a functional role in the regulation of OCSCs, while cells with ATP11B overexpression and with CDC50A and ATP11B co-expression could give rise to spheres. Therefore, we speculate that CDC50A play the role of regulating OCSCs by coordinating with ATP11B. The specific aims are to identify the interaction between CDC50A and ATP11B in vivo and in vitro, to study the role of ATP11B in regulating OCSCs, to find the signaling pathway associated with ATP11B to maintain the role of OCSCs and chemotherapy resistance by next generation sequencing, to analyze the value of ATP11B in diagnosis and treatment of ovarian cancer. The proposed research will not only lead to a better understanding of OCSCs regulation and chemotherapy resistance, but also provide theoretical basis for ovarian cancer diagnosis and treatment.

卵巢上皮癌是致死性第一位的妇科恶性肿瘤，化疗耐药是卵巢癌复发致死的根本原因。本课题组通过微量蛋白组学技术发现SP卵巢癌细胞中高表达蛋白CDC50A，前期研究已证实该蛋白是新发现的卵巢癌潜在干细胞标记物。CDC50A是ATP11B蛋白的亚基，前期研究发现过表达CDC50A不增加卵巢癌干细胞特性，而过表达ATP11B及共表达CDC50A和ATP11B能够增加卵巢癌细胞干性。因此我们推测CDC50A通过与ATP11B的协调作用而维持肿瘤干细胞特性。本研究旨在利用体内外实验验证CDC50A与ATP11B的蛋白相互作用；体内外实验验证ATP11B的卵巢癌干细胞生物学特性；高通量测序寻找ATP11B维持卵巢癌干细胞作用的信号通路及机制；临床分析ATP11B在卵巢癌诊治中的价值。本课题将探讨ATP11B参与维持卵巢癌干细胞的生物学特性及介导卵巢癌铂类耐药的作用和机制，为卵巢癌的诊治提供理论依据。

卵巢上皮癌是致死性第一位的妇科恶性肿瘤，化疗耐药是卵巢癌复发致死的根本原因。本课题组通过微量蛋白组学技术发现SP卵巢癌细胞中高表达蛋白CDC50A，前期研究已证实该蛋白是新发现的卵巢癌潜在干细胞标记物。CDC50A是ATP11B蛋白的亚基，前期研究发现过表达CDC50A不增加卵巢癌干细胞特性，而过表达ATP11B及共表达CDC50A和ATP11B能够增加卵巢癌细胞干性。因此我们推测CDC50A通过与ATP11B的协调作用而维持肿瘤干细胞特性。本研究首先通过Co-IP实验发现CDC50A和ATP11B的慢病毒表达载体共感染卵巢上皮癌细胞系SKOV3后，pullCDC50A的同时有ATP11B的表达，说明在卵巢癌细胞系SKOV3中CDC50A和ATP11B存在相互作用。其次，体外实验证实过表达ATP11B的SKOV3细胞系Sphere形成能力、传代能力、铂类耐药能力显著增强，而敲低ATP11B后，SKOV3细胞系Sphere形成能力和铂类耐药能力显著减弱。体内实验发现过表达ATP11B的SKOV3细胞系在小鼠中成瘤能力、传代能力较空载体增强，而敲低ATP11B的SKOV3细胞系在小鼠中成瘤能力减弱，且顺铂能在小鼠体内富集ATP11B表达阳性的卵巢癌细胞。体内外实验均证实ATP11B在卵巢癌细胞的更新能力和铂类耐药中发挥着重要作用。随后对原代卵巢上皮癌分选的ATP11B+/-的细胞进行RNA-seq发现ATP11B在脂质及NAD+代谢、蛋白质定位、碳酸氢盐运输等信号通路中发挥调节作用，为研究ATP11B的分子机制奠定基础。最后，通过在人原代卵巢癌标本中的免疫组化发现ATP11B对卵巢上皮癌的预后有一定预测价值，但仍需大样本前瞻性研究进行验证。通过本研究，探索了ATP11B作为潜在干细胞标记物的价值，且发现其高表达对患者预后的预测作用，为临床转化奠定了基础。