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（NO）生成的增加也会影响细胞的氧化还原状态，诱导蛋白质、脂质和DNA修饰。我们实验室最近的研究得出了一项新颖且具有临床意义的观察结果，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er阴性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EGFR，与先前在肺癌中的发现一致，并在er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信号的下游靶点是一般er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。这些发现目前正在与NCI的David Wink博士的实验室合作进行进一步的研究。他的团队正在研究雌激素受体阴性乳腺癌细胞系中的NO信号通路，以及这些通路如何在这些细胞中诱导基底样和干细胞样表型。此外，我们的临床研究结果可能会作为NCI重大机遇计划的一部分进行跟进，其中临床中心（PI: Elise Kohn）将在三阴性和炎症性乳腺癌患者中测试葛兰素史克提供的no_2特异性抑制剂。在2012财年，我们继续全面检查非裔美国人和欧裔美国患者er阳性和er阴性乳腺肿瘤的代谢组、蛋白质组和转录组，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。360种生化物质的浓度在er阴性和正常周围组织之间存在显著差异。其中311种在er阳性和正常周围组织中的浓度不同。非裔美国人和欧裔美国人乳腺癌亚型和肿瘤之间的代谢物水平存在显著差异，特别是在er阴性肿瘤中。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。我们与芝加哥大学的Olopade博士以及其他学术中心的研究人员合作，参与了一项非洲裔女性乳腺癌的GWAS研究。本研究旨在确定非洲裔美国妇女和西非妇女的遗传易感性位点。最初的结果表明，许多最初在欧洲和欧美女性身上发现的疾病易感基因位点无法在非洲裔女性身上复制。未来的研究将集中于尚未在其他人群（欧洲或亚洲后裔）中发现的新的易感位点。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 (NO) 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 ER-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 ER-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 EGFR, consistent with previous findings in lung cancer, and induces proteins, such as CD44 and c-Myc, in ER-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. NO 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 ER-negative breast cancer in general, and more specifically for basal-like breast cancer and the triple-negative disease. These findings are currently further pursued in collaboration with the laboratory of Dr. David Wink at the NCI. His group is studying the NO signaling pathways in ER-negative breast cancer cell lines and how these pathways induce a basal-like and stem cell-like phenotype in these cells. In addition, our clinical findings are possibly followed up as part of a Major Opportunity initiative at the NCI in which the Clinical Center (PI: Elise Kohn) will test a NOS2-specific inhibitor provided by GlaxoSmithKline in patients with triple-negative and inflammatory breast cancer. In FY12, we continued to comprehensively examine the metabolome, proteome and transcriptome of ER-positive and ER-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 ER-negative and normal surrounding tissue. 311 differed in their concentrations between ER-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 ER-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.In a collaboration with Dr. Olopade at the University of Chicago and investigators from other academic centers, we are participating in a GWAS study of breast cancer in women of African ancestry. This study is aimed to identify genetic susceptibility loci in African-American women and women from West Africa. First results show that many of the disease susceptibility loci first discovered for European and European-American women cannot be replicated in women of African descent. Future research will focus on novel susceptibility loci that have not been detected in other population groups (of European or Asian descent).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.