Characterization of the hormone response of breast cancer by metabolic flux analysis

As a more complete picture of the genetic and enzymatic composition of cells becomes apparent, there is a growing need to describe how cellular regulatory elements interact with the cellular environment to affect cell physiology. Concurrently measuring multiple metabolic pathways and their interactions describes intracellular regulatory mechanisms including changes in enzyme and substrate concentrations, enzyme activation or inhibition, and ultimately genetic control. The response of the central metabolism of MCF7 breast cancer cells to estradiol was quantified to characterize their phenotype and identify potential enzyme targets for drugs to treat tumors unresponsive to anti-estrogen therapy.;Carbon fluxes were calculated from 13C-NMR isotopomer data using a rapid and novel solution method that employs isotopomer path tracing to significantly reduce the number of variables and hence the solution time. Reversible reactions were described by a new parameter, the association factor, which scales hyperbolically with the rate of metabolite exchange. The automated solution method evaluated numerous models and generated confidence intervals for the flux results. The method is capable of determining the intracellular metabolic fluxes in all types of cells, and can incorporate any hypothesized pathway model. The pathway model utilized in this work contained seven independent intracellular branches in addition to mitochondrial compartmentalization.;The flux results showed that 0.5 muM estradiol stimulated many metabolic pathways in MCF7 cells including glycolysis, the TCA cycle, the pentose phosphate pathway, and glutaminolysis. By inhibiting fatty acid synthase, it was determined that this enzyme's demand for NADPH does not affect the flux through the pentose phosphate pathway. The results also showed that the malate-aspartate shuttle and intra-mitochondrial malic enzyme are inactive in MCF7 cells. The inability of cancer cells to transport reducing equivalents into the mitochondria provides a link between aerobic glycolysis, glutaminolysis, and increased proliferation and may explain the long sought after Warburg effect. From our results we hypothesize that the elevated NADH level in the cytosol increases mitochondrial uptake of glutamine and production of aspartate and citrate, which are both biosynthetic precursors.;A few enzymes are over-expressed in cancer cells that induce glycolysis and raise NADH levels. These potential therapeutic targets include inducible phosphofructokinase-II (Chesney et al., 1999); the carbohydrate-response element (ChoRE), which shares a consensus binding-site with the hypoxia-inducible growth factor (HIF-1) and the cMYC oncogene (Dang and Semenza, 1999); and hexokinase II. Two enzymes necessary for glutamine consumption, glutaminase and the glutamate/aspartate anti-porter, are also potential targets.;These flux results represent the first attempt to analyze metabolic fluxes in breast cancer cells and to quantify the metabolic effects of estradiol. Many decades of research have shown that the mechanisms of cancer cell proliferation are highly multivariate and interdependent, and cannot be fully explained by reductive investigations of single pathways. Because metabolic flux analysis is an integrative approach that compares multiple functions, it can identify predominant functions that are critical for cancer cell proliferation. As more integrative methods are employed and a better understanding of cancer cell proliferation is determined, improved treatments may soon be developed.