Metabolic Prolifing & Biochemical Analysis for Anti-Cancer Target Identification

描述(申请人提供)：由于细胞酶、转录因子和它们各自的基因以一种复杂和相互依赖的方式相互作用，细胞生理学并不仅仅由基因组决定。因此，靶向转化细胞的遗传异常可能不如直接靶向肿瘤改变的生理学那么有效。代谢谱通过确定主要代谢途径中的底物通量，可以揭示癌细胞的表型转化，并提供比信号转导和遗传研究单独的更详细的信息。转化剂通常会诱导高戊糖途径活性。快速增殖的乳腺癌细胞有很高的有氧糖酵解和谷氨酰胺分解速率，并增强了脂肪酸合成活性。这些改变的代谢通量表明，被认为对癌细胞增殖至关重要的酶和途径可能是药物开发的极佳靶点。在拟议的项目中，人乳腺癌细胞的细胞内代谢产物的浓度和通量将通过13C葡萄糖和谷氨酰胺生长的细胞的13C核磁共振谱来确定。生化系统分析，通过提供代谢产物流动的定量描述，将被用来揭示生长信号和代谢过程之间的联系。MCF7乳腺癌细胞的代谢图谱将通过使用13C核磁共振测定细胞内的通量来完成，条件与肿瘤中发现的条件相似。假设是转化的人类细胞中负责改变代谢通量的关键酶为新的抗癌药物提供了靶点。具体目的是：(1)识别在雌激素受体阳性(ER+)和阴性细胞(ER-)乳腺癌细胞中在模拟肿瘤条件下起作用的主要代谢途径。(2)定量测定乳腺癌细胞在有无他莫昔芬()和雌激素作用下的主次代谢通量和酶控制系数，包括那些可能被雌激素浓度升高逆转的代谢通量(“雌激素挽救”)，以及那些与ER结合无关的的作用。(3)通过研究脂肪酸合成、磷酸戊糖途径活性和其他需要NADP+/NADPH的途径之间的关系来研究雌激素和三苯氧胺的作用模式，并确定在雌激素和三苯氧胺存在的情况下，脂肪酸合成受阻(由脂肪酸合成酶抑制剂蓝蓝蛋白引起)对其他代谢途径的影响。

DESCRIPTION (provided by applicant): Because cellular enzymes, transcription factors, and their respective genes interact in a complex and interdependent manner, cellular physiology is not dictated by the genome alone. Targeting the genetic aberrations of transformed cells may therefore not be as effective as directly targeting the altered physiology of tumors. Metabolic profiling, by determining the substrate fluxes in major metabolic pathways, can reveal phenotypic transformations in cancer cells, and provide more detailed information than signal transduction and genetic studies alone. Transforming agents typically induce high pentose pathway activity. Rapidly proliferating breast cancer cells have high rates of aerobic glycolysis and glutaminolysis, and enhanced fatty acid synthesis activity. These altered metabolic fluxes suggest that enzymes and pathways identified as critical for cancer cell proliferation may be excellent targets for drug development. In the proposed project, the concentrations and fluxes of intracellular metabolites of human breast cancer cells will be determined from 13C NMR spectroscopy of cells grown on 13C glucose and glutamine. Biochemical systems analysis, by providing a quantitative description of metabolite flows, will be employed to reveal the link between growth signaling and metabolic processes. Metabolic profiling of MCF7 breast cancer cells will be accomplished by determination of intracellular fluxes using 13C NMR, under conditions mimicking those found in tumors. The hypothesis is that the crucial enzymes responsible for the altered metabolic fluxes in transformed human cells provide targets for new anti-cancer drugs. Specific aims are to: (1) Identify the primary metabolic pathways that are operative in estrogen receptor positive (ER+) and negative cells (ER-) breast cancer cells under conditions that mimic those of a tumor. (2) To quantify the primary and secondary metabolic fluxes and enzymes control coefficients in breast cancer cells in the presence and absence of tamoxifen (TAM) and estrogen, including those fluxes which may be reversed by increased concentrations of estrogen ("estrogen rescue"), and those effects of TAM that are not related to ER binding. (3) To examine modes of estrogen and tamoxifen action by investigating the relationship between fatty acid synthesis, pentose phosphate pathway activity, and other pathways requiring NADP+/NADPH, and to determine the effects of impaired fatty acid synthesis (resulting from the addition of the fatty acid synthase inhibitor, cerulenin) on other metabolic pathways in the presence of estrogen and tamoxifen.