Much of the meeting highlighted signaling pathways that regulate or mediate the EMT, focusing both on refinement and extension of known pathways, but also on the discovery of new regulators and novel pathways ().
Figure 1. Signaling events during EMT. The major signaling events that were reported in the meeting are summarized. Cleavage of E-cadherin (yellow) by MMP-3 resulted in activation of Snail1 through ROS. Snail1 localization to the nucleus is controlled by phosphorylation (more ...)
One of the first cell surface receptors identified that was able to stimulate scattering of epithelial cells was the Met receptor tyrosine kinase. Activation of Met by its ligand, hepatocyte growth factor, enhances the migration of multiple cell lines in vitro, and scattering of cultured multicystic dysplastic kidney cells is a classical EMT assay. Morag Park (McGill University, Montreal, Quebec, Canada) reported that transgenic mice expressing wild-type or active variants of Met under the control of the mouse mammary tumor virus promoter develop nodal and ductal hyperplasia and spontaneous mammary tumors, albeit with a long latency period (~1.5 yr). Park suggested that Met cooperates with the Her2/neu oncogene in activating EMT, and that the Crk family of SH2 and SH3 adaptor proteins are critical in Met-mediated EMT. Crk proteins are highly expressed in human breast tumors, and Park reported that small interfering RNA (siRNA) ablation of Crk inhibits Met-dependent cell migration and EMT.
Although the Met receptor-mediated signaling results in cell scattering, it has not been made clear whether Met signaling also has a more permanent effect on the expression or localization of some of the effectors of EMT, such as E-cadherin and β-catenin. Recent work by Walter Birchmeier (Max Delbruck Center, Berlin, Germany) suggests that Met also regulates intracellular localization of β-catenin. β-Catenin has a dual role in the EMT; it enhances cell–cell adhesion when bound to cadherin complexes in adherens junctions and also functions as a transcriptional coactivator upon entry into the nucleus (van Es et al., 2003
). The ability of β-catenin to enhance cadherin-dependent adhesion depends on β-catenin binding to α-catenin and on α-catenin binding to the cadherin (Chu et al., 2004
). Phosphorylation of β-catenin residue Y142 prevents α-catenin interaction and enhances the binding of β-catenin to BCL9-2, which is the vertebrate homologue of the Drosophila melanogaster
legless gene (Brembeck et al., 2004
). Interaction of β-catenin with BCL9-2 enhances nuclear accumulation of both proteins, simultaneously decreasing cadherin-mediated adhesion and activating catenin target gene transcription. Ectopic BCL9-2 expression is sufficient to induce EMT in cultured cells, and siRNA-mediated BCL9-2 inactivation drives the reverse mesenchymal–epithelial transition (MET). Birchmeier reported that Y142 can be phosphorylated by the Met tyrosine kinase, indicating the existence of an EMT activation pathway where Met induces β-catenin nuclear translocation by enhancing BCL9-2 interaction. This pathway satisfactorily links these two well known EMT regulators.
Interestingly, Pez/PTPN14, which is a tyrosine phosphatase that is frequently mutated in colorectal tumors (Wang et al., 2004
), induces Snail1 expression and can also activate cell migration (Yeesim Khew-Goodall, Hanson Institute, Adelaide, Australia). Pez can dephosphorylate β-catenin on tyrosine residues that regulate its interaction with the adherens junction complex, suggesting that Pez mutations contribute to EMT by preventing cytoplasmic β-catenin–cadherin interaction and enhancing its nuclear translocation. However, Pez overexpression in MDCK and MDA-MB468 cells was shown to be sufficient to cause EMT, and knockdown in zebrafish causes multiple developmental abnormalities, including aberrant pigmentation and craniofacial deformation. These defects are broadly consistent with dysfunctional neural crest EMT in the absence of Pez.
Cancer-relevant insights into EGF signaling were provided by Erik Thompson (University of Melbourne, Melbourne, Australia), who has identified EGF as a novel EMT inducer in human breast cancer, as measured by EGF's ability to decrease E-cadherin and increase vimentin production in PMC42 cells. Interestingly, EMT may influence the response of certain cancers to EGF receptor (EGFR)–targeted therapeutics. John Haley (OSI Pharmaceuticals, Melville, NY) presented data showing that the sensitivity of nonsmall cell lung cancer cell lines to erlotinib, which is an EGFR-targeted monoclonal antibody, did not correlate with EGFR levels, but rather depended on their EMT status, with those having undergone EMT showing resistance (Thomson et al., 2005
An interesting and novel aspect of EGFR signaling was presented by Mien-Chie Hung (The University of Texas MD Anderson Cancer Center, Houston, TX), who reported that EGFR, which is a transmembrane receptor tyrosine kinase, complexes with the STAT3 transcription factor in the nucleus and can be immunoprecipitated from the EGF-responsive iNos promoter (Lo et al., 2005a
). The role that promoter-complexed EGFR has in EMT is uncertain, but high nuclear EGFR is associated with a poor prognosis in breast carcinoma (Lo et al., 2005b
). The observation that a transmembrane receptor is found in functional promoter complexes in the nucleus was one of the meeting's most surprising observations, and it will be of great interest to characterize the topological and structural mechanisms through which a membrane receptor enters the nucleus and activates transcription (Giri et al., 2005
TGF-β is a major regulator of EMT and has been implicated in skin cancer development (Zavadil and Bottinger, 2005
). Jiri Zavadil (New York University School of Medicine, New York, NY) reported that TGF-β activates EMT through Smad-3–dependent activation of the HEY1 gene, a member of the Hairy/Enhancer-of-split family of transcriptional repressors. Zavadil used extensive gene expression profiling to identify HEY1 targets that are important in EMT induction (Zavadil et al., 2004
). He reported on the profiling of EMT in the following three different contexts: HaCaT human keratinocyte EMT in response to TGF-β, mouse model of aristolochic acid nephropathy, and human kidney-proximal tubule cells. Satisfyingly, one of these targets is Dishevelled 2
, which is a gene that regulates EMT by repressing the production of Notch, GSK3β, and β-catenin. Another HEY1 target seen in all three systems was the polycomb family histone methyltransferases EZH1/EZH2, suggesting that TGF-β–activated EMT could be controlled through structural histone modification. Other TGF-β targets include integrins β4 and α6. Richard Bates (University of Massachusetts, Worcester, MA) reported that the integrin αvβ6 is up-regulated during colon cancer development and highly expressed in metastatic samples (Bates, 2005
Christopher Gebeshuber showed that TGF-β induced Smad-2 tyrosine phosphorylation and that TGF-β–induced EMT was blocked upon expression of nonphosphorylatable Smad-2 mutant, the expression of which inhibited metastases formation. Gebeshuber also reported that this mutant had a reduced ability to interact with the Tcf–Lef1 transcription factor. This suggests that tyrosine phosphorylation of Smad-2 may potentiate Tcf–Lef1 interaction and stimulate both EMT and metastatic induction. Ali Nawshad (University of Nebraska, Lincoln, NE) and Elizabeth Hay reported a similar noncanonical role for TGF-β in the EMT of mouse palatal epithelial seam and kidney-proximal tubule cells. They reported that Smad-2/4 repressed E-cadherin transcription through Tcf–Lef1 (Masszi et al., 2004
; Nawshad et al., 2005
One of the functions of TGF-β is to stimulate expression of ECM proteins. Do ECM proteins initiate EMT? Andre Menke (University of Ulm, Ulm, Germany) showed that extracellular collagen that is deposited during a fibrotic disease can be an initiator of EMT. Menke reported that pancreatic cancer cell lines cultured on collagen I have a reduced capacity to cluster E-cadherin at points of cell–cell contact and have a more mesenchyme-like morphology. Menke postulated an EMT pathway where collagen induces both the recruitment of FAK to cadherin adhesion complexes and the phosphorylation of β-catenin. Phosphorylated β-catenin then translocates to the nucleus, activating EMT target genes. Conceptually, this may be similar to work by Mina Bissell describing the capacity of mechanical forces or the shape of the cell to initiate EMT.