The selective aryl hydrocarbon receptor modulator 6-methyl-1,3,8-trichlorodibenzofuran inhibits prostate tumor metastasis in TRAMP mice
Introduction
Prostate cancer is the most common non-cutaneous cancer diagnosed annually and the second leading cause of cancer death in American men [1]. There is considerable speculation that exposure to environmental pollutants may increase susceptibility to prostate cancer. One group of chemicals that is believed to have an association between exposure and cancer are dioxins, including the prototype 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Institute of Medicine has found “limited or suggestive evidence” of an association between Agent Orange (a herbicide contaminated with TCDD) exposure and human prostate cancer susceptibility [2]. It was further reported that US Air Force veterans with the greatest serum dioxin concentrations had the greatest prostate cancer risk [3]. Another study reported no overall increased risk of prostate cancer in the Agent Orange-exposed group but that prostate cancer was associated with high TCDD exposure [4]. And a recent study of more than 13,000 veterans found that Agent Orange-exposed men had a increased incidence of prostate cancer, developed prostate cancer at an earlier age, and had more aggressive prostate cancer than did unexposed veterans [5].
In mice, we recently identified an association between exposure to TCDD during early critical stages of prostate development and altered prostate histology later in life. In utero and lactational (IUL) TCDD exposure not only resulted in ventral prostate agenesis but also in a greater incidence of cribriform structures in dorsolateral prostates of senescent C57BL/6J mice [6]. Cribriform structures are hyperplastic lesions found in aging rats and mice, and are often considered to be pre-cancerous lesions in mouse strains susceptible to prostate carcinogenesis [7], [8]. However, C57BL/6J mice are not naturally susceptible to prostate cancer. The implications that early developmental exposure to TCDD may increase the prevalence of lesions associated with greater prostate cancer susceptibility in mice supports the epidemiological evidence that TCDD exposure may also increase prostate cancer risk, although epidemiology studies only correlated adult TCDD exposure with prostate cancer risk.
TCDD binds to the aryl hydrocarbon receptor (AhR), a basic-helix-loop-helix/Per-Arnt-Sim ligand-activated transcription factor. Upon ligand binding, the TCDD/AhR complex translocates to the nucleus and dimerizes with the AhR nuclear translocator (ARNT). The AhR-ARNT complex alters the transcription of a number of genes containing dioxin response elements [9], [10]. Using mice lacking the AhR (Ahr−/−), we demonstrated that effects of IUL TCDD exposure on the mouse prostate, including ventral prostate agenesis, were AhR dependent [11]. Dorsolateral and anterior prostate lobe weights in untreated AhR null mice were also reduced compared to prostates from wild-type littermates at various ages [11]. Even though androgen-dependent prostate lobe weights were reduced, circulating testosterone concentrations were unaltered [11]. This suggested that the AhR signaling pathway, in the absence of TCDD, is in some way involved in normal development of these two prostate lobes.
A commonly utilized model for experimental prostate cancer research is the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse [12], [13], [14], [15], [16]. The TRAMP model uses the minimal rat probasin gene promoter to drive expression of simian virus 40 (SV40) large T and small t antigens in the prostate epithelium in a hormonally and developmentally regulated manner. TRAMP mice characteristically express the T antigen oncoprotein by 8 weeks of age and develop distinct pathology as mild to severe hyperplasia precedes focal carcinoma. Further cytologic and immunohistochemical characterization revealed that the TRAMP tumor consisted of chromogranin A immunopositive and/or synaptophysin immunopositive neuroendocrine cells and that the architecture of these tumors resembled that of poorly differentiated prostate carcinoma [7], [17]. These fast growing neuroendocrine tumors soon lose their localization in the prostate and tumor cells metastasize to adjacent pelvic lymph nodes as early as 12 weeks of age.
When the effect of AhR was investigated in a C57BL/6J TRAMP mouse model of prostate carcinogenesis, diffuse prostatic epithelial hyperplasia characteristic of the TRAMP model was observed in all mice by 105 days of age. Yet Ahr+/+ TRAMP mice rarely developed prostate tumors, while Ahr−/− TRAMP mice did so with much greater frequency [18]. Quantitative RT-PCR and immunohistochemical analysis indicated that the tumors expressed molecular markers indicative of a neuroendocrine phenotype, suggesting that the Ahr regulated prostate carcinogenesis by inhibiting development of neuroendocrine tumors [18]. These results suggest that the Ahr has neuroendocrine tumor suppressor properties in the C57BL/6J TRAMP model.
Since mice lacking the Ahr were more susceptible to prostate carcinogenesis, we hypothesized that moderate activation of the AhR might confer protection. A 1999 study reporting that testosterone-induced cell proliferation in LNCaP prostate cancer cells was inhibited by TCDD [19] supports this hypothesis. While adverse effects of AhR activation by TCDD would negate any potential therapeutic value, less potent AhR ligands have emerged as potential candidates for protection against certain cancers. These selective AhR modulators (SAhRMs) bind to the AhR, but do so with little of the toxicity caused by TCDD exposure [20], [21], [22]. SAhRMs are being developed for the treatment of breast cancer and other hormone-dependent cancers [23], [24], [25]. The focus of the present study is on 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), a SAhRM whose structure is similar to that of the prototypical AhR agonist TCDD. 6-MCDF inhibited carcinogen-induced mammary tumor growth in rats [21], [22], [26], and inhibited pancreatic cancer cell proliferation in vitro [27]. The finding that 6-MCDF could also inhibit LNCaP prostate cancer cell growth in vitro [28] prompted the present study, which is the first to investigate whether 6-MCDF can inhibit prostate cancer development in vivo. We utilized the TRAMP mouse model to investigate effects of this SAhRM on prostate neuroendocrine tumorigenesis and on metastasis to the adjacent lymph nodes.
Section snippets
Transgenic mice
All experiments were conducted in accordance with University of Wisconsin Animal Care and Use Committee guidelines and the NIH Guide for the Care and Use of Laboratory Animals. Mice were housed in clear plastic cages with heat-treated chipped aspen bedding in rooms maintained at 24 ± 1 °C and with 12 h light and dark cycles. C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mice originating from Jackson Laboratory (Bar Harbor, ME, USA) were obtained from Dr. George Wilding (Department of Medicine, University of
Determination of 6-MCDF concentrations in the diet that did not alter weights of reproductive organs, liver, or thymus
An initial experiment was conducted to determine concentrations of 6-MCDF that would not influence weights of androgen-dependent reproductive organs. This minimized the likelihood that possible effects of 6-MCDF on tumorigenesis could be caused simply by increasing or decreasing androgen-dependent transgene expression. Likewise, it was important to determine 6-MCDF concentrations that would not alter weights of organs typically associated with the toxicity of a full AhR agonist like TCDD. In
Discussion
The AhR is best known for regulating biological responses to environmental chemicals including polycyclic aromatic hydrocarbons and dioxins [9], [10]. Studies with Ahr−/− mice have implicated the AhR in normal developmental processes [30], [31], [32], [33], including those for the anterior and dorsolateral prostate [11]. We subsequently demonstrated that the Ahr also has tumor suppressor activity, in that Ahr−/− TRAMP mice developed prostate tumors with far greater frequency than Ahr+/+ TRAMP
Acknowledgements
Portions of this work were supported by National Cancer Institute grant CA095751 and National Institutes of Health grants ES01332 and ES12352. Wayne Fritz was supported by the Molecular and Environmental Toxicology Postdoctoral Training Grant number T32 ES007015 from the NIEHS, NIH. The authors thank Dr. Terry Oberley, Pathology Department, School of Medicine, University of Wisconsin, for assistance with histological characterization of TRAMP prostates. We thank Heather Hardin for assistance
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Stephen Safe was granted United States patent numbers 5,516,790 for “Synthesis and application of alkyl-substituted dibenzofurans as antitumorigenic agents” and 6,136,845 for “Endocrine therapy for breast cancer: combined treatment with tamoxifen plus alkyl PCDFs”.