Nitric oxide and protein phosphatase 2 A provide novel therapeutic opportunities in ER-negative breast cancer

Basal-like breast cancer is an aggressive disease with limited therapeutic options because these tumors frequently express the ‘triple-negative’ phenotype. We have recently reported that inducible nitric oxide synthase (NOS2) is a strong predictor of survival in patients with estrogen receptor negative [ER(−)] breast cancer, and that NOS2 expression is correlated with a basal-like phenotype. Recent reports also describe the pro-tumor effects of NO in breast and many other types of cancer. NO promotes cancer progression by activating several oncogenic signaling pathways such as extracellular signal-regulated kinases (ERK)-1/2, phosphoinositide 3-kinases (PI3K)/Akt, and c-Myc. Protein phosphatase 2A (PP2A) is a tumor suppressor that negatively regulates the same cancer-related signaling pathways that are activated by NO. PP2A activity is suppressed in tumor cells, but potential pharmacological agents have recently been described to increase PP2A activity in ER(−) breast cancer cells. We examine here the various functions of NO and PP2A in breast cancer and propose a novel mechanism by which activation of PP2A antagonizes NO signaling that promotes ER(−) breast cancer. NO signaling in breast cancer The role of NO in cancer was one of the earliest biological effects described for this endogenously synthesized, unique signaling molecule [1]. Interestingly, NO has both tumor suppressing and tumor promoting effects [2]. For example, NO is essential in the macrophage-mediated eradication of leukemia cells and activates p53-mediated apoptosis [1,3]. p53 is a tumor suppressor protein that is activated by multiple cellular stresses and possesses multiple anti-cancer functions such as inducing cell apoptosis, cell-cycle arrest and activating DNA repair enzymes [4]. By contrast, NO increases genotoxicity and inhibits DNA repair enzymes [1,3]. Endogenous production of NO is catalyzed by a family of NOS enzymes, and the inducible isoform (NOS2) is associated with immune cell function and inflammation [5]. NOS2 overexpression in tumor cells is generally associated with increased proliferation and migration. For example, NOS2 expression promotes colon and breast cancer cell growth and invasiveness [3]. The relationship between NOS2 and p53 provides insight into the divergent properties of NO in cancer [6]. Wild-type p53 antagonizes NOS2 via two mechanisms. p53 binds to the Corresponding author: Wink, D.A. (wink@mail.nih.gov). NIH Public Access Author Manuscript Trends Pharmacol Sci. Author manuscript; available in PMC 2012 November 01. Published in final edited form as: Trends Pharmacol Sci. 2011 November ; 32(11): 644–651. doi:10.1016/j.tips.2011.07.001. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript TATA-binding element in the promoter region of the NOS2 gene to inhibit expression [7]. Furthermore, p53 binds directly to the NOS2 protein, which attenuates NOS activity [8]. By contrast, NO stabilizes p53, leading to apoptosis [9,10]. NO-mediated apoptosis of leukemia cells requires wild-type p53, because NO does not induce apoptosis in p53 null cells [11]. In addition, p53 mutant carcinoma cells expressing NOS2 have accelerated tumor growth and increased vascular endothelial growth factor (VEGF) production compared to wild-type p53 tumors [12], suggesting that p53 status plays a significant role in determining the proor anti-tumorigenic role of NO. These studies revealed an important relationship between p53 status, NO, and seemingly disparate cancer outcomes. NOS2 has been shown to have a role in a wide variety of cancers. High NOS2 expression is associated with favorable prognoses in ovarian [13] and lung cancers [14]. By contrast, many clinical studies have correlated elevated NOS2 expression with poor patient survival in various cancers, including breast, colon, gastric, esophageal, prostate, cervical, squamous cell carcinoma, hepatocellular carcinoma, melanoma, ovarian and leukemia [3]. Moreover, these findings are often linked with other inflammatory biomarkers. For example, NOS2 and COX-2 were associated with increased microvessel density in non-small-cell lung cancer [15]. Therefore, NOS2 is emerging as a biomarker of poor cancer patient prognosis (i.e. decreased patient survival). In this review we first discuss the clinical relevance of NOS2 expression and the pro-tumor signaling effects of NO. Because NOS2 expression is strongly associated with poor patient survival for many cancer types, it is a potential therapeutic target. We discuss how NO activates multiple signaling pathways associated with tumor progression and metastasis. Then we briefly review the tumor suppressor protein phosphatase 2A (PP2A), which negatively regulates the same signaling pathways that are activated by NO. Evidence is growing for the potential of PP2A agonists as a therapeutic approach that could counteract the pro-tumor effects of NO signaling (Figure 1). Clinical markers of NOS2 in breast cancer NO signaling is emerging as a key factor that enhances breast cancer aggressiveness. An early report showed that NOS2 expression is significantly associated with a high tumor grade in invasive ductal carcinoma patients [16]. More recent studies revealed that NOS2 is expressed in about 70% of all breast tumors, and that NOS2 expression correlates with markers of poor prognosis such nuclear epidermal growth factor receptor (EGFR) and STAT3 (signal transducer and activator of transcription 3), phospho-Akt and phosphoSTAT3 [17-19]. Increased NOS2 expression is significantly associated with phospho-Akt in human breast tumors regardless of breast cancer subtype, indicating that NO is clinically associated with increased Akt activation, consistent with cell-culture-based data [17]. This suggests that oncogenic pathways associated with Akt signaling are probably activated by NOS2. Recently NOS2 was shown to be a novel prognostic marker in ER(−) breast cancer patients [19] (Box 1). The association between high NOS2 expression and decreased ER(−) patient survival is remarkably strong (hazard ratio of 6.19 for survival after five years), suggesting that NOS2 expression is an important biomarker for ER(−) diseases [19]. Furthermore, high NOS2 expression is correlated with decreased survival among patients with basal-like breast cancer, another form of aggressive disease (Box 1) [19]. The NOS2 association with prognosis is independent of other predictive factors, such as tumor grade, lymph node involvement, and degree of metastasis, suggesting that early induction of metastasis is perhaps favored by proinflammatory NOS2 overexpression [19]. In addition, a recent report indicates that cyclooxygenase 2 (COX-2) is also a predictor of poor survival among ER(−) patients [20]. It is possible that the activities of NOS2 and COX-2 synergize to form a Switzer et al. Page 2 Trends Pharmacol Sci. Author manuscript; available in PMC 2012 November 01. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript tumor-induced inflammatory response that results in an aggressive tumor phenotype in ER(−) breast cancer patients. A signature set of 44 genes is associated with NOS2 overexpression, and 15 of these upregulated genes are well-established markers of basal-like breast cancer, such as Pcadherin, cytokeratins, Wnt5A, and KLF5 [19]. Furthermore, these basal-like markers are individually associated with aggressiveness and poor survival in patients. For example, Wnt5A has recently been shown to be associated with brain metastasis [21]. High KLF5 expression predicts shorter disease-free survival and overall survival than breast cancer patients with lower KLF5 expression [22]. KLF5 expression is higher in younger patients, consistent with triple-negative (Box 1) and/or basal-like breast cancer [22]. Other genes associated with NOS2 expression, such as CD44 and inflammatory molecules such as IL-6, IL-8 and S100A8 are associated with metastasis and poor outcome [19]. Therefore, the function of NOS2 as a predictor of ER(−) patient survival is perhaps mediated by NOdependent induction of these markers in ER(−) breast tumors. Although NOS2 expression is correlated with poor clinical outcomes in ER(−) breast cancers, it is possible that NO is not the NOS2 end-product that elicits the malignant phenotype. All three isoforms of NOS have been reported to produce superoxide under conditions of substrate (argi-nine) or cofactor (tetrahydrobiopterin) depletion [23]; therefore it is possible that superoxide generation can contribute to the pro-tumor environment provided by NOS2 expression. Although superoxide has cancer-promoting signaling effects [24], it does not account for the wide array of signaling effects seen with both NOS2 activity and exogenous NO-donor compounds described below. However, this alternative role of NOS2 could increase the tumorigenic effects of NOS2. NO activates multiple pro-tumor pathways NO signaling in breast cancer results in altered expression of many genes implicated in breast cancer proliferation, cellular invasion, tumor angiogenesis and metastasis. Thus, NO signaling affects multiple signaling pathways. For example, NO signaling in breast cancer cells is known to activate many cancer-related pathways, and NO increases hypoxia inducible factor (HIF), ERK-1/2 and phosphatidylinositol 3-kinase (PI3K/Akt) [25]. These signaling pathways have strong links to cancer proliferation, angiogenesis and tumor metastasis [3]. Recent reports show that discrete concentrations of NO over different time frames induce specific signal transduction pathways (Figure 2) [25]. NOS2 is capable of generating concentrations in a range from nanomolar to micromolar NO, and this range includes both cell regulatory functions and immunotoxic properties [5,25]. Here we briefly review some of the cancer-related signaling pathways associated with increasing levels of NO. Soluble guanylate cyclase Levels of NO that activate soluble guanylate cyc