ORIGINAL ARTICLE Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression MV Cronauer 1,2 , Y Ince 3 , R Engers 4 , L Rinnab 5 , W Weidemann 2 , CV Suschek 6,8 , M Burchardt 1,9 , H Kleinert 7 , J Wiedenmann 2 , H Sies 3 , R Ackermann 1 and K-D Kro¨ncke 3 1 Department of Urology, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany; 2 Department of General Zoology and Endocrinology, University Ulm, Ulm, Germany; 3 Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Du¨sseldorf,Du¨sseldorf,Germany; 4 InstituteofPathology, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany; 5 Department of Urology and Pediatric Urology, University Ulm, Ulm, Germany; 6 Institute of Biochemistry and Molecular Biology II, Heinrich- Heine-University Du¨sseldorf, Du¨sseldorf, Germany and 7 Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitroty- rosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA- binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-indepen- dently, thereby promoting prostate tumor progression. Oncogene (2007) 26, 1875–1884. doi:10.1038/sj.onc.1209984; published online 18 September 2006 Keywords: androgen receptor; nitric oxide; prostate cancer; prostate specific antigen; zinc-finger Introduction Inducible nitric oxide synthase (iNOS) is expressed in a variety of acute or chronic inflammatory human diseases as well as in various types of cancers including prostate cancer (Kro¨ncke et al., 2000). Once expressed, iNOS produces high amounts of nitric oxide (NO) for prolonged periods of time which causes nitrosative stress. In the prostatic gland, expression of iNOS is already detectable in precancerous high-grade prostatic intraepithelial neoplasia (Wang etal., 2003). In prostate cancer iNOS is expressed in 80–100% of prostate cancer specimens (Klotz et al., 1998; Aaltoma et al., 2001; Baltaci et al., 2001), showing highest expression in locally advanced and metastasized tumors (Aaltoma et al., 2001). Within the tumor, the majority of cells staining positive for iNOS are cancer cells (Klotz et al., 1998; Aaltoma et al., 2001; Baltaci et al., 2001; Wang et al., 2003; Bronte et al., 2005). Moreover, a variable amount of inflammatory cells, presumably macro- phages, inside and around the tumor equally express iNOS (Klotz et al., 1998; Aaltoma et al., 2001). In contrast to prostate cancer, normal putatively undi- seased prostatic tissue as well as tissue from benign prostatic hyperplasia stain negative or only weakly positive for iNOS (Klotz et al., 1998; Gradini et al., 1999; Aaltoma et al., 2001; Baltaci et al., 2001). Although strong iNOS expression has been correlated to a rapid cancer cell proliferation rate, dedifferentiation and prediction of poor survival (Aaltoma et al., 2001), the role of iNOS activity in prostate cancer remains unclear. Prostate cancer is the second most common cause of tumor-associated death in men in the United States and accounts for 9% of all cancer-related deaths among men in the European Union. The prostate gland is the prototype of an androgen-responsive organ, because androgen plays a pivotal role in regulating growth and differentiation of normal as well as of malignant prostatic epithelial cells. As prostate tumor cells depend on androgens for growth and survival, androgen ablation by orchiectomy, anti-androgen treatment or treatment with luteinizing hormone releasing Received 7 March 2006; revised 12 July 2006; accepted 3 August 2006; published online 18 September 2006 Correspondence: Dr K-D Kro¨ncke, Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Du¨ sseldorf, Universi- ta¨tsstr. 1, 40225 Du¨sseldorf, Germany. E-mail: kroencke@uni-duesseldorf.de 8 Current address: Department of Plastic and Reconstructive Surgery, Hand Surgery, and Burn Center, University Hospital of the RWTH- Aachen, 52057 Aachen, Germany. 9 Current address: Department of Urology and Pediatric Urology, Hannover Medical School, 30625 Hannover, Germany. Oncogene (2007) 26, 1875–1884 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc