Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth Ario Takeuchi a , Masaki Shiota a , Eliana Beraldi a , Daksh Thaper a , Kiyoshi Takahara a , Naokazu Ibuki a , Michael Pollak b , Michael E. Cox a , Seiji Naito c , Martin E. Gleave a , Amina Zoubeidi a,⇑ a The Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada b Department of Medicine and Oncology, McGill University, Montreal, Quebec, Canada c Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan article info Article history: Received 19 June 2013 Received in revised form 27 December 2013 Accepted 14 January 2014 Available online 1 February 2014 Keywords: Clusterin Insulin-like growth factor-I Prostate cancer STAT3 Twist1 abstract Clusterin (CLU) is cytoprotective molecular chaperone that is highly expressed in castrate-resistant pros- tate cancer (CRPC). CRPC is also characterized by increased insulin-like growth factor (IGF)-I responsive- ness which induces prostate cancer survival and CLU expression. However, how IGF-I induces CLU expression and whether CLU is required for IGF-mediated growth signaling remain unknown. Here we show that IGF-I induced CLU via STAT3–Twist1 signaling pathway. In response to IGF-I, STAT3 was phos- phorylated, translocated to the nucleus and bound to the Twist1 promoter to activate Twist1 transcrip- tion. In turn, Twist1 bound to E-boxes on the CLU promoter and activated CLU transcription. Inversely, we demonstrated that knocking down Twist1 abrogated IGF-I induced CLU expression, indicating that Twist1 mediated IGF-I-induced CLU expression. When PTEN knockout mice were crossed with lit/lit mice, the resultant IGF-I deficiency suppressed Twist1 as well as CLU gene expression in mouse prostate glands. Moreover, both Twist1 and CLU knockdown suppressed prostate cancer growth accelerated by IGF-I, sug- gesting the relevance of this signaling not only in an in vitro, but also in an in vivo. Collectively, this study indicates that IGF-I induces CLU expression through sequential activation of STAT3 and Twist1, and sug- gests that this signaling cascade plays a critical role in prostate cancer pathogenesis. Ó 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Prostate cancer is the most common solid malignant tumor among males in Western countries (Jemal et al., 2010). A series of epidemiological and biological studies demonstrate that the insulin-like growth factor (IGF) axis is a critical regulator of growth, survival, and metastatic potential in a variety of malignan- cies and is closely implicated in prostatic carcinogenesis and pros- tate cancer progression as well as resistance to castration therapy (Chan et al., 1998; Krueckl et al., 2004; Nickerson et al., 2001; Wolk et al., 1998). We have previously demonstrated that IGF-I pro- moted human prostate cancer cell growth and that increased IGF-I receptor (IGF-IR) expression and signaling are components of castrate resistant progression (Krueckl et al., 2004; Takahara et al., 2011). IGFs bind to the IGF-IR, which is a heterotetrameric type I receptor protein-tyrosine kinase composed of two ligand-binding a-subunits and two transmembrane b-subunits. The binding of li- gand to IGF-IR induces auto-phosphorylation of the b-subunits of the receptor complex and further activation of the protein-tyrosine kinase activity (Hubbard et al., 1994; Weiss and Schlessinger, 1998). Once activated, IGF-IR recruits and phosphorylates various downstream targets such as the insulin receptor substrate-1 and -2 which activate many signaling pathways, including Ras/Raf/ mitogen-activated protein kinase (MAPK) and PI3K/Akt, as well as signal transducer and activator of transcription 3 (STAT3; Zong et al., 2000) resulting in cell growth and survival. Clusterin (CLU) is a stress-induced cytoprotective chaperone, and involved in many biological processes such as sperm matura- tion, tissue differentiation, tissue remodeling, membrane recycling, lipid transportation, cell proliferation and cell death. CLU has been shown expressed in many human cancers (Zhong et al., 2010). In- creased levels of CLU have been reported in breast, colon, lung, bladder, prostate and other cancers (July et al., 2004; Kevans et al., 2009; Miyake et al., 2002; So et al., 2005; Steinberg et al., 1997). In prostate, CLU levels are low in benign prostate epithelial http://dx.doi.org/10.1016/j.mce.2014.01.012 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved. ⇑ Corresponding author. Address: The Vancouver Prostate Centre and Depart- ment of Urologic Sciences, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, Canada. Tel.: +1 604 875 4818; fax: +1 604 875 5654. E-mail address: azoubeidi@prostatecentre.com (A. Zoubeidi). Molecular and Cellular Endocrinology 384 (2014) 117–125 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce