[CANCER RESEARCH Vol. 62, 1966 –1970, April 1, 2002 Metabolic Markers of Breast Cancer: Enhanced Choline Metabolism and Reduced Choline-Ether-Phospholipid Synthesis 1 Rachel Katz-Brull, 2, 3 Dalia Seger, Dalia Rivenson-Segal, Edna Rushkin, and Hadassa Degani Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel ABSTRACT Specific genetic alterations during malignant transformation may in- duce the synthesis and breakdown of choline phospholipids, mediating transduction of mitogenic signals. The high level of water-soluble choline metabolites in cancerous breast tumors, relative to benign lesions and normal breast tissue, has been used as a diagnostic marker of malignancy. To unravel the biochemical pathways underlying this phenomenon, we used tracer kinetics and 13 C and 31 P magnetic resonance spectroscopy to compare choline transport, routing, and metabolism to phospholipids in primary cultures of human mammary epithelial cells and in MCF7 human breast cancer cells. The rate of choline transport under physiological choline concentrations was 2-fold higher in the cancer cells. The phospho- rylation of choline to phosphocholine and oxidation of choline to betaine yielded 10-fold higher levels of these metabolites in the cancer cells. However, additional incorporation of choline to phosphatidylcholine was similar in both cell types. Thus, enhanced choline transport and aug- mented synthesis of phosphocholine and betaine are dominant pathways responsible for the elevated presence of choline metabolites in cancerous breast tumors. Uniquely, reduced levels and synthesis of a choline-ether- phospholipid may also serve as a metabolic marker of breast cancer. INTRODUCTION Elevated concentrations of choline and choline metabolites (com- posite choline) were observed by MRS 4 in a variety of malignancies (1, 2). This elevation has been particularly useful for differentiating between malignant and benign breast lesions, because the former contain a significantly higher level of composite choline than the latter (3– 8). However, to fully exploit the diagnostic potential of breast MRS, it is necessary to elucidate the underlying biochemical mech- anisms leading to this metabolic phenomenon. Choline, a quaternary amine, is an essential nutrient supplied pre- dominantly by the diet (9, 10). The capacity to take up and secrete high levels of choline and choline metabolites is a central function of mammary epithelial cells. During lactation, these cells are capable of concentrating choline from the plasma and, subsequently, secreting milk that is rich in choline-containing metabolites, primarily PCho and GPCho (11). Active transport and diffusion are major mechanisms in the uptake of choline across cellular membranes. The diffusion capacity through membranes is related to the composition and special assembly of lipids, predominantly phospholipids and cholesterol, as well as of proteins. Routing of choline through its various metabolic pathways is cell and tissue specific (12). The intracellular metabolism of choline in the breast is partitioned among two major pathways: (a) synthesis of PtdCho; and (b) oxidation to produce the methyl donor betaine. Choline metabolism and choline-derived metabolites can undergo extensive alterations as a result of malignant transformations. Pro- gression of HMECs from a normal to a malignant phenotype was shown recently to be associated with a reversal in the ratio of PCho to GPCho, as well as an overall increase in the content of these two metabolites (13). PCho is a precursor of choline-derived phospholipids, as well as a product of their hydrolysis. The synthesis and degradation of phos- pholipids may be induced by growth factors that play a major role in malignant transformations (14, 15). The level of PCho in human breast cancer cells was found to be 10-fold higher than in HMECs (13, 16, 17). Moreover, high levels of PCho and other phosphomo- noesters were detected in human breast cancer biopsies and in patients in vivo (1, 18). The high levels of PCho correlated with up-regulation and increased activity of choline kinase, and choline kinase inhibitors exhibited antitumor activity (19, 20). High choline transport was suggested as the cause for the elevated levels of PCho in breast cancer (21). However, the exact role of PCho in malignant transformation and the involvement of other choline metabolites in this transforma- tion are not well understood. We present herein comparative studies of the metabolic steps that determine the nature and distribution of choline metabolites in normal and cancerous mammary epithelial cells. Detailed radioactive tracer measurements and model-based analyses yielded the mechanisms and kinetic parameters of choline transport. Additional metabolic studies using 13 C- and 31 P-MRS and 13 C-labeled choline enabled us to monitor the synthesis of PCho and betaine and characterize the composition and turnover of choline-derived phospholipids. Our results demonstrated enhanced transport of choline, augmented syn- thesis of PCho and betaine, and suppression of the synthesis of choline-derived ether lipids in breast cancer cells. MATERIALS AND METHODS Tissue Culture. MCF7 human breast cancer cells were cultured routinely in DMEM supplemented with 6% FCS and antibiotics, as described previously (21). T47D-clone 11 human breast cancer cells were routinely cultured in RPMI 1640 supplemented with 10% FCS, as described previously (17). Both media contained 28 M choline, with an additional 2 M choline from the serum. Primary cultures of HMECs were obtained from two sources: Clonetics Corp., San Diego, CA, which provided HMECs isolated from epithelial or- ganoids of human breast tissue; and our laboratory. The cells from Clonetics were cultured in serum-free mammary epithelial cell growth medium supple- mented with bovine pituitary extract, insulin, human epidermal growth factor, hydrocortisone, and antibiotics. For the second source of HMECs (HMEC*s), we isolated, separated, and cultivated the cells in our laboratory by processing breast tissue obtained from mammoplastic reduction surgery, as described previously (17). Choline Transport. MCF7 and HMEC cells were preincubated for 3 h in choline-free DMEM supplemented with 2% FCS. Transport experiments were initiated by adding various concentrations of choline (1– 400 M) to the above medium and trace amounts (1–2 Ci) of [methyl- 14 C]choline chloride (Sigma Received 8/24/01; accepted 1/23/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Israel Science Foundation and The Susan G. Komen Breast Cancer Foundation. H. D. is the incumbent of the Fred and Andrea Fallek Professorial Chair for Breast Cancer Research and heads the Willner Family Center for Vascular Biology. 2 To whom requests for reprints should be addressed, at Center for Advanced Imaging, W/CC-090, Beth Israel Deaconess Medical Center, One Deaconess Road, Boston, MA 02215. Phone: (617) 754-2009; Fax: (617) 754-2010; E-mail: rkatzbr@caregroup. harvard.edu. 3 Present address: Department of Radiology, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA 02215. 4 The abbreviations used are: MRS, magnetic resonance spectroscopy; PCho, phos- phocholine; GPCho, glycerophosphocholine; PtdCho, phosphatidylcholine; HMEC, hu- man mammary epithelial cell; NMR, nuclear magnetic resonance; NTP, nucleoside triphosphate. 1966 Research. on November 27, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from