Farnesyltransferase Inhibitor SCH66336 Induces Rapid

Phosphorylation of Eukaryotic Translation Elongation

Factor 2 in Head and Neck Squamous Cell
Carcinoma Cells
Hening Ren,
1
Shyh-Kuan Tai,
1,3
Fadlo Khuri,
4
Zuming Chu,
1
and Li Mao
1,2
1
Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center;
2
Cancer
Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas;
3
Department
of Otolaryngology, National Yang Ming University, Taipei Veteran General Hospital, Taipei, Taiwan; and
4
Department
of Oncology/Hematology, Winship Cancer Institute, Emory University, Atlanta, Georgia
Abstract
Farnesyltransferase inhibitors (FTIs) 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 phosphoryla-
tion (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, activa-
tion 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 administra-
tion. 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.
(Cancer Res 2005; 65(13): 5841-7)
Introduction
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-phos-
phare 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 trans-
form normal cells in both in vitro and in vivo models thereby
leading 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 (1012).
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 f24 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
G
1
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.
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 Hams F12 medium
supplemented with heat inactivated 5% fetal bovine serum and antibiotics
at 37jC in a humidified atmosphere consisting of 95% air and 5% CO
2
. For
synchronized culture, cells were grown exponentially to 40% confluence
and starved in serum-free DMEM/Hams 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
Requests for reprints: Li Mao, Molecular Biology Laboratory, Department of
Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson
Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363;
Fax: 713-796-8655; E-mail: lmao@mdanderson.org.
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 cells
were 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% 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 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 with
antibodies. Specific antibody binding was detected using an enhanced
chemiluminescence kit (Pierce, Rockford, IL) according to the manufac-
turers 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 (Thr
56
), eEF2, phospho-eEF2K (Ser
366
), eEF2K, mitogen-
activated protein kinase kinase (MEK), phosphor-MEK1/2 (Ser
217/221
), and
phospho-p70S6K/p-85S6K (Thr
389
). 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 10
5
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
18jC 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 manufacturers 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 AL of 25 mmol/L
ammonium carbonate containing 2 Ag/mL sequencing grade modified
trypsin (Roche Applied Science) at 37jC overnight. The digest products
were purified using C
18
microbed chromatography (ZipTip, Millipore,
Billerica, MA) according to the manufacturers 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 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
ACi [
35
S] 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.
Results
The replication cycle of the HNSCC cell line UMSCC38 was f30
hours when cultured in DMEM and Hams F12 with 5% serum. We
treated these cells with 8 Amol/L SCH66336 when most of them
had completed one replication after synchronization. We observed
a slowed cell accumulation at the G
1
phase of the cell cycle after 6
hours following SCH66336 treatment but a prolonged G
1
phase
(Fig. 1). We did not observe an emergence of a sub-G
1
population
in the drug-treated cells but an early accumulation of G
2
-M phase
(data not shown). These results suggest a delayed G
1
entry and G
1
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
f100 kDa with an isoelectric point (pI) of 7.1 and another
protein of f100 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
Figure 1. Change of G
1
phase distribution after SCH66336 treatment.
UMSCC38 cell growth was synchronized by serum starvation. The cells then
cultured with 5% serum and treated with 8 Amol/L SCH66336 when most of
them were rolled out from the G
1
phase. The percentage of cells in the G
1
phase
was determined using flow cytometry. Treatment with SCH66336 (FTI)
slowed entry of cells into G
1
and subsequent prolonged accumulation in the G
1
phase compared with cells treated with DMSO (Control ).
5
http://www.matrixscience.com/references/725.html
Cancer Research
Cancer Res 2005; 65: (13). July 1, 2005 5842 www.aacrjournals.org
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 phosphor-
ylation 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 (Thr
56
). The protein spot with a pI of 7.1
reacted with anti-phospho-eEF2 antibody, which increased after
Figure 2. Changes in protein levels
and modifications after SCH66336
treatment. UMSCC38 cells at 60%
confluence were treated with DMSO
(Control ) or 8 Amol/L SCH66336 for
24 hours. Total protein (5 10
5
cell
equivalent) was subjected to
two-dimensional gel electrophoresis
analysis, and separated proteins
were transferred to nitrocellulose
membranes. Total proteins on the
membranes were visualized by
staining with dye (A and B);
phosphorylated proteins were
detected by using antibodies specific
to phosphorylated substrates of PKA
and PKC (C and D). Total eEF2 and
P-eEF2 were detected by using
specific antibodies (E). The thinner
solid arrows (right ) indicate
unphosphorylated eEF2 whereas the
thicker solid arrows (left ) indicate
P-eEF2. F, peptides after trypsin
digestion measured by MALDI-TOF.
Matched peptides are listed based on
the sizes and their locations within
the protein.
FTI Induces eEF2 Phosphorylation
www.aacrjournals.org 5843 Cancer Res 2005; 65: (13). July 1, 2005
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 Amol/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 Amol/L SCH66336 (Fig. 3B). These
data indicate SCH66336 can induce rapid phosphorylation of Thr
56
in eEF2 in majority of HNSCC cell lines.
Because phosphorylation at Thr
56
inactivates eEF2 (18), we next
examined whether the increased P-eEF2 (Thr
56
) expression after
SCH66336 treatment inhibits protein synthesis. By adding
35
S-
labeled methionine and cystine into the culture medium of
UMSCC38 cells in the G
1
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 Thr
56
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 Ser
217
and
Ser
221
by ras-activated Raf-1 activity (23). The levels of P-eEF2K
(Ser
336
) 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 (Thr
56
) and P-eEF2K (Ser
336
) 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 (Ser
336
) level and increase in P-eEF2 (Thr
56
) 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 Thr
389
in
p70S6K is critical for its kinase activity in vivo (24, 25). Therefore,
we analyzed P-p70S6K (Thr
389
) 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.
Discussion
In synchronized UMSCC38 cells, SCH66336 treatment induced
a delay of the G
1
-phase entry starting at about 6 hours and
subsequent G
1
arrest. A previous study showed that SCH66336
induced G
1
arrest in cells transformed by H-ras or cells with an
activated H-ras but induced G
2
-M phase accumulation in cells
Figure 3. Induction of eEF2 phosphorylation in HNSCC
cell lines after SCH66336 treatment. HNSCC cells with
40% confluence were treated with 1 to 8 Amol/L SCH66336
for 24 hours. Total eEF2 and P-eEF2 were measured by
Western blot using specific antibodies. eEF2 and P-eEF2
levels at different time points with different SCH66336
concentrations in UMSCC38 cells. h-Actin was used as a
loading control. SF, serum free.
Cancer Research
Cancer Res 2005; 65: (13). July 1, 2005 5844 www.aacrjournals.org
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,
6
suggesting
that the G
1
effect we observed in the current study was
independent of H-ras mutation status. Chun et al. (27) recently
reported a G
2
-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 Ca
2+
/calmodulin-dependent kinase (1820). The principle
site of phosphorylation by eEF2 is Thr
56
(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 (2125,
2931). Phosphorylation of eEF2K at Ser
366
inactivates the kinase,
leading to dephosphorylation of eEF2 (Thr
56
) 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 (Thr
56
)
level, accompanied by substantial decrease in the level of P-MEK
(Ser
217/221
), P-p70S6K (Thr
389
), and P-eEF2K (Ser
336
).
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 mole-
cules 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 unde-
ectable quantities, presumably because of the use of high-affinity
antibodies. The antibodies we used are specific to the phosphor-
ylated 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 dephosphory-
lation. 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
Figure 4. Reduction of protein synthesis
in UMSCC38 cells after SCH66336
treatment. Synchronized cells were grown
to 30% confluence before treatment with
DMSO (control) or 8 Amol/L SCH66336 for
1 hour. Medium was then changed to
cystine- and methionine-free DMEM with
100 ACi
35
S and cultured for 4 hours. Total
protein was extracted and separated by
two-dimensional gel electrophoresis.
Proteins on the two-dimensional gel
electrophoresis gels were visualized by
either silver staining (A and B;
representing total protein) or
autoradiography (C and D; representing
newly synthesized proteins). The total
protein quantity on each gel was quantified
and compared (E).
6
L. Mao, unpublished data.
FTI Induces eEF2 Phosphorylation
www.aacrjournals.org 5845 Cancer Res 2005; 65: (13). July 1, 2005
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 FTIs 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.
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 accordance
with 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 under
serum starvation were used as positive control of eEF2 signaling. Total protein was extracted at different time points and subjected to Western blot analysis using
specific antibodies. h-Actin served as a loading control.
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