Relatively few studies have explored the association between common genetic variations and disease outcome although the concept of gene-environment interactions strongly suggests that these inter-individual variations may influence cancer survival because of their modifying effects on tumor biology and therapeutic outcome. We examined a functional single nucleotide polymorphism (rs4880) in the manganese superoxide dismutase gene that leads to a substitution of valine by alanine (Val16Ala). Manganese superoxide dismutase is an enzyme that protects against oxidative damage and modulates the efficacy of chemotherapeutic drugs. We hypothesized that Val16Ala affects breast cancer survival of patients receiving chemotherapy. Two patient populations from the United States (n=248) and Norway (n=340) were genotyped for Val16Ala. Kaplan-Meier survival and Cox Proportional-Hazards regression analyses were used to examine the relationship between Val16Ala and disease-specific survival. We found that Val16Ala was significantly associated with breast cancer outcome in both patient populations. Carriers of the Ala allele had inferior survival rates. This association was significant for patients receiving adjuvant therapy (HR = 2.47;95% CI, 1.46-4.19), but not for patients without it (HR = 1.47;95% CI, 0.57-3.74). After further stratification by type of chemotherapy, the effect of the Ala allele was mostly restricted to cyclophosphamide-containing chemotherapy regimens (HR = 22.0;95% CI, 5.22-92.9;Ala/Ala versus Val/Val). The finding provides the first evidence pointing toward a mechanism for cyclophosphamide resistance in breast cancer patients and may have important clinical implications because 20% to 25% of the general population in the United States and Europe is carrying this genotype. Although preliminary, these data suggest that patients with the Ala/Ala genotype should be considered for alternative treatment. Inducible nitric oxide synthase (NOS2) and cyclooxygenase-2 are signature genes of the inflammation response. A major physiological role of NOS2 is the release of nitric oxide to support the wound healing process. It has been hypothesized that the wound healing properties of nitric oxide could turn NOS2 into an oncogene that promotes the metastatic spread of human cancer. Previous research has shown that NOS2 is expressed in breast tumors. We specifically examined the function of NOS2 in estrogen receptor (ER)-negative breast cancer because of the urgent need of identifying novel targets for therapy in the estrogen receptor-negative disease. Immunohistochemistry of 248 breast tumors showed that NOS2 was moderately to strongly expressed in 173 of them (70%). We next examined the effect of NOS2 expression on predicting patient survival, and how this would be affected by the tumor estrogen receptor status. While NOS2 was not associated with breast cancer survival overall, we found that the estrogen receptor status modified the association between NOS2 and breast cancer survival, with high NOS2 expression being significantly associated with poor survival in the estrogen receptor-negative disease. We further investigated why NOS2 is associated with poor survival in estrogen receptor-negative but not estrogen receptor-positive breast cancer and analyzed the gene expression profiles of 32 microdissected breast tumors. We did not find a NOS2 gene signature in estrogen receptor-positive tumors, suggesting that NOS2 may not lead to significant gene expression changes in these tumors. In contrast, we found that 49 transcripts corresponding to 44 genes were differentially expressed in estrogen receptor-negative tumors comparing NOS2 high with NOS2 low expressing tumors. Among the genes most highly up-regulated were cytokeratins 6 and 17, and P-cadherin, which are marker genes of the basal-like breast cancer phenotype. To determine whether this 44-gene signature had further links with basal-like breast cancer, we examined previously published basal-like breast cancer gene signatures for similarities. Cross referencing the NOS2/estrogen receptor-negative gene signature with these data revealed that many transcripts in the NOS2 signature have previously been identified as being associated with basal-like breast cancer. In addition, we evaluated the association of the 44-gene signature with breast cancer survival in publicly available gene expression data sets of breast cancer. This analysis showed that the 44-gene signature was significantly associated with poor outcome in those data sets. Validation experiments were conducted to further determine whether the gene expression profile of NOS2 in estrogen receptor-negative breast tumor is at least partly caused by nitric oxide and examined the effects of nitric oxide on the protein expression of four genes: interleukin-8 (IL-8), S100 calcium binding protein A8 (S100A8) and P-cadherin (CDH3), which are both markers of basal-like breast cancer, and the hyaluronate receptor (CD44), which is a marker of breast cancer stem cells. Both IL-8 and S100A8 are known poor outcome markers for breast cancer. Using the slow release nitric oxide donor, DETA/NO, we found that nitric oxide induces IL-8, S100A8, CDH-3 and CD44 protein expression in the estrogen receptor-negative breast cancer cell lines, but not in the estrogen receptor-positive cell lines, suggesting that these effects of nitric oxide in breast cancer are restricted to estrogen receptor-negative tumors. Finally, we reintroduced the estrogen receptor into the estrogen receptor-negative breast cancer cell lines and examined whether receptor expression would repress up-regulation of these proteins. A protein expression analysis showed that the receptor completely inhibited nitric oxide-mediated induction of CD44 but not of the other markers. The finding is consistent with biological effects of nitric oxide in estrogen receptor-negative cells that are independent of the estrogen receptor status. Finally, we studied the relationship between NOS2 expression and activation of the EGF receptor. This receptor is frequently expressed in basal-like breast cancers, a poor outcome subtype. Statistical analyses revealed that NOS2 expression is associated with poor outcome in basal-like breast cancer and correlates with EGF receptor phosphorylation in the tumors. Exposure of breast cancer cells to nitric oxide also lead to increased receptor phosphorylation which activates receptor signaling. In summary, inducible nitric oxide synthase expression is a marker of poor outcome in estrogen receptor-negative breast cancer and inhibition of this enzyme should be pursued for therapy. We have previously reported an association between up-regulation of cyclooxygenase-2 and an increased phosphorylation of Akt in breast cancer. As an extension of this study, we tested the hypothesis that cyclooxgenase-2 is associated with breast cancer survival because of Akt pathway activation and also examined whether the association is influenced by the tumor estrogen and HER2 receptor status. Cyclooxygenase-2 was found to be significantly associated with survival in estrogen receptor-negative [Hazard ratio (HR) = 2.72;95% confidence interval (CI), 1.36-5.41] and HER2 over-expressing breast cancer (HR = 2.84;95% CI, 1.07-7.52). However, its association with survival was strongest among patients who were both estrogen receptor-negative and HER2-positive (HR = 5.95;95% CI, 1.01-34.9). Notably, cyclooxgenase-2 expression in this distinct breast cancer subtype sig [summary truncated at 7800 characters]
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