Clk2促进非BRCA缺陷型上皮性卵巢癌耐药的机制和临床意义

BRCA1 / 2 mutation leading to DNA damage repair pathway defection is an important incentive of ovarian cancer, and is closely related to patients’ sensitivity of chemotherapy drugs. Applicants found early: High expression of protein kinase Clk2 promotes the occurrence of platinum-resistant in ovarian cancer. Clk2 has the function of phosphorylation activating BRCA1, and knocking down Clk2 can reduce the activity of BRCA1, so as to weaken cancer cells’ ability of damaged DNA repair and promote cancer cells' apoptosis. Moreover, the combined use of Clk2 and PARP inhibitors can form a "synergistic lethal effect" and significantly enhance the DNA damage and chemosensitivity of drug-resistant cells in non-BRCA-deficient ovarian cancer cells. Therefore, we intend to combine in vitro and in vivo experiments and clinical large sample data: 1) to elucidate the role and molecular mechanism of Clk2 in regulating BRCA1 phosphorylation and mediating drug resistance in ovarian cancer; 2) to reveal key regulatory molecules and mechanism of Clk2 up-regulation in ovarian cancer; 3) to investigate the clinical feasibility of reversing ovarian cancer drug resistance by combining Clk2-BRCA signal interference with PARP inhibitor. The results will help clarify the mechanism of Clk2 promoting drug resistance in ovarian cancer patients,and provide new strategies to reverse drug resistance of non-BRCA deficient ovarian cancer patients.

BRCA1/2突变引发的DNA损伤修复通路缺陷是卵巢癌的重要诱因，并与患者对化疗药物的敏感性密切相关。申请人前期发现：蛋白激酶Clk2高表达促进上皮性卵巢癌发生铂类耐药。Clk2具有磷酸化激活BRCA1的功能，敲低Clk2能够降低BRCA1活性，从而减弱癌细胞修复损伤DNA的能力，促进细胞凋亡。而且，联合运用Clk2及PARP抑制剂可形成“协同致死效应”，明显增强非BRCA缺陷型卵巢癌耐药细胞的DNA损伤及其对化疗药物的敏感性。由此，我们拟结合体内外实验及临床大样本数据：1）阐明Clk2调控BRCA1磷酸化并介导卵巢癌耐药的作用及其分子机制；2）揭示Clk2表达上调的关键调控分子及其机制；3）探讨干扰Clk2-BRCA信号联合PARP抑制剂治疗卵巢癌耐药的临床可行性。所得结果有助于阐明Clk2促进卵巢癌发生耐药的机制，结合“协同致死效应”，为逆转非BRCA缺陷型卵巢癌患者耐药提供新策略。

我们明确了Clk2高表达是上皮性卵巢癌患者的不良预后因素，Clk2上调与卵巢癌铂耐药正相关。同时我们还通过一系列体内外实验证实了CLK2通过磷酸化BRCA1的Ser1423位点进而促进DNA损伤修复最终参与诱导铂耐药；应用多种与铂刺激相关的信号通路抑制剂，我们明确了顺铂通过 p38增强CLK2的蛋白稳定性；运用免疫共沉淀实验我们进一步发现在铂类刺激下p38结合CLK2的Thr343位点增强其蛋白稳定性；泛素化是蛋白降解最常见的途径，我们通过过使用蛋白酶体抑制剂MG13、数据库预测及免疫沉淀等一系列方法发现了E3泛素连接酶RNF8可能参与了p38抑制的CLK2泛素化过程。最后，我们通过体外细胞实验及体内PDX模型证实了：PARP抑制剂联合CLK2抑制剂能够在BRCA1野生型的卵巢癌中实现协同作用，显著增强铂类敏感性。