Stable isotopomer analysis of anabolic metabolic pathways in breast cancer

描述（由申请人提供）：尽管在技术和筛查方面取得了许多进展，但乳腺癌仍然是人类癌症的主要形式（终身风险约为100%）。1/8）。 肿瘤发生的一个被忽视的方面是表达生物化学表型的细胞代谢谱。 转化的细胞与其正常对应物的不同之处在于丧失了导致永生化的关键调节功能，避免了细胞凋亡和细胞周期失调。 一个分裂的细胞必须在每个周期中加倍其内容，需要摄入的材料既用于生物合成，并提供能量来驱动吸能合成代谢反应。 因此，癌细胞必须增加代谢途径的活性，以产生能量和增加免疫力。 不同来源的癌细胞应显示细胞类型的代谢特征，并反映增殖的特定方面，以及这些如何取决于外部条件，如营养物质和氧气的供应。 我们建议通过NMR和MS使用稳定同位素辅助代谢组学，在细胞培养物和原位小鼠肿瘤模型（异种移植物）中确定特定癌症相关代谢途径在乳腺癌细胞（四种细胞系）的发展和进展中的相对重要性，如下所示。 目标1.通过定量同位素分析培养的正常和癌性人乳腺上皮细胞对缺氧和特定营养素的反应，确定能量产生途径和合成代谢的相对重要性。 前体（[U-13 C]-葡萄糖、[13 C-1]和[13 C-2]葡萄糖）和13 C/15 N谷氨酰胺可以通过糖酵解、戊糖磷酸途径、柠檬酸循环和主要回补反应、氨基酸代谢、核碱基和磷脂生物合成来区分相对通量。 将通过非雌激素敏感性MDAMB 231和雌激素敏感性ZR-75-1和MCF-7细胞中的这些主要途径的相对通量与正常人乳腺上皮细胞（HMEC）进行比较。 目标2.确定细胞培养物中鉴定的特异性通路活性在异种移植小鼠模型的实际肿瘤中存在的程度。 对携带源自非MDAMB 231和ZR-75-1细胞的乳腺脂肪垫肿瘤的雌性裸鼠静脉内给予13 C葡萄糖或13 C/15 N谷氨酰胺，并在经过足够的时间进行代谢后切除肿瘤。 分析血浆以监测标记前体的摄取和再分布。 通过与目标1中相同的技术提取和分析代谢物。 肿瘤模型与细胞培养的比较显示了肿瘤环境和组织相互作用如何决定不同途径对乳腺肿瘤进展的相对重要性。 生化信息将用于解释人类乳腺癌的代谢谱。 这些方法的发展将提供有关乳腺癌的基本生物化学信息，并且对于以改善早期肿瘤诊断为目标的未来人类受试者研究的设计至关重要。 公共卫生相关性：尽管在技术和筛查方面取得了许多进展，但乳腺癌仍然是人类癌症的主要形式（终身风险约为100%）。1/8）。 然而，尽管癌基因如Myc和Ras或肿瘤抑制因子如p53在癌症中经常上调，但这些因子在疾病发生和进展中的确切作用尚不完全清楚。 更深入地了解癌细胞的生化表型和它们在限定条件下的正常对应物是癌细胞基因组学分析的基本对应物。 我们正在开发技术来测量癌细胞中生化途径的活性，最终可以在临床环境中应用于人类肿瘤。 体内肿瘤模型与细胞培养的比较将显示肿瘤环境和组织相互作用如何决定不同代谢途径对乳腺肿瘤进展的相对重要性。 这些肿瘤生物化学的基础知识为肿瘤进展提供了丰富的假设来源，这些假设可以直接在实际乳腺癌组织中进行测试。 将这种方法应用于临床环境将为识别用于早期诊断、预后和最终药物反应的标记物集奠定基础。

DESCRIPTION (provided by applicant): Despite the numerous advances in technology and screening breast cancer remains the major form of human cancers (lifetime risk ca. 1/8). A neglected aspect of tumorigenesis is the metabolic profile of cells, which expresses the biochemical phenotype. Transformed cells differ from their normal counterparts by loss of critical regulatory functions that lead to immortalization, avoidance of apoptosis and deregulated cell cycling. A dividing cell must double its contents during each cycle, requiring intake of materials both for biosynthesis, and to provide the energy to drive endergonic anabolic reactions. Thus, cancer cells must increase the activity of metabolic pathways for energy production and anabolism. Cancer cells of different origin should display metabolism characteristic of the cell type, and reflect specific aspects of proliferation, and how these depend on the external conditions such as the supply of nutrients and oxygen. We propose to determine the relative importance of specific cancer-relevant metabolic pathways in the development and progression of breast cancer cells (four lines) both in cell culture and in an orthotopic mouse tumor model (xenografts), using stable isotope assisted metabolomics by NMR and MS, as follows. Aim 1. To determine the relative importance of energy producing pathways and anabolic metabolism by quantitative isotopomer analysis of normal and cancerous human breast epithelial cells in culture in response to hypoxia and specific nutrients. The precursors ([U-13C]-glucose, [13C-1] and [13C-2] glucose), and 13C/15N glutamine can differentiate relative fluxes through glycolysis, pentose phosphate pathways, citric acid cycle and major anaplerotic reactions, amino acid metabolism, nucleobase and phospholipid biosynthesis. Relative fluxes through these major pathways in non-estrogen-sensitive MDAMB231 and estrogen sensitive ZR-75-1 and MCF-7 cells will be compared with normal human mammary epithelial cells (HMEC). Aim 2. To determine the extent that the specific pathway activities identified in cell culture are present in actual tumors in a xenograft mouse model. Female nude mice bearing mammary fat pad tumors deriving from non MDAMB231 and ZR-75-1 cells are given 13C glucose or 13C/15N glutamine i.v. and the tumors excised after sufficient time has elapsed for metabolism to occur. Plasma is analyzed to monitor uptake and redistribution of labeled precursors. Metabolites are extracted and analyzed by the same techniques as in Aim 1. The comparison of the tumor model with cell culture shows how the tumor environment and tissue interactions determine the relative importance of different pathways to breast tumor progression. The biochemical information will be used to interpret the metabolic profiles of human breast cancers. The development of these approaches will provide basic biochemical information about breast cancer, and is essential for the design of future studies with human subjects, with the goal of improved diagnosis of early stage tumors. PUBLIC HEALTH RELEVANCE: Despite the numerous advances in technology and screening breast cancer remains the major form of human cancers (lifetime risk ca. 1/8). However, although oncogenes such as Myc and Ras or tumor suppressors such as p53 are frequently upregulated in cancers, the precise roles of these factors in disease initiation and progression is not completely clear. A deeper understanding of the biochemical phenotype of cancerous cells and their normal counterparts under defined conditions is an essential counterpart to genomics analyses of cancer cells. We are developing the technology to measure the activity of biochemical pathways in cancer cells that ultimately can be applied to human tumors in a clinical setting. The comparison of the in vivo tumor model with cell culture will show how the tumor environment and tissue interactions may determine the relative importance of different metabolic pathways to breast tumor progression. This fundamental knowledge of tumor biochemistry then provides a rich source of hypotheses about tumor progression that can be tested directly in actual breast cancer tissue. Application of this approach to a clinical setting will set the stage for identifying sets of markers for early diagnosis, prognosis and ultimately drug responses.