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Investigating the Role of NOS2 in Breast Cancer

Nitric oxide synthase (NOS2) participates in an extensive network driving cancer progression and metastasis in ER- breast cancer. Heinecke and colleagues show that NOS2 derived nitric oxide (NO) perpetuates inflammatory loops in vivo and in vitro. The genes regulated by NO correlate with those in patient cohorts and are predictive of poor outcome. Image courtesy of Julie Heinecke and Lisa Ridnour.
Nitric oxide synthase (NOS2) participates in an extensive network driving cancer progression and metastasis in ER- breast cancer. Heinecke and colleagues show that NOS2 derived nitric oxide (NO) perpetuates inflammatory loops in vivo and in vitro. The genes regulated by NO correlate with those in patient cohorts and are predictive of poor outcome. Image courtesy of Julie Heinecke and Lisa Ridnour.

Inducible nitric oxide synthase (NOS2) is often elevated in breast tumors that lack expression of the estrogen receptor (ER) and predicts a poor prognosis for patients with these tumors. However, it is unclear whether NOS2 directly contributes to ER-negative breast cancer aggressiveness or how NOS2 expression is controlled within the tumor microenvironment. To tease apart the regulatory pathways upstream and downstream of NOS2, David Wink, Jr., Ph.D., Senior Investigator in CCR’s Radiation Biology Branch, along with colleagues from CCR’s Pediatric Oncology Branch, Laboratory of Human Carcinogenesis, and Laboratory of Experimental Immunology and from the Prostate Cancer Institute in Ireland, carried out studies in cell culture and mouse models.

First, the researchers determined the effect of inhibiting NOS2 activity with amminoguanidine (AG) on ER-negative breast tumor progression. They implanted human MDA-MB-231 (MB231) cells into the breasts of female immunodeficient mice. Half of these mice received water supplemented with AG. The investigators found that treatment with AG blunted tumor growth by 59 percent after 37 days. Likewise, exposure to AG dramatically reduced brain metastases at day 45, demonstrating that NOS2 activity drives tumor progression in this system.

Patients with ER-negative tumors and high NOS2 levels also express a collection of genes that provide important prognostic information. To determine whether inhibition of NOS2 affected expression of these genes, the scientists analyzed the tumors from their mouse model. They observed significantly reduced mRNA levels of COX2, TLR4, S100A8, CD44, IL-6, and IL-8 in the tumors from AG treated mice. Thus, blocking NOS2 limited the expression of this clinically relevant gene signature.

Since nitric oxide (NO) has been implicated in the mobility of ER-negative breast cancer cells, the researchers decided to examine the role of NO in cell migration induced by serum withdrawal, a model of nutrient deprivation in rapidly growing tumors. Monitoring MB231 cell movement from serum-free to serum-containing media in real time, they found that adding AG reduced cell motility. The addition of a low dose of the NO donor DETA/NO increased the migration of AG-treated cells to a level above that of untreated cells at 24 hours. High dose DETA/NO, however, caused sustained elevation of cell movement in the presence of AG, suggesting the importance of NO flux in the regulation of ER-negative breast cancer cell mobility.

Developing resistance to chemotherapy is another feature of aggressive cancers. To investigate the role of NOS2 in drug resistance, the scientists incubated 231 cells for 24 hours in serum-free media with or without AG or DETA/NO and determined the cells’ sensitivity to the microtubule inhibitor Taxol. When compared to cells treated with Taxol alone, serum withdrawal led to reduced Taxol-induced cell death. While the addition of AG did not affect survival, the combination of AG and low dose DETA/NO enhanced the survival of cells in serum-free media and Taxol, further implicating NO supplied by NOS2 in the progression of ER-negative breast cancer.

To understand how NOS2 expression and activity are regulated within a tumor, the investigators examined the effects of factors present in the tumor microenvironment on three human breast cancer cell lines: ER-negative MB231 and MDA-MB-468 (MB468) cells, as well as ER-positive MCF7. They found that serum withdrawal enhanced NOS2 protein expression, promoter activity, and enzyme activity in all three lines to different extents. Likewise, hypoxia promoted NOS2 protein production in all three lines but only when combined with serum withdrawal.

Cytokine stimulation, however, was more cell line dependent. Treating with a mixture of cytokines increased NOS2 protein levels in MB231 and MCF7 cells but not MDA-MB-468 (MB468) cells. Surprisingly, the mixture increased NOS2 mRNA and promoter activity in all three cell lines but with varied timing and intensity. Incubation with interferon-γ (IFN-γ) or tumor necrosis factor-α, on the other hand, enhanced NOS2 enzyme activity only in MDA-MB-468 (MB468) cells. The researchers also found that IFN-γ could increase NOS2 mRNA in MB231 cells and that IFN-γ treatment followed by serum withdrawal stimulated NOS2 protein levels and activity in these cells. Together, these results suggest that endogenous factors within the tumor microenvironment can alter NOS2 production and activity, which drive cancer progression.

Because NO is known to play both positive and negative roles in NOS2 expression, the scientists analyzed NOS2 levels in MB231 cells following IFN-γ or serum withdrawal with or without AG. They found that AG caused a slight reduction in NOS2 protein from IFN-γ but a dramatic decrease from serum withdrawal. In contrast, AG significantly reduced NOS2 mRNA from both IFN-γ and serum withdrawal. Adding back NO with high-dose DETA/NO restored NOS2 mRNA and protein levels from serum withdrawal in the presence of AG, suggesting a low level of NO flux is necessary for feed-forward NOS2 regulation in ER-negative breast cancer cells.

Finally, the researchers investigated the downstream targets of NOS2. After treating MB231 cells with IFN-γ followed by serum withdrawal, they saw enhanced mRNA expression of S100A8 and IL-6, which was blocked by addition of AG. Likewise, IFN-γ and serum withdrawal increased the protein level and nitration of TIMP1, another poor prognostic factor. AG treatment blocked TIMP1 protein induction and decreased basal levels of TLR-4 and IL-8. Adding a high dose of DETA/NO restored S100A8 mRNA levels in the AG-treated MB231 cells.

Taken together, these studies demonstrate that NOS2 is a key driver of signaling pathways in ER-negative breast that promote cancer growth, metastasis, and drug resistance. Inhibiting NOS2 activity may benefit patients with this aggressive form of cancer.

Reference
Heinecke JL, Ridnour LA, Cheng RYS, Switzer CH, Lizardo MM, Khanna C, Glynn S, Hussain SP, Young H, Ambs S, and Wink DA. Tumor Microenvironment-Based Feed-Forward Regulation of NOS2 in Breast Cancer Progression. PNAS. April 14, 2014. PubMed Link