Vimentin

Abstract

Epithelial-to-mesenchymal transition (EMT) is a critical event in the induction of cell motility and increased survival both under physiological situations like wound healing and during development as well as in malignant cells undergoing invasion and metastasis. Vimentin is an intermediate filament protein which is characteristically upregulated in cells undergoing EMT. For decades vimentin has been considered as a marker for EMT but its functional contribution to the process has remained unclear. Our data demonstrate that vimentin contributes to EMT via upregulating the gene expression of several EMT-linked genes. Especially, we find that vimentin regulates EMT associated induced migration via upregulation of the expression of receptor tyrosine kinase Axl. In addition to our data, several other exciting recent studies support the notion that vimentin in fact functions as a positive regulator of EMT and upregulation of vimentin appears to be a prerequisite for EMT induction.

EMT can be triggered in cells in many ways. Hyperactivation of the Ras-MAPK pathway can lead to cell transformation due to induced proliferation, survival and cell motility. This pathway is deregulated in more that 30% of human cancers¹ and typically this is due to deregulation of signaling components upstream of ERK1/2.² Activating mutations in the small GTPase Ras have been shown to induce EMT like changes in non-malignant MCF10A breast epithelial cells in recent studies.^3,4 These changes include upregulation of vimentin, N-cadherin, fibronectin, tyrosine kinase Axl and transcription factor slug. Conversely, E-cadherin and β-catenin are downregulated in H-RasV12 expressing cells.³ Therefore, Ras-transformed MCF10A cells are an attractive model for the analysis of EMT.

It has been demonstrated recently that silencing of vimentin in mammary epithelial cells results in downregulation of several EMT associated genes like tyrosine kinase Axl, integrin β4-subunit (ITGB4) and plasminogen activator urokinase (PLAU).³ Importantly, a positive correlation between vimentin and these genes was also observed in hundreds of breast cancer patient samples and 92 breast cancer cell lines. This underlines the universality of these findings and places vimentin expression at the heart of the entire process of EMT induction. These data also show that vimentin is critical for EMT-linked increased cell migration and that a significant proportion of vimentin induced cell migration is due to the ability of vimentin to upregulate expression of the pro-migratory kinase Axl.³

Axl is a receptor tyrosine kinase which is activated by its ligand Gas6, often in an auto-crine manner in cancer.⁵ Axl has been recently shown to be important in the metastasis of breast cancer cells in vivo.^6,7 Axl expression also predicts poor overall survival of breast cancer patients.⁸ Recent data indicate that induction of EMT in MCF10A cells by the loss of confluency or overexpression of H-RasV12 or Slug results in upregulation of Axl in a vimentin dependent manner.³ Therefore, the novel link between vimentin and Axl expression provides some mechanistic insight into the important role of vimentin is regulation of cell motility in mesenchymal and cancer cells.

Vimentin has been shown in many studies to be an important regulator of cell motility. In cell culture conditions, vimentin is upregulated at the wound edge in mammary epithelial cells and breast cancer cells.^3,9,10 Fibroblasts lacking vimentin migrate poorly¹¹ and mice lacking vimentin have delayed wound healing.¹² Furthermore, a very elegant recent paper describes how vimentin is a critical regulator of cell shape and motility in a number of different cell types.¹³ Finally, vimentin has been shown to bind to Scribble, a protein of the polarity complex Scribble/Dlg/Lgl. Interaction with vimentin protects Scribble from proteosomal degradation and thus positively regulate its stability. The interaction is mediated by the PDZ-domains of Scribble and results in redistribution of Scribble from filaments to the plasma membrane in MDCK cells and supports anterior-posterior polarization in migrating cells.¹⁴ Impaired migration and cell aggregation are observed upon vimentin or Scribble silencing.¹⁴ Therefore, it is becoming increasingly obvious that vimentin is a master regulator of cell architecture upon induction of EMT.

Recent data³ demonstrates that vimentin is required for EMT and Axl induction downstream of several stimuli. These include H-RasV12, overexpression of Slug and loss of cell confluency upon wounding. Furthermore, MDA-MB-231 basal-like breast cancer cells have significantly higher levels of vimentin and Axl compared to non-malignant MCF10A cells. This appears to be functionally relevant since silencing of vimentin and Axl in these cells lead to loss of cell motility in vitro. Finally, shRNA mediated downregulation of Axl in these cells impairs their ability to extravasate the lung parenchyma from the blood stream in mice in vivo. Taken together, these data have lead to a proposed model where vimentin induction is a critical prerequisite for EMT induction downstream of H-RasV12 and slug (Fig. 1A).

Figure 1.

(A) Vimentin is necessary for EMT induction down-stream of Slug- and H-Rasv12. A model indicating an important role for vimentin as a positive regulator of gene expression and morphological changes in EMT.³ (B) An extended view of vimentin as a regulator of EMT. Akt1 and PKCε associate with vimentin and phosphorylate it to induce cell migration. Akt1 phosphorylation contributes to stability of vimentin protein and induction of migration whereas PKCε phosphorylation enables recycling of endocytosed β1-integrin to the plasma membrane to facilitate migration.¹⁴,¹⁷ Vimentin associates with Scribble and protects it from proteosomal degradation.¹⁴ This supports maintenance of anterior-posterior cell polarity in migrating epithelial cells. In both panels black arrows indicate established cellular signals, black lines indicate protein-protein association and P indicates phosphorylation. Grey lines in (A) indicate EMT-related signals which may also be regulated by vimentin. However, this remains to be shown.

However, many open questions remain. How can vimentin regulate gene expression as it is unlikely to function as a transcription factor? Which signaling pathways, in addition to Axl, are dependent on vimentin? How is vimentin itself induced and regulated? Many intriguing clues to these open questions have emerged recently. It seems that vimentin regulates EMT on at least three different levels: (1) Gene expression. Vimentin (indirectly) regulates gene expression of proteins required for execution of EMT like Axl³ (2) Protein-protein interactions. Vimentin regulates anterior-posterior cell polarity and directional motility by directly binding to Scribble and stabilizing it on protein level.¹⁴ This supports the ability of MDCK cells to exhibit anterior-posterior polarization during directional cell migration. Vimentin also interacts with plectin isoform 1f¹⁵ and integrin α2β1.¹⁶ This links the intermediate filaments to focal adhesions and regulates their stability and turnover. (3) Phosphorylation. Vimentin can be phosphorylated by Akt1, a kinase activated downstream of Ras and PI3K, and phosphorylation of vimentin protects it from caspase-induced proteolysis and enhances cell migration and metastasis.¹⁷ The data published in this paper imply that increased stability of vimentin protein would function as a positive regulator on EMT which could be linked to its ability to influence gene expression.³ In addition, older data has linked vimentin phosphorylation by PKCε to the efficient recycling of β1-integrin to the cell surface, a process important for cell motility.¹⁴ Interestingly, in both of these cases it remains unclear whether Akt1 and PKCε are in fact directly phosphorylating vimentin in cells or are their actions mediated via regulation of another yet unidentified kinase. Based on these important pieces of information the emerging picture seems far more complicated than our recently published simplified model and could be modified to include several feed back loops. These could involve the ability of vimentin to regulate stability of signaling proteins which in turn induce transcription factors involved in EMT and upregulation of vimentin expression (Fig. 1B).

Extra View to: Vuoriluoto K, Haugen H, Kiviluoto S, Mpindi JP, Nevo J, Gjerdrum C, et al. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene. 2011;30:1436–1448. doi: 10.1038/onc.2010.509.