Novel Markers for Disease Outcome in Breast Cancer

尽管基因-环境相互作用的概念强烈表明，这些个体间的变异可能会影响癌症的生存，因为它们对肿瘤生物学和治疗结果有修饰作用，但探索常见遗传变异与疾病结局之间关系的研究相对较少。我们检测了锰超氧化物歧化酶基因的功能单核苷酸多态性（rs4880），该基因导致缬氨酸被丙氨酸取代（Val16Ala）。锰超氧化物歧化酶是一种防止氧化损伤和调节化疗药物疗效的酶。我们假设Val16Ala影响接受化疗的乳腺癌患者的生存。来自美国（n=248）和挪威（n=340）的两组患者对Val16Ala进行基因分型。Kaplan-Meier生存和Cox比例风险回归分析用于检验Val16Ala与疾病特异性生存之间的关系。我们发现，在两种患者群体中，Val16Ala与乳腺癌预后显著相关。携带Ala等位基因的人生存率较低。这种关联在接受辅助治疗的患者中具有显著性（HR = 2.47; 95% CI, 1.46-4.19），但在未接受辅助治疗的患者中无显著性（HR = 1.47; 95% CI, 0.57-3.74）。进一步按化疗类型分层后，Ala等位基因的作用主要局限于含环磷酰胺的化疗方案（HR = 22.0; 95% CI, 5.22-92.9; Ala/Ala vs . Val/Val）。这一发现提供了第一个指向乳腺癌患者环磷酰胺耐药机制的证据，可能具有重要的临床意义，因为在美国和欧洲，20%至25%的普通人群携带这种基因型。虽然这些数据是初步的，但这些数据表明Ala/Ala基因型患者应该考虑替代治疗。诱导型一氧化氮合酶（NOS2）和环氧化酶-2是炎症反应的标志基因。NOS2的一个主要生理作用是释放一氧化氮来支持伤口愈合过程。据推测，一氧化氮的伤口愈合特性可以将NOS2转化为促进人类癌症转移扩散的致癌基因。先前的研究表明，NOS2在乳腺肿瘤中表达。我们专门研究了NOS2在雌激素受体（ER）阴性乳腺癌中的功能，因为迫切需要找到治疗雌激素受体阴性疾病的新靶点。248例乳腺肿瘤的免疫组化结果显示，NOS2在其中173例（70%）中至强表达。接下来，我们研究了NOS2表达对预测患者生存的影响，以及肿瘤雌激素受体状态如何影响NOS2表达。虽然NOS2总体上与乳腺癌生存率无关，但我们发现雌激素受体状态改变了NOS2与乳腺癌生存率之间的关系，在雌激素受体阴性的疾病中，NOS2的高表达与低生存率显著相关。我们进一步研究了为什么NOS2与雌激素受体阴性而非雌激素受体阳性乳腺癌的低生存率相关，并分析了32例微解剖乳腺肿瘤的基因表达谱。我们在雌激素受体阳性肿瘤中未发现NOS2基因标记，提示NOS2可能不会导致这些肿瘤中基因表达的显著变化。对比NOS2高表达与NOS2低表达的肿瘤，我们发现44个基因对应的49个转录本在雌激素受体阴性肿瘤中存在差异表达。其中上调最多的基因是细胞角蛋白6和17，以及p-钙粘蛋白，它们是基底样乳腺癌表型的标记基因。为了确定这44个基因标记是否与基底样乳腺癌有进一步的联系，我们检查了先前发表的基底样乳腺癌基因标记的相似性。交叉参考NOS2/雌激素受体阴性基因标记与这些数据显示，NOS2标记中的许多转录本先前已被确定与基底样乳腺癌相关。此外，我们在公开的乳腺癌基因表达数据集中评估了44个基因特征与乳腺癌生存的关系。该分析表明，44个基因的特征与这些数据集中的不良预后显著相关。为了进一步确定NOS2在雌激素受体阴性乳腺肿瘤中的基因表达谱是否至少部分由一氧化氮引起，我们进行了验证实验，并检测了一氧化氮对四个基因蛋白表达的影响：白细胞介素-8 （IL-8）、S100钙结合蛋白A8 （S100A8）和p -钙粘蛋白（CDH3）是基底样乳腺癌的标志物，透明质酸受体（CD44）是乳腺癌干细胞的标志物。IL-8和S100A8都是乳腺癌的不良预后指标。使用缓释一氧化氮供体DETA/NO，我们发现一氧化氮在雌激素受体阴性的乳腺癌细胞系中诱导IL-8、S100A8、CDH-3和CD44蛋白的表达，而在雌激素受体阳性的乳腺癌细胞系中则没有，提示一氧化氮在乳腺癌中的这些作用仅限于雌激素受体阴性的肿瘤。最后，我们将雌激素受体重新引入雌激素受体阴性乳腺癌细胞系，并检查受体的表达是否会抑制这些蛋白的上调。蛋白表达分析表明，该受体完全抑制一氧化氮介导的CD44诱导，但不抑制其他标志物。这一发现与一氧化氮在独立于雌激素受体状态的雌激素受体阴性细胞中的生物学效应是一致的。最后，我们研究了NOS2表达与EGF受体激活的关系。这种受体经常在基底样乳腺癌中表达，这是一种预后差的亚型。统计分析显示，NOS2表达与基底样乳腺癌预后不良相关，并与肿瘤中EGF受体磷酸化相关。乳腺癌细胞暴露于一氧化氮也会导致受体磷酸化增加从而激活受体信号。综上所述，诱导型一氧化氮合酶表达是雌激素受体阴性乳腺癌预后不良的标志，应寻求抑制该酶的治疗。我们之前报道了环氧化酶-2上调与乳腺癌中Akt磷酸化增加之间的关联。作为本研究的延伸，我们验证了环氧化酶-2由于Akt通路激活而与乳腺癌存活相关的假设，并研究了这种关联是否受到肿瘤雌激素和HER2受体状态的影响。雌激素受体阴性的患者，环氧化酶-2与生存率显著相关[危险比(HR) = 2.72；95%可信区间（CI）， 1.36-5.41)和HER2过表达乳腺癌（HR = 2.84; 95% CI, 1.07-7.52）。然而，在雌激素受体阴性和her2阳性的患者中，其与生存率的相关性最强（HR = 5.95; 95% CI, 1.01-34.9）。值得注意的是，环氧化酶-2在这种独特的乳腺癌亚型中的表达[摘要截短于7800个字符]

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]