Abstract
Abnormal circulation in solid tumors results in hypoxia, which modulates both tumor intrinsic malignant properties as well as anti-tumor immune responses. Given the importance of hypoxia in glioblastoma (GBM) biology and particularly in shaping anti-tumor immunity, we analyzed which immunomodulatory genes are differentially regulated in response to hypoxia in GBM cells. Gene expression analyses identified the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2) as the second most downregulated gene in GBM cells cultured under hypoxic conditions. TDO2 catalyses the oxidation of tryptophan to N-formyl kynurenine, which is the first and rate-limiting step of Trp degradation along the kynurenine pathway (KP). In multiple GBM cell lines hypoxia reduced TDO2 expression both at mRNA and protein levels. The downregulation of TDO2 through hypoxia was reversible as re-oxygenation rescued TDO2 expression. Computational modeling of tryptophan metabolism predicted reduced flux through the KP and lower intracellular concentrations of kynurenine and its downstream metabolite 3-hydroxyanthranilic acid under hypoxia. Metabolic measurements confirmed the predicted changes, thus demonstrating the ability of the mathematical model to infer intracellular tryptophan metabolite concentrations. Moreover, we identified hypoxia inducible factor 1α (HIF1α) to regulate TDO2 expression under hypoxic conditions, as the HIF1α-stabilizing agents dimethyloxalylglycine (DMOG) and cobalt chloride reduced TDO2 expression. Knockdown of HIF1α restored the expression of TDO2 upon cobalt chloride treatment, confirming that HIF1α controls TDO2 expression. To investigate the immunoregulatory effects of this novel mechanism of TDO2 regulation, we co-cultured isolated T cells with TDO2-expressing GBM cells under normoxic and hypoxic conditions. Under normoxia TDO2-expressing GBM cells suppressed T cell proliferation, while hypoxia restored the proliferation of the T cells, likely due to the reduction in kynurenine levels produced by the GBM cells. Taken together, our data suggest that the regulation of TDO2 expression by HIF1α may be involved in modulating anti-tumor immunity in GBM.
Highlights
More than 60 years ago, Thomlinson and Gray postulated the occurrence of hypoxic regions in solid tumors [1]
To investigate if hypoxia differentially regulates genes that play a role in anti-tumor immune responses in GBM cells, we performed microarray analysis of A172 GBM cells exposed to 5 days of hypoxia (1% O2) as compared to cells cultured in normoxia (18.6% O2) (GSE138535)
Reduced O2 concentrations under hypoxia would be expected to affect the enzymatic activity of TDO2, our microarray data revealed that the expression of TDO2 may be reduced upon hypoxia in GBM cells
Summary
More than 60 years ago, Thomlinson and Gray postulated the occurrence of hypoxic regions in solid tumors [1]. Initial interest in studying hypoxia in tumors was due to the realization that hypoxic cells are more resistant to radiotherapy [2] resulting in adverse clinical outcomes for the patients. Cells under hypoxic conditions are known to shut down non-essential processes by chromatin modifications and a global downregulation of gene expression [4], while simultaneously genes needed for cell survival under oxygen limitation are upregulated through hypoxia inducible transcription factors (HIFs) [5]. The most well-known HIF family member HIF1α is degraded under normoxic conditions by the action of prolyl-hydroxylase (PHD) enzymes. Under hypoxic conditions PHD function is inhibited, stabilizing HIF1α and activating the expression of its target genes [6]. HIF1α can inhibit the expression of genes during hypoxia [7]
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