Novel Markers for Disease Outcome in Breast Cancer

过去，大多数分子流行病学研究考察了基因型和癌症风险之间的关系。尽管基因-环境相互作用的概念强烈表明基因型可能影响疾病结局，因为它们对肿瘤生物学和治疗结果的修饰作用，但相对较少的研究探索了常见遗传变异与疾病结局之间的关系。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，基因多态性影响治疗反应和乳腺癌生存，但需要更多的研究来确定基因多态性对治疗后不良预后的相对贡献。其中一些多态性可以成为指导癌症治疗的生物标志物。锰超氧化物歧化酶（SOD2）基因的一个功能性单核苷酸多态性（SNP），以密码子16 （rs4880）的T到C转换为特征，导致缬氨酸被丙氨酸（Val16Ala）取代。这种替代是常见的，20%至25%的普通人群具有Ala/Ala基因型。研究发现，携带Ala等位基因的人如果摄入少量抗氧化剂，患乳腺癌和前列腺癌的风险会增加。我们在两个独立的人群中研究了Val16Ala基因型与乳腺癌生存的关系，一个来自马里兰州巴尔的摩地区（n=244），另一个来自挪威（n=329）。我们观察到Ala等位基因与两种患者群体的不良疾病预后之间存在关联，并存在Ala等位基因的加性效应。在联合分析中，与Val等位基因纯合携带者相比，Ala等位基因纯合携带者的不良预后风险是Val等位基因纯合携带者的两倍（校正后HR = 2.19; 95% CI, 1.40-3.34）。按治疗类型分层显示，当Ala等位基因纯合携带者接受含阿霉素或环磷酰胺的治疗时，与Val等位基因纯合携带者相比，Ala等位基因的低生存率风险更大。在未接受化疗的患者中，Ala等位基因与疾病结局无关。我们的初步结果表明SOD2 Val16Ala基因型是化疗反应的潜在预测因子。诱导型一氧化氮合酶（NOS2）是炎症反应的标志基因，在宿主防御中起关键作用。这种酶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。一氧化氮（NO）也可能选择突变的p53肿瘤状态，激活Akt和HIF1等致癌途径，导致细胞存活增加和对治疗的抵抗。我们假设NOS2的表达通过激活这些途径，并通过诱导不良结果基因标记导致乳腺癌的低生存率。先前的研究表明，NOS2在乳腺肿瘤中表达，并与预后不良标志物相关。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，因为我们实验室之前的数据表明这些肿瘤具有炎症特征。我们用免疫组织化学方法检测了248例手术切除肿瘤中NOS2的表达，并随访了存活情况。NOS2蛋白在248例乳腺肿瘤中有173例（70%）中至强表达。由于之前观察到的NOS2表达与肿瘤p53状态、肿瘤分级和肿瘤血管生成之间的关系，我们在我们的样本集中研究了这些关系。与先前的报道一致，NOS2表达与p53突变频率增加、肿瘤分级高和微血管密度增加显著相关。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（ER）状态如何影响NOS2表达。虽然在非分层分析中NOS2与乳腺癌生存率没有相关性，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乳腺癌患者中，NOS2的高表达与生存率差显著相关，而雌激素受体阳性的乳腺癌患者中，NOS2的高表达与生存率差显著相关。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（8例NOS2低/ er阴性，9例NOS2低/ er阳性，9例NOS2高/ er阴性，6例NOS2高/ er阳性）。对于雌激素受体阳性的肿瘤，我们无法以可接受的错误发现率生成差异表达基因列表，这表明NOS2可能不会在这些肿瘤中产生基因标记。相反，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。为了确定该基因标记是否与基底样乳腺癌有进一步的联系，我们检查了两个先前发表的基底样乳腺癌基因标记的相似性。将NOS2/雌激素受体阴性基因标记与这些数据交叉对照显示，NOS2标记中的许多转录本（19/44）先前已被确定与基底样乳腺癌相关。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，我们进行了进一步的实验，并检测了一氧化氮对四种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。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。IL-8和S100A8都是乳腺癌的不良预后指标。该项目目前正在完成额外的实验，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表型的影响，以及雌激素受体在er阴性细胞系中的表达是否可以逆转一氧化氮诱导的表型。

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.