Raf plus TGFbeta-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin

Oncogene. 2006 Nov 16;25(54):7117-30. doi: 10.1038/sj.onc.1209701. Epub 2006 Jun 5.

Abstract

Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor beta (TGFbeta) signaling to cause epithelial-mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGFbeta pathways cooperate in EMT induction, we introduced a tamoxifen-inducible version of Raf-1 (RafER) into fully polarized, mammary epithelial cells (EpH4). EMT characterized by loss of E-cadherin expression and upregulation of invasiveness-promoting genes was induced by TGFbeta plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGFbeta was detectable in total cell lysates after 48 h and much earlier in detergent-insoluble fractions of E-cadherin. Both pathways cooperated to strongly enhance endocytosis of E-cadherin, mainly via the clathrin-dependent route. Pulse-chase experiments showed decreased E-cadherin protein stability in cells stimulated with TGFbeta and 4HT and increased E-cadherin half-life in the presence of monensin. Monensin and chloroquine prevented E-cadherin degradation to different extent, but only monensin effectively blocked the loss of E-cadherin from the junctional complexes. Both lysosome inhibitors caused accumulation of E-cadherin vesicles, some of which were positive for Cathepsin D and lysosome-associated membrane protein 1 (LAMP-1). In addition, TGFbeta and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGFbeta favors lysosomal degradation of E-cadherin instead of its recycling. Our data indicate that early stages of EMT involve cooperative, post-translational downregulation of E-cadherin, whereas loss of E-cadherin via transcriptional repression is a late event in EMT.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Northern
  • Blotting, Western
  • Cadherins / metabolism*
  • Cell Line, Tumor
  • Cell Transformation, Neoplastic*
  • Down-Regulation
  • Endocytosis
  • Epithelial Cells / metabolism*
  • Epithelial Cells / pathology
  • Fluorescent Antibody Technique
  • Immunoprecipitation
  • Lysosomes / metabolism
  • Mice
  • Microscopy, Confocal
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins
  • Transforming Growth Factor beta / metabolism*
  • raf Kinases / metabolism*

Substances

  • Cadherins
  • Recombinant Fusion Proteins
  • Transforming Growth Factor beta
  • raf Kinases