Signal transduction mechanisms in epithelium-mesenchyme transitions of embryonic and tumor cells
02 / 2002 - unknown
Website Hubrecht Laboratorium
Epithelium to mesenchyme transition (EMT) is a multistep process in which cells detach from an epithelium, and become solitary and highly migratory. EMT is crucial for morphogenetic movements in the developing embryo, and deregulation of this process plays an important role in metastasis of transformed cells, since many of the cellular changes in EMT are also found in metastatic transformed cell types. For instance, loss of E-Cadherin expression, the major molecule involved in epithelial cell-cell adhesion, is associated with the transition of tumor cells from a relatively benign, stationary state to an aggressive, metastatic one. Vice versa, expression of E-Cadherin in many cases leads to reversal of the metastatic potential and hence reduction of tumorigenicity. We are studying factors regulating EMT and the molecular mechanisms involved. A very early example of EMT in murine embryogenesis can be found in the formation of the extraembryonic parietal endoderm (PE) from primitive and later visceral endoderm. We have established that Parathyroid Hormone related Peptide (PTHrP) and its G-protein coupled receptor play a crucial role in this process. These molecules efficiently induce PE differentiation through EMT in an in vitro model system, the embryonal carcinoma (EC) cell line F9. They act through activation of the adenylate cyclase/protein kinase A (PKA) signalling pathway. In addition, we have found that activation of the small GTP-binding protein ras/Erk pathway as well as activation of Transforming Growth Factor (TGF)beta signalling have a profound effect on the PTHrP-induced EMT. Ras- and TGFbeta signalling have been shown to influence EMT in other systems as well, i.e. in murine mammary gland cells. In an ongoing collaboration with the group of Peter ten Dijke at the Netherlands Cancer Institute (NKI) we are studying EMT in such cells, i.e. the murine NMuMG cell line. To determine the molecular levels at which these molecules exert their effects on EMT, we are measuring a variety of parameters that change during EMT, such as the expression of junctional proteins, cytoskeletal changes and cell motility. Therefore, we have started a collaboration with the group of Jean-Paul Thiery at the Institut Curie in Paris, who are experts in this field. We have observed strong expression of mRNA encoding the transcription factor snail immediately after PTHrP addition. Snail belongs to a family of zinc finger proteins, that are thought to play a crucial role in embryonic events involving EMT in organisms as diverse as Drosophila, Xenopus, chick and mice. Snail and its close relative slug are expressed at a number of sites in which EMT occurs during embryogenesis, e.g. in parietal endoderm, mesoderm and neural crest formation. In two publications in Nature Cell Biology, snail was shown to directly repress transcription of the E-Cadherin gene. Expression of snail in breast cancer cells correlates with their metastatic potential, and it was shown that interfering with snail's action partially reverted the invasive phenotype of mesenchymal tumor cells. Apart from repressing E-Cadherin expression, snail regulates the expression of genes involved in other important aspects of EMT, e.g. cellular motility. The expression of snail mRNA in response to PTHrP also occurs in the absence of protein synthesis, making snail an immediate early response gene to PTHrP-receptor activation. F9 is the first cell line in which such an effect on snail transcription has been found. The F9 cells thus provide a unique system to study the regulation of snail expression as well as the role of snail in the regulation of EMT. Another aspect of PTHrP-PKA signalling we are studying is a possible cross-talk with the canonical Wnt-beta-catenin signalling pathway. We found that PTHrP induces rapid nuclear translocation of beta-catenin and activation of the wnt-TCF signalling pathway in F9 and a variety of other cells. Furthermore, careful study of PE isolated from PTHrP-receptor -/- embryos has established that the distribution of beta-catenin is abnormal. We are studying the relevance of these findings for PE formation.