Novel Markers for Disease Outcome in Breast Cancer

诱导型一氧化氮合酶（NOS2）和环氧化酶-2参与伤口愈合、血管生成和癌变。NOS2的上调和一氧化氮生成的增加也会影响细胞的氧化还原状态，并诱导蛋白质、脂质和DNA修饰。我们实验室最近的研究得出了具有临床意义的新发现，即NOS2表达与预后基底样转录模式相关，并且是雌激素受体阴性乳腺肿瘤女性生存不良的独立预测因子。当分析仅限于基底样乳腺肿瘤患者时，NOS2仍然是不良生存的预测因子。这一结果表明，除了在雌激素受体阴性的肿瘤中诱导基底样信号外，NOS2还可能在这种信号存在的情况下进一步增强疾病的侵袭性。乳腺肿瘤中NOS2的增加还与其他不良预后标志物相关，如肿瘤血管化和p53突变频率的增加，以及EGFR的激活。p53突变和EGFR过表达在基底样乳腺肿瘤中比其他乳腺癌亚型更常见。进一步的研究表明，一氧化氮激活表皮生长因子受体，与先前在肺癌中的发现一致，并在雌激素受体阴性的人乳腺癌细胞中诱导CD44和c-Myc等蛋白质。这些标记与乳腺癌的胚胎干细胞样表型和疾病结果有关。最近，干细胞标记物在基底样肿瘤中的表达增加。其中包括CD44，它是透明质酸和骨桥蛋白的受体。CD44在乳腺癌中是一个较差的预后指标，CD44阳性的乳腺癌细胞具有更强的侵袭活性，对放疗和化疗的抵抗力增强。总之，这些新的观察结果将NOS2与具有干细胞样特征的低分化乳腺癌表型的发展联系起来。一氧化氮可能通过激活c-Myc或诱导释放干细胞更新因子（如IL-8）来诱导这种表型，正如我们所展示的那样，一氧化氮具有这种特性。我们认为NOS2和NOS2信号的下游靶点是雌激素受体阴性乳腺癌的新治疗靶点，特别是基底样乳腺癌和三阴性疾病。我们还完成了一项研究，重新检查环氧化酶-2 （COX2）作为乳腺癌预后不良的标志物。此前有报道称，COX2是雌激素受体阳性疾病的预后标志和治疗靶点，但最近的临床试验未能发现COX2抑制剂对雌激素受体阳性乳腺癌患者的明显益处。在我们对来自大巴尔的摩地区的248名女性乳腺癌患者的研究中，COX-2表达的增加分别与雌激素受体阴性和her2阳性肿瘤患者乳腺癌特异性生存率的降低相关。在雌激素受体阴性和her2阳性肿瘤患者中，通过多因素分析的风险比判断，肿瘤COX-2升高与所有患者组中最差的生存率相关。通过Akt和凋亡通路中两个关键下游靶点的磷酸化状态判断，这伴随着Akt通路激活的增加。这些发现可能对COX-2靶向治疗乳腺癌具有启示意义，并提示雌激素受体阴性和HER2阳性肿瘤患者将受益于COX-2靶向治疗，该治疗在雌激素受体阴性肿瘤状态和HER2扩增导致HER2高表达的患者中效果最强。在2011财年，我们继续全面检测非裔美国人和欧美患者雌激素受体阳性和雌激素受体阴性乳腺肿瘤的代谢组学、蛋白质组学和转录组学，以发现生物标志物。该项目获得了NCI主任创新奖的资助。这项研究的前景是发现新的预后生物标志物，并阐明可能导致非裔美国女性乳腺癌侵袭性的原因。到目前为止，我们已经完成了肿瘤和邻近非癌组织的代谢组和转录组分析，并收集了大约75%的组织的蛋白质组分析的原始数据。转录组数据正在进行分析，并与先前发表的一项试点研究的数据进行比较（Martin et al.）。PLoS One, 2009)，显示非裔美国人和欧裔美国人乳腺癌患者肿瘤之间存在显著的基因表达差异。对代谢组的分析获得了乳腺肿瘤及其周围正常组织中536种生化物质的初步信息。其中，333人的名字/身份得到确认。在雌激素受体阴性和正常周围组织中，360种生化物质的浓度有显著差异。在雌激素受体阳性和正常周围组织中，它们的浓度有311个不同。非裔美国人和欧裔美国人乳腺癌亚型和肿瘤患者的代谢物水平存在显著差异，尤其是雌激素受体阴性肿瘤。我们没有发现组织代谢物浓度与体重指数、绝经状态、肿瘤分期和肿瘤HER2状态有任何差异。患者的收入和受教育程度也可能存在差异，这一发现需要进一步研究。目前，我们正在一项随访研究中验证这些发现，该研究包括106例雌激素受体阴性肿瘤和邻近的非癌组织（如可用），这些肿瘤来自非裔美国人（n = 49）和欧美乳腺癌患者（n = 21）。在本研究中，我们将评估这些80-150种代谢物的丰度，这些代谢物在肿瘤和正常患者以及非裔美国人和欧美患者之间的初步研究中显示出最显著的差异。这项验证性研究是由贝勒医学院与Arun Sreekumar合作进行的，使用靶向分析。此外，我们正在研究候选代谢物对细胞培养系统中癌症表型的影响。最后，我们开始了一个新的项目，评估压力生活事件对肿瘤生物学的影响。在细胞培养系统中，我们将模拟β肾上腺素能信号对肿瘤基质相互作用的影响。在一项临床研究中，我们将对接受乳腺癌手术的乳腺癌患者进行一项简短的调查，评估他们在过去一个月内感受到的压力。我们还将收集这些患者的冷冻肿瘤和邻近的正常乳腺组织和血液样本，并评估乳腺组织或血液样本是否具有与他们感知到的压力相关的生物特征。我们假设，高感知压力暴露的患者具有与更具侵袭性疾病和较差生存率一致的生物学特征。该试点研究的目的是从同意的患者中收集100对肿瘤/正常配对，并完成调查。

Inducible nitric oxide synthase (NOS2) and cyclooxygenase-2 are involved in wound healing, angiogenesis, and carcinogenesis. NOS2 up-regulation and increased nitric oxide production also affects the redox state of cells and induces protein, lipid, and DNA modifications. Recent research by our laboratory led to the novel and clinically significant observation that NOS2 expression is associated with a prognostic basal-like transcription pattern and is an independent predictor of poor survival in women with estrogen receptor-negative breast tumors. NOS2 remained a predictor of poor survival when the analysis was restricted to only those patients with basal-like breast tumors. This result suggests that in addition to inducing a basal-like signature in estrogen receptor-negative tumors, NOS2 may further enhance disease aggressiveness in the presence of this signature. Increased NOS2 in breast tumors also correlated with other poor outcome markers, such as an increased tumor vascularization and p53 mutation frequency, and activated EGFR. Both p53 mutations and EGFR overexpression occur more commonly in basal-like breast tumors than other breast cancer subtypes. Additional work showed that nitric oxide activates epidermal growth factor receptor, consistent with previous findings in lung cancer, and induces proteins, such as CD44 and c-Myc, in estrogen receptor-negative human breast cancer cells. These markers have been linked to an embryonic stem cell-like phenotype in breast cancer and disease outcome. Recently, an increased expression of stem cell markers in basal-like tumors has been reported. Among them was CD44, which is a receptor for hyaluronan and osteopontin. CD44 is a poor outcome marker in breast cancer and CD44-positive breast cancer cells have an increased invasive activity, increased resistance to radiation therapy and chemotherapeutics. Together, these novel observations link NOS2 to the development of a poorly differentiated breast cancer phenotype with stem cell-like characteristics. Nitric oxide may induce this phenotype by activation of c-Myc or by inducing the release of stem cell renewal factors like IL-8, a property that nitric oxide has, as we have shown. We propose that NOS2 and downstream targets of NOS2 signaling are novel therapeutic targets for estrogen receptor-negative breast cancer in general, and more specifically for basal-like breast cancer and the triple-negative disease. We also completed a study re-examining cyclooxygenase-2 (COX2) as a marker of poor outcome in breast cancer. It has previously been reported that COX2 is a marker of outcome and a therapeutic target in the estrogen receptor-positive disease but very recent clinical trials failed to find a clear benefit of COX2 inhibitors for patients with estrogen receptor-positive breast cancer. In our study of 248 women with incident breast cancer from the Greater Baltimore area, increased expression of COX-2 was associated with decreased breast cancer-specific survival only in patients with estrogen receptor-negative and HER2-positive tumors, respectively. In patients with both estrogen receptor-negative and HER2-positive tumors, increased tumor COX-2 was associated with the most inferior survival among all patient groups, as judged by the hazard ratio in the multivariate analysis. This was accompanied by increased Akt pathway activation, as judged by the phosphorylation status of Akt and two key downstream targets in the apoptosis pathway. These findings could have implications for COX-2 targeted therapy in breast cancer and suggest that patients with estrogen receptor-negative and HER2-positive tumors would benefit from a COX-2 targeted therapy with the efficacy of this therapy being strongest in patients with both an estrogen receptor-negative tumor status and an amplification of HER2 leading to high HER2 expression. In FY11, we continued to comprehensively examine the metabolome, proteome and transcriptome of estrogen receptor-positive and estrogen receptor-negative breast tumors from African-American and European-American patients for biomarker discovery. This project received funds through a NCI Director Innovation Award. The promise of the study is the discovery of novel biomarkers for prognosis, and for elucidating what may drive the aggressiveness of breast cancer in African-American women. To date, we have completed the metabolome and transcriptome analysis of the tumors and the adjacent non-cancerous tissues and have collected the raw data from the proteome analysis for about 75% of the tissues. The transcriptome data are being analyzed and compared with data from a previously published pilot study (Martin et al. PLoS One, 2009), showing significant gene expression differences between tumors from African-American and European-American breast cancer patients. The analysis of the metabolome yielded preliminary information on 536 biochemicals in the breast tumors and the surrounding normal tissues. Of those, 333 are named/identified. 360 biochemicals differed significantly in their concentrations between estrogen receptor-negative and normal surrounding tissue. 311 differed in their concentrations between estrogen receptor-positive and normal surrounding tissue. There were significant differences in metabolite levels between breast cancer subtypes and tumors from African-American patients and European-American patients, specifically in estrogen receptor-negative tumors. We did not find any differences in tissue metabolite concentrations by body mass index, menopausal status, or by tumor stage and tumor HER2 status. There are perhaps also differences by income and education of the patients, a finding which will need further examination. Currently, we are validating these findings in a follow-up study consisting of 106 estrogen receptor-negative tumors and adjacent non-cancerous tissues (as available) from African-American (n = 49) and European-American breast cancer patients (n = 21). In this study, we will evaluate the abundance of those 80-150 metabolites that showed the most significant differences in the pilot study between tumor and normal and between African-American and European-American patients. This validation study is conducted at the Baylor College of Medicine in collaboration with Arun Sreekumar, using targeted assays. In addition, we are examining the influence of candidate metabolites on cancer phenotypes in cell culture systems. Lastly, we started a new project evaluating the impact of stressful life events on tumor biology. In cell culture systems, we will model the effects of beta adrenergic signaling on tumor stroma interactions. In a clinical study, we will give breast cancer patients, who have breast cancer surgery, a short survey evaluating their perceived stress within the last one month. We will also collect frozen tumor and adjacent normal breast tissue and blood samples from these patients and evaluate whether the breast tissue or the blood samples have a biological signatures related to their perceived stress. We hypothesize that patients with a high perceived stress exposure have a biological signature consistent with a more aggressive disease and poorer survival. The pilot study is designed to collect 100 tumor/normal pairs from consented patients with a completed survey.