Neddylation修饰催化酶UBC12作为新型抗肺癌分子靶点的鉴定及其机制研究

Neddylation is a novel post-translational modification which is overactivated in tumor tissues and facilities tumor initiation, while blockage of neddylation exerts remarkable anti-tumor effects. Recently, small molecule inhibitor MLN4924 targeting NEDD8 Activating Enzyme NAE (E1) has been successfully developed and advanced into I/II phase clinical trials due to its high efficiency and low toxicity, thus MLN4924 appears to have good application prospects. Nevertheless, recent studies have shown that NAE mutation triggered by MLN4924 causes drug resistance of tumor cancer cells against MLN4924. Thus, it is urgent to exploring new therapeutic targets aiming at neddylation pathway. Our recent studies have found that NEDD8 conjugating enzyme E2 (UBC12) is markedly overexpressed in lung cancer tissues which positively relates to whole neddylation level in tumor tissues and poor prognosis of patients. Hence, previous work indicates that given the significant role in promotion of lung cancer progression, UBC12 serves as a potential anticancer therapeutic target. In this project, we will investigate effects of UBC12 dysregulation on neddylation pathway in addition to malignant phenotype of lung cancer cells further evaluate anti-tumor efficacy via targeting UBC12 and finally illuminate the molecular mechanism. As a result, our work will provide scientific basis for UBC12 as a novel anti-tumor target against neddylation.

Neddylation是一种新型蛋白翻译后修饰方式，在肿瘤组织过度活化并促进肿瘤发生，而阻断此修饰通路具有显著的抗肿瘤效果。近年来，NEDD8活化酶NAE（E1）抑制剂MLN4924被成功开发并已进入I/II期临床试验阶段，显示出良好的应用前景。但最近研究表明，MLN4924可诱导肿瘤细胞发生耐药突变，因此针对Neddylation通路鉴定新的抗肿瘤靶点势在必行。我们最近发现，NEDD8结合酶UBC12(E2)在肺癌组织显著高表达，并且与肿瘤组织内总体Neddylation修饰水平及肺癌患者预后不良显著正相关，提示UBC12是一个潜在促肺癌发生发展的新型治疗靶点。本项目拟从研究UBC12失调对Neddylation通路总体活化和肿瘤细胞恶性表型的影响入手，评价靶向UBC12抗肿瘤效果并阐明相关分子机制，为最终鉴定UBC12作为针对Neddylation通路的新型抗肿瘤分子靶点提供科学依据。

Neddylation是一种新型蛋白翻译后修饰方式，在肿瘤组织过度活化并促进肿瘤发生，而阻断此修饰通路具有显著的抗肿瘤效果。近年来，NEDD8活化酶NAE(E1)抑制剂MLN4924被成功开发并已进入I/II期临床试验阶段，显示出良好的应用前景。但最近研究表明，MLN4924可诱导肿瘤细胞发生耐药突变，因此针对Neddylation通路鉴定新的抗肿瘤靶点势在必行。我们最近发现，NEDD8结合酶UBC12(E2)在肺癌组织显著高表达，并且与肿瘤组织内总体Neddylation修饰水平及肺癌患者预后不良显著正相关，提示UBC12是一个潜在促肺癌发生发展的新型治疗靶点。依托本课题基金支持，我们从研究UBC12失调对Neddylation通路总体活化和肿瘤细胞恶性表型的影响入手，评价靶向UBC12抗肿瘤效果并阐明相关分子机制，为最终鉴定UBC12作为针对Neddylation通路的新型抗肿瘤分子靶点提供科学依据。.研究发现，下调UBC12表达显著抑制总体蛋白的Neddylation修饰。并且，下调UBC12只对Cul1、Cul2、Cul3、Cul4的Neddylation修饰有抑制，而对受UBE2F调控的Cul5无影响。提示，靶向UBC12对Neddylation通路的抑制作用具有特异性。.ATPlite细胞增殖分析和细胞克隆形成实验均发现，下调UBC12显著抑制肺癌细胞生长。细胞迁移实验分析发现，下调UBC12显著抑制肺癌细胞迁移能力。.对细胞周期进行分析发现，肺癌细胞应答下调UBC12发生G2/M期细胞周期阻滞，WB检测发现Wee1表达上调而G2期向M期过渡的蛋白pH3表达下调，提示细胞周期阻滞发生在G2期。同时，肺癌细胞在下调UBC12后CRL经典底物p21、p27、CDT1、ORC1等均明显积聚。提示我们，下调UBC12通过诱导G2期细胞周期阻滞、DNA损伤应答等从而引起肺癌细胞发生细胞衰老和细胞凋亡导致细胞死亡。.裸鼠皮下瘤模型显示下调UBC12在体内具有良好的抗肿瘤效果。对裸鼠皮下瘤进行IHC染色发现，下调UBC12 CRL底物p27表达阳显著升高。提示，下调UBC12表达在体内亦通过引起CRL抑癌蛋白底物积聚，从而诱导G2期细胞周期阻滞、DNA损伤应答等从而引起肺癌细胞发生细胞衰老和细胞凋亡导致肺癌细胞死亡。.本研究提示UBC12为非常有前景的抗肺癌治疗靶点。