肾细胞癌中有机阳离子转运体2表达抑制的表观遗传机制及联合用药PD/PK研究

Renal cell carcinoma is the highest mortality rate of cancer tumor in urinary and reproductive system. The decrease of anticancer drug uptake in tumor cells is one of the important reasons leading to the current clinical renal cell carcinoma drug-resistance. Our previous study found that the organic cation transporter 2 (OCT2) expression levels in renal cell carcinoma was significantly decreased compared to its expression levels in normal tissue and the DNA methylation levels in the OCT2 promoter area in renal cell carcinoma were significantly high than that in normal kidney tissues. These results indicated that epigenetic modification might play an important role in this program. This project first adopted a variety of in vitro and in vivo models to study the effect of DNA methylation and histone modification on the expression of OCT2 in renal cell carcinoma, and their interaction with some transcription factors, such as Max protein, c-Myc, and MLL, and clarifies the epigenetic regulation mechanism. In addition, we found coadministration of epigenetic drugs can improve the expression of OCT2 in renal cell carcinoma, and increased the accumulation of anticancer drugs in cancer cells, and obtained synergistic anticancer effect. Therefore, this project intends to study the synergism between epigenetic drugs and OCT2 substrate anticancer drugs coadministered on renal cell cancer therapy, and clarify the PD/PK correlation between them, and to obtain good combination schemes.

肾细胞癌是泌尿生殖系肿瘤中死亡率最高的癌症，肿瘤细胞对抗癌药物的摄取减少是导致目前临床上肾细胞癌产生耐药性的重要原因之一。我们前期的预实验发现，肾细胞癌中有机阳离子转运体2（OCT2）的表达水平较正常组织显著降低，且肾细胞癌中OCT2基因启动子区域的DNA甲基化水平较正常组织明显上升，这提示表观遗传修饰在其中可能发挥了重要的作用。本课题拟采用多种体内外模型研究DNA甲基化和组蛋白修饰对OCT2在肾细胞癌中表达降低的作用，以及与Max蛋白、原癌基因、组蛋白甲基转移酶等转录调节因子的相互作用，阐明表观遗传调节机制。另外，发现通过合用表观遗传类药物可提高肾细胞癌中OCT2的表达,增加抗癌药物在癌细胞中的积聚，获得抗癌增效作用。为此，本课题拟在体内外进一步研究表观遗传类药物与OCT2底物类抗癌药物联合给药对肾细胞癌治疗的协同作用，阐明它们的PD/PK相关性，以期获得效果显著的联合用药方案。

肾细胞癌（Renal cell carcinoma,RCC）是泌尿系统中最常见的恶性肿瘤，约占成人恶性肿瘤的3%。RCC也是最容易产生多药耐药的肿瘤之一，其临床化疗有效率仅有7-10% ，是死亡率最高的泌尿系统肿瘤。有机阳离子转运体OCT2主要表达在肾小管上皮细胞基底侧或顶侧，参与分泌和重吸收的功能，是肾脏中含量最多的有机阳离子转运体。前期研究表明，OCT2转运体在RCC患者肿瘤组织中蛋白表达丢失，表观遗传修饰中的DNA甲基化和组蛋白修饰均在OCT2转录抑制中发挥重要作用，且两者之间存在紧密联系，初步推测的信号通路为：MLL1负责催化OCT2基因启动子区域组蛋白甲基化激活信号H3K4Me3，转录因子MYC能够招募MLL1，从而促使OCT2基因转录激活。我们的研究发现，DNA发生高甲基化后，MYC与OCT2基因启动子区E-Box位点结合受阻，MYC招募能力减弱引起MLL1募集降低，最终导致转录激活信号H3K4Me3组蛋白修饰在OCT2基因启动子区显著下降，抑制起始转录下调蛋白表达水平。前期研究表明在细胞水平上表观遗传药物DAC与化疗药物奥沙利铂联合用药能增加RCC细胞系对奥沙利铂的敏感性。为得到更可靠的结论，本研究采用联合用药治疗RCC细胞（786-O，CAKI-1）裸鼠模型，发现通过表观遗传药物DAC逆转RCC细胞中OCT2转运体的表达，能够增加奥沙利铂在肿瘤组织的蓄积从而对肿瘤的杀伤性。该研究成果发表在Science Translational Medicine（2016, 8(348):348ra97)，被编辑誉为“Opening a door into cancer cells”，同时Nature Reviews Nephrology期刊对该成果进行了亮点评述。

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