Nitric Oxide Synthase 2-Dependent Metabolic Dysregulation, Immunosuppression, and Genomic Instability in Metastatic Triple-Negative Breast Cancer

Chronic inflammation drives many diseases, including cancer, where inflammation is associated with metastasis, the cause of death in 90% of cancer fatalities. Tumor inflammation drives inducible nitric oxide synthase (NOS2 or iNOS) and cyclooxygenase 2 (COX2) expression, each of which is associated with poor outcomes in cancer. Here, we knocked out the NOS2 gene in the murine triple negative breast cancer allograft tumor model 4T1 and examined metastatic spread from primary mammary tumors to lung in BALB/c mice with intact immune systems. Remarkably, while the parental 4T1 tumors were highly metastatic, metastasis from 4T1 NOS2-/- tumors was nearly eliminated. In cell culture, we find that nitric oxide from NOS2 induces a glycolytic phenotype, epitrascriptomic dysregulation, DNA damage, and prostaglandin E2 synthesis by stimulating COX2. Human colorectal cancer cell line DLD1 yielded similar results. RNA sequencing revealed NOS2-dependent loss of mRNA associated with regulation of chromosome and DNA replication, including cell cycle checkpoints, as well as mRNA associated with RNA demethylation, including tRNA demethylation. Immunoprofiling of the tumor microenvironment revealed immunosuppressed tumors that were low in T cells and high in myeloid derived suppressor cells expressing interferon gamma, which induces NOS2 expression. Others have shown nitric oxide from immune cells suppresses tumor immunity. Here, we show nitric oxide from tumor cells plays a critical role in metastasis. Taken together, these data suggest high NOS2 in tumor cells drives metastasis through a broad accumulation of cancer-associated factors – including metabolic dysregulation, genetic instability, and immunosuppression – all of which contribute to aggressive metastasis.