新型内源性多肽CPAP1提高卵巢癌顺铂敏感性的作用与机制研究

In order to explore the protective molecules in ovarian cancer ascites, we used mass spectrometry to screen differentially expressed peptides in ascites of chemosensitive and chemoresistant ovarian cancer patients. We found that CPAP1 peptide could increase the cisplatin sensitivity of ovarian cancer cells in vivo and in vitro. Peptide pull-down assay found that URI which is located in the chromosomal region associated with platinum-resistance of ovarian cancer may be the target of CPAP1. Cisplatin induced activation of S6K1-BAD signaling pathway downstream of URI was significantly inhibited by adding CPAP1. So we hypothesized that CPAP1 can increase the chemosensitivity of ovarian cancer by targeting URI. In the future, we will verify the applicability of CPAP1 function in promoting cisplatin sensitivity to other platinum drugs, and comprehensively evaluated the CPAP1 function in promoting cisplatin sensitivity of ovarian cancer by using drug-resistant ovarian cancer cells, the ovarian cancer orthotopic model and the Patient Derived Xenograpt (PDX) model, and further elucidated the mechanism of CPAP1 function in increasing the cisplatin sensitivity by physical methods and rescue experiment. Besides, we will explore the modification methods to improve the stability and activity of CPAP1. If the study is successful, it is expected to provide a new adjuvant therapy for ovarian cancer clinical chemotherapy, so it has a strong innovation and transformation application prospects.

从探索卵巢癌腹水中可能存在的保护成分出发，我们利用质谱技术筛选了卵巢癌化疗敏感病人和耐药病人腹水中差异表达的多肽，发现其中敏感病人中丰度较高的CPAP1在体内和体外均可以提高卵巢癌的顺铂敏感性。pull-down实验发现位于卵巢癌铂类耐药相关染色体区域的URI可能是CPAP1的靶基因，CPAP1的加入可以显著抑制顺铂诱导的URI下游S6K1-BAD信号通路的激活，提示CPAP1可能通过靶向URI提高卵巢癌的顺铂敏感性。本研究拟验证CPAP1促顺铂敏感性作用对其他铂类药物的适用性；利用卵巢癌耐药细胞、原位模型和病人来源移植瘤(PDX)模型全面评估CPAP1提高卵巢癌顺铂敏感性的作用；以URI为机制主线，通过物理学方法、挽救实验方案深入阐明CPAP1的作用机制；探索提高CPAP1稳定性和活性的修饰方法。研究如获成功，有望为卵巢癌临床化疗提供新的辅助治疗手段，因此具有较强的创新性和转化前景。

卵巢癌腹水作为一种特殊的肿瘤微环境含有十分复杂的成分，它的出现虽然与卵巢癌进展相关，但其中也不乏抑癌成分。临床经验也显示部分合并腹水的卵巢癌患者预后反而较好。本研究从腹水中的多肽成分出发，利用质谱技术筛选卵巢癌化疗敏感患者和耐药患者腹水中差异表达的多肽，从中选择化疗敏感患者腹水中丰度显著增加的6条及丰度显著降低的2条多肽，检测IC50发现CPAP1能够提升人卵巢癌细胞对紫杉醇和顺铂的敏感性。FITC标记多肽入胞实验发现穿膜肽（TAT）修饰的CPAP1能够进入卵巢癌细胞内、主要定位于细胞核。CCK8、IC50、流式细胞术实验表明CPAP1能够抑制A2780、OVCAR3和我们自己建立的顺铂耐药卵巢癌细胞A2780DR的增殖能力，显著提升卵巢癌细胞对顺铂和卡铂的敏感性并增加顺铂处理后的细胞凋亡。多肽pull down实验发现CPAP1能够与NOLC1结合并在细胞内共定位，进一步的RNA测序、RT-qPCR和Western Blot等结果表明CPAP1可能通过与NOLC1结合、调控P53信号通路来调节细胞对DNA损伤的反应，从而提升卵巢癌细胞对铂类药物的敏感性。敲降NOLC1在体外可以抑制卵巢癌细胞的生长，增加卵巢癌细胞对铂类药物的敏感性，在体内可以协同卡铂抑制肿瘤的生长。综上，我们发现内源性多肽CPAP1可通过结合NOLC1，调控P53信号通路从而提升卵巢癌对铂类药物的敏感性。