Estradiol stimulates the biosynthetic pathways of breast cancer cells: Detection by metabolic flux analysis
Introduction
Selective estrogen receptor modulators (SERMs), of which tamoxifen is the most common, are successful breast cancer treatments (Katzenellenbogen and Frasor, 2004) that also decrease the incidence of breast cancer when administered preventatively (Fisher et al., 1998). However, SERMs are ineffective in patients with estrogen receptor (ER) negative (ER-negative) and SERM-resistant tumors (Hanstein et al., 2004; Howell et al., 1998; Lewis et al., 2004). Because tumors develop resistance to SERMs, adjuvant administration does not provide therapeutic benefit for longer than 5 years (Fisher et al., 2001; Lewis et al., 2004).
SERMs act primarily by suppressing endogenous estrogen stimulation of breast cancer cell proliferation (Katzenellenbogen and Frasor, 2004). Currently, the mechanisms by which estrogens stimulate proliferation are not completely known. However, much has been learned about the downstream effects of estrogens as a result of the sequencing of the human genome and advances in microarray technology (Frasor et al., 2003; Gruvberger et al., 2001). It has been shown that resistance to SERMs is correlated with an increased expression of ERs lacking the ligand binding region, rendering the receptors constituently active (Chaidarun and Alexander, 1998; Chan and Dowsett, 1997; Daffada et al., 1995; Desai et al., 1997). Understanding the mechanisms of estrogen stimulation may provide a route to design estrogen-independent therapies that could be effective in patients with ER-negative and SERM-resistant tumors (Katzenellenbogen and Frasor, 2004; Shen and Brown, 2003). Such therapies would be able to extend adjuvant therapy beyond 5 years (Lewis et al., 2004).
One of the difficulties in determining the mechanism of estrogen stimulation is that the expressions of many genes are modulated by estradiol (Frasor et al., 2003; Gruvberger et al., 2001), the most biologically active estrogen. While only a few genes are direct targets of the ER (Hanstein et al., 2004), several of the targets of the ER are transcription factors, including c-jun (Hyder et al., 1992, Hyder et al., 1998; Weisz and Rosales, 1990), c-fos (Hanstein et al., 2004; Weisz et al., 1990) and c-Myc (Dubik and Shiu, 1992; Hanstein et al., 2004; Rochefort, 1995), which each target an extensive list of genes.
Metabolic enzymes that are specifically stimulated by estradiol are potential drug targets. The stimulation of breast cancer cell proliferation by estradiol is known to be accompanied by an increase in intracellular metabolic activity (Neeman and Degani, 1989; Rivenzon-Segal et al., 2002), including the rates of glucose and glutamine consumption and the rate of lactate production (Furman et al., 1992). Estradiol also directly regulates the expression glucose-6-phosphate dehydrogenase (G6PDH; Thomas et al., 1990), the enzyme that directs glucose carbons into the pentose phosphate pathway. Several enzymes in the metabolic pathways stimulated by estradiol have been proposed as targets for anti-cancer agents (Boros et al., 2000), including transketolase (Cascante et al., 2002), G6PDH (Boren et al., 2002) and glutaminase (Lobo et al., 2000; Medina, 2001; Medina et al., 1992). For example, somatostatin, dehydroepiandrosterone-sulfate, and oxythiamine, which are inhibitors of G6PDH and transketolase, and antisense glutaminase mRNA have been shown to reduce cancer cell growth and are promising therapeutics (Boros et al., 1997, Boros et al., 1998, Boros et al., 2000; Cameron Smith et al., 2003; Lobo et al., 2000; Lora et al., 2004).
The objective of the present work is to use metabolic flux analysis to investigate the metabolic mechanisms of estradiol-stimulated breast cancer cell growth. To date, a comprehensive description of the intracellular pathways that are modulated by estrogenic stimulation is not available. Metabolic flux analysis is a technique used to quantify the intracellular metabolism of cells using: (1) a detailed description of intracellular metabolism; (2) the isotopic labeling patterns of intracellular metabolites; (3) the rates of nutrient consumption; and (4) a computer algorithm to interpret the isotopic data (Forbes et al., 2001). Isotope analysis has recently been used to identify enzymatic drug targets in cancer cells (Boren et al., 2002; Boros et al., 2000, Boros et al., 2003), to show that Gleevec inhibits ribose formation (Boren et al., 2001), to quantify the energy metabolism of tumor spheroids (Wehrle et al., 2000), and to quantify the metabolism of perfused livers (Lee et al., 2000, Lee et al., 2003b) and cultured hepatocytes (Chan et al., 2003a, Chan et al., 2003b, Lee et al., 2003a).
The metabolic pathway considered in the present work includes the reactions of primary energy metabolism and several pathways linked to biosynthesis, including the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. In addition, the metabolic pathway model incorporates both mitochondrial compartmentalization and reversible trans-membrane reactions to more accurately model the role of mitochondria in cancer cells. The computer algorithm used to calculate the intracellular fluxes was designed to employ isotopomer data and quantify reversible trans-membrane fluxes (Forbes et al., 2001). To determine the importance of fatty acid synthase (FAS) and lactate dehydrogenase, we inhibited each enzyme by addition of cerulenin and oxamate, respectively. In this work, we describe the use of these metabolic analysis techniques to determine the major enzymatic pathways that are triggered by estradiol.
Section snippets
Culture techniques
MCF-7, ER-positive, breast carcinoma cells were a kind gift from Dr. Gary Firestone (UC Berkeley). ER-positive cells were used to determine the effects of estradiol because ER-negative cells do not respond to estradiol stimulation. All cells were grown in Dulbecco's modified Eagle's medium with 5% fetal bovine serum (Sigma) in 850 cm2 roller bottles (Corning). A 5% CO2 headspace was maintained in the bottles, which were turned at 1 RPM on a roller apparatus (Bellco) and kept at 37 °C.
Cell growth
The average final cell concentrations (expressed as cells per cm2) after 156 h for the six treatments are shown in Fig. 3A. The increase in growth rate caused by estradiol addition was not significant over the time-scale of this experiment. However, in experiments of longer duration (data not shown), estradiol significantly increased cell growth, supporting the trends evident in Fig. 3A.
Extracellular fluxes
Specific metabolic production rates (in units of μmol/h/109 cell) at the time of extraction were determined by
Estradiol stimulation
Estradiol has a number of effects on the physiology of MCF-7 breast cancer cells. Here we have shown that it increases the consumption of glucose and glutamine, increases the production of lactate, and increases the flux through the pentose phosphate pathway. These metabolic effects result in a higher growth rate. Glucose is consumed to provide both biosynthetic precursors and the energy required for biosynthesis. Our results show that glutamine is consumed primarily to provide biosynthetic
Conclusions
We have shown that estradiol increases flux through the pentose phosphate pathway and increases the consumption of glutamine in MCF-7 breast cancer cells. We have also shown that in these cells intra-mitochondrial malic enzyme (f23) is inactive and the MAS (f9 and α1) is minimally active. Together these results show that glutamine is not oxidized in the mitochondria and suggest that it is instead consumed primarily to produce biosynthetic precursors. In these proliferating breast cancer cells,
Acknowledgments
The authors wish to thank Americo Peniflor, Astrid Oust, James Stapleton and Donald Boscoe for excellent technical assistance and are grateful to Drs. Camille Anderson, Brenta Fenton, and Christaino Migliorini for their many helpful discussions. This work was supported by the Department of Defense breast cancer program, Grant# DAMD 17-97-1-7053.
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