The Role of Inflammation and Nitric Oxide Synthase 2 in Triple-Negative Breast Cancer Metastasis

Abstract

High expression of inflammatory marker nitric oxide synthase 2 (NOS2) expression in tumor cells indicates poor prognosis, poor survival, and chemotherapeutic resistance in triple-negative breast cancer (TNBC) patients. The tumor microenvironment (TME) comprises various cell types that play a role in tumor survival and invasion by providing soluble factors such as pro-inflammatory cytokines IFN-γ, IL-1β, and TNF-α, which are known to drive NOS2 expression. NOS2 is an enzyme that generates nitric oxide (NO), which is suggested to drive tumor progression via increased tumor cell motility and invasion. This dissertation investigates a novel role for NOS2/NO in TNBC, focusing on the impact of NOS2 expression in tumor cells. By optimizing a hemoglobin-based NO detection assay, we determined that TNBC cell line produce NO at levels comparable to that of the RAW 264.7 macrophage cell line, reaching up to 1.3 pmol NO/min/g total cellular protein. We found that CRISPR/Cas9-mediated knockout of the NOS2 gene in the 4T1 parental cell line significantly suppresses lung metastasis from primary 4T1 tumors in Balb/c mice, providing direct evidence that inhibition of NOS2 in tumor cells can suppress metastatic progression in TNBC. We used flow cytometry to immune profile the primary orthotopic 4T1 mammary tumors, confirming the presence of inflammatory cytokine expression in the TME. Our findings suggest that tumoral NOS2/NO modulates the TME by promoting the polarization of tumor-associated macrophages (TAMs) toward a pro-tumorigenic and immunosuppressive M2-like phenotype. We also found that cytokine-induced NOS2/NO enhanced m6A-mRNA methylation, suggesting a potential mechanism by which NOS2/NO dysregulates the epitranscriptomic process by inhibiting demethylase FTO, thereby contributing to the metastatic progression of TNBC. These findings indicate that NOS2/NO is a key regulator of TNBC metastasis, thereby making it a promising therapeutic target for preventing the spread of this aggressive breast cancer subtype.