An endogenous ligand of the human aryl hydrocarbon receptor promotes tumor formation

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liverand neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO-AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology. DOI: https://doi.org/10.1038/nature10491 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-50509 Accepted Version Originally published at: Opitz, C A; Litzenburger, U M; Sahm, F; Ott, M; Tritschler, I; Trump, S; Schumacher, T; Jestaedt, L; Schrenk, D; Weller, M; Jugold, M; Guillemin, G J; Miller, C L; Lutz, C; Radlwimmer, B; Lehmann, I; von Deimling, A; Wick, W; Platten, M (2011). An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor. Nature, 478(7368):197-203. DOI: https://doi.org/10.1038/nature10491 An endogenous ligand of the human aryl hydrocarbon receptor promotes tumor formation. Christiane A. Opitz, Ulrike M. Litzenburger, Felix Sahm, Martina Ott, Isabel Tritschler, Saskia Trump, Theresa Schumacher, Leonie Jestaedt, Dieter Schrenk, Michael Weller, Manfred Jugold, Gilles J. Guillemin, Christine L. Miller, Christian Lutz, Bernhard Radlwimmer, Irina Lehman, Andreas von Deimling, Wolfgang Wick, Michael Platten Department of Neurooncology, Neurology Clinic and National Center for Tumor Diseases University Hospital of Heidelberg, Heidelberg, Germany; Experimental Neuroimmunology Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Neuropathology, Institute of Pathology, University Hospital of Heidelberg and Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany, Department of Neurology, University Hospital Zürich, Zurich, Switzerland; Department for Environmental Immunology, Helmholtz Center for Environmental Research, Leipzig, Germany; Department of Neuroradiology, University Hospital of Heidelberg, Germany; Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany; Small Animal Imaging Center, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Pharmacology, University of New South Wales, Sydney, Australia; Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA Heidelberg Pharma AG, Ladenburg, Germany; Department of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany * these authors contributed equally to this work Word count: 3086, summary paragraph: 170, figures: 6, references: 34 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxins, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. The identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxins, however, has remained enigmatic. We identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumor cells via tryptophan-2,3-dioxygenase (TDO), a liverand neuron-specific Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumor immune responses and promotes tumor cell survival and motility via the AHR in an autocrine/paracrine fashion. The TDO-AHR pathway is active in human brain tumors and associated with malignant progression and poor survival. As Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a novel pathophysiological function of the AHR with profound implications for cancer and immune biology. Degradation of Trp by indoleamine-2,3-dioxygenases 1 and 2 (IDO1/2) in tumors and tumordraining lymph nodes inhibits antitumor immune responses and is associated with a poor prognosis in various malignancies. Inhibition of IDO1/2 suppresses tumor formation in animal models and is currently tested in phase I/II clinical trials in cancer patients. The relevance of Trp catabolism for human tumor formation and progression however remains elusive. A screen of human cancer cell lines revealed constitutive degradation of Trp and release of high micromolar amounts of Kyn in brain tumor cells, namely glioma cell lines and glioma-initiating cells (GIC), but not human astrocytes (Fig. 1a). Surprisingly, IDO1 and 2 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor IDO2 did not account for the constitutive Trp catabolism in brain tumors (Supplementary Fig. 1a-e, Supplementary note 1). Conversely, tryptophan-2,3-dioxygenase (TDO), which is predominantly expressed in the liver and believed to regulate systemic Trp concentrations, was strongly expressed in human glioma cells (Supplementary Fig. 1b) and correlated with Kyn release (Fig. 1b; Supplementary note 2). Pharmacological inhibition or knockdown of TDO blocked Kyn release by glioma cells, while knockdown of IDO1 and IDO2 had no effect (Fig. 1c,d, Supplementary Fig. 2a, Supplementary note 3), thus confirming that TDO is the central Trp-degrading enzyme in human glioma cells. In human brain tumor specimens TDO protein expression increased with malignancy and correlated with the proliferation index (Fig. 1e-h, Supplementary Fig. 2b,c,3a,b; Supplementary note 4,5). As described previously, healthy human brain showed weak TDO staining in the neurons (Fig. 1e). TDO expression was not confined to gliomas but was also detected in other types of cancers (Supplementary Fig. 3b,c; Supplementary note 6). Reduced Trp concentrations were measured in the sera of glioma patients (Fig. 1i). This enhanced systemic Trp degradation, however, did not translate into increased Kyn levels (Fig. 1i), most likely because Kyn is taken up by other cells and metabolised to quinolinic acid. Indeed, accumulation of quinolinic acid was detected in TDOexpressing glioma tissue (Fig. 1j, Supplementary Fig. 3d; Supplementary note 7). Kyn suppresses allogeneic T cell proliferation. Allogeneic T cell proliferation inversely correlated with the Kyn formation by glioma-derived TDO (Fig. 2a, Supplementary Fig. 4a,b; Supplementary note 8). Knockdown of TDO in glioma cells (Supplementary Fig. 4c,d; Supplementary note 9) restored allogeneic T cell proliferation, while addition of Kyn to the TDO knockdown cells prevented the restoration of T cell proliferation (Fig. 2b). Kyn concentration-dependently inhibited the proliferation of T cell receptor stimulated CD4+ and CD8+ T cells (Supplementary Fig. 4e). In addition, knockdown of TDO resulted in enhanced lysis of glioma cells by alloreactive PBMC (Supplementary Fig. 4f). Finally, decreased 3 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor infiltration with leukocyte common antigen (LCA) positive and CD8+ immune cells was observed in sections of human glioma with high TDO expression in comparison to those with low TDO expression (Fig. 2c,d), indicating that Kyn formation by TDO may suppress antitumor immune responses. In vivo experiments in immunocompetent mice demonstrated that tumors expressing TDO grew faster than their TDO-deficient counterparts (Fig 2e; Supplementary Fig. 4g,h; Supplementary Note 10). In line with this result, TDO expressing tumors displayed a higher proliferation index than TDO-deficient controls (Fig. 2f; Suppl. Fig. 4i). TDO activity suppressed antitumor immune responses as evidenced by reduced interferon-gamma (IFN-) release of T cells and tumor cell lysis by spleen cells of mice bearing TDO-expressing tumors in comparison with mice bearing TDO-deficient tumors (Fig. 2g,h), thus underscoring that TDO activity suppresses antitumor immune responses in vivo. We next assessed the autocrine effects of Kyn on glioma cells. While no differences in cell cycle progression were detected between controls and glioma cells with TDO knockdown (Supplementary Fig. 5a), knockdown of TDO reduced motility and clonogenic survival (Fig. 3a-c, Supplementary Fig. 5b,c; Supplementary Note 11). This was mediated by Kyn as exogenous addition of Kyn restored motility and clonogenic survival in the absence of Trp (Fig. 3d,e; Supplementray Fig. 5d,e), suggesting that Kyn increases the motility of malignant glioma cells. In GIC sphere formation was enhanced in response to Kyn (Fig. 3f). Finally, tumor formation was impaired when TDO knockdown tumors were orthotopically implanted in the brains of nude mice, which are devoid of functional T cells (Fig. 3g, Supplementary Fig. 5f,g; Supplementary note 12). To analyse whether inhibition of antitumor NK cell responses, which are func