The aryl hydrocarbon receptor is a member of the basic-helix-loop-helix family of transcription factors. AhR mediates the biochemical and toxic effects of a number of polyaromatic hydrocarbons such as 2,3,7,8,-tetrachloro-dibenzo-p-dioxin (TCDD). AhR is widely known for regulating the transcription of drug metabolizing enzymes involved in the xenobiotic metabolism of carcinogens and therapeutic agents, such as cytochrome P450-1B1 (CYP1B1). Additionally, AhR has also been reported to interact with multiple signaling pathways during prostate development. Here we investigate the effect of sustained AhR signaling on androgen receptor function in prostate cancer cells. Immunoblot analysis shows that AhR expression is increased in androgen independent (C4-2) prostate cancer cells when compared to androgen sensitive (LNCaP) cells. RT-PCR studies revealed constitutive AhR signaling in C4-2 cells without the ligand induced activation required in LNCaP cells. A reduction of AhR activity by short RNA mediated silencing in C4-2 cells reduced expression of both AhR and androgen responsive genes. The decrease in androgen responsive genes correlates to a decrease in phosphorylated androgen receptor and androgen receptor expression in the nucleus. Furthermore, the forced decrease in AhR expression resulted in a 50% decline in the growth rate of C4-2 cells. These data indicates that AhR is required to maintain hormone independent signaling and growth by the androgen receptor in C4-2 cells. Collectively, these data provide evidence of a direct role for AhR in androgen independent signaling and provides insight into the molecular mechanisms responsible for sustained androgen receptor signaling in hormone refractory prostate cancer.

BackgroundDistant prostate cancers are commonly hormone refractory and exhibit increased growth no longer inhibited by androgen deprivation therapy. Understanding all molecular mechanisms contributing to uncontrolled growth is important to obtain effective treatment strategies for hormone refractory prostate cancers (HRPC). The aryl hydrocarbon receptor (AhR) affects a number of biological processes including cell growth and differentiation. Several studies have revealed that exogenous AhR ligands inhibit cellular proliferation but recent evidence suggests AhR may possess intrinsic functions that promote cellular proliferation in the absence of exogenous ligands.Methods/ResultsqRT-PCR and western blot analysis was used to determine AhR mRNA and protein expression in hormone sensitive LNCaP cells as well as hormone refractory DU145, PC3 and PC3M prostate cancer cell lines. LNCaP cells express AhR mRNA and protein at a much lower level than the hormone refractory cell models. Cellular fractionation and immunocytochemistry revealed nuclear localization of AhR in the established hormone refractory cell lines while LNCaP cells are devoid of nuclear AhR protein. qRT-PCR analysis used to assess basal CYP1B1 levels and a xenobiotic responsive element binding assay confirmed ligand independent transcriptional activity of AhR in DU145, PC3 and PC3M cells. Basal CYP1B1 levels were decreased by treatment with specific AhR inhibitor, CH223191. An in vitro growth assay revealed that CH223191 inhibited growth of DU145, PC3 and PC3M cells in an androgen depleted environment. Immunohistochemical staining of prostate cancer tissues revealed increased nuclear localization of AhR in grade 2 and grade 3 cancers compared to the well differentiated grade 1 cancers.ConclusionsTogether, these results show that AhR is constitutively active in advanced prostate cancer cell lines that model hormone refractory prostate cancer. Chemical ablation of AhR signaling can reduce the growth of advanced prostate cancer cells, an effect not achieved with androgen receptor inhibitors or growth in androgen depleted media.

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