ICANCER RESEARCH 56. 2745-2747. June 15. 19%| Advances in Brief Inhibition of Fatty Acid Synthesis Induces Programmed Cell Death in Human Breast Cancer Cells1 Ellen S. Pizer, Christian Jackisch, Fawn D. Wood, Gary R. Pasternack, Nancy E. Davidson, and Francis P. Kuhajda2 Division of Molecular Pathology. Department of Pathology ¡E.S.P.. F.D.W., G.R.P.. F.P.K.], ami Department of Oncology ¡C.J.,N.E.D.I. The Johns Hopkins Medical Institutions, Baltimore. Man-land 212X7 Abstract One of the key limiting factors in the treatment of advanced stage human epithelial malignancies is the lack of new, selective molecular targets for antineoplastic therapy. A substantial subset of human breast, ovarian, endometrial, coloréela!,and prostatic cancers express elevated levels of fatty acid synthase, the major enzyme required for endogenous fatty acid biosynthesis, and carcinoma lines are growth inhibited by cerulenin, a noncompetitive inhibitor of fatty acid synthase. We have shown previously that the difference in fatty acid biosynthesis between cancer and normal cells is an exploitable target for metabolic inhibitors in the in vitro setting and in vivo in a human ovarian carcinoma xenograft in nude mice. Here, we report that cerulenin treatment of human breast cancer cells inhibits fatty acid synthesis within 6 h after exposure, that loss of clonogenic capacity occurs within the same interval, and that DNA fragmentation and morphological changes characteristic of apoptosis ensue. Introduction The treatment of human epithelial malignancies is limited by drug resistance and toxic side effects of therapy, which contribute to ultimate treatment failure for the majority of advanced stage cancer victims. Identification of new, selective molecular targets for antine oplastic therapy provides an opportunity for therapeutic advancement. Although disordered intermediary metabolism in cancer cells has been known for the better part of this century ( 1), little attention has been paid to fatty acid metabolism. One early study showed elevated levels of fatty acid synthesis in tumor tissues, although the significance of the observations was not appreciated (2). We and others (3-10) have recently shown that some clinical human ovarian, endometrial, breast, colorectal, and prostatic cancers overexpress FAS.3 Tumor cells which express high levels of fatty acid synthesizing enzymes use endogenously synthesized fatty acids for membrane biosynthesis and also appear to export large amounts of lipid.4 In marked contrast, normal cells preferentially utilize dietary lipids. We have shown recently that these biochemical differences provide a selective target for metabolic inhibitors both in vitro and in vivo. Key observations are that: (a) inhibition of the ß-ketoacylsynthase site of FAS is selectively cytotoxic to cancer cells with increased fatty Received 2/12/96: accepted 4/25/96. The cosls of publication of this article were defrayed in part by Ihe 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. ' Supported by grants from the National Cancer Institute (CA57545), the American Institute for Cancer Research, the W. W. Smith Charitable Trust, and the Susan G. Komen Foundation. E. P. was supported by USPHS Grant T32 AI 07247 and by a grant from Ihe Steiler Research Fund. C. J. was supported by a fellowship from the Deutsche Forschungs- gemeinschap. 2 To whom requests for reprints should be addressed, at Department of Pathology. The Johns Hopkins Medical Institutions, 720 Rutland Avenue. Ross 512, Baltimore, MD 21205. 1The abbreviation used is: FAS, fatty acid synthase. 4 H. S. Heine. F. P. Kuhajda. F. D. Wood, A. Kayler, and G. R. Pasternack, Increased rates of fatty acid synthesis in breast cancer and identification of unique products, manuscript in preparation. acid biosynthesis but not to normal skin fibroblasts in vitro; (b) addition of palmitate, the major direct product of FAS, reverses the cytotoxic effects of its inhibition, demonstrating mechanistic spec ificity (11-14); (c) elevations in fatty acid synthase expression in carcinoma cell lines are comparable to levels in primary human tumors assessed by immunohistochemistry; (d) fatty acid synthetic activities of a carcinoma line grown in vitro or as a murine xenograft are similar, and are 4- to >20-fold higher than normal murine tissues; and (e) treatment with the specific FAS inhibitor, cerulenin, produces regression of established ascites tumor, reduc tion in ascites incidence, delay in onset of ascites, and significantly increased survival in a nude mouse xenograft model of human ovarian carcinoma (15). The mechanism by which inhibition of fatty acid synthesis pro duces its antitumor effect remains unexplained. The evidence pre sented here indicates that exposure of breast carcinoma cells to cerulenin produces inhibition of fatty acid synthesis within 6 h and produces irreversable lethal injury measured as a reduction of clono genic capacity in the same interval. Treated breast carcinoma cells subsequently undergo DNA fragmentation and morphological changes characteristic of apoptosis. Materials and Methods Cell Lines and Culture Conditions. ZR-75-1, SKBR3, and MCF-7 cells were maintained in RPMI 1640 with 10% fetal bovine serum except as otherwise indicated. Cultures were incubated at 37°Cin a humidified 5% CO2 atmosphere. A 5 mg/ml stock solution of cerulenin (Sigma Chemical Co.) in DMSO was diluted into experimental cultures to final concentrations of 5 or 10 fig/ml. Measurement of Endogenous Lipid Synthesis. ZR-75-1 cells were plated at 2 X IO5 cells/well in 24-well plates and incubated overnight prior to use. Cerulenin (Sigma Chemical Co.) in DMSO was added for indicated incubation times. Each well was pulse labeled with 0.1 /xCi [U-'4C]acetate during the final 2 hours of drug treatment and washed, and cellular lipids were extracted (13) and assayed for I4C by scintillation counting. Controls consisted of cells incubated with DMSO alone. All determinations were performed in triplicate with error bars representing the SE. Clonogenic Assay. After overnight incubation, 1 X 10" ZR-75-1, SKBR3, or MCF-7 cells were exposed to cerulenin (Sigma Chemical Co.) in DMSO (0.1-0.2%) for 6 h, washed, detached by trypsin digestion, counted, and replated at 1000 or 500 cells/60-mm plate in triplicate. Colonies were stained with crystal violet and counted 3-5 days after plating. Controls consisted of cells incubated with DMSO without cerulenin. Error bars represent the SE. DNA Fragmentation Assay and Morphological Evaluation. Exponen tially growing ZR-75-1 cells were plated at 1-5 x IO4 cells/cm2 in DMEM with 5% fetal bovine serum and 2 mM glutamine. After attachment, the medium was changed, and cells were incubated with or without cerulenin continuously until harvesting. At harvest, medium and trypsinized cells were combined, and DNA was extracted from pelleted cells and subjected to pulse field gel electrophoresis as described previously (16). Cells treated in parallel were spun onto glass slides, fixed in methanol, and stained with Hoechst 33342 for evaluation of morphological changes of apoptosis by fluorescence micros copy at 480 nm. 2745 Research. on September 20, 2015. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from