The Farnesyl Transferase Inhibitor (FTI) SCH66336 (lonafarnib) Inhibits Rheb Farnesylation and mTOR Signaling
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.m503763200
ISSN1083-351X
AutoresAndrea Basso, Asra Mirza, Gongjie Liu, Brian J. Long, W. Robert Bishop, Paul T. Kirschmeier,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoLonafarnib (SCH66336) is a farnesyl transferase inhibitor (FTI) that inhibits the post-translational lipid modification of H-Ras and other farnesylated proteins. K- and N-Ras are also substrates of farnesyl transferase; however, upon treatment with FTIs, they are alternatively prenylated by geranylgeranyl transferase-1. Despite the failure to abrogate prenylation of K- and N-Ras, growth of many tumors in preclinical models is inhibited by FTIs. This suggests that the anti-proliferative action of FTIs is dependent on blocking the farnesylation of other proteins. Rheb (Ras homologue enriched in brain) is a farnesylated small GTPase that positively regulates mTOR (mammalian target of rapamycin) signaling. We found that Rheb and Rheb2 mRNA were elevated in various tumor cell lines relative to normal cells. Peptides derived from the carboxyl termini of human Rheb and Rheb2 are in vitro substrates for farnesyl transferase but not geranylgeranyl transferase-1. Rheb prenylation in cell culture was completely inhibited by SCH66336, indicating a lack of alternative prenylation. SCH66336 treatment also inhibited the phosphorylation of S6 ribosomal protein, a downstream target of Rheb and mTOR signaling. SCH66336 did not inhibit S6 phosphorylation in cells expressing Rheb-CSVL, a mutant construct of Rheb designed to be geranylgeranylated. Importantly, expression of Rheb-CSVL also abrogated SCH66336 enhancement of tamoxifen- and docetaxel-induced apoptosis in MCF-7 breast cancer cells and ES-2 ovarian cancer cells, respectively. Further, inhibition of Rheb signaling by rapamycin treatment, small interfering RNA, or dominant negative Rheb enhanced tamoxifen- and docetaxel-induced apoptosis, similar to FTI treatment. These studies demonstrated that Rheb is modified by farnesylation, is not a substrate for alternative prenylation, and plays a role in SCH66336 enhancement of the anti-tumor response to other chemotherapeutics. Lonafarnib (SCH66336) is a farnesyl transferase inhibitor (FTI) that inhibits the post-translational lipid modification of H-Ras and other farnesylated proteins. K- and N-Ras are also substrates of farnesyl transferase; however, upon treatment with FTIs, they are alternatively prenylated by geranylgeranyl transferase-1. Despite the failure to abrogate prenylation of K- and N-Ras, growth of many tumors in preclinical models is inhibited by FTIs. This suggests that the anti-proliferative action of FTIs is dependent on blocking the farnesylation of other proteins. Rheb (Ras homologue enriched in brain) is a farnesylated small GTPase that positively regulates mTOR (mammalian target of rapamycin) signaling. We found that Rheb and Rheb2 mRNA were elevated in various tumor cell lines relative to normal cells. Peptides derived from the carboxyl termini of human Rheb and Rheb2 are in vitro substrates for farnesyl transferase but not geranylgeranyl transferase-1. Rheb prenylation in cell culture was completely inhibited by SCH66336, indicating a lack of alternative prenylation. SCH66336 treatment also inhibited the phosphorylation of S6 ribosomal protein, a downstream target of Rheb and mTOR signaling. SCH66336 did not inhibit S6 phosphorylation in cells expressing Rheb-CSVL, a mutant construct of Rheb designed to be geranylgeranylated. Importantly, expression of Rheb-CSVL also abrogated SCH66336 enhancement of tamoxifen- and docetaxel-induced apoptosis in MCF-7 breast cancer cells and ES-2 ovarian cancer cells, respectively. Further, inhibition of Rheb signaling by rapamycin treatment, small interfering RNA, or dominant negative Rheb enhanced tamoxifen- and docetaxel-induced apoptosis, similar to FTI treatment. These studies demonstrated that Rheb is modified by farnesylation, is not a substrate for alternative prenylation, and plays a role in SCH66336 enhancement of the anti-tumor response to other chemotherapeutics. Rheb (Ras homologue enriched in brain) is a GTPase identified as a gene up-regulated in rat brain by synaptic activity and growth factors (1Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). It is ubiquitously expressed in human tissue and found to be up-regulated in transformed cells (2Gromov P.S. Madsen P. Tomerup N. Celis J.E. FEBS Lett. 1995; 377: 221-226Crossref PubMed Scopus (57) Google Scholar). For example, Rheb mRNA is higher in SV40-transformed MRC-5 V2 fibroblast as compared with the parental cell line. Tuberous sclerosis (TSC) 1The abbreviations used are: TSC, tuberous sclerosis complex; FPT, farnesyl transferase; FTI, farnesyl transferase inhibitor; GGPT, geranylgeranyl transferase; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase; HA hemagglutinin; siRNA, small interfering RNA; RT, reverse transcription; Rheb, Ras homologue enriched in brain; mTOR, mammalian target of rapamycin. complex TSC1·TSC2 (hamartin·tuberin) serves as a GTPase-activating protein for Rheb, and overexpression of TSC1·TSC2 results in a reduction in activated Rheb-GTP (3Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 4Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1424) Google Scholar, 5Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 6Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (716) Google Scholar). Additionally, mutations in TSC1·TSC2 result in a loss of its tumor suppressor function. These mutations cause tuberous sclerosis, an autosomal dominant genetic disorder that occurs in 1 in 6000 people. TSC is characterized by benign tumors in the brain, heart, kidney, skin, and eyes, and common clinical symptoms of TSC include seizures, mental retardation, autism, and organ failure (7European Chromosome 16 Tuberous Sclerosis Consortium Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1511) Google Scholar, 8van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. Lindhout D. van den Ouweland A. Halley D. Young J. Burley M. Jeremiah S. Woodward K. Nahmias J. Fox M. Ekong R. Osborne J. Wolfe J. Povey S. Snell R.G. Cheadle J.P. Jones A.C. Tachataki M. Ravine D. Kwiatkowski D.J. Science. 1997; 277: 805-808Crossref PubMed Scopus (1397) Google Scholar). TSC1·TSC2 complex is negatively regulated by Akt-mediated phosphorylation of TSC2, and as a result, Rheb activity is increased (9Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 10Li Y. Inoki K. Yeung R. Guan K.L. J. Biol. Chem. 2002; 277: 44593-44596Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar, 12Liu M.Y. Cai S. Espejo A. Bedford M.T. Walker C.L. Cancer Res. 2002; 62: 6475-6480PubMed Google Scholar, 13Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar). The complex is positively regulated by LKB-stimulated AMP-activated protein kinase-dependent phosphorylation of tuberin, and as a result, Rheb signaling is inhibited (14Shaw R.J. Bardeesy N. Manning B.D. Lopez L. Kosmatka M. DePinho R.A. Cantley L.C. Cancer Cell. 2004; 6: 91-99Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar, 15Inoki K. Zhu T. Guan K.L. Cell. 2003; 115: 577-590Abstract Full Text Full Text PDF PubMed Scopus (3013) Google Scholar). Mutations in the LKB tumor suppressor are associated with Peutz-Jeghers syndrome, an autosomal dominant inherited disorder that leads to gastrointestinal polyps and predisposes people to various tumors (16Jenne D.E. Reimann H. Nezu J. Friedel W. Loff S. Jeschke R. Muller O. Back W. Zimmer M. Nat. Genet. 1998; 18: 38-43Crossref PubMed Scopus (984) Google Scholar, 17Hemminki A. Markie D. Tomlinson I. Avizienyte E. Roth S. Loukola A. Bignell G. Warren W. Aminoff M. Hoglund P. Jarvinen H. Kristo P. Pelin K. Ridanpaa M. Salovaara R. Toro T. Bodmer W. Olschwang S. Olsen A.S. Stratton M.R. de la Chapelle A. Aaltonen L.A. Nature. 1998; 391: 184-187Crossref PubMed Scopus (1351) Google Scholar). Rheb positively regulates mTOR signaling, and overexpression of Rheb induces phosphorylation of mTOR substrates, S6 kinase, and 4EBP-1 (18Castro A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 19Tabancay Jr., A.P. Gau C.L. Machado I.M. Uhlmann E.J. Gutmann D.H. Guo L. Tamanoi F. J. Biol. Chem. 2003; 278: 39921-39930Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Recently, Rheb was found to directly bind mTOR and, in a GTP-dependent manner, stimulate mTOR activity (20Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar, 21Smith E.M. Finn S.G. Tee A.R. Browne G.J. Proud C.G. J. Biol. Chem. 2005; 280: 18717-18727Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). As a consequence, Rheb overexpression in Drosophila causes overgrowth of multiple tissues due to increase in cell size (22Patel P.H. Thapar N. Guo L. Martinez M. Maris J. Gau C.L. Lengyel J.A. Tamanoi F. J. Cell Sci. 2003; 116: 3601-3610Crossref PubMed Scopus (130) Google Scholar, 23Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Nat. Cell Biol. 2003; 5: 566-571Crossref PubMed Scopus (535) Google Scholar, 24Stocker H. Radimerski T. Schindelholz B. Wittwer F. Belawat P. Daram P. Breuer S. Thomas G. Hafen E. Nat. Cell Biol. 2003; 5: 559-565Crossref PubMed Scopus (434) Google Scholar). Rheb is a farnesylated protein, and upon treatment with FTI-277, Rheb-induced phosphorylation of S6 kinase is inhibited (18Castro A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 25Clark G.J. Kinch M.S. Rogers-Graham K. Sebti S.M. Hamilton A.D. Der C.J. J. Biol. Chem. 1997; 272: 10608-10615Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Interestingly, the lack of Rheb results in increased arginine uptake in yeast, a phenotype also induced by mutation in farnesyl transferase (FPT) (26Urano J. Tabancay A.P. Yang W. Tamanoi F. J. Biol. Chem. 2000; 275: 11198-11206Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). FPT catalyzes the transfer of a 15-carbon isoprenyl lipid from farnesyl diphosphate onto a cysteine residue of various protein substrates. FPT recognizes the carboxyl-terminal CAAX box of the substrate (C, cysteine; A, aliphatic amino acid; X, carboxyl-terminal amino acid). Proteins terminating in serine, methionine, glutamine, and alanine are recognized by FPT. Proteins ending in leucine are modified with a 20-carbon isoprene by a related enzyme, geranylgeranyl protein transferase-1 (GGPT-1) (27Reiss Y. Stradley S.J. Gierasch L.M. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 732-736Crossref PubMed Scopus (311) Google Scholar, 28Yokoyama K. Goodwin G.W. Ghomashchi F. Glomset J.A. Gelb M.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5302-5306Crossref PubMed Scopus (217) Google Scholar). Farnesylated proteins include Ras, Pre-Lamin A, HDJ2, PTP-CAAX tyrosine phosphatases, CENP-E, and CENP-F (29Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8323-8327Crossref PubMed Scopus (780) Google Scholar, 30Wolda S.L. Glomset J.A. J. Biol. Chem. 1988; 263: 5997-6000Abstract Full Text PDF PubMed Google Scholar, 31Caplan A.J. Tsai J. Casey P.J. Douglas M.G. J. Biol. Chem. 1992; 267: 18890-18895Abstract Full Text PDF PubMed Google Scholar, 32Wang J. Kirby C.E. Herbst R. J. Biol. Chem. 2002; 277: 46659-46668Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 33Ashar H.R. James L. Gray K. Carr D. Black S. Armstrong L. Bishop W.R. Kirschmeier P. J. Biol. Chem. 2000; 275: 30451-30457Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Due to the frequency of activating Ras mutations in human cancer, farnesyl transferase inhibitors were originally investigated as a way to antagonize Ras function. SCH66336 is a selective, nonpeptidic CAAX-competitive inhibitor of FPT (FPT IC50, 1.9 nm; GGPT-1 IC50, >50,000 nm) (34Njoroge F.G. Taveras A.G. Kelly J. Remiszewski S. Mallams A.K. Wolin R. Afonso A. Cooper A.B. Rane D.F. Liu Y.T. Wong J. Vibulbhan B. Pinto P. Deskus J. Alvarez C.S. del Rosario J. Connolly M. Wang J. Desai J. Rossman R.R. Bishop W.R. Patton R. Wang L. Kirschmeier P. Ganguly A.K. J. Med. Chem. 1998; 41: 4890-4902Crossref PubMed Scopus (113) Google Scholar). It has demonstrated broad anti-tumor activity in animal models and is undergoing clinical investigation in human cancer. Preclinically, SCH66336 and other FTIs enhance the anti-tumor activity of taxanes (35Moasser M.M. Sepp-Lorenzino L. Kohl N.E. Oliff A. Balog A. Su D.S. Danishefsky S.J. Rosen N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1369-1374Crossref PubMed Scopus (200) Google Scholar, 36Shi B. Yaremko B. Hajian G. Terracina G. Bishop W.R. Liu M. Nielsen L.L. Cancer Chemother. Pharmacol. 2000; 46: 387-393Crossref PubMed Scopus (116) Google Scholar). Membrane localization of Ras requires its post-translation prenylation. Farnesylation is followed by proteolysis of the carboxyl AAX amino acids and carboxymethylation of the carboxyl-terminal farnesyl cysteine, rendering the carboxyl terminus more hydrophobic (37Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar, 38Gutierrez L. Magee A.I. Marshall C.J. Hancock J.F. EMBO J. 1989; 8: 1093-1098Crossref PubMed Scopus (330) Google Scholar). FTIs prevent H-Ras farnesylation, membrane localization, and reverse H-Ras-induced cellular transformation (39Kohl N.E. Mosser S.D. deSolms S.J. Giuliani E.A. Pompliano D.L. Graham S.L. Smith R.L. Scolnick E.M. Oliff A. Gibbs J.B. Science. 1993; 260: 1934-1937Crossref PubMed Scopus (619) Google Scholar). However, K- and N-Ras are also in vitro substrates of GGPT-1, and in cells treated with FTIs, their isoprene content switches to geranylgeranyl (40Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 41James G.L. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Despite the failure to abrogate prenylation of K- and N-Ras, mouse mammary tumor virus (MMTV)-K-Ras and MMTV-N-Ras tumors are inhibited by FTIs (42Mangues R. Corral T. Kohl N.E. Symmans W.F. Lu S. Malumbres M. Gibbs J.B. Oliff A. Pellicer A. Cancer Res. 1998; 58: 1253-1259PubMed Google Scholar, 43Omer C.A. Chen Z. Diehl R.E. Conner M.W. Chen H.Y. Trumbauer M.E. Gopal-Truter S. Seeburger G. Bhimnathwala H. Abrams M.T. Davide J.P. Ellis M.S. Gibbs J.B. Greenberg I. Koblan K.S. Kral A.M. Liu D. Lobell R.B. Miller P.J. Mosser S.D. O'Neill T.J. Rands E. Schaber M.D. Senderak E.T. Oliff A. Kohl N.E. Cancer Res. 2000; 60: 2680-2688PubMed Google Scholar). This suggests that the anti-proliferative action of FTIs is dependent on blocking the farnesylation of other proteins in addition to the Ras family. In the current studies, we found that both human Rheb and Rheb2 are in vitro substrates for FPT but not for GGPT-1. In cell culture, Rheb prenylation is completely inhibited by SCH66336. As a result, SCH66336 inhibits signaling downstream of Rheb and mTOR. This effect of FTIs is abrogated by the expression of Rheb-CSVL, a mutant engineered to be modified by geranylgeranylation, which remains prenylated in the presence of SCH66336. This mutant also abrogates the ability of SCH66336 to enhance the apoptotic effect of both tamoxifen and docetaxel. Further, inhibition of Rheb signaling by rapamycin treatment, siRNA, or a dominant negative Rheb protein mimics the effect of SCH66336 treatment, enhancing the action of tamoxifen and docetaxel. These studies suggest that inhibition of Rheb farnesylation plays a role in the SCH66336 enhancement of other chemotherapeutics. Materials—SCH66336, SCH66337 (synthesized by Schering-Plough) (34Njoroge F.G. Taveras A.G. Kelly J. Remiszewski S. Mallams A.K. Wolin R. Afonso A. Cooper A.B. Rane D.F. Liu Y.T. Wong J. Vibulbhan B. Pinto P. Deskus J. Alvarez C.S. del Rosario J. Connolly M. Wang J. Desai J. Rossman R.R. Bishop W.R. Patton R. Wang L. Kirschmeier P. Ganguly A.K. J. Med. Chem. 1998; 41: 4890-4902Crossref PubMed Scopus (113) Google Scholar), mevastatin, and 4-OH-tamoxifen (Sigma) were dissolved in Me2SO. Mevalonolactone, docetaxel, and estradiol (Sigma) were dissolved in ethanol. Heregulin (Sigma) was dissolved in phosphate-buffered saline. Rapamycin (Cell Signaling, Beverly, MA) was dissolved in methanol. FPT/GGPT Assay—Recombinant human FPT and GGPT-1 were expressed and purified as previously described (40Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). 1 nm FPTase and 225 nm [3H]FPP (16.1 Ci/mmol) (PerkinElmer Life Sciences) in assay buffer (50 mm Tris, 5 mm MgCl2, 5 μm ZnCl2, 0.1% Triton X-100, 5 mm dithiothreitol) were incubated together for 20 min, as were 3 nm GGPT-1 and 200 nm [3H]GPP (23 Ci/mmol) (PerkinElmer Life Sciences) in assay buffer (40 mm HEPES, 2 mm MgCl2, 5 mm dithiothreitol). Varying concentrations of peptide substrates were added to the reaction. Biotinylated peptide substrates (synthesized by Syn-Pep, Dublin, CA) were dissolved in Me2SO: Rheb (QGKSSCSVM), Rheb2 (GQERRCHLM), DESGPGCMSCKCVLS, and SGSGYRASNRSCAIL. After 1 h, the reaction was stopped with 750 μg of streptavidin-coated beads (Amersham Biosciences) in 0.25 m EDTA, and product formation ([3H]prenyl peptide) was measured using scintillation proximity assay. Cell Culture—The human cancer cell lines MCF-7, ES2 (American Type Culture Collection (ATCC, Manassas, VA), and MDA-435 (kindly provided by Dr. Janet Price, M. D. Anderson, Houston, TX) were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium:F-12. COS-7 (ATCC) cells were maintained in Dulbecco's modified Eagle's medium. The media were supplemented with 2 mm glutamine, 50 units/ml penicillin, 50 units/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Invitrogen) and incubated at 37 °C in 5% CO2. Transfections—Human Rheb proteins were expressed as fusion proteins containing HA tag at the amino terminus. The coding region was amplified by PCR from human brain cDNA (BD Biosciences) and subcloned into pHM6 vector (Roche Applied Science). HA-tagged Rheb-CSVL, Rheb-SSVM, Rheb-D60K, Rheb-D60V, and Rheb-D60V-CSVL were made by site-directed mutagenesis (Stratagene, La Jolla, CA). Identities of the plasmids were confirmed by DNA sequencing (Beckman CEQ2000 Dye Terminator Cycle Sequencing, Fullerton, CA). siRNA was obtained from Dharmacon (Lafayette, CO). Sequences used for siRNA studies were: Rheb siRNA, 5′-CCUCAGACAUACUCCAUAGUU-3′ and 5′-p-CUAUGGAGUAUGUCUGAGGUU-3′; Rheb2, 5′-GCCAAUUUGUGGACUCCUAUU-3′ and 5′-p-USGGAGUCCACAAAUUGGCUU-3′. Cells were transiently transfected with 20 μg of DNA or 100 nm siRNA and 50 μl of Lipofectamine 2000 (Invitrogen) for 5 h, at which time the culture medium was changed. The next day, the cells were drug-treated. For stable transfections, MCF-7 cells were transfected with 5 μg of mevalonate transporter (44Kim C.M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1992; 267: 23113-23121Abstract Full Text PDF PubMed Google Scholar) and 50 μl of Lipofectamine 2000. The cells were selected with geneticin (Invitrogen) and screened on the ability to survive in the presence of 20 μm mevastatin plus 100 μm mevalonolactone and the ability to uptake [3H]mevalonolactone (PerkinElmer Life Sciences). Protein Analysis—Cells were lysed in RIPA buffer (50 mm Tris-HCl, 50 mm NaCl, 1% Nonidet P-40, 0.5% Na-deoxycholate, 1 mm EDTA, 2.5 mm Na3VO4, 20 mm β-glycerol phosphate, and complete protease inhibitor) (Roche Applied Science) and cleared by centrifugation. Protein concentration was determined using BCA reagent (Pierce). Samples were separated by 8, 10, or 14% SDS-PAGE (Invitrogen), transferred to polyvinylidene difluoride, immunoblotted, and detected by chemiluminescence using ECL detection reagents (Amersham Biosciences). Polyclonal antibodies used were PARP (Promega, Madison, WI), S6, P-S6 (Ser-235/236), Akt, P-Akt (Ser-473), MAPK, and P-MAPK (Thr-202/Tyr-204) (Cell Signaling). Monoclonal antibodies used were HDJ-2 (Neomarkers, Fremont, CA), and HA (Roche Applied Science). Blots were scanned and quantified by densitometry. Mevalonate Labeling—COS-7 cells were electroporated with 20 μg of HA-Rheb and 5 μg of pMev transporter or MCF-7 pMev cells were transfected with 20 μg of HA-Rheb and 50 μl of Lipofectamine 2000. That day, the cells were treated with SCH66336. The next day, the cell medium was changed, and the cells were treated with SCH66336 and 20 μm mevastatin in medium containing dialyzed serum. Three hours later, the cells were treated with the same medium containing 100 μCi/ml [3H]mevalonolactone. After 18 h of labeling, the cells were lysed, and Rheb was immunoprecipitated and analyzed on SDS-polyacrylamide gels. The gels were fixed in 10% acetic acid/10% methanol and then incubated in Amplify (Amersham Biosciences), dried, and viewed by autoradiography. Duplicate gels were immunoblotted for HA. TaqMan Analysis—All breast, pancreatic, small cell lung tumor cell lines, and CRL5806, CRL5807, LnCap, and PCa-2b were obtained from the ATCC. All other tumor cells were obtained from the National Cancer Institute (Bethesda, MD). Primary cells (HMEC50, HMEC21, SAEC, NHBE258, PrEC, and CC39SK) were obtained from Clonetics (East Rutherford, NJ). RNA was isolated using TRIzol (Invitrogen) and then treated with DNase (Ambion, Austin, TX). RNA from normal colon, brain, ovary, and pancreas tissue was obtained from BD Biosciences. Quantitative reverse transcription-PCR (RT-PCR) was performed using Applied Biosciences (Foster City, CA) RT-PCR master mix. The primers (Genscript, Piscataway, NJ) for Rheb were 5′-TCGGTGATGTGATTCTGCT-3′ and 3′-GGAGCTCTGACCCAAATGAT-5′. The 5′Fam-3′ Tamra probe for Rheb was TCCCAGTGTCCTCAGGCTTTGC-5′. Primers for Rheb2 were 5′-AAGAAGCTGGCAGAGTCCTG-3′ and 3′-TCCTGGATGACTTTGGTAA-5′. The 5′Fam-3′ Tamra probe for Rheb2 was TGCCTTGAGTCAGCTGATTCTCTCG. The 23-kDa highly basic protein was used as a housekeeping gene to normalize the quantification of mRNA samples. Primers were 5′-CTGGAAGTACCAGGCAGTGACA-3′ and 3′-CCGGTAGTGGATCTTGGCTTT-5′. The 5′Fam-3′Tamra probe was CCACCCTGGAGGAGAAGAGGAAAGAGAA. Analysis was performed in triplicate. Caspase Activity—Activity was measured by cleavage of a fluorescent substrate (BD Biosciences). 5 μl of DEVD substrate labeled with 7-amino-4-trifluoromethyl coumarin and 50 μl of 2× reaction buffer was added to 50 μl of cell lysate. 7-Amino-4-trifluoromethyl coumarin substrate conjugate emits blue light (400 nm), but when cleaved, free 7-amino-4-trifluoromethyl coumarin fluoresces yellow-green (505 nm). Caspase activity was measured by a blue to green shift in the fluorescence upon cleavage of the 7-amino-4-trifluoromethyl coumarin fluorophore. Rheb mRNA Is Elevated in Tumor Cell Lines—Rheb is ubiquitously expressed in human tissue. Rheb expression is elevated in SV40-transformed MRC-5 V2 fibroblasts, as compared with the parental cell line (2Gromov P.S. Madsen P. Tomerup N. Celis J.E. FEBS Lett. 1995; 377: 221-226Crossref PubMed Scopus (57) Google Scholar). We found that both Rheb and Rheb2 mRNA were widely expressed with the highest expression in the brain. The two were overexpressed in most tumor cell lines, as compared with normal cell lines/tissue (Fig. 1). Rheb mRNA was analyzed by quantitative RT-PCR in 67 tumor cell lines and 10 normal cells for 8 different tissue types. For breast, lung, prostate, and skin, primary cells were used. For colon, brain, ovary, and pancreas, mRNA was obtained from normal tissue. Rheb mRNA expression was increased by at least 2-fold in various tumor cells: 2/9 breast, 7/12 lung, 2/4 prostate, 8/9 colon, 6/7 ovarian, 2/7 melanoma, and 7/7 pancreatic. Likewise, Rheb2 mRNA was increased by at least 2-fold in various tumor cells when compared with normal cells: 8/9 breast, 3/12 lung, 3/4 prostate, 4/9 colon, 4/7 ovarian, 7/7 melanoma, and 6/7 pancreatic. All colon, ovarian, melanoma, and pancreatic tumor cells in our panel had an elevated expression of Rheb, Rheb2, or both. Little or no overexpression of either was observed in glioma cell lines as compared with normal brain. These data suggest that, not only is activity of the Rheb pathway up-regulated in tumor cells by amplification and mutational activation of upstream signaling proteins, Rheb expression itself is elevated in many tumor cells. Rheb Is Modified by Farnesylation—K- and N-Ras are substrates for GGPT-1, and therefore, upon cellular treatment with FTIs, the isoprene content of these Ras isoforms switches to geranylgeranyl (40Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). We wanted to determine whether human Rheb and Rheb2 were solely farnesylated or subject to alternative prenylation. Peptides derived from the carboxyl termini of Rheb (QGKSSCSVM) and Rheb2 (GQERRCHLM) were tested as in vitro substrates for FPT and GGPT-1. H-Ras peptide ending in CVLS served as a positive control for FPT, and a peptide ending in CAIL served as a positive control for GGPT-1. Following incubation of peptides with FPT or GGPT-1, incorporation of [3H]isoprene was measured using scintillation proximity assay. Both Rheb and Rheb2 were substrates for FPT but not for GGPT-1 (Fig. 2). Further, Rheb peptides bound FPT with greater affinity than the H-Ras peptide. The Km for Rheb and Rheb2 were 102 + 33 and 42 + 14 nm, respectively, compared with 528 + 185 nm for H-Ras. These data indicate human Rheb and Rheb2 are modified by farnesylation and are not substrates for GGPT-1. SCH66336 Inhibits Rheb Farnesylation—Next, we wanted to determine whether Rheb was modified by farnesylation in vivo. Cells were transfected with a cDNA encoding the mevalonate transporter and treated with mevastatin to increase labeling of cellular isoprene pools with [3H]mevalonolactone. Mevalonolactone is a precursor for both geranylgeranyl diphosphate and farnesyl diphosphate. COS-7 cells were electroporated with cDNA encoding HA-Rheb and labeled with [3H]mevalonolactone. In untreated cells, immunoprecipitated HA-Rheb incorporated [3H]isoprene (Fig. 3A). SCH66336 treatment completely inhibited this modification at 1000 nm, with an IC50 between 10 and 100 nm. Although there was a 15% decline in HA-Rheb levels at 1000 nm, at 100 nm, there was no decline in HA-Rheb and a >50% decline in [3H]isoprene. SCH66336 is a selective FPTase inhibitor that has no effect on GGPT-1 at the concentrations used. Incorporation of [3H]mevalonolactone into Rheb would not be blocked by SCH66336 if Rheb was alternatively prenylated by GGPT-1. These data show that Rheb is a substrate for farnesylation and is not alternatively prenylated in FTI-treated cells. Additionally, upon treatment with SCH66336, HA-Rheb underwent a gel mobility shift on SDS-PAGE (Fig. 3A), similar to the shift seen when unprenylated HDJ-2 and H-Ras accumulate upon FTI treatment. A point mutation in Rheb changing the CAAX box from CSVM to CSVL was predicted to convert it into a substrate for GGPT-1, whereas mutation of the modified cysteine residue to serine (SSVM) will block all prenylation of Rheb. We found that prenylation of HA-Rheb-CSVL in cells was not inhibited by SCH66336, consistent with Rheb-CSVL geranylgeranylation (Fig. 3B). As expected, HA-Rheb-SSVM did not incorporate [3H]isoprene (Fig. 3B). HA-Rheb-SSVM migrated on SDS-PAGE with similar mobility to that of HA-Rheb in FTI-treated cells, whereas HA-Rheb-CSVL migrated between untreated and treated HA-Rheb (Fig. 3B). We expanded this finding to human cancer cells. MCF-7 cells that stably expressed mevalonate transporter were labeled with [3H]mevalonolactone. Similar to the COS-7 finding, HA-Rheb was labeled with [3H]isoprene, and this modification was completely inhibited by SCH66336 (Fig. 3C). SCH66336 Inhibits S6 Phosphorylation—Rheb has been shown to promote phosphorylation of S6 kinase, a downstream target of mTOR signaling. Rheb overexpression in 293T cells stimulates phosphorylation of S6 kinase, and this is inhibited by FTI-277 (18Castro A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). We found that SCH66336 inhibited phosphorylation of S6 ribosomal protein in the human breast cancer cell line MCF-7 (Fig. 4A). Several experiments with 1 μm SCH66336 resulted in an average 70% reduction in S6 phosphorylation. Interestingly, concentrations required to inhibit S6 phosphorylation were similar to those that blocked Rheb farnesylation. Additionally, heregulin-stimulated phosphorylation of S6 in MCF-7 cells was blocked by SCH66336 (Fig. 4B). Similar inhibition of S6 phosphorylation was observed in the human ovarian cancer cell line ES2 (data not shown). In contrast,
Referência(s)