Functions of the aryl hydrocarbon receptor (AHR) beyond the canonical AHR/ARNT signaling pathway.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor regulating adaptive and maladaptive responses toward exogenous and endogenous signals. Research from various biomedical disciplines has provided compelling evidence that the AHR is critically involved in the pathogenesis of a variety of diseases and disorders, including autoimmunity, inflammatory diseases, endocrine disruption, premature aging and cancer. Accordingly, AHR is considered an attractive target for the development of novel preventive and therapeutic measures. However, the ligand-based targeting of AHR is considerably complicated by the fact that the receptor does not always follow the beaten track, i.e. the canonical AHR/ARNT signaling pathway. Instead, AHR might team up with other transcription factors and signaling molecules to shape gene expression patterns and associated physiological or pathophysiological functions in a ligand-, cell- and micromilieu-dependent manner. Herein, we provide an overview about some of the most important non-canonical functions of AHR, including crosstalk with major signaling pathways involved in controlling cell fate and function, immune responses, adaptation to low oxygen levels and oxidative stress, ubiquitination and proteasomal degradation. Further research on these diverse and exciting yet often ambivalent facets of AHR biology is urgently needed in order to exploit the full potential of AHR modulation for disease prevention and treatment.

The Aryl Hydrocarbon Receptor in the Pathogenesis of Environmentally-Induced Squamous Cell Carcinomas of the Skin.

Cutaneous squamous cell carcinoma (SCC) is one of the most frequent malignancies in humans and academia as well as public authorities expect a further increase of its incidence in the next years. The major risk factor for the development of SCC of the general population is the repeated and unprotected exposure to ultraviolet (UV) radiation. Another important risk factor, in particular with regards to occupational settings, is the chronic exposure to polycyclic aromatic hydrocarbons (PAH) which are formed during incomplete combustion of organic material and thus can be found in coal tar, creosote, bitumen and related working materials. Importantly, both exposomal factors unleash their carcinogenic potential, at least to some extent, by activating the aryl hydrocarbon receptor (AHR). The AHR is a ligand-dependent transcription factor and key regulator in xenobiotic metabolism and immunity. The AHR is expressed in all cutaneous cell-types investigated so far and maintains skin integrity. We and others have reported that in response to a chronic exposure to environmental stressors, in particular UV radiation and PAHs, an activation of AHR and downstream signaling pathways critically contributes to the development of SCC. Here, we summarize the current knowledge about AHR's role in skin carcinogenesis and focus on its impact on defense mechanisms, such as DNA repair, apoptosis and anti-tumor immune responses. In addition, we discuss the possible consequences of a simultaneous exposure to different AHR-stimulating environmental factors for the development of cutaneous SCC.

Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response.

Polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs) and structurally-related environmental pollutants may contribute to the pathogenesis of various diseases and disorders, primarily by activating the aryl hydrocarbon receptor (AHR) and modulating downstream cellular responses. Accordingly, AHR is considered an attractive molecular target for preventive and therapeutic measures. However, toxicological risk assessment of AHR-modulating compounds as well as drug development is complicated by the fact that different ligands elicit remarkably different AHR responses. By elucidating the differential effects of PAHs and DLCs on aldo-keto reductase 1C3 expression and associated prostaglandin D2 metabolism, we here provide evidence that the epidermal growth factor receptor (EGFR) substantially shapes AHR ligand-induced responses in human epithelial cells, i.e. primary and immortalized keratinocytes and breast cancer cells. Exposure to benzo[a]pyrene (B[a]P) and dioxin-like polychlorinated biphenyl (PCB) 126 resulted in a rapid c-Src-mediated phosphorylation of EGFR. Moreover, both AHR agonists stimulated protein kinase C activity and enhanced the ectodomain shedding of cell surface-bound EGFR ligands. However, only upon B[a]P treatment, this process resulted in an auto-/paracrine activation of EGFR and a subsequent induction of aldo-keto reductase 1C3 and 11-ketoreduction of prostaglandin D2. Receptor binding and internalization assays, docking analyses and mutational amino acid exchange confirmed that DLCs, but not B[a]P, bind to the EGFR extracellular domain, thereby blocking EGFR activation by growth factors. Finally, nanopore long-read RNA-seq revealed hundreds of genes, whose expression is regulated by B[a]P, but not by PCB126, and sensitive towards pharmacological EGFR inhibition. Our data provide novel mechanistic insights into the ligand response of AHR signaling and identify EGFR as an effector of environmental chemicals.

Inhibition of 6-formylindolo[3,2-b]carbazole metabolism sensitizes keratinocytes to UVA-induced apoptosis: Implications for vemurafenib-induced phototoxicity.

Ultraviolet (UV) B irradiation of keratinocytes results in the formation of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) which is a high-affinity ligand for the aryl hydrocarbon receptor (AHR). The resulting activation of AHR signaling induces the expression of cytochrome P450 (CYP) 1A1 which subsequently metabolizes FICZ. Importantly, FICZ is also a nanomolar photosensitizer for UVA radiation. Here, we assess whether a manipulation of the AHR-CYP1A1 axis in human epidermal keratinocytes affects FICZ/UVA-induced phototoxic effects and whether this interaction might be mechanistically relevant for the phototoxicity of the BRAF inhibitor vemurafenib. Treatment of keratinocytes with an AHR agonist enhanced the CYP1A1-catalyzed metabolism of FICZ and thus prevented UVA photosensitization, whereas an inhibition of either AHR signaling or CYP1A1 enzyme activity resulted in an accumulation of FICZ and a sensitization to UVA-induced oxidative stress and apoptosis. Exposure of keratinocytes to vemurafenib resulted in the same outcome. Specifically, CYP phenotyping revealed that vemurafenib is primarily metabolized by CYP1A1 and to a lesser degree by CYP2J2 and CYP3A4. Hence, vemurafenib sensitized keratinocytes to UVA-induced apoptosis by interfering with the CYP1A1-mediated oxidative metabolism of FICZ. In contrast to this pro-apoptotic effect, a treatment of UVB-damaged keratinocytes with vemurafenib suppressed apoptosis, a process which might contribute to the skin carcinogenicity of the drug. Our results provide insight into the mechanisms responsible for the photosensitizing properties of vemurafenib and deliver novel information about its metabolism which might be relevant regarding potential drug-drug interactions. The data emphasize that the AHR-CYP1A1 axis contributes to the pathogenesis of cutaneous adverse drug reactions.

Extended Multicomponent Reactions with Indole Aldehydes: Access to Unprecedented Polyheterocyclic Scaffolds, Ligands of the Aryl Hydrocarbon Receptor.

The participation of reactants undergoing a polarity inversion along a multicomponent reaction allows the continuation of the transformation with productive domino processes. Thus, indole aldehydes in Groebke-Blackburn-Bienaymé reactions lead to an initial adduct which spontaneously triggers a series of events leading to the discovery of novel reaction pathways together with direct access to a variety of linked, fused, and bridged polyheterocyclic scaffolds. Indole 3- and 4-carbaldehydes with suitable isocyanides and aminoazines afford fused adducts through oxidative Pictet-Spengler processes, whereas indole 2-carbaldehyde yields linked indolocarbazoles under mild conditions, and a bridged macrocycle at high temperature. These novel structures are potent activators of the human aryl hydrocarbon receptor signaling pathway.

Vemurafenib acts as an aryl hydrocarbon receptor antagonist: Implications for inflammatory cutaneous adverse events.

BACKGROUND: In recent years, the BRAF inhibitor vemurafenib has been successfully established in the therapy of advanced melanoma. Despite its superior efficacy, the use of vemurafenib is limited by frequent inflammatory cutaneous adverse events that affect patients' quality of life and may lead to dose reduction or even cessation of anti-tumor therapy. To date, the molecular and cellular mechanisms of vemurafenib-induced rashes have remained largely elusive. METHODS: In this study, we deployed immunohistochemistry, RT-qPCR, flow cytometry, lymphocyte activation tests, and different cell-free protein-interaction assays. RESULTS: We here demonstrate that vemurafenib inhibits the downstream signaling of the canonical pathway of aryl hydrocarbon receptor (AhR) in vitro, thereby inducing the expression of proinflammatory cytokines (eg, TNF) and chemokines (eg, CCL5). In line with these results, we observed an impaired expression of AhR-regulated genes (eg, CYP1A1) and an upregulation of the corresponding proinflammatory genes in vivo. Moreover, results of lymphocyte activation tests showed the absence of drug-specific T cells in respective patients. CONCLUSION: Taken together, we obtained no hint of an underlying sensitization against vemurafenib but found evidence suggesting that vemurafenib enhances proinflammatory responses by inhibition of canonical AhR signaling. Our findings contribute to our understanding of the central role of the AhR in skin inflammation and may point toward a potential role for topical AhR agonists in supportive cancer care.

The Toll-like receptor agonist imiquimod is metabolized by aryl hydrocarbon receptor-regulated cytochrome P450 enzymes in human keratinocytes and mouse liver.

The Toll-like receptor 7 agonist imiquimod (IMQ) is an approved drug for the topical treatment of various skin diseases that, in addition, is currently tested in multiple clinical trials for the immunotherapy of various types of cancers. As all of these trials include application of IMQ to the skin and evidence exists that exposure to environmental pollutants, i.e., tobacco smoke, affects its therapeutic efficacy, the current study aims to elucidate the cutaneous metabolism of the drug. Treatment of human keratinocytes with 2.5 µM benzo[a]pyrene (BaP), a tobacco smoke constituent and aryl hydrocarbon receptor (AHR) agonist, for 24 h induced cytochrome P450 (CYP) 1A enzyme activity. The addition of IMQ 30 min prior measurement resulted in a dose-dependent inhibition of CYP1A activity, indicating that IMQ is either a substrate or inhibitor of CYP1A isoforms. Incubation of 21 recombinant human CYP enzymes with 0.5 µM IMQ and subsequent LC-MS analyses, in fact, identified CYP1A1 and CYP1A2 as being predominantly responsible for IMQ metabolism. Accordingly, treatment of keratinocytes with BaP accelerated IMQ clearance and the associated formation of monohydroxylated IMQ metabolites. A co-incubation with 5 µM 7-hydroxyflavone, a potent inhibitor of human CYP1A isoforms, abolished basal as well as BaP-induced IMQ metabolism. Further studies with hepatic microsomes from CD-1 as well as solvent- and ß-naphthoflavone-treated CYP1A1/CYP1A2 double knock-out and respective control mice confirmed the critical contribution of CYP1A isoforms to IMQ metabolism. Hence, an exposure to life style-related, dietary, and environmental AHR ligands may affect the pharmacokinetics and, thus, treatment efficacy of IMQ.