Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis

Abstract

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells

“…However, during malignant tumor progression, epithelium-derived tumor cells often escape TGF-b-induced growth control, and once this has occurred, TGF-b promotes tumor progression resulting in increased tumor cell invasion and metastasis . This is often associated with oncogenic Ras-signaling (Janda et al, 2002;Oft et al, 2002). In the present study, TGFb had no effect on proliferation of head and neck SCC cells in culture or on the levels of cyclin-dependent kinase inhibitors p15 (Ink4b) , p21 (Cip1/Waf1) , and p27 (Kip1) .…”

MFG-E8/lactadherin regulates cyclins D1/D3 expression and enhances the tumorigenic potential of mammary epithelial cells

“…To better understand the effect of MFG-E8 in NMuMG cells, we examined their growth pattern in collagen gels. Embedding of cells in a gel of an extracellular matrix protein allows studying their morphology and underlying biochemical properties in a three-dimensional (3D) environment that mimics the situation encountered by the cells in vivo (Janda et al, 2002). After 6 days in collagen gels, we observed that whereas control cells tended to form elongated structures with minimal side MFG-E8 promotes mammary tumor growth C Carrascosa et al Figure 6C).…”

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

“…In several epithelial cell models, cooperation of the Raf/ ERK/MAPK pathway and TGFb signaling is required for induction and maintenance of EMT in vitro and a metastatic phenotype in vivo (Lehmann et al, 2000;Janda et al, 2002a). To investigate the mechanism of how Raf and TGFb signaling cooperate in these processes, we generated EpH4 cells stably expressing an 4HT-inducible RafER construct (EGFP-DRaf1-hbER(YY)) (Woods et al, 1997).…”

“…In particular, the ability of transforming growth factor b (TGFb) to induce EMT has been correlated with a tumor-promoting role of TGFb during late-stage cancer development, contrasted by TGFb-induced cell cycle arrest and apoptosis induced in normal epithelial cells (Derynck et al, 2002;Grunert et al, 2003). Using fully polarized mammary epithelial cells (EpH4) transformed with oncogenic Ras (Ep2Ras), we showed that a Ras-induced hyperactive extracellular signal-regulated kinase (ERK)/ mitogen-activated protein kinase (MAPK) signaling cooperating with TGFb is required to cause EMT in vitro and in vivo and tumor metastasis in nude mice (Oft et al, 1996;Janda et al, 2002a). In contrast, Ras-induced phosphatidylinositol 3 0 -kinase signaling was required for efficient protection from TGFbinduced apoptosis as well as for rapid tumor growth (Janda et al, 2002b).…”