P1.03-08 RNF43 Ubiquitinates and Degrades Phosphorylated E-Cadherin by c-Src to Facilitate Epithelial-Mesenchymal Transition in Lung Adenocarcinoma

Abstract

In epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT). However, the precise mechanisms remain unclear. Protein antibody microarray was applied to identify the potential E3 ligase to downregulate E-cadherin. EMT was determined by morphology, epithelial-mesenchymal markers and aggressive behavior in vivo with the lung adenocarcinoma cell lines and tissues. The molecular biological methods including western blotting, immunoprecipitation, MALDI-Mass, immunohistochemistry, et al were applied to explore the signaling of RNF43-mediated EMT. Protein antibody microarray and E3 ligase profiling revealed that RING finger protein 43 (RNF43) is linked with E-cadherin downregulation within the context of c-Src activation in lung adenocarcinoma tissues. In addition, c-Src-Caspase-8 interaction markedly increased c-Src activity. Activated c-Src phosphorylated E-cadherin at the tyrosine 797 site to initiate RNF43-mediated E-cadherin ubiquitination at lysine 816 and subsequent degradation, thus freeing β-catenin into the nucleus to upregulate Vimentin and RNF43 in lung adenocarcinoma cell. Decreased E-cadherin and increased Vimentin induced EMT phenotype and promoted tumor metastasis. Frizzled 8 (Frz8)-RNF43 induced ubiquitination of phosphorylated E-cadherin was blocked by monoclonal antibody against the cysteine-rich domain (CRD) of Frz8 but not by antibodies against the protease domain (PA) of RNF43. Our data suggest that RNF43 participates in the regulation of EMT in the metastasis of lung adenocarcinoma, through ubiquitination and degradation of phosphorylated E-cadherin by activated c-Src.