1. ¹Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi "Magna Graecia", Campus Germaneto, Catanzaro, Italy
2. ²Signal Transduction Laboratories, Imperial Research Fund, London, UK
3. ³Research Institute of Molecular Pathology, Vienna, Austria

Correspondence: Professor M Grieco, Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi "Magna Graecia", Campus Germaneto – livello 7, Localita' Germaneto, Catanzaro 88100, Italy. E-mail: mgrieco@unicz.it

Received 9 May 2005; Revised 10 April 2006; Accepted 18 April 2006; Published online 5 June 2006.

Abstract

Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor (TGF) signaling to cause epithelial–mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGF 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 TGF plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGF 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 TGF 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, TGF and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGF 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.