Differential cooperation of MAPKs with TGF-β signaling in epithelial to mesenchymal transition (EMT)

© 2005 Dr. Bo WangTumor invasion and metastasis are the major causes of treatment failure in cancer patients and principal contributing factors to cancer morbidity and mortality. However, little is understood about either event at the molecular level. Epithelial to Mesenchymal Transition (EMT) has been recognized as a cellular mechanism for carcinoma progression towards invasion and metastasis. Transforming growth factor-β (TGF-β) signaling and mitogen-activated protein kinases (MAPKs) have been identified as important molecular contributors in EMT. However, how TGF-β signaling cooperates with MAPKs in EMT at the molecular level is yet to be fully understood. EMT is a multifaceted event and ill-defined. To define functional molecular markers of specific aspects of EMT, MDCK cells were chosen as an epithelial model while v-Ha-Ras transformed MDCK cells were used as a mesenchymal model. To investigate the roles of temporal and activation levels of Ras-MAPK during the EMT processes, cell line with inducible v-Ha-Ras expression in MDCK cells was established. Here, we demonstrate that long-term TGF-β treatment leads to early stages of EMT without progression through to the late stages of EMT. Weak activation of ERK1/2 by epidermal growth factor (EGF) is not sufficient to induce EMT, but can enhance/speed up the effect of TGF-β on early stage (or partial) EMT. During the initiation and progression of EMT, long-term hyper-activation of Ras-ERK1/2 and its cooperation with TGF-β is required to drive EMT to near completion. More importantly, we have shown for the first time that mild but not hyper - or diminished Ras-ERK1/2 activation at late stage EMT further enhances mesenchymal properties of MDCK cells. Partial reduction of Ras-ERK1/2 results in robust expression of the mesenchymal marker vimentin and greatly enhances cell motility and invasive growth in vitro. Thus, the requirement of Ras activation during various stages of EMT is time and magnitude specific: long-term hyper-activation of Ras-ERK1/2 cooperating with TGF-β signaling efficiently initiates EMT and progresses to late stages, while mild-activation of the Ras-ERK1/2 together with TGF-β signaling completes the late phases of EMT. To uncover changes of signaling pathways during different stages of EMT, the effect of Ras-ERK1/2 activation on TGF-β-Smad signaling was investigated. We demonstrate that, in context of EMT in MDCK cells, Ras activation enhances TGF-β signaling through Smad phosphorylation, nuclear translocation and target gene expression. However, the sensitization of TGF-β-Smad signaling caused by Ras appears to be indirect. Long-term (when at least partial EMT is achieved) but not short-term Ras activation enhances the phosphorylation and translocation of Smad2/3, inhibits Smad2 shuttling between cytoplasm and nucleus, and increases (CAGA)12 reporter gene activation. Interestingly, our study demonstrates that TGF-β-Smad signaling is modulated by Ras activation in time-dependent and magnitude specific manner. The sensitization of Smad2/3 signaling during early EMT requires hyper- and sustained Ras activation. In contrast, reduced Ras-ERK1/2 activation at late stages of EMT results in further enhancement of sensitization of Smad2/3 signaling. p38 MAPK activation induces some specific early events of EMT, such as disruption of adherens junction and actin stress fibres at basal level in MDCK cells. Disruption of the tight junction and supporting actin cytoskeleton requires cooperation with TGF-β. Contrary to the positive effect of p38 MAPK during early stages of EMT, we find that inhibition of p38 MAPK by its specific inhibitor, SB203580, enhances Ras-induced mesenchymal cell migration and invasion in vitro at late stages of EMT, suggesting a negative effect of p38 MAPK on late phases of EMT. In summary, this thesis demonstrates that specific levels of Ras-ERK1/2 activation cooperate with TGF-β signaling in driving different phases of EMT, Ras activation sensitizes TGF-β-Smad2/3 signaling, while p38 MAPK cooperates with TGF-β both positively and negatively in the initiation and invasion phases of EMT, respectively