The Farnesyl Transferase Inhibitor

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 35, Issue of September 2, pp. 31101–31108, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Farnesyl Transferase Inhibitor (FTI) SCH66336 (lonafarnib) Inhibits Rheb Farnesylation and mTOR Signaling ROLE IN FTI ENHANCEMENT OF TAXANE AND TAMOXIFEN ANTI-TUMOR ACTIVITY*

Received for publication, April 6, 2005, and in revised form, June 20, 2005 Published, JBC Papers in Press, July 8, 2005, DOI 10.1074/jbc.M503763200

Andrea D. Basso, Asra Mirza, Gongjie Liu, Brian J. Long, W. Robert Bishop, and Paul Kirschmeier‡ From the Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033

Lonafarnib (SCH66336) is a farnesyl transferase inhib- sclerosis (TSC)1 complex TSC1⅐TSC2 (hamartin⅐tuberin) serves itor (FTI) that inhibits the post-translational lipid mod- as a GTPase-activating protein for Rheb, and overexpression of ification of H-Ras and other farnesylated proteins. K- TSC1⅐TSC2 results in a reduction in activated Rheb-GTP (3–6). and N-Ras are also substrates of farnesyl transferase; Additionally, mutations in TSC1⅐TSC2 result in a loss of its however, upon treatment with FTIs, they are alterna- tumor suppressor function. These mutations cause tuberous tively prenylated by geranylgeranyl transferase-1. De- sclerosis, an autosomal dominant genetic disorder that occurs spite the failure to abrogate prenylation of K- and N-Ras, in 1 in 6000 people. TSC is characterized by benign tumors in growth of many tumors in preclinical models is inhib- the brain, heart, kidney, skin, and eyes, and common clinical ited by FTIs. This suggests that the anti-proliferative symptoms of TSC include seizures, mental retardation, autism, action of FTIs is dependent on blocking the farnesyla- and organ failure (7, 8). tion of other proteins. Rheb (Ras homologue enriched in TSC1⅐TSC2 complex is negatively regulated by Akt-mediated brain) is a farnesylated small GTPase that positively phosphorylation of TSC2, and as a result, Rheb activity is regulates mTOR (mammalian target of rapamycin) sig- increased (9–13). The complex is positively regulated by LKB- naling. We found that Rheb and Rheb2 mRNA were elevated in various tumor cell lines relative to normal cells. stimulated AMP-activated protein kinase-dependent phospho- Peptides derived from the carboxyl termini of human rylation of tuberin, and as a result, Rheb signaling is inhibited Rheb and Rheb2 are in vitro substrates for farnesyl (14, 15). Mutations in the LKB tumor suppressor are associated transferase but not geranylgeranyl transferase-1. Rheb with Peutz-Jeghers syndrome, an autosomal dominant inher- prenylation in cell culture was completely inhibited by ited disorder that leads to gastrointestinal polyps and predis- SCH66336, indicating a lack of alternative prenylation. poses people to various tumors (16, 17). SCH66336 treatment also inhibited the phosphorylation Rheb positively regulates mTOR signaling, and overexpres- of S6 ribosomal protein, a downstream target of Rheb sion of Rheb induces phosphorylation of mTOR substrates, S6 and mTOR signaling. SCH66336 did not inhibit S6 phos- kinase, and 4EBP-1 (18, 19). Recently, Rheb was found to phorylation in cells expressing Rheb-CSVL, a mutant directly bind mTOR and, in a GTP-dependent manner, stimu- construct of Rheb designed to be geranylgeranylated. late mTOR activity (20, 21). As a consequence, Rheb overex- Importantly, expression of Rheb-CSVL also abrogated pression in Drosophila causes overgrowth of multiple tissues SCH66336 enhancement of tamoxifen- and docetaxel-in- due to increase in cell size (22–24). Rheb is a farnesylated duced apoptosis in MCF-7 breast cancer cells and ES-2 protein, and upon treatment with FTI-277, Rheb-induced phos- ovarian cancer cells, respectively. Further, inhibition of phorylation of S6 kinase is inhibited (18, 25). Interestingly, the Rheb signaling by rapamycin treatment, small interfering lack of Rheb results in increased arginine uptake in yeast, a RNA, or dominant negative Rheb enhanced tamoxifen- and phenotype also induced by mutation in farnesyl transferase docetaxel-induced apoptosis, similar to FTI treatment. (FPT) (26). These studies demonstrated that Rheb is modified by far- FPT catalyzes the transfer of a 15-carbon isoprenyl lipid nesylation, is not a substrate for alternative prenylation, from farnesyl diphosphate onto a cysteine residue of various and plays a role in SCH66336 enhancement of the anti- tumor response to other chemotherapeutics. 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 ser- Rheb (Ras homologue enriched in brain) is a GTPase identi- ine, methionine, glutamine, and alanine are recognized by fied as a gene up-regulated in rat brain by synaptic activity and FPT. Proteins ending in leucine are modified with a 20-carbon growth factors (1). It is ubiquitously expressed in human tissue isoprene by a related enzyme, geranylgeranyl protein transfer- and found to be up-regulated in transformed cells (2). For ase-1 (GGPT-1) (27, 28). Farnesylated proteins include Ras, example, Rheb mRNA is higher in SV40-transformed MRC-5 Pre-Lamin A, HDJ2, PTP-CAAX tyrosine phosphatases, V2 fibroblast as compared with the parental cell line. Tuberous CENP-E, and CENP-F (29–33). Due to the frequency of activating Ras mutations in human cancer, farnesyl transferase inhibitors were originally investigated as a way to antagonize * 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 1 The abbreviations used are: TSC, tuberous sclerosis complex; FPT, indicate this fact. farnesyl transferase; FTI, farnesyl transferase inhibitor; GGPT, gera- ‡ To whom correspondence should be addressed: Schering-Plough nylgeranyl transferase; MAPK, mitogen-activated protein kinase; Research Institute, 2015 Galloping Hill Rd., K-15-4950, Kenilworth, PARP, poly(ADP-ribose) polymerase; HA hemagglutinin; siRNA, small NJ 07033. Tel.: 908-740-7327; Fax: 908-740-3918; E-mail: paul. interfering RNA; RT, reverse transcription; Rheb, Ras homologue en- [email protected]. riched in brain; mTOR, mammalian target of rapamycin.

This paper is available on line at http://www.jbc.org 31101 31102 SCH66336 Inhibits mTOR Signaling

Ras function. SCH66336 is a selective, nonpeptidic CAAX-com- Identities of the plasmids were confirmed by DNA sequencing (Beck- petitive inhibitor of FPT (FPT IC , 1.9 nM; GGPT-1 IC , man CEQ2000 Dye Terminator Cycle Sequencing, Fullerton, CA). 50 50 siRNA was obtained from Dharmacon (Lafayette, CO). Sequences used Ͼ50,000 nM) (34). It has demonstrated broad anti-tumor activ- for siRNA studies were: Rheb siRNA, 5Ј-CCUCAGACAUACUCCAUA- ity in animal models and is undergoing clinical investigation in GUU-3Ј and 5Ј-p-CUAUGGAGUAUGUCUGAGGUU-3Ј; Rheb2, 5Ј-GC- human cancer. Preclinically, SCH66336 and other FTIs en- CAAUUUGUGGACUCCUAUU-3Ј and 5Ј-p-USGGAGUCCACAAAUU- hance the anti-tumor activity of taxanes (35, 36). GGCUU-3Ј. Cells were transiently transfected with 20 ␮g of DNA or Membrane localization of Ras requires its post-translation 100 nM siRNA and 50 ␮l of Lipofectamine 2000 (Invitrogen) for 5 h, at prenylation. Farnesylation is followed by proteolysis of the which time the culture medium was changed. The next day, the cells carboxyl AAX amino acids and carboxymethylation of the car- were drug-treated. For stable transfections, MCF-7 cells were transfected with 5 ␮g of mevalonate transporter (44) and 50 ␮l of Lipo- boxyl-terminal farnesyl cysteine, rendering the carboxyl termi- fectamine 2000. The cells were selected with geneticin (Invitrogen) and nus more hydrophobic (37, 38). FTIs prevent H-Ras farnesyla- screened on the ability to survive in the presence of 20 ␮M mevastatin tion, membrane localization, and reverse H-Ras-induced plus 100 ␮M mevalonolactone and the ability to uptake [3H]mevalono- cellular transformation (39). However, K- and N-Ras are also in lactone (PerkinElmer Life Sciences). vitro substrates of GGPT-1, and in cells treated with FTIs, 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, their isoprene content switches to geranylgeranyl (40, 41). De- ␤ 2.5 mM Na3VO4,20mM -glycerol phosphate, and complete protease spite the failure to abrogate prenylation of K- and N-Ras, inhibitor) (Roche Applied Science) and cleared by centrifugation. mouse mammary tumor virus (MMTV)-K-Ras and MMTV-N- Protein concentration was determined using BCA reagent (Pierce). Ras tumors are inhibited by FTIs (42, 43). This suggests that Samples were separated by 8, 10, or 14% SDS-PAGE (Invitrogen), the anti-proliferative action of FTIs is dependent on blocking transferred to polyvinylidene difluoride, immunoblotted, and de- the farnesylation of other proteins in addition to the Ras tected by chemiluminescence using ECL detection reagents (Amer- family. sham Biosciences). Polyclonal antibodies used were PARP (Promega, Madison, WI), S6, P-S6 (Ser-235/236), Akt, P-Akt (Ser-473), MAPK, In the current studies, we found that both human Rheb and and P-MAPK (Thr-202/Tyr-204) (Cell Signaling). Monoclonal antibod- Rheb2 are in vitro substrates for FPT but not for GGPT-1. In ies used were HDJ-2 (Neomarkers, Fremont, CA), and HA (Roche cell culture, Rheb prenylation is completely inhibited by Applied Science). Blots were scanned and quantified by densitometry. SCH66336. As a result, SCH66336 inhibits signaling down- Mevalonate Labeling—COS-7 cells were electroporated with 20 ␮gof stream of Rheb and mTOR. This effect of FTIs is abrogated by HA-Rheb and 5 ␮g of pMev transporter or MCF-7 pMev cells were ␮ ␮ the expression of Rheb-CSVL, a mutant engineered to be mod- transfected with 20 g of HA-Rheb and 50 l of Lipofectamine 2000. That day, the cells were treated with SCH66336. The next day, the cell ified by geranylgeranylation, which remains prenylated in the medium was changed, and the cells were treated with SCH66336 and presence of SCH66336. This mutant also abrogates the ability 20 ␮M mevastatin in medium containing dialyzed serum. Three hours of SCH66336 to enhance the apoptotic effect of both tamoxifen later, the cells were treated with the same medium containing 100 and docetaxel. Further, inhibition of Rheb signaling by rapa- ␮Ci/ml [3H]mevalonolactone. After 18 h of labeling, the cells were lysed, mycin treatment, siRNA, or a dominant negative Rheb protein and Rheb was immunoprecipitated and analyzed on SDS-polyacryl- mimics the effect of SCH66336 treatment, enhancing the action amide gels. The gels were fixed in 10% acetic acid/10% methanol and then incubated in Amplify (Amersham Biosciences), dried, and viewed of tamoxifen and docetaxel. These studies suggest that inhibi- by autoradiography. Duplicate gels were immunoblotted for HA. tion of Rheb farnesylation plays a role in the SCH66336 en- TaqMan Analysis—All breast, pancreatic, small cell lung tumor cell hancement of other chemotherapeutics. lines, and CRL5806, CRL5807, LnCap, and PCa-2b were obtained from the ATCC. All other tumor cells were obtained from the National EXPERIMENTAL PROCEDURES Cancer Institute (Bethesda, MD). Primary cells (HMEC50, HMEC21, Materials—SCH66336, SCH66337 (synthesized by Schering-Plough) SAEC, NHBE258, PrEC, and CC39SK) were obtained from Clonetics (34), mevastatin, and 4-OH-tamoxifen (Sigma) were dissolved in (East Rutherford, NJ). RNA was isolated using TRIzol (Invitrogen) and

Me2SO. Mevalonolactone, docetaxel, and estradiol (Sigma) were dis- then treated with DNase (Ambion, Austin, TX). RNA from normal solved in ethanol. Heregulin (Sigma) was dissolved in phosphate-buff- colon, brain, ovary, and pancreas tissue was obtained from BD Bio- ered saline. Rapamycin (Cell Signaling, Beverly, MA) was dissolved sciences. Quantitative reverse transcription-PCR (RT-PCR) was per- in methanol. formed using Applied Biosciences (Foster City, CA) RT-PCR master FPT/GGPT Assay—Recombinant human FPT and GGPT-1 were mix. The primers (Genscript, Piscataway, NJ) for Rheb were 5Ј-TCG- expressed and purified as previously described (40). 1 nM FPTase and GTGATGTGATTCTGCT-3Ј and 3Ј-GGAGCTCTGACCCAAATGAT-5Ј. 225 nM [3H]FPP (16.1 Ci/mmol) (PerkinElmer Life Sciences) in assay The 5ЈFam-3Ј Tamra probe for Rheb was TCCCAGTGTCCTCAGGCTT- ␮ Ј Ј Ј buffer (50 mM Tris, 5 mM MgCl2,5 M ZnCl2, 0.1% Triton X-100, 5 mM TGC-5 . Primers for Rheb2 were 5 -AAGAAGCTGGCAGAGTCCTG-3 dithiothreitol) were incubated together for 20 min, as were 3 nM and 3Ј-TCCTGGATGACTTTGGTAA-5Ј. The 5ЈFam-3Ј Tamra probe for GGPT-1 and 200 nM [3H]GPP (23 Ci/mmol) (PerkinElmer Life Sciences) Rheb2 was TGCCTTGAGTCAGCTGATTCTCTCG. The 23-kDa highly

in assay buffer (40 mM HEPES, 2 mM MgCl2,5mM dithiothreitol). basic protein was used as a housekeeping gene to normalize the quanti- Varying concentrations of peptide substrates were added to the reac- fication of mRNA samples. Primers were 5Ј-CTGGAAGTACCAGGCAG- tion. Biotinylated peptide substrates (synthesized by Syn-Pep, Dublin, TGACA-3Ј and 3Ј-CCGGTAGTGGATCTTGGCTTT-5Ј. The 5ЈFam-3ЈTa-

CA) were dissolved in Me2SO: Rheb (QGKSSCSVM), Rheb2 (GQER- mra probe was CCACCCTGGAGGAGAAGAGGAAAGAGAA. Analysis RCHLM), DESGPGCMSCKCVLS, and SGSGYRASNRSCAIL. After was performed in triplicate. 1 h, the reaction was stopped with 750 ␮g of streptavidin-coated beads Caspase Activity—Activity was measured by cleavage of a fluores- (Amersham Biosciences) in 0.25 M EDTA, and product formation cent substrate (BD Biosciences). 5 ␮l of DEVD substrate labeled with ([3H]prenyl peptide) was measured using scintillation proximity assay. 7-amino-4-trifluoromethyl coumarin and 50 ␮lof2ϫ reaction buffer Cell Culture—The human cancer cell lines MCF-7, ES2 (American was added to 50 ␮l of cell lysate. 7-Amino-4-trifluoromethyl coumarin Type Culture Collection (ATCC, Manassas, VA), and MDA-435 (kindly substrate conjugate emits blue light (400 nm), but when cleaved, free provided by Dr. Janet Price, M. D. Anderson, Houston, TX) were main- 7-amino-4-trifluoromethyl coumarin fluoresces yellow-green (505 tained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium:F-12. nm). Caspase activity was measured by a blue to green shift in the COS-7 (ATCC) cells were maintained in Dulbecco’s modified Eagle’s fluorescence upon cleavage of the 7-amino-4-trifluoromethyl couma- medium. The media were supplemented with 2 mM glutamine, 50 rin fluorophore. units/ml penicillin, 50 units/ml streptomycin, and 10% heat-inactivated RESULTS fetal bovine serum (Invitrogen) and incubated at 37 °C in 5% CO2. Transfections—Human Rheb proteins were expressed as fusion pro- Rheb mRNA Is Elevated in Tumor Cell Lines—Rheb is ubiq- teins containing HA tag at the amino terminus. The coding region was uitously expressed in human tissue. Rheb expression is ele- amplified by PCR from human brain cDNA (BD Biosciences) and sub- cloned into pHM6 vector (Roche Applied Science). HA-tagged Rheb- vated in SV40-transformed MRC-5 V2 fibroblasts, as compared CSVL, Rheb-SSVM, Rheb-D60K, Rheb-D60V, and Rheb-D60V-CSVL with the parental cell line (2). We found that both Rheb and were made by site-directed mutagenesis (Stratagene, La Jolla, CA). Rheb2 mRNA were widely expressed with the highest expres- SCH66336 Inhibits mTOR Signaling 31103

FIG.1.Rheb mRNA is elevated in tumor cell lines. mRNA was isolated from a panel of cell lines, and quantitative RT-PCR analysis was performed to determine the relative level of Rheb and Rheb2 mRNA. Rheb expression is plotted on a linear scale, whereas that of Rheb2 is plotted on a logarithmic scale. Normal cells are graphed in black, and tumor cells are graphed in blue. sion in the brain. The two were overexpressed in most tumor man Rheb and Rheb2 were solely farnesylated or subject to cell lines, as compared with normal cell lines/tissue (Fig. 1). alternative prenylation. Peptides derived from the carboxyl Rheb mRNA was analyzed by quantitative RT-PCR in 67 tu- termini of Rheb (QGKSSCSVM) and Rheb2 (GQERRCHLM) mor cell lines and 10 normal cells for 8 different tissue types. were tested as in vitro substrates for FPT and GGPT-1. H-Ras For breast, lung, prostate, and skin, primary cells were used. peptide ending in CVLS served as a positive control for FPT, For colon, brain, ovary, and pancreas, mRNA was obtained and a peptide ending in CAIL served as a positive control for from normal tissue. Rheb mRNA expression was increased by GGPT-1. Following incubation of peptides with FPT or at least 2-fold in various tumor cells: 2/9 breast, 7/12 lung, 2/4 GGPT-1, incorporation of [3H]isoprene was measured using prostate, 8/9 colon, 6/7 ovarian, 2/7 melanoma, and 7/7 pancre- scintillation proximity assay. Both Rheb and Rheb2 were sub- atic. Likewise, Rheb2 mRNA was increased by at least 2-fold in strates for FPT but not for GGPT-1 (Fig. 2). Further, Rheb various tumor cells when compared with normal cells: 8/9 peptides bound FPT with greater affinity than the H-Ras pep- ϩ ϩ breast, 3/12 lung, 3/4 prostate, 4/9 colon, 4/7 ovarian, 7/7 mel- tide. The Km for Rheb and Rheb2 were 102 33 and 42 14 anoma, and 6/7 pancreatic. All colon, ovarian, melanoma, and nM, respectively, compared with 528 ϩ 185 nM for H-Ras. These pancreatic tumor cells in our panel had an elevated expression data indicate human Rheb and Rheb2 are modified by farne- of Rheb, Rheb2, or both. Little or no overexpression of either sylation and are not substrates for GGPT-1. was observed in glioma cell lines as compared with normal SCH66336 Inhibits Rheb Farnesylation—Next, we wanted to brain. These data suggest that, not only is activity of the Rheb determine whether Rheb was modified by farnesylation in vivo. pathway up-regulated in tumor cells by amplification and mu- Cells were transfected with a cDNA encoding the mevalonate tational activation of upstream signaling proteins, Rheb ex- transporter and treated with mevastatin to increase labeling of pression itself is elevated in many tumor cells. cellular isoprene pools with [3H]mevalonolactone. Mevalono- Rheb Is Modified by Farnesylation—K- and N-Ras are sub- lactone is a precursor for both geranylgeranyl diphosphate and strates for GGPT-1, and therefore, upon cellular treatment farnesyl diphosphate. COS-7 cells were electroporated with with FTIs, the isoprene content of these Ras isoforms switches cDNA encoding HA-Rheb and labeled with [3H]mevalonolac- to geranylgeranyl (40). We wanted to determine whether hu- tone. In untreated cells, immunoprecipitated HA-Rheb incor- 31104 SCH66336 Inhibits mTOR Signaling

FIG.2.Rheb is modified by farnesylation. The indicated amount of peptides from the carboxyl termini of Rheb, Rheb2, H-Ras, and lamin B (CAAX containing a methionine to leucine substitution at the X FIG.3.SCH66336 inhibits Rheb farnesylation. A and B, COS-7 position) were tested as substrates for FPT and GGPT-1 enzymes in the cells were electroporated with HA-Rheb and mevalonate (Mev) trans- presence of [3H]FPP or [3H]GGPP. Incorporation of [3H]isoprene was porter. C, MCF-7/pMev cells were transfected with HA-Rheb. A–C, cells detected using scintillation proximity assay. The average of four exper- were labeled with 100 ␮Ci/ml [3H]mevalonolactone for 18 h in the iments is shown with S.E. presence of 20 ␮M mevastatin and varying concentrations of SCH66336. Rheb was immunoprecipitated with anti-HA beads. Gels were viewed porated [3H]isoprene (Fig. 3A). SCH66336 treatment com- by autoradiography or immunoblotted for HA. Experiments were repeated in both COS-7 and MCF-7 cells, and a representative experi- pletely inhibited this modification at 1000 nM, with an IC50 ment is displayed. WT, wild type; DMSO,Me2SO. 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 3 HA-Rheb and a Ͼ50% decline in [ H]isoprene. SCH66336 is a by FTI-277 (18). We found that SCH66336 inhibited phospho- selective FPTase inhibitor that has no effect on GGPT-1 at the rylation of S6 ribosomal protein in the human breast cancer cell 3 concentrations used. Incorporation of [ H]mevalonolactone into line MCF-7 (Fig. 4A). Several experiments with 1 ␮M Rheb would not be blocked by SCH66336 if Rheb was alterna- SCH66336 resulted in an average 70% reduction in S6 phos- tively prenylated by GGPT-1. These data show that Rheb is a phorylation. Interestingly, concentrations required to inhibit substrate for farnesylation and is not alternatively prenylated S6 phosphorylation were similar to those that blocked Rheb in FTI-treated cells. Additionally, upon treatment with farnesylation. Additionally, heregulin-stimulated phosphoryla- SCH66336, HA-Rheb underwent a gel mobility shift on SDS- tion of S6 in MCF-7 cells was blocked by SCH66336 (Fig. 4B). PAGE (Fig. 3A), similar to the shift seen when unprenylated Similar inhibition of S6 phosphorylation was observed in the HDJ-2 and H-Ras accumulate upon FTI treatment. human ovarian cancer cell line ES2 (data not shown). In con- A point mutation in Rheb changing the CAAX box from trast, stimulation of Akt and MAPK phosphorylation in MCF-7 CSVM to CSVL was predicted to convert it into a substrate for cells was unaffected by SCH66336 treatment. Surprisingly, GGPT-1, whereas mutation of the modified cysteine residue to basal phosphorylation of MAPK was slightly increased in FTI- serine (SSVM) will block all prenylation of Rheb. We found that treated MCF-7 cells. prenylation of HA-Rheb-CSVL in cells was not inhibited by SCH66336, consistent with Rheb-CSVL geranylgeranylation Ectopic expression of HA-Rheb-CSVL, a mutant designed to (Fig. 3B). As expected, HA-Rheb-SSVM did not incorporate be modified by geranylgeranylation, abrogated SCH66336 in- [3H]isoprene (Fig. 3B). HA-Rheb-SSVM migrated on SDS- hibition of S6 phosphorylation (Fig. 4C). MCF-7 and ES-2 cells ␮ PAGE with similar mobility to that of HA-Rheb in FTI-treated were treated with 1 M SCH66336 for 24 h. In both cell types, cells, whereas HA-Rheb-CSVL migrated between untreated SCH66336 induced a complete gel shift of HA-Rheb. In vector- and treated HA-Rheb (Fig. 3B). and HA-Rheb MCF-7- and ES2-transfected cells, SCH66336 We expanded this finding to human cancer cells. MCF-7 cells reduced levels of S6 phosphorylation by ϳ80%. However, in that stably expressed mevalonate transporter were labeled HA-Rheb-CSVL-transfected MCF-7 cells levels of S6 phospho- with [3H]mevalonolactone. Similar to the COS-7 finding, HA- rylation were unaffected in the presence of 1 ␮M SCH66336. In Rheb was labeled with [3H]isoprene, and this modification was HA-Rheb-CSVL-transfected ES2 cells, 1 ␮M SCH66336 re- completely inhibited by SCH66336 (Fig. 3C). sulted in only a 15% decline in S6 phosphorylation. These data SCH66336 Inhibits S6 Phosphorylation—Rheb has been suggest that inhibition of Rheb farnesylation is responsible for shown to promote phosphorylation of S6 kinase, a downstream SCH66336-induced reduction in S6 phosphorylation. target of mTOR signaling. Rheb overexpression in 293T cells Inhibition of Rheb Function Enhances the Effects of Tamox- stimulates phosphorylation of S6 kinase, and this is inhibited ifen—SCH66336 enhances the anti-tumor response to both ta- SCH66336 Inhibits mTOR Signaling 31105

FIG.5. Inhibition of Rheb function enhances the effects of tamoxifen. MCF-7 cells were drug-treated in the presence of 1 nM estradiol in estrogen-depleted medium containing 10% dialyzed serum for 24 h. Cell lysates were immunoblotted for the p85 PARP fragment, FIG.4. SCH66336 inhibits S6 phosphorylation. Levels of total P-S6, S6, HDJ2, and HA. Experiments were repeated three times, and and phosphorylated S6, Akt, and MAPK were determined by Western a representative experiment is shown. A, cells transfected with HA- blot analysis. Experiments were repeated a minimum of three times, Rheb were treated with Me SO, 1 ␮M SCH66336, 10 ␮M 4-OH-tamox- and a representative experiment is shown. A, MCF-7 cells were treated 2 ifen, or both. B, cells were treated with increasing concentrations of with SCH66336 in the presence of 0.1% serum for 24 h. B, MCF-7 cells rapamycin with or without 10 ␮M 4-OH-tamoxifen. C, HA-Rheb-trans- were treated with 1 ␮M SCH66336 for 72 h, of which during the last fected cells were treated with 0, 5, and 10 ␮M 4-OH-tamoxifen. WT, 24 h, the cells were starved. The cells were then stimulated with 10 wild type. ng/ml heregulin (HRG) for 10 min. C, cells were transfected with HA- Rheb and treated with 1 ␮M SCH66336 in the presence of 0.1% serum for 24 h. WT, wild type. The combination of these agents enhanced this effect and in-

2 duced a 3.5-fold increase. This enhancement of tamoxifen-in- moxifen and taxanes in tumor xenograft models (36). In cell duced apoptosis was abrogated by expression of geranylgera- ␮ culture, we found that 24-h treatment with 1 M SCH66336 or nylated HA-Rheb-CSVL. In these cells, SCH66336 induced a ␮ 10 M tamoxifen reduced the levels of S6 phosphorylation in 1.3-fold increase in p85 PARP fragment. Tamoxifen alone or in MCF-7 cells (Fig. 5A). Combination treatment completely combination with SCH66336 induced a 2.0- and 2.1-fold in- blocked S6 phosphorylation. Similar results were observed in crease, respectively. These data suggest that SCH66336 en- both vector- and HA-Rheb-transfected cells. Expression of HA- hancement of tamoxifen-induced pro-apoptotic effects is a con- Rheb-CSVL rescued the loss of S6 phosphorylation in response sequence of inhibiting Rheb farnesylation. to FTI alone or in response to the combination. SCH66336 We hypothesized that inhibition of Rheb signaling may aug- equally inhibited farnesylation of the co-chaperone protein ment the effects of tamoxifen. To test this hypothesis, other HDJ2 in both control and Rheb-transfected cells. measures of inhibiting Rheb function were tested. We first SCH66336 treatment augmented tamoxifen-induced apopto- treated cells with increasing concentrations of rapamycin to sis, as demonstrated by accumulation of the p85 PARP frag- block mTOR signaling downstream of Rheb. MCF-7 cells were ment (Fig. 5A). Similar results were seen in control and HA- treated with rapamycin with or without 10 ␮M tamoxifen for Rheb-transfected cells. SCH66336 and tamoxifen induced a 24 h. Rapamycin, similar to SCH66336, enhanced tamoxifen 1.5- and 2.0-fold increase in p85 PARP fragment, respectively. induced PARP cleavage and loss of S6 phosphorylation (Fig. 5B). Tamoxifen and rapamycin (0.1 nM) alone induced a 50 2 B. J. Long, manuscript in preparation. and 80% reduction in S6 phosphorylation, respectively. The 31106 SCH66336 Inhibits mTOR Signaling

combination of these two agents induced an almost complete loss. Further, the p85 PARP fragment was barely detectable in tamoxifen and rapamycin (0.1 nM) single agent-treated cells. In cells treated with the combination, there was a 2.5-fold increase in the p85 PARP fragment. This was further enhanced to 7.0-fold in the presence of 1 nM rapamycin, a concentration that alone induced a 1.8-fold increase. Dominant negative mutant Rheb-D60K lacks the ability to bind both GTP and GDP, whereas the Rheb-D60V dominant negative preferentially binds GDP (19). We found that, in MCF-7 cells, expression of either of these two dominant negative proteins reduced basal levels of phosphorylated S6 (Fig. 5C). Expression of HA-Rheb-D60K and HA-Rheb-D60V induced a 70 and 50% reduction in S6 phosphorylation, respectively. When treated with tamoxifen for 24 h, cells expressing dominant negative Rheb were more sensitive to tamoxifen- induced PARP cleavage and loss of phosphorylated S6 (Fig. 5C). Tamoxifen (10 ␮M) treatment resulted in a 75% reduction in S6 phosphorylation. This was enhanced to an almost complete loss of S6 phosphorylation in the presence of dominant negative Rheb. The p85 PARP fragment was barely detected in vector- and HA-Rheb-transfected cells treated with 5 ␮M tamoxifen but was easily detected in cells expressing dominant negative Rheb. Further, p85 PARP accumulation was enhanced by Ͼ2-fold in cells expressing dominant negative Rheb as compared with vector- and HA-Rheb-transfected cells. These data show rapamycin treatment or expression of dominant FIG.6. Inhibition of Rheb function enhances docetaxel-induced caspase activity. Caspase-3 activity was measured by cleavage negative HA-Rheb proteins mimicked SCH66336 with respect of fluorescent DEVD peptide. The average of three experiments in ES2 to enhancing tamoxifen-induced effects. cells is shown with S.E. Similar results were observed in MDA-435 Inhibition of Rheb Function Enhances the Effects of Do- cells. A, ES2 cells transfected with HA-Rheb were treated with Me2SO ␮ cetaxel—Similar to the results with tamoxifen, we demon- (DMSO), 1 M SCH66336, 10 nM docetaxel, or both for 4 h followed by Me2SO or SCH66336 for the remainder of the 48-h treatment. Signifi- strated that SCH66336 enhances the consequences of docetaxel cant difference in vector and wild type (WT) cells between single agent treatment, including induction of caspase-3 activity.3 We and combination treatments; p value Ͻ 0.01. B, the ability of various sought to determine the effects of HA-Rheb-CSVL, dominant Rheb inhibitory methods to enhance docetaxel was examined. Cells negative HA-Rheb, and rapamycin on the response to FTI in were treated with 100 nM rapamycin with or without 10 nM docetaxel for 4 h followed by rapamycin for the remainder of the 48-h treatment. combination with docetaxel. ES2 ovarian cancer cells were Cells were also transfected with siRNA or HA-Rheb and then exposed to transfected with vector, HA-Rheb, or HA-Rheb-CSVL, and docetaxel for 4 h, the media was changed, and the cells were incubated caspase-3 activity was measured (Fig. 6A). The cells were for the remainder of the 48-h treatment. Significant differences be- treated with Me SO, 1 ␮M SCH66336, 10 nM docetaxel, or both tween rapamycin and vehicle, D60V and vector, combination siRNA 2 and control are shown; p value Ͻ 0.05. for 4 h followed by Me2SO or SCH66336 for the remainder of the 48-h treatment. Single agent SCH66336 treatment had Rheb-transfected cells. Further, a HA-Rheb-D60V Rheb mu- minor effects on caspase-3 activity (1.3-fold increase). Treat- tant terminating in CSVL also functioned as a dominant neg- ment of cells with docetaxel caused a 2-fold increase in ative, and docetaxel induced a 3.1-fold increase in cells caspase-3 activity. Combination therapy resulted in a 3.4-fold expressing this protein (Fig. 6B). induction in caspase activity. A similar pattern was seen in Rheb siRNA also enhanced docetaxel-induced caspase activ- cells transfected with wild-type HA-Rheb. Ectopic expression of ity. In control siRNA-transfected cells, docetaxel induced a HA-Rheb-CSVL abrogated SCH66336 enhancement of do- 2.3-fold increase. Docetaxel induced a 2.2- and 2.8-fold increase cetaxel-induced caspase activity. In these cells, docetaxel and in cells transfected with either Rheb or Rheb2 siRNA, respec- combination treatment induced a 1.8- and 2-fold increase in tively (Fig. 6B). Rheb siRNA reduced Rheb mRNA levels by caspase activity, respectively. Similar results were observed in 86% as determined by quantitative RT-PCR, whereas Rheb2 MDA-435 breast cancer cells (data not shown). siRNA reduced Rheb2 mRNA levels by 62% (data not shown). To determine whether SCH66336 enhancement of docetaxel- Each specific siRNA had no effect on mRNA expression of the induced caspase activity was a result of inhibiting Rheb, we other family member. In cells transfected with both Rheb blocked Rheb signaling by rapamycin treatment, expression of siRNA plus Rheb2 siRNA, Rheb and Rheb2 mRNA were re- dominant negative HA-Rheb-D60V, or siRNA. Cells were duced by 90 and 79%, respectively. As commercially available treated with vehicle or 100 nM rapamycin with or without 10 nM Rheb antibody detects endogenous Rheb poorly, we could not docetaxel for 4 h followed by vehicle or rapamycin alone for the confirm knockdown of the Rheb protein. We therefore trans- remainder of the 48-h treatment. Docetaxel induced a 3.3-fold fected cells with HA-Rheb with or without Rheb siRNA and increase in caspase-3 activity in rapamycin-treated cells, com- found HA-Rheb was depleted completely in the presence of the pared with a 2.2-fold increase in vehicle-treated cells (Fig. 6B). siRNA (data not shown). We found that docetaxel induced a Similar to rapamycin treatment, ectopic expression of HA- 4.8-fold increase in caspase activity in these cells, suggesting Rheb-D60V enhanced docetaxel-induced caspase activity. Do- that both siRNA against Rheb and Rheb2 was needed to sen- cetaxel induced a 3.5-fold increase in HA-Rheb-D60V cells, as sitize cells to docetaxel-induced caspase activity (Fig. 6B). compared with a 2.2- and 2.3-fold increase in vector- and HA- DISCUSSION SCH66336 (lonafarnib) is a farnesyl transferase inhibitor 3 A. D. Basso and B. J. Long, manuscript submitted. (FTI) that blocks post-translational processing of farnesylated SCH66336 Inhibits mTOR Signaling 31107 proteins including H-Ras. Upon treatment of cells with FTIs, and Rheb2, docetaxel induced a 4.8-fold increase in caspase some farnesylated proteins, including K- and N-Ras, become activity, as compared with a 2.2-fold increase in control-trans- alternatively prenylated by geranylgeranyl transferase-1 (40, fected cells. Cells transfected with siRNA against either Rheb 45). Despite the failure to abrogate prenylation of K- and N- or Rheb2 alone did not display enhanced sensitivity to do- Ras, growth of many tumors in preclinical models is inhibited cetaxel, indicating that inhibition of both family members by FTIs. This suggests the anti-proliferative action of FTIs is is needed. dependent on blocking farnesylation of proteins in addition to We have reproduced these experiments using structurally the Ras family. In this report, we investigated the role of distinct FTIs, including R115777 (Tipifarnib) and BMS-214662 farnesylation of Rheb (Ras homologue-enriched in brain), a (data not shown). We also tested SCH66337, an enantiomer of small GTPase that positively regulates mTOR signaling. Rheb SCH66336 that does not inhibit FPT in vitro and fails to induce overexpression induces phosphorylation of mTOR substrates, a HDJ-2 mobility shift in treated cells. We found that the FTIs an effect blocked by treatment with FTI-277, suggesting that (but not SCH66337) inhibited Rheb farnesylation, as detected Rheb function is regulated by farnesylation (18). Clark et al. by a mobility shift on SDS-PAGE. Similar to SCH66336, (25) previously reported that GST-Rheb is labeled by farnesyl R115777 and BMS-214662 (but not SCH66337) enhanced apo- diphosphate (but not geranylgeranyl diphosphate) when incu- ptosis in response to both tamoxifen and docetaxel. bated with rabbit reticulocyte lysates. Rheb may represent one of the critical elusive targets of We demonstrated that human Rheb and Rheb2 are farnesy- farnesyl transferase inhibitors. We found that Rheb and Rheb2 lated and are not substrates for alternative prenylation. In expression were elevated in tumor cell lines relative to their vitro, peptides derived from the carboxyl termini of Rheb and normal counterparts. Rheb plays a key role in cell growth in Rheb2 were substrates for FPT but not GGPT-1. In cell culture, that it activates signaling via the mTOR pathway. The Rheb ⅐ Rheb protein was labeled by mevalonolactone and was thus a GTPase-activating protein hamartin tuberin complex is nega- substrate for prenylation. This modification was completely tively regulated by Akt-mediated phosphorylation of tuberin, blocked by SCH66336, a selective FPT inhibitor, indicating resulting in increased Rheb activity (9). Akt signaling is ele- that, similar to H-Ras, Rheb and Rheb2 are not alternatively vated in tumors by Akt amplification, phosphatidylinositol prenylated in FTI-treated cells. Post-translational processing 3-kinase amplification, loss of heterozygosity of phosphatase of both Rheb family members is inhibited by SCH66336. and tensin homologue, HER2 amplification, and EGFR-activat- As a result of inhibiting Rheb farnesylation, SCH66336 ing mutations (52–59). Our data show that Rheb activity may blocks S6 phosphorylation. FTI L-744,832 was previously re- also be increased in tumor cells by elevated mRNA expression. ported to mimic rapamycin by inhibiting DNA synthesis and S6 Our data demonstrated that Rheb is overexpressed in tumor cells, is modified by farnesylation, is not a substrate for alter- kinase activation in response to serum. Law et al. (46) further native prenylation, and plays a role in SCH66336 enhancement demonstrated that a rapamycin-resistant mutant of S6 kinase of other chemotherapeutics. To date, FTIs have demonstrated is resistant to FTI-induced dephosphorylation. The mechanism efficacy in hematologic malignancies (60) but have shown lim- whereby FTI inhibits S6 kinase activation was undefined in ited single agent activity in solid tumors. Ongoing clinical trials these studies. Recently, it was found that Rheb overexpression with lonafarnib and other FTIs in combination with taxanes induces phosphorylation of mTOR substrates S6 kinase and and hormonal therapies in solid tumors are in progress. Inhi- 4E-BP1. Rheb-induced phosphorylation of S6 is blocked by bition of other farnesylated proteins in addition to Rheb may rapamycin, FTI-277, or its upstream regulator TSC (3, 18, 19). contribute to the anti-tumor effects of FTIs. Candidate proteins Similar to Law et al., we showed that SCH66336 blocked include RhoB and the centromere-associated proteins CENP-E heregulin-induced phosphorylation of S6, although having no and CENP-F (33, 61, 62). Defining the relative importance of effect on the activation of Akt and MAPK. Further, basal phos- these various proteins in the anti-tumor activity of FTIs re- phorylation of S6 was reduced in SCH66336-treated cells. quires further study. Our current results suggest that Rheb These effects were abrogated by the expression of HA-Rheb- may be one of the critical FTI targets and that inhibition of CSVL, a mutant Rheb that is geranylgeranylated and thus not Rheb farnesylation by FTIs may play a critical role in enhanc- effected by SCH66336. These data indicate that inhibition of ing the anti-tumor effect of docetaxel and tamoxifen. Rheb farnesylation is responsible for FTI-induced reduction in S6 phosphorylation. Acknowledgments—We thank Rosalinda Syto and Bill Windsor for SCH66336 synergizes with various chemotherapy agents, expression and purification of FPT and GGPT. We appreciate everyone including taxanes and cisplatin, to enhance their anti-tumor who contributed to the RNA panel, especially Suxing Liu, Asra Mirza, Sandra Koseoglu, Priya Dayanath, Xiaoying Wang, and Irma Oliva. We activity (47, 48). We found that single agent FTI treatment has thank Dr. Janet Price for kindly providing MDA-435 cells. We are little effect on apoptosis, but FTI treatment enhances tamox- grateful for the helpful discussions and insightful input from all of the ifen- and taxane-induced apoptosis.3 Rapamycin was recently members of the tumor biology group at Schering-Plough Research reported to synergistically interact in combination with pacli- Institute. taxel and carboplatin (49). Further, rapamycin restores sensi- REFERENCES tivity to Akt-induced tamoxifen-resistant cells (50). Our data 1. Yamagata, K., Sanders, L. K., Kaufmann, W. E., Yee, W., Barnes, C. A., suggest that FTI enhancement of tamoxifen- and taxane-in- Nathans, D., and Worley, P. F. (1994) J. Biol. Chem. 269, 16333–16339 2. Gromov, P. S., Madsen, P., Tomerup, N., and Celis, J. E. (1995) FEBS Lett. duced apoptosis is a consequence of the inhibition of Rheb 377, 221–226 signaling. Expression of HA-Rheb-CSVL abrogated SCH66336 3. Tee, A. R., Manning, B. D., Roux, P. P., Cantley, L. C., and Blenis, J. (2003) Curr. Biol. 13, 1259–1268 enhancement of both tamoxifen- and docetaxel-induced apopto- 4. Inoki, K., Li, Y., Xu, T., and Guan, K. L. (2003) Genes Dev. 17, 1829–1834 sis. Further, inhibition of Rheb signaling by rapamycin treat- 5. Garami, A., Zwartkruis, F. J., Nobukuni, T., Joaquin, M., Roccio, M., Stocker, ment or expression of dominant negative Rheb proteins mim- H., Kozma, S. C., Hafen, E., Bos, J. L., and Thomas, G. (2003) Mol. Cell 11, 1457–1466 icked FTI treatment and sensitized cells to tamoxifen- and 6. Zhang, Y., Gao, X., Saucedo, L. J., Ru, B., Edgar, B. A., and Pan, D. (2003) Nat. docetaxel-induced apoptosis. We found that Rheb-Asp-60 mu- Cell Biol. 5, 578–581 7. European Chromosome 16 Tuberous Sclerosis Consortium (1993) Cell 75, tations created dominant negative proteins, supporting the 1305–1315 findings of Tabancay et al. (19) and conflicting with the findings 8. van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S., Lindhout, D., van den Ouweland, A., Halley, D., Young, J., of Li et al. (51). Rheb siRNA also enhanced docetaxel-induced Burley, M., Jeremiah, S., Woodward, K., Nahmias, J., Fox, M., Ekong, R., caspase activity. In cells transfected with siRNA against Rheb Osborne, J., Wolfe, J., Povey, S., Snell, R. G., Cheadle, J. P., Jones, A. C., 31108 SCH66336 Inhibits mTOR Signaling

Tachataki, M., Ravine, D., and Kwiatkowski, D. J. (1997) Science 277, Graham, S. L., Smith, R. L., Scolnick, E. M., Oliff, A., and Gibbs, J. B. (1993) 805–808 Science 260, 1934–1937 9. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002) Nat. Cell Biol. 4, 40. Zhang, F. L., Kirschmeier, P., Carr, D., James, L., Bond, R. W., Wang, L., 648–657 Patton, R., Windsor, W. T., Syto, R., Zhang, R., and Bishop, W. R. (1997) 10. Li, Y., Inoki, K., Yeung, R., and Guan, K. L. (2002) J. Biol. Chem. 277, J. Biol. Chem. 272, 10232–10239 44593–44596 41. James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 11. Potter, C. J., Pedraza, L. G., and Xu, T. (2002) Nat. Cell Biol. 4, 658–665 6221–6226 12. Liu, M. Y., Cai, S., Espejo, A., Bedford, M. T., and Walker, C. L. (2002) Cancer 42. Mangues, R., Corral, T., Kohl, N. E., Symmans, W. F., Lu, S., Malumbres, M., Res. 62, 6475–6480 Gibbs, J. B., Oliff, A., and Pellicer, A. (1998) Cancer Res. 58, 1253–1259 13. Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J., and Cantley, L. C. (2002) 43. Omer, C. A., Chen, Z., Diehl, R. E., Conner, M. W., Chen, H. Y., Trumbauer, Mol. Cell 10, 151–162 M. E., Gopal-Truter, S., Seeburger, G., Bhimnathwala, H., Abrams, M. T., 14. Shaw, R. J., Bardeesy, N., Manning, B. D., Lopez, L., Kosmatka, M., DePinho, Davide, J. P., Ellis, M. S., Gibbs, J. B., Greenberg, I., Koblan, K. S., Kral, R. A., and Cantley, L. C. (2004) Cancer Cell 6, 91–99 A. M., Liu, D., Lobell, R. B., Miller, P. J., Mosser, S. D., O’Neill, T. J., Rands, 15. Inoki, K., Zhu, T., and Guan, K. L. (2003) Cell 115, 577–590 E., Schaber, M. D., Senderak, E. T., Oliff, A., and Kohl, N. E. (2000) Cancer 16. Jenne, D. E., Reimann, H., Nezu, J., Friedel, W., Loff, S., Jeschke, R., Muller, Res. 60, 2680–2688 O., Back, W., and Zimmer, M. (1998) Nat. Genet. 18, 38–43 44. Kim, C. M., Goldstein, J. L., and Brown, M. S. (1992) J. Biol. Chem. 267, 17. Hemminki, A., Markie, D., Tomlinson, I., Avizienyte, E., Roth, S., Loukola, A., 23113–23121 Bignell, G., Warren, W., Aminoff, M., Hoglund, P., Jarvinen, H., Kristo, P., 45. James, G., Goldstein, J. L., and Brown, M. S. (1996) Proc. Natl. Acad. Sci. Pelin, K., Ridanpaa, M., Salovaara, R., Toro, T., Bodmer, W., Olschwang, S., U. S. A. 93, 4454–4458 Olsen, A. S., Stratton, M. R., de la Chapelle, A., and Aaltonen, L. A. (1998) 46. Law, B. K., Norgaard, P., and Moses, H. L. (2000) J. Biol. Chem. 275, Nature 391, 184–187 10796–10801 18. Castro, A. F., Rebhun, J. F., Clark, G. J., and Quilliam, L. A. (2003) J. Biol. 47. Liu, M., Bryant, M. S., Chen, J., Lee, S., Yaremko, B., Lipari, P., Malkowski, Chem. 278, 32493–32496 M., Ferrari, E., Nielsen, L., Prioli, N., Dell, J., Sinha, D., Syed, J., Korfma- 19. Tabancay, A. P., Jr., Gau, C. L., Machado, I. M., Uhlmann, E. J., Gutmann, cher, W. A., Nomeir, A. A., Lin, C. C., Wang, L., Taveras, A. G., Doll, R. J., D. H., Guo, L., and Tamanoi, F. (2003) J. Biol. Chem. 278, 39921–39930 Njoroge, F. G., Mallams, A. K., Remiszewski, S., Catino, J. J., Girijavallab- 20. Long, X., Lin, Y., Ortiz-Vega, S., Yonezawa, K., and Avruch, J. (2005) Curr. han, V. M., and Bishop, W. R. (1998) Cancer Res. 58, 4947–4956 Biol. 15, 702–713 48. Nielsen, L. L., Shi, B., Hajian, G., Yaremko, B., Lipari, P., Ferrari, E., Gurnani, 21. Smith, E. M., Finn, S. G., Tee, A. R., Browne, G. J., and Proud, C. G. (2005) M., Malkowski, M., Chen, J., Bishop, W. R., and Liu, M. (1999) Cancer Res. J. Biol. Chem. 280, 18717–18727 59, 5896–5901 22. Patel, P. H., Thapar, N., Guo, L., Martinez, M., Maris, J., Gau, C. L., Lengyel, 49. Mondesire, W. H., Jian, W., Zhang, H., Ensor, J., Hung, M. C., Mills, G. B., and J. A., and Tamanoi, F. (2003) J. Cell Sci. 116, 3601–3610 Meric-Bernstam, F. (2004) Clin. Cancer Res. 10, 7031–7042 23. Saucedo, L. J., Gao, X., Chiarelli, D. A., Li, L., Pan, D., and Edgar, B. A. (2003) 50. deGraffenried, L. A., Friedrichs, W. E., Russell, D. H., Donzis, E. J., Middleton, Nat. Cell Biol. 5, 566–571 A. K., Silva, J. M., Roth, R. A., and Hidalgo, M. (2004) Clin. Cancer Res. 10, 24. Stocker, H., Radimerski, T., Schindelholz, B., Wittwer, F., Belawat, P., Daram, 8059–8067 P., Breuer, S., Thomas, G., and Hafen, E. (2003) Nat. Cell Biol. 5, 559–565 51. Li, Y., Inoki, K., and Guan, K. L. (2004) Mol. Cell. Biol. 24, 7965–7975 25. Clark, G. J., Kinch, M. S., Rogers-Graham, K., Sebti, S. M., Hamilton, A. D., 52. Bellacosa, A., de Feo, D., Godwin, A. K., Bell, D. W., Cheng, J. Q., Altomare, and Der, C. J. (1997) J. Biol. Chem. 272, 10608–10615 26. Urano, J., Tabancay, A. P., Yang, W., and Tamanoi, F. (2000) J. Biol. Chem. D. A., Wan, M., Dubeau, L., Scambia, G., Masciullo, V., Ferrandina, G., 275, 11198–11206 Benedetti-Panici, P., Mancuso, S., Neri, G., and Test, J. R. (1995) Int. J. 27. Reiss, Y., Stradley, S. J., Gierasch, L. M., Brown, M. S., and Goldstein, J. L. Cancer 64, 280–285 (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 732–736 53. Shayesteh, L., Lu, Y., Kuo, W. L., Baldocchi, R., Godfrey, T., Collins, C., Pinkel, 28. Yokoyama, K., Goodwin, G. W., Ghomashchi, F., Glomset, J. A., and Gelb, D., Powell, B., Mills, G. B., and Gray, J. W. (1999) Nat. Genet. 21, 99–102 M. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5302–5306 54. Bose, S., Wang, S. I., Terry, M. B., Hibshoosh, H., and Parsons, R. (1998) 29. Casey, P. J., Solski, P. A., Der, C. J., and Buss, J. E. (1989) Proc. Natl. Acad. Oncogene 17, 123–127 Sci. U. S. A. 86, 8323–8327 55. Feilotter, H. E., Nagai, M. A., Boag, A. H., Eng, C., and Mulligan, L. M. (1998) 30. Wolda, S. L., and Glomset, J. A. (1988) J. Biol. Chem. 263, 5997–6000 Oncogene 16, 1743–1748 31. Caplan, A. J., Tsai, J., Casey, P. J., and Douglas, M. G. (1992) J. Biol. Chem. 56. Pesche, S., Latil, A., Muzeau, F., Cussenot, O., Fournier, G., Longy, M., Eng, 267, 18890–18895 C., and Lidereau, R. (1998) Oncogene 16, 2879–2883 32. Wang, J., Kirby, C. E., and Herbst, R. (2002) J. Biol. Chem. 277, 46659–46668 57. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., 33. Ashar, H. R., James, L., Gray, K., Carr, D., Black, S., Armstrong, L., Bishop, Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and Press, M. F. (1989) W. R., and Kirschmeier, P. (2000) J. Biol. Chem. 275, 30451–30457 Science 244, 707–712 34. Njoroge, F. G., Taveras, A. G., Kelly, J., Remiszewski, S., Mallams, A. K., 58. Paez, J. G., Janne, P. A., Lee, J. C., Tracy, S., Greulich, H., Gabriel, S., Wolin, R., Afonso, A., Cooper, A. B., Rane, D. F., Liu, Y. T., Wong, J., Herman, P., Kaye, F. J., Lindeman, N., Boggon, T. J., Naoki, K., Sasaki, H., Vibulbhan, B., Pinto, P., Deskus, J., Alvarez, C. S., del Rosario, J., Connolly, Fujii, Y., Eck, M. J., Sellers, W. R., Johnson, B. E., and Meyerson, M. (2004) M., Wang, J., Desai, J., Rossman, R. R., Bishop, W. R., Patton, R., Wang, L., Science 304, 1497–1500 Kirschmeier, P., and Ganguly, A. K. (1998) J. Med. Chem. 41, 4890–4902 59. Sordella, R., Bell, D. W., Haber, D. A., and Settleman, J. (2004) Science 305, 35. Moasser, M. M., Sepp-Lorenzino, L., Kohl, N. E., Oliff, A., Balog, A., Su, D. S., 1163–1167 Danishefsky, S. J., and Rosen, N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 60. Alsina, M., Fonseca, R., Wilson, E. F., Belle, A. N., Gerbino, E., Price-Troska, 1369–1374 T., Overton, R. M., Ahmann, G., Bruzek, L. M., Adjei, A. A., Kaufmann, 36. Shi, B., Yaremko, B., Hajian, G., Terracina, G., Bishop, W. R., Liu, M., and S. H., Wright, J. J., Sullivan, D., Djulbegovic, B., Cantor, A. B., Greipp, Nielsen, L. L. (2000) Cancer Chemother. Pharmacol. 46, 387–393 P. R., Dalton, W. S., and Sebti, S. M. (2004) Blood 103, 3271–3277 37. Hancock, J. F., Cadwallader, K., and Marshall, C. J. (1991) EMBO J. 10, 61. Ashar, H. R., James, L., Gray, K., Carr, D., McGuirk, M., Maxwell, E., Black, 641–646 S., Armstrong, L., Doll, R. J., Taveras, A. G., Bishop, W. R., and Kir- 38. Gutierrez, L., Magee, A. I., Marshall, C. J., and Hancock, J. F. (1989) EMBO schmeier, P. (2001) Exp. Cell Res. 262, 17–27 J. 8, 1093–1098 62. Lebowitz, P. F., Casey, P. J., Prendergast, G. C., and Thissen, J. A. (1997) 39. Kohl, N. E., Mosser, S. D., deSolms, S. J., Giuliani, E. A., Pompliano, D. L., J. Biol. Chem. 272, 15591–15594