ORIGINAL ARTICLE The RNA-binding protein Sam68 contributes to proliferation and survival of human prostate cancer cells R Busa` 1,3 , MP Paronetto 1,3 , D Farini 1 , E Pierantozzi 1 , F Botti 1 , DF Angelini 3 , F Attisani 2 , G Vespasiani 2 and C Sette 1,3 1 Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy; 2 Department of Urology, University of Rome Tor Vergata, Rome, Italy and 3 Institute for Neuroscience IRCSS Fondazione Santa Lucia, Rome, Italy The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and its activation correlates with tyrosine phosphorylation of the RNA- binding protein Sam68. Herein, we have investigated the expression and function of Sam68 in human prostate cancer cells. Analysis of specimens obtained from 20 patients revealed that Sam68 is upregulated at the protein level in 35% of the samples. Real-time polymerase chain reaction confirmed the results at the mRNA level in most patients. Downregulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression and reduced the proliferation rate. Moreover, depletion of Sam68 sensitized cells to apoptosis induced by DNA- damaging agents. Similarly, stable cell lines expressing a truncated GFP-Sam68 GSG protein displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Our results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents. Oncogene (2007) 26, 4372–4382; doi:10.1038/sj.onc.1210224; published online 22 January 2007 Keywords: prostate cancer; Sam68; cell proliferation; apoptosis; RNA metabolism Introduction Prostate carcinoma (PCa) originates as an androgen- dependent hyper-proliferation of the epithelial cells of the gland and it evolves in an androgen-independent, highly aggressive cancer for which no cure is available yet (Feldman and Feldman, 2001). As androgen- refractoriness is associated with poor prognosis, it is of primary importance to identify the molecular pathways that can be targeted by therapies alternative to andro- gen-depletion. A predominant role in the development of androgen-refractoriness is played by the upregula- tion of signal transduction pathways that allow prostate cancer cells to autonomously produce their own requirements of growth factors and nutrients (Grossmann etal., 2001). In many cases, these autocrine loops trigger the activation of tyrosine kinase pathways. In this regard, it was shown that the tyrosine kinase Src is required for proliferation and migration of prostate cancer cells (Migliaccio etal., 2000; Lee etal., 2001) and that inhibition of Src blocks their adhesion to the extracellular matrix and invasiveness (Nam etal., 2005). Src is the prototype of a class of tyrosine kinases that have been intensively studied due to their impact on cell tranformation and tumour development (Irby and Yeatman, 2000). The activity of Src-related tyrosine kinases is increased in a multitude of primary tumours and metastatic lesions and Src-specific inhibitors may have a therapeutic application in inhibiting tumour progression and/or metastasis (Nam etal., 2005). In line with the role of Src in PCa, the tumour suppressor DOC2/DAB2, an endogenous Src inhibitory protein, is frequently downregulated in this cancer (Zhou et al., 2003), whereas an activator of Src, the truncated c-Kit protein tr-Kit, is aberrantly expressed in prostate tumours at advanced stage of the disease. Activation of Src in these PCas correlated with tyrosine phosphory- lation of Sam68 (Src substrate in Mitosis, 68 kDa) (Paronetto et al., 2004), an RNA-binding protein that acts as a post-transcriptional regulator of gene expres- sion (Lukong and Richard, 2003). Sam68 belongs to the signal transduction and activation of RNA metabolism (STAR) family of RNA-binding proteins, which appear to link signal transduction pathways with the regulation of RNA metabolism (Lukong and Richard, 2003). They are cha- racterized by a GSG (Gpr33-Sam68-GLD-1) domain, which is required for RNA binding and homodimeri- zation, flanked by regions involved in protein–protein interactions and post-translational modifications, which affect the affinity and specificity of RNA bind- ing. In particular, Sam68 interacts with the SH2 and SH3 domains of several signalling proteins acting as a scaffold molecule in response to different stimuli (Richard et al., 1995; Paronetto et al., 2003). Physical Received 8 August 2006; revised 27 October 2006; accepted 13 November 2006; published online 22 January 2007 Correspondence: Professor C Sette, Department of Public Health and Cell Biology, University of Rome ‘Tor Vergata’, Via Montpellier, 1, Rome 00133, Italy. E-mail: claudio.sette@uniroma2.it Oncogene (2007) 26, 4372–4382 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc