TF/PAR2 Signaling Axis Supports the Protumor Effect of Neutrophil Extracellular Traps (NETs) on Human Breast Cancer Cells

Simple Summary Neutrophil extracellular traps (NETs) contribute to tumor progression at different stages, such as primary growth, metastasis, angiogenesis, and cancer-associated thrombosis. The knowledge of biomolecular mechanisms of this process is crucial to developing strategies to mitigate tumor progression, mainly, metastasis. NETs promote the activation of proinflammatory pathways and trigger the epithelial-mesenchymal transition (EMT) process. In this work, we demonstrate that NETs enhance the expression of tissue factor (TF) in breast tumor cells, thus increasing the procoagulant activity. NETs also promote protease-activated receptor 2 (PAR2) signaling, leading to the expression of pro-tumor cytokines and factors associated with EMT. These phenomena are supported by in silico gene correlation analysis of TF/PAR2 and pro-tumor genes analyzed in samples from breast cancer patients. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.Abstract Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.