[CANCER RESEARCH 60, 5125–5133, September 15, 2000] Chemoprevention of Prostate Carcinogenesis by -Difluoromethylornithine in TRAMP Mice 1 Sanjay Gupta, Nihal Ahmad, Susan R. Marengo, Gregory T. MacLennan, Norman M. Greenberg, and Hasan Mukhtar 2 Departments of Dermatology [S. G., N. A., H. M.], Urology [S. R. M.], and Pathology [G. T. M.], Case Western Reserve University, Cleveland, Ohio 44106, and Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 [N. M. G.] ABSTRACT Development of effective chemopreventive agents for human consump- tion requires conclusive evidence of their efficacy in animal models that have relevance to human diseases. Transgenic adenocarcinoma mouse prostate (TRAMP) is an excellent model of prostate cancer that mimics progressive forms of human disease inasmuch as 100% of males develop histological PIN by 8 –12 weeks of age that progress to adenocarcinoma with distant site metastases by 24 –28 weeks of age. In these animals, ornithine decarboxylase (ODC) activity (>3-fold) as well as protein ex- pression (>4-fold) was found to be markedly higher in the dorsolateral prostate as compared with the nontransgenic littermates, suggesting their suitability to determine the chemopreventive effect of -difluoromethylo- rnithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, against prostate cancer. Using male TRAMP mice, we studied the effect of oral consumption of DFMO on development of prostate carcinogenesis and surrogate end point biomarkers related to prostate cancer progres- sion. In two independent experiments, each consisting of 8 animals on test, the cumulative incidence of prostatic cancer development at 28 weeks of age in 16 untreated TRAMP mice was 100% (16 of 16), whereas 94% (15 of 16) and 69% (11 of 16) of the animals exhibited distant site metastases to lymph nodes and lungs, respectively. Oral consumption of 1% DFMO (w/v) in the drinking water to TRAMP mice from 8 to 28 weeks of age resulted in a significant decrease in (a) weight (59%) and volume (66%) of prostate, (b) genitourinary weight (63%), and (c) ODC enzyme activity (52%) in the dorsolateral prostate. Importantly, in none of the DFMO-fed TRAMP mice were any distant metastases to lymph node and lungs observed. Furthermore, DFMO treatment resulted in the marked reduc- tion in the protein expression of proliferation cell nuclear antigen, ODC, and probasin in the dorsolateral prostate. The protein expression of antimetastases markers, i.e., E-cadherin and - and -catenin, was found to be restored in DFMO-fed animals as compared with the non-DFMO- fed mice. These chemopreventive effects of DFMO were further confirmed by immunohistochemical analysis of the dorsolateral prostate. Histologi- cal analysis of the dorsolateral prostate of DFMO-fed animals displayed marginal epithelial stratification, a small number of cribriform structures, elongated hyperchromatic epithelial nuclei, and a significant increase in apoptotic index. Non-DFMO-fed animals, on the other hand, displayed extensive epithelial stratification with profound cribriform structures ac- companied with marked thickening, remodeling, and hypercellularity of the fibromuscular stroma. In nontransgenic littermates fed with DFMO, no significant alterations in the above parameters were evident. These data demonstrate that ODC represents a promising and rational target for chemoprevention of human prostate cancer and that TRAMP mice are excellent models for screening of novel drugs and chemopreventive regi- mens for potential human use. INTRODUCTION CaP 3 is the most commonly diagnosed solid tumor and the second leading cause of cancer mortality in American men (1). According to an estimate by the American Cancer Society, 18% of the male population in the United States will develop invasive CaP during their lifetime (1, 2). The development of CaP in humans has been viewed as a multistage process, involving the onset as small latent carcinoma of low histological grade to large metastatic lesion of higher grade (3). Unfortunately, there are limited treatment options available for this disease because chemotherapy and radiation therapy are largely inef- fective, and metastatic disease frequently develops even after poten- tially curative surgery (4 – 6). Chemoprevention could be an effective approach to reduce CaP incidence (7, 8). Indeed, CaP is an excellent candidate disease for chemoprevention because of its diagnosis in elderly men, and therefore even a modest delay in the neoplastic development achieved through pharmacological or nutritional inter- vention could result in a substantial reduction in the incidence of this clinically detectable disease (9). For relevance to human population, chemoprevention studies should be conducted in animal models that mimic progressive forms of human disease and possess surrogate end point biomarkers for rapid screening of drugs (10 –12). In recent years, genetically manip- ulated animal models have emerged as resources for developing strategies against many pathological conditions, including cancer (13– 15). Advantages of these genetically manipulated animals for chemo- prevention studies include induction of carcinogenesis by discrete genetic changes and the ability to modulate oncogenes or tumor suppressor genes implicated in human cancers (16 –18). One strength of transgenic models is that in these animals, cancer arises from normal cells in their natural tissue microenvironment and progress through multiple stages, as does human cancer (19). For CaP very few genetically manipulated models are available (20, 21). One such model is TRAMP (22). TRAMP mice express a PB-Tag transgene consisting of the minimal -426/+28-bp regulatory element of the rat probasin promoter directing prostate-specific epithelial expression of the SV40 early genes (T/t antigens; Refs. 22 and 23). TRAMP is an excellent model that serves as a general prototype of the pathways, parameters, and molecular mechanisms of multistage pros- tate tumorigenesis (22). TRAMP males develop spontaneous multi- stage prostate carcinogenesis that exhibits both histological and mo- lecular features similar to that of human CaP (24). TRAMP males characteristically express the PB-Tag transgene by 8 weeks of age and develop distinct pathology in the epithelium of the dorsolateral pros- tate by 10 weeks of age. Distant site metastasis can be detected as early as 12 weeks of age in male TRAMP mice, and by 28 weeks of age, 100% of the animals harbor CaP that metastasizes to the lymph nodes and lungs (22, 24). In the present study, we determined the use of TRAMP mice for Received 2/14/00; accepted 7/10/00. 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 United States Public Health Service Grants CA RO1 78809 (to H. M.), CA58204 and CA84296 (to N. M. G.), by funds from American Institute for Cancer Research (to H. M.), Department of Defense DAMD 17-00-1-0527 (to H. M.), and Cancer Research Foundation of America (to S. G. and N. A.). 2 To whom requests for reprints should be addressed, at Department of Dermatology, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106. Phone: (216) 368-1127; Fax: (216) 368-0212; E-mail: hxm4@po.cwru.edu. 3 The abbreviations used are: CaP, prostate cancer; DFMO, -difluoromethylornithine; ODC, ornithine decarboxylase; mPB-1, murine probasin-1; PCNA, proliferating cell nuclear antigen; HRP, horseradish peroxidase; PIN, prostatic intraepithelial neoplasia; GU, genitourinary. 5125 Research. on October 26, 2018. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from