In this study, we examined whether NOS2 influences breast cancer survival and investigated mechanisms by which NOS2 and NO may cause a poor outcome phenotype. The research led to the clinically significant observation that NOS2 expression is associated with a prognostic basal-like transcription pattern and is a predictor of inferior survival in women with ER-negative tumors. Moreover, the presence of NOS2 correlated with other poor outcome markers, such as an increased microvessel density and p53 mutation frequency as well as activated EGFR. Additional work found that NO induces basal-like genes and IL-8 only in ER-negative breast cancer cells. Some of these genes that were found to be induced by NO, such as CD44 and c-Myc, have been linked to an embryonic stem cell–like phenotype in breast cancer that is enriched in basal-like breast tumors (39
), raising the possibility that the expression of NOS2 and increased NO release into the tumor microenvironment lead to poorly differentiated and aggressive tumors with a distinct gene signature, enhanced angiogenesis, and the inactivation of the p53 tumor suppressor gene.
Previous reports indicated that elevated NOS2 expression may be linked to a high grade and poor prognosis in breast cancer (12
), and also to poor outcome in other human epithelial cancers. In addition, mammary tumor latency is increased in NOS2-knockout mice (43
). Aberrant expression of NOS2 has been observed in human breast (12
), colon (14
), stomach (44
), non–small cell lung (23
), esophageal (46
), and squamous cell head and neck cancer (47
). In the two breast cancer studies, increased NOS2 correlated with tumor grade (12
) and tumor angiogenesis and p53 protein accumulation (13
). Our observations are consistent with these reports. NO has proangiogenic activities (48
) and also promotes carcinogenesis through the inactivation of wild-type p53 function, by either causing loss of DNA-binding activity (51
) and/or selecting for mutant p53 (26
). Thus, we also evaluated in the current study to what extent tumor grade, tumor p53 status, and tumor microvessel density may influence the association of NOS2 with survival among the ER-negative breast cancer patients. This analysis showed that high NOS2 is the single most significant predictor among them and predicts poor outcome of ER-negative breast cancer patients largely independent of these 3 factors (Supplemental Figure 7). Two other studies of breast cancer analyzed NOS2 expression in tumors and examined the relationship between NOS2 and patient outcome (41
). In agreement with our findings and the study by Vakkala et al. (13
), NOS2 was found to be commonly expressed by the cancer cells themselves. However, the two studies were relatively small-sized, with mainly ER-positive patients, which did not allow an independent assessment of NOS2 in ER-negative breast cancer. Both studies found a significant association of NOS2 with high-grade tumors, as we did in the present study, and a borderline association of high NOS2 with decreased survival in the univariate but not multivariable analysis. From their results, it appears that NOS2 expression may have some prognostic value in ER-positive breast tumors, although our study did not find this.
In an effort to determine why high NOS2 expression is associated with poor survival in ER-negative breast cancer, we performed a gene expression analysis of microdissected breast tumor epithelium and also assayed phenotypes of ER-negative breast cells that were exposed to NO. These approaches led to the discovery of a distinct and robust basal-like gene expression signature for high NOS2 in ER-negative tumor epithelium and NO-induced phenotypic alterations in ER-negative cells that indicated increased aggressiveness. Moreover, the NOS2 signature per se is prognostic, as found by us with the analysis of an independent sample set with gene expression data from 77 ER-negative breast tumors (32
). An oncogenic effect of NO in the ER-negative MDA-MB-231 breast cancer cell line has been observed by others (21
), consistent with a NO-induced poor outcome phenotype and our data. The global gene expression analysis did not find a major influence of NOS2 status on gene expression in ER-positive tumors, and furthermore, NO did not induce an aggressive phenotype in ER-positive breast cancer cells, as indicated by the cell migration and invasion results.
It is likely that both the difference in ER expression and intrinsic differences between ER-negative and ER-positive breast cancer cells independent of the tumor ER status contribute to this diminished responsiveness to NO in ER-positive cancer cells. We found that ER expression inhibited NO-induced upregulation of the stem cell marker CD44, and also S100A8 and P-cadherin, but not of IL-8. IL-8 is preferentially expressed by ER-negative breast tumors and breast cancer cell lines (34
). Atypical methylation of the IL8
gene is a candidate mechanism for the differential expression of IL-8 in breast tumors (52
). Regulation of IL-8 expression by NO has previously been observed in pancreatic cancer cells, human monocytes, and lung epithelial cells (53
). IL-8 is of particular importance in breast tumor biology and is associated with increased breast cancer cell invasion, neutrophil infiltration, and microvessel density (35
). IL-8 mediates metastasis in breast cancer and other cancers, and its secretion correlates with early dissemination and poor survival (56
). Moreover, it has been shown that IL-8 supports breast cancer stem cell renewal and invasion (57
). Our findings suggest that NOS2 may lead to poor survival among ER-negative patients partly because it induces IL-8 selectively in tumors of these patients and not in ER-positive tumors, leading to increased stem cell renewal, cell invasion, angiogenesis, and metastasis. This hypothesis is supported by our two findings that NO induces IL-8 only in ER-negative breast cancer cells and that IL-8 expression is increased in ER-negative tumors with high NOS2 but not in ER-positive tumors with high NOS2. Whether ER expression regulates IL-8 levels in breast cancer cells remains controversial. We did not find that IL-8 secretion is influenced by transient ER expression in ER-negative cells. In contrast, it was reported that an MDA-MB-231 clone that constitutively expressed an ER transgene had lower IL-8 production than the parent cell line (35
). However, the same authors did not find that estrogen influenced IL-8 expression in the ER-expressing cells, arguing that perhaps clonal selection rather than ER expression may have contributed to this finding.
Alternatively, NOS2 may not produce NO as efficiently in ER-positive tumors as in ER-negative tumors, or NO signaling may occur differentially in ER-positive and ER-negative tumors because of differences in the tumor microenvironment. Effects of estrogen on NOS activity have been described; however, these appear to be mainly restricted to the endothelial isoform. Estrogen has been found to alter NOS2 expression in murine macrophages, splenocytes, and vascular smooth muscle cells by mechanisms involving ER (58
), but because of the known differences in the promoter regulation of human and murine NOS2 (61
), we cannot be sure that estrogen would have similar effects on human NOS2. Differences in the tumor microenvironment between ER-positive and ER-negative tumors have been observed that may affect NO signaling. ER-negative tumors tend to have more tumor-associated macrophages than ER-positive tumors (62
), and many proinflammatory cytokines are expressed at a higher level in ER-negative tumors than ER-positive tumors (63
). Macrophages and these cytokines will alter the tumor microenvironment and may lead to a proinflammatory state. Thus, the NO biochemistry and signaling, which are greatly influenced by reactive oxygen species, may be different in ER-negative and ER-positive breast cancer.
The gene signature induced by NOS2 in ER-negative breast tumors showed some striking similarities to two gene signatures associated with basal-like breast cancer (1
). Basal-like breast cancer is an ER-negative breast cancer subtype with an aggressive phenotype and limited therapy options (1
). Our evaluation of NOS2 in basal-like tumors indicated that its expression is associated with poor outcome. This suggests that NOS2 is a novel candidate prognostic marker for basal-like breast cancer and possibly a therapeutic target, whose activity could be selectively inhibited. Recently, increased expression of stem cell markers in basal-like tumors has been reported (40
). Among them was CD44, which is the major receptor for hyaluronan. Expression of CD44 is a poor outcome marker in breast cancer (65
), and CD44-positive breast cancer cells exhibit increased invasive properties (66
), increased resistance to radiotherapy (67
), and increased resistance to chemotherapeutics (68
). We observed that CD44 protein is induced by NO in ER-negative breast cancer cells and its gene expression was significantly elevated in ER-negative tumors with high NOS2. This observation links NOS2 and increased NO production to the development of a poorly differentiated breast cancer phenotype with stem cell–like characteristics. NO may induce this phenotype by activation of transcription factors, such as c-Myc, or by inducing the release of stem cell renewal factors like IL-8 (57
), a property that NO has, as we have shown in this study. To gain an understanding of what may drive the NOS2 gene signature in ER-negative tumors, we used a bioinformatics tool to identify candidate factors that would induce these genes. c-Myc advanced as the top candidate from this analysis. Because of this finding, and because c-Myc is a part of an embryonic stem cell–like gene expression signature in breast cancer (39
), we tested whether NO induces this transcription factor in ER-negative breast cancer cells. Indeed, NO upregulated c-Myc in MDA-MB-468 cells, which have basal-like characteristics. We consider this finding as preliminary evidence that NO may induce the basal-like signature in ER-negative breast tumors in part by upregulation of c-Myc.
EGFR is one of the signature genes in basal-like breast cancer (31
). NO has previously been shown to activate EGFR in lung cancer cells (69
). Furthermore, nuclear EGFR signaling has been shown to lead to upregulation of NOS2 in breast cancer cells (70
). Because of the association of the NOS2 signature with the basal-like gene expression signature and the role of the EGFR in basal-like breast cancer, we explored the hypothesis that NO may activate this receptor through phosphorylation in EGFR-positive MDA-MB-468 cells and breast tumors. This work led to the finding that NO induces EGFR phosphorylation at Tyr1045 and Tyr1173 in breast cancer cells and of a significant association between EGFR phosphorylation and NOS2 expression in breast tumors. Together, these early mechanistic data indicate that besides inducing a poor prognosis signature and enhancing angiogenesis, NOS2 may also cause a poor outcome phenotype by activating the oncogenic EGFR pathway.
In conclusion, our study provides evidence that NOS2 is predictor of survival and determinant of disease aggressiveness associated with ER-negative breast cancer, and as such is a candidate target for therapy. The underlying mechanisms that lead to an NO-induced poor outcome phenotype may include a combination of events, such as the induction of a basal-like phenotype, activation of the EGFR pathway, increased angiogenesis, and selection for mutant p53 cells. We propose that selective NOS2 inhibitors may be particularly efficacious in patients with basal-like breast cancer.