BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

Hepatoprotection mediated by free radical scavenging molecules such as dimethyl sulfoxide (Me₂SO) arose the question as to whether this effect involved one or several anti-apoptotic signals. Here, using primary cultures of rat hepatocytes and in vivothioacetamide-induced liver failure, we showed that Me₂SO failed to prevent any cleavage of initiator caspase-8 and -9 but constantly inhibited procaspase-3 maturation and apoptosis execution, pointing to an efficient inhibition of cleaved initiator caspase activities. Evidence was recently provided that apoptosis might require both caspase and ASK1/JNK-p38 activities. We demonstrated that this kinase pathway was strongly inhibited in the presence of Me₂SO whereas overexpression of ASK1 was able to restore caspase-3 activity and apoptosis. Interestingly, we also found that GST Μ1/2 and GST Α1/2 dropped under apoptotic conditions; furthermore transfection of GST Μ1, A1, or P1 to cells overexpressing ASK1, abolished caspase-3 activity and restored viability. This role of GSTs was further assessed by showing that their high expression level was tightly associated with inhibition of ASK1 activity in Me₂SO-protected hepatocytes. Together, these results demonstrate that Me₂SO-mediated hepatoprotection involves a dual inhibition of cleaved initiator caspase and ASK1/JNK-p38 activities. Furthermore, in highlighting the control of apoptosis by GSTs, these data provide new insights for analyzing the complex mechanisms of hepatoprotection.

The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B). Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C. AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca²⁺) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was designed to characterize this link between Ca²⁺ and the AhR pathway. We report that fast elevation of intracellular Ca²⁺ in TCDD-exposed mammary MCF-7 cells was associated with transient enhanced activity of the Ca²⁺/calmodulin (CaM)-dependent protein kinase (CaMK) pathway. Chemical inhibition of this pathway using the CaM antagonist W7 or the CaMK inhibitor KN-93 strongly reduced TCDD-mediated induction of the AhR target gene CYP1A1. Small interfering RNA (siRNA)-mediated knockdown expression of CaMKIα, one of the CaMK isoforms, similarly prevented CYP1A1 up-regulation. Both KN-93 and siRNA targeting CaMKIα were found to abolish TCDD-mediated activation of CYP1A1 promoter and TCDD-triggered nuclear import of AhR, a crucial step of the AhR signaling pathway. TCDD-mediated inductions of various AhR targets, such as the drug metabolizing CYP1B1, the cytokine interleukin-1β, the chemokines interleukin-8 and CCL1, the adhesion molecule β7 integrin, and the AhR repressor, were also prevented by KN-93 in human macrophages. Taken together, these data identified the Ca²⁺/CaM/CaMKIα pathway as an important contributing factor to AhR-mediated genomic response.