The selective aryl hydrocarbon receptor modulator 6-methyl-1,3,8-trichlorodibenzofuran inhibits prostate tumor metastasis in TRAMP mice

Abstract

The aryl hydrocarbon receptor (AhR) is a basic-helix-loop-helix transcription factor that binds halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, and endogenous compounds. We previously reported that AhR null (Ahr^−/−) transgenic adenocarcinoma of the mouse prostate (TRAMP) mice on a C57BL/6J background develop prostate tumors with much greater frequency than AhR wild-type (Ahr^+/+) TRAMP mice, suggesting that the AhR has tumor suppressor properties. Because AhR signaling pathway inactivation increased susceptibility to prostate tumorigenesis, we tested the hypothesis that a selective AhR modulator (SAhRM), 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), can protect against prostate tumorigenesis. TRAMP mice on the standard C57BL/6J × FVB genetic background were fed 0, 10, or 40 mg 6-MCDF/kg diet beginning at 8 weeks of age. Tumor incidence, pelvic lymph node metastasis, and serum vascular endothelial growth factor (VEGF) concentrations were determined at 140 days of age. Prostate tumor incidence and size were not significantly reduced in mice fed 6-MCDF. However, the frequency of pelvic lymph node metastasis was reduced fivefold in mice fed the 40 mg 6-MCDF/kg diet. Serum VEGF concentrations were also reduced by 6-MCDF treatment, particularly in mice without prostate tumors, and 6-MCDF was shown to act directly on cultured prostates to inhibit VEGF secretion. Together, these results suggest that 6-MCDF inhibits metastasis, in part, by inhibiting prostatic VEGF production prior to tumor formation. This is the first report that 6-MCDF can confer protection against prostate cancer in vivo.

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.