Estradiol stimulates the biosynthetic pathways of breast cancer cells: Detection by metabolic flux analysis

Abstract

Selective estrogen receptor (ER) modulators are highly successful breast cancer therapies, but they are not effective in patients with ER negative and selective estrogen receptor modulator (SERM)-resistant tumors. Understanding the mechanisms of estrogen-stimulated proliferation may provide a route to design estrogen-independent therapies that would be effective in these patients. In this study, metabolic flux analysis was used to determine the intracellular fluxes that are significantly affected by estradiol stimulation in MCF-7 breast cancer cells. Intracellular fluxes were calculated from nuclear magnetic resonance (NMR)-generated isotope enrichment data and extracellular metabolite fluxes, using a specific flux analysis algorithm. The metabolic pathway model used by the algorithm includes glycolysis, the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. The pathway model also incorporates mitochondrial compartmentalization and reversible trans-mitochondrial membrane reactions to more accurately describe the role of mitochondria in cancer cell proliferation. Flux results indicate that estradiol significantly increases carbon flow through the pentose phosphate pathway and increases glutamine consumption. In addition, intra-mitochondrial malic enzyme was found to be inactive and the malate-aspartate shuttle (MAS) was only minimally active. The inactivity of these enzymes indicates that glutamine is not oxidized within mitochondria, but is consumed primarily to provide biosynthetic precursors. The excretion of glutamine carbons from the mitochondria has the secondary effect of limiting nicotinamide adenine dinucleotide (NADH) recycle, resulting in NADH buildup in the cytosol and the excretion of lactate. The observed dependence of breast cancer cells on pentose phosphate pathway activity and glutamine consumption for estradiol-stimulated biosynthesis suggests that these pathways may be targets for estrogen-independent breast cancer therapies.

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.