ORIGINAL ARTICLE Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer K Vuoriluoto 1,2 , H Haugen 3 , S Kiviluoto 1 , J-P Mpindi 1 , J Nevo 1,2 , C Gjerdrum 3 , C Tiron 3 , JB Lorens 3 and J Ivaska 1,2,4 1 Medical Biotechnology, VTT Technical Research Centre of Finland, Turku, Finland; 2 Turku Centre for Biotechnology, University of Turku, Turku, Finland; 3 Department of Biomedicine, University of Bergen, Bergen, Norway and 4 Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Epithelial-to-mesenchymal transition (EMT) is a critical event in the progression toward cancer metastasis. The intermediate filament protein vimentin is an important marker of EMT and a requisite regulator of mesenchymal cell migration. However, it is not known how vimentin functionally contributes to cancer cell invasion. Here, we report that ectopic expression of oncogenic H-Ras-V12G and Slug induces vimentin expression and migration in pre-malignant breast epithelial cells. Conversely, vimentin expression is necessary for Slug- or H-Ras-V12G-induced EMT-associated migration. Furthermore, silencing of vimentin in breast epithelial cells results in specific changes in invasiveness-related gene expression including upregulation of RAB25 (small GTPase Rab25) and down- regulation of AXL (receptor tyrosine kinase Axl), PLAU (plasminogen activator, urokinase) and ITGB4 (integrin b4-subunit). Importantly, gene expression profiling ana- lyses reveal that vimentin expression correlates positively/ negatively with these genes also in multiple breast cancer cell lines and breast cancer patient samples. Focusing on the tyrosine kinase Axl, we show that induction of vimentin by EMT is associated with upregulation of Axl expression and that Axl enhances the migratory activity of pre-malignant breast epithelial cells. Using null and knock-down cells and overexpression models, we also show that regulation of breast cancer cell migration in two- and three-dimensional matrices by vimentin is Axl- dependent and that Axl functionally contributes to lung extravasation of breast cancer cells in mice. In conclusion, our data show that vimentin functionally contributes to EMT and is required for induction of Axl expression. Moreover, these results provide a molecular explanation for vimentin-dependent cancer cell migration during EMT by identifying Axl as a key proximal component in this process. Oncogene advance online publication, 8 November 2010; doi:10.1038/onc.2010.509 Keywords: vimentin; EMT; Axl Introduction Epithelial-to-mesenchymal transition (EMT) is a unique process in which epithelial cells lose their cell–cell contacts and apical-basal polarity, and transdifferentiate into fibroblastic migratory cells with mesenchymal characteristics. It has been proposed that EMT is involved in cancer progression, particularly during invasion and intravasation when tumor cells migrate to distant organs to form metastases (Thiery, 2002; Gupta and Massague, 2006). Intermediate filaments are a family of proteins that form one of the tripartite components of the cytoske- leton. They are expressed in a highly regulated manner in terms of tissue and developmental stage specificity and have important functions in human health and disease (Toivola et al., 2005). Vimentin is an inter- mediate filament protein normally expressed in cells of mesenchymal origin (Steinert and Roop, 1988). How- ever, vimentin can also be expressed in epithelial cells undergoing EMT both under physiological and patho- logical situations (Franke et al., 1982; Thiery, 2002; Gupta and Massague, 2006). Vimentin regulates cell migration in many cell types. In fibroblasts, vimentin filaments connect the nucleus to the plasma membrane and contribute to the formation of vimentin-associated matrix adhesions that are dyna- mically turned over in migrating cells (Ivaska et al., 2007). Notably, vimentin null cells display reduced mechanical stability and motility in vitro (Eckes et al., 1998) and vimentin null mice show an impaired ability to heal wounds in vivo (Eckes et al., 2000). Vimentin is also critical for the recycling of endocytosed cell adhesion receptors, integrins, to the plasma membrane and disruption of vimentin function results in impaired migration (Ivaska et al., 2005). However, the mechan- ism(s) underlying the functional contribution of vimen- tin to epithelial cell migration remain unclear. Many aggressive breast cancer cell lines express vimentin (Neve et al., 2006). Moreover, overexpres- sion of vimentin in a vimentin-negative, non-invasive MCF7 breast cancer cell line increases integrin traffic, migration and invasiveness (Hendrix et al., 1997; Ivaska et al., 2005). Furthermore, several studies have reported overexpression of vimentin in breast cancer (reviewed in Kokkinos et al., 2007). In particular, vimentin expression Received 19 April 2010; revised 3 September 2010; accepted 28 September 2010 Correspondence: Professor J Ivaska, VTT Medical Biotechnology, University of Turku, Centre for Biotechnology, Ita¨inen Pitka¨katu 4C, Turku 20520, Finland. E-mail: Johanna.Ivaska@vtt.fi Oncogene (2010) 1–13 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc