Tryptophan Catabolism Pathway As A Target For Antitumor Compound Identification

L-tryptophan is an essential amino acid that is required for protein synthesis and for physiological activities.Inadequate intake or abnormal tryptophan metabolism can lead to infant growth retardation and mental depression.Tumor cells sequester tryptophan for tumor growth and tumor evasion of immune surveillance.Tryptophan undergoes catabolism catalyzed by indoleamine 2,3 dioxygenase-1(IDO1)and tryptophan 2,3-dioxygenase(TDO2)in tumor cells.One of the product,L-kynurenine,has recently been identified as an endogenous ligand of the aryl hydrocarbon receptor(AHR)under physiological conditions.Ligation of Kyn to AHR results signal transduction that promotes tumor cell proliferation,tumor cell metastasis,angiogenesis and tumor immune evasion.Therefore,inhibitors of IDO are currently under clinical trials as adjuvant for antitumor therapy.We undertook a project to identify antitumor agents by targeting the Kyn-AHR axis.The gliomas include astrocytic,oligodendroglial,oligoastrocytic,ependymal and choroid plexus and other neuroepithelial tumors according to the World Health Organization(WHO)classification of tumors in the CNS.The current standard of care in elderly patients with glioblastoma or anaplastic astrocytoma is resection or biopsy followed by involved-field radiotherapy.The effective treatment of chemotherapy is limited by BBB and drug side effects.Recent research revealed that human aryl hydrocarbon receptor(AHR),a receptor of the tryptophan catabolite kynurenine,implicated in cancer biology suppressing antitumor immune responses and promoting tumor cell survival and motility in an autocrine/paracrine fashion.Ligand-AhR complexes directly induced cytochrome family proteins such as CYP1A1 and CYP1B1 resulting in cellular damage and carcinogenesis.Additionally,AHR proteins functionally cooperated with NF-κB elements to upregulate inflammatory gene expression for example IL-6 and IL-8 that can lead to a cancer promoting in tissue microenvironment.In our investigation,we took advantage of CRISPR/Cas9 to establish an AHR gene defect stable glioma cell line.Deletion of AHR leaded to the reduction of AHR-regulated gene expression resulted in the decrease of tumor proliferation both in vitro and in vivo.Kyn-AHR pathway play a crucial role in human brain tumors and is associated with malignant progression.We screened and identified kynurenine-AHR pathway inhibitors using the CYP1A1 promoter-driving luciferase reporter assay.We demonstrated that#97 and#52 inhibit CYP1A1 promoter activity induced by Kyn in both 293T and U87 cells.Furthermore,the compounds prevented Kyn-induced nuclear translocation of AHR and down regulated the mRNA levels of the AHR-regulated gene cytochrome P450 1B1(CYP1B1)and IL-8.The compounds exerted antitumor effect against U87 cells with AHR expression while no influence on U251 cells lacking in AhR.The results were validated by CRISP-Cas9 mediated knock out AHR gene in U87 cells.Oral administration of#97 not only prevented tumor growth in implanted subcutaneous tumors but also revealed the same outcome in orthotopically implanted model of human glioma cells.Even the tumor tissue had severely developed,the compound also significantly improved survival rate of#97-treated mice compared to control.In summary,our findings emphasize the important role of AHR played on tumor development and uncover a therapeutic approach to decline growth and tumorigenicity of glioma tumor by inhibitors targeting the Kyn-AHR pathway.