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Lee JT, Steelman LS, Chappell WH, McCubrey JA.. Akt inactivates ERK causing
decreased response to chemotherapeutic drugs in advanced CaP cells. Cell Cycle
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[Cell Cycle 7:5, 631-636; 1 March 2008]; ©2008 Landes Bioscience
Report
Akt inactivates ERK causing decreased response to chemotherapeutic
drugs in advanced CaP cells
John T. Lee,1 Linda S. Steelman,1 William H. Chappell1 and James A. McCubrey1,2,*
1Department of Microbiology and Immunology; 2Leo Jenkins Cancer Center; Brody School of Medicine at East Carolina University; Greenville, North Carolina USA
Key words: Akt, Raf, PTEN, prostate cancer
Activation of the PI3K/Akt signaling cascade is often associ-
ated with advanced forms of prostatic carcinoma (CaP). This
is likely explained by the common loss of the PTEN gene in
a majority of CaP patients. Conversely, activation of the Raf/
MEK/ERK pathway is seldom linked with prostatic disease. The
interplay between these two pathways in advanced CaP has not
been established. The following manuscript demonstrates that
Akt can directly associate with Raf-1 causing its inactivation via
phosphorylation of a negative regulatory residue (serine 259).
Inhibition of PI3K with either LY294002 and wortmannin was
sufficient to cause upregulation of ERK activity as measured
by immunoblotting. Prolonged treatment with two commonly-
ON
used chemotoxic compounds, doxorubicin and paclitaxel, caused
.D
increased activation of ERK in PTEN-positive DU145 cells,
but not PTEN-negative PC3 cells. Others have reported that
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ERK activation is essential for drug-induced death, which, when
combined with these data, supports the notion that Akt plays an
IEN
integral role in the response of prostate cancer cells to chemothera-
peutic drugs. These results demonstrate that, in prostate cancer
SC
cells, the efficacy of chemotherapy may be limited by its effects
on the intracellular signaling pathways found within the cell. The
BIO
genotype of the tumor must be considered for an effective response
to these and other antineoplastic drugs.
Introduction
ES
The advent of hormone relapse in patients with advanced carci-
ND
noma of the prostate (CaP) leaves the afflicted with relatively few
therapeutic options. Most often, CaP patients are administered a
variety of chemotherapeutic compounds to combat the disease.1-
LA
5 Employing the use of these drugs, however, has been met with
only modest success due to issues of toxicity and the drug-resistant
08
nature of CaP. Consequently, the life expectancy of individuals
with hormone-refractory prostate cancer (HRPC) is less than 18
20
months;6 efforts to minimize drug resistance should prove effective
in extending the life expectancy of HRPC patients.
©
*Correspondence to: James A. McCubrey; East Carolina University; Microbiology
& Immunology; 600 Moye Boulevard; Brody Building; Greenville, North Carolina
27858 USA; Tel.: (252).744.2704; Fax: (252).744.3104; Email: mccubreyj@mail.
ecu.edu
Submitted: 11/30/07; Accepted: 12/06/07
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/article/5416
www.landesbioscience.com
E .
UT
Drug resistance is manifested via a multitude of biomolecular
RIB
events including inhibiting entrance of chemotoxic agents into the
cell, loss of enzymatic activities involved in metabolizing the drug,
IST
and overexpression of ABC (ATP-Binding Cassette) drug efflux
pumps, among others.7-9 Recently, we and others have shown that
aberrant activity of the PI3K/PTEN/Akt/mTOR signaling cascade
D
can augment drug resistance in advanced CaP cells.10-17 These
OT
reports detail several potential mechanisms responsible for increased
chemoresistance including PI3K-induced overexpression of the
MRP-1 drug pump as well as activation of mTOR (mTORC1
complex) that is downstream of PI3K/Akt. Consequently, targeted
inhibition of the PI3K/Akt signaling pathway is quickly gaining
interest with regards to chemotherapeutic intervention in an array
of cell types.18-19
The PI3K/Akt signaling pathway is well-characterized for its role
in mediating cellular survival.20-23 The PTEN/MMAC (Phosphatase
and Tensin homologue deleted on chromosome Ten/Mutated in
Multiple Advanced Cancers) phosphatase is a negative regulator
of this pathway and is reported to be mutated in approximately
50–80% of patients with CaP.23-25 Consequently, it stands to reason
that dysregulated PI3K/Akt/PTEN/mTOR activity may play a major
role in the progression of this disease.
While the PI3K/Akt/PTEN/mTOR cascade is known for its
ability to confer survival benefits to the cell, the classical MAPK
(mitogen-activated protein kinase) pathway is widely regarded to be
responsible for cellular proliferation and differentiation. The MAPK
signaling cascade relays signals from the extracellular space through a
series of proteins, including Raf, MEK and ERK.26-29 Together, these
kinases contribute to a proliferative phenotype, which can often lead
to malignancies of different origins. Contrary to its role in normal
development, MAPK signaling is also believed to be essential for
chemotoxic drug-induced apoptosis. Several studies have outlined
that the pro-apoptotic action of various chemotherapeutics is depen-
dent upon ERK activation.30-32 These data strongly suggest that
Raf/MEK/ERK signaling must be fully functional to potentiate the
effects of anti-neoplastic agents.
In this study, we report that the elevated levels of activated Akt
present in PC3 cells, which lack PTEN, directly phosphorylate
and causes the inactivation of Raf-1. While this event has been
­documented in other cell types,33-35 it has not been reported in cells
of prostatic origin. Furthermore, we show that chemotoxic drugs have
the capacity to induce phosphorylation of ERK, but only when the
Cell Cycle 631
 Akt-mediated ERK inactivation in CaP cells

PI3K/Akt signaling cascade is under strict regulation by PTEN. This

latter observation underscores the importance of considering tumor
genotype when designing therapeutic regimens for CaP patients. The
results presented herein have implications for drug resistance mecha-
nisms as well as explain events associated with CaP etiology.

Results
Akt phosphorylation is elevated in PTEN-negative CaP cells. In
a previous report, we determined that PC3 cells are PTEN-negative,
while DU145 cells possess functional PTEN.12 To verify whether
the presence of PTEN was sufficient to maintain low levels of Akt
activation, western blotting analysis was performed and probed with

.
antibodies specific for both total and phospho-Akt. Figure 1 demon-

E
strates that PTEN negative PC3 cells have elevated levels of activated

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Akt, while DU145 cells have minimal endogenous Akt phosphoryla- Figure 1. Status of Akt Activation in DU145 and PC3 CaP Cells. PTEN-
tion. These lysates were prepared from cells grown normally, (RPMI positive DU145 and PTEN-negative PC3 CaP cells were lysed and cell

+ 10% FBS). These data emphasize the importance of PTEN in
minimizing aberrant signaling through PI3K and Akt.
Inhibition of PI3K activity activates ERK. During attempts to
RIB
extracts separated via gel electrophoresis. Cells were grown under normal
growth conditions (RPMI + 10% FBS) for 24 hrs and lysates were prepared.
Western blots are depicted for total and phospho-Akt (T308 and S473) for

IST
each cell line.
modulate PI3K activity through the use of the PI3K-specific inhib-
itor, LY294002, we discovered that inhibition of PI3K was directly

D
correlated with ERK phosphorylation (Fig. 2). To ensure that this
was due to PI3K inhibition and not merely to non-specific effects of

OT
LY294002 treatment, these experiments were repeated using another
PI3K inhibitor, wortmannin. Indeed, inhibition of PI3K increased ON
ERK phosphorylation in both cell lines tested. The results from these
assays demonstrate that inhibition of PI3K via chemical inhibitors is
.D
sufficient to upregulate ERK activation and suggest that these path-
ways may intersect in advanced CaP cells.
Active Akt increases levels of phospho-Raf-1(S259). Other
CE

studies have reported that Akt is able to directly phosphorylate and

IEN

inactivate Raf-1 on a serine residue (S259).39-40 While the regulation

of Raf is very complicated with some phosphorylation/dephosphory-
lation events associated with activation and some associated with
SC

inactivation,41-42 it is well-accepted that phosphorylation of Raf-1

on S259 can result in inactivation of the protein. Consequently, we
BIO

sought to determine the levels of phospho-Raf-1 (S259) in PC3 and

DU145 cells. These cell lines were treated with epidermal growth
factor (EGF) in the presence and absence of various inhibitors.
ES

Figure 3 demonstrates that inactive Raf-1 is found in much higher

levels in PC3 cells (lane 6), where Akt activity is constitutively-acti-
ND

vated; conversely, phospho-Raf-1 (S259) levels are at a minimum in

the DU145 cells (lane 1) which harbors a functional PTEN gene Figure 2. Effects of PI3K Inhibition on ERK Phosphorylation in CaP Cells.
LA

product. Likewise, administration of EGF in the presence or absence DU145 and PC3 cells were starved for 24 hrs and treated with the PI3K-
of inhibitors yielded similar patterns of Raf-1 phosphorylation on specific inhibitors, LY294002 (10 μM) and wortmannin (100 nM) for 1 hour
S259 (PC3 > DU145). Collectively, it appears that constitutive Akt before extracting cellular lysates. Lysates were electrophoresed and western
08

blots performed with antibodies against total and activated ERK.

activation, via loss of PTEN, in PC3 cells is sufficient to increase
20

endogenous levels of inactivated Raf-1. Akt may phosphorylate Raf-1

on S259 in advanced CaP cells in an indirect or direct fashion. complex in PC3 cells (lane 2, top and bottom), but much less so in
©

Akt and Raf-1 co-immunoprecipitate in a single complex.
Results from the previous studies indicated that Akt exerts a role in
the inactivation of Raf-1. To test this whether this occurs in advanced
prostate cancer cells, EGF was administered to both DU145 and PC3
cells to activate the PI3K/Akt and Raf/MEK/ERK pathways43 and
reciprocal co-immunoprecipitation (Co-IP) assays were performed to
determine whether these molecules were complexed within the cell.
Figure 4 demonstrates that Akt and Raf-1 can be found in a single
DU145 cells (lane 4, top and bottom). This data suggest that Raf-1
is inactivated via direct phosphorylation by activated Akt.
Treatment of PTEN positive CaP cells with chemotoxic drugs
induces ERK phosphorylation. During the process of isolating drug-
resistant mutants of both DU145 and PC3 cells, it was discovered
that exposure of DU145 to either doxorubicin or paclitaxel caused
activation of ERK (Fig. 5A). Previously, we have reported that
ERK activation is not capable of rescuing cells from paclitaxel- or

632 Cell Cycle 2008; Vol. 7 Issue 5

 Akt-mediated ERK inactivation in CaP cells
Figure 3. Comparison of Inactivated Raf-1 Levels in Advanced CaP Cells.
DU145 and PC3 cells were starved for 24 hours and EGF was administered
(100 ng/ml) in complete media. One hour after EGF-treatment, cellular
extracts were isolated and immunoblotting performed. Antibodies against total
and inactivated Raf-1 were used to assay levels of each of these respective
proteins.
ON
.D
CE
IEN
SC
Figure 4. Co-immunoprecipitation of Raf-1 and Akt. Advanced CaP cells
were starved for 24 hours, treated with EGF (100 ng/ml) for 1 hour, lysed
BIO
and then immunoprecipitated with antibodies specific for either Akt or Raf-
1 (labeled “IP antibody”). After immunoprecipitation with agarose beads,
reciprocal antibodies, Raf-1 and Akt respectively, were added to pull down
Raf-1/Akt complexes.
ES
doxorubicin-induced death.44 Since activation of ERK was not
ND
observed to confer chemoresistant properties to CaP cells previously,
it was hypothesized that the observed ERK phosphorylation was a
LA
consequence of exposure to the drugs.
To test this hypothesis, cells were treated with doxorubicin over a
08
24-hour period and the protein lysates were probed with antibodies
for phospho- and total ERK. These results are presented in Figure
20
5B. Administration of doxorubicin caused an early increase in the
amount of ERK phosphorylation in DU145 cells. However, in PC3
©
cells that were treated with doxorubicin no increase in activated ERK
was detected, indicating that the constitutive Akt activity of this cell
line was able to maintain the inactivation of Raf-1 and, hence, ERK.
Collectively, these data further suggest that Akt activation is related
to ERK inactivation in PTEN negative CaP cells.
www.landesbioscience.com
Discussion
In previous publications, we have reported that the PI3K/Akt/
PTEN and Raf/MEK/ERK signaling pathways have synergistic
effects which lead to the transformation of hematopoietic cells.45-
52 Here, we report that Akt activation can lead to inactivation of
the MAPK pathway via Raf-1. The results of these studies have
importance in many arenas of prostatic research: disease progres-
sion and etiology, therapeutic efficacy and resistance to chemotoxic
compounds.
PTEN, the negative regulator of PI3K signaling, is lost in a large
percentage of prostatic carcinomas. Consequently, many patients
with CaP harbor tumors with high levels of Akt signaling.53-54
E .
Our previous work showed that aberrant Akt signaling can lead
UT
to heightened levels of multidrug-resistance protein-1 (MRP-1)
expression thereby causing increased resistance to doxorubicin and
RIB
paclitaxel, two drugs commonly used to treat CaP patients.12 This
subsequent report outlines a secondary role for Akt in the progression
of CaP, namely inactivation of the Raf/MEK/ERK cascade.
IST
Signals transduced by the Raf/MEK/ERK cascade are generally
thought to induce cellular growth, differentiation and/or prolifera-
D
tion.26 Given that chemotoxic drugs target cells which divide rapidly,
one would then hypothesize that cells with high levels of ERK activity
OT
would be more effectively targeted by these compounds. Conversely,
the PI3K/Akt pathway is known for its role in cell survival—as such,
cells with heightened Akt activity may not be as affected by treatment
with chemotherapeutic drugs. Our previous reports12,44 support this
hypothesis.
Inactivation of ERK has been reported to coincide with higher
Gleason grades and poorly differentiated CaP.54-55 Due to its role in
inducing differentiation, one hypothesis is that, in normal prostatic
epithelial cells, ERK acts to induce cellular differentiation; however
upon tumorigenesis, ERK is inactivated causing cells to differen-
tiate much less efficiently, which is a hallmark of advanced CaP.54
The data presented in this report support the notion that ERK is
inactivated in advanced CaP. Furthermore, evidence in this manu-
script indicates that loss of PTEN and subsequent Akt activation is
responsible for ERK inactivation via phosphorylation of Raf-1 on
S259 (Figs. 3 and 4). Lastly, others have shown that ERK activation
is necessary for drug-induced death;30-32 the findings herein suggest
that administration of chemotoxic drugs may have minor deleterious
effects in cells with heightened Akt activity (i.e., those with PTEN
deletions), as they will serve to downregulate signaling within the
Raf/MEK/ERK pathway.
Resistance to chemotherapeutic compounds is a problem that
continues to impede the success of modern drug regimens.56-57
Circumventing this resistance is an area of intense investigation due
to the short life-span of patients who are afflicted with hormone-
refractory prostate cancer. Previously, we and others have identified
the PI3K/Akt pathway as a primary mediator of drug resistance in
advanced CaP.10,58-59 This information, combined with knowledge
of the Raf/MEK/ERK pathway, lends support to the mechanistic
model shown in Figure 6, which is also supported by similar studies
in ovarian cancer cells.33 This model depicts the loss of PTEN as a
major event in CaP tumorigenesis. After loss or mutation of PTEN,
Akt becomes constitutively active thereby causing phosphorylation
and inactivation of Raf-1 on S259. Since this event is one that
Cell Cycle 633
 Akt-mediated ERK inactivation in CaP cells

then lysed in gold lysis buffer (GLB) containing 20 mM

Tris (pH~7.5), 137 mM NaCl, 5 mM ethylenediamine-
tetraacetic acid (EDTA), 1% (v/v) Triton X-100, 15%
(v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM
sodium orthovanadate, 1 mM ethylene glycol-bis(β-
aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 10
mM sodium fluoride, 1 mM tetrasodium pyrophosphate
and 0.1 mM β-glycerophosphate. Lysates were clarified
by centrifugation at 14,000 RPM for 15 minutes and the
total protein was quantified using the bicinchoninic acid
(BCA) method (Pierce Biochemicals, Rockford, IL).
Western blotting analysis. Twenty-five μg of cellular

E .
protein was resolved on 10% SDS polyacrylamide gels and

UT
then transferred to PVDF membranes. The membranes
were first incubated with primary antibodies against Akt,

RIB
phospho-Akt (S473 and T308), total Raf-1, phospho Raf-
1 (S259), total ERK and phospho-ERK and subsequently
incubated with horseradish peroxidase-linked secondary

IST
antibodies from Santa Cruz (Santa Cruz Biotechnologies,
Santa Cruz, CA). All primary antibodies were purchased

D
from Cell Signaling (New England Biolabs, Beverly, MA).
Figure 5. Relationship of Chemotoxic Drug Treatment and Activation of Signaling The western blot procedure was performed as previously

OT
Pathways. (A) DU145 and PC3 cells were treated and maintained in doxorubicin
described elsewhere.36-38
(100 nM) and paclitaxel (10 nM) over 3 months. After this period, resistant cells
Co-immunoprecipitation assay. DU145 and PC3 cells
ON
(denoted with an “R” superscript) were lysed and western blots were performed using
antibodies specific for total and activated Akt and ERK. (B) DU145 and PC3 cells
were treated with 100 nM doxorubicin for the times indicated and western blots were
were lysed in a buffer containing 50 mM HEPES (pH~9.5)
(Sigma), 150 mM NaCl, 10% glycerol (w/v), 1% Triton
performed. The immunoblots were then probed with total- and phospho-ERK. X-100 (w/v), 1 mM EGTA, 1.5 mM MgCl2, 0.4 mM
.D
PMSF, 2 μM pepstatin, 0.1 mg/ml aprotinin and 1 mg/ml
inactivates Raf-1, signals ordinarily transduced by the Raf/MEK/ leupeptin. Lysis was carried out at 4°C with constant agitation
CE

ERK cascade are negated. Consequently, the cell loses its ability to over 15 minutes. Cellular debris was subsequently sedimented by
further differentiate, while heightened Akt activity confers a survival centrifugation at 16,000 g for 15 minutes. After the supernatant
IEN

advantage to the cell. Together, these events may lead to a tumor was isolated, it was precleared by adding 60 μl of protein agarose A
microenvironment where the individual cells are not only resistant beads (Invitrogen) to each sample for 30 minutes at 4°C with gentle
SC

to chemotherapeutic compounds, but also fail to differentiate effi- rocking. After sedimentation (16,000 g for 15 minutes), precleared
ciently. lysates were transferred to sterile microcentrifuge tubes and an
BIO

Further tests will be necessary to validate the conclusions drawn appropriate antibody was added to each sample (1.5 μg mouse IgG
in Figure 6; however, this report lays the groundwork for studies control, 2 μg total Akt or 2 μg total Raf-1) and mixed overnight
that may ultimately lead to a more complete understanding of the at 4°C with gentle rocking. 75 μl of protein agarose A beads were
ES

biomechanistic events that cause CaP as well as the inherent drug added to each sample after overnight rocking and incubated for
resistant nature of these tumors. Such studies will be invaluable for an additional 1 hour at 4°C before centrifugation at 10,000 g (5
ND

developing more effective treatment options, drug regimens and minutes). Supernatants were discarded and immunoprecipitates
should lead to a better overall understanding of the disease. were washed twice in lysis buffer prior to mixing the pellets in
LA

60 μl of SDS-PAGE sample buffer. Samples were then separated

Materials and Methods using SDS-PAGE analysis. Antibodies against total Raf-1 and total
Cell lines and reagents. DU145 and PC3 cells (American Type Akt were used to probe the appropriate membranes to determine
08

Culture Collection, Rockville, MD) were grown in RPMI 1640 (Life whether reciprocal immunoprecipitation occurred.
20

Technologies, Inc., Bethesda, MD) supplemented with 10% fetal Acknowledgements

bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), 2 mM gluta- JTL, LSS, WHC and JAM were supported in part by a grant from
©

mine, 100 units/liter penicillin and 100 μg/ml streptomycin. All cells the NIH (NCI) R01CA098195. This research was supported by the
were cultured at 37°C in a humidified incubator with a 5% CO2 Brody Brothers Medical Foundation (#997729).
atmosphere. All chemicals were purchased from Sigma Chemical
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E .
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D
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OT
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