Metabolic Prolifing & Biochemical Analysis for Anti-Cancer Target Identification

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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.