Abstract
Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor β (TGFβ) signaling to cause epithelial–mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGFβ 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 TGFβ plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGFβ 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 TGFβ 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, TGFβ and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGFβ 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.
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Acknowledgements
We thank Enrico Avvedimento and Lorenzo Chiariotti (University of Naples ‘Federico II’, Italy) for stimulating discussions and valuable suggestions. Heather Bond (University of Catanzaro, Italy) for critical reading of the manuscript. We also thank Simona Polo (European Institute of Oncology, Milan, Italy) for reagents and helpful hints on experiments concerning monoubiquitination Pasquale Spataro (University of Messina) for help with confocal microscopy, and Yasuyuki Fujita (MRC, London, UK) for kind gift of mouse Hakai cDNA. This work was supported by grants from MIUR, Progetto 12, Cluster-04.
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Janda, E., Nevolo, M., Lehmann, K. et al. Raf plus TGFβ-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin. Oncogene 25, 7117–7130 (2006). https://doi.org/10.1038/sj.onc.1209701
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DOI: https://doi.org/10.1038/sj.onc.1209701
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