Raf plus TGFbeta-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin.
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.
Janda
E
E
Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi Magna Graecia, Campus Germaneto, Catanzaro, Italy.
Nevolo
M
M
Lehmann
K
K
Downward
J
J
Beug
H
H
Grieco
M
M
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D015536:Down-Regulation; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D048490:raf Kinases / Q000378:metabolism*
D015820:Cadherins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D048490:raf Kinases