Snail and Slug, key regulators of TGF-b-induced EMT, are sufficient for the induction of single-cell invasion Hildegonda P.H. Naber a , Yvette Drabsch a , B. Ewa Snaar-Jagalska b , Peter ten Dijke a,c, * , Theo van Laar a a Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Centre, Leiden, The Netherlands b Institute of Biology, Leiden University, Leiden, The Netherlands c Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden article info Article history: Received 28 March 2013 Available online 22 April 2013 Keywords: TGF-b Single cell invasion EMT Slug Snail abstract TGF-b plays a dual role in cancer; in early stages it inhibits tumor growth, whereas later it promotes inva- sion and metastasis. TGF-b is thought to be pro-invasive by inducing epithelial-to-mesenchymal transi- tion (EMT) via induction of transcriptional repressors, including Slug and Snail. In this study, we investigated the role of Snail and Slug in TGF-b-induced invasion in an in vitro invasion assay and in an embryonic zebrafish xenograft model. Ectopic expression of Slug or Snail promoted inva- sion of single, rounded amoeboid cells in vitro. In an embryonic zebrafish xenograft model, forced expres- sion of Slug and Snail promoted single cell invasion and metastasis. Slug and Snail are sufficient for the induction of single-cell invasion in an in vitro invasion assay and in an embryonic zebrafish xenograft model. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Transforming Growth Factor-b (TGF-b) is a pleiotropic cytokine involved a multitude of biological processes. Deregulation of TGF-b signaling has been observed in several diseases, such as fibrosis and cancer [1]. In cancer, TGF-b plays a dual role; in early stages it inhibits tumor growth, whereas in later stages it promotes inva- sion and metastasis [2]. In line with its oncogenic role, TGF-b is fre- quently overexpressed in breast cancer [3–5]. Furthermore, inhibition of TGF-b signaling in breast cancer reduces metastasis in several mouse models of breast cancer [6–9]. TGF-b signals through a heteromeric receptor complex com- posed of the TGF-b type I receptor Activin-receptor like kinase (ALK) 5 and the TGF-b type II receptor (TGF-bRII). Within this complex, the TGF-bRII phosphorylates ALK5, which on its turn phosphorylates Smad2 and Smad3. Phosphorylation of Smad2 and Smad3 induces a conformational change, which allows these Smad proteins to form a heteromeric complex with Smad4. This complex translocates to the nucleus, where it affects tran- scription of target genes [2,10–12]. In addition, receptor activation also results in non-Smad signaling, such as the mitogen activated protein kinases (MAPK) pathway [13]. TGF-b is thought to be pro-invasive by inducing epithelial-to- mesenchymal transition (EMT). During EMT, carcinoma cells ac- quire a more motile, mesenchymal phenotype. This process is marked by the loss of epithelial markers such as E-cadherin, zona occludens (ZO)-1, EPCAM and keratin 18 (KRT18), and induction of mesenchymal markers such as a-smooth muscle actin (a- SMA), vimentin and N-cadherin. Several transcription factors, such as Snail and Slug, are able to downregulate E-cadherin expression by binding to E-boxes in the E-cadherin promoter. Snail and Slug are zinc finger proteins of the Snail family that recognize E2 box type elements (CAGGTG/CACCTG). Furthermore, the bHLH factor Twist is also able to induce EMT, although it does not directly reg- ulate E-cadherin [14]. Besides E-cadherin, Snail and Slug transcrip- tion factors downregulate components of the adherens junctions: desmosomes, polarity proteins and miRNAs [15]. TGF-b regulates EMT by inducing these transcription regulators. Furthermore, Smad proteins also are able to interact with these transcription fac- tors and jointly regulate target genes [16]. Although studies in vitro clearly have demonstrated that cancer cells are able to undergo EMT, the question if EMT also occurs in cancer in vivo has been subject of debate. The presence of more mesenchymal cells at the invasive edge suggested that EMT might occur in this area of the tumor [17]. Furthermore, cancer cells which have undergone TGF-b-induced EMT closely resemble can- cer stem cells, of which a few cells can give rise to a whole new tu- mor [18]. Further studies revealed that Snail is partially responsible for the TGF-b-induced stem cell phenotype [19]. These studies strongly suggest the occurrence of EMT in cancer. 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.04.037 ⇑ Corresponding author. Address: Department of Molecular Cell Biology and Centre for Biomedical Genetics, Cancer Genomics Centre Netherlands, Leiden University Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Fax: +31 (0) 715268270. E-mail addresses: P.ten_Dijke@lumc.nl (P. ten Dijke), T.van_Laar@lumc.nl (T. van Laar). Biochemical and Biophysical Research Communications 435 (2013) 58–63 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc