Abstract
A novel role of the dihydroorotatedehydrogenase (DHODH) inhibitor leflunomide as a potential anti-melanoma therapy was recently reported (Nature 471∶518-22, 2011). We previously reported that leflunomide strongly activates the transcriptional activity of the Aryl Hydrocarbon Receptor (AhR). We therefore tested whether the AhR regulates the anti-proliferative effects of leflunomide in melanoma. We first evaluated the expression of AhR in melanoma cells and found that AhR is highly expressed in A375 melanoma as well as in several other cancer cell types. To evaluate whether AhR plays a role in regulating the growth inhibitory effects of leflunomide in A375 cells, we generated a stable cell line from parental A375 cells expressing a doxycycline (DOX) inducible AhR shRNA. Using these cells in the absence or presence of DOX (normal AhR levels or AhR-knockdown, respectively) we found that the anti-proliferative effects of leflunomide, but not its metabolite A771726, were strongly dependent upon AhR expression. It has been well established that supplementation of cells with exogenous uridine completely rescues the anti-proliferative effects due to DHODH inhibition. Thus, we performed uridine rescue experiments in A375 cells to determine whether the anti-proliferative effects of leflunomide are solely due to DHODH inhibition as previously reported. Interestingly, saturating levels of uridine only modestly rescued A375 cells from the anti-proliferative effects of both leflunomide and A771726, indicating additional mechanism(s), apart from DHODH inhibition are responsible for the anti-proliferative effects of leflunomide in melanoma cells. Uridine also did not rescue MDA-MB-435S melanoma cell proliferation after leflunomide treatment. Our results reveal that the AhR is a molecular target of leflunomide and support the feasibility of the clinical application of leflunomide for treating melanoma. Furthermore, analysis of expression data from 967 cancer cell lines revealed that AhR is expressed in multiple different cancer types supporting the intriguing possibility of targeting the AhR for therapy in a number of cancers.
Highlights
The Aryl Hydrocarbon Receptor (AhR) is a ligand activated transcription factor belonging to the basic helix-loop-helix PER/ ARNT/SIM family of transcription factors and regulates a wide range of biological activities ranging from transcriptional modulation of a battery of genes involved in xenobiotic metabolism, most notably members of the cytochrome P450 family, to cell cycle progression through both ligand dependent and independent mechanisms. [1,2,3,4,5,6,7] The AhR is localized in the cytosol, and upon activation by a ligand translocates to the nucleus where it binds its obligate heterodimeric partner AhR Nuclear Translocator protein (ARNT)
A few FDA approved drugs have recently been shown to activate AhR transcription. [17,18,19] For example, we recently reported that AhR activation by leflunomide, a well known immunosuppressive agent used to treat rheumatoid arthritis, alters cell proliferation and tissue regeneration in a context-specific manner. [18,20] Leflunomide is converted to its primary metabolite A771726 via isoxazole ring cleavage, and whereas metabolism of leflunomide to A771726 is required for dihydroorotatedehydrogenase (DHODH) inhibition, [21] this conversion significantly abrogates the AhR-activating properties of leflunomide. [18]
AhR Signaling is Intact in A375 Melanoma Cells In the present study, we hypothesized that the effects of leflunomide in A375 melanoma cells are mediated by a previously unappreciated activation of the AhR
Summary
The Aryl Hydrocarbon Receptor (AhR) is a ligand activated transcription factor belonging to the basic helix-loop-helix PER/ ARNT/SIM (bHLH/PAS) family of transcription factors and regulates a wide range of biological activities ranging from transcriptional modulation of a battery of genes involved in xenobiotic metabolism, most notably members of the cytochrome P450 family, to cell cycle progression through both ligand dependent and independent mechanisms. [1,2,3,4,5,6,7] The AhR is localized in the cytosol, and upon activation by a ligand translocates to the nucleus where it binds its obligate heterodimeric partner AhR Nuclear Translocator protein (ARNT). [1,2,3,4,5,6,7] The AhR is localized in the cytosol, and upon activation by a ligand translocates to the nucleus where it binds its obligate heterodimeric partner AhR Nuclear Translocator protein (ARNT) This complex proceeds to bind AhR/xenobiotic response elements to regulate the transcription of a battery of target genes in a ligand dependent manner. [8,9] Selective AhR modulators that interfere with estrogen receptor transcription have been shown to inhibit breast cancer cell proliferation. [22] Our results revealed that the AhR is essential in mediating the anti-proliferative effects of leflunomide in melanoma cells and that the inhibition of DHODH by leflunomide’s active metabolite A771726 can only partially account for inhibition of melanoma cells. Analysis of expression data from 967 cancer cells revealed that AhR is broadly expressed in several cancer types including lung, breast, liver, stomach and pancreas. [26]
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