COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer.

BACKGROUND: Inducible cyclooxgenase-2 (COX-2) is commonly overexpressed in breast tumors and is a target for cancer therapy. Here, we studied the association of COX-2 with breast cancer survival and how this association is influenced by tumor estrogen and HER2 receptor status and Akt pathway activation. METHODS: Tumor COX-2, HER2 and estrogen receptor α (ER) expression and phosphorylation of Akt, BAD, and caspase-9 were analyzed immunohistochemically in 248 cases of breast cancer. Spearman's correlation and multivariable logistic regression analyses were used to examine the relationship between COX-2 and tumor characteristics. Kaplan-Meier survival and multivariable Cox proportional hazards regression analyses were used to examine the relationship between COX-2 and disease-specific survival. RESULTS: COX-2 was significantly associated with breast cancer outcome in ER-negative [Hazard ratio (HR) = 2.72; 95% confidence interval (CI), 1.36-5.41; comparing high versus low COX-2] and HER2 overexpressing breast cancer (HR = 2.84; 95% CI, 1.07-7.52). However, the hazard of poor survival associated with increased COX-2 was highest among patients who were both ER-negative and HER2-positive (HR = 5.95; 95% CI, 1.01-34.9). Notably, COX-2 expression in the ER-negative and HER2-positive tumors correlated significantly with increased phosphorylation of Akt and of the two Akt targets, BAD at Ser136 and caspase-9 at Ser196. CONCLUSIONS: Up-regulation of COX-2 in ER-negative and HER2-positive breast tumors is associated with Akt pathway activation and is a marker of poor outcome. The findings suggest that COX-2-specific inhibitors and inhibitors of the Akt pathway may act synergistically as anticancer drugs in the ER-negative and HER2-positive breast cancer subtype.

Rrp1b, a new candidate susceptibility gene for breast cancer progression and metastasis.

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

A stromal gene signature associated with inflammatory breast cancer.

The factors that determine whether a breast carcinoma will develop into inflammatory breast cancer (IBC) remain poorly understood. Recent evidence indicates that the tumor stroma influences cancer phenotypes. We tested the hypotheses that the gene expression signature of the tumor stroma is a distinctive feature of IBC. We used laser capture microdissection to obtain enriched populations of tumor epithelial cells and adjacent stromal cells from 15 patients with IBC and 35 patients with invasive, noninflammatory breast cancer (non-IBC). Their mRNA expression profiles were assessed using Affymetrix GeneChips. In addition, a previously established classifier for IBC was evaluated for the resulting data sets. The gene expression profile of the tumor stroma distinguished IBC from non-IBC, and a previously established IBC prediction signature performed better in classifying IBC using the gene expression profile of the tumor stroma than it did using the profile of the tumor epithelium. In a pathway analysis, the genes differentially expressed between IBC and non-IBC tumors clustered in distinct pathways. We identified multiple pathways related to the endoplasmic stress response that could be functionally significant in IBC. Our findings suggest that the gene expression in the tumor stroma may play a role in determining the IBC phenotype.

Association of MTHFR gene polymorphisms with breast cancer survival.

BACKGROUND: Two functional single nucleotide polymorphisms (SNPs) in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, C677T and A1298C, lead to decreased enzyme activity and affect chemosensitivity of tumor cells. We investigated whether these MTHFR SNPs were associated with breast cancer survival in African-American and Caucasian women. METHODS: African-American (n = 143) and Caucasian (n = 105) women, who had incident breast cancer with surgery, were recruited between 1993 and 2003 from the greater Baltimore area, Maryland, USA. Kaplan-Meier survival and multivariate Cox proportional hazards regression analyses were used to examine the relationship between MTHFR SNPs and disease-specific survival. RESULTS: We observed opposite effects of the MTHFR polymorphisms A1298C and C677T on breast cancer survival. Carriers of the variant allele at codon 1298 (A/C or C/C) had reduced survival when compared to homozygous carriers of the common A allele [Hazard ratio (HR) = 2.05; 95% confidence interval (CI), 1.05-4.00]. In contrast, breast cancer patients with the variant allele at codon 677 (C/T or T/T) had improved survival, albeit not statistically significant, when compared to individuals with the common C/C genotype (HR = 0.65; 95% CI, 0.31-1.35). The effects were stronger in patients with estrogen receptor-negative tumors (HR = 2.70; 95% CI, 1.17-6.23 for A/C or C/C versus A/A at codon 1298; HR = 0.36; 95% CI, 0.12-1.04 for C/T or T/T versus C/C at codon 677). Interactions between the two MTHFR genotypes and race/ethnicity on breast cancer survival were also observed (A1298C, pinteraction = 0.088; C677T, pinteraction = 0.026). CONCLUSION: We found that the MTHFR SNPs, C677T and A1298C, were associated with breast cancer survival. The variant alleles had opposite effects on disease outcome in the study population. Race/ethnicity modified the association between the two SNPs and breast cancer survival.

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties.

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.

Household income is associated with the p53 mutation frequency in human breast tumors.

BACKGROUND: A study from Scotland reported that the p53 mutation frequency in breast tumors is associated with socio-economic deprivation. METHODS: We analyzed the association of the tumor p53 mutational status with tumor characteristics, education, and self-reported annual household income (HI) among 173 breast cancer patients from the greater Baltimore area, United States. RESULTS: p53 mutational frequency was significantly associated with HI. Patients with < $15,000 HI had the highest p53 mutation frequency (21%), followed by the income group between $15,000 and $60,000 (18%), while those above $60,000 HI had the fewest mutations (5%). When dichotomized at $60,000, 26 out of 135 patients in the low income category had acquired a p53 mutation, while only 2 out of 38 with a high income carried a mutation (P < 0.05). In the adjusted logistic regression analysis with 3 income categories (trend test), the association between HI and p53 mutational status was independent of tumor characteristics, age, race/ethnicity, tobacco smoking and body mass. Further analyses revealed that HI may impact the p53 mutational frequency preferentially in patients who develop an estrogen receptor (ER)-negative disease. Within this group, 42% of the low income patients (< $15,000 HI) carried a mutation, followed by the middle income group (21%), while those above $60,000 HI did not carry mutations (Ptrend < 0.05). CONCLUSIONS: HI is associated with the p53 mutational frequency in patients who develop an ER-negative disease. Furthermore, high income patients may acquire fewer p53 mutations than other patients, suggesting that lifetime exposures associated with socio-economic status may impact breast cancer biology.

Increased NOS2 predicts poor survival in estrogen receptor-negative breast cancer patients.

Inducible nitric oxide synthase (NOS2) is involved in wound healing, angiogenesis, and carcinogenesis. NOS2 upregulation and increased nitric oxide (NO) production affect the redox state of cells and can induce protein, lipid, and DNA modifications. To investigate whether NOS2 levels influence survival of breast cancer patients, we examined NOS2 expression and its association with tumor markers and survival in 248 breast tumors. In multivariable survival analysis, increased NOS2 predicted inferior survival in women with estrogen receptor α-negative (ER-negative) tumors. Microdissected tumor epithelium from ER-negative tumors with high NOS2 had increased IL-8 and a gene expression signature characteristic of basal-like breast cancer with poor prognosis. In cell culture, NO only induced selected signature genes in ER-negative breast cancer cells. ER transgene expression in ER-negative cells inhibited NO-induced upregulation of the stem cell marker CD44 and other proteins encoded by signature genes, but not of IL-8. Exposure to NO also enhanced cell motility and invasion of ER-negative cells. Last, pathway analysis linked the tumor NOS2 gene signature to c-Myc activation. Thus, NOS2 is associated with a basal-like transcription pattern and poor survival of ER-negative patients.

Differences in the tumor microenvironment between African-American and European-American breast cancer patients.

BACKGROUND: African-American breast cancer patients experience higher mortality rates than European-American patients despite having a lower incidence of the disease. We tested the hypothesis that intrinsic differences in the tumor biology may contribute to this cancer health disparity. METHODS AND RESULTS: Using laser capture microdissection, we examined genome-wide mRNA expression specific to tumor epithelium and tumor stroma in 18 African-American and 17 European-American patients. Numerous genes were differentially expressed between these two patient groups and a two-gene signature in the tumor epithelium distinguished between them. To identify the biological processes in tumors that are different by race/ethnicity, Gene Ontology and disease association analyses were performed. Several biological processes were identified which may contribute to enhanced disease aggressiveness in African-American patients, including angiogenesis and chemotaxis. African-American tumors also contained a prominent interferon signature. The role of angiogenesis in the tumor biology of African-Americans was further investigated by examining the extent of vascularization and macrophage infiltration in an expanded set of 248 breast tumors. Immunohistochemistry revealed that microvessel density and macrophage infiltration is higher in tumors of African-Americans than in tumors of European-Americans. Lastly, using an in silico approach, we explored the potential of tailored treatment options for African-American patients based on their gene expression profile. This exploratory approach generated lists of therapeutics that may have specific antagonistic activity against tumors of African-American patients, e.g., sirolimus, resveratrol, and chlorpromazine in estrogen receptor-negative tumors. CONCLUSIONS: The gene expression profiles of breast tumors indicate that differences in tumor biology may exist between African-American and European-American patients beyond the knowledge of current markers. Notably, pathways related to tumor angiogenesis and chemotaxis could be functionally different in these two patient groups.

A mitochondrial target sequence polymorphism in manganese superoxide dismutase predicts inferior survival in breast cancer patients treated with cyclophosphamide.

PURPOSE: Manganese superoxide dismutase protects against oxidative damage and modulates the efficacy of chemotherapeutic drugs. A functional single-nucleotide polymorphism in codon 16 of SOD2 (rs4880), which encodes manganese superoxide dismutase, results in a substitution of valine by alanine (Val16Ala). We hypothesized that this single-nucleotide polymorphism affects breast cancer survival of patients receiving chemotherapy. EXPERIMENTAL DESIGN: 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. RESULTS: Val16Ala was significantly associated with breast cancer outcome in both patient populations. Carriers of the Ala allele had inferior survival rates in the multivariate analysis [hazard ratio (HR), 2.44 and 95% confidence interval (95% CI), 1.11-5.37 in U.S. cohort; HR, 1.91 and 95% CI, 1.06-3.45 in Norway cohort for Ala/Ala versus Val/Val]. In an analysis of the combined cohorts, 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). CONCLUSION: The Val16Ala polymorphism affects survival of patients receiving cyclophosphamide-containing chemotherapy. The findings provide the first evidence pointing toward a mechanism for cyclophosphamide resistance in breast cancer patients.

The mannose-binding lectin (MBL2) haplotype and breast cancer: an association study in African-American and Caucasian women.

Common genetic variants in cancer-related genes contribute to breast cancer. The innate immune system plays a crucial role in the immune surveillance against malignancies, thus it is plausible that genetic variations in key genes of the innate immunity such as the mannose-binding lectin (MBL), MBL2, could influence the risk for breast cancer. We investigated the association of MBL2 genotypes with breast cancer and conducted a comprehensive genotype and haplotype analysis of 26 MBL2 single nucleotide polymorphisms (SNPs) in a case-control study of breast cancer [166 African-American (AA) case patients versus 180 controls and 127 Caucasian (CAU) case patients versus 137 controls]. We observed that the A allele of the 3'-UTR SNP Ex4-1067 (NCBI SNP ID: rs10824792) was significantly associated with a decreased disease risk in AA women [odds ratio (OR) = 0.47, 95% confidence interval (CI) = 0.27-0.81]. Haplotype analysis of MBL2 showed that the frequency of the corresponding 3' haplotype TATAAC (Ex4-1483, Ex4-1067, Ex4-1047, Ex4-901, Ex4-710, 3238bp 3' STP) was lower in cases than controls among AA women (0.15 versus 0.21; P = 0.02) suggesting a protective effect after adjusting for covariates (OR = 0.51, 95% CI = 0.29-0.88, P = 0.018). In conclusion, this study presents preliminary evidence that common genetic variants in the 3'-UTR of MBL2 might influence the risk for breast cancer in AA women, probably in interaction with the 5' secretor haplotypes that are associated with high concentrations of MBL.

Inflammation and IGF-I activate the Akt pathway in breast cancer.

Akt signaling may promote breast cancer progression and poor disease outcome. We hypothesized that serum insulin-like growth factor I (IGF-I) and a proinflammatory tumor environment induce phosphorylation of Akt and downstream targets of Akt in breast cancer. We studied the relationship between Akt pathway activation, IGF-I and markers of inflammation, e.g., nitric oxide synthase-2 (NOS2), cyclooxygenase-2 (COX2) and tumor phagocyte density, in 248 breast tumors. We also examined the association of Akt phosphorylation with breast cancer survival. We observed that phosphorylation of Akt, BAD and caspase-9 correlated strongly with the expression of the 2 proinflammatory enzymes, NOS2 and COX2, in breast tumors (p < 0.001; Spearman rank correlation). Both NOS2 and COX2 expression were independently associated with Akt phosphorylation in the multivariate analysis. Serum IGF-I concentrations and the IGF-I/IGFBP3 ratio correlated with Akt phosphorylation at Thr308 and Ser473 in breast tumors (p

Association of breast cancer outcome with status of p53 and MDM2 SNP309.

BACKGROUND: A common single-nucleotide polymorphism (SNP) in the promoter region of the MDM2 gene, known as T-309G and referred to as SNP309 for this study, leads to increased expression of Mdm2 protein and attenuated function of the p53 tumor suppressor protein. We investigated whether genetic variants in MDM2 were associated with breast cancer incidence and survival and whether the variant status could interact with the tumor p53 status to modify breast cancer survival. METHODS: We used multivariable logistic and Cox regression analyses to study the relationship of SNP309 status and the status of a second MDM2 SNP in exon 12 at codon 354 (SNP354) with breast cancer incidence and with disease-specific survival among 293 case patients and 317 cancer-free control subjects. Survival analysis included 248 of the 293 case patients who had known tumor p53 status. All statistical tests were two-sided. RESULTS: We did not observe an association between SNP309 status and breast cancer incidence in the unstratified analysis, but we did find a statistically significant association between SNP354 status and breast cancer incidence (odds ratio = 3.34, 95% confidence interval [CI] = 1.88 to 5.93). We also discovered a statistically significant interaction between SNP309 status and tumor p53 expression for breast cancer survival (P(interaction) = .002). Among homozygous carriers of the common MDM2 SNP309 allele (T/T), a mutant p53 status (risk ratio [RR] of death = 2.33, 95% CI = 1.08 to 5.03) and aberrant p53 protein expression (RR = 2.61, 95% CI = 1.22 to 5.57) in breast tumors were associated with poor survival. Tumor p53 status was not associated with breast cancer survival among carriers of the variant MDM2 SNP309 allele (G/T or G/G), which is consistent with a dominant effect of the variant allele. CONCLUSION: A strong interaction between SNP309 status and tumor p53 status appears to modify the association between p53 status and breast cancer survival.