Download Involvement of PI3K/Akt pathway in cell cycle

Leukemia (2003) 17, 590–603
& 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00
www.nature.com/leu
MOLECULAR TARGETS FOR THERAPY (MTT)
Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic
transformation: a target for cancer chemotherapy
F Chang1,4, JT Lee1,4, PM Navolanic1, LS Steelman1, JG Shelton1, WL Blalock2, RA Franklin1,3 and JA McCubrey1,3
1
Department of Microbiology & Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA;
Shands Cancer Center, University of Florida, Gainesville, FL, USA; and 3Leo Jenkins Cancer Center, Brody School of Medicine
at East Carolina University, Greenville, NC, USA
2
The PI3K/Akt signal transduction cascade has been investigated extensively for its roles in oncogenic transformation.
Initial studies implicated both PI3K and Akt in prevention of
apoptosis. However, more recent evidence has also associated
this pathway with regulation of cell cycle progression. Uncovering the signaling network spanning from extracellular
environment to the nucleus should illuminate biochemical
events contributing to malignant transformation. Here, we
discuss PI3K/Akt-mediated signal transduction including its
mechanisms of activation, signal transducing molecules, and
effects on gene expression that contribute to tumorigenesis.
Effects of PI3K/Akt signaling on important proteins controlling
cellular proliferation are emphasized. These targets include
cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors. Furthermore, strategies used to inhibit the
PI3K/Akt pathway are presented. The potential for cancer
treatment with agents inhibiting this pathway is also addressed.
Leukemia (2003) 17, 590–603. doi:10.1038/sj.leu.2402824
Keywords: PI3K; PTEN; Akt; cell cycle; apoptosis; targeted
therapy; inhibition
Introduction
An understanding of normal cellular function has been gained
from research characterizing various signal transduction pathways. Despite these advances, processes responsible for
neoplastic transformation of normal cells remain largely
undefined. For this reason, many chemotherapeutic regimens
currently available are relatively nonspecific and have many
toxic side effects. The ultimate goal of signal-transduction-based
research is to develop chemotherapeutic drugs that target
cancerous cells without affecting nontransformed cells.
Signaling through the Raf/MEK/ERK cascade is relatively well
understood1–9 and much progress has been achieved in the
inhibition of this signaling pathway.10,11 In many cases,
however, activation of the Raf/MEK/ERK pathway alone is not
responsible for oncogenic transformation. Recent evidence has
suggested that the PI3K/Akt pathway is a viable target for novel
antineoplastic drugs. This review will describe PI3K/Akt pathway signaling and its effects on cell cycle progression,
prevention of apoptosis, and malignant transformation. Lastly,
several potential anticancer therapies targeting this cascade are
discussed.
Correspondence: JA McCubrey, Department of Microbiology &
Immunology, Brody School of Medicine at East Carolina University,
Greenville, NC 27858, USA; Fax: 1 252 816 3104
4
The first two authors contributed equally to this work.
Received 3 September 2002; accepted 23 January 2002
Aberrant PI3K activity and oncogenic transformation
The relation between dysregulated PI3K activity and cancer is
well documented. For example, a mutated form of the p85
subunit of PI3K has recently been isolated in a Hodgkin’s
lymphoma-derived cell line (CO).12 The PI3K/Akt pathway has
been shown to be the predominant growth-factor-activated
pathway in LNCaP human prostate carcinoma cells.13 PI3K
activity has been linked to a variety of human tumors including
breast cancer,14 lung cancer,15 melanomas,16 and leukemia17
among others. Further evidence has shown that Akt (protein
kinase B, PKB), a downstream kinase of PI3K, is also involved in
malignant transformation.18 Inhibition of this pathway is a
promising approach for novel chemotherapeutic agents.
Cytokine activation of PI3K/Akt pathway
After cytokine activation of a specific receptor, PI3K can be
activated by two mechanisms. First, a phosphorylated tyrosine
(Y) residue on the receptor serves as a docking site for the p85
regulatory subunit of PI3K.1 This recruits the catalytic subunit of
PI3K, p110, to this complex. Alternatively, upon activation of
the receptor by the cognate cytokine, Shc binds the receptor to
enable Grb-2 and Sos to form a complex that activates Ras.19–22
Ras can then induce membrane localization and activation of
the p110 subunit of PI3K, as determined by crystal structure
studies.23 Specifically, this interaction occurs between regions
on Ras, known as switch I (residues 33–41) and switch II
(residues 63, 64, and 73), which bind to a sequence on PI3K-g
referred to as the Ras-binding domain (RBD).24 It was recently
shown that the PI3K regulatory subunit, p85, contains two
additional regulatory domains: a GTPase-responsive domain
(GRD) that can mediate PI3K activation through binding small
GTPases (Ras, Rac1), and an inhibitory domain that can block
these binding events. PI3K inhibition can be lifted when certain
tyrosine kinases such as Src, Lck, and Abl phosphorylate the
S668 residue contained within the inhibitory domain.25
Different Ras isoforms have also been shown to have varying
abilities to activate PI3K. Although Ras molecules are commonly
believed to activate both PI3K/Akt and Raf/MEK/ERK cascades,
Ha-Ras preferentially activates PI3K and Ki-Ras preferentially
activates Raf.26
Activated PI3K converts phosphatidylinositol (4,5)-phosphate [PI(4,5)P2] into phosphatidylinositol (3,4,5)-phosphate
[PI(3,4,5)P3], which results in membrane localization of
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
591
Figure 1
Activation of the PI3K/Akt pathway. Two mechanisms by which cytokine receptor activation can result in activation of the PI3K/Akt
pathway are indicated. Note that significant activation of this pathway occurs in close proximity to the cell membrane. SH2: Src homology-2
domain; PH: pleckstrin homology domain.
phosphotidylinositol-dependent kinase-1 (PDK1) via its pleckstrin homology (PH) domain. Akt is also recruited to the lipidrich plasma membrane by its PH domain and is phosphorylated
at residues T308 and S473 by PDK1 and an unidentified kinase,
respectively (Figure 1).
Akt is the primary mediator of PI3K-initiated signaling and has
a number of downstream substrates that may contribute to
malignant transformation. Some of these substrates are Bad,
procaspase-9, I-kB kinase (IKK), CREB, the forkhead family of
transcription factors (FKHR/AFX/FOX), glycogen synthase kinase-3 (GSK-3), p21Cip1, and Raf.18 IKK and CREB are activated
by Akt phosphorylation, whereas Raf, Bad, procaspase-9, FKHR,
and GSK-3 are inactivated. Mechanisms by which Akt modulates activity of these proteins to promote cell survival and cell
cycle progression will be discussed in the following sections.
Activity of the PI3K/Akt pathway is negatively regulated by
phosphatases. Phosphatase and tensin homologue deleted on
chromosome 10 (PTEN), which is also known as mutated in
multiple advanced cancers-1 (MMAC1), as well as Src
homology 2 (SH2) containing phosphatases 1 and 2 (SHIP-1
and SHIP-2) remove phosphates from PI(3,4,5)P3.27–31 Mutations in these phosphatases that eliminate their activity can lead
to tumor progression. Consequently, genes encoding these
phosphatases are referred to as antioncogenes or tumor
suppressor genes. An overview of the PI3K/Akt pathway is
presented in Figure 2.
Cell cycle overview
Uncontrolled cell division can lead to tumorigenesis. Cell
division is coordinated by a number of proteins including
cyclins and cyclin-dependent kinases (Cdks). Cdks exist as in-
active serine/threonine kinase monomers that become activated
when bound to specific cyclins. Cyclins are nuclear proteins that
are transiently expressed in order to activate its cognate Cdk.
These proteins form complexes that facilitate cell cycle
progression through various cell cycle stages (Figure 3). Cdk
activities are inhibited by two families of Cdk inhibitors (CKIs).
The first CKI family is composed of inhibitors of Cdk4 (INK4),
which are p16INK4a, p15INK5b, p18INK4c, and p19INK4d. INK4
family CKIs inhibit kinase activity of Cdk4/cyclin D and Cdk6/
cyclin D complexes.32 The second family is Cip/Kip CKIs, which
includes p21Cip1, p27Kip1, and p57Kip2. Cip/Kip family CKIs
inhibit a broader set of Cdks, including Cdk2, Cdk4, and
Cdk6.33,34 The roles that these regulatory proteins play in cell
cycle progression can be modulated by PI3K signaling.
p21Cip1 and PI3K/Akt: cell cycle regulatory effects
As previously discussed, the PI3K/Akt pathway is important for
the regulation of cell cycle progression.35–40 For instance, the
PI3K/Akt pathway is crucial for DNA synthesis in mesenchymal
cells35 and expression of dominant negative (DN) Akt blocks
platelet-derived-growth-factor (PDGF)-induced DNA synthesis
and cell proliferation.40 p21Cip1 was initially thought to be a CKI
because it inhibited cell proliferation when overexpressed in
human fibroblasts. Evidence gathered by in vitro reconstitution
studies showed that p21Cip1 inhibited activity of most Cdk
isoforms.41,42 Further investigation has suggested that p21Cip1
has a more complex role in regulating Cdk activity. Under
physiological conditions, p21Cip1 can bind to Cdk4/cyclin D and
Cdk6/cyclin D complexes, and induce their kinase activities
from early G1 until the middle of S phase.43 In contrast, p21Cip1
can bind to and inhibit kinase activities of Cdk2/cyclin A and
Cdk2/cyclin E complexes from the late G1 until the S phase.44,45
Leukemia
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
592
Figure 2
Overview of the PI3K/Akt pathway. The PI3K/Akt pathway is activated either by the p85 PI3K regulatory subunit binding to an
activated tyrosine residue on the activated IL-3 receptor or by the PI3K p110 subunit binding to activated Ras. Both result in membrane localization
of PI3K. Some downstream effects of PI3K activation pathway are shown. PI(4,5)P2 is phosphorylated by PI3K to yield PI(3,4,5)P3, which promotes
membrane localization of PDK1 through its PH domain. PDK1 then phosphorylates and activates Akt. Note that, depending on the residue
phosphorylated, proteins can either be activated or inactivated by phosphorylation. Proteins activated by S/T or Y phosphorylation are indicated
with a black P and a clear circle. Proteins inactivated by S/T or Y phosphorylation are shown with a white P in a black circle. Transcription factors
are indicated by diamonds. Phosphatases are indicated by rectangles. Phosphatases that inhibit activity, such as PTEN and SHIP, are indicated in
black rectangles. The PP2A phosphatase, which activates Raf, is shown in a white rectangle. Open arrows indicate phosphate groups hydrolyzed
by phosphatases.
The function of p21Cip1 during cell cycle progression appears to
ensure appropriate Cdk activation.
p21Cip1 may also promote cell survival. Insulin-like growth
factor-I (IGF-I) improved survival of C3 myoblasts by inducing
p21Cip1 through an Akt-dependent mechanism.46 IGF-I induction of cell survival was significantly reduced by expression of
antisense p21Cip1 cDNA. In summary, p21Cip1 has both positive
and negative regulatory effects on cell cycle progression, and is
also a regulator of cell survival.
The PI3K/Akt pathway is required for p21Cip1 induction in
human ovarian carcinoma cells.47 Inactivation of this pathway
by drug treatment or DN Akt expression decreased levels of
p21Cip1.47,48 In contrast, expression of a constitutively active Akt
mutant increased p21Cip1 expression.47 Mechanisms by which
the PI3K/Akt pathway regulates p21Cip1 protein expression were
further explored by several groups. It was shown that Akt could
directly phosphorylate both T145 and S146 near the carboxyl
terminus of p21Cip1.49–52 As a result of these phosphorylation
events, DNA synthesis and Cdk activity were stimulated, and
cellular proliferation increased.
In normal cells, p21Cip1 exists as a complex with a cyclin, a
Cdk, and proliferating cell nuclear antigen (PCNA) (Figure 4).
p21Cip1 binds PCNA directly and inhibits DNA pol d holoLeukemia
enzyme activity.53,54 Phosphorylation of T145 by Akt causes
PCNA release from the PCNA/p21Cip1/Cdk/cyclin quaternary
complex (Figure 4). PCNA can then form a complex with the
DNA polymerase d holoenzyme and activator protein-1 (AP-1)
to induce DNA synthesis.49,52 Cytoplasmic relocalization of
p21Cip1 was observed in fibroblasts upon phosphorylation by
Akt;49 however, this relocalization was not observed in
endothelial cells.52
Furthermore, T145 phosphorylation also decreased binding
affinity of p21Cip1 for Cdk2 by more than 50% in endothelial
cells. As a result, inhibition of Cdk2 by p21Cip1 decreased.52
Interestingly, it appears that modulation of Cdk activity by Aktinduced p21Cip1 phosphorylation at T145 was specific for Cdk2
because it had little effect on Cdk4 activity.
Akt can also phosphorylate S146 on p21Cip1 (Figure 4)50,51.
Phosphorylation at this site is associated with increased p21Cip1
protein stability. p21Cip1 is normally a short-lived protein with a
high proteosomal degradation rate.55 Phosphorylation of S146
in glioblastoma cell lines significantly prolonged the half-life of
p21Cip1.50 Since Akt increases protein levels of both p21Cip1 and
cyclin D1, it consequently induces assembly of p21Cip1/Cdk4/
cyclin D and p21Cip1/Cdk6/cyclin D complexes as well as their
associated kinase activities.50
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
p21Cip1 on T57 (Figure 5). This phosphorylation event is
associated with a higher degradation rate of p21Cip1. Furthermore, if GSK-3-induced T57 phosphorylation is inhibited
by PI3K/Akt activation, the half-life of p21Cip1 protein is
significantly increased.51 Interestingly, GSK-3 does not phosphorylate p27Kip1, which suggests functional diversity in
regulation of these two Cip/Kip family CKIs.64
593
PI3K/Akt pathway and p27Kip1
Figure 3
Cell cycle overview. The cell cycle is divided into G1
(gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis) stages. The
point after which cellular division must occur is referred to as the
restriction point (R) and is located at the latter portion of G1.
Progression through the cell cycle is promoted by formation of Cdk/
cyclin complexes. The relative activity of each complex is represented
by the thickness of gray arcs. Regulators of Cdk/cyclin complexes are
shown in black.
Figure 4
Phosphorylation of p21Cip1 by Akt regulates cell cycle
progression. Phosphorylation of p21Cip1 at T145 and S14b by Akt can
affect both DNA synthesis and cell cycle progression.
PI3K and GSK-3
GSK-3 is a substrate of the PI3K/Akt pathway that is constitutively active in unstimulated cells. GSK-3 phosphorylates
numerous proteins, such as glycogen synthase, cyclin D, and
c-Myc, which maintains these proteins in an inactive state.56
Activation of PI3K or Akt can lead to GSK-3 inactivation.57
Many kinases, including protein kinase C (PKC), p90RSK, p70SbK,
and Akt, phosphorylate GSK-3 in vitro. However, Akt appears
to be the kinase primarily responsible for phosphorylation of
GSK-3 at S9 in vivo (Figure 5).57,58
Recently, it has been shown that like Akt, GSK-3 also
regulates p21Cip1 protein stability (see Figure 5). p21Cip1 can
be degraded through ubiquitin-mediated59 or ubiquitin-independent pathways.60,61 The C8 a subunit of the 20S proteosome
directly binds to the carboxy terminus of p21Cip1, which is
necessary for proteosomal degradation.61 GSK-3, in its constitutively active state, causes decreased p21Cip1 protein levels in
endothelial cells.51 Moreover, specific inhibition of GSK-3 with
lithium chloride62,63 prevents p21Cip1 degradation and increases
its protein level 10-fold.51 GSK-3 can directly phosphorylate
The PI3K/Akt pathway has also been implicated in altering
p27KIP1 activity. Expression of DN Akt in mesenchymal cells
caused transcriptional induction of p27Kip1, which led to the
inhibition of both Cdk2 activity and DNA synthesis.65 However,
these effects were not observed in an endothelial cell model.48
Therefore, the effects of PI3K/Akt on p27Kip1 expression may be
cell type specific.
The forkhead family of transcription factors (TFs) has recently
been categorized into the forkhead box factor (FOX) family.66
The FOX family includes AFX/FOXO4, FKHR/FOXO1, and
FKHR-L1/FOXO3a. Akt can inactivate FOX TFs through direct
phosphorylation of multiple S/T residues38,67,68 (Figure 6). As a
result of these phosphorylation events, proapoptotic genes
induced by FOX family transcription factors are downregulated,
including both p27Kip1 and Fas. Thus, the PI3K/Akt pathway
prevents cell death by both inducing and repressing gene
expression.
Recently, three laboratories almost simultaneously demonstrated that Akt can directly phosphorylate p27Kip1 on T157 and
subsequently abolish its inhibitory activity against Cdk-2.69–71 In
order to inhibit Cdk-2 kinase activity, p27Kip1 requires translocation from cytosol to the nucleus so as to bind with Cdk-2.
Wild-type p27Kip1 was observed in both the cytosol and
nucleus. A T157A p27Kip1 mutant, however, exclusively
localized in the nucleus was found to be associated with cell
cycle arrest. This suggested that T157 of p27Kip1 is important for
nuclear localization and Cdk-2 inhibition. Furthermore, T157 is
located within the nuclear localization sequence of p27Kip1
(amino acids 151–166) and is contained in a putative Akt
consensus phosphorylation site (RXXRXXT157D). Using breast
cell lines as a model, it was shown that Akt could phosphorylate
p27Kip1 on T157 both in vitro and in vivo. Akt-induced T157
phosphorylation caused retention of p27Kip1 in the cytoplasm
and thus, prevention of cell cycle arrest induced by p27Kip1.
PI3K/Akt pathway and cyclin D1
The PI3K/Akt pathway is also a strong activator of cyclin D1, a
critical player in cell cycle progression. In a manner similar to
that described for p21Cip1, cyclin D1 protein levels are also
regulated by GSK-3. Cyclin D1 degradation occurs mainly
through the ubiquitin-dependent proteolysis pathway triggered
by phosphorylation36 (Figure 7). In order to be recognized by
ubiquitin-conjugating enzymes E1 and E2 for proteolysis, cyclin
D1 must be phosphorylated at T286 near its carboxyl terminus.36
GSK-3 is the primary kinase that phosphorylates cyclin D1 at this
residue.36 Moreover, cyclin D1 phosphorylation at T286 by
GSK-3 is also associated with the translocation of cyclin D1 from
the nucleus to the cytoplasm. Rapid cyclin D1 degradation
induced by GSK-3 is inhibited by activation of PI3K/Akt
pathway because Akt directly phosphorylates and inactivates
GSK-3.
Leukemia
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
594
Figure 5
Regulation of p21Cip1 protein stability by the PI3K/Akt pathway. Effects of Akt on GSK-3 and stability of p21Cip1 are indicated. Akt can
induce GSK-3 phosphorylation, which renders it inactive. Akt can phosphorylate p21Cip1, which stabilizes the protein.
Figure 6
Effects of the PI3K/Akt pathway on FOX transcription factors. Akt can phosphorylate and inactivate FOX transcription factors. DN Akt
can block this effect to increase expression of p27Kip1 and Fas, which inhibit cell cycle progression and cause apoptosis, respectively.
Figure 7
Regulation of cyclin D1 protein stability by the PI3K/Akt pathway. Cyclin D1 is phosphorylated by GSK-3, which can be prevented by
phosphorylation of GSK-3 by Akt. Phosphorylated cyclin D is translocated from the nucleus to the cytoplasm and targeted for degradation by
ubiquitination.
PI3K/Akt pathway and c-Fos
c-Fos is a transcriptional regulator that can elevate expression of
many proliferatory genes. Consequently, its ability to enhance
transcription of these genes is strictly controlled. Transfection of
NIH-3T3 mouse fibroblast cells with a constitutively active PI3K
p110 mutant induced transcription from the c-Fos promoter,
which was blocked by cotransfection with DN Ras.65 Moreover,
Leukemia
transfection of rat mesenchymal cells with a DN Akt mutant
decreased c-Fos transcription.67 Induction of c-Fos transcription
by PI3K or Akt was mediated by Elk-1.67 These data suggest that
the PI3K/Akt pathway executes some of its antiapoptotic effects
through transcription factors such as Elk-1 and c-Fos. These
results are intriguing because ERK also phosphorylates Elk-1,
which places this transcription factor downstream of both Raf/
MEK/ERK and PI3K/Akt pathways (Figure 8).
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
595
Figure 8
Effects of the PI3K/Akt signaling on Elk-1 and c-Fos
transcription factors. PI3K/Akt and Raf/MEK/ERK pathways can
cooperate to induce c-Fos transcription.
Figure 9
PI3K/Akt and Raf/MEK/ERK pathways cooperate to
regulate bad phosphorylation. Phosphorylation and inactivation of
the proapoptotic Bad protein by cooperation between PI3K/Akt and
Raf/MEK/ERK pathways.
PI3K/Akt pathway and NF-jB
NF-kB is a transcription factor that can be induced by a variety
of signals. Inhibitor of kB (I-kB) binds to NF-kB and inactivates it
by localizing NF-kB to the cytosol where it is unable to regulate
transcription. Phosphorylation of I-kB targets it for ubiquitination and degradation. In the absence of I-kB, NF-kB’s nuclear
localization signal is exposed and NF-kB localizes to the
nucleus where it is able to induce transcription. The role of
NF-kB was originally described as a factor associated with
apoptosis. Many agents, which induce apoptosis, such as TNF-a,
also induce NF-kB activation.72,73 However, it was subsequently shown that inhibition of NF-kB activation potentiates
apoptosis.74–77 In addition, inhibition of I-kB expression by
oligonucleotides led to cell transformation.78 The data now
support the concept that NF-kB is an antiapoptotic molecule.
Consistent with this role, NF-kB has been shown to induce
transcription of antiapoptotic proteins.79–82 IKK is known to
phosphorylate and induce the degradation of I-kB.83 Akt is
reported to phosphorylate and activate IKK.84,85 Activation of
IKK causes phosphorylation and degradation of I-kB, which
leads to localization of NF-kB to the nucleus, where it can
induce transcription of antiapoptotic genes.85 Thus, Akt can
inhibit apoptosis by activating NF-kB.
PI3K/Akt signaling and the Bcl-2 family of proteins
The Bcl-2 family of proteins are structurally related molecules
that play an instrumental role in the regulation of apoptosis. This
family includes both proapoptotic proteins such as Bad and Bax
as well as antiapoptotic proteins such as Bcl-2 and Bcl-XL. The
balance of antiapoptotic to proapototic proteins is believed to
dictate whether a cell will survive or undergo apoptosis. This
concept has been termed the rheostat theory.86
Raf has been shown to directly phosphorylate Bad and Bcl-2
to exert antiapoptotic effects.87,88 However, the role of Raf in
apoptosis regulation remains controversial. Depending on cell
type as well as apoptotic stimulus, Raf can inhibit89–96 or
promote97–99 apoptosis. Mitochondrial Raf-1 inactivates Bad by
phosphorylating Bad at S136100 (Figure 9). Mitochondrial Raf-1
is unable to activate ERK1 or ERK2 and appears to inactivate Bad
by a mechanism dependent upon MEK and Akt. In rat
hepatocytes, A-Raf has been found to be localized to inner
and outer mitochondrial membrane protein import receptors
hTOM and hTIM, which suggests that A-Raf may also exert
antiapoptotic effects.101
Akt can modulate Bad activity by phosphorylating S112.102
Phosphorylated Bad then dissociates from Bcl-2 to form a
complex with the 14-3-3 adaptor protein. Dissociation of Bad
from Bcl-2 is associated with cell survival. Recent studies have
also shown that p70S6K, which is also a target of the PI3K/Akt
pathway, can also phosphorylate Bad at S112.103–105 Furthermore, Raf/MEK/ERK and PI3K/Akt pathways can synergize to
induce cell survival and cellular transformation.5,6 This may
result from phosphorylation of proapoptotic Bad at these two
serine residues. It is worth noting that a third residue of Bad is
phosphorylated by the PKA pathway.105
Interactions between Raf/MEK/ERK and PI3K/Akt pathways
It has been shown that PI3K/Akt and Raf/MEK/ERK pathways can
interact in multiple ways. In some cells, Akt can contribute to
Raf-1 inactivation (see Figures 2 and 10). However, phosphorylation of Raf-1 by Akt has not been universally observed and
complete inactivation of Raf-1 also requires phosphorylation at
other residues such as S621. Since Raf can regulate p21Cip1
activity, inhibition of Raf by Akt would be expected to reduce
p21Cip1 activity, which could affect cell cycle progression64,65,67,106–109 (see Figure 10).
The Raf/MEK/ERK cascade can also activate the PI3K/Akt
pathway. We have observed that Raf can activate Akt activity in
hematopoietic cells transformed to grow in response to activated
Raf in the absence of exogenous cytokines (unpublished data).
Others have observed that some antiapoptotic effects of the
PI3K/Akt pathway are dependent upon the Raf/MEK/ERK pathway. Even though both pathways can be regulated by Ras, they
Leukemia
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
596
Figure 10
Interactions between PI3K/Akt and Raf/MEK/ERK pathways. Potential sites of interaction between these two pathways are indicated.
These pathways interact and cross-regulate each other by both direct and indirect mechanisms. These pathways regulate gene expression by
transcriptional, post-transcriptional, and post-translational mechanisms.
cross-regulate one another to control both apoptosis and cell
cycle progression.
Ectopic overexpression of Raf and MEK oncoproteins led to
abrogation of cytokine dependence of murine FDC-P1 and
human TF-1 hematopoietic cells.1,93–96,110–112 It has been
postulated that Raf/MEK/ERK signaling can activate the PI3K/
Akt pathway to induce cell survival. The PI3K inhibitor
LY294002 and the downstream p70S6K inhibitor rapamycin are
potent inhibitors of Raf-mediated proliferation, suggesting that
some effects of Raf on cell growth are mediated by the PI3K/Akt/
p70S6K pathway as discussed later. The issue is complicated
because many Raf-transformed cells express autocrine cytokines
that activate the PI3K/Akt pathway. However, we have observed
that Raf could induce PI3K acivity in cytokine-dependent cells
that do not secrete autocrine growth factors.
Crosstalk between these two pathways has been demonstrated. It has been shown that Akt can directly phosphorylate
Raf isoforms within their CR2 amino-terminal regulatory
domain. These sites include S259 on Raf-1 and both S364 and
S428 on B-Raf. Serine-phosphorylated Raf molecules then
become inactivated by binding to the 14-3-3 adaptor protein.113–115 The inhibitory effect of Akt on Raf activity appears to
be dependent upon cell type and stage of differentiation116,117
because some investigators have not observed this effect.
Certain differentially expressed mediators may be essential for
association of Akt and Raf.
Recently, a negative feedback loop has been identified
involving Raf/MEK/ERK and PI3K/SGK pathways.118 Serum and
glucocorticoid-inducible kinase (SGK) shares a high homology
with Akt.119 Even though physiological targets of SGK and their
function(s) remain largely unknown, regulation of SGK has been
documented. SGK shares a regulatory pathway with Akt. Like
Leukemia
Akt, SGK can be activated by PI3K120–122 even though it lacks a
PH domain. Secondly, both Akt and SGK are directly
phosphorylated and activated by PDK1.119,122 Interestingly,
SGK and Akt phosphorylate different residues of FKHR-L1 and
synergistically inactivate B-Raf.
Using murine FDC-P1 hematopoietic cells as a model, we
have determined that Raf activation led to G1 progression in
association with downregulation of p27Kip1 and upregulation of
p21Cip1 protein levels.64 Although Cdk4/cyclin D/p21Cip1
complex formation was detected, no binding was observed
between p21Cip1 and Cdk2/cyclin E or between p27Kip1 and
Cdk4/cyclin D or Cdk2/cyclin E. The increases in p21Cip1
protein levels and binding of p21Cip1 with Cdk4/cyclin D
complexes induced by Raf were correlated with increased Cdk4
kinase activity.64 Our results suggest that, in hematopoietic
cells, Raf signaling increases levels of p21Cip1 protein, which
then specifically binds to and activates the Cdk4/cyclin D
complex. The fact that p27Kip1 was found not to associate with
Cdk2 or Cdk4 suggested that, at least in our model system,
p27Kip1 has little effect on Cdk2 or Cdk4 kinase activity. This
finding is interesting because it suggests that Raf activation
differentially regulates p21Cip1 and p27Kip1 activities. Thus,
p21Cip1 and p27Kip1 appear to have similar, yet unique
functions.
Involvement of the PI3K/Akt pathway in neoplasia
The PI3K pathway is negatively regulated by phosphatases.
PTEN is considered a tumor suppressor gene.123 PTEN is a dual
specificity lipid and protein phosphatase that removes the 3phosphate from the PI3K lipid product PI(3,4,5)P3 to yield
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
597
PI(4,5)P2, which prevents Akt activation.124–127 Recently, SHIP2, has been shown to remove the 5-phosphate from PI(3,4,5)P3
to yield PI(3,4)P2.128,129
Aberrant proliferative and antiapoptotic signals transduced by
the PI3K/Akt pathway have been linked to malignant transformation.130 Ras can activate PI3K and some Ras mutations cause
deregulated activation of PI3K and Akt.131–133 Since PTEN and
SHIP-2 negatively regulate PI3K activity, loss of their phosphatase activity may also induce Akt activation.134–136 Mutations
and hemizygous deletions of PTEN have been detected in
primary acute leukemia and non-Hodgkin’s lymphoma. In
addition, many hematopoietic cell lines lack or have low PTEN
protein expression.134,135 Increased Akt expression has also
been linked with tumor progression. Akt-2 gene amplification
has been observed in some cervical, ovarian, and pancreatic
cancers as well as non-Hodgkin’s lymphomas.136–139 SHIP-1
may also affect Akt activity by controlling levels of PI(3,4,5)P3
and PI(3,4)P2. If SHIP-1 activitiy is lost, PI(3,4,5)P3 levels would
be elevated, leading to Akt phosphorylation at T308 by PDK1.
Conversely, PI(3,4)P2 levels would be decreased, which would
prevent activation of the unidentified kinase responsible for Akt
phosphorylation at S473.140 Thus, this pathway is regulated
such that cetain mutations would cause partial, but not
complete, Akt activation.
PI3K/Akt pathway as a target for chemotherapy
Inhibition of the PI3K/Akt pathway could prevent uncontrolled
cellular proliferation. Consequently, antiapoptotic signals transduced by PI3K and downstream molecules have become a focus
of recent drug discovery research. This section will discuss
strategies that have been used to inhibit this pathway.
Small molecule inhibitors (SMIs) have been discovered for
many kinases involved in various signal transduction pathways
(Figure 11). For example, quercetin is marketed by Calbiochem
(San Diego, CA, USA) as an inhibitor of PI3K. However, its
specificity has been questioned because it has also been
found to inhibit other serine/threonine kinases such as AMPK,
CK2, p90RSK2, and p70S6K1. 1412-(4-Morpholinyl)-8-phenyl-4H1-benzopyran-4-one (LY294002) and wortmannin are two
widely used PI3K inhibitors. Of these two, LY294002 is more
specific and is therefore more commonly used than wortmannin.141 LY294002 is a quercetin derivative with a reported IC50
value of 1.4 mm.142 Wortmannin is a fungal metabolite with an
IC50 value of 5 nm.143
Akt inhibitors have been recently discovered. One is 1L-6hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, which is marketed by Calbiochem. It has been
reported to inhibit selectively Akt with an IC50 value of
approximately 5 mm, which is much less than its IC50 value of
90 mm for PI3K inhibition.144 Additionally, it was recently
reported that the tyrosine phosphorylation inhibitor (tyrphostin)
AG957, which is also named NSC 654705, caused dephosphorylation of Akt.145 This report is intriguing because AG957
was previously described as an inhibitor specific for the Bcr-Abl
chromosomal translocation gene product implicated in development of chronic myelogenous leukemia (CML).
p70S6K is another viable target for chemotherapeutic intervention within the PI3K/Akt pathway. Rapamycin is commonly
used to inhibit p70S6K activity. It is a macrolide antibiotic
produced by the filamentous bacterium Streptomyces hygroscopicus.146 Rapamycin is used as a powerful immunosuppressant, which has been effective in a variety of clinical settings
such as organ transplantation. Rapamycin dephosphorylates
Figure 11
Signal transduction inhibitors targeting the PI3K/Akt
pathway. Structures of some small molecular weight membrane
permeable drugs targeting this pathway are indicated: (a) LY294002
(PI3K inhibitor), (b) wortmannin (PI3K inhibitor), (c) 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Akt
Inhibitor), (d) rapamycin (p70S6K inhibitor), and (e) quercetin (PI3K
inhibitor).
p70S6K at sites different from those associated with activation.147
As a consequence, phosphorylation of the S6 ribosomal protein
necessary for cell growth does not occur.
Transfection with plasmid-based antisense vectors or treatment with synthetic antisense oligonucleotides have also been
used to disrupt signaling by the PI3K/Akt pathway. Antisense
nucleic acids inhibit translation by hybridizing to complementary mRNA transcripts to prevent ribosome binding. Skorski
et al148 showed that expression of antisense cDNA or treatment
with antisense oligonucleotides downregulated p85 gene
expression and abrogated PI3K signaling. Antisense RNAs
were found to drastically reduce tumorgenicity of PANC1
cells, which overexpress Akt2, in nude mice.149 Antisense
oligonucleotides targeted to Akt1 had several effects on a variety
of cancer cell lines, including reduced ability to grow on soft
agar, induction of apoptosis, and enhanced susceptibility to
various chemotherapeutic agents.150 Similar results were
obtained from studies of antisense oligonucleotides targeted to
PDK1.151
Earlier in this review, it was discussed that PI3K is a known
downstream substrate of the GTPase Ras. Disruption of Ras
activity may also be effective for inhibition of the PI3K/Akt
pathway. Three approaches that have been taken to inhibit Ras
signaling include prevention of Ras membrane localization,
antisense strategies, and ectopic expression of DN proteins.
Membrane localization of Ras has been inhibited with
farnesyltransferase inhibitors (FTIs). Ras becomes active only
after post-translational addition of farnesyl or geranylgeranyl
isoprenoid moieties to its carboxy terminus. Without such
Leukemia
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
598
modification, Ras remains in the cytosol where it cannot
activate PI3K. Enzymes catalyzing transfer of these lipid
moieties to Ras include farnesyltransferase (FT), geranylgeranyltransferase type I (GGTI), and geranylgeranyltransferase type II
(GGTII). An abundance of FTIs are currently available for
laboratory use. Chemical structures of some FTIs are shown in
Figure 12. For example, arglabin-DMA is a recently discovered
FTI that is derived from a species of wormwood endemic to
central Asia.152 There are currently four FTIs undergoing clinical
trials: R115777, SCH66336, L-778,123, and BMS214662.153,154 Jiang et al155 reported that apoptosis caused by
FTI-277 could be rescued through expression of a constitutively
active form of Akt, suggesting that FTIs may mediate some
inhibitory effects not only through the Ras/Raf/MEK/ERK pathway, but also the PI3K/Akt signaling cascade.
Antisense strategies have also been used to disrupt Ras.
Expression of antisense K-Ras cDNA suppressed the malignant
phenotype of large cell lung carcinomas156 and inhibited growth
of H460a lung cancer cells nearly three-fold.157 ISIS 2503 is a
20-base antisense phosphorothioate oligodeoxyribonucleotide
targeting H-Ras that is currently in phase 2 clinical trials.158,159
Lastly, expression of DN proteins has been used to inhibit Ras
signaling. DN proteins bind and inhibit function of endogenous
proteins. Two DN forms of Ras have been described. The first,
Ras17N, blocks endogenous Ras GTPase activity, which is
essential for signal transduction.160,161 Ras17N has been
described to inhibit Akt activation elicited by the v-Crk
oncogene.162 Another DN mutant binds endogenous Ras to
form inactive complexes localized to the cytoplasm. Signals
from the plasma membrane are not transduced by Ras because
its localization to the plasma membrane is disrupted.163
Inhibition of both PI3K and Akt has also been achieved with
DN proteins. Expression of a DN form of p85, Dp85, decreased
cell growth in Bcr-Abl-transformed cells.164 Another report
Figure 12
Leukemia
described use of a DN form of p110, the catalytic subunit of
PI3K.165 Furthermore, Mabuchi et al166 reported that ovarian
cancer cells become sensitized to paclitaxel by DN Akt
expression.
Lastly, PI3K signaling can be abrogated through the activity of
two known phosphatases. SHIP-2 overexpression has been
observed to cause Akt inactivation and cell cycle arrest in
glioblastoma cells.167 Overexpression of PTEN inhibits proliferation and malignant progression of prostate cancer cells.168
Taken together, these data indicate that phosphatases may have
therapeutic promise by abrogating signaling transduced through
PI3K. Strategies for inhibiting the PI3K/Akt pathway are outlined
in Figure 13.
Summary
In this review, we have discussed the effects of the PI3K/Akt
pathway on cell cycle progression and apoptosis. This pathway
plays critical roles in cell proliferation, oncogenesis, and its
activity is regulated positively by Ras and negatively by PTEN.
The PI3K/Akt pathway can induce cell cycle progression by
modulating the protein stability of cyclin D and p21Cip1. Akt can
directly phosphorylate p21Cip1 on S146 and increase its protein
stability. Alternatively, Akt can also prolong the half-life of both
p21Cip1 and cyclin D through inactivation of GSK-3, which
phosphorylates p21Cip1 and cyclin D to induce their degradation. Akt facilitates this inactivation event via phosphorylation of
GSK-3 on residue S9. The PI3K/Akt pathway can also induce
DNA synthesis. When T145 of p21Cip1 is phosphorylated by Akt,
DNA pol d is released from p21Cip1 and binds with AP-1 to
induce DNA synthesis. Recently, Akt has also been shown to
directly phosphorylate p27Kip1 on T157 to cause its retention in
the cytosol. As a result, p27Kip1 loses its inhibitory effect on
Farnesyltransferase inhibitors. Chemical structures of commonly used FTIs are shown.
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
599
Figure 13
Sites of inhibition of the PI3K/Akt pathway. Schematic overview of the potential negative regulators of PI3K/Akt signaling. Black
boxes indicate mechanisms of inhibition.
Cdk2. Moreover, phosphorylation of FKHR-L1 by Akt also leads
to reduced p27Kip1 transcription. The PI3K/Akt pathway also
regulates activity of Bcl-2 family members. Both Akt and p70S6K
can phosphorylate proapoptotic Bad, which promotes association of Bad with 14-3-3 adaptor protein and allows excess Bcl-2
or Bcl-XL to out compete Bax, there by preventing apoptosis.
It is becoming increasingly evident that interactions between
the PI3K/Akt and Raf/MEK/ERK pathwys are important for the
regulation of cell cycle progression and apoptosis. Some
scientists have observed that Akt phosphorylates Raf-1 on
S259 to inhibit its kinase activity, and these inhibitory effects
may be released by removal of this phosphate by protein
phosphatase 2A. However, these interactions remain controversial because they have not been unequivocably confirmed by
other investigators. Future studies into these types of biomolecular interactions are, therefore, warranted.
Despite the tremendous amount of research that remains
uncompleted, it is evident that signal transduction pathways
interact to regulate cell cycle progression and apoptosis. An
understanding of how these different cascades control expression of cell cycle regulatory genes by transcriptional and posttranscriptional mechanisms may reveal novel strategies to
prevent their aberrant activation in transformed cells. It is
hopeful that further characterization of pathways regulating
cell cycle progression and apoptosis will facilitate development of chemotherapeutic inhibitors that will suppress hyperactivity of these cascades and thus prove effective for cancer
treatment.
Acknowledgements
We thank Ms Catherine Spruill for the excellent artwork. This
work was supported by grants from the United States National
Institutes of Health (R01CA51025) and grants from the North
Carolina Biotechnology Center (9805-ARG-0006, 2000-ARG0003) to JAM. RAF was supported in part by grants from the
American Cancer Society (IRG-97-149), American Heart Associa-
tion (9930099N), and the North Carolina Biotechnology Center
(9705-ARG-0009).
References
1 Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE,
Wang XY, Algate PA et al. Signal transduction, cell cycle
regulatory, and anti-apoptotic pathways regulated by IL-3 in
hematopoietic cells: possible sites for intervention with antineoplastic drugs. Leukemia 1999; 13: 1109–1166.
2 Franklin RA, McCubrey JA. Kinases: positive and negative
regulators of apoptosis. Leukemia 2000; 14: 2019–2034.
3 White MK, McCubrey JA. Suppression of apoptosis: role in cell
growth and neoplasia. Leukemia 2001; 15: 1011–1021.
4 McCubrey JA, Steelman LS, Blalock WL, Lee JT, Moye PW, Chang
F et al. Synergistic effects of PI3K/Akt on abrogation of cytokinedependency induced by oncogenic Raf. Adv Enzyme Regul 2001;
41: 289–323.
5 McCubrey JA, Lee JT, Steelman LS, Blalock WL, Moye PW, Chang
F et al. Interactions between the PI3K and Raf signaling pathways
can result in the transformation of hematopoietic cells. Cancer
Detect Prev 2001; 25: 375–393.
6 McCubrey JA, Blalock WL, Chang F, Steelman LS, Pohnert SC,
Navolanic PM et al. Signal transduction pathways: cytokine
model. In: Blagosklonny M. (ed). Cell Cycle Checkpoints and
Cancer. Austin, TX: Landes Bioscience Press, 2001, pp 17–51.
7 McCubrey JA, May WS, Duronio V, Mufson A. Serine/threonine
phosphorylation in cytokine signal transduction. Leukemia 2000;
14: 9–21.
8 McCubrey JA, Blalock WL, Saleh O, Pearce M, Burrows C,
Steelman LS et al. Enhanced ability of daniplestim and
myelopoietin-1 to suppress apoptosis in human hematopoietic
cells. Leukemia 2001; 15: 1203–1216.
9 Srinivasa SP, Doshi PD. Extracellular signal-regulated kinase and
p38 mitogen-activated protein kinase pathways cooperate in
mediating cytokine-induced proliferation of a leukemic cell line.
Leukemia 2002; 16: 244–253.
10 Lee Jr JT, McCubrey JA. The Raf/MEK/ERK signal transduction
cascade as a target for chemotherapeutic intervention in
leukemia. Leukemia 2002; 16: 486–507.
11 Lee Jr JT, McCubrey JA. Targeting the Raf kinase cascade in
cancer therapyFnovel molecular targets and therapeutic strategies. Expert Opin Ther Targets 2002 6: 659–678.
Leukemia
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
600
12 Jucker M, Sudel K, Horn S, Sickel M, Wegner W, Fiedler W et al.
Expression of a mutated form of the p85alpha regulatory subunit
of phosphatidylinositol 3-kinase in a Hodgkin’s lymphomaderived cell line (CO). Leukemia 2002; 16: 894–901.
13 Lin J, Adam RM, Santiestevan E, Freeman MR. The phosphatidylinositol 3’-kinase pathway is a dominant growth factor-activated
cell survival pathway in LNCaP human prostate carcinoma cells.
Cancer Res 1999; 59: 2891–2897.
14 Fry MJ. Phosphoinositide 3-kinase signalling in breast cancer:
how big a role might it play? Breast Cancer Res 2001; 3: 304–
312.
15 Lin X, Bohle AS, Dohrmann P, Leuschner I, Schulz A, Kremer B
et al. Overexpression of phosphatidylinositol 3-kinase in human
lung cancer. Langenbecks Arch Surg 2001; 386: 293–301.
16 Krasilnikov M, Adler V, Fuchs SY, Dong Z, Haimovitz-Friedman
A, Herlyn M et al. Contribution of phosphatidylinositol 3-kinase
to radiation resistance in human melanoma cells. Mol Carcinog
1999; 24: 64–69.
17 Martinez-Lorenzo MJ, Anel A, Monleon I, Sierra JJ, Pineiro A,
Naval J et al. Tyrosine phosphorylation of the p85 subunit of
phosphatidylinositol 3-kinase correlates with high proliferation
rates in sublines derived from the Jurkat leukemia. Int J Biochem
Cell Biol 2000; 32: 435–445.
18 Nicholson KM, Anderson NG. The protein kinase B/Akt signalling
pathway in human malignancy. Cell Signal 2002; 14: 381–395.
19 Rodriguez-Viciana P, Marte BM, Warne PH, Downward J.
Phosphatidylinositol 3’kinase: one of the effectors of Ras. Philos
Trans Roy Soc London Ser B: Biol Sci 1996; 351: 225–231.
20 Scheid MP, Woodgett JR. PKB/AKT: functional insights from
genetic models. Nat Rev Mol Cell Biol 2001; 2: 760–768.
21 Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD.
Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci 1997; 22: 267–272.
22 Duronio V, Scheid MP, Ettinger S. Downstream signalling events
regulated by phosphatidylinositol 3-kinase activity. Cell Signall
1998; 10: 233–239.
23 Pacold ME, Suire S, Perisic O, Lara-Gonzalez S, Davis CT,
Walker EH et al. Crystal structure and functional analysis of Ras
binding to its effector phosphoinositide 3-kinase gamma. Cell
2000; 103: 931–943.
24 Djordjevic S, Driscoll PC. Structural insight into substrate
specificity and regulatory mechanisms of phosphoinositide 3kinases. Trends Biochem Sci 2002; 27: 426–432.
25 Chan TO, Rodeck U, Chan AM, Kimmelman AC, Rittenhouse SE,
Panayotou G et al. Small GTPases and tyrosine kinases coregulate
a molecular switch in the phosphoinositide 3-kinase regulatory
subunit. Cancer Cell 2002; 1: 181–191.
26 Yan J, Roy S, Apolloni A, Lane A, Hancock JF. Ras isoforms vary
in their ability to activate Raf-1 and phosphoinositide 3-kinase. J
Biol Chem 1998; 273: 24052–24056.
27 Vazquez F, Sellers WR. The PTEN tumor suppressor protein: an
antagonist of phosphoinositide signaling. Biochim Biophys Acta
2000; 1470: M21–M35.
28 Wu X, Senechal K, Neshat MS, Whang YE, Sawyers CL. The
PTEN/MMAC1 tumor suppressor phosphatase functions as a
negative regulator of the phosphoinositide 3-kinase/Akt pathway.
Proc Natl Acad Sci USA 1998; 95: 15587–15591.
29 Sakai A, Thieblemont C, Wellmann A, Jaffe ES, Raffeld M. PTEN
gene alterations in lymphoid neoplasms. Blood 1998; 92:
3410–3415.
30 Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM
et al. 50 Phospholipid phosphatase SHIP-2 causes protein kinase B
inactivation and cell cycle arrest in glioblastoma cells. Mol Cell
Biol 2000; 20: 6860–6871.
31 Muraille E, Pesesse X, Kuntz C, Erneux C. Distribution of the srchomology-2-domain-containing inositol 5-phosphatase SHIP-2 in
both non-haemopoietic and haemopoietic cells and possible
involvement in SHIP-2 in negative signaling of B-cells. Biochem J
1999; 342: 697–705.
32 Carnero A, Hannon GJ. The INK4 family of CDK inhibitors. Curr
Top Microbiol Immunol 1998; 227: 43–55.
33 Morgan DO. Cyclin-dependent kinases: engines, clocks, and
microprocessors. Annu Rev Cell Dev Biol 1997; 13: 261–291.
34 Peter M. The regulation of cyclin-dependent kinase inhibitors
(CKIs). Prog Cell Cycle Res 1997; 3: 99–108.
Leukemia
35 Choudhury GG, Karamitsos C, Hernandez J, Gentilini A,
Bardgette J, Abbound HE. PI-3-kinase and MAPK regulate
mesangial cell proliferation and migration in response to PDGF.
Am J Physiol 1997; 273: F931–F938.
36 Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase
kinase-3b regulates cyclin D1 proteolysis and subcellular
localization. Genes Dev 1998; 12: 3499–3511.
37 Gille H, Downward J. Multiple ras effector pathways contribute
to G1 cell cycle progression. J Biol Chem 1999; 274: 22033–
22040.
38 Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like forkhead
transcription factors mediate cell-cycle regulation by Ras and
PKB through p27Kip1. Nature 2000; 404: 782–787.
39 Muise-Helmericks RC, Grimers HL, Bellacosa A, Malstrom SE,
Tsichlis PN, Rosen N. Cyclin D expression is controlled posttranscriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J Biol Chem 1998; 273: 29864–29872.
40 Choudhury GG. Akt serine threonine kinase regulates plateletderived growth factor-induced DNA synthesis in glomerular
mesangial cells: regulation of c-fos and p27Kip1 gene expression. J
Biol Chem 2002; 276: 35636–35643.
41 Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D.
P21 is a universal inhibitor of cyclin kinases. Nature 1993; 366:
701–704.
42 Harper JW, Elledge SJ, Keyomarsi K, Dynlacht B, Tsai LH, Zhang
P et al. Inhibition of cyclin-dependent kinases by p21. Mol Biol
Cell 1995; 6: 387–400.
43 Sherr CJ, Roberts MJ. CDK inhibitors: positive and negative
regulators of G1-phase progression. Genes Dev 1999; 13: 1501–
1512.
44 Rank KB, Evans DB, Sharma SK. The N-terminal domains of
cyclin-dependent kinase inhibitory proteins block the phosphorylation of cdk2/cyclin E by the CDK-activating kinase. Biochem
Biophys Res Commun 2000; 271: 469–473.
45 Shimizu T, Takahashi N, Tachibana K, Takeda K. Complex
regulation of CDK2 and G1 arrest during neuronal differentiation
of human prostatic cancer TSU-Prl cells by staurosporine.
Anticancer Res 2001; 21: 893–898.
46 Lawlor MA, Rotwein P. Insulin-like growth factor-mediated
muscle cell survival: central roles for Akt and cyclin-dependent
kinase inhibitor p21. Mol Cell Biol 2000; 23: 8983–8995.
47 Mitsuuchi Y, Johnson SW, Selvakumaran M, Williams SJ,
Hamilton TC, Testa JR. The phosphatidylinositol 3-kinase/AKT
signal transduction pathway plays a critical role in the expression
of p21WAF1/CIP1/SDI1 induced by cisplatin and paclitaxel. Cancer
Res 2000; 60: 5390–5394.
48 Schönherr E, Levkau B, Schaefer L, Kresse H, Walsh K. Decorinmediated signal transduction in endothelial cells. J Biol Chem
2001; 276: 40687–40692.
49 Zhou B, Liao Y, Xia W, Spohn B, Lee M, Hung M. Cytoplasmic
localization of p21Cip1/WAF1 by Akt-induced phosphorylation
in HER-2/neu-overexpressing cells. Nat Cell Biol 2001; 3:
245–252.
50 Li Y, Dowbenko D, Lasky LA. AKT/PKB phosphorylation of
p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and
promotes cell survival. J Biol Chem 2002; 277: 11352–11361.
51 Rössig L, Badorff C, Holzmann Y, Zeiher AD, Dimmeler S.
Glycogen synthase kinase-3 couples AKT-dependent signaling to
the regulation of p21Cip1 degradation. J Biol Chem 2002; 277:
9684–9689.
52 Rössig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, Dimmeler S.
Akt-dependent phosphorylation of p21Cip1 regulates PCNA
binding and proliferation of endothelial cells. Mol Cell Biol
2001; 21: 5644–5657.
53 Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of
cyclin-dependent kinases controls DNA replication by interaction
with PCNA. Nature 1994; 369: 574–578.
54 Flores-Rozas H, Kelman Z, Dean FB, Pan ZQ, Harper JW, Elledge
SJ et al. Cdk-interacting protein 1 directly binds with proliferating
cell nuclear antigen and inhibits DNA replication catalyzed by
the DNA polymerase delta holoenzyme. Proc Natl Acad Sci USA
1994; 91: 8655–8659.
55 Blagosklonny MV, Wu GS, Omura S, el-Deiry WS. Proteasomedependent regulation of p21WAF1/CIP1 expression. Biochem
Biophys Res Commun 1996; 227: 564–569.
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
601
56 Cantley LC. The phosphoinositide 3-kinase pathway. Science
2002; 296: 1655–1657.
57 van Weeren PC, de Bruyn KM, de Vries-Smits AM, van Lint J,
Burgering BM. Essential role for protein kinase B (PKB) in insulininduced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 1998; 273:
13150–13156.
58 Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA.
Inhibition of glycogen synthase kinase-3 by insulin mediated by
protein kinase B. Nature 1995; 378: 785–789.
59 Maki CG, Howley PM. Ubiquitination of p53 and p21 is
differentially affected by ionizing and UV radiation. Mol Cell
Biol 1997; 17: 355–363.
60 Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM,
Clurman BE. Proteasomal turnover of p21Cip1 does not require
p21Cip1 ubiquitination. Mol Cell 2000; 5: 403–410.
61 Touitou R, Richardson J, Bose S, Nakanishi M, Rivett J, Allday MJ.
A degradation signal located in the C-terminus of p21WAF1/CIP1
is a binding site for the C8 alpha-subunit of the 20S proteasome.
EMBO J 2001; 20: 2367–2375.
62 Stambolic V, Ruel L, Woodgett JR. Lithium inhibits glycogen
synthase kinase-3 activity and mimics wingless signalling in
intact cells. Curr Biol 1996; 6: 1664–1668.
63 Klein PS, Melton DA. A molecular mechanism for the effect
of lithium on development. Proc Natl Acad Sci USA 1996; 93:
8455–8459.
64 Chang F, McCubrey JA. p21Cip1 induced by Raf is associated with
increased Cdk4 activity in hematopoietic cells. Oncogene 2001;
20: 4353–4364.
65 Hu Q, Klippel A, Muslin AJ, Fantl WJ, Williams LT. Rasdependent induction of cellular responses by constitutively active
phosphatidylinositol-3 kinase. Science 1995; 268: 100–102.
66 Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for
the winged helix/forkhead transcription factors. Genes Dev 2000;
14: 142–146.
67 Choudhury GG, Kim YS, Simon M, Wozney J, Harris S,
Choudhury GN et al. Bone morphogenetic protein 2 inhibits
platelet-derived growth factor-induced c-fos gene transcription and DNA synthesis in mesangial cells. Involvement of
mitogen-activated protein kinase. J Biol Chem 1999; 274:
10897–10902.
68 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. Akt
promotes cell survival by phosphorylating and inhibiting a
Forkhead transcription factor. Cell 1999; 96: 857–868.
69 Viglietto G, Motti ML, Bruni P, Melillo RM, D’Alessio A,
Califano D et al. Cytoplasmic relocalization and inhibition of
the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Aktmediated phosphorylation in breast cancer. Nat Med 2002; 8:
1136–1144.
70 Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J et al. PKB/
Akt mediates cell-cycle progression by phosphorylation of
p27Kip1 at threonine 157 and modulation of its cellular
localization. Nat Med 2002; 8: 1145–1152.
71 Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K
et al. PKB/Akt phosphorylates, p27, impairs nuclear import of
p27 and opposes p27-mediated G1 arrest. Nat Med 2002; 8:
1153–1160.
72 Beg AA, Baldwin Jr AS. Activation of multiple NF-kappa B/Rel
DNA-binding complexes by tumor necrosis factor. Oncogene
1994; 9: 1487–1492.
73 Beg AA, Finco TS, Nantermet PV, Baldwin Jr AS. Tumor necrosis
factor and interleukin-1 lead to phosphorylation and loss of I
kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell
Biol 1993; 13: 3301–3310.
74 Beg AA, Baltimore D. An essential role for NF-kappaB in
preventing TNF-alpha-induced cell death. Science 1996; 274:
782–784.
75 Wang CY, Mayo MW, Baldwin Jr AS. TNF- and cancer therapyinduced apoptosis: potentiation by inhibition of NF-kappaB.
Science 1996; 274: 784–787.
76 Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM.
Suppression of TNF-alpha-induced apoptosis by NF-kappaB.
Science 1996; 274: 787–789.
77 Liu ZG, Hsu H, Goeddel DV, Karin M. Dissection of TNF receptor
1 effector functions: JNK activation is not linked to apoptosis
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
while NF-kappaB activation prevents cell death. Cell 1996; 87:
565–576.
Beauparlant P, Kwan I, Bitar R, Chou P, Koromilas AE, Sonenberg
N et al. Disruption of I kappa B alpha regulation by antisense
RNA expression leads to malignant transformation. Oncogene
1994; 9: 3189–3197.
Chen C, Edelstein LC, Gelinas C. The Rel/NF-kappaB family
directly activates expression of the apoptosis inhibitor Bcl-x(L).
Mol Cell Biol 2000; 20: 2687–2695.
Zong WX, Edelstein LC, Chen C, Bash J, Gelinas C. The
prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional
target of NF-kappaB that blocks TNFalpha-induced apoptosis.
Genes Dev 1999; 13: 382–387.
Lee HH, Dadgostar H, Cheng Q, Shu J, Cheng G. NF-kappaBmediated up-regulation of Bcl-x and Bfl-1/A1 is required for
CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci
USA 1999; 96: 9136–9141.
Wang CY, Guttridge DC, Mayo MW, Baldwin Jr AS. NF-kappaB
induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol
1999; 19: 5923–5929.
Karin M, Delhase M. The I kappa B kinase (IKK) and NF-kappa B:
key elements of proinflammatory signalling. Semin Immunol
2000; 12: 85–98.
Romashkova JA, Makarov SS. NF-kappaB is a target of
AKT in anti-apoptotic PDGF signalling. Nature 1999; 401:
86–90.
Madrid LV, Wang CY, Guttridge DC, Schottelius AJ, Baldwin Jr
AS, Mayo MW. Akt suppresses apoptosis by stimulating the
transactivation potential of the RelA/p65 subunit of NF-kappaB.
Mol Cell Biol 2000; 20: 1626–1638.
Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes
in vivo with a conserved homolog, Bax, that accelerates
programmed cell death. Cell 1993; 74: 609–619.
Wang HG, Rapp UR, Reed JC. Bcl-2 targets the protein kinase
Raf-1 to mitochondria. Cell 1996; 87: 629–638.
Blagosklonny MV, Schulte T, Nguyen P, Trepel J, Neckers LM.
Taxol-induced apoptosis and phosphorylation of Bcl-2 protein
involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res 1996; 56: 1851–1854.
Lau QC, Brusselbach S, Muller R. Abrogation of c-Raf expression
induces apoptosis in tumor cells. Oncogene 1998; 16: 1899–
1902.
Salomoni P, Wasik MA, Riedel RF, Reiss K, Choi JK, Skorski T
et al. Expression of constitutively active Raf-1 in the mitochondria
restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant. J Exp Med 1998; 187: 1995–
2007.
Majewski M, Nieborowska-Skorska M, Salomoni P, Slupianek A,
Reiss K, Trotta R et al. Activation of mitochondrial Raf-1 is
involved in the antiapoptotic effects of Akt. Cancer Res 1999; 59:
2815–2819.
Peruzzi F, Prisco M, Dews M, Salomoni P, Grassilli E, Romano G
et al. Multiple signaling pathways of the insulin-like growth factor
1 receptor in protection from apoptosis. Mol Cell Biol 1999; 19:
7203–7215.
McCubrey JA, Steelman LS, Hoyle PE, Blalock WL, WeinsteinOppenheimer C, Franklin RA et al. Differential abilities of
activated Raf oncoproteins to abrogate cytokine-dependency,
prevent apoptosis and induce autocrine growth factor
synthesis in human hematopoietic cells. Leukemia 1998; 12:
1903–1929.
McCubrey JA, Steelman LS, Moye PW, Hoyle PE, WeinsteinOppenheimer C, Chang F et al. Effects of deregulated Raf and
MEK1 expression on the cytokine-dependency of hematopoietic
cells. Adv Enzyme Regul 2000; 40: 305–337.
Moye PW, Blalock WL, Hoyle PE, Chang F, Franklin RA,
Weinstein-Oppenheimer C et al. Synergy between Raf and
BCL2 in abrogating the cytokine-dependency of hematopoietic
cells. Leukemia 2000; 14: 1060–1079.
Hoyle PE, Moye PW, Steelman LS, Blalock WL, Franklin RA,
Pearce M et al. Differential abilities of the Raf family of protein of
kinases to abrogate cytokine-dependency and prevent apoptosis
in murine hematopoietic cells by a MEK1-dependent mechanism.
Leukemia 2000; 14: 642–656.
Leukemia
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
602
97 Blagosklonny MV, Schulte TW, Nguyen P, Mimnaugh EG, Trepel
J, Neckers L. Taxol induction of p21Waf1 and p53 requires c-raf-1.
Cancer Res 1995; 55: 4623–4626.
98 Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C,
Coffer P, Downward J et al. Suppression of c-Myc-induced
apoptosis by Ras signalling through PI3K and PKB. Nature 1997;
385: 544–548.
99 Basu S, Bayoumy S, Zhang Y, Lozano J, Kolesnick R. BAD enables
ceramide to signal apoptosis via Ras and Raf-1. J Biol Chem 1998;
273: 30419–30426.
100 von Gise A, Lorenz P, Wellbrock C, Hemmings B, BerberichSiebelt F, Rapp UR et al. Apoptosis suppression by Raf-1 and
MEK1 requires MEK and phosphatidylinositol 3-kinase dependent
signals. Mol Cell Biol 2001; 21: 2324–2336.
101 Yuryev A, Ono M, Goff SA, Macaluso F, Wennogle LP. Isoformspecific localization of A-RAF in mitochondria. Mol Cell Biol
2000; 20: 4870–4878.
102 Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y et al. Akt
phosphorylation of BAD couples survival signals to the cellintrinsic death machinery. Cell 1997; 91: 231–241.
103 Harada H, Andersen JS, Mann M, Terada N, Korsmeyer SJ. P70S6
kinase signals cell survival as well as growth, inactivating the proapoptotic molecule Bad. Proc Natl Acad Sci USA 2001; 98:
9666–9670.
104 Fang X, Yu S, Eder A, Mao M, Bast Jr RC, Boyd D et al. Regulation of BAD phosphorylation at serine 112 by the Rasmitogen-activated protein kinase pathway. Oncogene 1999; 18:
6635–6640.
105 Virdee K, Parone PA, Tolkovsky AM. Phosphorylation of the proapoptotic protein BAD on serine 155, a novel site, contributes to
cell survival. Curr Biol 2000; 10: 1151–1154.
106 Chang F, Steelman LS, McCubrey JA. Raf-induced cell cycle
progression in human TF-1 hematopoietic cells. Cell Cycle 2002;
1: 220–226.
107 Dijkers PF, Medema RH, Pals C, Banerji L, Thomas SH, Lam EWF
et al. Forkhead transcription factor FKHR-L1 modulates cytokinedependent transcriptional regulation of p27Kip1. Mol Cell Biol
2000; 20: 9138–9148.
108 Woods D, Parry D, Cherwinski H, Bosch E, Lees E, McMahon M.
Raf-induced proliferation or cell cycle arrest is determined by the
level of Raf activity with arrest mediated by p21Cip. Mol Cell Biol
1997; 17: 5598–5611.
109 Lloyd AC, Obermuller F, Staddon S, Barth CF, McMahon M,
Land H. Cooperating oncogenes converge to regulate cyclin/Cdk
complexes. Genes Dev 1997; 11: 663–677.
110 Blalock WL, Pearce M, Steelman LS, Franklin RA, McCarthy SA,
Cherwinski H et al. A conditionally-active form of MEK1 results
in autocrine transformation of human and mouse hematopoietic
cells. Oncogene 2000; 19: 526–536.
111 Blalock WL, Moye PW, Chang F, Pearce M, Steelman LS,
McMahon M et al. Combined effects of aberrant MEK1 activity
and BCL-2 overexpression on relieving the cytokine-dependency
of human and murine hematopoietic cells. Leukemia 2000; 14:
1080–1096.
112 Blalock WL, Pearce M, Chang F, Lee J, Pohnert SC, Burrows C
et al. Effects of inducible MEK1 activation on the cytokinedependency of lymphoid cells. Leukemia 2001; 15: 794–807.
113 Zimmermann S, Moelling K. Phosphorylation and regulation of
Raf by Akt (protein kinase B). Science 1999; 286: 1741–1744.
114 Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD et al.
Negative regulation of the serine/threonine kinase B-Raf by Akt. J
Biol Chem 2000; 275: 27354–27359.
115 Reusch JP, Zimmermann S, Schaefer M, Paul M, Moelling K.
Regulation of Raf by Akt controls growth and differentiation in
vascular smooth muscle cells. J Biol Chem 2001; 276: 7644–7655.
116 Rommel C, Clarke BA, Zimmermann S, Nuñez L, Rossamn R,
Reid K et al. Differentiation stage-specific inhibition of the Raf–
MEK–ERK pathway by Akt. Science 1999; 286: 1738–1741.
117 Moelling K, Schad K, Bosse M, Zimmermann S, Schweneker M.
Regulation of Raf–Akt cross-talk. J Biol Chem 2002; 277:
31099–31106.
118 Mizuno H, Nishida E. The ERK MAP kinase pathway mediates
induction of SGK (serum- and glucocorticoid-inducible kinse) by
growth factors. Genes Cells 2001; 6: 261–268.
Leukemia
119 Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, Hemmings
BA. Serum and glucocorticoid-inducible kinase (SGK) is a target
of the PI3-kinase-stimulated signaling pathway. EMBO J 1999;
18: 3024–3033.
120 Kobayashi T, Deak M, Morrice N, Cohen P. Characterization of
the structure and regulation of two novel isoforms of serum- and
glucocorticoid-induced protein kinase. Biochem J 1999; 344:
189–197.
121 Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL,
Richards JS. Follicle-stimulating hormone (FSH) stimulates
phosphorylation and activation of protein kinase B (PKB/Akt)
and serum and glucocorticoid-induced kinase (SGK): evidence
for a kinase-independent signaling by FSH in granulosa cells. Mol
Endocrinol 2000; 14: 1283–1300.
122 Prasad N, Topping RS, Zhou D, Decker SJ. Oxidative stress and
vanadate induce tyrosine phosphorylation of phosphoinositidedependent kinase 1 (PDK1). Biochemistry 1998; 39: 6929–6935.
123 Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI et al. PTEN,
a putative protein tyrosine phosphatase gene mutated in human
brain, breast, and prostate cancer. Science 1997; 275: 1943–
1947.
124 Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C,
Sasaki T et al. Negative regulation of PKB/Akt-dependent cell
survival by the tumor suppressor PTEN. Cell 1998; 95: 29–39.
125 Gu J, Tamura M, Yamada KM. Tumor suppressor PTEN inhibits
integrin- and growth factor-mediated mitogen-activated protein
(MAP) kinase signaling pathways. J Cell Biol 1998; 143:
1375–1383.
126 Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1,
dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998; 273: 13375–13378.
127 Myers MP, Pass I, Batty IH, Van der Kaay J, Stolarov JP,
Hemmings BA et al. The lipid phosphatase activity of PTEN is
critical for its tumor supressor function. Proc Natl Acad Sci USA
1998; 95: 13513–13518.
128 Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM
et al. 50 Phospholipid phosphatase SHIP-2 causes protein kinase B
inactivation and cell cycle arrest in glioblastoma cells. Mol Cell
Biol 2000; 20: 6860–6871.
129 Muraille E, Pesesse X, Kuntz C, Erneux C. Distribution of the srchomology-2-domain-containing inositol 5-phosphatase SHIP-2 in
both non-haemopoietic and haemopoietic cells and possible
involvement in SHIP-2 in negative signaling of B-cells. Biochem J
1999; 342: 697–705.
130 Baserga R, Morrione A. Differentiation and malignant transformation: two roads diverged in a wood. J Cell Biochem 1999; 32:
(Suppl) 68–75.
131 Suzuki J, Kaziro Y, Koide H. An activated mutant of R-Ras inhibits
cell death caused by cytokine deprivation in BaF3 cells in the
presence of IGF-1. Oncogene 1997; 15: 1689–1697.
132 McIlroy J, Chen D, Wjasow C, Michaeli T, Backer JM. Specific
activation of p85–p110 phosphatidylinositol 30 -kinase stimulates
DNA synthesis by Ras- and p70 S6 kinase-dependent pathways.
Mol Cell Biol. 1997; 17: 248–255.
133 Rodriguez-Viciana P, Marte BM, Warne PH, Downward J.
Phosphatidylinositol 3’kinase: one of the effectors of Ras. Philos
Trans Roy Soc London Ser B: Biol Sci 1996; 351: 225–231.
134 Dahia PL, Aquiar RC, Alberta J, Kum JB, Caron S, Sill H et al.
PTEN is inversely correlated with the cell survival factor Akt/PKB
and is inactivated via multiple mechanism in haematological
malignancies. Hum Mol Genet 1999; 8: 185–193.
135 Sakai A, Thieblemont C, Wellmann A, Jaffe ES, Raffeld M. PTEN
gene alterations in lymphoid neoplasms. Blood 1998; 92: 3410–
3415.
136 Graff JR, Konicek BW, McNulty AM, Wang Z, Houck K, Allen S
et al. Increased Akt activity contributes to prostate cancer
progression by dramatically accelerating prostate tumor growth
and diminishing p27Kip-1 expression. J Biol Chem 2000; 275:
24500–24505.
137 Staal SP. Molecular cloning of the akt oncogene and its human
homologues AKT1 and AKT2: amplification of AKT1 in a primary
human gastric adenocarcinoma. Proc Natl Acad Sci USA 1987;
84: 5034–5037.
138 Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF,
Hamilton TC et al. AKT2, a putative oncogene encoding a
P13K/Akt pathway as a target for cancer chemotherapy
F Chang et al
603
139
140
141
142
143
144
145
146
147
148
149
150
151
152
member of a subfamily of protein-serine/threonine kinases, is
amplified in human ovarian carcinomas. Proc Natl Acad Sci USA.
1992; 89: 9267–9271.
Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA,
Watson DK et al. Amplification of AKT2 in human pancreatic
cells and inhibiton of AKT2 expression and tumorigenicity
by anti-sense RNA. Proc Natl Acad Sci USA 1996; 93:
3636–3641.
Scheid MP, Huber M, Damen JE, Hughes M, Kang V, Neilsen P
et al. Phosphatidylinositol (3,4,5)P3 is essential but not sufficient
for PKB activation: phosphatidylinositol (3,4)P2 is required for
PKB phosphorylation at Ser473, studies using cells from SHIP-/knockout mice. J Biol Chem 2002; 277: 9027–9035.
Davies SP, Reddy H, Caivano M, Cohen P. Specificity and
mechanism of action of some commonly used protein kinase
inhibitors. Biochem J 2000; 351(Part 1): 95–105.
Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor
of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994; 269:
5241–5248.
Arcaro A, Wymann MP. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5trisphosphate in neutrophil responses. Biochem J 1993; 296(Part
2): 297–301.
Hu Y, Qiao L, Wang S, Rong SB, Meuillet EJ, Berggren M et al.
3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid
analogues and carbonate surrogates block PI3-K, Akt, and cancer
cell growth. J Med Chem 2000; 43: 3045–3051.
Urbano A, Gorgun G, Foss F. Mechanisms of apoptosis by the
tyrphostin AG957 in hematopoietic cells. Biochem Pharmacol
2002; 63: 689–692.
Dumont FJ, Su Q. Mechanism of action of the immunosuppressant rapamycin. Life Sci 1996; 58: 373–395.
Ferrari S, Pearson RB, Siegmann M, Kozma SC, Thomas G. The
immunosuppressant rapamycin induces inactivation of p70s6k
through dephosphorylation of a novel set of sites. J Biol Chem
1993; 268: 16091–16094.
Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ,
Wen SC, Zon G et al. Phosphatidylinositol-3 kinase activity is
regulated by BCR/ABL and is required for the growth of
Philadelphia chromosome-positive cells. Blood 1995; 86:
726–736.
Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA,
Watson DK et al. Amplification of AKT2 in human pancreatic
cells and inhibition of AKT2 expression and tumorigenicity by
antisense RNA. Proc Natl Acad Sci USA 1996; 93: 3636–3641.
Liu X, Shi Y, Han EK, Chen Z, Rosenberg SH, Giranda VL et al.
Downregulation of Akt1 inhibits anchorage-independent cell
growth and induces apoptosis in cancer cells. Neoplasia 2001; 3:
278–286.
Flynn P, Wongdagger M, Zavar M, Dean NM, Stokoe D.
Inhibition of PDK-1 activity causes a reduction in cell proliferation and survival. Curr Biol 2000; 10: 1439–1442.
Shaikenov TE, Adekenov SM, Williams RM, Prashad N, Baker FL,
Madden TL et al. Arglabin-DMA, a plant derived sesquiterpene,
inhibits farnesyltransferase. Oncol Rep 2001; 8: 173–179.
153 Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J
Natl Cancer Inst 2001; 93: 1062–1074.
154 Johnston SR. Farnesyl transferase inhibitors: a novel targeted
therapy for cancer. Lancet Oncol 2001; 2: 18–26.
155 Jiang K, Coppola D, Crespo NC, Nicosia SV, Hamilton AD, Sebti
SM et al. The phosphoinositide 3-OH kinase/AKT2 pathway as a
critical target for farnesyltransferase inhibitor-induced apoptosis.
Mol Cell Biol 2000; 20(1): 139–148.
156 Zhang Y, Mukhopadhyay T, Donehower LA, Georges RN,
Roth JA. Retroviral vector-mediated transduction of K-ras
antisense RNA into human lung cancer cells inhibits expression of the malignant phenotype. Hum Gene Ther 1993; 4:
451–460.
157 Mukhopadhyay T, Tainsky M, Cavender AC, Roth JA. Specific
inhibition of K-ras expression and tumorigenicity of lung cancer
cells by antisense RNA. Cancer Res 1991; 51: 1744–1748.
158 Cunningham CC, Holmlund JT, Geary RS, Kwoh TJ, Dorr A,
Johnston JF et al. A Phase I trial of H-ras antisense oligonucleotide
ISIS 2503 administered as a continuous intravenous infusion
in patients with advanced carcinoma. Cancer 2001; 92:
1265–1271.
159 Morse MA. Technology evaluation: ISIS-2503, Isis Pharmaceuticals. Curr Opin Mol Ther 2001; 3: 589–594.
160 Feig LA, Cooper GM. Inhibition of NIH 3T3 cell proliferation by a
mutant ras protein with preferential affinity for GDP. Mol Cell
Biol 1988; 8: 3235–3243.
161 Feig LA. Tools of the trade: use of dominant-inhibitory mutants of
Ras-family GTPases. Nat Cell Biol 1999; 1: E25–E27.
162 Akagi T, Murata K, Shishido T, Hanafusa H. v-Crk activates the
phosphoinositide 3-kinase/AKT pathway by utilizing focal adhesion kinase and H-Ras. Mol Cell Biol 2002; 22: 7015–7023.
163 Fiordalisi JJ, Holly SP, Johnson II RL, Parise LV, Cox AD. A
distinct class of dominant negative Ras mutants: cytosolic GTPbound Ras effector domain mutants that inhibit Ras signaling and
transformation and enhance cell adhesion. J Biol Chem 2002;
277: 10813–10823.
164 Sonoyama J, Matsumura I, Ezoe S, Satoh Y, Zhang X, Kataoka Y
et al. Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of
BCR/ABL in K562 cells. J Biol Chem 2002; 277: 8076–8082.
165 Takuwa N, Fukui Y, Takuwa Y. Cyclin D1 expression mediated
by phosphatidylinositol 3-kinase through mTOR-p70(S6K)-independent signaling in growth factor-stimulated NIH 3T3 fibroblasts. Mol Cell Biol 1999; 19: 1346–1358.
166 Mabuchi S, Ohmichi M, Kimura A, Hisamoto K, Hayakawa J,
Nishio Y et al. Inhibition of phosphorylation of BAD and Raf-1 by
Akt sensitizes human ovarian cancer cells to Paclitaxel. J Biol
Chem 2002; 277: 33490–33500.
167 Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM
et al. 50 Phospholipid phosphatase SHIP-2 causes protein kinase B
inactivation and cell cycle arrest in glioblastoma cells. Mol Cell
Biol 2000; 20: 6860–6871.
168 Murillo H, Huang H, Schmidt LJ, Smith DI, Tindall DJ. Role of
PI3K signaling in survival and progression of LNCaP prostate
cancer cells to the androgen refractory state. Endocrinology
2001; 142: 4795–4805.
Leukemia