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Stable isotopomer analysis of anabolic metabolic pathways in breast cancer

乳腺癌合成代谢途径的稳定同位素分析

基本信息

批准号：
7876986
负责人：
ANDREW N. LANE
金额：
$ 16.28万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7876986
关键词：
Accounting Aerobic Amino Acids Anabolism Apoptosis Biochemical Biochemical Pathway Biochemistry Biomass Breast Breast Cancer Cell Breast Cancer Detection Cancerous Cell Culture Techniques Cell Cycle Cell Proliferation Cells Characteristics Citric Acid Cycle Clinical Data Development Diagnostic Neoplasm Staging Disease Early Diagnosis Environment Epithelial Cells Estrogens Fatty acid glycerol esters Female Future Genomics Genotype Glucose Glutamine Glycolysis Goals Human Hypoxia Intake Knowledge Label Laboratories Lead MCF7 cell Malignant Neoplasms Mammary Neoplasms Mammary gland Mass Spectrum Analysis Measures Messenger RNA Metabolic Metabolic Pathway Metabolism Modeling Monitor Mus Nude Mice Nutrient Oncogenes Oxygen Pathway interactions Pentosephosphate Pathway Pharmaceutical Preparations Phenotype Phospholipid Metabolism Phospholipids Plasma Production Proteins Reaction Regulation Relative (related person)Role Sampling Solid Neoplasm Source Staging System TP53 gene Techniques Technology Testing Time Tissues Tracer Tumor Suppressor Proteins Tumor stage Tumor-Derived Warburg Effect Xenograft Model Xenograft procedure aminoacid biosynthesis cancer cell cell transformation cell type design human subject improved in vivo interest lifetime risk malignant breast neoplasm metabolomics mouse model neglect nucleobase outcome forecast public health relevance response stable isotope tumor tumor progression tumorigenesis uptake

项目摘要

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.

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，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新陈代谢，它表达了生化表型。转化细胞与正常细胞的不同之处在于失去了导致永生化的关键调控功能，避免了细胞凋亡，并放松了对细胞周期的调控。分裂细胞必须在每个周期内将其含量增加一倍，这既需要摄入用于生物合成的物质，也需要提供能量来驱动促合成反应。因此，癌细胞必须增加代谢途径的活性，以产生能量和合成代谢。不同来源的癌细胞应该表现出细胞类型的代谢特征，并反映出特定的增殖方面，以及这些方面如何依赖于外部条件，如营养物质和氧气的供应。我们建议使用核磁共振和质谱仪的稳定同位素辅助代谢组学，确定特定的癌症相关代谢途径在乳腺癌细胞(四个系)在细胞培养和原位小鼠肿瘤模型(异种移植)中的发展和进展中的相对重要性，如下所示。目的1.通过对培养的正常和癌变乳腺上皮细胞对缺氧和特定营养物质的反应进行定量同位素分析，确定能量产生途径和合成代谢途径的相对重要性。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、柠檬酸循环和主要的逆转录反应、氨基酸代谢、碱基和磷脂的生物合成来区分相对通量。在对雌激素不敏感的MDAMB231和对雌激素敏感的ZR-75-1和MCF-7细胞中，通过这些主要途径的相对通量将与正常的人乳腺上皮细胞(HMEC)进行比较。目的2.在异种移植小鼠模型中，确定在细胞培养中发现的特定通路活性在实际肿瘤中的存在程度。给荷有非MDAMB231和ZR-75-1乳腺脂肪垫肿瘤的裸鼠静脉注射13C葡萄糖或13C/15N谷氨酰胺。而肿瘤在新陈代谢发生足够的时间后被切除。分析血浆以监测标记前体的摄取和再分布。代谢产物的提取和分析采用与目标1相同的技术。肿瘤模型与细胞培养的比较表明，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。生化信息将被用来解释人类乳腺癌的代谢情况。这些方法的发展将提供有关乳腺癌的基本生化信息，对于设计未来的人类受试者研究至关重要，目的是改善早期肿瘤的诊断。与公共健康相关：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，虽然癌基因如Myc和RAS或肿瘤抑制基因如P53在癌症中经常上调，但这些因素在疾病发生和发展中的确切作用尚不完全清楚。更深入地了解癌细胞及其正常细胞在特定条件下的生化表型，是对癌细胞进行基因组学分析的关键。我们正在开发一项技术，以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。这种肿瘤生物化学的基础知识提供了丰富的关于肿瘤进展的假说来源，可以在实际的乳腺癌组织中直接测试。将这种方法应用到临床环境中，将为确定早期诊断、预后和最终药物反应的标志物集奠定基础。