An Sirna Screen Identifies RSK1 As a Key Modulator of Lung Cancer
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Oncogene (2011) 30, 3513–3521 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE An siRNA screen identifies RSK1 as a key modulator of lung cancer metastasis R Lara1,7, FA Mauri2, H Taylor3, R Derua4, A Shia3, C Gray5, A Nicols5, RJ Shiner2, E Schofield6, PA Bates6, E Waelkens4, M Dallman3, J Lamb3, D Zicha5, J Downward7, MJ Seckl1 and OE Pardo1 1Department of Oncology, Hammersmith Campus, Cyclotron Building, London, UK; 2Histopathology Imperial College London, Hammersmith Campus, London, UK; 3Division of Cell and Molecular Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, UK; 4Labo Proteı¨ne Fosforylatie en Proteomics, Katholieke Universiteit Leuven, Leuven, Belgium; 5Light Microscopy Department, London Research Institute, London, UK; 6Biomolecular Modelling Laboratory, London Research Institute, London, UK and 7Signal Transduction Laboratory, Cancer Research UK, London Research Institute, London, UK We performed a kinome-wide siRNA screen and identified Introduction 70 kinases altering cell migration in A549 lung cancer cells. In particular, ribosomal S6 kinase 1 (RSK1) Lung cancer is the most common cancer killer with silencing increased, whereas RSK2 and RSK4 down- a 5-year survival rate o5%. Non-small cell lung cancer regulation inhibited cell motility. In a secondary collagen- (NSCLC) accounts for 80% of cases of which adeno- based three-dimensional invasion screen, 38 of our hits carcinoma represents the majority. Most patients cross-validated, including RSK1 and RSK4. In two further present with metastatic lesions and are incurable. Hence, lung cancer cell lines, RSK1 but not RSK4 silencing a better understanding of the biological processes showed identical modulation of cell motility. We therefore underlying lung cancer cell motility, invasion and selected RSK1 for further investigation. Bioinformatic metastasis is needed to improve patient survival. analysis followed by co-immunoprecipitation-based vali- The 90-kDa ribosomal S6 kinase (RSK) family is dation revealed that the actin regulators VASP and Mena activated downstream of the Ras/MEK/Erk signalling interact with RSK1. Moreover, RSK1 phosphorylated pathway (Blenis et al., 1991). Four human isoforms VASP on T278, a site regulating its binding to actin. In (RSK1–4) exist (Anjum and Blenis 2008). RSKs are addition, silencing of RSK1 enhanced the metastatic characterised by the existence of two kinase domains potential of these cells in vivo using a zebrafish model. that come into close proximity following activating Finally, we investigated the relevance of this finding in phosphorylation events. Their downstream substrates human lung cancer samples. In isogenically matched include a number of cytoplasmic and nuclear targets tissue, RSK1 was reduced in metastatic versus primary (CREB, Fos, Jun, TSC2 and filamin A (Anjum and lung cancer lesions. Moreover, patients with RSK1- Blenis 2008)) that explain their involvement in diverse negative lung tumours showed increased number of cellular processes, such as cell proliferation and survival. metastases. Our results suggest that the findings of our Increased expression of RSKs was shown in breast high-throughput in vitro screen can reliably identify (Smith et al., 2005) and prostate cancer (Clark et al., relevant clinical targets and as a proof of principle, 2005), whereas RSK2 activity has been linked to cell RSK1 may provide a biomarker for metastasis in lung transformation (Cho et al., 2007; Kang et al., 2007). cancer patients. RSKs have been shown to phosphorylate filamin A Oncogene (2011) 30, 3513–3521; doi:10.1038/onc.2011.61; (Woo et al., 2004) and p27Kip (Larrea et al., 2009), with published online 21 March 2011 RSK1 being implicated in promoting melanoma cell migration in vitro (Larrea et al., 2009). However, the Keywords: ribosomal S6 kinase 1; metastasis; lung relevance of these findings to cancer metastasis in vivo or cancer; siRNA screen; VASP 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- Correspondence: Professor MJ Seckl and Dr OE Pardo, Department of lated by both homo- (Zimmermann et al., 2002) or Oncology, Cancer Medicine, Imperial College London, Hammersmith heterotetramerisation (Gertler et al., 1996) and phos- Campus, Cyclotron Building, Du Cane Road, London, W12 0NN, phorylation events (Benz et al., 2009) that control UK. E-mail: [email protected] or [email protected] their effect on actin polymerisation. Increased expres- Received 5 July 2010; revised 29 November 2010; accepted 2 February sion of Ena/VASP family members enhances cancer 2011; published online 21 March 2011 cell invasiveness in vitro and in vivo (Han et al., 2008; RSK1 in lung cancer cell metastasis R Lara et al 3514 Philippar et al., 2008) and correlates with tumour 779 Targets 1.8 progression in several malignancies (Gurzu et al., Motility screen 1.6 2008; Hu et al., 2008; Toyoda et al., 2009), including 1.4 RSK1 Scramble lung adenocarcinoma (Dertsiz et al., 2005). 160 Hits 1.2 ERBB2 Here, we performed an siRNA kinome library screen Toxicity screen 1 0.8 RSK2 0.6 in A549 human lung adenocarcinoma cells and demon- 70 Hits SRC RSK4 0.4 strated that RSK1 silencing increased migration and Mean speed ratio MST1R Publication 0.2 invasion. This could potentially be explained by VASP 0 phosphorylation on T278, a site regulating binding 57 Hits Individual Targets to actin (Blume et al., 2007). Knockdown of RSK1 enhanced the metastatic potential of A549 cells in vivo. Sc RSK1 RSK2 RSK4 Moreover, immunohistochemical staining revealed that 2 2 2 2 1 1 1 1 RSK1 was downregulated in metastatic lung cancer 0 0 0 0 samples compared with isogenically matched primary Units) -1 -1 -1 -1 Distamce (Arbitrary -2 -2 -2 -2 tumours. Thus, in lung cancer, our results suggest that -2 -10 1 2 -2-10 1 2 -2-1 0 1 2 -2 -1 0 1 2 RSK1 expression inhibits metastasis and might be a predictive biomarker for disease dissemination. RSK1 RSK2 RSK4 8 *** 7 2 2 6 ** 5 *** 1.5 1.5 * Results ** * * * 4 * * ** ** Identification of novel regulators of cell motility in lung 3 1 1 cancer cells 2 ** ** Speed (Arbitrary Units) Using the well-characterised A549 NSCLC cell line, we 1 0.5 0.5 established a high-throughput random-walk motility screen in which 60 cells per condition were automatically tracked ScP 1 2 3 4 ScPSc41 2 3 4 P 1 2 3 using Metamorph software (Molecular Devices, Chicago, RT RSK1 RSK2 RSK4 IL, USA). The resulting trajectory plots were analysed with a Mathematica notebook (Katso et al., 2006) and analysis GAPDH GAPDH GAPDH of variance performed to assess the significance of the changes observed with Po0.05 used as a cutoff. Our Figure 1 RSK family members regulate cell migration. screen identified 160 proteins that when downregulated, (a) Overview of the migration screen. (b) Distribution of the mean speed ratio to scramble for individual targets. (c) Trajectory plots significantly altered A549 cell migration (Figures 1a of representative fields of view (n ¼ 15) following transfection and b). To exclude cellular toxicity as a cause for with RSK1, 2, 4 or non-targeting (Sc) SmartPool siRNAs. decreased cell motility, we used data from our pre- (d) Representative validation experiment for RSK1, 2 and 4. P; viously published toxicity screen (Swanton et al., 2007) pool, 1–4; deconvoluted single oligonucleotides. The average random migration speed distribution of 60 cells per condition is performed with the same cells and RNAi library. represented as a box plot. Box; 75% and side bar; 12.5% of the cell Cell motility hits that showed 410% toxicity were population. Transverse bar; median speed. Analysis of variance discarded, thereby reducing the number of our candi- with Sc as reference: *Po0.05, **Po0.005, ***Po0.001. Repre- dates to 70 proteins (Figure 1a). Reassuringly, several of sentative reverse transcriptase–PCR (RT) for the target mRNAs these were already known modulators of cell migration, with GAPDH as a loading control. including MAP4K4 (Collins et al., 2006), AKT2 (Irie et al., 2005), EphB6 (Fox and Kandpal, 2009) and CDC42 (Nobes and Hall, 1995). However, 57 of our hits As metastasis involves both enhanced motility and were novel modulators of migration (Figure 1a and invasiveness, we next performed a secondary three- Supplementary Figure 1 for a full hit list). Four kinase dimensional high-throughput collagen-based invasion families were found, with several members capable of screen on our 70 motility hits. Thirty-eight of these altering migration as follows (Supplementary Figure 2): cross-validated in the two screens (Supplementary the ribosomal protein S6 kinase , casein kinase , CDC- Figure 4). Figure 2a shows side views of representative like kinase and Arf-domain containing kinase families. stacks in which knockdown of 4.1B was used as a To validate our hits, a second toxicity assay and positive control (Cavanna et al., 2007). We decided to SmartPool (Dharmacon, Lafayette, CO, USA) decon- focus on the RSK family as a paradigm to validate the volution were performed. We confirmed that changes in importance of our screen and because of our previous cell viability did not account for the observed motility interest in S6 kinases (Pardo et al., 2001, 2006). effects (Supplementary Figure 2) and that 14 of the 17 In keeping with our migration data, silencing of RSK1 targets validated with at least 2 of the 4 deconvoluted enhanced, whereas RSK4 knockdown suppressed inva- individual sequences (Supplementary Figure 2 and sion (Figures 2a and b). However, unlike the motility Figure 1d). These effects correlated with target down- screen, RSK2 silencing failed to suppress invasion. regulation at the protein and/or mRNA levels (Figure 1d Hence, silencing of RSK1 and RSK4 alter invasion in a and Supplementary Figure 3).