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A Fatty Acid Synthase Blockade Induces Tumor Cell-cycle Arrest by Down-regulating Skp2

2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês

10.1074/jbc.m405061200

1083-351X

Lynn M. Knowles, Fumiko Axelrod, Cecille D. Browne, Jeffrey W. Smith,

Cancer, Hypoxia, and Metabolism

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins. In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins. Breast cancer is the second-leading cause of cancer death and morbidity for women in the United States (1Weir H.K. Thun M.J. Hankey B.F. Ries L.A. Howe H.L. Wingo P.A. Jemal A. Ward E. Anderson R.N. Edwards B.K. J. Natl. Cancer Inst. 2003; 95: 1276-1299Crossref PubMed Scopus (768) Google Scholar, 2McDonald J.A. Quade B.J. Broekelmann T.J. LaChance R.R. Forsman K. Hasegawa E. Akiyama S. J. Biol. Chem. 1987; 262: 2957-2967Abstract Full Text PDF PubMed Google Scholar). Although advances in early detection and treatment have led to a decline in mortality, the survival rate for patients with advanced-stage breast cancer is still low (1Weir H.K. Thun M.J. Hankey B.F. Ries L.A. Howe H.L. Wingo P.A. Jemal A. Ward E. Anderson R.N. Edwards B.K. J. Natl. Cancer Inst. 2003; 95: 1276-1299Crossref PubMed Scopus (768) Google Scholar). Consequently, there is still a great need to identify and validate new molecular targets for antitumor therapy. This study focuses on mammary carcinoma, where fatty acid synthase (FAS) 1The abbreviations used are: FAS, fatty acid synthase; siRNA, small interfering RNA; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; FP-PEG-TAMRA, fluorophosphonate-poly-ethyleneglycol-tetramethyl rhodamine.1The abbreviations used are: FAS, fatty acid synthase; siRNA, small interfering RNA; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; FP-PEG-TAMRA, fluorophosphonate-poly-ethyleneglycol-tetramethyl rhodamine. has attracted considerable attention as a potential drug target. Much of the interest in FAS stems from the fact that the enzyme is up-regulated in about 50% of breast cancers and is an indicator of poor prognosis (3Alo P.L. Visca P. Marci A. Mangoni A. Botti C. Di Tondo U. Cancer. 1996; 77: 474-482Crossref PubMed Scopus (273) Google Scholar, 4Alo P.L. Visca P. Trombetta G. Mangoni A. Lenti L. Monaco S. Botti C. Serpieri D.E. Di Tondo U. Tumori. 1999; 85: 35-40Crossref PubMed Scopus (85) Google Scholar, 5Wang Y. Kuhajda F.P. Li J.N. Pizer E.S. Han W.F. Sokoll L.J. Chan D.W. Cancer Lett. 2001; 167: 99-104Crossref PubMed Scopus (93) Google Scholar, 6Milgraum L.Z. Witters L.A. Pasternack G.R. Kuhajda F.P. Clin. Cancer Res. 1997; 3: 2115-2120PubMed Google Scholar, 7Alo P.L. Visca P. Botti C. Galati G.M. Sebastiani V. Andreano T. Di Tondo U. Pizer E.S. Am. J. Clin. Pathol. 2001; 116: 129-134Crossref PubMed Scopus (67) Google Scholar). FAS is the enzyme responsible for cellular synthesis of palmitate, the precursor of long-chain nonessential fatty acids (8Tsukamoto Y. Wong H. Mattick J.S. Wakil S.J. J. Biol. Chem. 1983; 258: 15312-15322Abstract Full Text PDF PubMed Google Scholar, 9Chirala S.S. Jayakumar A. Gu Z.W. Wakil S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3104-3108Crossref PubMed Scopus (67) Google Scholar, 10Rangan V.S. Joshi A.K. Smith S. Biochemistry. 2001; 40: 10792-10799Crossref PubMed Scopus (54) Google Scholar, 11Joshi A.K. Witkowski A. Smith S. Biochemistry. 1997; 36: 2316-2322Crossref PubMed Scopus (61) Google Scholar). FAS, which contains seven separate enzymatic pockets, is situated as a head-to-tail dimer with the ketoacyl synthase and malonyl/acetyl transferase domains of one monomer working together with the dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase domains on the adjacent monomer (8Tsukamoto Y. Wong H. Mattick J.S. Wakil S.J. J. Biol. Chem. 1983; 258: 15312-15322Abstract Full Text PDF PubMed Google Scholar, 9Chirala S.S. Jayakumar A. Gu Z.W. Wakil S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3104-3108Crossref PubMed Scopus (67) Google Scholar, 10Rangan V.S. Joshi A.K. Smith S. Biochemistry. 2001; 40: 10792-10799Crossref PubMed Scopus (54) Google Scholar, 11Joshi A.K. Witkowski A. Smith S. Biochemistry. 1997; 36: 2316-2322Crossref PubMed Scopus (61) Google Scholar). These enzymatic domains act sequentially to condense acetyl-CoA with malonyl-CoA to form a four-carbon intermediate. Six additional turns of the cycle of the enzyme convert this intermediate to palmitate, which is then liberated from FAS by the action of the thioesterase domain (12Mattick J.S. Nickless J. Mizugaki M. Yang C.Y. Uchiyama S. Wakil S.J. J. Biol. Chem. 1983; 258: 15300-15304Abstract Full Text PDF PubMed Google Scholar). Because FAS functions as a head-to-tail dimer, targeted inhibition of one of the enzymatic domains of FAS can ablate the activity of one or both FAS subunits (10Rangan V.S. Joshi A.K. Smith S. Biochemistry. 2001; 40: 10792-10799Crossref PubMed Scopus (54) Google Scholar, 11Joshi A.K. Witkowski A. Smith S. Biochemistry. 1997; 36: 2316-2322Crossref PubMed Scopus (61) Google Scholar). Cerulenin, a natural product, is an antagonist of the ketoacyl synthase domain (the condensing enzyme) of FAS and functions by covalently modifying the active site cysteine, resulting in dead-end inhibition (13Moche M. Schneider G. Edwards P. Dehesh K. Lindqvist Y. J. Biol. Chem. 1999; 274: 6031-6034Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). c75, a synthetic analog of cerulenin, also targets the condensing enzyme and inhibits fatty acid synthesis (14Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Crossref PubMed Scopus (501) Google Scholar). The inhibition of FAS by either cerulenin or c75 can suppress tumor cell proliferation and, in some cases, can induce tumor cell apoptosis (14Kuhajda F.P. Pizer E.S. Li J.N. Mani N.S. Frehywot G.L. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3450-3454Crossref PubMed Scopus (501) Google Scholar, 15Kuhajda F.P. Jenner K. Wood F.D. Hennigar R.A. Jacobs L.B. Dick J.D. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6379-6383Crossref PubMed Scopus (569) Google Scholar, 16Furuya Y. Akimoto S. Yasuda K. Ito H. Anticancer Res. 1997; 17: 4589-4593PubMed Google Scholar, 17Pizer E.S. Jackisch C. Wood F.D. Pasternack G.R. Davidson N.E. Kuhajda F.P. Cancer Res. 1996; 56: 2745-2747PubMed Google Scholar, 18Pizer E.S. Chrest F.J. DiGiuseppe J.A. Han W.F. Cancer Res. 1998; 58: 4611-4615PubMed Google Scholar, 19Li J.N. Gorospe M. Chrest F.J. Kumaravel T.S. Evans M.K. Han W.F. Pizer E.S. Cancer Res. 2001; 61: 1493-1499PubMed Google Scholar, 20Heiligtag S.J. Bredehorst R. David K.A. Cell Death Differ. 2002; 9: 1017-1025Crossref PubMed Scopus (48) Google Scholar). These observations support the contention that FAS is a relevant drug target in oncology. However, both cerulenin and c75 are now known to have other molecular targets (21Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9498-9502Crossref PubMed Scopus (212) Google Scholar, 22Lawrence D.S. Zilfou J.T. Smith C.D. J. Med. Chem. 1999; 42: 4932-4941Crossref PubMed Scopus (79) Google Scholar, 23De Vos M.L. Lawrence D.S. Smith C.D. Biochem. Pharmacol. 2001; 62: 985-995Crossref PubMed Scopus (27) Google Scholar), so searches for additional antagonists of FAS with better selectivity and distinct mechanisms of action are certainly warranted. We recently reported (24Kridel S.J. Axelrod F. Rozenkrantz N. Smith J.W. Cancer Res. 2004; 64: 2070-2075Crossref PubMed Scopus (448) Google Scholar) that Orlistat, a drug approved for treating obesity, is a rather potent and selective inhibitor of FAS in prostate carcinoma cells. The drug elicits its effects by inhibiting the thioesterase domain of FAS, which is responsible for releasing palmitate from the acyl carrier protein of the enzyme. By virtue of this activity, Orlistat slowed the growth of xenograft tumors of PC-3 prostate carcinoma cells in mice (24Kridel S.J. Axelrod F. Rozenkrantz N. Smith J.W. Cancer Res. 2004; 64: 2070-2075Crossref PubMed Scopus (448) Google Scholar). The objective of the present study was two-fold. First, we sought to use an independent strategy to confirm that the antiproliferative effects of Orlistat result from inhibition of FAS. Second, we sought to elucidate the mechanism by which a FAS blockade interferes with tumor cell proliferation. Both Orlistat and small interfering RNA (siRNA)-targeting FAS cause a dramatic down-regulation of Skp2, a component of an E3 ubiquitin ligase that tags p27Kip1 for degradation by the proteasome. These findings mechanistically connect two biochemical pathways being explored as drug targets in cancer, fatty acid biosynthesis, and ubiquitin-dependent proteolysis. Cell Lines—The MDA-MB-435 cell line was obtained from Janet Price at the University of Texas Southwestern Medical Center. MDA-MB-231 and MCF7 were purchased from American Type Culture Collection (Manassas, VA). Tumor cells were maintained in minimal Eagle's media, Earle's salts (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (Irvine Scientific), 2 mm l-glutamine (Invitrogen), minimal Eagle's media vitamins (Invitrogen), nonessential amino acids (Irvine Scientific) and antibiotics (Omega Scientific, Inc., Tarzana, CA). Profiling Serine Hydrolase Activity in Mammary Epithelial Cells— Cultured cells were washed with ice-cold phosphate-buffered saline (PBS), harvested with a cell scraper, and collected by centrifugation. Cell pellets were resuspended in 50 mm Tris-HCl (pH 8.0) and lysed by sonication as described previously (25Liu Y. Patricelli M.P. Cravatt B.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14694-14699Crossref PubMed Scopus (805) Google Scholar, 26Kidd D. Liu Y. Cravatt B. Biochemistry. 2001; 40: 4005-4015Crossref PubMed Scopus (305) Google Scholar). The soluble and insoluble cell fractions were separated by ultracentrifugation at 64,000 rpm for 1 h at 4 °C. Protein concentrations were determined by using the BCA assay kit from Pierce. The resulting extracts were diluted in lysis buffer to yield a 1 mg/ml final protein concentration. The fluorophosphonate probes FP-PEG-TAMRA (fluorophosphonate-poly-ethyleneglycol-tetramethyl rhodamine), FP-PEG-BODIPY, and FP-PEG-biotin were synthesized and generously provided by Activx Biosciences (La Jolla, CA). Serine hydrolase activity was examined by incubating the soluble cell fractions (40 μl) with FP-PEG-TAMRA (2 μm) for 1 h at room temperature. Nonspecific labeling was monitored by denaturing samples for 10 min at 100 °C prior to labeling with FP-PEG-TAMRA. The reaction was terminated by the addition of 2× Laemmli sample buffer, boiled for 5 min, and resolved by SDS-10% PAGE. Fluorescent-labeled hydrolases were visualized at 605 nm using a Hitachi flatbed scanner and quantitated with Image Analysis (MiraiBio, Alameda, CA). Purification and Identification of Serine Hydrolases by Avidin-biotin Affinity Chromatography and Matrix-assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry—Serine hydrolases were identified using the FP-PEG-biotin probe (26Kidd D. Liu Y. Cravatt B. Biochemistry. 2001; 40: 4005-4015Crossref PubMed Scopus (305) Google Scholar). Briefly, cell lysates were preabsorbed with avidin-agarose to reduce nonspecific binding of proteins during purification. Lysates were labeled with FP-PEG-biotin (5 μm) at room temperature for 1 h, after which proteins were separated from unincorporated FP-PEG-biotin by gel filtration on Nap 25 columns. After the addition of 0.5% SDS, the eluate was boiled for 10 min to denature proteins. Samples were diluted with 50 mm Tris (pH 7.5) and 150 mm NaCl and absorbed with avidin-agarose for 1 h at room temperature. Avidin-agarose beads were pelleted by centrifugation and washed 8× with 50 mm Tris (pH 7.5), 150 mm NaCl, and 1% Tween 20. Labeled proteins were eluted with 2× sample buffer, resolved by SDS-10% PAGE, and detected by silver staining. Specific bands were extracted and subjected to in-gel trypsin digestion and peptide mass fingerprinting with MALDI-TOF using methods described previously (27Landry F. Lombardo C.R. Smith J.W. Anal. Biochem. 2000; 279: 1-8Crossref PubMed Scopus (83) Google Scholar, 28Harvey S. Zhang Y. Landry F. Miller C. Smith J.W. Physiol. Genomics. 2001; 5: 129-136Crossref PubMed Scopus (30) Google Scholar). Inhibition of Serine Hydrolase Activity with Orlistat—Orlistat was extracted from Xenical™ capsules (Roche Applied Science) by solubilizing each pill in 1 ml of ethanol. Insoluble product was removed by centrifugation (14,000 rpm for 5 min). The supernatant yielded a solution of Orlistat (250 mm), which was aliquoted and stored at -80 °C. Soluble cell extracts (40 μl) were incubated with Orlistat (0–1 μm) for 20 min prior to FP-PEG-TAMRA addition. Lysates were labeled with FP-PEG-TAMRA, and reactions were terminated and processed as described above. The final concentration of Me2SO or ethanol in each reaction was 10%. Gene Silencing Using siRNA—FAS siRNA sequences corresponding to 5′-CAA CTA CGG CTT TGC CAA T (nucleotides 6213–6231), 5′-GCA ACT CAC GCT CCG GAA A (nucleotides 6657–6675), 5′-GCC CTG AGC TGG ACT ACT T (nucleotides 6146–6164), and 5′-GGT ATG CGA CGG GAA AGT A (nucleotides 7515–7533) were custom designed and pooled together by Dharmacon (Lafayette, CO). MDA-MB-435 cells were plated at 3.125 × 104/cm2 in 6-cm plates for 24 h prior to transfection with 100 nm FAS, Skp2 (Dharmacon Smartpool M-003324–01) or scrambled control (Dharmacon D-001206–13) siRNA in Opti-MEM medium (Invitrogen) using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were placed into normal culture medium 6 h post-transfection and grown for an additional 42 h. Fatty Acid Biosynthesis—The incorporation of [14C]malonyl CoA into cellular fatty acids was measured according to published methods (15Kuhajda F.P. Jenner K. Wood F.D. Hennigar R.A. Jacobs L.B. Dick J.D. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6379-6383Crossref PubMed Scopus (569) Google Scholar). Briefly, MDA-MB-435 cells were harvested with a cell scraper and centrifuged at 2,000 rpm for 5 min and then frozen at -80 °C. Cell pellets were hypotonically lysed in 20 mm Tris (pH 7.5), 1 mm dithiothreitol, and 1 mm EDTA, and insoluble material was removed by centrifugation (14,000 rpm) for 15 min at 4 °C. Lysates (380 μg) were exposed to Orlistat (0.1–10 μm) or vehicle at 25 °C for 1 h. Lysates in 80-μl volumes were added to 520 μl of solution containing 581 μm NADPH, 193 μm acetyl CoA, and 116 mm KCl (pH 6.6). Reactions were mixed with 0.4 μCi [2-14C]malonyl CoA (Amersham Biosciences) for 25 min at 37 °C. Cold malonyl CoA (208 μm) was added to reaction mixtures, which were incubated for an additional 15 min at 37 °C. Reactions were terminated by the addition of chloroform:methanol (1:1). The chloroform extracts were dried under N2 and extracted with water-saturated butanol. The butanol extract was evaporated under N2, and labeled fatty acids were quantified by scintillation counting. The identity of the labeled fatty acid was verified by comparison to a palmitate standard on thin layer chromatography. Briefly, lipid extracts from Orlistat and vehicle-treated lysates of MDA-MB-435 cells were resuspended in 40 μl of chloroform, spotted on silica gel (EM Science), and chromatographed in hexane/diethyl ether/acetic acid (45:5:1). Tritiated palmitate (PerkinElmer Life Sciences) and cold palmitate (Sigma) were used as standards. Chromatographed lipids were detected by exposing the plate in iodine vapor and on Biomax film (Kodak). Cell Proliferation Assays—Cells were plated at 6.25 × 104/cm2 in 96-well plates for 24 h. Cells were washed with PBS and incubated in serum-free RPMI 1640 medium for 24 h prior to the addition of Orlistat (0–100 μm). Cell proliferation was measured 72 h later using the cell proliferation BrdU ELISA kit (Roche) according to the manufacturer's directions. Cell Synchronization and Cell-cycle Analysis—Tumor cells were plated at 6.25 × 104/cm2 in 6-well plates for 24 h, washed with PBS, and serum-starved for an additional 24 h. M phase synchronization was achieved by treating cells with 100 nm nocodazole (Sigma) for 16 h. Synchronized cells were treated with Orlistat (0–50 μm) immediately after release from the block and harvested at various times over 24 h. Cells were collected by trypsinization, rinsed in cold PBS, fixed in 70% ethanol, and stored at -20 °C. Cellular DNA was stained by the addition of PBS containing 200 units/ml of RNase (Roche) and 18 μg/ml of propidium iodide (Molecular Probes, Eugene, OR). Fluorescence was monitored on 15,000 cells per sample using a BD FACSort tabletop cytometer (BD Biosciences). Data were analyzed with Modfit LT software (Verity Software House, Topsham, ME). Western Blot Analysis—Cells were synchronized with nocodazole, treated with Orlistat (0–50 μm), harvested by trypsinization, and rinsed with PBS. Pellets were lysed in 2× SDS sample buffer, passed 20 times through an 18-gauge needle, boiled for 5 min, and stored at -80 °C. Insoluble material was removed by centrifugation (14,000 rpm) for 10 min at 4 °C, and protein was separated by SDS-PAGE. After electrophoresis, protein was transferred onto nitrocellulose and probed overnight at 4 °C with anti-FAS (BD Transduction Laboratories, San Jose, CA), anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-tubulin (Santa Cruz Biotechnology), anti-Rb (Santa Cruz Biotechnology), anti-phospho-Rb (Ser-780; Cell Signaling Technology, Beverly, MA), anti-phospho-Rb (Ser-795; Cell Signaling Technology), anti-phospho-Rb (Ser-907/811; Cell Signaling Technology), and anti-p45 Skp2 (Zymed Laboratories, Inc., South San Francisco, CA). Immunoreactivity was detected by using anti-mouse or anti-rabbit IgG-conjugated peroxidase and visualized by enhanced chemiluminescence. Serine Hydrolase Profile of Normal and Neoplastic Mammary Epithelial Cells—Activity-based protein profiling was used to visualize the profile of active serine hydrolases present in mammary carcinoma cells. We used an activity-based probe of serine hydrolases consisting of a fluorophosphonate moiety linked to tetramethyl rhodamine (FP-PEG-TAMRA). The fluorophosphonate warhead tags the active site serine of serine hydrolases, forming an adduct that is stable to SDS gel electrophoresis (29Patricelli M.P. Giang D.K. Stamp L.M. Burbaum J.J. Proteomics. 2001; 1: 1067-1071Crossref PubMed Scopus (235) Google Scholar). Activity profiles of three human breast cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-435, were compared (Fig. 1). In each case, more than 20 active serine hydrolases were detected as fluorescent bands on the SDS gel. Pre-heating the sample to denature the enzymes in the lysate eliminated reaction with the FP-PEG-TAMRA probe. The identity of many of these enzymes was determined with mass spectrometry. They included, among others, dipeptidyl peptidases 7 and 9, prolyloligopeptidase, lysophospholipase-1, and FAS. Orlistat Suppresses Tumor Cell Proliferation by Interfering with G1/S Progression—Our previous work (24Kridel S.J. Axelrod F. Rozenkrantz N. Smith J.W. Cancer Res. 2004; 64: 2070-2075Crossref PubMed Scopus (448) Google Scholar) showed that Orlistat inhibits FAS in prostate carcinoma cells and that such inhibition slows their growth in vivo. Given the presence of FAS in mammary carcinoma cells (Fig. 1), we conducted studies to determine whether Orlistat would inhibit their proliferation. Studies were conducted to determine whether Orlistat interferes with the proliferation of mammary carcinoma cells. Cells were incubated with Orlistat for 72 h, and DNA synthesis was measured by incorporation of bromodeoxyuridine. Orlistat inhibited proliferation of the MCF7, MDA-MB-231, and MDA-MB-435 cell lines (Fig. 2A). Slight differences were observed in the response of each cell line to Orlistat. Although proliferation of the MDA-MB-231 cells was completely inhibited by Orlistat, proliferation of the MDA-MB-435 cells was knocked down by 70–80%, and proliferation of the MCF7 cells was suppressed by ∼50%. To determine the effect of Orlistat on cell-cycle progression, we used synchronized cultures of MDA-MB-435 cells. Cells were synchronized in the M phase using a nocodazole block. After release of the block, cells were exposed to a saturating concentration of Orlistat and analyzed for the distribution of cells in the G1 and S phases every 4 h by flow cytometry. Orlistat dramatically slowed the entry of the cells into S phase compared with untreated cells (Fig. 2B). The amplitude of the G1 peak in Orlistat-treated cells declines slowly after a period of several hours. This decline in the G1 population results partially from apoptosis 2L. M. Knowles and J. W. Smith, unpublished observation. and also partially because the blockade is leaky. Similar results were obtained when cells were synchronized at the G1/S border using thymidine, released from the block, and allowed to progress through the next cell cycle (data not shown). Orlistat and Anti-FAS siRNA Block Fatty Acid Biosynthesis—We sought an independent means of inhibiting FAS to solidify the role of this enzyme in regulating tumor cell proliferation. Therefore, we compared Orlistat and siRNA-targeting FAS for the ability to knock down the activity of the enzyme in MDA-MB-435 mammary carcinoma cells (Fig. 3). As expected, Orlistat was without effect upon the level of FAS (Fig. 3A, left panel) but did ablate the activity of the thioesterase domain as indicated with the activity-based probe FP-PEG-TAMRA (Fig. 3A, right panel). The siRNA-targeting FAS reduced the level of FAS protein (Fig. 3B, left panel) and thereby reduced the labeling of the enzyme with the activity-based probe (Fig. 3B, right panel). Both antagonists also blocked the synthesis of palmitate, the end product of FAS, as indicated by reductions to the incorporation of [14C]malonyl CoA into fatty acids (Fig. 3, C and D). An FAS Blockade Alters the Key Regulatory Steps in the Retinoblastoma Protein Pathway—We examined the effects of a FAS blockade on key regulatory steps in the retinoblastoma protein (Rb) protein pathway, a primary regulator of the G1/S transition (30Brown V.D. Phillips R.A. Gallie B.L. Mol. Cell. Biol. 1999; 19: 3246-3256Crossref PubMed Scopus (94) Google Scholar). This analysis included measures of (i) the phosphorylation status of Rb, a parameter that governs the interaction of this protein with E2F-1 and subsequent entry into S phase (30Brown V.D. Phillips R.A. Gallie B.L. Mol. Cell. Biol. 1999; 19: 3246-3256Crossref PubMed Scopus (94) Google Scholar); (ii) p27Kip1, which negatively regulates cyclin-dependent kinase activity (31Loden M. Nielsen N.H. Roos G. Emdin S.O. Landberg G. Oncogene. 1999; 18: 2557-2566Crossref PubMed Scopus (45) Google Scholar); and (iii) Skp2, a protein component of the E3 ubiquitin ligase that regulates degradation of p27Kip1 (32Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell. Biol. 1999; 1: 193-199Crossref PubMed Scopus (1323) Google Scholar). The effect of a concentration range of Orlistat on each of these parameters was measured by Western blotting (Fig. 4A). Orlistat reduced phosphorylation of the Rb protein, up-regulated p27Kip1, and down-regulated Skp2. These effects were evident at levels of the drug consistent with the cellular IC50 of Orlistat for inhibition of the FAS thioesterase (∼1–3 μm). To independently verify that the effects of Orlistat could be attributed to its ability to block FAS, similar experiments were conducted with siRNA-targeting FAS (Fig. 4B). Like Orlistat, the siRNA-targeting FAS decreased the phosphorylation of Rb, increased the level of p27Kip1, and reduced the level of Skp2. An siRNA-targeting Skp2 had identical effects upon its downstream target, p27Kip1, and upon the phosphorylation status of the Rb protein. Together, these findings provide strong support for the idea that a FAS blockade acts upon the Rb pathway via regulation of Skp2 and also indicate that a FAS blockade is likely to have effects similar to an Skp2 blockade. Results from this study reinforce the idea that FAS is a relevant drug target in oncology and further support the notion that FAS is the relevant target for Orlistat in tumor cells. The inhibitory effects of Orlistat on FAS are rather unexpected, because the drug has been studied for more than 15 years with no mention of the effects upon fatty acid synthesis. The effects of the drug on FAS are likely to have been overlooked because Orlistat is administered orally but is not significantly absorbed into the bloodstream. The drug acts in preventing absorption of dietary fat by inhibiting pancreatic lipase (another serine hydrolase) in the digestive tract (33Hadvary P. Sidler W. Meister W. Vetter W. Wolfer H. J. Biol. Chem. 1991; 266: 2021-2027Abstract Full Text PDF PubMed Google Scholar, 34Hauptman J.B. Jeunet F.S. Hartmann D. Am. J. Clin. Nutr. 1992; 55: 309S-313SCrossref PubMed Scopus (173) Google Scholar). The results of the present study indicate that Orlistat and other β-lactones should be considered to be a promising class of thioesterase antagonists that could be exploited for antitumor therapy. The evidence supporting the conclusion that the antiproliferative effects of Orlistat are mediated by inhibition of FAS is as follows: first, our activity-based protein-profiling experiments indicate that FAS is the only serine hydrolase target for Orlistat in the breast cancer cell lines. We have noted three other bands with Mr ∼ 90–150 kDa that are also inhibited by Orlistat, but these seem to be breakdown products of FAS because they can be immunoprecipitated with anti-FAS antibody and because they are knocked down with siRNA-targeting FAS (see Fig. 3B). Second, Orlistat blocks the incorporation of [14C]malonyl CoA into palmitate, a biosynthetic reaction mediated by FAS. Third, the effects of Orlistat on tumor cell proliferation and regulation of Rb, p27Kip1, and Skp2 all occur at concentrations of the drug that approximate its cellular IC50 for FAS (between 1 and 3 μm). Fourth, our prior work shows that Orlistat directly inhibits the activity of the recombinant thioesterase of FAS (24Kridel S.J. Axelrod F. Rozenkrantz N. Smith J.W. Cancer Res. 2004; 64: 2070-2075Crossref PubMed Scopus (448) Google Scholar). Fifth, the effects of Orlistat on tumor cell proliferation and on regulation of Rb and p27Kip1 are mimicked by siRNA-targeting FAS. The present study also provides a new insight into the mechanisms underlying the connection between FAS and tumor cell proliferation. Orlistat arrests the cell cycle at the G1/S transition. This effect was noted in all breast cancer cells we tested, along with tumor cells derived from the prostate (not shown). These findings suggest that the block in G1/S progression is a common mechanism mediating the antiproliferative effects of Orlistat. Substantial decreases in DNA synthesis have likewise been noted in response to cerulenin and c75, other antagonists of FAS (19Li J.N. Gorospe M. Chrest F.J. Kumaravel T.S. Evans M.K. Han W.F. Pizer E.S. Cancer Res. 2001; 61: 1493-1499PubMed Google Scholar). The G1/S cell-cycle arrest elicited by a FAS blockade is mediated through the Rb pathway. Cells treated with either Orlistat or siRNA-targeting FAS show decreased phosphorylation of Rb and increased levels of the complex between Rb and E2F-1 (data not shown). The increased association of these two proteins prevents the transcriptional activity of E2F-1 essential for entry into S phase. We have traced the effects of a FAS blockade upstream of Rb and found such a blockade to alter the levels of p27Kip1 and Skp2. p27Kip1 acts as a negative regulator of the cyclin-dependent kinases that ultimately phosphorylate Rb and, therefore, p27Kip1 acts as a negative regulator of G1/S transition (35Rank K.B. Evans D.B. Sharma S.K. Biochem. Biophys. Res. Commun. 2000; 271: 469-473Crossref PubMed Scopus (30) Google Scholar). Inhibition of FAS led to increases in p27Kip1 protein levels without affecting its transcription (data not shown), indicating a stabilization of p27Kip1. Interestingly, inhibition of FAS also substantially reduces the levels of Skp2, an F-box protein essential for proteasome degradation of p27Kip1. Like siRNA-targeting FAS, an siRNA-targeting Skp2 also increased p27Kip1 levels and blocked phosphorylation of Rb. Consequently, we conclude that inhibition of FAS acts upstream of the proteasome to control p27Kip1 levels and ultimately blocks cell-cycle progression. The mechanistic connections between the FAS blockade by Orlistat and reductions in Skp2 are the subject of current investigations. The observations in this report are different from prior work in which cerulenin and c75 were employed as FAS inhibitors. These compounds block both G1/S and G2/M progression (16Furuya Y. Akimoto S. Yasuda K. Ito H. Anticancer Res. 1997; 17: 4589-4593PubMed Google Scholar, 19Li J.N. Gorospe M. Chrest F.J. Kumaravel T.S. Evans M.K. Han W.F. Pizer E.S. Cancer Res. 2001; 61: 1493-1499PubMed Google Scholar, 36Pizer E.S. Wood F.D. Pasternack G.R. Kuhajda F.P. Cancer Res. 1996; 56: 745-751PubMed Google Scholar). We found no evidence that FAS blockade with Orlistat affected the G2/M transition point. There are two potential explanations for this difference in effect. One possibility is that inhibition of the different enzymatic pockets of FAS elicits distinct downstream effects. Although we cannot definitively exclude this possibility, it is difficult to envision how antagonists of distinct enzymatic pockets, each leading to inhibition of product formation, could elicit different effects. Another possibility is that cerulenin and c75 bind to additional molecular targets that account for the effects upon G2/M. In this regard, we have begun to analyze clonal variants of MDA-MB-435 cells resistant to Orlistat. We have found these cells to retain sensitivity to cerulenin (data not shown), a finding that lends support to the idea that these two compounds have different mechanisms of action. It is now clear that several avenues for antitumor therapy converge at the G1/S transition. Recent work indicates that Skp2 is one of the important G1/S regulatory points because it is necessary for ubiquitin-dependent degradation of p27Kip1 (32Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell. Biol. 1999; 1: 193-199Crossref PubMed Scopus (1323) Google Scholar). Consequently, Skp2 is a positive regulator of cell-cycle progression. In fact, Skp2 has even been suggested as a potential drug target (37Schulman B.A. Carrano A.C. Jeffrey P.D. Bowen Z. Kinnucan E.R. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. Nature. 2000; 408: 381-386Crossref PubMed Scopus (483) Google Scholar), an idea that is significantly strengthened by the recent observation that Skp2 levels are associated with reduced survival in prostate cancer (38Yang G. Ayala G. Marzo A.D. Tian W. Frolov A. Wheeler T.M. Thompson T.C. Harper J.W. Clin. Cancer Res. 2002; 8: 3419-3426PubMed Google Scholar). The present study shows that a FAS blockade ultimately decreases Skp2 levels. Therefore, FAS represents an upstream leverage point for targeting Skp2. Identifying and understanding the mechanism and molecular players that make this connection is an important direction for future investigation.