Nitric Oxide, a Mediator of Inflammation, Regulates Tumorigenesis

Hussain SP, Trivers GE, Hofseth LJ, He P, Shaikh I, Mechanic LE, Doja S, Jiang W, Subleski J, Shorts L, Haines D, Laubach VE, Wiltrout RH, Djurickovic D, and Harris CC. Nitric oxide, a mediator of inflammation, suppresses tumorigenesis. Cancer Res 64: 6849–53, 2004.

itric oxide (NO·) is a major signaling molecule with diverse physiological functions that include neurotransmission, vasodilation, immune regulation, and host defense against pathogenic microorganisms. Intriguingly, recent evidence has implicated NO· in the development of human cancer, which can be explained by the discoveries linking NO· with the critical cellular processes that maintain genetic stability (e.g., apoptosis, DNA repair, and the cell cycle). However, the ultimate outcome of NO· action depends on its concentration, redox status, the type of cells it is affecting, and the availability of metals in those cells (Hussain SP et al. Nat Rev Cancer 3: 276–85, 2003). One of the most interesting discoveries about the p53 tumor suppressor protein is that it transrepresses the inducible form of nitric oxide synthase (NOS2), which further connects NO· regulation with tumorigenesis. NOS2 catalyzes the conversion of arginine to citrulline and produces micromolar quantities of NO· for a prolonged period, unlike other members of the NOS family (e.g., NOS1 and NOS3). We earlier showed that p53-deficient mice have a higher expression of NOS2 and produce a higher level of NO· when compared with p53 wild-type mice.

We used a genetic strategy to determine if NO· and p53 cooperatively regulate tumorigenesis in p53-deficient mice, an animal model of cancer-prone Li-Fraumeni syndrome. We generated mice that were deficient in both p53 and NOS2 and also eight other combinations. To make a valid comparison, these mice were crossbred to be more than 99% C57BL6. Lymphomas developed more rapidly in TP53–/–NOS2–/– or TP53–/–NOS2+/– mice than in TP53–/–NOS2+/+ mice. Likewise, sarcoma and lymphomas developed faster in TP53+/–NOS2–/– or TP53+/–NOS2+/– than in TP53+/–NOS2+/+ mice. These observations suggest that NO· suppresses tumorigenesis in this model, but how? The balance of cellular proliferation and cell death can influence tumor development, and NO· is involved in both of these processes. Therefore, we asked whether there are differences in apoptosis and proliferation in mice with different NOS2 status. When we compared double-knockout mice, we found that TP53–/–NOS2+/+ mice showed a higher apoptotic index and a decreased proliferation index with an increased expression of the death receptor ligands, CD95 and TRAIL, and the cell cycle checkpoint protein, p21waf1, in the spleen and thymus prior to tumor development.

The role of NO· in immune modulation argues in favor of a different immune profile in mice deficient in both p53 and NOS2 when compared with p53-deficient mice. We found that TP53–/–NOS2–/– mice produced a four-fold higher level of anti-inflammatory interleukin 10 (IL-10), when compared with TP53–/– mice. The increased level of IL-10 may also contribute to the rapid tumor development in the p53 and NOS2 double-knockout mice. IL-10 favors the type 2 T-helper cell (TH2) response and induces antigen-activated CD4+ cells to become CD4+/CD25+ T-regulatory cells. T-regulatory cells can also produce IL-10 and are potently immunosuppressive. IL-10 also inhibits the maturation of dendritic cells. Therefore, the loss of NO·-related TH1-antitumor response and IL-10–mediated inhibition of specific immune recognition by impairment of dendritic cell function and suppression of immune function through T-regulatory cells may enhance tumorigenesis. Therefore, we propose two models: a cell cycle arrest and increased apoptosis model, and an immune suppression model for NO·-mediated suppression of tumorigenesis (Figure 1). We are currently testing the hypothesis that IL-10 accelerates tumor development in TP53 knockout mice by generating TP53/IL-10/NOS2 triple-knockout mice. Depending on their intestinal microbial flora, IL-10 mice can develop inflammatory colitis and colon cancer. TP53/IL-10/NOS2 triple-knockout mice may have increased tumor latency of lymphoma, leukemia, and sarcoma, but decreased tumor latency and increased incidence of colon cancer as compared with TP53–/–NOS2–/– mice. However, the question remains of how does NO· regulate IL-10 production? We are currently investigating the possible mechanisms that might be responsible for NO·-mediated reduction of IL-10 in TP53-deficient mice.

Figure 1. Mechanistic models of nitric oxide (NO·)–mediated inhibition of tumorigenesis in p53-deficient mice.
A) NO·-mediated regulation of cellular proliferation and apoptosis in p53-deficient mice. Elevated levels of NO· production in p53-deficient mice with wild-type nitric oxide synthase 2 (NOS2) increase the expressions of the death receptor ligands, TRAIL and CD95-L (Fas-L), and the cell cycle checkpoint protein, p21waf1, to decrease tumorigenicity. B) Immune suppression model: The loss of NO·-related type 1 T-helper cell (TH1)–antitumor response and interleukin 10 (IL-10)–mediated inhibition of specific immune recognition and suppression of immune function can enhance tumorigenesis in mice deficient in both p53 and NOS2. Th2, type 2 T-helper cell; T-Reg, T-regulatory cells.

Is NO·-mediated regulation of tumorigenesis in p53-deficient mice altered under an inflammatory microenvironment? We have preliminary evidence that higher levels of NO·, produced by treating these mice with heat-inactivated Corynebacterium parvum, a potent inflammatory agent, increase spontaneous tumorigenesis. Because the p53-deficient mice primarily develop spontaneous lymphoma, leukemia, sarcoma, and rarely carcinoma, future studies to investigate the role of NO· in the modulation of carcinoma are warranted.

S. Perwez Hussain, PhD
Staff Scientist
hussainp@mail.nih.gov

Curtis C. Harris, MD
Chief, Laboratory of Human Carcinogenesis
NCI-Bethesda, Bldg. 37/Rm. 3068
Tel: 301-496-2048
Fax: 301-496-0497
curtis_harris@nih.gov