EZH2调控铜转运蛋白表达影响铂蓄积参与上皮性卵巢癌获得性顺铂耐药机制

Cytoreductive surgery combined with platinum-based chemotherapy represents the standard treatment strategy of epithelial ovarian cancer (EOC), but the recurrences due to acquired resistance against chemotherapy agents occur so frequently that the 5-years overall survival in patients with EOC is about 35%. Our previous study showed that EZH2 is an essential regulator of cell sensitivity to cisplatin in EOC, and cisplatin stimulates both polycomb repressive complex 2 (PRC2) pathway (H3K27me3) and non-PRC2 dependent pathway (phosphorylation of EZH2). The applicant demonstrated in a project of NFSC, for the first time, down-regulation of EZH2 resulted in increased platinum accumulation in EOC cells and enhanced the cytotoxicity of cisplatin. We further showed that copper transporters (CTRs) were dysregulated in cisplatin resistant EOC which are responsible for the active transport of platinum, and depletion of EZH2 altered CTRs expression levels in vitro. Thus, we propose that EZH2 induces acquired cisplatin resistance in epithelial ovarian cancer cells through targeting copper transporters via PRC2 and/or non-PRC2 dependent pathway and impacting intracellular platinum accumulation. The present object focuses on the clarification of the molecular mechanisms and attempts to prevent EOC from acquired cisplatin resistance by using drugs targeting the mechanisms through which EZH2 regulates CTRs as a novel chemotherapy strategy for EOC.

肿瘤细胞减灭术辅以铂为基础的联合化疗是上皮性卵巢癌(EOC)标准治疗方案，而化疗耐药导致的复发是EOC死亡的主要原因。我们前期发现EZH2是调控EOC顺铂敏感性的重要因子，且顺铂可激活EZH2多梳抑制复合体2(PRC2)通路(组蛋白甲基化)和非PRC2通路(EZH2磷酸化)。申请人在国家自然科学青年基金研究中首次发现下调EZH2能促进EOC中铂的蓄积并增强顺铂的抑瘤作用。进一步研究提示负责铂主动转运的铜转运蛋白 (CTRs)在耐药肿瘤中表达异常，且细胞中EZH2下调可引起CTRs表达变化。因此我们假设：EZH2通过PRC2和/或非PRC2依赖机制调控CTRs表达，影响铂类药物的转运，调节铂在肿瘤细胞中的蓄积，从而参与EOC耐药机制。本项目将进一步阐明EOC获得性顺铂耐药的分子机制，并尝试以EZH2对CTRs的调控机制为靶点，联合用药阻断获得性顺铂耐药的产生，为EOC临床化疗提供新的策略。

根据研究计划，我们完成了以下研究内容：一、铂介导的上皮性卵巢癌获得性耐药过程中EZH2和CTRs表达变化的相关研究：① 数据库生信分析及Meta分析研究了TCGA和GEO数据库中EZH2及CTRs在卵巢癌患者中的表达情况，与铂药耐药的相关性，结果提示EZH2的表达和CTR1呈负相关，EZH2高表达的患者铂药耐药的可能性较高，且预后较差，而CTR1高表达的患者铂药耐药的概率较低，且预后较好。② 卵巢癌标本库患者组织芯片结果也提示与①部分相似的结果。③ 模拟周期临床化疗，建立卵巢癌铂药耐药细胞模型，观测到耐药细胞的EZH2/H3K27me3的水平逐渐升高。二、EZH2调控CTRs影响铂药转运和EOC耐药：①下调EZH2的水平，卵巢癌细胞内铂药蓄积增多，对顺铂的敏感性增加；上调EZH2的水平，卵巢癌细胞内铂药蓄积减少，对顺铂敏感性降低；②BODIPY-Pt荧光铂模拟铂药转运示踪，下调EZH2的水平，卵巢癌细胞内荧光铂蓄积增多，对荧光铂的敏感性增加；③免疫荧光标记卵巢癌细胞内的CTR1，下调EZH2，荧光铂与CTR1的共定位系数增加，提示EZH2阻碍了CTR1对铂药的转运。三、EZH2调控CTR1转运铂药介导卵巢癌铂药耐药的机制：①PRC2依赖性机制的探索：我们在探索EZH2和CTR1的表达关系时发现，下调EZH2、抑制H3K27me3的水平，CTR1的整体表达并无明显变化，提示EZH2并不通过直接调控CTR1的表达影响CTR1的功能；进一步探索发现EZH2/H3K27me3主要通过影响卵巢癌细胞DNA损伤修复而介导耐药②非PRC2依赖性机制的探索—EZH2影响铂介导的CTR1的降解：CTR1在转运铂药的过程中会发生降解，从而限制铂药的有效转运，下调EZH2能够有效减少铂介导的CTR1降解；③EZH2促进铂介导的CTR1的泛素化：通过对CTR1蛋白周期及泛素化情况的研究，发现EZH2能够促进CTR1的泛素化，进而加快CTR1的降解，下调EZH2，CTR1的泛素化程度降低。四、联合用药阻断EZH2作用通路改善EOC顺铂耐药。基于EZH2的PRC2依赖途径和PRC2非依赖途径介导卵巢癌耐药，我们发现EZH2抑制剂GSK126和PARP抑制剂奥拉帕尼通过联用通过在细胞水平和动物水平均取得明显疗效。综上，EZH2或可称为上皮性卵巢癌铂药耐药的一个治疗靶点。