Tryptophan (Trp) metabolism has been studied for its immunomodulatory effects in cancer, with increase in metabolites that suppress immune response against cancer. However, the intrinsic metabolic contributions of Trp catabolism to cancer cell biosynthesis and survival are poorly understood. Herein, we investigated for the first time the role of Trp as a source of one-carbon metabolism through formate production in triple-negative breast cancer (TNBC) cells and its effect on serine synthesis pathway via phosphoglycerate dehydrogenase (PHGDH) expression, the rate-limiting enzyme in serine biosynthesis.
CRISPR-Cas9 system was used to generate tryptophan 2,3-dioxygenase (TDO) and/or indoleamine 2,3-dioxygenase (IDO) knockout TNBC cell lines. Multi-omics approach using mass spectrometry Q-TOF system and RNA-seq was employed to integrate targeted and untargeted metabolomics, 13C fluxomics, and transcriptomics in TNBC cell lines. For fluxomics studies, cells were cultured with 13C-labeled tryptophan to trace the metabolic flux of carbon atoms through Trp metabolism into kynurenine pathway.
While serine is known as the main source of one-carbon metabolism, our fluxomics data revealed that one-carbon unit is released during the conversion of N-formylkynurenine to kynurenine. To this end, labelled metabolites pertinent to tetrahydrofolate and methionine cycles were identified, which showed one-carbon unit was subsequently incorporated into these pathways to generate purines. Furthermore, integrated transcriptome and metabolomic analysis of tryptophan dioxygenase KO versus WT, as well as formylkynurenine and kynurenine treated cells, revealed the link between Trp catabolism and serine anabolism via alteration in PHGDH expression. Moreover, KO cells deprived one-carbon units derived from Trp metabolism showed upregulation of PHGDH gene expression. Interestingly, KO cells showed lower growth rate in Serine-Glycine free and PHGDH-inhibition conditions by NCT-503.
This study provides evidence that Trp metabolism contributes to one-carbon metabolism in TNBC beyond its immunosuppressive functions. We also show that disruption of Trp metabolism upregulates PHGDH as a compensatory mechanism. This study identifies simultaneous inhibition of both pathways as a promising dual-targeting therapeutic strategy for TNBC treatment.