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

Abstract

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

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

“…TGF-␤ is a key regulator of epithelial-mesenchymal transition, which is characterized by loss of polarity and increased cell motility. T␤R/SMAD and RTK/RAS signaling pathways cooperate to regulate epithelial-mesenchymal transition through several different mechanisms (11,31). We have uncovered a cooperative interaction between RTK/RAS and TGF-␤ pathways through RIN1-mediated receptor endocytosis.…”

EMT, the cytoskeleton, and cancer cell invasion

“…Moreover, integrinactivated focal adhesion kinase (FAK) can phosphorylate β-catenin and thus induce its ubiquitylation and degradation and the disassembly of the E-cadherin cell adhesion complex [59]. Endocytosis of E-cadherin can occur via clathrin or caveolin-dependent mechanisms [60][61][62]. A key player in clathrin-mediated E-cadherin endocytosis is Arf6, a Ras-related small GTPase.…”

Rack1 promotes epithelial cell–cell adhesion by regulating E-cadherin endocytosis

“…These processes are fine-tuned and facilitate junctional remodeling during normal physiological processes such as wound healing (Bryant and Stow, 2004;Kowalczyk and Reynolds, 2004;Yap et al, 2007;Delva and Kowalczyk, 2009;Schill and Anderson, 2009). The homeostasis is however disrupted in pathological states, including cancer, and could result from excessive signaling by growth factors such as epidermal growth factor, transforming growth factor-b, hepatocyte growth factor (HGF) or aberrant activation of non-receptor tyrosine kinases like Src (Lu et al, 2003;Janda et al, 2006;Mosesson et al, 2008;Shen et al, 2008;Orlichenko et al, 2009;Saitoh et al, 2009). For instance, Src regulates various facets of signaling to AJs as well as E-cadherin stability, and is a key contributor to epithelial-mesenchymal transition, which drives tumor metastasis (Frame, 2004;Yeatman, 2004).…”