Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligand(s) that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl-3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including: CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an ~ 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target.The aryl hydrocarbon receptor (AHR) 1 is a ligand-activated transcription factor that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors, a class of proteins that are considered environmental sensors. Prior to activation, the AHR is located in the cytoplasm of the cell, complexed with two heat-shock protein 90 molecules, X-associated

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The pleiotropic cytokine interleukin 6 (IL-6) is involved in immune cell homeostasis. Additionally, IL-6 expression and signaling in tumor cells have been shown to elicit both protumor and antitumor properties. There is a plethora of mechanistic knowledge regarding how IL-6 signal transduction translates to biological responses. However, there is little understanding as to what factors control IL-6 expression within a tumor cell environment. The studies presented herein show that, in MCF-7 breast and ECC-1 endocervical cancer cells, the stimulation of aryl hydrocarbon receptor (AHR) activity, in combination with IL-1B or phorbol 12-myristate 13-acetate (PMA) treatment, results in a marked synergistic induction of IL-6 levels over what is seen without AHR activation. Chromatin immunoprecipitation experiments suggest that the regulation of IL-6 mRNA expression occurs at the chromatin level, as AHR presence on the IL-6 promoter was observed in response to treatment with AHR ligand. Synergistic induction of IL-6 expression was sustained for 72 hours, with accumulation of IL-6 protein reaching levels 4.8-fold above IL-1B treatment alone. In addition, transcriptional regulation of the prototypic AHR responsive gene Cyp1a1 was negatively regulated by PMA and IL-1B treatment. Silencing of RELA expression alleviated IL-1B-mediated repression of AHR transcriptional activity, whereas PMA-mediated repression was maintained. Additionally, small interfering RNA studies reveal that AHR and RELA are necessary for synergistic induction of IL-6. The findings presented here reveal the AHR as a potential therapeutic target for selective modulation of IL-6 expression in some tumor cell types. The data also suggest a possible previously unrecognized mechanism of AHRmediated tumor promotion. [Cancer Res 2008;68(10):3609-17]

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, the role of the AHR in normal physiology is still an area of intense investigation. For example, this receptor plays an important role in certain immune responses. We have previously determined that the AHR can mediate repression of acute-phase genes in the liver. For this observation to be therapeutically useful, selective activation of the AHR would likely be necessary. Recently, the selective estrogen receptor ligand WAY-169916 has also been shown to be a selective AHR ligand. WAY-169916 can efficiently repress cytokine-mediated acute-phase gene expression (e.g. SAA1), yet fail to mediate a dioxin response element-driven increase in transcriptional activity. The goals of this study were to structurally modify WAY-169916 to block binding to the estrogen receptor and increase its affinity for the AHR. A number of WAY-169916 derivatives were synthesized and subjected to characterization as AHR ligands. The substitution of a key hydroxy group for a methoxy group ablates binding to the estrogen receptor and increases its affinity for the AHR. The compound 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA 360), in particular, exhibited essentially no AHR agonist activity, yet was able to repress cytokine-mediated SAA1 gene expression in Huh7 cells. SGA 360 was tested in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated ear inflammatory edema model using C57BL6/J and Ahr−/− mice. Our findings indicate that SGA 360 significantly inhibits TPA-mediated ear swelling and induction of a number of inflammatory genes (e.g. Saa3, Cox2, Il6) in C57BL6/J mice. In contrast, SGA 360 had no effect on TPA-mediated ear swelling or inflammatory gene expression in Ahr−/− mice. Collectively, these results indicate that SGA 360 is a selective Ah receptor modulator (SAhRM) that exhibits anti-inflammatory properties in vivo.

The aryl hydrocarbon receptor (AHR) is regarded as an important homeostatic transcriptional regulator within physiological and pathophysiological processes, including xenobiotic metabolism, endocrine function, immunity, and cancer. Agonist activation of the AHR is considered deleterious based on toxicological evidence obtained with environmental pollutants, which mediate toxic effects through AHR. However, a multitude of plant-derived constituents, e.g., polyphenols that exhibit beneficial properties, have also been described as ligands for the AHR. It is conceivable that some of the positive aspects of such compounds can be attributed to suppression of AHR activity through antagonism. Therefore, we conducted a dioxin response element reporter-based screen to assess the AHR activity associated with a range of flavonoid compounds. Our screen identified two flavonoids (5-methoxyflavone and 7,4Ј-dimethoxyisoflavone) with previously unidentified AHR agonist potential. In addition, we have identified and characterized 6,2Ј,4Ј-trimethoxyflavone (TMF) as an AHR ligand that possesses the characteristics of an antagonist having the capacity to compete with agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene, thus effectively inhibiting AHR-mediated transactivation of a heterologous reporter and endogenous targets, e.g., CYP1A1, independent of cell lineage or species. Furthermore, TMF displays superior action by virtue of having no partial agonist activity, in contrast to other documented antagonists, e.g., ␣-napthoflavone, which are partial weak agonists. TMF also exhibits no species or promoter dependence with regard to AHR antagonism. TMF therefore represents an improved tool allowing for more precise dissection of AHR function in the absence of any conflicting agonist activity.

The concept of selective receptor modulators has been established for the nuclear steroid hormone receptors. Such selective modulators have been used therapeutically with great success in the treatment of cancer. However, this concept has not been examined with regard to the aryl hydrocarbon receptor (AHR) because of the latent toxicity commonly associated with AHR activation. AHR-mediated toxicity is primarily derived from AHR binding to its dioxin response element (DRE) and driving expression of CYP1 family members, which have the capacity to metabolize procarcinogens to genotoxic carcinogens. Recent evidence using a non-DRE binding AHR mutant has established the DRE-independent suppression of inflammatory markers by the AHR. We wished to determine whether such DRE-independent repression with wild-type AHR could be dissociated from canonical DRE-dependent transactivation in a ligand-dependent manner and, in doing so, prove the concept of a selective AHR modulator (SAhRM). Here, we identify the selective estrogen receptor (ER) modulator Way-169916 as a dually selective modulator, binding both ER and AHR. Inflammatory gene expression associated with the cytokine-inducible acute-phase response (e.g., SAA1 and CRP) are diminished by Way-169916 in an AHR-dependent manner. Furthermore, activation of AHR by Way-169916 fails to stimulate canonical DREdriven AHR-mediated CYP1A1 expression, thus eliminating the potential for AHR-mediated genotoxic stress. Such anti-inflammatory activity in the absence of DRE-mediated expression fulfills the major criteria of an SAhRM, which suggests that selective modulation of AHR is possible and renders the AHR a therapeutically viable drug target for the amelioration of inflammatory disease.

There is increasing evidence that the aryl hydrocarbon receptor (AHR) plays a role in tumor progression through numerous mechanisms. We have previously shown that, in certain cancer cell lines that are typically non-responsive to cytokine-mediated IL6 induction, activation of the AHR with the agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin derepresses the IL6 promoter and allows for synergistic induction following IL1β treatment. The mechanism by which this occurs involves liganded AHR binding upstream from the transcription start site and dismissing HDAC-containing corepressor complexes, giving rise to a promoter structure that is more amenable to NF-κB activation. This fact, combined with observations of multiple endogenously-produced chemicals activating the AHR, led us to study its role in basal expression among high cytokine-producing cancer cell lines. The current study provides evidence that several head and neck squamous cell carcinoma cell lines have a level of constitutively bound AHR at the IL6 promoter, allowing for higher basal and readily inducible IL6 transcription. Treatment of these cell lines with an AHR antagonist led to dismissal of the AHR from the IL6 promoter and recruitment of corepressor complexes, thus diminishing cytokine expression. Head and neck squamous cell carcinoma is typically a high cytokine-producing tumor type, with IL6 expression levels correlating with disease aggressiveness. For this reason, AHR antagonist treatment could represent a novel adjuvant therapy for patients, lowering pro-growth and anti-apoptotic signaling with minimal systemic side effects.