Nitric oxide (NO) donors have been shown to stimulate and inhibit the proliferation, migration, and differentiation of endothelial cellsin vitroand angiogenesisin vivo. Recently, we have shown distinct thresholds for NO to regulate p53-Ser-15P, phosphorylated extracellular signal-regulated kinase (pERK), and hypoxia inducible factor 1α in tumor cells. Because these signaling pathways also promote the growth and survival of endothelial cells, we examined their roles in angiogenic responses of venous endothelial cells and vascular outgrowth of muscle explants elicited by NO. An additional protein involved in the regulation of angiogenesis is thrombospondin-1 (TSP1), a matricellular glycoprotein known to influence adhesion, migration, and proliferation of endothelial cells. Here we demonstrate a triphasic regulation of TSP1 mediated by a slow and prolonged release of NO that depends on ERK phosphorylation. Under conditions of 5% serum, a 24-h exposure of NO donor (0.1–1,000 μM) mediated a triphasic response in the expression of TSP1 protein: decreasing at 0.1 μM, rebounding at 100 μM, and decreasing again at 1,000 μM. Under the same conditions, we observed a dose-dependent increase in P53 phosphorylation and inverse biphasic responses of pERK and mitogen-activated protein kinase phosphatase-1. Both the growth-stimulating activity of low-dose NO for endothelial cells and suppression of TSP1 expression were ERK-dependent. Conversely, exogenous TSP1 suppressed NO-mediated pERK. These results suggest that dose-dependent positive- and negative-feedback loops exist between NO and TSP1. Limiting TSP1 expression by positive feedback through the ERK mitogen-activated protein kinase pathway may facilitate switching to a proangiogenic state at low doses of NO.

Control of cellular metabolism is critical for efficient cell function, although little is known about the interplay between cell subset–specific metabolites in situ, especially in the tumor setting. Here, we determined how a macrophage-specific (Mϕ-specific) metabolite, itaconic acid, can regulate tumor progression in the peritoneum. We show that peritoneal tumors (B16 melanoma or ID8 ovarian carcinoma) elicited a fatty acid oxidation–mediated increase in oxidative phosphorylation (OXPHOS) and glycolysis in peritoneal tissue–resident macrophages (pResMϕ). Unbiased metabolomics identified itaconic acid, the product of immune-responsive gene 1–mediated (Irg1-mediated) catabolism of mitochondrial cis-aconitate, among the most highly upregulated metabolites in pResMϕ of tumor-bearing mice. Administration of lentivirally encoded Irg1 shRNA significantly reduced peritoneal tumors. This resulted in reductions in OXPHOS and OXPHOS-driven production of ROS in pResMϕ and ROS-mediated MAPK activation in tumor cells. Our findings demonstrate that tumors profoundly alter pResMϕ metabolism, leading to the production of itaconic acid, which potentiates tumor growth. Monocytes isolated from ovarian carcinoma patients’ ascites fluid expressed significantly elevated levels of IRG1. Therefore, IRG1 in pResMϕ represents a potential therapeutic target for peritoneal tumors.

The dual or biphasic responses of cancer to nitric oxide (NO) arise from its concentration dependent ability to regulate tumor growth, migration, invasion, survival, angiogenesis, and metastasis. The outcome of these various NO-dependent processes is dictated by several factors including NO flux, the chemical redox environment, and the duration of NO exposure. Further, it was recently discovered that an NO-induced redox flux in vascular endothelial cells hypersensitizes these cells to the antiangiogenic effects of thrombospondin-1. This suggests a novel treatment paradigm for targeting tumor-driven angiogenesis that combines redox modulation with mimetic derivatives of thrombospondin-1. This article discusses the biphasic nature of NO in cancer biology and the implications of NO-driven redox flux for modulation of tumor-stimulated angiogenesis, growth, and metastasis.

Matrix metalloproteinases (MMPs) are of central importance in the proteolytic remodeling of matrix and the generation of biologically active molecules. MMPs are distinguished by a conserved catalytic domain containing a zinc ion, as well as a prodomain that regulates enzyme activation by modulation of a cysteine residue within that domain. Because nitric oxide (NO) and derived reactive nitrogen species target zinc ions and cysteine thiols, we assessed the ability of NO to regulate MMPs. A dose-dependent, biphasic regulatory effect of NO on the activity of MMPs (MMP-9, -1, and -13) secreted from murine macrophages was observed. Low exogenous NO perturbed MMP/tissue inhibitor of metalloproteinase (TIMP)-1 levels by enhancing MMP activity and suppressing the endogenous inhibitor TIMP-1. This was cGMP-dependent, as confirmed by the cGMP analog 8-bromo-cGMP, as well as by the NO–soluble guanylyl cyclase–cGMP signaling inhibitor thrombospondin-1. Exposure of purified latent MMP-9 to exogenous NO demonstrated a concentration-dependent activation and inactivation of the enzyme, which occurred at higher NO flux. These chemical reactions occurred at concentrations similar to that of activated macrophages. Importantly, these results suggest that NO regulation of MMP-9 secreted from macrophages may occur chemically by reactive nitrogen species-mediated protein modification, biologically through soluble guanylyl-cyclase-dependent modulation of the MMP-9/TIMP-1 balance, or proteolytically through regulation of MMP-1 and -13, which can cleave the prodomain of MMP-9. Furthermore, when applied in a wound model, conditioned media exhibiting peak MMP activity increased vascular cell migration that was MMP-9-dependent, suggesting that MMP-9 is a key physiologic mediator of the effects of NO in this model.

Introduction: The Ets-1 transcription factor is a candidate breast cancer oncogene that regulates the expression of genes involved in tumor progression and metastasis. Ets-1 signaling has also been linked to the development of a basal-like breast cancer phenotype. We recently described a nitric oxide (NO)-induced gene signature that is associated with poor disease outcome in estrogen receptor-negative (ER-) breast cancer and contains both stem cell-like and basal-like components. Thus, we examined the role of Ets-1 in NO signaling and NO-induced phenotypes in ER- human breast cancer cells. Methods: Promoter region analyses were performed on genes upregulated in inducible nitric oxide synthase (NOS2) high expressing tumors for Ets-binding sites. In vitro mechanisms were examined in human basal-like breast cancer cells lines. NO signaling effects were studied using either forced NOS2 expression or the use of a chemical NO-donor, diethlylenetriamine NONOate (DETANO). Results: Promoter region analysis of genes that are up-regulated in human ER-negative breast tumors with high NOS2 expression revealed that the Ets-binding sequence is the only common promoter element present in all of these genes, indicating that Ets-1 is the key transcriptional factor down-stream of oncogenic NOS2-signaling. Accordingly, both forced NOS2 over-expression and exposure to NO-donors resulted in significant Ets-1 transcriptional activation in ER- breast cancer cells. Functional studies showed that NO activated Ets-1 transcriptional activity via a Ras/MEK/ERK signaling pathway by a mechanism that involved Ras S-nitrosylation. RNA knock-down of Ets-1 suppressed NO-induced expression of selected basal-like breast cancer markers such as P-cadherin, S100A8, IL-8 and αβ-crystallin. Additionally, Ets-1 knock-down reduced NO-mediated cellular proliferation, matrix metalloproteinase and cathepsin B activities, as well as matrigel invasion. Conclusions: These data show that Ets-1 is a key transcriptional mediator of oncogenic NO signaling that promotes the development of an aggressive disease phenotype in ER- breast cancer in an Ets-1 and Ras-dependent manner, providing novel clues of how NOS2 expression in human breast tumors is functionally linked to poor patient survival.