RNA Interference–Mediated Silencing of the Acetyl-CoA- Carboxylase-a Gene Induces Growth Inhibition and Apoptosis of Prostate Cancer Cells Koen Brusselmans, Ellen De Schrijver, Guido Verhoeven, and Johannes V. Swinnen Laboratory for Experimental Medicine and Endocrinology, University of Leuven, Leuven, Belgium Abstract Overexpression of lipogenic enzymes is a common character- istic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)–mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-A (ACC-A), the enzyme providing the malonyl- CoA substrate. Silencing of the ACC-a gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP pros- tate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-A or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prereq- uisite for cytotoxicity induced by inhibition of tumor- associated lipogenesis and suggest that in addition to FAS, ACC-A is a potential target for cancer intervention. (Cancer Res 2005; 65(15): 6719-25) Introduction Enhanced expression of lipogenic enzymes is increasingly recognized as a common characteristic of a wide variety of tumors (1–3). Overexpression of fatty acid synthase (FAS), a key lipogenic enzyme that catalyzes the terminal steps in the de novo biosynthesis of long-chain fatty acids (4), is found already in the earliest stages of tumor development (5, 6). In many tumor types, FAS overexpression is further pronounced as the tumor progresses towards a more advanced stage (2, 5, 7). In breast and prostate cancers, similar changes have been found in the expression of acetyl-CoA-carboxylase-a (ACC-a), the rate-limiting enzyme of fatty acid synthesis that catalyzes the condensation of malonyl-CoA using acetyl-CoA and CO 2 as precursors (1, 8). The finding that lipogenesis is low in nearly all nonmalignant adult tissues (9, 10), whereas it is up-regulated in many tumors, has led to the exploration of endogenous lipogenesis as a novel target for prevention and/or treatment of cancer. Up to now, nearly all attempts to examine this possibility have focused on FAS. Both chemical inhibitors of FAS (cerulenin, c75, Orlistat, EGCG) and the use of more selective approaches such as gene silencing with small interfering RNA (siRNA) targeting FAS have resulted in growth arrest and cell death in tumor cells (2, 11–21). Although it has been suggested that cytotoxicity induced by FAS inhibition is the result of accumulation of the toxic intermediate malonyl-CoA (22, 23), the exact mechanism by which inhibition of FAS induces tumor cell death remains a matter of debate. In the present work, we examined the effect of siRNA-mediated knockdown of ACC-a on prostate cancer cells and compared the effects with those of knockdown of FAS. It is shown that inhibition of ACC-a induces a similar growth arrest and tumor cell death as observed after blockage of FAS, despite the fact that there is no accumulation of malonyl-CoA. Materials and Methods Cell culture. The human LNCaP prostate cancer cell line was obtained from the American Type Culture Collection (Manassas, VA). Nonmalignant skin fibroblasts were provided by Prof. Dr. J.J. Cassiman (K.U. Leuven, Belgium). Cells were cultured at 37jC in a humidified incubator with a 5% CO 2 /95% air atmosphere in RPMI 1640 supplemented with 3 mmol/L L- glutamine and 10% FCS (Invitrogen, Carlsbad, CA). For experiments examining biological effects in the absence of androgens, charcoal-treated FCS was used to reduce background steroid levels. For analyzing cellular responses to androgens, R1881 (methyltrienolone; DuPont/NEN, Boston, MA) was dissolved in ethanol and added to the cultures. For culturing cells in the presence of palmitate-bovine serum albumin (BSA) complex, palmitate (Sigma, St. Louis, MO) was first complexed to fatty acid–free BSA (Invitrogen) as described (24, 25). Briefly, 4 volumes of a 4% BSA solution in 0.9% NaCl were added to 1 volume of 5 mmol/L palmitate in ethanol and incubated at 37jC for 1 hour, to obtain a 1 mmol/L stock solution of BSA-complexed palmitate. RNA interference. Transfection procedures with siRNA using Oligofect- amine (Invitrogen) have been described previously (17); siRNA oligonu- cleotides were purchased from Dharmacon (Lafayette, CO). Sequences of siRNA oligonucleotides targeting FAS and luciferase (Luc) have been described (17). The siRNA oligonucleotides targeting ACC-a were sense, CAAUGGCAUUGCAGCAGUGdTdT and antisense, CACUGCUGCAAUGC- CAUUGdTdT. RNA analysis. An 840-bp cDNA probe for ACC was synthesized by PCR on human cDNA (generated by reverse transcription as described; ref. 26) using 5V -TTCTCAGAGCTTCCGAACTTGCT and 5V -CTAACCTGGCTCTAC- CAACCAC as forward and reverse primer, respectively. PCR products were cloned into the pGEM-T vector (Promega, Madison, WI). Probes for FAS and 18S rRNA, RNA preparation, and Northern blot procedures have been described previously (26). 2- 14 C-acetate incorporation assay and TLC analysis. At 24, 48, 72, 96, or 120 hours after transfection with siRNA, 2- 14 C-labeled acetate (57 mCi/ mmol; 2 ACi/dish; Amersham International, Aylesbury, United Kingdom) was added to the cell culture medium. After 4 hours incubation, cells were collected by centrifugation and resuspended in 0.8 mL PBS. Lipids were extracted using the Bligh Dyer method as previously described (17); Note: K. Brusselmans and E. De Schrijver contributed equally to this work. Requests for reprints: Johannes V. Swinnen, Laboratory for Experimental Medicine and Endocrinology, Gasthuisberg, O&N niv 9, Herestraat 49, bus 902, B-3000, Leuven, Belgium. Phone: 32-16-345974; Fax: 32-16-345934; E-mail: johan.swinnen@med.kuleuven.be. I2005 American Association for Cancer Research. www.aacrjournals.org 6719 Cancer Res 2005; 65: (15). 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