ORIGINAL ARTICLE An siRNA screen identifies RSK1 as a key modulator of lung cancer metastasis R Lara 1,7 , FA Mauri 2 , H Taylor 3 , R Derua 4 , A Shia 3 , C Gray 5 , A Nicols 5 , RJ Shiner 2 , E Schofield 6 , PA Bates 6 , E Waelkens 4 , M Dallman 3 , J Lamb 3 , D Zicha 5 , J Downward 7 , MJ Seckl 1 and OE Pardo 1 1 Department of Oncology, Hammersmith Campus, Cyclotron Building, London, UK; 2 Histopathology Imperial College London, Hammersmith Campus, London, UK; 3 Division of Cell and Molecular Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, UK; 4 Labo Proteı¨ne Fosforylatie en Proteomics, Katholieke Universiteit Leuven, Leuven, Belgium; 5 Light Microscopy Department, London Research Institute, London, UK; 6 Biomolecular Modelling Laboratory, London Research Institute, London, UK and 7 Signal Transduction Laboratory, Cancer Research UK, London Research Institute, London, UK We performed a kinome-wide siRNA screen and identified 70 kinases altering cell migration in A549 lung cancer cells. In particular, ribosomal S6 kinase 1 (RSK1) silencing increased, whereas RSK2 and RSK4 down- regulation inhibited cell motility. In a secondary collagen- based three-dimensional invasion screen, 38 of our hits cross-validated, including RSK1 and RSK4. In two further lung cancer cell lines, RSK1 but not RSK4 silencing showed identical modulation of cell motility. We therefore selected RSK1 for further investigation. Bioinformatic analysis followed by co-immunoprecipitation-based vali- dation revealed that the actin regulators VASP and Mena interact with RSK1. Moreover, RSK1 phosphorylated VASP on T278, a site regulating its binding to actin. In addition, silencing of RSK1 enhanced the metastatic potential of these cells in vivo using a zebrafish model. Finally, we investigated the relevance of this finding in human lung cancer samples. In isogenically matched tissue, RSK1 was reduced in metastatic versus primary lung cancer lesions. Moreover, patients with RSK1- negative lung tumours showed increased number of metastases. Our results suggest that the findings of our high-throughput in vitro screen can reliably identify relevant clinical targets and as a proof of principle, RSK1 may provide a biomarker for metastasis in lung cancer patients. Oncogene advance online publication, 21 March 2011; doi:10.1038/onc.2011.61 Keywords: ribosomal S6 kinase 1; metastasis; lung cancer; siRNA screen; VASP Introduction Lung cancer is the most common cancer killer with a 5-year survival rate o5%. Non-small cell lung cancer (NSCLC) accounts for 80% of cases of which adeno- carcinoma represents the majority. Most patients present with metastatic lesions and are incurable. Hence, a better understanding of the biological processes underlying lung cancer cell motility, invasion and metastasis is needed to improve patient survival. The 90-kDa ribosomal S6 kinase (RSK) family is activated downstream of the Ras/MEK/Erk signalling pathway (Blenis et al., 1991). Four human isoforms (RSK1–4) exist (Anjum and Blenis 2008). RSKs are characterised by the existence of two kinase domains that come into close proximity following activating phosphorylation events. Their downstream substrates include a number of cytoplasmic and nuclear targets (CREB, Fos, Jun, TSC2 and filamin A (Anjum and Blenis 2008)) that explain their involvement in diverse cellular processes, such as cell proliferation and survival. Increased expression of RSKs was shown in breast (Smith et al., 2005) and prostate cancer (Clark et al., 2005), whereas RSK2 activity has been linked to cell transformation (Cho et al., 2007; Kang et al., 2007). RSKs have been shown to phosphorylate filamin A (Woo et al., 2004) and p27Kip (Larrea et al., 2009), with RSK1 being implicated in promoting melanoma cell migration in vitro (Larrea et al., 2009). However, the relevance of these findings to cancer metastasis in vivo or in patients has not been established. The Ena/VASP family of actin-binding proteins is not known to mediate RSK1 effects, but is involved in various processes, including cell migration (Krause et al., 2003). Three human family members exist; Mena, VASP and EVL. Their overlapping function is modu- lated by both homo- (Zimmermann et al., 2002) or heterotetramerisation (Gertler et al., 1996) and phos- phorylation events (Benz et al., 2009) that control their effect on actin polymerisation. Increased expres- sion of Ena/VASP family members enhances cancer cell invasiveness in vitro and in vivo (Han et al., 2008; Received 5 July 2010; revised 29 November 2010; accepted 2 February 2011 Correspondence: Professor MJ Seckl and Dr OE Pardo, Department of Oncology, Cancer Medicine, Imperial College London, Hammersmith Campus, Cyclotron Building, Du Cane Road, London, W12 0NN, UK. E-mail: m.seckl@imperial.ac.uk or o.pardo@imperial.ac.uk Oncogene (2011) 1–9 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc