ZNF750调控E2F2抑制口腔鳞癌恶性进展的分子机制及靶点确认

Molecularly targeted drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. ZNF750 is the only known gene residing in focal deletion in head and neck squamous cancers, and is key factor to turn on the terminal epidermal differentiation gene program. Our preliminary studies showed that ZNF750 expression was decreased or deleted in the OSCC tissues and cells. The expression of the E2F2 was increased in OSCC tissues. Over-expression of ZNF750 up-regulated the expression of differentiation genes but down-regulated E2F2 expression, blocked the cell cycle progression and cell proliferation. The present studies indicated that the loss of function of ZNF750 resulted in the cell differentiation damage, reduced the E2F2 expression which regulated the cell cycle G1/S check point, leading to OSCC cells proliferation. The present study is to explore the function of ZNF750 in OSCC malignant progression and its biological function, identification of the E2F2 transcriptional activity Cis-elements regulated by ZNF750, and confirming the key molecular functional domain of ZNF750 and the recruitment cofactor by ZNF750 for regulation the E2F2, to reveal the molecular mechanisms of ZNF750 for inhibiting oral squamous cell carcinoma malignant progression. This study will provide a theoretical foundation for treatment of OSCC and develope the molecularly targeted drugs upon ZNF750.

分子靶向药用于多种恶性肿瘤的治疗，但针对口腔鳞癌(OSCC)分子靶向新疗法的发展远滞后于其它癌症。锌指蛋白750（ZNF750）是头颈部鳞癌中唯一所知位于局灶性缺失的基因，是启动细胞分化的关键。预实验发现OSCC组织及细胞中ZNF750降低或缺失、组织中细胞周期转录激活因子E2F2增多；过表达ZNF750可上调OSCC细胞分化基因并下调E2F2表达、阻滞细胞周期进程与细胞增殖等，推测ZNF750功能缺失可使细胞分化受损，对调控细胞周期G1/S检测点的E2F2抑制减弱，导致OSCC细胞增殖。本课题拟研究ZNF750的功能状态在OSCC恶性进展中的意义及生物学功能，确定ZNF750调控E2F2转录活性的顺式作用元件，并明确ZNF750调控E2F2的关键分子功能区及招募的辅助因子，揭示ZNF750抑制口腔鳞癌恶性进展的分子机制，为OSCC治疗及研发以ZNF750为靶点的分子靶向药奠定基础。

本项目阐明了抑癌基因锌指蛋白750（ZNF750）调控E2F2对口腔鳞癌（OSCC）体内外抗肿瘤的作用及机制。主要取得以下成果：（1）明确了OSCC组织中ZNF750表达变化规律及临床意义。（2）证明了ZNF750促分化抑增殖及对细胞周期的调控。ZNF750可抑制始祖基因KRT8、MMP28但促进分化基因LCE3A、SPRR1a的表达；敲减ZNF750促进KRT8、MAK2L1表达及细胞增殖而抑制KLF4表达。RNA-seq显示差异表达基因主要涉及细胞增殖及细胞周期信号通路，尤其是DNA复制通路中的所有基因均被ZNF750抑制。此外，ZNF750可抑制与增殖、转移、粘附相关的FGFBP1、VCAN、MELK、TNC VTCN1及S100A9的表达。然而，敲减ZNF750则促进上述基因的表达。（3）体内外揭示了ZNF750抑制E2F2及Ezh2-p21-G1/s通路发挥抗肿瘤作用的机制。证明了过表达或敲减ZNF750可分别抑制或促进CAL-27细胞和异种移植瘤中与表观遗传调控相关的基因（PHF19、EZH2、EED、SUZ12），UBE2C及E2F2的表达。ZNF750可使裸鼠肿瘤重量、肿瘤体积、Ki67、PCNA、MMPs蛋白或基因表达降低，而MMPs抑制剂TIMP1表达升高。然而，敲减ZNF750引起相反结果；活体生物荧光成像技术显示ZNF750组裸鼠体内肺转移瘤生长较sh-ZNF750组明显降低。(4)双荧光素酶报告分析ZNF750对E2F2系列截短体的调控，发现ZNF750对E2F2截断体8（-154→+100）具有显著负调控作用，其调控E2F2转录活性区位于-154bp→130bp。通过预先过表达或敲减E2F2，明确了ZNF750通过E2F2调控G1/S转换中的核心作用和抑癌机制，揭示了ZNF750负调控E2F2的保守模序（C2H2及PLNLS），突变ZNF750模序结构C2H2及PLNLS则丧失其对E2F2的负调控及恶性生物学行为的抑制。（5）在ZNF750对肿瘤微环境及肿瘤干细胞方面进行拓展研究，明确ZNF750促肿瘤血管正常化及抑制CSC-like细胞的作用。本项目发表论文5篇，修稿论文1篇，在ZNF750抑制E2F2调控Ezh2-p21-G1/S转换、发挥抗肿瘤作用方面取得了特色鲜明的研究成果，并对分子靶向药物研究具有重要意义。