Epithelial to mesenchymal transition occurs in the later stages of epithelial tumor progression, with cells expressing mesenchymal markers. Of these, the intermediate filament protein vimentin is frequently upregulated in metastatic carcinomas. Previously, microarray studies showed that the gene encoding vimentin is highly upregulated in metastatic HN12 cells compared to a related primary tumor cell line. In this study, we confirmed this difference using real-time quantitative PCR, western blot analysis, and immunostaining. Furthermore, EGF and TGF-β, growth factors that induce migration and invasion of HN12 cells, produced synergistic increases in vimentin expression. To assess the contribution of vimentin to the biological properties, HN12 cells were stably transfected with a plasmid that directs synthesis of vimentin shRNA. Clones expressing decreased amounts of vimentin were isolated and characterized. These cells showed significantly reduced proliferation compared to non-targeting controls. Moreover, downregulation of vimentin led to a decrease in cell motility, as well as reducing their ability to invade through a basement membrane substitute. Using transient transfection assays, vimentin promoter activity was determined in HN12 cells to define regulatory elements important for controlling vimentin upregulation in the absence or presence of EGF and TGF-β. Taken together, the data indicate that overexpression of vimentin is important for proliferation and invasion of metastatic HN12 cells, and suggest that EGF- dependent pathways target binding elements in the proximal vimentin promoter, while TGF-β is likely to act in an AP1-dependent manner. Furthermore, both growth factors appear to synergize by stimulating promoter activation through the ASE site, suggesting involvement of Stat-dependent pathways in regulation of vimentin expression in HN12 cells.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-2083 |
| Date | 01 January 2005 |
| Creators | Paccione, Rachel J. |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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