Targeted Degradation of KRAS by an Engineered Ubiquitin Ligase Suppresses Pancreatic Cancer Cell Growth In Vitro and In Vivo
Targeted Degradation of KRAS by an Engineered Ubiquitin Ligase Suppresses Pancreatic Cancer Cell Growth In Vitro and In Vivo
复制标题
工程化泛素连接酶靶向降解 KRAS 可抑制体外和体内胰腺癌细胞生长
DOI：
10.1158/1535-7163.mct-12-0650
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发表时间：
2013-03-01
影响因子：
5.7
通讯作者：
Chen, Jie
中科院分区：
文献类型：
--
作者：
Ma, Yihui;Gu, Yumei;Chen, Jie
KRAS是一个有吸引力的胰腺导管腺癌（PDAC）治疗靶点。E3连接酶被认为是直接负责底物识别的泛素缀合系统的组分。在这项研究中，工程E3泛素连接酶（RC-U）产生的目标KRAS癌蛋白的泛素化和降解。构建工程化的E3泛素连接酶（RC-U）（pRC-U和表达慢病毒的RC-U）。将pRC-U质粒转染人胰腺癌细胞后，测定KRAS表达水平。还在用pRC-U转染并用MG-132或放线菌酮处理的细胞中评价KRAS表达。研究了RC-U与KRAS之间的相互作用以及RC-U是否可以泛素化KRAS。检测转染pRC-U的胰腺癌细胞中细胞外信号调节激酶1/2（ERK 1/2）和磷酸化ERK 1/2（pERK 1/2）的水平。评价RC-U对胰腺癌细胞生长的影响。RC-U降低KRAS蛋白水平。pRC-U转染后，在MG-132存在下KRAS稳定性增加。用突变KRAS构建体与pRC-U一起转染HEK 293 T细胞，并用放线菌酮孵育以抑制新蛋白质合成。外源突变KRAS癌蛋白降解更快。RC-U可以结合KRAS，并且KRAS可以被RC-U泛素化。pERK 1/2蛋白水平降低。RC-U导致体外和体内细胞增殖减少。RC-U通过泛素依赖性蛋白酶体介导的降解途径破坏KRAS。RC-U抑制胰腺癌细胞生长在体外和体内。Mol Cancer Ther; 12（3）; 286-94.©2012 AACR。
KRAS is an attractive pancreatic ductal adenocarcinoma (PDAC) therapeutic target. E3 ligase is thought to be the component of the ubiquitin conjugation system that is directly responsible for substrate recognition. In this study, an engineered E3 ubiquitin ligase (RC-U) was generated to target the KRAS oncoprotein for ubiquitination and degradation. The engineered E3 ubiquitin ligases (RC-U) were constructed (pRC-U and lentivirus-expressing RC-U). After transfecting the pRC-U plasmid into human pancreatic cancer cells, KRAS expression levels were determined. KRAS expression was also evaluated in cells transfected with pRC-U and treated with MG-132 or cycloheximide. Interactions between RC-U and KRAS as well as whether RC-U could ubiquitinate KRAS were investigated. Extracellular signal–regulated kinase 1/2 (ERK1/2) and phosphorylated ERK 1/2 (pERK1/2) levels were examined in pancreatic cancer cells transfected with pRC-U. The effects of RC-U on pancreatic cancer cell growth were assessed. RC-U decreased KRAS protein levels. After pRC-U transfection, KRAS stability was increased in the presence of MG-132. HEK 293T cells were transfected with a mutant KRAS construct together with pRC-U and incubated with cycloheximide to inhibit new protein synthesis. The exogenous mutant KRAS oncoprotein was degraded more quickly. RC-U can bind KRAS and KRAS can be ubiquitinated by RC-U. pERK1/2 protein levels were decreased. RC-U resulted in reduced cell proliferation in vitro and in vivo. KRAS destruction by RC-U occurred through a ubiquitin-dependent, proteasome-mediated degradation pathway. RC-U inhibited pancreatic cancer cell growth in vitro and in vivo. Mol Cancer Ther; 12(3); 286–94. ©2012 AACR.