Novel Markers for Disease Outcome in Breast Cancer

乳腺癌疾病结果的新标志物

• 批准号: 7965798
• 负责人: Stefan Ambs
• 金额: $ 19.26万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965798
• 关键词: Adjuvant Therapy; Affect; Alanine; Alleles; Biological; Breast Cancer Cell; CD44 gene; CDH3 gene; Calgranulin A; Cancer Patient; Cancer Research Project; Cancer cell line; Cell Line; Cells; Chemotherapy-Oncologic Procedure; Clinical; Confidence Intervals; Cyclophosphamide; Cytokeratin; Data; Data Set; Disease; Disease Marker; Disease Outcome; ERBB2 gene; Enzyme Inhibition; Enzymes; Epidermal Growth Factor Receptor; Estrogen Receptor Status; Estrogen Receptors; Estrogen receptor negative; Estrogen receptor positive; Estrogens; Europe; Gene Expression; Gene Expression Profile; General Population; Genes; Genetic Variation; Genotype; Goals; Human; Immunohistochemistry; Individual; Inferior; Inflammation; Interleukin-8; Lead; Link; Malignant Neoplasms; Mammary Neoplasms; Manganese Superoxide Dismutase; Mediating; Molecular Profiling; NOS2A gene; Nitric Oxide; Nitric Oxide Donors; Norway; Oncogenes; Outcome; P-Cadherin; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Physiological; Process; Property; Proteins; Publishing; Receptor Signaling; Regression Analysis; Reporting; Research; Resistance; Role; Single Nucleotide Polymorphism; Stratification; Survival Rate; Testing; Therapeutic; Transcript; Tumor Biology; United States; Up-Regulation; Validation; Valine; Variant; Wound Healing; alanylalanine; alternative treatment; cancer stem cell; chemotherapy; cyclooxygenase 2; gene environment interaction; hazard; human NOS2A protein; hyaluronate; malignant breast neoplasm; novel; novel marker; oxidative damage; patient population; protein expression; receptor; receptor expression; research study; response; tumor; valylvaline

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]

相对较少的研究探讨了常见的遗传变异与疾病结果之间的关联，尽管基因环境相互作用的概念强烈表明这些个体间的变化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的影响改变了。我们检查了锰超氧化物歧化酶基因中的功能性单核苷酸多态性（RS4880），该基因导致丙氨酸（Val16ala）替代Valine。锰超氧化物歧化酶是一种酶，可预防氧化损伤并调节化学治疗药物的功效。我们假设Val16ala会影响接受化疗的患者的乳腺癌存活。 VAL16ALA对美国的两个患者人群（n = 248）和挪威（n = 340）进行了基因分型。 Kaplan-Meier生存期和Cox比例危害回归分析用于检查Val16ala与疾病特异性生存之间的关系。我们发现Val16ala在两个患者人群中都与乳腺癌结局显着相关。 ALA等位基因的载体的存活率较低。这种关联对于接受辅助治疗的患者很重要（HR = 2.47; 95％CI，1.46-4.19），但对没有IT的患者而言并不重要（HR = 1.47; 95％CI，0.57-3.74）。通过化疗类型进一步分层后，ALA等位基因的作用主要仅限于含环磷酰胺的化学疗法方案（HR = 22.0; 95％CI，5.22-92.9; ALA/ALA与Val/Val/Val/val）。该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，并且可能具有重要的临床意义，因为美国和欧洲的20％至25％的总人群的20％至25％携带了这种基因型。尽管初步，但这些数据表明应考虑患有ALA/ALA基因型的患者进行替代治疗。诱导型一氧化氮合酶（NOS2）和环氧酶-2是炎症反应的特征基因。 NOS2的主要生理作用是释放一氧化氮以支持伤口愈合过程。据推测，一氧化氮的伤口愈合特性可以将NOS2变成一种癌细胞，从而促进人类癌症的转移性扩散。先前的研究表明，NOS2在乳腺肿瘤中表达。我们特别检查了NOS2在雌激素受体（ER）阴性乳腺癌中的功能，因为迫切需要鉴定雌激素受体阴性疾病中治疗的新靶标。 248种乳腺肿瘤的免疫组织化学表明，NOS2在其中173个（70％）中适度地表达。接下来，我们检查了NOS2表达对预测患者存活的影响，以及这将如何受肿瘤雌激素受体状态的影响。虽然NOS2与总体上的乳腺癌生存无关，但我们发现雌激素受体状态改变了NOS2与乳腺癌生存之间的关联，高NOS2表达与雌激素受体阴性疾病中的生存率差显着相关。我们进一步研究了为什么NOS2与雌激素受体阴性但不存在雌激素受体阳性乳腺癌的生存率不佳，并分析了32种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没有发现NOS2基因特征，这表明NOS2可能不会导致这些肿瘤中的显着基因表达变化。相反，我们发现在雌激素受体阴性肿瘤中差异表达了49种与NOS2高的NOS2低表达肿瘤相对的转录本。在最高度上调的基因中，有细胞角蛋白6和17和p-钙粘着蛋白，它们是基础样乳腺癌表型的标记基因。为了确定这个44基因的签名是否与基础样的乳腺癌有了进一步的联系，我们检查了先前发表的基础样乳腺癌基因标志的相似性。交叉引用NOS2/雌激素受体阴性基因信号，并显示了这些数据表明，NOS2特征中的许多转录本以前已被确定为与基底样乳腺癌有关。此外，我们评估了公开可用的乳腺癌基因表达数据集与乳腺癌生存的关联。该分析表明，在这些数据集中，44基因的签名与不良结果显着相关。进行了验证实验，以进一步确定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是由一氧化氮引起的，并检查了一氧化氮对四个基因蛋白表达的影响：interleukin-8（IL-8），s100 calling calligin caliun calium calling Protin a8（s100 calling protigin caligin of ccding蛋白A8（s100 calling）（S100）的影响基底样的乳腺癌和透明质酸受体（CD44），它是乳腺癌干细胞的标志。 IL-8和S100A8均为乳腺癌的结果标记不佳。 使用缓慢释放的一氧化氮供体DETA/NO，我们发现一氧化一氧化物诱导雌激素受体阴性乳腺癌细胞系中的IL-8，S100A8，CDH-3和CD44蛋白表达，但并未在雌激素受体阳性细胞系中，但在乳腺癌中均具有乳腺癌的抑制作用，这表明了这些影响乳腺癌的作用。最后，我们将雌激素受体重新引入到雌激素受体阴性乳腺癌细胞系中，并检查受体表达是否会抑制这些蛋白的上调。 蛋白质表达分析表明，受体完全抑制了一氧化氮介导的CD44的诱导，而不是其他标记物的诱导。该发现与一氧化氮在雌激素受体阴性细胞中的生物学作用一致，这些细胞与雌激素受体状态无关。最后，我们研究了NOS2表达与EGF受体的激活之间的关系。该受体经常以基础样的乳腺癌表达，这是较差的结果亚型。统计分析表明，NOS2表达与基础样乳腺癌的预后不良有关，并且与肿瘤中的EGF受体磷酸化相关。乳腺癌细胞暴露于一氧化氮还会导致受体磷酸化增加，从而激活受体信号。总而言之，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，应采用该酶的抑制作用进行治疗。我们先前已经报道了环氧酶-2上调与乳腺癌中AKT磷酸化增加之间的关联。作为这项研究的扩展，我们检验了以下假设：由于AKT途径的激活，环氧酶-2与乳腺癌的存活有关，还研究了该关联是否受肿瘤雌激素和HER2受体状态的影响。发现环氧合酶2与雌激素受体阴性[危险比（HR）= 2.72的存活显着相关； 95％置信区间（CI），1.36-5.41]和HER2过表达的乳腺癌（HR = 2.84; 95％CI，1.07-7.52）。 但是，在雌激素受体阴性和HER2阳性的患者中，其与生存的关联最强（HR = 5.95; 95％CI，1.01-34.9）。值得注意的是，在这种独特的乳腺癌亚型SIG中的环氧酶-2表达[汇总以7800个字符]