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 previously 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 provided a 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. Our data suggest that patients with the Ala/Ala genotype should be considered for alternative treatment. Inducible nitric oxide synthase (NOS2) is involved in wound healing, angiogenesis, and carcinogenesis. NOS2 up-regulation and increased nitric oxide (NO) production affects the redox state of cells and induces protein, lipid, and DNA modifications. It has been hypothesized that the wound healing properties of NO could turn NOS2 into an oncogene that promotes the metastatic spread of human cancer. We hypothesized that NOS2 influences survival of breast cancer patients by altering cancer cell characteristics. We examined immunohistochemical expression of NOS2 in 248 breast tumors and assessed its association with tumor markers and survival. The immunohistochemistry showed that NOS2 was moderately to strongly expressed in 173 of them (70%). While NOS2 was not associated with breast cancer survival overall, we found that the estrogen receptor status alpha (ER) modified the association between NOS2 and breast cancer survival, with high NOS2 expression being significantly associated with poor survival in the ER-negative disease. In the multivariate survival analysis, increased NOS2 predicted inferior survival in women with ER-negative tumors. Gene expression analysis of microdissected tumor epithelium found increased interleukin-8 and a poor prognosis signature characteristic of basal-like breast cancer in ER-negative tumors with high NOS2. In cell culture, NO induced signature genes only in ER-negative breast cancer cells. ER transgene expression in these cells inhibited NO-induced up-regulation of the stem cell marker CD44 and other proteins encoded by signature genes, but not of interleukin-8. Exposure to NO also enhanced cell motility and invasion of ER-negative cells and led to increased epidermal growth factor receptor phosphorylation which activates receptor signaling. Lastly, a pathway analysis linked the tumor NOS2 gene signature to c-Myc activation and NO induces c-Myc in basal-like MDA-MB-468 human breast cancer cells. In summary, we provide evidence that NOS2 causes poor survival among ER-negative patients because it induces a prognostic basal-like transcription pattern in these tumors. In summary, NOS2 is a marker of poor outcome in ER-negative breast cancer and inhibition of this enzyme should be pursued for therapy. In FY10, we started to comprehensively examine the metabolome, proteome and transcriptome of ER-positive and ER-negative breast tumors from African-American and European-American patients for biomarker discovery. This project was partly funded by a NCI Director Innovation Award. It is the great advantage of this study design that the readout of the combined analysis of these datasets can reveal true biological differences on the pathway level, e.g. comparing the two patient groups, or ER-positive vs. ER-negative disease, or good vs. poor outcome. The promise of the study is the discovery of novel biomarkers for prognosis, and for elucidating what may drive the aggressiveness of breast cancer in African-American women. Only the metabolome analysis has been completed (but without validation), as of September 2010. This analysis yielded preliminary information on 536 biochemicals in the breast tumors and the surrounding normal tissues. Of those, 333 are named/identified. 360 biochemicals differed significantly in their concentrations between ER-negative and normal surrounding tissue. 311 differed in their concentrations between ER-positive and normal surrounding tissue. There were significant differences in metabolite levels between tumors from African-American patients and European-American patients, specifically in ER-negative tumors. We did not find any differences in tissue metabolite concentrations by body mass index, menopausal status, or by tumor stage and tumor HER2 status. There are perhaps significant differences by income and education of the patients but these findings have to be verified to exclude potential confounding effects. Some of the most significant differences between tumor and normal tissue, and between African-American and European-American patients, are currently being validated in collaboration with Tim Veenstra's LPAT. We are confirming our findings for the following metabolites: sarcosine, kynurenine, 2-hydroxyglutarate, s-adenosylmethionine, N-acetyl-aspartate, N-acetyl-mannosamine, and dimethylarginine. As a next step, we will test the seven metabolites in cell culture models to examine if they increase migration and invasion of human breast cancer cell lines.
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