Metallothionein 3: An Androgen-Upregulated Gene Enhances Cell Invasion and Tumorigenesis of Prostate Carcinoma Cells Horng-Heng Juang, 1,2 Li-Chuan Chung, 1 Hsin-Ching Sung, 1 Tsui-Hsia Feng, 3 Yi-Hua Lee, 1 Phei-Lang Chang, 2,4 and Ke-Hung Tsui 2,4 * 1 Department of Anatomy,College of Medicine,Chang Gung University, Kwei-Shan,Tao-Yuan,Taiwan 2 Bioinformation Center,Chang Gung Memorial Hospital, Kwei-Shan,Tao-Yuan,Taiwan 3 School of Nursing,College of Medicine,Chang Gung University, Kwei-Shan,Tao-Yuan,Taiwan 4 Department of Urology,Chang Gung Memorial Hospital, Kwei-Shan,Tao-Yuan,Taiwan BACKGROUND. Metallothioneins (MT1, MT2, MT3, and MT4) are regarded as modulators regulating a number of biological processes including cell proliferation, differentiation, and invasion. We determined the effects of androgen, cadmium, and arsenic on MT1/2 and MT3 in prostate carcinoma cells, and evaluated the functional effects of MT3 on cell proliferation, invasion, and tumorigenesis. METHODS. We determined the expression of MT1/2 and MT3 in prostate carcinoma cells by immunoblotting assays or real-time reverse transcription-polymerase chain reactions. The effects of ectopic MT3 overexpression or MT3-knockdown on cell proliferation, invasion, and tumorigenesis were determined by 3 H-thymidine incorporation, matrigel invasion, and murine xenograft studies. The effects of androgen, cadmium, and arsenic on target genes were assessed using immunoblotting and reporter assays. RESULTS. Androgen, cadmium, and arsenic treatments enhanced gene expression of MT1/2 and MT3 in prostate carcinoma LNCaP cells. Results of immunohistochemical staining indicated MT3 overexpression was found predominantly in the nuclear areas of PC-3 cells overexpressing MT3. Overexpression of MT3 significantly increased cell proliferation, invasion, and tumorigenic activities in PC-3 cells in vitro and in vivo. MT3 overexpression downregulated the gene expressions of N-myc downstream regulated gene 1 (Ndrg1) and maspin, and attenuated blocking effects of doxorubicin in PC-3 cells on cell proliferation. MT3- knockdown enhanced Ndrg1 and maspin expressions in LNCaP cells. CONCLUSIONS. The experiments indicate that MT3 is an androgen-upregulated gene, and promotes tumorigenesis of prostate carcinoma cells. The downregulation of Ndrg1 and maspin gene expressions appears to account for the enhancement of proliferative and invasive functions of MT3 in PC-3 cells. Prostate 73:1495–1506, 2013. # 2013 Wiley Periodicals, Inc. KEY WORDS: prostate; metallothionein; androgen; Ndrg1; maspin INTRODUCTION The mammalian metallothioneins are a family of cysteine-rich proteins, central to the binding of metals, which fall into at least four subgroups, namely MT1, MT2, MT3, and MT4 [1]. MT1 encodes multiple iso- forms, MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, and MT1X, while MT2 includes a single isoform, MT2A. MT1/2 (MT1 and MT2) are detected by the Grant sponsor: Chang Gung Memorial Hospital; Grant numbers: CMRPD-190543; CMRPD-190613; CMRPG-392143; Grant sponsor: National Science Council, Taiwan, ROC; Grant numbers: NSC 101- 2320-B-182-002; NSC 101-2314-B-182A-099-MY3. *Correspondence to: Dr. Ke-Hung Tsui, Department of Urology, Chang Gung Memorial Hospital, 5 Fu-Shing Street, Kwei-Shan, Tao-Yuan 333, Taiwan. E-mail: t2130@adm.cgmh.org.tw Received 3 July 2012; Accepted 15 May 2013 DOI 10.1002/pros.22697 Published online 21 June 2013 in Wiley Online Library (wileyonlinelibrary.com). The Prostate 73:1495^1506 (2013) ß 2013 Wiley Periodicals, Inc.