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步，但乳腺癌仍然是人类癌症的主要形式（终生风险约为1/8）。肿瘤发生的一个被忽视的方面是细胞的代谢谱，它表达生化表型。转化细胞与正常细胞的不同之处在于，它们失去了导致永生化、避免细胞凋亡和细胞周期失控的关键调控功能。一个分裂的细胞必须在每个循环中加倍它的含量，这既需要摄入生物合成的物质，也需要提供驱动内源性合成代谢反应的能量。因此，癌细胞必须增加能量产生和合成代谢的代谢途径的活性。不同来源的癌细胞应显示出细胞类型的代谢特征，并反映出增殖的特定方面，以及这些方面如何依赖于外部条件，如营养和氧气的供应。我们建议在细胞培养和原位小鼠肿瘤模型（异种移植物）中，使用稳定同位素辅助代谢组学（NMR和MS）确定特定癌症相关代谢途径在乳腺癌细胞（四系）发育和进展中的相对重要性，具体方法如下。目的1。通过定量同位素分析培养的正常和癌乳腺上皮细胞对缺氧和特定营养物质的反应，确定能量产生途径和合成代谢的相对重要性。前体（[U-13C]-葡萄糖、[13C-1]和[13C-2]葡萄糖）和13C/15N谷氨酰胺可以通过糖酵解、戊糖磷酸途径、柠檬酸循环和主要复变反应、氨基酸代谢、核碱基和磷脂生物合成来区分相对通量。非雌激素敏感的MDAMB231和雌激素敏感的ZR-75-1和MCF-7细胞通过这些主要途径的相对通量将与正常人乳腺上皮细胞（HMEC）进行比较。目标2。确定在细胞培养中鉴定的特定途径活性在异种移植小鼠模型的实际肿瘤中存在的程度。携带非MDAMB231和ZR-75-1细胞的乳腺脂肪垫肿瘤的雌性裸鼠给予13C葡萄糖或13C/15N谷氨酰胺静脉注射，并在足够的时间使代谢发生后切除肿瘤。分析血浆以监测标记前体的摄取和再分配。代谢物的提取和分析采用与Aim 1相同的技术。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。这些生化信息将用于解释人类乳腺癌的代谢特征。这些方法的发展将提供有关乳腺癌的基本生化信息，并且对于设计未来的人类受试者研究至关重要，其目标是改善早期肿瘤的诊断。公共卫生相关性：尽管在技术和筛查方面取得了许多进步，但乳腺癌仍然是人类癌症的主要形式（终生风险约为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。深入了解癌细胞及其正常细胞在特定条件下的生化表型是对癌细胞基因组学分析的必要对应。我们正在开发一种技术来测量癌细胞中生化途径的活性，最终可以应用于临床环境中的人类肿瘤。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。这种肿瘤生物化学的基本知识为肿瘤进展提供了丰富的假设来源，可以直接在实际的乳腺癌组织中进行测试。将这种方法应用于临床，将为早期诊断、预后和最终的药物反应确定一系列标记物奠定基础。

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.