Lactate from glycolysis regulates inflammatory macrophage polarization in breast cancer.

Globally, breast cancer is one of the leading causes of cancer death in women. Metabolic reprogramming and immune escape are two important mechanisms supporting the progression of breast cancer. Lactate in tumors mainly comes from glycolysis and glutaminolysis. Using multiomics data analysis, we found that lactate is mainly derived from glycolysis in breast cancer. Single-cell transcriptome analysis found that breast cancer cells with higher malignancy, especially those in the cell cycle, have higher expression levels of glycolytic metabolic enzymes. Combined with clinical data analysis, it was found that the expression of the lactate transporter SLC16A3 is correlated with breast cancer molecular subtypes and immune infiltration. Among 22 immune cells, macrophages are the most abundant immune cells in breast cancer tissues, and the proportion of M1 macrophages is lower in the high SLC16A3 expression group. Finally, in vitro experiments confirmed that lactate could inhibit the expression of M1 macrophage markers at both RNA and protein levels. In conclusion, we found that lactate produced by glycolysis regulates the polarization of inflammatory macrophages in breast cancer.

Tryptophan metabolism regulates inflammatory macrophage polarization as a predictive factor for breast cancer immunotherapy.

Metabolic reprogramming plays a pivotal role in regulating macrophage polarization and function. However, the impact of macrophage tryptophan metabolism on polarization within the breast cancer microenvironment remains elusive. In this study, we used single-cell transcriptome analysis and found that macrophages had the highest tryptophan metabolic activity in breast cancer, melanoma, and head and neck squamous cell carcinoma (HNSC). Further analysis revealed that the tryptophan metabolic activity of macrophages was positively correlated with the M1 macrophage scores in breast cancer. Pancancer analysis found positive correlations between tryptophan metabolism and the M1 macrophage score in almost all tumor types. Spatial transcriptome analysis revealed higher tryptophan metabolism in regions with higher M1 macrophage score in breast cancer tissues. Immune infiltration analysis revealed that the high tryptophan metabolism group exhibited a higher immune score, an increased proportion of CD8+ T cells, augmented cytolytic activity mediated by CD8+ T cells, and elevated expression of immune checkpoint molecules. Spatial immunophenotype cohort analysis exhibited that breast cancer patients expected to respond to immunotherapy had stronger tryptophan metabolism, with a 73.8 % area under the ROC curve. Single-cell transcriptome analysis of the immunotherapy cohort found that patients responding to immunotherapy had higher macrophage tryptophan metabolism prior to treatment initiation. Finally, in vitro experiments demonstrated elevated expression of tryptophan metabolic enzymes in M1 macrophages. Moreover, tryptophan facilitated the expression of M1 polarization markers, whereas inhibitors of tryptophan metabolic enzymes, such as NLG919, LM10, and Ro 61-8048, inhibited the expression of M1 polarization markers. In conclusion, this study identified a dual role for macrophage tryptophan metabolism in breast cancer; on the one hand, it promotes macrophage M1 polarization, while on the other hand, it serves as a promising predictor for the effectiveness of immunotherapy in breast cancer.

The SMYD3-MTHFD1L-formate metabolic regulatory axis mediates mitophagy to inhibit M1 polarization in macrophages.

BACKGROUND: SMYD3 (protein 3 containing SET and MYND structural domains) belongs to the SMYD methylesterase family and is a histone lysine methyltransferase that promotes gene transcription mainly by catalysing the trimethylation of lysine at position 4 of histone subunit 3 (H3K4me3). Studies have shown that SMYD3 plays a key role in tumour cell proliferation and differentiation; however, its role in macrophage polarization is unclear. METHODS: We screened the M1- and M2-polarized macrophage differential histone modifying enzyme using bioinformatics analysis. The SMYD3 overexpression plasmid was transfected into M1 macrophages, and the SMYD3-regulated target gene was analysed by RNA-seq and ChIP-Seq. The effect of knocking down MTHFD1L on M1 polarization and the change of the intracellular metabolite formic acid content were investigated. M1 macrophages were stimulated with different concentrations of formic acid (2, 10 and 40 mM) to detect the expression of M1-related genes, ROS production, and changes in the expression of the mitophagy-related proteins LC3, PINK1 and p-Parkin. RESULTS: Here, we used bioinformatics to analyse SMYD3, a histone methyltransferase associated with M1 polarization; overexpression of SMYD3 significantly suppressed the LPS/IFN-γ-induced M1 phenotype in macrophages. RNA-seq analysis demonstrated that SMYD3 significantly activated the one-carbon folate metabolic pathway in M1 macrophages. In addition, we used ChIP-seq analysis to identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) as the target gene of the transcriptional activation by SMYD3 through H3K4me3 histone modification. Activation of MTHFD1L causes the accumulation of the intracellular metabolite formate. Exogenous stimulation with different concentrations of formate increased the expression of key genes involved in the mitochondrial respiratory chain complex, ROS production, and the expression of autophagy-related proteins LC3, PINK1, and p-Parkin and suppressed the expression of M1-related genes. CONCLUSIONS: Our study demonstrates that SMYD3 regulates the activity of the mitochondrial metabolic enzyme MTHFD1L through H3K4me3 histone methylation modification, promotes formate synthesis and induces mitophagy, which inhibits M1 polarization in macrophages.