Targeted Degradation of KRAS by an Engineered Ubiquitin Ligase Suppresses Pancreatic Cancer Cell Growth In Vitro and In Vivo

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 ERK1/2 (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. (C)2012 AACR.