Expression of p66 Shc protein correlates with proliferation of human prostate cancer cells Suresh Veeramani 1,5 , Tsukasa Igawa 1,5,6 , Ta-Chun Yuan 1 , Fen-Fen Lin 1 , Ming-Shyue Lee 1,7 , Jamie S Lin 1,8 , Sonny L Johansson 2,3 and Ming-Fong Lin* ,1,3,4 1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198, USA; 2 Department of Pathology, University of Nebraska Medical Center, Omaha, NE 68198, USA; 3 Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198, USA; 4 Section of Urologic Surgery, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA p66 Shc , an isoform of Shc adaptor proteins, is shown to mediate various signals, including cellular stress. How- ever, little is known about its involvement in carcino- genesis. We previously showed that p66 Shc protein level is upregulated by steroid hormones in human carcinoma cells and is higher in prostate cancer (PCa) specimens than adja- cent noncancerous cells. In this study, we investigated the role of p66 Shc protein in PCa cell proliferation. Among different PCa cell lines tested, p66 Shc protein level showed positive correlation with cell proliferation, that is, rapid- growing cells expressed higher p66 Shc protein than slow- growing cells. Exposure of slow-growing LNCaP C-33 cells to epidermal growth factor (EGF) and 5a-dihydro- testosterone (DHT) led to upregulation of proliferation and p66 Shc protein level. Conversely, growth suppression of fast-growing cells by cellular form of prostatic acid phosphatase (cPAcP) expression, a negative growth regulator, downregulated their p66 Shc protein level. Additionally, increased expression of p66 Shc protein by cDNA transfection in LNCaP C-33 cells resulted in increased cell proliferation. Cell cycle analyses showed higher percentage of p66 Shc -overexpressing cells at S phase (24%) than control cells (17%), correlating with their growth rates. On the other hand, transient knock-down of p66 Shc expression by RNAi in rapidly growing cells decreased their proliferation as evidenced by the reduced cell growth as well as S phase in p66 Shc -knocked down cells. The p66 Shc signaling in cell growth regulation is apparently mediated by extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK). Thus, our results indicate a novel role for p66 Shc in prostate carcinogenesis, in part, promoting cell proliferation. Oncogene (2005) 24, 7203–7212. doi:10.1038/sj.onc.1208852; published online 19 September 2005 Keywords: p66 Shc ; prostatic acid phosphatase; prostate cancer; cell proliferation Introduction Shc (Src homolog and collagen homolog) proteins are adaptor molecules that contain SH2 and PTB domains (Ravichandran, 2001). They exist in three different isoforms with molecular masses of 46, 52 and 66 kDa. Shc proteins are conventionally known to transduce the mitogenic signals from receptor tyrosine kinases to downstream targets, such as, extracellular signal- regulated kinases/mitogen-activated protein kinases (ERK/MAPK) (Ravichandran, 2001). All the isoforms contain three functional domains – an SH2 domain, a PTB domain and a CH1 domain with three conserved tyrosine residues that are phosphorylated with respect to various signals (Ravichandran, 2001). Additionally, p66 Shc has a unique CH2 domain at the N-terminus of the protein, which contains a serine residue that could be phosphorylated under stress signals (Migliaccio et al., 1999). Different members of the Shc proteins exhibit distinct expression patterns and biological functions. For example, p52 Shc and p46 Shc are expressed in most of the cells, while p66 Shc protein is expressed predominantly in epithelial cells (Migliaccio et al., 1997). Both p52 Shc and p66 Shc are distributed throughout the cytosol, whereas p46 Shc localizes to mitochondria (Ventura et al., 2004). Nevertheless, recent studies indicate that in response to stress signals, such as, H 2 O 2 treatment, a fraction of cytosolic p66 Shc is translocated to mitochondria, where it is associated with heat–shock protein to mediate apoptotic response (Orsini et al., 2004). Despite that p66 Shc , like p52 Shc /p46 Shc , is phosphorylated at its tyrosine residues during epidermal growth factor (EGF) treatment and forms complexes with Grb-2, there are certain functional differences between p66 Shc and the other two Shc members. First, p66 Shc , unlike p52 Shc , could not transform NIH3T3 mouse fibro- blast cells in vitro (Migliaccio et al., 1997). Second, Received 17 December 2004; revised 4 May 2005; accepted 4 May 2005; published online 19 September 2005 *Correspondence: M-F Lin; E-mail: mlin@unmc.edu 5 These two authors contributed equally to this article 6 Current address: Department of Urology, Nagasaki University School of Medicine, Sakamoto 1-7-1 Nagasaki 852-8501, Japan 7 Current address: Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA 8 Current address: School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA Oncogene (2005) 24, 7203–7212 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc