Farnesyltransferase Inhibitor SCH66336 Induces Rapid Phosphorylation of Eukaryotic Translation Elongation Factor 2 in Head and Neck Squamous Cell Carcinoma Cells

Farnesyltransferase inhibitors (FTI) are a class of therapeutic agents designed to target tumors with mutations of the ras oncogene. However, the biological effect of FTIs is often independent of ras mutation status, which suggests the existence of additional mechanisms. In this study, we investigated the molecular effects of SCH66336, an FTI, in head and neck squamous cell carcinoma cells using proteomic approaches. We showed that SCH66336 induced phosphorylation (inactivation) of eukaryotic translation elongation factor 2 (eEF2), an important molecule for protein synthesis, as early as 3 hours after SCH66336 administration. Protein synthesis was subsequently reduced in the cells. Paradoxically, activation of eEF2 kinase (eEF2K), the only known kinase that regulates eEF2, was observed only at 12 hours after SCH66336 treatment. Consistent with this observation, the inhibition of phosphorylated-MEK and phosphorylated-p70S6K, the two key signaling molecules responsible for activation of eEF2K, also occurred at least 12 hours after SCH66336 administration. Our data suggest that inhibition of protein synthesis through inactivation of eEF2 is a novel mechanism of SCH66336-mediated growth inhibition and that this effect is independent of ras-MEK/p70S6K-eEF2K signaling cascades.

Protein prenylation is a posttranslational modification in which a farnesyl or geranylgeranyl isoprenoid is linked to a specific cystine residue of proteins through a thioether bond (1). The housekeeping enzymes farnesyltransferase and glycerol-3-phosphare cytidylyltransferase I and II catalyze the addition of a prenyl group to a conserved cystine residue in proteins that contain the motif CaaX, CC, or CxC at or near the COOH terminal of their nascent 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 small GTP-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 signals from cell surface receptors to cytosol and nucleus. Activation mutations of ras are frequently detected in various types of human cancers (5, 6) and its constitutive activation helps transform normal cells in both in vitro and in vivo models thereby leading to tumor formation (7, 8). The discovery that prenylation is a necessary step in the functional maturation of ras (9) prompted the development of farnesyltransferase inhibitors (FTI) as targeted therapeutic agents in cancers with a ras mutation (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 vitro and in vivo for other tumors with or without a ras mutation (14, 15). However, the mechanism of this activity is poorly understood. The effect of SCH66336 on cell growth inhibition is often observed within a few hours after administration, although the cellular half-life of ras is ∼24 hours (16). In light of this discrepancy and the possibility that FTIs may inhibit the activity of farnesylated proteins other than ras, FTIs may work through more than one pathway for their antitumor activity. Using proteomic approaches, we explored how SCH66336 affects the growth of HNSCC cells and whether the effects were dependent on ras signaling. We found that SCH6636 may induce growth inhibition of HNSCC cells by delaying their entry into and accumulation in the G₁ phase of the cell cycle. Evidence emerged that SCH66336 induces rapid inactivation of eukaryotic translation elongation factor 2 (eEF2) through its phosphorylation and subsequent reduction of protein synthesis. Furthermore, the inactivation of eEF2 was independent of ras-MEK-eEF2 kinase (eEF2K) and ras-PI3K/p70S6K-eEF2K signaling cascades.

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 37°C 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 and grown for 18 hours in medium with 5% serum and with serum-free medium for 24 hours. Serum-containing medium was then added back, and cells were harvested at different times, fixed in 70% cold ethanol, and stored at 4°C 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 μmol/L/mL propidium iodide in PBS buffer containing 50 μg/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% Triton X-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 μg) were separated through a 10% polyacrylamide 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 with antibodies. Specific antibody binding was detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL) according to the manufacturer's protocol.

For Western blotting, antibodies were obtained from Cell Signaling Technology (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 (Ser^217/221), 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 were washed 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 gradient strip (pH 5 to 8, Bio-Rad) for 12 hours and focused under 48,000 V hours at 18°C in an IPGphor isoelectric focusing unit (Amershan Biosciences, Piscataway, NJ). After focusing, the immobilized pH gradient strips were treated 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 gel electrophoresis Western blotting, the separated proteins were transferred to nitrocellulose membranes, blocked, and probed with antibodies as described 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 followed by exposure to autoradiography. To quantitate the level of protein expression by two-dimensional gel electrophoresis, the autoradiography or gel image was scanned using Amersham-Pharmacia ImageScanner. The integrated absorbance of all recognized protein spots was obtained by analyzing the gel image with ImageMaster 2D image analysis software (Amersham Biosciences).

Peptide mapping for protein identification. After two-dimensional gel 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 were excised, destained in 50% methanol, and dehydrated in acetonitrile. The dried gel slots were rehydrated and digested in 25 μL of 25 mmol/L ammonium carbonate containing 2 μg/mL sequencing grade modified trypsin (Roche Applied Science) at 37°C overnight. The digest products were 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 α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, St. Louis, MO), and 1.5 μL 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 (http://www.matrixscience.com/references/725.html).

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

The replication cycle of the HNSCC cell line UMSCC38 was ∼30 hours when cultured in DMEM and Ham's F12 with 5% serum. We treated these cells with 8 μmol/L SCH66336 when most of them had completed one replication after synchronization. We observed a slowed cell accumulation at the G₁ phase of the cell cycle after 6 hours following SCH66336 treatment but a prolonged G₁ phase (Fig. 1). We did not observe an emergence of a sub-G₁ population in 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 electrophoresis analysis to compare expression levels in UMSCC38 cells before and after treatment. The expression levels of the most of the proteins remained similar. The exceptions were a protein of ∼100 kDa with an isoelectric point (pI) of 7.1 and another protein of ∼100 kDa with a pI of 7.4, which increased and decreased, respectively, after SCH66336 treatment (Fig. 2A and B). In a separate attempt to identify proteins whose phosphorylation was affected by FTI treatment, we stained two-dimensional protein blots with several phosphorylation-specific antibodies, including antibodies specific to phosphorylated substrates of PKA and PKC. We found that the level of protein phosphorylation was generally reduced after SCH66336 treatment, but the phosphorylation of a few protein spots, including a 100-kDa protein with a pI 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) protein spot seeing increased after FTI treatment by chromogenic staining. Using mass spectrometer-based peptide fingerprinting, we identified both 100-kDa proteins as eEF2 (Fig. 3). To confirm the proteins were indeed eEF2, we did two-dimensional gel electrophoresis and Western blotting using antibodies specific to eEF2 and phospho-eEF2 (Thr⁵⁶). The protein spot with a pI of 7.1 reacted with anti-phospho-eEF2 antibody, which increased after SCH66336 treatment, whereas both protein spots of pI 7.1 and 7.4 reacted with anti-eEF2 antibody (Fig. 2E). These data show that eEF2 was predominantly unphosphorylated in exponentially growing UMSCC38 cells and that SCH66336 induced its phosphorylation.

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 as the SCH66336 concentration increased from 2 to 8 μmol/L (Fig. 4A). Because UMSCC38 is among the most sensitive ones to SCH66336 induced eEF2 phosphorylation, it was selected for further analysis to determine potential mechanism of the phosphorylation. Using UMSCC38 cells as a model, we then analyzed how soon eEF2 phosphorylation occurs after SCH66336 treatment. Increased P-eEF2 level was observed as early as 3 hours after administration of 5 to 8 μmol/L SCH66336 (Fig. 3B). These data 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 after SCH66336 treatment inhibits protein synthesis. By adding ³⁵S-labeled methionine and cystine into the culture medium of UMSCC38 cells in the G₁ phase 1 hour after SCH66336 treatment, we found that the amount of newly synthesized proteins in the following 4 hours was substantially reduced compared with that in cells not treated with SCH66336 but that the reduction of total proteins 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 by phosphorylation through the ras-MEK signaling pathway (21, 22), one may expect that the inhibition of ras activity would result in the activation of eEF2K by reducing eEF2K phosphorylation, thereby increasing the P-eEF2 level. To determine whether the effect of SCH66336 on eEF2 is through the ras-MEK-eEF2K pathway, we analyzed the levels of MEK and eEF2K proteins and their phosphorylated forms after SCH66336 treatment. Activation of 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 (Ser^217/221) were decreased at 12 and 24 hours after SCH66336 administration, respectively, whereas the P-eEF2 level sharply increased as early as 3 hours after SCH66336 administration, and this increased level was maintained for up to 30 hours (Fig. 5). We found it interesting that the total eEF2 level did not changed over time but that the levels of MEK and eEF2K were reduced at 24 and 30 hours after the treatment. The P-MEK level was transiently reduced at 3 hours but rebounded by 6 hours with 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(Ser^217/221) were seen. Furthermore, in the serum-starved cells, the level of P-MEK 1/2 (Ser^217/221) reduced dramatically, with corresponding decrease in P-eEF2K (Ser³³⁶) level and increase in P-eEF2 (Thr⁵⁶) level (Fig. 5). These results suggest SCH66336 induced eEF2 phosphorylation 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 in p70S6K is critical for its kinase activity in vivo (24, 25). Therefore, we analyzed P-p70S6K (Thr³⁸⁹) status in UMSCC38 cells after treatment with SCH66336. The level of P-p70S6K was transiently reduced at 3 hours but rebounded by 6 hours before declining at 12 hours and 30 hours. Similar changes were seen in cells treated with vehicle (DMSO). In contrast, the changes of P-p70S6K level were greatly reduced in serum-starved cells (Fig. 5). These results suggest the induction of P-eEF2 is also independent of the ras-PI3K/p70S6K-eEF2K pathway.

In synchronized UMSCC38 cells, SCH66336 treatment induced a delay of the G₁-phase entry starting at about 6 hours and subsequent G₁ arrest. A previous study showed that SCH66336 induced G₁ arrest in cells transformed by H-ras or cells with an activated H-ras but induced G₂-M phase accumulation in cells without activated H-ras (26). However, mutations of H-ras are rare in HNSCC, and no mutation in H-ras and K-ras genes was identified in any of the HNSCC cell lines analyzed,⁵

suggesting that the G₁ effect we observed in the current study was independent of H-ras mutation status. Chun et al. (27) recently reported a G₂-M arrest in a HNSCC cell line (SqCC/Y1) after SCH66336 treatment. These data suggest the presence of two distinct 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 HNSCC cells. eEF2, also known as polypeptidyl-tRNA translocase, is a key enzyme in protein biosynthesis. It catalyzes the translocation of peptidyl tRNA from the A site to the P site on the ribosome, and the activity of eEF2 is regulated through phosphorylation by eEF2K, a unique Ca²⁺/calmodulin-dependent kinase (18–20). The principle site of phosphorylation by eEF2 is Thr⁵⁶ (28). The phosphorylation inactivates eEF2 activity by preventing it from binding to ribosome (18), resulting in reduced protein synthesis. The eEF2K activity is regulated by growth factors through either the MEK/extracellular signal-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 protein synthesis (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 after treatment, whereas the increased P-eEF2 level was detected at as early as 3 hours and the high level was maintained thereafter. The reduced P-eEF2K level after 12 hours may be explained by SCH66336-mediated inhibition of ras signaling. Consistent with this view, P-MEK and P-p70S6K levels were also reduced 12 hours or 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 for eEF2 phosphorylation because P-eEF2K level was not reduced before 12 hours and the high P-eEF2 level did not fluctuate between 3 and 30 hours. Because eEF2K is the only known kinase for eEF2, our data suggest the presence of a novel mechanism mediated by SCH66336 to inactivate eEF2 and thereby inhibit protein synthesis. In supporting of our hypothesis, we found that serum starvation leads to an increase in P-eEF2 (Thr⁵⁶) level, accompanied by substantial decrease in the level of P-MEK (Ser^217/221), P-p70S6K (Thr³⁸⁹), and P-eEF2K (Ser³³⁶).

Two possibilities may explain our observations. First, SCH66336 might affect farnesyl-dependent proteins other than ras and result in increased eEF2 phosphorylation through an unidentified kinase. The substantial changes in phosphorylation of proteins other than eEF2 after SCH66336 treatment observed suing two-dimensional gel electrophoresis and Western blotting indicate the involvement of other signaling molecules responsible for the cellular response to SCH66336. Identification and characterization of these molecules may help reveal the precise mechanism of the signaling cascade affecting eEF2 after SCH66336 treatment. The combined two-dimensional gel electrophoresis and Western blot approach allowed us to observe proteins at very low and otherwise undetectable quantities, presumably because of the use of high-affinity antibodies. The antibodies we used are specific to the phosphorylated substrates of PKA and PKC; thus, the unknown protein kinase 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 dephosphorylation. Previous studies have shown that P-eEF2 may be reduced by growth stimuli (32, 33), but inhibition of serine-threonine protein phosphatase 2A (PP2A), a complicated protein complex, may attenuate the reduction of P-eEF2 level (34). If SCH66336 inhibits PP2A activity, then the P-eEF2 level may be elevated despite the lack of activation of eEF2K. However, inhibition of PP2A has been shown to increase cell proliferation and tumorigenicity (35), which is inconsistent with the phenotypic functions of SCH66336 in cancer cells (12, 13). Furthermore, PP2A function has been found to be impaired in some human cancers (36), which supports its role in antiproliferation and antitransformation. Nevertheless, the functional status of the PP2A complex in HNSCC may help elucidate the involvement of this complex in SCH66336-induced cellular responses.

The reduced protein synthesis after SCH66336 treatment is consistent with the increased level of P-eEF2, which affects only the synthesis of new proteins. Although we have not determined the identity of the proteins whose synthesis was affected by SCH66336 treatment, we can predict that their reduced level have effected the cellular functions, which may be part of the underlying mechanism of the FTI's antitumor activity. A better understanding how eEF2 function is controlled and which proteins are affected by eEF2 may allow us to develop novel strategies to target protein synthesis for treating or preventing HNSCC and other human cancers.

Grant support: Department of Defense grant DAMD17-01-1-01689-1 and National Cancer Institute grants PO1 CA106451, PO1 CA91844, and U01 CA 86390.

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