Novel Markers for Disease Outcome in Breast Cancer

乳腺癌疾病结果的新标志物

• 批准号: 7733307
• 负责人: Stefan Ambs
• 金额: $ 22.43万
• 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/7733307
• 关键词: Adriamycin PFS; Affect; Alanine; Alleles; Antioxidants; Area; Baltimore; Biological Markers; Breast; Breast Cancer Cell; CD44 gene; CDH3 gene; Calgranulin A; Cancer Patient; Cancer Research Project; Cancer cell line; Cell Line; Cell Survival; Codon Nucleotides; Cyclophosphamide; Cytokeratin; Data; Disease Marker; Disease Outcome; Enzymes; Estrogen Receptor Status; Estrogen Receptors; Estrogen receptor negative; Estrogen receptor positive; Frequencies; Gene Expression; Gene Expression Profile; General Population; Genes; Genetic Polymorphism; Genetic Variation; Genotype; Goals; Host Defense; Human; Inflammation; Interleukin-8; Laboratories; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Mammary Neoplasms; Manganese Superoxide Dismutase; Maryland; Molecular Epidemiology; Molecular Profiling; Mutation; NOS2A gene; Nitric Oxide; Nitric Oxide Donors; Norway; Oncogenes; Oncogenic; Outcome; P-Cadherin; Pathway interactions; Patients; Personal Satisfaction; Phenotype; Physiological; Population; Process; Property; Proteins; Publishing; Rate; Relative (related person); Reporting; Research; Resected; Risk; Role; SOD2 gene; Sampling; Single Nucleotide Polymorphism; Stratification; TP53 gene; Therapeutic; Transcript; Tumor Angiogenesis; Tumor Biology; Valine; Wound Healing; alanylalanine; cancer risk; cancer stem cell; cancer therapy; chemotherapy; concept; density; epidemiology study; follow-up; gene environment interaction; human NOS2A protein; hyaluronate; hypoxia inducible factor 1; malignant breast neoplasm; mutant; novel; protein expression; receptor; research study; response; therapy resistant; tumor

In the past, most molecular epidemiology studies examined the association between genotypes and cancer risk. Relatively few studies have explored the association between common genetic variations and disease outcome, although the concept of gene-environment interactions strongly suggests that genotypes may influence disease outcome because of their modifying effects on tumor biology and therapeutic outcome. It is well known that large differences exist in patient response to treatment. Several recent studies showed that polymorphisms influence therapy response and breast cancer survival, but more studies are needed to define the relative contribution of gene polymorphisms to poor outcome after therapy. Some of these polymorphisms could become biomarkers to guide cancer therapy. A functional single nucleotide polymorphism (SNP) in the manganese superoxide dismutase (SOD2) gene, characterized by a T to C transition in codon 16 (rs4880), leads to a substitution of valine by alanine (Val16Ala). This substitition is common and 20% to 25% of the general population have the Ala/Ala genotype. Carriers of the Ala allele were found to have an increased risk of breast and prostate cancer if they consume low amounts of antioxidants. We studied the association of the Val16Ala genotype with breast cancer survival in two independent populations, one from the greater Baltimore, Maryland area (n=244) and one from Norway (n=329). We observed an association between the Ala allele and poor disease outcome in both patient populations with an additive effect of the Ala allele. In the combined analysis, homozygous carriers of the Ala allele had twice the risk of a poor outcome when compared with homozygous carriers of the Val allele (adjusted HR = 2.19; 95% CI, 1.40-3.34). Stratification by therapy type showed that homozygous carriers of the Ala allele had a greater risk of poor survival, when compared with homozygous carriers of the Val allele, when they received therapies containing either adriamycin or cyclophosphamide. The Ala allele was not associated with disease outcome among those patients who did not receive chemotherapy. Our preliminary results indicate that the SOD2 Val16Ala genotype is a potential predictor of response to chemotherapy. Inducible nitric oxide synthase (NOS2) is a signature gene of the inflammation response and has key functions in host defense. A major physiological role of this enzyme 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. Nitric oxide (NO) may also select for a mutant p53 tumor status and activate oncogenic pathways such the Akt and HIF1 pathways, leading to increased cell survival and resistance to therapy. We hypothesized that NOS2 expression leads to poor survival in breast cancer by activating these pathways, and by inducing a poor outcome gene signature. Previous research has shown that NOS2 is expressed in breast tumors and is associated with poor outcome markers. We aimed our research to understand the function of NOS2 in estrogen receptor (ER)-negative breast cancer because previous data from our laboratory indicated that these tumors have an inflammation signature. We examined immunohistochemically the expression of NOS2 in 248 surgically resected tumors with follow-up for survival. NOS2 protein was moderately to strongly expressed in 173 of the 248 breast tumors (70%). Because of the previously observed relationships between NOS2 expression and tumor p53 status, tumor grade, and tumor angiogenesis, we examined those relationships in our samples set. Consistent with the previous reports, NOS2 expression was found to significantly correlate with an increased p53 mutation frequency, high tumor grade, and increased microvessel density. We next examined the effect of NOS2 expression on predicting patient survival, and how this would be affected by the tumor estrogen receptor (ER) status. While NOS2 was not associated with breast cancer survival in the unstratified analysis, 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 estrogen receptor-negative breast cancer patients, but not estrogen receptor-positive breast cancer patients. 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 (8 NOS2 low/ER-negative; 9 NOS2 low/ER-positive; 9 NOS2 high/ER-negative; 6 NOS2 high/ER-positive). We could not generate a list of differentially expressed genes at an acceptable false-discovery rate for the estrogen receptor-positive tumors, suggesting that NOS2 may not produce a gene signature in these tumors. In contrast, we found that 49 transcripts corresponding to 44 genes were differentially expressed in estrogen receptor-negative tumors. Among the genes most highly up-regulated were the cytokeratins 6 and 17, and P-cadherin, which are marker genes of the basal-like breast cancer phenotype. To determine whether this gene signature had further links with basal-like breast cancer, we examined two 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 (19/44) in the NOS2 signature have previously been identified as being associated with basal-like breast cancer. We conducted additional experiments 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. 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. Both IL-8 and S100A8 are known poor outcome markers for breast cancer. This project is currently being completed with additional experiments that examine the effect of nitric oxide on the phenotype of estrogen receptor-negative and estrogen receptor-positive breast cancer cells, and whether expression of the estrogen receptor in ER-negative cell lines can reverse the nitric oxide-induced phenotype.

过去，大多数分子流行病学研究检查了基因型与癌症风险之间的关联。尽管基因 - 环境相互作用的概念强烈表明基因型可能会影响疾病的结果，但相对较少的研究探讨了常见遗传变异与疾病结果之间的关联，因为它们对肿瘤生物学和治疗结果的影响改变了。众所周知，患者对治疗的反应存在很大差异。最近的几项研究表明，多态性会影响治疗反应和乳腺癌的存活，但是需要更多的研究来定义基因多态性对治疗后不良预后的相对贡献。这些多态性中的一些可能成为指导癌症治疗的生物标志物。锰超氧化物歧化酶（SOD2）基因中的功能性单核苷酸多态性（SNP），其特征在于密码子16（rs4880）中的t向C转变，导致丙氨酸（val16ala）替换valine。 这种取代很常见，20％至25％的总人群具有ALA/ALA基因型。如果ALA等位基因的载体消耗少量的抗氧化剂，则发现乳腺癌和前列腺癌的风险增加。我们研究了两个独立人群中Val16ala基因型与乳腺癌存活的关联，一个来自大巴尔的摩，马里兰州地区（n = 244），一个来自挪威（n = 329）。我们观察到两个患者人群中ALA等位基因与疾病不良结果之间的关联与ALA等位基因的加性作用。在组合分析中，与Val等位基因的纯合子载体相比，ALA等位基因的纯合载体的预期风险不佳（调整后的HR = 2.19; 95％CI，1.40-3.34）。通过治疗类型的分层表明，与Val等位基因的纯合载体相比，ALA等位基因的纯合载体的生存风险更大，当时他们接受了含有含有阿霉素或环磷酰胺的疗法。在那些未接受化疗的患者中，ALA等位基因与疾病结果无关。我们的初步结果表明，SOD2 Val16Ala基因型是对化学疗法反应的潜在预测指标。诱导一氧化氮合酶（NOS2）是炎症反应的特征基因，在宿主防御中具有关键功能。该酶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。一氧化氮（NO）还可以选择突变体p53肿瘤状态并激活致癌途径，例如AKT和HIF1途径，从而导致细胞存活率增加和对治疗的耐药性。我们假设NOS2表达通过激活这些途径并诱导较差的结果基因特征来导致乳腺癌的存活不良。先前的研究表明，NOS2在乳腺肿瘤中表达，并且与不良预后标记有关。 我们的研究目的是了解NOS2在雌激素受体（ER）阴性乳腺癌中的功能，因为我们实验室的先前数据表明这些肿瘤具有炎症特征。我们在248种手术切除的肿瘤中检查了NOS2的免疫组织化学检查，并随访。 NOS2蛋白在248种乳腺肿瘤中的173份（70％）中适度地表达。由于NOS2表达与肿瘤p53状态，肿瘤等级和肿瘤血管生成之间的关系，我们检查了样品集中的这些关系。与先前的报道一致，发现NOS2表达与p53突变频率，高肿瘤等级和微血管密度增加显着相关。接下来，我们研究了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（ER）状态的影响。虽然NOS2与乳腺癌的生存无关，但我们发现雌激素受体状态改变了NOS2和乳腺癌生存之间的关联，高NOS2表达与雌激素受体阴性乳腺癌患者的生存不良显着相关，但没有雌激素受体受体阳性乳腺癌患者。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相关，并分析了32个微解析乳腺肿瘤的基因表达谱（8个NOS2低/ER-ER-阴性； 9 NOS2低/ER-ER-ER-ER-ER-ER-ER-ER阳性； 9 NOS2高/ER; 9 NOS2高/er-ne-ne-Nenegative; 6 Nos2高/ER-er-er-er-er-er-er-er-er-Posive）。我们无法以雌激素受体阳性肿瘤可接受的假差速率生成差异表达的基因列表，这表明NOS2可能不会在这些肿瘤中产生基因特征。相比之下，我们发现在雌激素受体阴性肿瘤中差异表达了与44个基因相对应的49种转录本。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。为了确定该基因特征是否与基础样的乳腺癌有进一步的联系，我们检查了两个先前发表的基础样乳腺癌基因特征的相似性。 交叉引用NOS2/雌激素受体阴性基因的信号，并显示了这些数据表明，NOS2特征中的许多转录本（19/44）以前已被确定为与基础样乳腺癌有关。 We conducted additional experiments 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。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。 IL-8和S100A8均为乳腺癌的结果标记不佳。目前正在通过其他实验完成该项目，以检查一氧化氮对雌激素受体阴性和雌激素受体阳性乳腺癌细胞的表型的影响，以及在ER阴性细胞系中雌激素受体的表达是否可以逆转硝酸氧化物诱导的表型。