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

Abstract

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 oncoproteinwasdegradedmorequickly. RC-Ucan bindKRASand KRAScan beubiquitinatedbyRC-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. Ó2012 AACR.

Direct Modulation of Small GTPase Activity and Function

“…This category includes screening for Ras-destabilizing molecules which reduce functional Ras protein levels in cells, [163] or engineering aE 3u biquitin ligase,w hich targets K-Ras for ubiquitination and degradation. [164] These ideas might not solve the problem entirely,but they can lead the way to new technologies and fresh ideas.…”

Engineered ubiquitin ligase PTB-U-box targets insulin/insulin-like growth factor receptor for degradation and coordinately inhibits cancer malignancy

“…Over the past decade, several groups have successfully utilized such approaches to eradicate the disease-associated proteins [43]. On the other hand, different chimeric E3 ubiquitin ligases, by fusing an “interacting domain” with an “E3 catalytic domain”, have been reported to specifically degrade cancer-causing proteins, including β-catenin [46], KRAS [47] and ErbB1-3 [44], thus successfully inhibiting the related tumor growth in cultured cells and animal models. Our study presented here further strengthens the proof-of-concept of targeted ubiquitination and degradation of oncogenic RTKs.…”

miR-134 Functions as a Tumor Suppressor in Cell Proliferation and Epithelial-to-Mesenchymal Transition by Targeting KRAS in Renal Cell Carcinoma Cells

“…The downregulation of KRAS reduced tumor growth in pancreatic cancer, glioma, and oral cancer, among others (Yu et al, 2010;Shin et al, 2011;Wang et al, 2012;Ma et al, 2013;Tanaka et al, 2013). Recently, KRAS was found to inhibit EMT (Buonato et al, 2014).…”