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 functional in nude mice, by TDO may account for impaired formation of TDO knockdown tumors, we compared subcutaneous tumor growth in the presence or absence of NK cells. NK cell depletion (Supplementary Fig. 5h) enhanced the growth of both control and TDO knockout tumors but did not restore the growth of TDO knockout tumors to 4 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor that of controls (Fig. 3h). Histochemical analyses revealed that TDO knockout tumors showed less mitoses and Ki-67 staining than control tumors (Fig. 3i). Collectively, these data suggest that Kyn generated by constitutive TDO activity enhances the malignant phenotype of human gliomas in an autocrine manner in the absence of functional antitumor T cell and NK cell responses. To understand the molecular mechanisms underlying the autocrine effects of Kyn on glioma cells, we performed a microarray analysis of Kyn-treated glioma cells. Detailed pathway analysis revealed broad induction of AHR response genes by Kyn (Fig. 4a ; Supplementary Fig. 6a,b, 7; Supplementary note 13). TCDD-inducible poly [ADP-ribose] polymerase (TIPARP) and plasminogen activator inhibitor 2 (referred to as PAI-2 or SERPINB2), two direct AHR target genes, showed the strongest upregulation after 24 h (Supplementary Fig. 6a). These two AHR target genes in addition to another direct AHR target gene, cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) were also among the 6 most upregulated genes after 8 h (Supplementary Fig. 6a). The 25 genes with the lowest (array) p-values in response to 8 h and 24 h Kyn are regulated by the AHR (Fig. 4a; Supplementary. Fig. 6b). The AHR is a transcription factor of the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family, which is activated by xenobiotics such as benzo[a]pyrene and 2,3,7,8-tetrachlordibenzodioxin (TCDD). Malignant glioma cell lines as well as GIC express the AHR constitutively (Supplementary Fig. 6c), and upregulation of AHR target genes by Kyn was confirmed in two different glioma cell lines (Supplementary Fig. 6d,e). Kyn led to translocation of the AHR into the nucleus after 1 h, thus showing an immediate effect of Kyn on the AHR (Fig. 4b,c, Supplementary Fig. 8a). In accordance, Western blot analyses of Kyn-activated tumor cells showed reduced cytoplasmic localisation paralleled by increased nuclear accumulation of the AHR comparable to that induced by TCDD (Fig. 4d). In the nucleus the AHR forms a heterodimer with the AHR nuclear 5 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor translocator (ARNT) that interacts with the core binding motif of the dioxin-response elements (DRE) located in regulatory regions of AHR target genes. Kyn concentrationdependently induced DRE-luciferase activity in glioma cells with an EC50 of 36.6 μM (Fig. 4e). AHR activation was unique to Kyn in a panel of Trp catabolites (Supplementary Table 1). An ethoxyresorufin-O-deethylase (EROD) assay confirmed the induction of the functional AHR target gene cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) with an EC50 of 12.3 μM for Kyn (Supplementary Fig. 8b). Radioligand binding assays using mouse liver cytosol from Ahr-proficient and Ahr-deficient mice demonstrated that Kyn binds to the AHR with a KD of ≈ 4 μM (Fig. 4f). The activation of the AHR and upregulation of AHR-regulated gene expression in response to Kyn was inhibited by the AHR antagonist 3,4-DMF (Fig. 4g, Supplementary Fig. 8f,g; Supplementary note 14) or knockdown of the AHR (Fig. 4h,i, Supplementary Fig. 8h,i), indicating that Kyn is a specific agonist of the AHR. The involvement of the same or similar AHR residues in the binding to Kyn, TCDD and 3-MC was confirmed by the fact that activation of the AHR by all three ligands was inhibited by 3,4-DMF (Supplementary Fig. 8f,g; Supplementary note 14). Importantly, the endogenous Kyn production of glioma cells was sufficient to activate the AHR, as knockdown of TDO decreased the expression of AHR regulated genes (Fig. 4j, Supplementary Fig. 8j,k; Supplementary note 15). As mean Kyn concentrations of 37.01 +/13.4 μM were measured in U87 xenografts (n=6), sufficient Kyn concentrations to activate the AHR were also reached in vivo (Suplementary note 15). We next assessed whether the tumor-promoting effects of Kyn are mediated by the AHR. Kyn failed to induce motility of glioma cells after AHR knockdown (Fig. 5a). Also, the increase in clonogenic survival in response to Kyn was abolished in glioma cells with a knockdown of the AHR (Fig. 5b,c). Interleukin 1, beta (IL1B), epiregulin (EREG), aldehyde dehydrogenase 1 family, member A3 (ALDH1A3), interleukin 8 (IL8) and interleukin 6 (IL6) are regulated by 6 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor Kyn via the AHR in glioma cells and have been reported to promote invasiveness and tumor growth, suggesting that these genes may contribute to the autocrine tumor-promoting effects of TDO-derived Kyn (Supplementary Note 16). In summary these data indicate that AHR signaling is indeed responsible for the autocrine effects of TDO-derived Kyn on tumor cells. To determine whether TDO also influences antitumor immune responses via the AHR we analysed the infiltration of LCA+ and CD8+ immune cells in human glioma sections in relation to their AHR expression (Fig. 5d,e). Infiltration by LCA+ and CD8+ immune cells was decreased in sections of human glioma with high AHR expression compared to those with low AHR expression (Fig. 5d,e), suggesting that AHR signalling may be involved in reducing immune cell infiltration. In accordance with an activation of the AHR by TDOderived Kyn, expression of the AHR target gene TIPARP in LCA+ immune cells was observed only in sections expressing TDO (Fig. 5f). To analyse the contribution of host AHR expression to tumor growth, we compared the growth of murine tumors with and without Tdo expression in Ahr-deficient and Ahr-proficient mice. In Ahr-proficient mice Tdo expression strongly enhanced tumor growth in comparison to tumors not expressing Tdo (Fig. 5g). The same effect was observed in Ahr-deficient mice, albeit to a much lower extent (Fig. 5g). As murine glioma cells express functional AHR (Supplementary Fig. 9a, Supplementary note 17), these results suggest that the increase in tumor growth mediated by TDO in Ahr-deficient mice is due to autocrine effects of TDO on the tumor cells themselves. The finding that TDO leads to stronger tumor growth in Ahr-proficient than in Ahr-deficient mice (Fig. 5g), indicates that AHR-mediated host effects enhance tumor growth. Staining of LCA+ immune cells in the tumors revealed that expression of TDO reduced the infiltration with LCA+ immune cells in Ahr-proficient mice, but not in Ahr-deficient mice (Fig. 5h,i), suggesting that TDO-mediated suppression of anti-tumor immune responses via the AHR contributes to the host effects enhancing the growth of Tdo-expressing tumors. In summary, these results 7 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor indicate that TDO enhances tumor growth by suppressing the antitumor immune response via the AHR. To assess the effect of AHR activity on the tumor cells themselves, we performed in vivo experiments using AHR-proficient and AHR-deficient human glioma cells (Fig 5j). Knockdown of the AHR in the glioma cells inhibited tumor growth (Fig.5j, Supplementary note 18), underscoring the importance of AHR signaling for the autocrine effects of Trp degradation. In line, the AHR knockdown tumors showed less mitoses and a reduced proliferative index in comparison to the AHR-proficient control tumors (Fig.5k). Next we aimed to investigate whether TDO-derived Kyn activates the AHR in human brain tumor tissue. Indeed, TDO expression correlated with the expression of the AHR and AHR target genes in human glioma tissue (Fig. 6a-d, Supplementary Fig.9d,e; Supplementary note 19), indicating that constitutive TDO expression in glioma cells produced sufficient Kyn levels to activate the AHR. To address whether the TDO-Kyn-AHR signalling pathway is also activated in cancers other than gliomas, we analysed microarray data of diverse human tumor entities (Fig. 6e; Supplementary Fig. 10a). Interestingly, TDO expression correlated with the expression of the AHR target gene CYP1B1 not only in glioma (Fig. 6d), but also in B cell lymphoma, Ewing sarcoma, bladder carcinoma, cervix carcinoma, colorectal carcinoma, lung carcinoma and ovarian carcinoma (Fig. 6e; Supplementary. Fig. 10a; Supplementary note 20). This finding indicates that the TDO-Kyn-AHR pathway is not confined to brain tumors but appears to be a common trait of cancers. Analysis of the Rembrandt database revealed that the overall survival of glioma patients (WHO grade II-IV) with an upregulation of TDO, the AHR or the AHR target gene CYP1B1 was reduced compared to patients with intermediate or downregulated expression of these genes (Fig. 6f; Supplementary Fig. 10b; Supplementary note 21). Finally, in patients with 8 OPITZ et al., An endogenous tryptophan catabolite activates the aryl hydrocarbon receptor glioblastoma (WHO grade IV), the expression of the AHR targets CYP1B1, IL1B, IL6 and IL8, which are regulated by TDO-derived Kyn in glioma cells (Fig. 4j; Supplementary Fig. 6d,e), were found to predict survival even independent of WHO grade (Fig. 6g; Supplementary Fig. 10c), thus further underscoring the importance of AHR activation for the malignant phenotype of gliomas. In summary these data suggest that endogenous tumorderived Kyn activates the AHR in an autocrine/paracrine fashion to promote tumor progression. Cancer-associated immunosuppression by Trp degradation has to date been attributed solely to the enzymatic activity of IDO in cancer cells and tumor-draining lymph nodes. Thus, IDO inhibition is currently being evaluated as a therapeutic strategy to treat cancer in clinical trials. We show that TDO is strongly expressed in cancer and equally capable of producing immunosuppressive Kyn. In IDO-negative glioma cells, TDO appears to be the sole determinant of constitutive Trp degradation, indicating that TDO represents a novel therapeutic target in glioma therapy. Furthermore we delineate the importance of constitutive Trp degradation to sustain the malignant phenotype of cancer by acting on the tumor cells themselves. Emerging evidence points towards a tumor-promoting role of the AHR. AHR activation promotes clonogenicity and invasiveness of cancer cells. Transgenic mice with a constitutively active AHR spontaneously develop tumors and the repressor of the AHR (AHRR) is a tumor suppressor gene in multiple human cancers. The aberrant phenotype of Ahr-deficient mice points to the existence of endogenous AHR ligands. While different endogenously produced metabolites such as arachidonic acid metabolites, bilirubin, cAMP, tryptamine and 6-formylindolo[3,2-b]carbazole (FICZ) have been shown to be agonists of the AHR, their functionality has not been convincingly demonstrated in a pathophysiological