Taki 9_8

Epithelial-mesenchymal transition (EMT) is a crucial event in cancer progression. We previously reported that EMT up-regulates matrix metalloproteinase-2 (MMP-2) expression in squamous cell carcinoma (SCC) cells. In this study, we showed that Tet Off-induced expression of Snail or SIP1, and treatment with TGF-ß1 induced EMT in terms of down-regulation of E-cadherin, and up-regulation of vimentin and MMP-2 expression with morphological changes. In SCC cells, SIP1 expression was induced by Snail and TGF-ß1, but Snail expression was not induced by SIP1 or TGF-ß1. However, expression of Snail but not SIP1 was strongly increased by TGF-ß1 in highly invasive SCC cells with mesenchymal phenotypes. Analysis of the MMP-2 promoter revealed that an Ets-1 binding site, located between position -1255 and -1248 relative to the transcriptional start site, was critical for the activation by Snail, SIP1 and TGF-ß1 in SCC cells. Induced expression of Snail and SIP1 resulted in the increased expression of Ets-1 and DNA-binding activities of nuclear proteins to the Ets-1-binding site and strong Ets-1 expression was detected in highly invasive SCC cells. Furthermore, overexpression of Ets-1 induced the promoter-activation and expression of MMP-2 without EMT. These results indicate that EMT induces Ets-1 expression, which activates the MMP-2 promoter, but Ets-1 by itself has no activity to induce EMT in SCC cells. Introduction The loss of epithelial characteristics and acquisition of a mesenchymal phenotype are important events in the progression towards more invasive and metastatic cancerous cells. This transformation is referred to as epithelial-mesenchymal transition (EMT), which was originally found to occur during embryonic development including gastrulation and neuroepithelium formation (1,2). E-cadherin is an adhesion molecule of epithelial cells whose expression is frequently down-regulated in invasive cancers. Genes encoding transrepressors of E-cadherin, through binding to E-box sequences in the E-cadherin promoter, have been reported to closely associate with EMT. Snail and Zeb families of zinc-finger proteins and basic helix-loop-helix transcription factors including E12/E47 (3) and Twist (4) have been identified as repressors of E-cadherin and triggers of EMT. An inverse correlation between Snail and E-cadherin expression has been reported in many types of cancer including squamous cell carcinoma (SCC) (5-8). Overexpression of Snail results in the dramatic down-regulation of E-cadherin, conversion to a fibroblastic phenotype, and acquisition of more invasive properties (9-12). The Zeb zinc-finger homeobox family includes Zeb-1/‰EF1 and Zeb-2/Smad-interacting protein 1 (SIP1) (13). Increased expression of SIP1 in invasive cancers without expression of E-cadherin has been also reported (14,15). SIP1 is expressed in response to TGF-ß1 and binds to Smads, the TGF-ß1 signaling proteins (16-18). TGF-ß1 also induces EMT in several epithelial cell types (19-21). Different effects of TGF-ß1 on the expression of the Snail or SIP1 gene have been reported and the correlation of Snail with SIP1 expression is still not clear (14,22-24). Besides E-cadherin, other epithelial cell-specific molecules are repressed by EMT, such as desmoplakins (9) cytokeratin 18 and MUC1 (23), claudin, occludin, · and ß-catenin (25-27). The up-regulation of mesenchymal markers including vimentin and fibronectin (9,28) has also been reported. We previously INTERNATIONAL JOURNAL OF ONCOLOGY 28: 487-496, 2006 487 Involvement of Ets-1 transcription factor in inducing matrix metalloproteinase-2 expression by epithelial-mesenchymal transition in human squamous carcinoma cells MASAYUKI TAKI1, KRISTIN VERSCHUEREN2, KAZUHIRO YOKOYAMA3, MASARU NAGAYAMA3 and NOBUYUKI KAMATA1 1Department of Oral and Maxillofacial Surgery, Division of Cervico-Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan; 2Department of Cell Growth, Differentiation, and Development VIB and Laboratory of Molecular Biology (Celgen), University of Leuven, Herestraat 49, B-3000 Leuven, Belgium; 3Department of Oral and Maxillofacial Surgery, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan Received August 9, 2005; Accepted September 19, 2005 _________________________________________ Correspondence to: Dr Nobuyuki Kamata, Department of Oral and Maxillofacial Surgery, Division of Cervico-Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan E-mail: [email protected]