Original Article
Oncogene (2006) 25, 7117–7130. doi:10.1038/sj.onc.1209701; published online 5 June 2006
Raf plus TGF
-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin
E Janda1, M Nevolo1, K Lehmann2, J Downward2, H Beug3 and M Grieco1
- 1Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi "Magna Graecia", Campus Germaneto, Catanzaro, Italy
- 2Signal Transduction Laboratories, Imperial Research Fund, London, UK
- 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.
Keywords:
E-cadherin, Raf, TGFbeta, EMT, endocytosis, monoubiquitination
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