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.
References
[1]
Beischlag TV, Luis Morales J, Hollingshead BD, Perdew GH (2008) The aryl hydrocarbon receptor complex and the control of gene expression. Crit Rev Eukaryot Gene Expr 18: 207–250.
[2]
Kolluri SK, Weiss C, Koff A, Gottlicher M (1999) p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev 13: 1742–1753.
[3]
Ma Q, Whitlock JP Jr (1996) The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Mol Cell Biol 16: 2144–2150.
[4]
Puga A, Barnes SJ, Dalton TP, Chang C, Knudsen ES, et al. (2000) Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. J Biol Chem 275: 2943–2950.
[5]
Puga A, Xia Y, Elferink C (2002) Role of the aryl hydrocarbon receptor in cell cycle regulation. Chem Biol Interact 141: 117–130.
[6]
Weiss C, Kolluri SK, Kiefer F, Gottlicher M (1996) Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the Ah receptor. Exp Cell Res 226: 154–163.
[7]
Kolluri SK, Balduf C, Hofmann M, Gottlicher M (2001) Novel target genes of the Ah (dioxin) receptor: transcriptional induction of N-myristoyltransferase 2. Cancer Res 61: 8534–8539.
[8]
Fritz WA, Lin TM, Cardiff RD, Peterson RE (2007) The aryl hydrocarbon receptor inhibits prostate carcinogenesis in TRAMP mice. Carcinogenesis 28: 497–505.
[9]
Fritz WA, Lin TM, Safe S, Moore RW, Peterson RE (2009) The selective aryl hydrocarbon receptor modulator 6-methyl-1,3,8-trichlorodibenzofuran inhibits prostate tumor metastasis in TRAMP mice. Biochem Pharmacol 77: 1151–1160.
[10]
Safe S, McDougal A (2002) Mechanism of action and development of selective aryl hydrocarbon receptor modulators for treatment of hormone-dependent cancers (Review). Int J Oncol 20: 1123–1128.
[11]
Safe S, Qin C, McDougal A (1999) Development of selective aryl hydrocarbon receptor modulators for treatment of breast cancer. Expert Opin Investig Drugs 8: 1385–1396.
[12]
Zhang S, Lei P, Liu X, Li X, Walker K, et al. (2009) The aryl hydrocarbon receptor as a target for estrogen receptor-negative breast cancer chemotherapy. Endocr Relat Cancer 16: 835–844.
[13]
Fan Y, Boivin GP, Knudsen ES, Nebert DW, Xia Y, et al. (2010) The aryl hydrocarbon receptor functions as a tumor suppressor of liver carcinogenesis. Cancer Res 70: 212–220.
[14]
Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, et al. (2008) The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest 118: 640–650.
[15]
Marlowe JL, Puga A (2005) Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis. J Cell Biochem 96: 1174–1184.
[16]
Pang PH, Lin YH, Lee YH, Hou HH, Hsu SP, et al. (2008) Molecular mechanisms of p21 and p27 induction by 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, involved in antiproliferation of human umbilical vascular endothelial cells. J Cell Physiol 215: 161–171.
[17]
Hu W, Sorrentino C, Denison MS, Kolaja K, Fielden MR (2007) Induction of cyp1a1 is a nonspecific biomarker of aryl hydrocarbon receptor activation: results of large scale screening of pharmaceuticals and toxicants in vivo and in vitro. Mol Pharmacol 71: 1475–1486.
[18]
O’Donnell EF, Saili KS, Koch DC, Kopparapu PR, Farrer D, et al. (2010) The anti-inflammatory drug leflunomide is an agonist of the aryl hydrocarbon receptor. PLoS One 5.
[19]
DuSell CD, Nelson ER, Wittmann BM, Fretz JA, Kazmin D, et al. (2010) Regulation of aryl hydrocarbon receptor function by selective estrogen receptor modulators. Mol Endocrinol 24: 33–46.
[20]
Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, et al. (1999) Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol 93: 198–208.
[21]
Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA (1996) The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 35: 1270–1273.
[22]
White RM, Cech J, Ratanasirintrawoot S, Lin CY, Rahl PB, et al. (2011) DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471: 518–522.
[23]
Jerant AF, Johnson JT, Sheridan CD, Caffrey TJ (2000) Early detection and treatment of skin cancer. Am Fam Physician 62: 357–368, 375-356, 381-352.
[24]
Jux B, Kadow S, Luecke S, Rannug A, Krutmann J, et al. (2011) The aryl hydrocarbon receptor mediates UVB radiation-induced skin tanning. J Invest Dermatol 131: 203–210.
[25]
Luecke S, Backlund M, Jux B, Esser C, Krutmann J, et al. (2010) The aryl hydrocarbon receptor (AHR), a novel regulator of human melanogenesis. Pigment Cell Melanoma Res 23: 828–833.
[26]
Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, et al. (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483: 603–607.
[27]
Bisson WH, Koch DC, O’Donnell EF, Khalil SM, Kerkvliet NI, et al. (2009) Modeling of the aryl hydrocarbon receptor (AhR) ligand binding domain and its utility in virtual ligand screening to predict new AhR ligands. J Med Chem 52: 5635–5641.
[28]
Kolluri SK, Zhu X, Zhou X, Lin B, Chen Y, et al. (2008) A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell 14: 285–298.
[29]
Denison MS, Rogers JM, Rushing SR, Jones CL, Tetangco SC, et al. (2002) Analysis of the aryl hydrocarbon receptor (AhR) signal transduction pathway. Curr Protoc Toxicol Chapter 4: Unit4 8.
[30]
Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, et al. (2000) Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet 24: 227–235.
[31]
Atienza JM, Zhu J, Wang X, Xu X, Abassi Y (2005) Dynamic monitoring of cell adhesion and spreading on microelectronic sensor arrays. J Biomol Screen 10: 795–805.
[32]
Greene S, Watanabe K, Braatz-Trulson J, Lou L (1995) Inhibition of dihydroorotate dehydrogenase by the immunosuppressive agent leflunomide. Biochem Pharmacol 50: 861–867.
[33]
Nair RV, Cao W, Morris RE (1995) Inhibition of smooth muscle cell proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized by uridine. Immunol Lett 48: 77–80.
[34]
Williamson RA, Yea CM, Robson PA, Curnock AP, Gadher S, et al. (1995) Dihydroorotate dehydrogenase is a high affinity binding protein for A77 1726 and mediator of a range of biological effects of the immunomodulatory compound. J Biol Chem 270: 22467–22472.
[35]
Rae JM, Creighton CJ, Meck JM, Haddad BR, Johnson MD (2007) MDA-MB-435 cells are derived from M14 melanoma cells–a loss for breast cancer, but a boon for melanoma research. Breast Cancer Res Treat 104: 13–19.
[36]
Jin DQ, Jung JW, Lee YS, Kim JA (2004) 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits cell proliferation through arylhydrocarbon receptor-mediated G1 arrest in SK-N-SH human neuronal cells. Neurosci Lett 363: 69–72.
[37]
Yang X, Liu D, Murray TJ, Mitchell GC, Hesterman EV, et al. (2005) The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells. Oncogene 24: 7869–7881.
[38]
Goodale BC, La Du JK, Bisson WH, Janszen DB, Waters KM, et al. (2012) AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish. PLoS One 7: e29346.
[39]
Spodnik JH, Wozniak M, Budzko D, Teranishi MA, Karbowski M, et al. (2002) Mechanism of leflunomide-induced proliferation of mitochondria in mammalian cells. Mitochondrion 2: 163–179.
