farnesyltransferase inhibitor sch66336 induces rapid

Farnesyltransferase Inhibitor SCH66336 Induces Rapid

Phosphorylation of Eukaryotic Translation Elongation

Factor 2 in Head and Neck Squamous Cell

Carcinoma Cells

Hening Ren,1Shyh-Kuan Tai,

1,3Fadlo Khuri,

4Zuming Chu,

1and Li Mao

1,2

1Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center; 2CancerBiology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas; 3Departmentof Otolaryngology, National Yang Ming University, Taipei Veteran General Hospital, Taipei, Taiwan; and 4Departmentof Oncology/Hematology, Winship Cancer Institute, Emory University, Atlanta, Georgia

Abstract

Farnesyltransferase inhibitors (FTIs) are a class of therapeuticagents designed to target tumors with mutations of the rasoncogene. However, the biological effect of FTIs is oftenindependent of ras mutation status, which suggests theexistence of additional mechanisms. In this study, weinvestigated the molecular effects of SCH66336, an FTI, inhead and neck squamous cell carcinoma cells using proteomicapproaches. We showed that SCH66336 induced phosphoryla-tion (inactivation) of eukaryotic translation elongation factor2 (eEF2), an important molecule for protein synthesis, as earlyas 3 hours after SCH66336 administration. Protein synthesiswas subsequently reduced in the cells. Paradoxically, activa-tion of eEF2 kinase (eEF2K), the only known kinase thatregulates eEF2, was observed only at 12 hours after SCH66336treatment. Consistent with this observation, the inhibition ofphosphorylated-MEK and phosphorylated-p70S6K, the twokey signaling molecules responsible for activation of eEF2K,also occurred at least 12 hours after SCH66336 administra-tion. Our data suggest that inhibition of protein synthesisthrough inactivation of eEF2 is a novel mechanism ofSCH66336-mediated growth inhibition and that this effect isindependent of ras-MEK/p70S6K-eEF2K signaling cascades.(Cancer Res 2005; 65(13): 5841-7)

Introduction

Protein prenylation is a posttranslational modification in whicha farnesyl or geranylgeranyl isoprenoid is linked to a specificcystine residue of proteins through a thioether bond (1). Thehousekeeping enzymes farnesyltransferase and glycerol-3-phos-phare cytidylyltransferase I and II catalyze the addition of a prenylgroup to a conserved cystine residue in proteins that contain themotif CaaX, CC, or CxC at or near the COOH terminal of theirnascent proteins (2).Comprising up to 0.5% of all proteins in mammalian tissues (3),

prenylated proteins have diverse functions in cell growth,differentiation, cytoskeleton structure, and vesicle trafficking(2, 4). Examples of such proteins are the ras family of smallGTP-binding proteins, Rho family proteins, certain phosphatases

and protein kinases, nuclear lamins, and centromere protein F (4).The ras protein plays a critical role in transducing growth signalsfrom cell surface receptors to cytosol and nucleus. Activationmutations of ras are frequently detected in various types ofhuman cancers (5, 6) and its constitutive activation helps trans-form normal cells in both in vitro and in vivo models therebyleading to tumor formation (7, 8). The discovery that prenyla-tion is a necessary step in the functional maturation of ras (9)prompted the development of farnesyltransferase inhibitors(FTIs) as targeted therapeutic agents in cancers with a ras muta-tion (10–12).In a clinical study of patients with head and neck squamous cell

carcinoma (HNSCC), we observed antitumor activity of SCH66336,a potent nonpeptide tricyclic inhibitor of farnesyltransferase (13).This FTI has also been shown to have antitumor activity in vitroand in vivo for other tumors with or without a ras mutation(14, 15). However, the mechanism of this activity is poorlyunderstood. The effect of SCH66336 on cell growth inhibition isoften observed within a few hours after administration, althoughthe cellular half-life of ras is f24 hours (16). In light of thisdiscrepancy and the possibility that FTIs may inhibit the activity offarnesylated proteins other than ras, FTIs may work through morethan one pathway for their antitumor activity. Using proteomicapproaches, we explored how SCH66336 affects the growth ofHNSCC cells and whether the effects were dependent on rassignaling. We found that SCH6636 may induce growth inhibition ofHNSCC cells by delaying their entry into and accumulation in theG1 phase of the cell cycle. Evidence emerged that SCH66336induces rapid inactivation of eukaryotic translation elongationfactor 2 (eEF2) through its phosphorylation and subsequentreduction of protein synthesis. Furthermore, the inactivation ofeEF2 was independent of ras-MEK-eEF2 kinase (eEF2K) and ras-PI3K/p70S6K-eEF2K signaling cascades.

Materials and Methods

Cell lines and culture conditions. Eight human HNSCC cell lines

(UMSCC14B, UMSCC17B, UMSCC21A, UMSCC22A, UMSCC38, MDA1186,

MDA886, and TR146) were used in this study. The cells were grown in

monolayer culture in a 1:1 mixture of DMEM and Ham’s F12 medium

supplemented with heat inactivated 5% fetal bovine serum and antibiotics

at 37jC in a humidified atmosphere consisting of 95% air and 5% CO2. For

synchronized culture, cells were grown exponentially to 40% confluence

and starved in serum-free DMEM/Ham’s F12 medium for 24 hours before

serum-containing medium was added back.

Cell cycle analysis. UMSCC38 cells were grown to 30% confluence andgrown for 18 hours in medium with 5% serum and with serum-free medium

Requests for reprints: Li Mao, Molecular Biology Laboratory, Department ofThoracic/Head and Neck Medical Oncology, The University of Texas M.D. AndersonCancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363;Fax: 713-796-8655; E-mail: [email protected].

I2005 American Association for Cancer Research.

www.aacrjournals.org 5841 Cancer Res 2005; 65: (13). July 1, 2005

Research Article

for 24 hours. Serum-containing medium was then added back, and cellswere harvested at different times, fixed in 70% cold ethanol, and stored at

4jC until cell cycle analysis. The FTI SCH66336 dissolved in DMSO was

added to the cell culture medium, and cells were harvested at different

times. The cells were then stained with 50 Amol/L/mL propidium iodide in

PBS buffer containing 50 Ag/mL RNase A. DNA content was measured

using an EPICS 752 flow cytometer (Coulter Corp., Hialeah, FL). Data

analysis was done using the Multi series (Phoenix Flow Systems, San

Diego, CA) and Summit software (Cytomation, Fort Collins, CO).Protein extraction and Western blot analysis. Cells were washed in

cold PBS and incubated for 15 minutes on ice in a buffer containing

50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% SDS, and 1% TritonX-100 supplemented with a protease inhibitor cocktail (Roche Applied

Science, Indianapolis, IN). The cell lysates were spun in a centrifuge at

12,000 � g for 5 minutes. The protein concentration of the supernatant was

determined using a detergent-compatible protein assay kit (Bio-Rad,Hercules, CA). Proteins (10 Ag) were separated through a 10% polyacryl-

amide gel in a Mini-Protean II apparatus (Bio-Rad) and transferred to a

nitrocellulose membrane (BA83; Schleicher & Shuell BioScience, Keene,

NH). Membranes were blocked with 2% casein in PBS and probed withantibodies. Specific antibody binding was detected using an enhanced

chemiluminescence kit (Pierce, Rockford, IL) according to the manufac-

turer’s protocol.

For Western blotting, antibodies were obtained from Cell SignalingTechnology (Beverly, MA) against phospho-(serine/threonine) protein kinase

A (PKA) substrate, phospho-(serine) protein kinase C (PKC) substrate,

phospho-eEF2 (Thr56), eEF2, phospho-eEF2K (Ser366), eEF2K, mitogen-activated protein kinase kinase (MEK), phosphor-MEK1/2 (Ser217/221), and

phospho-p70S6K/p-85S6K (Thr389). Monoclonal anti-actin antibody (AC-15)

was obtained from Sigma Chemical (St. Louis, MO).

Two-dimensional gel electrophoresis. Cells grown in monolayer werewashed in cold PBS thrice, and proteins were extracted by the addition of

two-dimensional gel electrophoresis sample buffer containing 8 mol/L urea,4% CHAPS, and 25 mmol/L DTT. An aliquot of cell lysates containing an

equivalent of 5 � 105 cells was applied to a 17-cm immobilized pH gradientstrip (pH 5 to 8, Bio-Rad) for 12 hours and focused under 48,000 V hours at

18jC in an IPGphor isoelectric focusing unit (Amershan Biosciences,

Piscataway, NJ). After focusing, the immobilized pH gradient strips weretreated sequentially with 2% DTT followed by 2.5% iodoacetamide in SDS-

PAGE equilibration buffer [6 mol/L urea, 0.375 mol/L Tris (pH 8.8), 2% SDS,and 20% glycerol] for 15 minutes each. Focused proteins were then

separated in a 10% SDS-polyacrylamide gel. For two-dimensional gelelectrophoresis Western blotting, the separated proteins were transferred to

nitrocellulose membranes, blocked, and probed with antibodies asdescribed above. For analysis of newly synthesized (radiolabeled) proteins,

the gels were fixed and stained with a Silver Stain Plus kit (Bio-Rad)

according to the manufacturer’s protocol and dried on filter paper followedby exposure to autoradiography. To quantitate the level of protein

expression by two-dimensional gel electrophoresis, the autoradiographyor gel image was scanned using Amersham-Pharmacia ImageScanner. The

integrated absorbance of all recognized protein spots was obtained byanalyzing the gel image with ImageMaster 2D image analysis software

(Amersham Biosciences).

Peptide mapping for protein identification. After two-dimensionalgel electrophoresis separation of cellular proteins, the gels were stained

using colloidal Coomassie brilliant blue (Bio-Rad) in 17% ammonium

sulfate and 15% methanol, as previously described (17). Protein spots wereexcised, destained in 50% methanol, and dehydrated in acetonitrile. The

dried gel slots were rehydrated and digested in 25 AL of 25 mmol/L

ammonium carbonate containing 2 Ag/mL sequencing grade modified

trypsin (Roche Applied Science) at 37jC overnight. The digest productswere purified using C18 microbed chromatography (ZipTip, Millipore,

Billerica, MA) according to the manufacturer’s protocol. The purified

peptides were eluted in 50% acetonitrile and 0.1% trifluoroacetic acid

saturated with a-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, St. Louis,MO), and 1.5 AL of the peptide mix were spotted on a sample plate for

analysis. Peptide fragments were determined by using a matrix-assisted

laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), AXIMA-CFR (Kratos Analytical, Manchester, United Kingdom).

Protein identification based on peptide fingerprints was achieved using

online search engine: Mascot.5

Metabolic labeling. Cells were cultured in 60-mm plastic dishes to30% confluence, synchronized by serum starvation for 24 hours, andgrown in serum-containing medium for another 24 hours. Thesynchronized cells were then treated with SCH66336 in full culturemedium for 1 hour. The medium was changed to cystine- andmethionine-free DMEM with 5% serum for 30 minutes and then 100ACi [35S] trans-label mixture (Amersham Biosciences) was added. Cellswere harvested 4 hours later, and proteins were extracted in the two-dimensional gel electrophoresis sample buffer. The extracted cellularproteins were analyzed for total proteins and newly synthesized proteinsas described above.

Results

The replication cycle of the HNSCC cell line UMSCC38 was f30hours when cultured in DMEM and Ham’s F12 with 5% serum. Wetreated these cells with 8 Amol/L SCH66336 when most of themhad completed one replication after synchronization. We observeda slowed cell accumulation at the G1 phase of the cell cycle after 6hours following SCH66336 treatment but a prolonged G1 phase(Fig. 1). We did not observe an emergence of a sub-G1 populationin the drug-treated cells but an early accumulation of G2-M phase(data not shown). These results suggest a delayed G1 entry and G1

arrest by SCH66336 in UMSCC38 cells.To identify which protein expression were affected by

SCH66336 treatment, we did two-dimensional gel electrophoresisanalysis to compare expression levels in UMSCC38 cells beforeand after treatment. The expression levels of the most of theproteins remained similar. The exceptions were a protein off100 kDa with an isoelectric point (pI) of 7.1 and anotherprotein of f100 kDa with a pI of 7.4, which increased anddecreased, respectively, after SCH66336 treatment (Fig. 2A and B).In a separate attempt to identify proteins whose phosphorylation

Figure 1. Change of G1 phase distribution after SCH66336 treatment.UMSCC38 cell growth was synchronized by serum starvation. The cells thencultured with 5% serum and treated with 8 Amol/L SCH66336 when most ofthem were rolled out from the G1 phase. The percentage of cells in the G1 phasewas determined using flow cytometry. Treatment with SCH66336 (FTI)slowed entry of cells into G1 and subsequent prolonged accumulation in the G1

phase compared with cells treated with DMSO (Control ).

5 http://www.matrixscience.com/references/725.html

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Cancer Res 2005; 65: (13). July 1, 2005 5842 www.aacrjournals.org

was affected by FTI treatment, we stained two-dimensionalprotein blots with several phosphorylation-specific antibodies,including antibodies specific to phosphorylated substrates of PKAand PKC. We found that the level of protein phosphorylation wasgenerally reduced after SCH66336 treatment, but the phosphor-ylation of a few protein spots, including a 100-kDa protein with apI of 7.1 was increased (Fig. 2C and D). The 100-kDa (pI = 7.1)protein spot recognized by anti-phospho-PKA and anti-phospho-

PKC substrate motif coincided with the 100-kDa (pI = 7.1) proteinspot seeing increased after FTI treatment by chromogenicstaining. Using mass spectrometer-based peptide fingerprinting,we identified both 100-kDa proteins as eEF2 (Fig. 3). To confirmthe proteins were indeed eEF2, we did two-dimensional gelelectrophoresis and Western blotting using antibodies specific toeEF2 and phospho-eEF2 (Thr56). The protein spot with a pI of 7.1reacted with anti-phospho-eEF2 antibody, which increased after

Figure 2. Changes in protein levelsand modifications after SCH66336treatment. UMSCC38 cells at 60%confluence were treated with DMSO(Control ) or 8 Amol/L SCH66336 for24 hours. Total protein (5 � 105 cellequivalent) was subjected totwo-dimensional gel electrophoresisanalysis, and separated proteinswere transferred to nitrocellulosemembranes. Total proteins on themembranes were visualized bystaining with dye (A and B);phosphorylated proteins weredetected by using antibodies specificto phosphorylated substrates of PKAand PKC (C and D ). Total eEF2 andP-eEF2 were detected by usingspecific antibodies (E). The thinnersolid arrows (right ) indicateunphosphorylated eEF2 whereas thethicker solid arrows (left ) indicateP-eEF2. F, peptides after trypsindigestion measured by MALDI-TOF.Matched peptides are listed based onthe sizes and their locations withinthe protein.

FTI Induces eEF2 Phosphorylation


SCH66336 treatment, whereas both protein spots of pI 7.1 and 7.4reacted with anti-eEF2 antibody (Fig. 2E). These data show thateEF2 was predominantly unphosphorylated in exponentiallygrowing UMSCC38 cells and that SCH66336 induced itsphosphorylation.To determine whether the treatment effect is universal in

HNSCC, we examined the effect on seven other HNSCC cell lines.Increased P-eEF2 was observed in all but the UMSCC17B line asthe SCH66336 concentration increased from 2 to 8 Amol/L(Fig. 4A). Because UMSCC38 is among the most sensitive ones toSCH66336 induced eEF2 phosphorylation, it was selected forfurther analysis to determine potential mechanism of thephosphorylation. Using UMSCC38 cells as a model, we thenanalyzed how soon eEF2 phosphorylation occurs after SCH66336treatment. Increased P-eEF2 level was observed as early as 3 hoursafter administration of 5 to 8 Amol/L SCH66336 (Fig. 3B). Thesedata indicate SCH66336 can induce rapid phosphorylation of Thr56

in eEF2 in majority of HNSCC cell lines.Because phosphorylation at Thr56 inactivates eEF2 (18), we next

examined whether the increased P-eEF2 (Thr56) expression afterSCH66336 treatment inhibits protein synthesis. By adding 35S-labeled methionine and cystine into the culture medium ofUMSCC38 cells in the G1 phase 1 hour after SCH66336 treatment,we found that the amount of newly synthesized proteins in thefollowing 4 hours was substantially reduced compared with that incells not treated with SCH66336 but that the reduction of totalproteins was insubstantial (Fig. 4).Because phosphorylation of eEF2 at Thr56 is mainly caused by

the activity of eEF2K (19, 20) which itself is negatively regulated byphosphorylation through the ras-MEK signaling pathway (21, 22),one may expect that the inhibition of ras activity would result inthe activation of eEF2K by reducing eEF2K phosphorylation,thereby increasing the P-eEF2 level. To determine whether theeffect of SCH66336 on eEF2 is through the ras-MEK-eEF2Kpathway, we analyzed the levels of MEK and eEF2K proteins andtheir phosphorylated forms after SCH66336 treatment. Activationof MEK1 and MEK2 occurs through phosphorylation at Ser217 and

Ser221 by ras-activated Raf-1 activity (23). The levels of P-eEF2K(Ser336) and P-MEK1/2 (Ser217/221) were decreased at 12 and24 hours after SCH66336 administration, respectively, whereas theP-eEF2 level sharply increased as early as 3 hours after SCH66336administration, and this increased level was maintained for up to30 hours (Fig. 5). We found it interesting that the total eEF2 leveldid not changed over time but that the levels of MEK and eEF2Kwere reduced at 24 and 30 hours after the treatment. The P-MEKlevel was transiently reduced at 3 hours but rebounded by 6 hourswith corresponding change in P-eEF2K level (Fig. 5).In contrast to the dramatic changes in the FTI treated cells, the

levels of P-eEF2 (Thr56) and P-eEF2K (Ser336) level in vehicle(DMSO)–treated cells did not change significantly over time,whereas the transient depression of P-MEK 1/2(Ser217/221) wereseen. Furthermore, in the serum-starved cells, the level of P-MEK1/2 (Ser217/221) reduced dramatically, with corresponding decreasein P-eEF2K (Ser336) level and increase in P-eEF2 (Thr56) level(Fig. 5). These results suggest SCH66336 induced eEF2 phosphor-ylation is independent of ras-MEK-eEF2K pathway.Phosphorylation of eEF2 could also occur through the ras-PI3K/

p70S6K-eEF2K pathway (12, 22). Phosphorylation of Thr389 inp70S6K is critical for its kinase activity in vivo (24, 25). Therefore,we analyzed P-p70S6K (Thr389) status in UMSCC38 cells aftertreatment with SCH66336. The level of P-p70S6K was transientlyreduced at 3 hours but rebounded by 6 hours before declining at12 hours and 30 hours. Similar changes were seen in cells treatedwith vehicle (DMSO). In contrast, the changes of P-p70S6K levelwere greatly reduced in serum-starved cells (Fig. 5). These resultssuggest the induction of P-eEF2 is also independent of the ras-PI3K/p70S6K-eEF2K pathway.

Discussion

In synchronized UMSCC38 cells, SCH66336 treatment induceda delay of the G1-phase entry starting at about 6 hours andsubsequent G1 arrest. A previous study showed that SCH66336induced G1 arrest in cells transformed by H-ras or cells with anactivated H-ras but induced G2-M phase accumulation in cells

Figure 3. Induction of eEF2 phosphorylation in HNSCCcell lines after SCH66336 treatment. HNSCC cells with40% confluence were treated with 1 to 8 Amol/L SCH66336for 24 hours. Total eEF2 and P-eEF2 were measured byWestern blot using specific antibodies. eEF2 and P-eEF2levels at different time points with different SCH66336concentrations in UMSCC38 cells. h-Actin was used as aloading control. SF , serum free.

Cancer Research


without activated H-ras (26). However, mutations of H-ras are rarein HNSCC, and no mutation in H-ras and K-ras genes wasidentified in any of the HNSCC cell lines analyzed,6 suggestingthat the G1 effect we observed in the current study wasindependent of H-ras mutation status. Chun et al. (27) recentlyreported a G2-M arrest in a HNSCC cell line (SqCC/Y1) afterSCH66336 treatment. These data suggest the presence of twodistinct mechanisms of this FTI in cell growth inhibition in HNSCC.In this study, we showed that SCH66336 induced a rapid

inactivation of eEF2 and inhibition of protein synthesis in HNSCCcells. eEF2, also known as polypeptidyl-tRNA translocase, is a keyenzyme in protein biosynthesis. It catalyzes the translocation ofpeptidyl tRNA from the A site to the P site on the ribosome, andthe activity of eEF2 is regulated through phosphorylation by eEF2K,a unique Ca2+/calmodulin-dependent kinase (18–20). The principlesite of phosphorylation by eEF2 is Thr56 (28). The phosphorylationinactivates eEF2 activity by preventing it from binding to ribosome(18), resulting in reduced protein synthesis. The eEF2K activity isregulated by growth factors through either the MEK/extracellularsignal-regulated kinase or PI3K/p70S6K signaling pathways (21–25,29–31). Phosphorylation of eEF2K at Ser366 inactivates the kinase,leading to dephosphorylation of eEF2 (Thr56) and increased proteinsynthesis (21, 25).The increased P-eEF2 level observed in this study cannot be

simply explained by the decreased P-eEF2K level in SCH66336-treated cells, because this decrease was observed 12 hours aftertreatment, whereas the increased P-eEF2 level was detected at asearly as 3 hours and the high level was maintained thereafter. Thereduced P-eEF2K level after 12 hours may be explained bySCH66336-mediated inhibition of ras signaling. Consistent withthis view, P-MEK and P-p70S6K levels were also reduced 12 hoursor later after SCH66336 treatment. The transient reduction of P-MEK and P-p70S6K levels at 3 hours is interesting and warrants

further investigation, but it is unlikely to be the mechanism foreEF2 phosphorylation because P-eEF2K level was not reducedbefore 12 hours and the high P-eEF2 level did not fluctuatebetween 3 and 30 hours. Because eEF2K is the only known kinasefor eEF2, our data suggest the presence of a novel mechanismmediated by SCH66336 to inactivate eEF2 and thereby inhibitprotein synthesis. In supporting of our hypothesis, we found thatserum starvation leads to an increase in P-eEF2 (Thr56)level, accompanied by substantial decrease in the level of P-MEK(Ser217/221), P-p70S6K (Thr389), and P-eEF2K (Ser336).Two possibilities may explain our observations. First, SCH66336

might affect farnesyl-dependent proteins other than ras and resultin increased eEF2 phosphorylation through an unidentified kinase.The substantial changes in phosphorylation of proteins other thaneEF2 after SCH66336 treatment observed suing two-dimensionalgel electrophoresis and Western blotting indicate the involvementof other signaling molecules responsible for the cellular responseto SCH66336. Identification and characterization of these mole-cules may help reveal the precise mechanism of the signalingcascade affecting eEF2 after SCH66336 treatment. The combinedtwo-dimensional gel electrophoresis and Western blot approachallowed us to observe proteins at very low and otherwise unde-ectable quantities, presumably because of the use of high-affinityantibodies. The antibodies we used are specific to the phosphor-ylated substrates of PKA and PKC; thus, the unknown proteinkinase might belong to PKA or PKC signaling cascades.The other possibility is that SCH66336 inhibits the activity of a

protein phosphatase and reduces the rate of eEF2 depho‘sphory-lation. Previous studies have shown that P-eEF2 may be reduced bygrowth stimuli (32, 33), but inhibition of serine-threonine proteinphosphatase 2A (PP2A), a complicated protein complex, mayattenuate the reduction of P-eEF2 level (34). If SCH66336 inhibitsPP2A activity, then the P-eEF2 level may be elevated despite thelack of activation of eEF2K. However, inhibition of PP2A has beenshown to increase cell proliferation and tumorigenicity (35), whichis inconsistent with the phenotypic functions of SCH66336 in

Figure 4. Reduction of protein synthesisin UMSCC38 cells after SCH66336treatment. Synchronized cells were grownto 30% confluence before treatment withDMSO (control) or 8 Amol/L SCH66336 for1 hour. Medium was then changed tocystine- and methionine-free DMEM with100 ACi 35S and cultured for 4 hours. Totalprotein was extracted and separated bytwo-dimensional gel electrophoresis.Proteins on the two-dimensional gelelectrophoresis gels were visualized byeither silver staining (A and B ;representing total protein) orautoradiography (C and D ; representingnewly synthesized proteins). The totalprotein quantity on each gel was quantifiedand compared (E ).

6 L. Mao, unpublished data.



cancer cells (12, 13). Furthermore, PP2A function has been foundto be impaired in some human cancers (36), which supports itsrole in antiproliferation and antitransformation. Nevertheless, thefunctional status of the PP2A complex in HNSCC may helpelucidate the involvement of this complex in SCH66336-inducedcellular responses.The reduced protein synthesis after SCH66336 treatment is

consistent with the increased level of P-eEF2, which affects onlythe synthesis of new proteins. Although we have not determinedthe identity of the proteins whose synthesis was affected bySCH66336 treatment, we can predict that their reduced level haveeffected the cellular functions, which may be part of the

underlying mechanism of the FTI’s antitumor activity. A betterunderstanding how eEF2 function is controlled and whichproteins are affected by eEF2 may allow us to develop novelstrategies to target protein synthesis for treating or preventingHNSCC and other human cancers.

Acknowledgments

Received 8/31/2004; revised 4/1/2005; accepted 4/13/2005.Grant support: Department of Defense grant DAMD17-01-1-01689-1 and National

Cancer Institute grants PO1 CA106451, PO1 CA91844, and U01 CA 86390.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Figure 5. Effect of SCH66336 on eEF2 signaling in UMSCC38 cells. UMSCC38 cells at 40% confluence were treated with 8 Amol/L SCH66336 or DMSO. Cells underserum starvation were used as positive control of eEF2 signaling. Total protein was extracted at different time points and subjected to Western blot analysis usingspecific antibodies. h-Actin served as a loading control.

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farnesyltransferase inhibitor sch66336 induces rapid

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