RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma

BMC Cancer. 2019 Jul 8;19(1):670. doi: 10.1186/s12885-019-5880-1.

Abstract

Background: 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.

Methods: Protein antibody microarray analysis and E3 ligase profiling were performed to detect the unique E3 ligase underlying E-cadherin downregulation in lung adenocarcinoma tissues. Gene knockdown was performed using viral shRNA. Immunoblotting, immunofluorescence, immunoprecipitation, and xenograft models in vivo were integratively applied to explore RNF43-induced EMT in lung adenocarcinoma cell lines.

Results: Protein antibody microarray analysis and E3 ligase profiling revealed that the RING finger protein 43 (RNF43) was linked to E-cadherin downregulation within the context of c-Src activation in lung adenocarcinoma tissues. In addition, the 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 allowing the nuclear translocation of β-catenin and upregulation of Vimentin and RNF43 expression in lung adenocarcinoma cells. Decreased E-cadherin expression and increased Vimentin expression induced the EMT phenotype and promoted tumor metastasis. The Frizzled 8 (Frz8)-RNF43-induced ubiquitination of phosphorylated E-cadherin was blocked by a monoclonal antibody against the cysteine-rich domain (CRD) of Frz8 but not by antibodies against the protease domain (PA) of RNF43.

Conclusions: Our data suggest that RNF43 participates in the regulation of EMT in the metastasis of lung adenocarcinoma through the ubiquitination and degradation of phosphorylated E-cadherin by activated c-Src.

Keywords: C-Src; Caspase-8; EMT; RNF43.

MeSH terms

  • A549 Cells
  • Adenocarcinoma of Lung / metabolism
  • Adenocarcinoma of Lung / secondary*
  • Animals
  • Antibodies, Monoclonal
  • Antigens, CD / metabolism*
  • CSK Tyrosine-Protein Kinase / metabolism*
  • Cadherins / metabolism*
  • Caspase 8 / metabolism
  • Cells, Cultured
  • DNA-Binding Proteins / immunology
  • DNA-Binding Proteins / metabolism*
  • Epithelial-Mesenchymal Transition*
  • Follow-Up Studies
  • Humans
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / pathology*
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Oncogene Proteins / immunology
  • Oncogene Proteins / metabolism*
  • Phosphorylation
  • Proteolysis
  • Receptors, Cell Surface / metabolism
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination
  • Vimentin / metabolism
  • beta Catenin / metabolism

Substances

  • Antibodies, Monoclonal
  • Antigens, CD
  • CDH1 protein, human
  • CTNNB1 protein, human
  • Cadherins
  • DNA-Binding Proteins
  • Fzd8 protein, human
  • Oncogene Proteins
  • Receptors, Cell Surface
  • VIM protein, human
  • Vimentin
  • beta Catenin
  • RNF43 protein, human
  • Ubiquitin-Protein Ligases
  • CSK Tyrosine-Protein Kinase
  • CSK protein, human
  • Caspase 8