[CANCER RESEARCH 62, 1370 –1376, March 1, 2002] Toremifene Prevents Prostate Cancer in the Transgenic Adenocarcinoma of Mouse Prostate Model 1 Sharan Raghow, 2 Massoumeh Z. Hooshdaran, 3 Sanjay Katiyar, 3 and Mitchell S. Steiner University of Tennessee Urologic Research Laboratories, Memphis, Tennessee 38163 ABSTRACT The chemopreventive efficacy of toremifene, an antiestrogen, was eval- uated in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. TRAMP mice were segregated into three groups: (a) the low-dose toremifene group (6.6 mg/kg/day); (b) the high-dose toremifene group (33 mg/kg/day); and (c) the control placebo group. Efficacy of treatment was measured by the absence of palpable tumor. To extend these studies using more sensitive techniques, TRAMP mice were then treated with placebo, flutamide (an antiandrogen; 33 mg/kg/day), or toremifene (10 mg/kg/day). Animals from each treatment group were sacrificed at 7, 10, 15, 20, 25, and 30 weeks of age, and prostate tissues and seminal vesicles were harvested. Tissues from animals (n  5) in each group were evaluated by wholemount dissections of genitourinary tracts, histology, immunohisto- chemistry, and Western blot analyses. Blood was pooled per group to measure estradiol and testosterone hormonal levels. Tumors formed at week 17 in the placebo group (n  10), at week 21 in the high-dose toremifene group (n  12), and at week 29 in the low-dose toremifene group (n  12). This represents an increased tumor latency of up to 12 weeks. By 33 weeks, all animals in the placebo group had tumors com- pared with only 35% of the animals treated with toremifene. Although both flutamide and toremifene decreased tumor incidence compared with the placebo, toremifene was more effective than flutamide. High-grade prostatic intraepithelial neoplasia was observed in animals in the placebo group, but not in animals treated with toremifene. Moreover, toremifene- treated animals had prolonged survival compared with placebo-treated animals. By 33 weeks of age, 100% of the placebo-treated animals had developed palpable tumors and died, whereas 60% of the toremifene- treated animals were tumor free. T antigen levels in the prostate of toremifene-treated animals were similar to those of placebo-treated, age- matched animals. Whereas serum estradiol levels remained unchanged, the total and free testosterone levels were elevated in the toremifene- treated group. Toremifene treatment did not affect androgen receptor levels. Because toremifene prevented prostate cancer in a milieu of ele- vated blood free testosterone levels with no change in prostate androgen receptor expression, the mechanism of toremifene’s chemopreventive ac- tivity may be through nonandrogenic pathways, such as estrogen receptor signaling. INTRODUCTION Prostate cancer is the most frequently diagnosed noncutaneous cancer and the second leading cause of cancer deaths in men (1). Changes in androgen and estrogen levels with age are thought to be involved in prostate cancer because its incidence rises sharply with age (2). The focus of chemoprevention is not on the treatment of the disease (cancer) but rather on the oncogenic process [carcinogenesis (3)]. High-grade PIN 4 is considered a precursor of adenocarcinoma of the prostate because about 60% of men with high-grade PIN develop prostate cancer within 2 years (4, 5). Androgen deprivation by flut- amide and LHRH agonists, but not by finasteride, reduced high-grade PIN (5–7). Unfortunately, the serious side effects of testosterone- lowering drugs are not acceptable to men without prostate cancer. Increasing serum estrogens and decreasing serum androgens and 5-reductase activity with age lead to stromal hyperproliferation in the prostate (2). Rising estrogens appear to increase sensitivity of the prostate tissue to androgens by up-regulation of the AR (8 –10). Estradiol in the presence of androgens has been shown to stimulate carcinoma in situ and adenocarcinoma of the prostate in Noble rats (11–14). Estradiol is also capable of inducing high-grade PIN and prostate cancer in the aging dog (2, 15). Thus, estrogenic stimulation with decreasing androgen levels contributes to the genesis of prostatic dysplasia and subsequent prostate cancer (16 –18). Both the prostatic stroma and epithelium express ERs, and estro- gens are important for prostate growth (19, 20). Recently, a new ER, ER, was cloned from a rat prostatic cDNA library and is present in murine and human prostates (21–24). Consequently, the previous ER is now designated as ER. ER and ER are highly homologous, have similar affinity for estradiol, and can hetero- or homodimerize to form a signaling dimeric complex (21, 22). Although estradiol acti- vates both ER and ER, ER stimulates transcription and cellular proliferation, whereas ER quenches ER activation (25). ER is localized predominantly in the prostatic stroma (26), whereas ER is found in the secretory epithelial cells of the prostate (21, 22). In the TRAMP model, the PB-Tag transgene is expressed specifi- cally in the epithelial cells of the prostate. The probasin promoter contains an ARE. All TRAMP mice express the transgene in an androgen-dependent manner and eventually develop prostate cancer that mirrors human prostate cancer progression (27, 28). The TRAMP model has several advantages over currently existing models: (a) mice develop progressive forms of PIN as early as 10 weeks and develop invasive adenocarcinoma by 18 weeks of age; (b) metastatic spread of prostate cancer in TRAMP mice to lymph node, lung, kidney, adrenal gland, and bone resembles human disease; (c) development and pro- gression of prostate cancer can be followed within a relatively short period of 10 –30 weeks; (d) prostate tumors arise with 100% fre- quency; and (e) animals may be screened for the presence of the prostate cancer transgene before the onset of clinical prostate cancer. Thus, TRAMP transgenic mice represent a reliable model to directly test the efficacy of chemopreventive agents that may alter prostate carcinogenesis. SERMs are structurally diverse nonsteroidal compounds that func- tionally mimic estradiol in their action but also possess cancer- suppressing activity. Tamoxifen, a SERM, has been widely used to treat breast cancer. Toremifene is a chlorinated derivative of tamox- ifen that lacks the DNA adduct forming ability of tamoxifen and has lower genotoxicity than tamoxifen (29 –31). Toremifene inhibited 7,12-dimethylbenz(a)anthracene-induced rat mammary cancer (32). Toremifene has been used for breast cancer treatment in 27 countries and used for as long as 13 years in Finland (33). Consequently, Received 8/7/01; accepted 1/2/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Department of Defense Grant DAMD17-98-1-8642, the Assisi Foun- dation, and the J. R. Hyde III Family Foundation, (Memphis, TN). 2 To whom requests for reprints should be addressed, at Department of Urology, F210 Coleman, University of Tennessee Health Science Center, 956 Court Avenue, Memphis, TN 38163. Phone: (901) 448-2636; Fax: (901) 448-1476; E-mail: sraghow@utmem.edu. 3 M. Z. H. and S. K. contributed equally to this work. 4 The abbreviations used are: PIN, prostatic intraepithelial neoplasia; TRAMP, trans- genic adenocarcinoma of mouse prostate; Tag, T antigen; PB-Tag, probasin promoter SV40 large T antigen; AR, androgen receptor; ER, estrogen receptor; SERM, selective estrogen receptor modulator; ARE, androgen response element; HRP, horseradish perox- idase; EIA, enzyme immunoassay. 1370 Research. on November 26, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from