AKT/PKB signaling mechanisms in cancer and chemoresistance

Article (PDF Available)inFrontiers in Bioscience 10:975-87 · February 2005with126 Reads
Source: PubMed
During the past decade, Akt (also known as protein kinase B, PKB) has been extensively studied. It regulates a variety of cellular processes by mediating extracellular (mitogenic growth factor, insulin and stress) and intracellular (altered tyrosine receptor kinases, Ras and Src) signals. Activation of Akt by these signals is via its pleckstrin homology (PH) domain binding to products of phosphatidylinositol 3-kinase (PI3K). This process is negatively regulated by a dual phosphatase PTEN tumor suppressor. Today, more than 30 Akt substrates have been identified. These phosphorylation events mediate the effects of Akt on cell survival, growth, differentiation, angiogenesis, migration and metabolism. Further, PI3K/PTEN/Akt pathway is frequently altered in many human malignancies and overexpression of Akt induces malignant transformation and chemoresistance. Thus, the Akt pathway is a major target for anti-cancer drug development. This review focuses on Akt signaling mechanism in oncogenesis and chemoresistance, and ongoing translational efforts to therapeutically target Akt.
[Frontiers in Bioscience 10, 975-987, January 1, 2005]
Donghwa Kim, Han C. Dan, Sungman Park, Lin Yang, Qiyuan Liu, Satoshi Kaneko, Jinying Ning, Lili He, Hua Yang,
Mei Sun, Santo V. Nicosia, Jin Q. Cheng
Departments of Pathology and Interdisciplinary Oncology, University of South Florida College of Medicine and H. Lee Moffitt
Cancer Center, Tampa, Florida 33612
1. Abstract
2. Background
3. Comparison of Akt isoforms
3.1. Sequence comparison and expression pattern
3.2. Phenotype of the knock-out mouse
4. Akt oncogenic activity and its role in chemoresistance
4.1. Mechanism of Akt activation
4.2. Transforming activity of Akt
4.3. Alterations of Akt pathway in human cancer
4.4. Akt and chemoresistance
5. Normal cellular function of Akt
5.1. Anti-apoptosis
5.1.1. Apoptotic proteins
5.1.2. Other molecules related cell survival
5.2. Cell cycle progression
5.2.1. Cell cycle regulators
5.2.2. mTOR and TSC2
5.2.3. Others
6. Akt pathway as potential therapeutic target for cancer intervention
6.1. Biological approaches
6.2. Pharmacological approaches for inhibition of upstream Akt
6.3. Akt inhibitors
7. Perspective
8. Acknowledgments
9. References
During the past decade, Akt (also known as
protein kinase B, PKB) has been extensively studied. It
regulates a variety of cellular processes by mediating
extracellular (mitogenic growth factor, insulin and stress)
and intracellular (altered tyrosine receptor kinases, Ras and
Src) signals. Activation of Akt by these signals is via its
pleckstrin homology (PH) domain binding to products of
phosphatidylinositol 3-kinase (PI3K). This process is
negatively regulated by a dual phosphatase PTEN tumor
suppressor. Today, more than 30 Akt substrates have been
identified. These phosphorylation events mediate the
effects of Akt on cell survival, growth, differentiation,
angiogenesis, migration and metabolism. Further,
PI3K/PTEN/Akt pathway is frequently altered in many
human malignancies and overexpression of Akt induces
malignant transformation and chemoresistance. Thus, the
Akt pathway is a major target for anti-cancer drug
development. This review focuses on Akt signaling
mechanism in oncogenesis and chemoresistance, and
ongoing translational efforts to therapeutically target Akt.
Akt was originally identified as the oncogene
transduced by AKT8 acute transforming retrovirus. The
AKT8 retrovirus was isolated from an AKR mouse
thymoma in 1977. This virus induces malignant
transformation in the mink lung epithelial cell line CCL-64
and tumor formation, specifically thymic lymphoma, in
nude mice (1). A decade later, its defective retrovirus was
Akt in cancer and chemoresistance
Figure 1. Identification and comparison of Akt. (A) v-Akt
was cloned from mink lung cells transformed with AKT8
retrovirus. The AKT8 virus was isolated from a thymoma
in AKR mouse. (B) Domain structure of Akt. The
chromosomal localization of Akt isoforms is listed on left
and the percentage of homology is shown on the bottom.
identified from mink lung epithelial cells infected with
AKT8 virus, and was shown to contain a cell-derived
oncogenic sequence, which was termed Akt (2, Figure 1).
In early 1990, sequence analysis of the Akt viral
oncogene and its cellular homolog revealed that it encodes
a serine-threonine protein kinase, composed of a carboxy-
terminal kinase domain very similar to that of PKC and
PKA and an amino terminal PH domain (3). Akt was also
cloned based on its homology with PKC or PKA by two
additional groups, who named it RAC or PKB (4, 5). To
date, the protein is most commonly referred to as Akt/PKB.
3.1. Sequence and expression pattern
Three major isoforms of Akt encoded by three
separate genes have been identified in mammalian cells.
Akt1/PKBα and Akt2/PKBβ were the first isolated
isoforms (3-7). Akt3/PKBγ was subsequently cloned
through homology screening (8, 9). While Akt1 is the true
human homologue of v-akt (98% identity at the amino acid
level), Akt2 and Akt3 are v-akt closely related kinases (6,
The three isoforms of Akt/PKB are highly homologous
to v-akt. The overall homology between these three
isoforms is >85%. They share a very similar structure,
which contains an N-terminal PH domain, a central kinase
domain, and a serine/threonine-rich C-terminal region. All
three Akt/PKB isoforms possess conserved threonine and
serine residues (T308/S473 in Akt1, T309/S474 in Akt2
and T305/S472 in Akt3) that together with the PH domain
are critical for Akt/PKB activation. The C-terminal regions
between these three isoforms are more diverse (homology
73%~84%) as compared to the kinase domain (homology
90%~95%), suggesting that C-terminal regions may
represent functional difference between Akt1, Akt2 and
Akt3 (Figure 1).
Although Akt1, Akt2, and Akt3 display high
sequence homology, there are clear differences between
them in terms of biological and physiological function: 1)
overexpression of wild type (WT)-Akt2, but not Akt1 and
Akt3, transforms NIH 3T3 cells and induces invasion and
metastasis in human breast and ovarian cancer cells (10,
11); 2) Akt2, but not Akt1 and Akt3, is frequently amplified
in certain types of human cancer even though alterations of
three isoforms of Akt have been detected at kinase and
protein levels in human malignancies (6, 12-19); 3) Akt1
expression is relatively uniform in various normal organs
whereas high levels of Akt2 and Akt3 mRNA are detected
in skeletal muscle, heart, placenta and brain (6, 9, 20, 21);
4) Akt2 but not Akt1 plays un unique role in muscle
differentiation (22, 23) and 5) Akt1-, Akt2- and Akt3-
deficient mice displayed different phenotypes (24-27).
3.2. Phenotype of the Akt knock-out mouse
A knockout study demonstrated that mice
deficient in Akt2 are impaired in the ability of insulin to
lower blood glucose because of defects in the action of the
hormone on skeletal muscle and liver. Akt2
mice are born
without apparent defects, but develop peripheral insulin
resistance and nonsuppressible hepatic glucose production,
resulting in hyperglycemia accompanied by inadequate
compensatory hyperinsulinemia (24), similar in some
important features to type 2 diabetes in human. These
phenotypic characteristics are not compensated by the
presence of Akt1 and Akt3, reflecting differences of
substrate specificity in insulin-responsive tissues. In
contrast, Akt1-deficient mice do not display a diabetic
phenotype. The mice are viable but display impairment in
organismal growth with smaller organs than wild type
littermates (25, 26). Such relatively subtle phenotypic
changes in Akt1
mice suggest that Akt2 and Akt3 may
substitute to some extent for Akt1 (26). In contrast, a
recent report shows that Akt3 knock out mice exhibit
uniformly reduced brain size, affecting all major brain
regions, suggesting a central role of Akt3 in postnatal
development of the brain (27). Nevertheless, these data
indicate that there are non-redundant functions between 3
isoforms of Akt in certain tissues and/or cell types.
4.1. Mechanism of Akt activation
Akt is activated by a variety of stimuli, including
growth factors, protein phosphatase inhibitors, and cellular
Akt in cancer and chemoresistance
Figure 2. Schematic representation of Akt activation. Extracellular (growth factors) and intracellular (active Ras and Src)
stimuli activate phosphatidylinositol 3-kinase, which results in production of phosphatidylinositol-3,4,5-trisphosphate (PIP3). PH
domains of Akt and PDK1 subsequently bind to PIP3 leading to phosphorylation of Thr-308 and Ser-473 and activation of Akt.
stress in a PI3K-dependent manner (28-31). Activation of
Akt depends on the integrity of the PH domain, which
binds to PI3K products PtdIns-3,4-P2 and PtdIns-3,4,5-P3,
and on the phosphorylation of Thr
in Akt2 and
in Akt3) in the activation loop and Ser
Akt2 and Ser
in Akt3) in the C-terminal activation
domain by PDK1 and ILK or DNA-PK (Figure 2, ref. 32-
34). The activity of Akt is negatively regulated by PTEN, a
tumor suppressor gene that is mutated in a number of
human malignancies. PTEN encodes a dual-specificity
protein and lipid phosphatase that reduces intracellular
levels of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 in cells by
converting them to PtdIns-4-P1 and PtdIns-4,5-P2,
respectively, thereby inhibiting the PI3K/Akt pathway (35,
4.2. Transforming activity of Akt
Previous studies demonstrated that
overexpression of WT-Akt2, but not WT-Akt1, in NIH 3T3
cells resulted in malignant transformation (10). Ahmed et
al. also showed that Akt1 is not tumorigenic when
overexpressed in the nontumorigenic rat T cell lymphoma
cell line 5675. In contrast, v-akt-expressing 5675 cells and
active forms of Akt-expressing chicken embryo fibroblasts
were highly tumorigenic (37, 38). Since v-akt arose by
way of an in-frame fusion of the viral Gag and Akt, the
oncogenic difference between v-akt and Akt1 may be due
to myristoylation of the amino-terminus of v-akt (3, 20,
38). Several lines of evidence show that the PH domain of
Akt is required for its membrane translocation and
activation, and that attachment of a membrane-targeting
sequence (myristoylation/palmitoylation) to the amino-
terminus of Akt is sufficient to induce its maximal
activation (39). Recent data also show that overexpression
of constitutively active Akt1 and Akt3, but not kinase-dead
Akt1 (Myr-Akt1-K179M) and Akt3, in NIH 3T3 cells leads
to oncogenic transformation (14 and unpublished data).
These results suggest that the kinase activity of Akt
contributes to the control of cell transformation.
4.3. Alterations of Akt pathway in human cancer
Akt2 locates at chromosome 19q13, which it is
frequently overpresented in human cancers. Amplification
of the Akt2 has been observed in 15% of human ovarian
carcinomas and 20% of human pancreatic cancers (6, 12,
17, 19). In contrast to Akt2, Akt1 has been reported to be
amplified in only a single human gastric carcinoma (2).
Because of its location at chromosome band 14q32,
proximal to the IGH locus, Akt1 had been proposed as a
candidate gene targeted by 14q32 chromosome
rearrangements in human T-cell malignancies,
prolymphocytic leukemias, and mixed lineage childhood
leukemia. However, no such alteration of Akt1 was
detected in more than 30 hematologic specimens examined
(unpublished data). Accumulated studies have shown
frequent overexpression and/or activation of Akt in
different human malignancies (12-19, 40-42). Alterations
of Akt were predominantly observed in late stage and high-
grade tumors, suggesting that Akt plays an important role
in tumor progression rather than initiation.
4.4. Akt and chemoresistance
Recent studies indicate that overexpression of
HER-2/neu or Xiap renders tumor cells resistant to TNFα
Akt in cancer and chemoresistance
Figure 3. Mmechanism of Akt involvement in human
oncogenesis and chemoresistance.
or to chemotherapeutic agents through activation of the
PI3K/Akt pathway (43-45). Cancer cells either expressing
constitutively active Akt or containing Akt gene
amplification are also far more resistant to paclitaxel than
cancer cells expressing low levels of Akt (46). We have
recently observed that cisplatin-sensitive ovarian cancer
cells (A2780s and OV2008) transfected with constitutively
active Akt2 become resistant to cisplatin, whereas
overexpression of dominant-negative (DN) Akt2 renders
cisplatin-resistant ovarian cancer cells (A2780cp and C13)
susceptible to cisplatin-induced apoptosis (47, 48). In
addition, we previously reported inhibition of
tumorigenecity and invasiveness of pancreatic cancer cell
lines by antisense Akt2 (12) and recently demonstrated
that PI3K/Akt is a critical target for farnesyltransferase
inhibitor (FTI) and geranylgeranyltransferase I inhibitor
(GGTI) -induced apoptosis. Constitutively active Akt
overcame FTI-277 and GGTI298-induced programmed cell
death (49, 50). Taken together, these data indicate that the
Akt pathway is a critical target for cancer intervention and
that activation of this pathway is associated with
chemoresistance in human cancer.
The molecular mechanism by which Akt induces
transformation and drug resistance is still not fully
understood. It is believed that Akt anti-apoptotic activity
and induction of cell cycle progression largely contribute to
these processes (Figure 3).
5.1. Anti-apoptosis
5.1.1 . Apoptotic proteins
In numerous cell types, it has been shown that
Akt induces cell survival and suppresses apoptotic death
induced by a variety of stimuli. A major identified target of
Akt is BAD, which is a BH3 domain-containing
proapoptotic protein that binds Bcl-2 and Bcl-XL and
inhibits their anti-apoptotic potential (51). When BAD is
phosphorylated on Ser1-36 by Akt, it does not exhibit
proapoptotic activity in cells. It has also been shown that
Akt activates PAK1, which in turn phosphorylates BAD at
Ser-112 resulting in its release from Bcl-
complex (52).
Once phosphorylated, BAD is released from a complex
with Bcl-2/Bcl-x
that is localized on the mitochondrial
membrane, and forms a complex with 14-3-3 proteins (51-
BAX is a 21-kDa protein that is important in
controlling cell
death, particularly in hematopoietic cells.
Cells that overexpress
BAX show enhanced apoptosis (54),
whereas BAX-null cells display
resistance to both
spontaneous and induced apoptosis. The BAX protein is
normally found in the
cytoplasm heterodimerized to anti-
apoptotic Bcl-2 family members
such as Mcl-1 and Bcl-x
however, once the cell is exposed
to an apoptotic stimulus,
BAX translocates to the mitochondria
(55–57), where it is
thought to form oligomers. These
promote apoptosis by
forming large transmembranous pores, resulting in the loss
of mitochondrial membrane potential and the release of
c (58, 59).
We and others have shown that
ectopic expression of Akt inhibits BAX conformational
change and mitochondrial translocation induced by
chemotherapeutic reagents. A recent study suggests that
Akt might phosphorylate BAX at Ser-184 (60).
A previous study shows Akt inhibition of
apoptosis at the postmitochondrial level (61). An X-linked
inhibitor of apoptosis protein (XIAP) has been recognized
as an important antiapoptotic protein by direct interaction
and inhibition of activated caspases 9, 3 and 7 at
postmitochondrial level (62-68). It is known that a number
of chemotherapeutic reagents induce XIAP degradation
leading to programmed cell death (69-71). Elevated level
of XIAP rendered cells resistant to cisplatin whereas
knockdown XIAP sensitized cells to apoptosis induced by
cisplatin and trail (70, 72, 73). We have recently
demonstrated that XIAP is a direct substrate of Akt. Akt
phosphorylates XIAP and inhibits XIAP
ubiquitination/degradation induced by cisplatin (74).
Knockdown XIAP by RNA interference or antisense XIAP
largely abrogates Akt-induced cisplatin resistance (74).
Therefore, XIAP is a major target of Akt at
postmitochondrial level.
Human caspase-9 has been reported to be phosphorylated
by Akt, resulting in attenuation of its activity (75).
However, the phosphorylation site is not conserved in other
mammalian species, suggesting that this regulation of Akt
is not likely to be a major physiological regulatory
5.1.2. Other cell survival- related molecules
Accumulated evidence has shown that JNK is
activated by a number of chemotherapeutic drugs and plays
an essential role in anti-tumor reagents-induced
programmed cell death (76, 77). Knockout JNK renders
cells resistant to DNA damage-stimulated apoptosis (78).
It has been shown that JNK mediates chemotherapeutic
Akt in cancer and chemoresistance
drug-induced apoptosis by phosphorylation of Bim, Bmf
and Bid (79). We and others have previously demonstrated
that Akt inhibited JNK activation induced by cisplatin
through phosphorylation of apoptosis signal regulating
kinase ASK1. The phosphorylation of ASK1 by Akt
inhibited its kinase activity and failed to stimulate JNK
activation (59, 80, 81).
Forkhead transcription factor (FoxO1, FoxO3,
FoxO4, previously known as FKHR, FKHRL1 and AFX) is
important in the induction of apoptosis (82, 83). Their
target genes include FasL and Bim, which plays a pivotal
role in death receptor and mitochondrial pathways. Akt
phosphorylates FoxO at three serine/threonine sites (84-
86). Upon phosphorylation of FoxO proteins by Akt, FoxO
binds to 14-3-3 proteins which results in translocation of
FoxO to the cytosol from the nucleus and consequently
inactivation of its function as a transcription factor (84, 85).
Akt has been demonstrated to phosphorylate Yes-
associated protein (YAP) and induce its association with
14-3-3 proteins. As is the case for FoxO, this results in the
localization of YAP into the cytosol (87). YAP is a
transcriptional co-activator which binds to p73 and
promotes the transcription of its target genes. As p73 is a
member of p53 family that plays an important role in the
induction of apoptosis, Akt phosphorylation of YAP
impairs the transcriptional activity of p73 and attenuates the
induction of pro-apoptotic gene expression in response to
DNA damaging agents.
There have been some indications that Akt can
induce the expression of pro-survival genes, including IAPs
and Bcl2. This may be due to positive cross-talk between
the Akt and NFκB pathways. Activation of NFκB is
dependent on the phosphorylation and degradation of IκB,
an inhibitor of NFκB, by the IκB kinase (IKK) complex.
Akt has been shown to regulate IKK activity in both direct
and indirect manner. It has been shown that Akt interacts
with and phosphorylates IKKα on Thr-23 (88, 89). Several
studies have also provided evidence that Akt
phosphorylates Ser/Thr kinase Tpl-2 (or Cot) on Ser-400,
resulting in IKK complex activation (90, 91).
5.2. Cell cycle progression
5.2.1. Cell cycle regulators
Akt targets several key cell cycle regulators
including p21
, p27
, and MDM2. Akt
phosphorylates p21
on residues Thr-145 and Ser-146
(92-94). The phosphorylation of Thr-145 inhibits p21
nuclear localization and affinity to Cdk2, Cdk4 and PCNA
leading to activation of cyclin/CDK and DNA replication.
However, phosphorylation of Ser-146 enhances protein
stability of p21 that may result in cell survival (93, 94).
Human p27
, another major cyclin/CDK inhibitor, is also
phsophorylated by Akt on Thr-157, even though this site is
not conserved in other species (95-97). As Thr-157 resides
within a nuclear localization signal (NLS) region, Akt
phosphorylation of Thr-157 leads to p27 exclusion from the
nucleus. Our laboratory has shown that Akt decreases
TSC1/TSC2 tumor complex by phosphorylation of TSC2
resulting in the destabilization of p27 (98). In addition, Akt
phosphrylation of forkhead protein inhibits p27 at
transcriptional level (99). Taken collectively, Akt
downregulates p27 at different levels leading to activation
of cyclin/Cdk and cell cycle progression (95-99)
MDM2 is a major negative regulator of p53
tumor suppressor. Loss of p53 function has been thought
to be a major mechanism of chemoresistance (100).
MDM2 has ubiquitin E3 ligase activity, directly binds to
p53 and targets it for ubiquitination and proteasome
degradation (101, 102). Akt has been shown to
phosphorylate MDM2 on Ser-166 and Ser-186 and induce
MDM2-mediated p53 ubiquitination, even though there is
still controversial regarding subcellular localization of Akt
phosphorylated MDM2 (103-105).
5.2.2. mTOR and TSC2
The mammalian target of rapamycin (mTOR) is a
serine/threonine protein kinase and is best known as a
regulator of cell cycle progression and cell proliferation by
integrating signals from nutrients (amino acids and energy)
and growth factors (106-108). The best known
biochemical function of mTOR is to regulate protein
translation by initiation of mRNA translation and ribosome
synthesis leading to an increased rate of cell growth (an
increase in cell mass and size), which is required for
supporting the rapid proliferation (an increase in cell
number). A model for mTOR function suggests that
mTOR regulates primarily the rate of cell growth and
secondarily cell cycle progression. The mTOR-dependent
downstream effectors, S6K1, 4E-BP1, and eIF4E, also
regulate the rate of cell cycle progression. When quiescent
U2OS cells are stimulated with serum to enter G
from G
, overexpression of S6K1 and eIF4E accelerates S
phase entry, while reduced expression of S6K1 with RNAi
or overexpression of a dominant mutant of 4E-BP1 inhibits
the rate of S phase entry (109, 110). Moreover,
overexpression of rapamycin-resistant mutants of S6K1 or
overexpression of eIF4E partially rescues the rapamycin-
induced delay in G
progression to S phase, indicating that
S6K1 and eIF4E are downstream mediators of mTOR-
dependent cell division (109, 110).
It has been shown that Akt mediates insulin and
nutrient activation of mTOR pathway through both direct
and indirect mechanism. Akt phosphorylates mTOR at
serine-2448 and possible serine-2446, which fit the Akt
phosphorylation consensus motif (RXRXXS/T) in 3T3-L1
adipocytes, HEK293 cells and intact skeletal muscle (111-
115). Although one might expect that Akt
phosophorylation of serine-2448 would activate mTOR,
there is no direct evidence to support such an effect. In
fact, when expressed in HEK293 cells,
nonphosphorylatable mTOR-S2448A (converting serine to
alanine) or mTOR-S2446/2448A exhibit the same effect as
overexpressed wild type mTOR in mediating insulin-
stimulated phosphorylation of S6K1 and 4E-BP1. These
data suggest that mTOR is not a direct target of Akt. We
and others have demonstrated that Akt interacts with and
phosphorylates tuberin, a product of tumor suppressor
tuberous sclerosis complex (TSC) 2 gene. A possible
mechanism for Akt activation of mTOR has been proposed
by finding of tumor suppressor TSC2 linking Akt to
Akt in cancer and chemoresistance
Tuberous sclerosis is an autosomal dominant
disorder developing hamartomas in multiple organs and is
caused by mutation of either the TSC1 or the TSC2 tumor
suppressor gene. TSC1 and TSC2 function as a complex
to inhibit cell growth. Overexpression of TSC2 and TSC1
inhibited mTOR activity and blocked the increase in
phosphorylation of S6K1 and 4E-BP1 in response to
nutrients or growth factor stimulation (116-118). We and
others have shown that Akt phosphorylates TSC2 at
multiple serine/threonine sites and causes to degradation of
TSC2 and TSC1 and disruption of TSC1/TSC2 complex
(98, 11-123), resulting in release of its inhibition of mTOR.
Further, expression of nonphosphorylatable TSC2 mutants
with alanine substitutions at Akt phosphorylation sites
blocks growth factor-induced S6K1 activation. TSC2 has
been shown to interact with overexpressed TOR in
Drosophila but this interaction does not occur in
mammalian cells (124).
Several studies have shown TSC2/TSC1
inhibition of mTOR through TSC2 GAP activity to
hydrolyze Rheb-GTP to inactive Rheb-GDP form. Rheb is
a GTP-binding protein and overexpresing Rheb increases
S6K1 and 4E-BP1 phosphorylation but does not induce the
activity of a rapamycin-resistant form of S6K1, suggesting that
Rheb signaling to S6K1 is through mTOR and not through a
parallel pathway. However, no evidence shows that Rheb
directly activates mTOR. Disruption of Rheb in S. cerevisiae
leads to an increase in the uptake of arginine and lysine by the
amino acid permease Can 1p (125-128). This implies that
Rheb may control mTOR indirectly by changing amino acid
level. Recent studies have demonstrated that mTOR functions
as part of a larger signaling complex. Two mTOR-associated
proteins, Raptor and GβL, have been identified by sequencing
proteins that coimmunoprecipitated with mTOR. The GβL
binds to kinase domain of mTOR whereas Raptor links mTOR
to S6K1 and 4E-BP1 by binding to their TOR signaling (TOS)
motifs, leading to mTOR-dependent phosphorylation of S6K
and 4E-BP1 in response
to nutrients or growth factors (129-
134). However, there is no evidence that these two proteins
are involved in Akt regulation of mTOR.
In addition, a number of transcriptional factors
that associate with cell cycle control are regulated by Akt.
Cyclic AMP (cAMP)-response element binding protein
(CREB), is phosphorylated by Akt on Ser-133. This
process results in increased affinity of CREB to its co-
activator CRB, leading to transcriptional upregulation of
cell cycle associated genes such as cyclin D1 (135).
Estrogen receptor (ER)α is also phosphorylated by Akt
(136). The phosphorylated ERα will induce its target gene
expression which is thought to involve anti-estrogen
resistance (136, 137).
Finally, angiogenesis induced by Akt may also
associate with its transforming activity and
chemoresistance. Accumulated evidence shows that Akt
plays a central role in the sprouting of new blood vessels by
mediating many angiogenic growth factors and regulating
downstream target molecules that are potentially involved
in blood vessel growth. It is known that VEGF has various
functions on endothelial cells, the most prominent of which
is the induction of proliferation and differentiation by
selectively binding to the Flk-1/KDR receptor and
subsequent activation of Akt pathway (138).
Constitutively active Akt also induces VEGF mRNA
expression by stabilization (139) and enhanced translation
(140) of HIF1α through regulation of mTOR pathway.
Moreover, Akt phosphorylates eNOS on Ser-1177,
resulting in enzymatic activation of eNOS (141), which
leads to production of NO and angiogenesis.
Although cytotoxic chemotherapeutic drugs are
first-line agents for cancer, chemoresistance remains a major
therapeutic hurdle. The prospect of gene targeted anti-
tumor agents as a therapeutic approach for cancer,
particularly for the chemoresistant disease, has generated
considerable excitement.
As described above, Akt pathway is essential for
cell survival, cell cycle progression and angiogenesis.
Amplification/overexpression/activation of PIK3CA
(p110α) enzymatic subunit of PI3K and Akt as well as
somatic mutation of gene encoding p110α are frequently
detected in human malignancy (142-146). Inhibition of
PI3K and/or Akt induces programmed cell death in cancer
cells (144). Expression of constitutively active Akt results
in cancer cells
resistance to cisplatin and taxol-induced
apoptosis, whereas dominant negative Akt sensitizes the
cells to chemotherapeutic drugs (147, 148). Thus,
PI3K/Akt pathway is a critical target for cancer
intervention and inhibition of PI3K and/or Akt could
overcome a subset of chemoresistant cancers.
6.1. Biological approaches
Biological approaches include antisense,
dominant-negative, antibody of PI3K and Akt as well as
peptides to mimic and compete pleckstrin-homology (PH)
domain of Akt binding to PI3K products, PtdIns-3,4-P2 and
PtdIns-3,4,5-P3. We and other have previously
demonstrated that the introduction of antisense Akt2 or
DN-Akt into several Akt-overexpressing cancer cell lines
abrogates endogenous Akt expression and diminishes their
invasiveness and tumor formation in nude mice (12, 149).
Antisense oligonucleotides of Akt can inhibit Akt pathway
and induce apoptosis in different cell lines
and cell
growth and survival can also be inhibited by the expression
of dominant negative (DN) forms of PI3K and Akt (150,
151). Our recent data show that expression of DN-Akt in
NIH3T3 cells remarkably reduces v-H-ras-induced colony
formation and tumor formation (unpublished data).
Moreover, consistent with the tumor-inhibitory effects of
DN-PI3K and DN-Akt is the demonstration of the
inhibition of Ras and BCR/ABL malignant transformation
with p85
iSH2 and DN-Akt, respectively (152, 153).
Microinjection of AKT2 antibody into myoblasts can also
specifically block their function, i.e., induction of myotube
(154). Further studies are required to investigate the effects
of antibodies of PI3K and Akt on human cancer cell
Akt in cancer and chemoresistance
6.2. Pharmacological approaches for upstream
inhibition of Akt
Although wortmanin and LY294002 efficiently
abrogate PI3K activity and have been widely used in the
cell culture system (155, 156), they have not been applied
for clinical trails due to either toxicity (LY294002) or a
short of half-live (wortmanin). We have demonstrated that
farnesyltransferase inhibitor (FTI)-277, originally designed
to block Ras oncoprotein, inhibits PI3K/Akt pathway and
induces apoptosis in a number of human cancer cell lines
(49, 157). FTIs are highly effective at inhibiting tumor
growth without toxicity to normal cells. However, the
mechanism by which they inhibit tumor growth is not well
understood (158-160). FTIs are unable to induce apoptosis
in Raf transformed NIH 3T3 cells even though MAPK
pathway is inhibited by FTIs (158, 159), indicating that
FTIs may target other cell survival pathway(s) regulated by
Ras or other farnesylated proteins. Interestingly, our data
showed that FTI-277 induces apoptosis only in Akt2-
overexpressing human cancer cell lines. Furthermore,
overexpression of Akt2, but not oncogenic H-Ras,
sensitizes NIH 3T3 cells to FTI-277; and a high serum level
prevents FTI-277-induced apoptosis in H-Ras- but not
Akt2-transformed NIH 3T3 cells (49, 157). These data
suggest that FTIs specifically target the PI3K/Akt pathway
to inhibit tumor cell growth and may be candidate agents
for reversing resistance of human cancer to cytotoxic
chemotherapeutic drugs.
6.3. Akt inhibitors
The importance of Akt in cell survival, growth,
cell transformation and human malignancy has prompted
the search for specific and safe pharmacological inhibitors
for Akt. Five recent reports including ours have identified
the compounds as potential Akt inhibitors that reduce Akt
kinase activity in a number of cancer cell lines (161-165).
Hu et al. synthesized a phosphatidylinositol analogue (1L-
6-hydroxy-methyl-chiro-inositol 2(R)-2-O-methyl-3-O-
octadecylcarbonate) and showed that it inhibited Akt by
compete with phosphatidylinositol (161). This compound
reduces the resistance of human leukemia cells to
chemotherapeutic drugs and ionizing radiation (161).
Chaudhary et al. demonstrated that the plant-derived
pigment curcumin reduces Akt activity resulting in cell
growth arrest in several prostate cancer cell lines (162). A
compound synthesized from the natural plant compound
rotenone (degeulin) has also been identified as a potential
Akt and PI3K inhibitor in malignant human bronchial
epithelial cells (163). Meuillet et al. have used a novel
strategy to identify a group of D-3-deoxy-phosphatidyl-
myo-inositols that bind to the PH domain of Akt, trapping
it in the cytosol and preventing its activation in response to
growth factors (164). We have recently identified a small
molecule inhibitor of Akt, API (Akt/P
KB signaling
inhibitor)-2/TCN, by screening the National Cancer
Institute Diversity Set (165). API-2 inhibited the kinase
activity of Akt resulting in suppression of cell growth and
induction of apoptosis in human cancer cells harboring
constitutively activated Akt. API-2 is highly
selective for
Akt and does not inhibit the activation of PI3K, PDK1,
PKC, SGK , PKA, STAT3, Erk-1/2, or JNK. Furthermore,
API-2 potently inhibited tumor growth in nude mice of
human cancer cells where Akt is aberrantly
expressed/activated but not of those cancer cells where it is
not. These findings suggest that API-2 exerts anti-tumor
activity largely by inhibition of Akt (165).
In the past decade, the mechanism for Akt
activation, lipid second messenger-mediated
phosphorylation of Akt, has been well characterized.
However, the physiological and pathological difference of
three isoforms of Akt remains elusive. While Akt is a key
molecule in cell survival, downstream targets that mediate
this action are still obscure. As Akt plays a pivotal role in
human cancer development and chemoresistance, it is
essential that future work is aimed at developing
pharmacological reagents as well as genetic and
biochemical approaches that not only identify novel roles
for Akt but also verify the physiological functions
previously ascribed. The generation of a potent and
specific Akt inhibitors, especially isoform-specific Akt
inhibitors, would certainly revolutionize the study of the
processes mediated by Akt in the same way inhibitors of
MAP kinase kinase 1 activation (e.g., PD98059,
PD184352, U0126) have on our understanding of processes
regulated by the classical MAP kinase pathway. More
importantly, such drugs or in combination with
conventional chemotherapeutic agents would reasonably
improve the outcome of human cancer.
This work was supported by grants from National
Cancer Institute Grants and Department of Defense (J.Q.C.)
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Key Words: Akt/PKB, Apoptosis, Cell Growth, Inhibitor,
Chemoresistance, Review
Send correspondence to: Dr Jin Q. Cheng, Department of
Pathology, University of South Florida College of
Medicine and H. Lee Moffitt Cancer Center, 12901 Bruce
B. Downs Blvd., MDC Box 11, Tampa, Florida 33612,
Tel.: 813-974-8595; Fax: 813-974-5536; E-mail:
    • "The PI3K-Akt-mTOR pathway plays a pivotal role in apoptosis/survival signaling and is involved in chemo- resistance [28]. Phosphorylated mTOR and its downstream target kinase p70S6K were inhibited in both cell lines after Rapa treatment. "
    [Show abstract] [Hide abstract] ABSTRACT: While combined chemotherapy (CT) with an autophagy inducer and an autophagy inhibitor appears paradoxical, it may provide a more effective perturbation of autophagy pathways. We used two dissimilar cell lines to test the hypothesis that autophagy is the common denominator of cell fate after CT. HA22T cells are characterized by CT-induced apoptosis and use autophagy to prevent cell death, while Huh7.5.1 cells exhibit sustained autophagic morphology after CT. Combined CT and rapamycin treatment resulted in a better combination index (CI) in Huh7.5.1 cells than combined CT and chloroquine, while the reverse was true in HA22T cells. The combination of 3 drugs (triplet drug treatment) had the best CI. After triplet drug treatment, HA22T cells switched from protective autophagy to mitochondrial membrane permeabilization and endoplasmic reticulum stress response-induced apoptosis, while Huh7.5.1 cells intensified autophagic lethality. Most importantly, both cell lines showed activation of Akt after CT, while the triplet combination blocked Akt activation through inhibition of phospholipid lipase D activity. This novel finding warrants further investigation as a broad chemosensitization strategy.
    Article · Jul 2016
    • "After C35 was identified and characterised as a novel protein binding partner of DNp73 that has a significant role in cancer cell progression and chemo-resistance, we studied the molecular mechanisms underlying the enhancement of ovarian cancer progression by the C35–DNp73 interaction. It is well known that the AKT signalling pathway is associated with chemo-resistance in human cancers (Brognard et al, 2001; Li et al, 2001; Fraser et al, 2003; Dan et al, 2004; Pommier et al, 2004; Kim et al, 2005; Abedini et al, 2010). TAp73 and DNp73 have also been suggested to have important roles in the sensitivity of cancer cells to druginduced apoptosis (Irwin et al, 2003; Vayssade et al, 2005; Al-Bahlani et al, 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The purpose of this study was to characterise the oncogenic roles of C35, a novel protein binding partner of ΔNp73, in ovarian cancer and to investigate the functional significance of C35–ΔNp73 interaction in the regulation of chemo-resistance. Methods: C35 expression was evaluated by quantitative real-time PCR in human ovarian cancer tissues and cell lines. The aggressiveness of ovarian cancer cells overexpressing C35 was examined by cell proliferation, migration, soft agar and nude mouse xenograft. The significance of C35–ΔNp73 interaction in chemo-resistance was evaluated by apoptosis assays and cell viability after cisplatin treatment. Results: The expression of C35 was significantly enhanced in human ovarian cancer tissues. Overexpression of C35 augmented proliferation, migration and tumourigenicity in ovarian cancer cell lines. C35 knockdown inhibited cell motility and cell growth. The co-expression of C35 and ΔNp73 by transient or stable transfection in ovarian cancer cells induced greater resistance to cisplatin treatment than did transfection with C35 or ΔNp73 alone. The cisplatin resistance was demonstrated to be caused by increased AKT and NFκB activity induced by C35–ΔNp73. Conclusion: Our results suggest that ΔNp73 might cooperate with C35 to promote tumour progression and contribute to cisplatin resistance in ovarian cancer cells. Future studies of the functional roles of ΔNp73 and C35 will provide insight that will aid in the establishment of new strategies and more effective therapies.
    Full-text · Article · Jul 2013
    • "Previous studies (4,16) have shown that CDDP resistance is associated with AKT overexpression. The activation of AKT promotes the development of resistance to chemotherapy treatment (17–19). In the present study, JAK2, activated by CDDP-induced ROS, was associated with STAT3 phosphorylation and the transactivation of a STAT-targeted AKT gene promoter. "
    [Show abstract] [Hide abstract] ABSTRACT: The use of chemotherapy drugs for the treatment of cancer is an effective therapeutic measure. However, chemoresistance affects the effectiveness of the treatment. AKT overexpression has been observed in chemoresistance. AKT expression in colon cells induced cisplatin resistance. The present study demonstrated the role of reactive oxygen species (ROS) in the induction of AKT regulation by cisplatin through the activation of JAK2/STAT3 at the transcriptional level in colon cancer cells. HCT-116 cells treated with cisplatin exhibited increased JAK2 and STAT3 activities. Reducing the expression of JAK2 in colon cancer cells using small interfering RNA (siRNA) decreased AKT expression. The present study demonstrated that AKT activation is closely associated with chemoresistance in human tumors. The inhibition of ROS decreased the levels of AKT in colon cancer cell lines. The JAK2/STAT3 pathway was also shown to mediate AKT expression and represents a potential target for overcoming cisplatin resistance in human tumors.
    Full-text · Article · Mar 2013
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