Abstract 3710: Serum tryptophan metabolites mediate constitutive AHR activity in head and neck squamous cell carcinoma cells

Abstract The aryl hydrocarbon receptor (AHR) is transcription factor, that is activated upon binding to various exogenous and endogenous ligands. AHR activation leads to induction of target gene expression, including the members of cytochrome P450 enzyme family that metabolize many xenobiotics. Numerous studies have shown that the AHR is involved in multiple regulatory pathways important during carcinogenesis. High level of AHR protein and activation was observed in head and neck squamous cell carcinoma (HNSCC) and has been correlated with aggressive tumor phenotypes and migratory potential. Endogenous compounds derived from host tryptophan metabolism such as kynurenine, as well as the derivatives of indole derived from bacterial tryptophan metabolism such as indole-3-propionic acid are known to activate AHR. A fundamental question not previously addressed is whether these circulating tryptophan (Tryp) metabolites can mediate persistent AHR activation within tumor tissues and its effect on tumorigenesis. The focus of our research is to determine whether the continuous exposure of Tryp metabolites at relevant concentrations leads to sustained AHR activation in HNSCC cells and its subsequent effect on tumor cell phenotypes. Here, we present data demonstrating sustained AHR activation by Tryp metabolites in two HNSCC cell lines. A total of 6 tryptophan (Tryp) metabolites present at significant levels in serum with known AHR agonist activity were identified and quantified from 40 healthy individuals on a controlled diet by LC/MS/MS. HN30 and OSC19 cells were treated with a representative pool of 6 Tryp metabolites in a cell culture system to achieve continuous exposure to the Tryp metabolites, thus mimicking blood circulation in vivo. Treated cells were collected at six different time points and AHR activation within cells was determined by measuring mRNA and protein expression of AHR target genes. The effect of Tryp pool on HN30 and OSC19 cell viability was determined. The presence of Tryp metabolites within the cells and media was quantitated by LC/MS/MS at six time points over the course of 24 h. Continuous exposure of Tryp metabolites resulted in sustained AHR activation in HN30 and OSC19 cells, confirmed by induced mRNA expression of target genes and subsequent protein levels. The LC/MS data revealed the presence of all six Tryp metabolites in media collected at all time points, suggesting no spontaneous degradation of metabolites. Tryp metabolites were detected inside the cells starting from 2 h post-treatment to the last time point 24 h. Viability assays indicated no cytotoxic effects of Tryp metabolites on cells. Overall, our work has demonstrated a pattern of sustained AHR activation in HNSCC cells resulting from continuous exposure of Tryp metabolites, supporting the concept that endogenous Tryp metabolites in HNSCC patients would impact tumor progression, metastasis, and treatment outcomes. Citation Format: Dhwani Patel, Ethan W. Morgan, Fangcong Dong, Iain A. Murray, Gary H. Perdew. Serum tryptophan metabolites mediate constitutive AHR activity in head and neck squamous cell carcinoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3710.