Enhanced nitric oxide synthase 2 (NOS2) expression correlates with poor patient survival in invasive triple-negative breast cancer (TNBC). (A) Higher NOS2 mRNA expression in invasive TNBC versus non-TNBC (P = 3.85 × 10−5). (B) High NOS2 expression correlates with death at 5 years in invasive breast carcinoma (P = 0.037). Kaplan-Meier survival analyses in (C) Van de Vijver (n = 69; P = 0.04) and (D) Curtis (n = 260; P = 0.01) breast databases show that high NOS2 expression correlates with worse overall survival of patients with TNBC. (E) Immunohistochemical analysis of TNBC human samples for inducible nitric oxide synthase (iNOS) protein expression. Weak-to-moderate (3 or 4), moderate-to-strong (5 or 6), and strong (7) were the cutoffs established for further analysis of survival. Several samples showed expression in both tumor (T) and stromal (S) cells (original optical objective: 20×). MDA-MB-231 cells transfected with either NOS2-directed shRNA (shRNA1) or empty vector (EV) were used as negative and positive control of iNOS staining, respectively (original optical objective: 10×). Counterstain: hematoxylin. (F) Increased iNOS expression is associated with less patient survival when compared with low iNOS expression. Kaplan-Meier survival analysis of TNBC human patient samples (n = 83) (P = 0.05). shRNA1, small hairpin RNA 1; TCGA, The Cancer Genome Atlas.