IRG1-itaconate axis regulates lung tumorigenesis via pentose phosphate pathway metabolism

Abstract

TAMs use different nutrients in different metabolic pathways to support their function during tumor development. Although there has been some progress in understanding TAM metabolism, the involvement of TAM metabolism in tumor growth and especially in the immune response to the tumor remains unexplored. A recent discovery is the metabolite itaconate, which plays an important role in immune regulation during inflammatory responses. Itaconate is mainly produced by anti-tumor M1-like Mɸ. However, its role in lung cancer development is still largely unexplored. In this study, we have shown that mice lacking Irg1 as source of itaconate are more susceptible to lung cancer development. The proliferation rate was significantly increased in Irg1-deficient lung tumors. Our scRNA-seq analysis of human and mouse lung tumors identified Mɸ as the primary immune cells responsible for Irg1 expression. Accordingly, transplantation of Irg1-deficient bone marrow cells into wild-type mice supported the anti-tumor function of Irg1 findings, as recipient mice of Irg1-deficient bone marrow cells exhibited a higher incidence of lung tumors. The absence of Irg1 in the bone marrow cells not only led to a higher proliferation rate, but also to an increased presence of pro-tumor Mɸ in the lung tumors. Considering itaconate as the main product of IRG1, we showed that Octyl Ita, a permeable form of itaconate, reduced lung tumor formation in various lung cancer mouse models in vivo by decreasing tumor proliferation rates and inducing an anti-tumor phenotype in TAMs. Interestingly, application of Octyl Ita in human tPCLS, a novel human ex vivo system, also resulted in decreased cancer cell proliferation and a shift of TAMs towards an anti-tumor phenotype. We have also shown that the proliferation inhibitory effect of Octyl Ita/itaconate can be recapitulated in various lung cancer cell lines in vitro. To uncover the underlying mechanisms of the antiproliferative properties of octyl-Ita, metabolomic analysis of A549 lung cancer cells revealed a significant down-regulation of PPP metabolism upon Octyl Ita treatment. Further transcriptomic and proteome analysis revealed dysregulation of metabolic pathways related to PPP metabolism, including oxidative stress response. In addition, we identified G6PD as the major target of Octyl Ita/itaconate in PPP metabolism. Octyl Ita/Itaconate can directly bind to G6PD and inhibit its activity without affecting its mRNA and protein levels. Interestingly, Octyl Ita loses its antiproliferative functions in G6PD-deficient cancer cells. Conversely, the antiproliferative function of Octyl Ita is restored when cancer cells are supplemented with downstream G6PD metabolites. We found that Octyl Ita can reprogram pro-tumoral Mɸ into anti-tumoral Mɸ by downregulating PPP metabolism and reducing G6PD activity. Transcriptomic and proteomic analysis have also demonstrated the effects of Octyl Ita treatment on signaling pathways associated with PPP metabolism in pro-tumor Mɸ. However, our analysis suggests that G6PD is only partially involved in the anti-tumor function of Octyl Ita in pro-tumor Mɸ. We also showed that IRG1 is essential for the anti-tumor function of Mɸ, as depletion of IRG1 enhances the pro-tumor phenotype and functions in anti-tumor Mɸ. Finally, our study showed that ablation of Irg1 leads to increased G6PD activity in mouse lung tumors, while treatment with Octyl Ita reduces G6PD activity in mice with lung tumors. Higher G6PD activity was found in human lung tumors compared to normal tissue. Similarly, increased G6PD mRNA expression has shown a negative correlation with survival of lung cancer patients. In conclusion, our findings suggest that Octyl Ita possesses significant potential as an antitumor metabolite with potential therapeutic applications in the treatment of lung cancer. It not only inhibits the proliferation of cancer cells, but also induces an anti-tumor phenotype within tumor microenvironment, providing a promising avenue for further research and development of lung cancer treatment.