Biochimica et Biophysica Acta 1813 (2011) 19781986

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r

Review

Akt, FoxO and regulation of apoptosis

Xinbo Zhang b,d, Naimei Tang b,d, Timothy J. Hadden a,d, Arun K. Rishi a,b,c,
a
John D. Dingell V.A. Medical Center, Wayne State University, Detroit, MI 48201, USA
b
Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA
c
Department of Oncology, Wayne State University, Detroit, MI 48201, USA
d
Department of Internal Medicine, Wayne State University, Detroit, MI 48201, USA

a r t i c l e i n f o a b s t r a c t

Article history: Forkhead box O (FoxO) transcription factors are downstream targets of the serine/threonine protein kinase B
Received 7 January 2011 (PKB)/Akt. The Akt kinase regulates processes of cellular proliferation and survival. Phosphorylation of FoxOs
Received in revised form 9 March 2011 by Akt inhibits transcriptional functions of FoxOs and contributes to cell survival, growth and proliferation.
Accepted 11 March 2011
Emerging evidence suggests involvement of FoxOs in diverse intracellular signaling pathways with critical
Available online 31 March 2011
roles in a number of physiological as well as pathological conditions including cancer. The FoxO signaling is
Keywords:
regulated by their interactions with other intracellular proteins as well as their post-translational
PKB/Akt modications such as phosphorylation. FoxOs promote cell growth inhibitory and/or apoptosis signaling by
FoxO either inducing expression of multiple pro-apoptotic members of the Bcl2-family of mitochondria-targeting
Tumor suppression proteins, stimulating expression of death receptor ligands such as Fas ligand and tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL), or enhancing levels of various cyclin-dependent kinase inhibitors (CDKIs).
Coupled with their ability to cross-talk with p53, FoxOs represent an important class of tumor suppressors in a
variety of cancers. This review summarizes our current understanding of mechanisms by which Akt and FoxOs
regulate cell growth and survival that in turn offers opportunities for development of novel strategies to
combat cancer. This article is part of a Special Issue entitled: P13K-AKT-FOxO axis in cancer and aging.
2011 Elsevier B.V. All rights reserved.

1. Introduction domain in the nucleus, and their nucleocytoplasmic shuttling is regulated

Forkhead box transcription factors were named after the Drosophila
forkhead gene, and this superfamily consists of 19 subclasses of Fox genes,
FoxAFoxS [1,2]. The members of this superfamily share a highly
conserved DNA-binding FOX domain. Although some of the FOX genes
are ubiquitously expressed, many members are expressed in a spatial and
temporal manner. The FOX transcription factors that belong to the other
(O) subfamily (FoxO) include only one member each in Caenorhabditis
elegans (dauer formation-16) and Drosophila (dFoxO), and four members
(FoxO1, 3, 4, 6) in mammals [1]. The FoxO transcription factors regulate
cellular differentiation, growth, survival, cell cycle, metabolism, stress and
tumor suppression pathways. FoxO1 (aka FKHR), FoxO3 (aka FKHRL1),
and FoxO4 (aka AFX) are all expressed ubiquitously in mammals. The
FoxO1 and FoxO4 transcripts however are highly expressed in adipose
tissue and skeletal muscle, respectively, while FoxO3 is abundant in
various tissues including brain, heart, kidney, and spleen. FoxO6 mRNA on
the other hand is expressed predominantly in the developing and adult
brain, suggesting a role in nervous system [3]. FoxO proteins activate or
repress transcription of target genes through their DNA-binding FOX
This article is part of a Special Issue entitled: P13K-AKT-FOxO axis in cancer and aging.
Corresponding author at: Room B4334, VA Medical Center, 4646 John R., Detroit, MI
48201, USA. Tel.: + 1 313 576 4492.
E-mail address: Rishia@Karmanos.org (A.K. Rishi).
by their specic nuclear export and import signal sequences. Subcellular
localization and transcriptional functions of FoxOs are often regulated by
their post-translational modications, such as phosphorylation, acetyla-
tion, and ubiquitination [3].
Protein kinase B (PKB)/Akt pathway is an essential pathway for
cell survival and growth during development and carcinogenesis. Akt
is a serine-threonine kinase that is regulated mainly following
activation of the second messenger phospholipid kinase phosphati-
dylinositol 3-kinase (PI3K). In mammals three closely related iso-
forms of Akt are encoded by distinct loci, and functional redundancy
among Akt isoforms exists as suggested by lack of developmental
defects in animals having targeted deletion of a specic isoform [4].
However, in spite of the functional redundancy, only Akt 2 and 3
isoforms are often pathologically amplied in human cancers. The
PI3K/Akt signaling regulates cell proliferation and survival in part by
phosphorylating FoxOs to promote their interaction with 14-3-3
protein that results in nuclear exclusion and eventual ubiquitin
proteasome pathway (UPP)-dependent degradation of FoxOs [5].
2. AKT-FoxO signaling axis
The Akt kinase remains a subject of diverse investigations due
mainly to its critical roles in a variety of pathways and cellular processes.
Although the Akt signaling pathways are activated by receptor tyrosine

0167-4889/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2011.03.010
 X. Zhang et al. / Biochimica et Biophysica Acta 1813 (2011) 19781986
kinases, cytokine receptors, G-protein coupled receptors, B and T cell
receptors, and integrins, two major cellular processes, insulin metab-
olism and cancer progression, are critically regulated by this kinase. Akt
plays an important role in metabolism by targeting glycogen synthase
kinase 3 (GSK3) to regulate glycogen synthesis. Akt regulates cell
cycle and proliferation by directly targeting CDKIs p21WAF1/CIP1 and
p27KIP1, and indirectly by modulating levels of cyclin D1 and p53. Akt
regulation of cell survival however involves direct inhibition (phos-
phorylation) of pro-apoptotic signals such as Bad and FoxOs. In the
discussion below we summarize information on the roles of Akt in
insulin metabolism and cell growth and survival pathways associated
with carcinogenesis.
Signaling through the insulin and insulin-like growth factor (IGF)
pathways is essential for nutrient homeostasis as well as for growth and
development. In mammals the IGF signaling system includes three
peptide hormones, insulin, IGF1 and IGF2, that are ligands for a series of
receptor isoforms that includes two insulin receptors (IRa and IRb), the
IGF1 receptor (IGF1r) and two receptor hybrids consisting of an IGF1r
monomer and either an IRa or IRb monomer. There is also an IGF2 receptor
(the mannose-6-phosphate receptor) that acts as a signaling antagonist.
IRb predominates in insulin sensitive target tissues such as muscle, fat and
liver and binds insulin with high afnity. IRa binds both insulin and IGF-2
with moderate afnity and is the predominant insulin receptor isoform in
fetal tissues [6]. Upon ligand binding, the insulin or IGF receptors undergo
tyrosine autophosphorylation, enhancing tyrosine kinase activity of the
receptor and propagate signals through common pathways that include
the insulin receptor substrate (IRS)/phosphatidylinositol 3-kinase (PI3K)
pathway [reviewed in 7]. The IRSs are recruited to the phosphorylated
receptor by their phosphotyrosine binding (PTB) and pleckstrin homology
(PH) domains. Receptor-bound IRS proteins that are phosphorylated by
action of the intrinsic receptor tyrosine kinase bind several proteins via
their SH2 domains. Among these is the p85 regulatory subunit of PI3K.
Recent work by Lim et al. [8] uncovered an important role for the
Connector Enhancer of KSR 1 (CNK1) scaffold protein in recruitment of
IRS1 to the membrane and downstream activation of the PI3K/Akt
pathway. It was shown that CNK 1 accumulates at the cell surface in
insulin-treated cells and participates in localization of cytohesins at the
cell surface. The cytohesins facilitate changes in the lipid environment of
the cell membrane, resulting in generation of a phosphoinositide(4,5)
bisphosphate (PIP2)-rich environment, essential for recruitment of IRS1
and signaling through PI3K [9]. Interaction between the IRS and both SH2
domains of p85 activates the catalytic (p110) subunit of PI3K. Activated
PI3K phosphorylates its lipid substrate PIP2 to produce phosphoinositide
(3,4,5)trisphosphate (PIP3), an important intracellular messenger. PIP3
recruits phosphatidylinositol-dependent kinase 1 (PDK1) and Akt, both
Ser/Thr kinases, to the plasma membrane, wherein Akt is activated by
PDK1-mediated phosphorylation. Signaling through the IRS/PI3K path-
way can be modulated by phospholipid phosphatases, such as SHIP2 and
PTEN (phosphatase and tensin homolog deleted from chromosome ten),
which mediate dephosphorylation of PIP3. It is noteworthy that recent
work has shown that CNK1 interacts with Akt, promoting cell
proliferation through activation of the Akt/FoxO signaling cascade [10].
FoxOs also mediate cell cycle arrest, DNA repair and apoptosis [11].
Phosphorylation by Akt inactivates FoxOs resulting in their accumulation
in the cytoplasm. Thus, signaling through the IRS/PI3K/Akt cascade, by
inactivating FoxOs, results in decreased expression of negative cell-cycle
regulators, such as retinoblastoma-related protein p130 and CDKI
p27Kip1. Akt phosphorylation of FoxOs promotes cell survival since the
FoxOs regulate the pro-apoptotic protein TRAIL. Cell survival is also
promoted by Akt phosphorylation of BAD, a component of the BAD/Bcl-
XL complex, resulting in dissociation of the complex [12]. Moreover, Akt
also has the capacity to phosphorylate and activate IKK a component of
the upstream kinase that regulates NF-B activation [13]. Activated IKK
phosphorylates IB, targeting it for proteasomal degradation. As a result,
NF-B is activated and translocated into the nucleus, where it activates
transcription of pro-survival genes. Activation of an alternative branch of
1979
the NF-B pathway and consequent expression of matrix metalloprotei-
nase 9 in a CNK1-dependent fashion is thought to promote invasiveness
of breast and cervical cancer cells [14].
Signaling through the insulin-like pathway thus results in
activation of Akt, which, in turn, activates expression of pro-survival
and anti-apoptotic genes. Situations may arise that will promote an
increase in the strength or duration of Akt-activating signals. For
example, mutations in PTEN may decrease dephosphorylation of PIP3;
mutant forms of PI3K catalytic subunit may also result in increased
abundance of PIP3. Mutations such as these are commonly encoun-
tered in a variety of cancers and not only highlight the importance of
the PI3K/Akt signaling cascade but relevance of the Akt activation as a
potential therapeutic target in cancer [15,16].
3. FOXO and suppression signaling
3.1. FoxO transcription factorsbona de tumor suppressors
Increasing evidence shows that members of the FoxO subfamily are
pivotal for the maintenance of tissue homeostasis in organs and are
frequently dysregulated in cancer. The role of FoxOs in tumorigenesis
was initially discovered by their involvement in chromosomal trans-
locations in human cancers [1719]. For example, FoxO1, FoxO3A, and
FoxO4 genes were found at chromosomal breakpoints in some forms of
leukemia such as myeloid/lymphoid or mixed lineage leukemia (MLL)
and alveolar rhabdomyosarcoma. The FoxO1 gene was identied in the
studies of t(2, 13)(q35;q14) and t(1, 13)(p36;q14) chromosomal
translocations found in rhabdomyosarcomas. The FoxO3 gene was
found at t(6;11)(q21;q23) chromosomal translocation from an acute
myeloid leukemia patient, while FoxO4 was present at t(x;11)(q13;q23)
translocation in acute lymphoblastic leukemia [20]. Further observation
indicated that the MLL-FoxO4 fusion resulted in transdominant
repression of wild-type FoxO expressed from the remaining intact allele
[21]. These initial ndings suggest that loss-of-function of FoxOs due to
gene translocations may play important roles in tumor development,
even though a single allele of FoxO genes may be sufcient to prevent
tumorigenesis [22].
Overexpression of FoxO inhibits tumor growth in vitro and tumor
size in vivo in breast cancer, and cytoplasmic localization of FoxO
correlated with poorer survival in breast cancer patients [23,24]. In
contrast, nuclear localization of FoxOs suspends cell cycle progression
[25], promotes apoptosis [26], and negatively regulates angiogenesis
[27]. The signicance of FoxO antitumoral activity is also underscored
by studies conducted in leukemia [28], prostate cancer [2931], and
glioblastoma [32]. Furthermore, triple knockout mouse models
proved the tumor suppressor properties of FoxOs, as mice simulta-
neously lacking the principal members of the mammalian FoxO
subfamily, FoxO1, FoxO3a, and FoxO4, are prone to develop
hemangiomas and lymphoproliferative diseases [21]. Although
implicated as tumor suppressors, genetic inactivation of FOXOs is
rarely encountered in human cancers. Recent studies found that
FoxO3 was deleted in carcinogen-induced human lung adenocarci-
noma (LAC) of mice and in human non-small cell lung cancer (NSCLC)
cell lines, suggesting that FoxO3 loss contributes to NSCLC pathogen-
esis [33,34]. FoxO3 gene was also identied as a novel target of
deletion in human lung adenocarcinoma. Biallelic or homozygous
deletion of FoxO3 was detected in 8 of 33 (24.2%) mostly early-stage
LAC of smokers [35]. FoxOs are inactivated in the majority of human
cancers, owing to the overactivation of the PI3K/Akt pathway and the
latter is due to mutations in RAS, PTEN or PI3K [36]. These
observations strongly dene FoxOs as bona de tumor suppressors
that may be useful as therapeutic targets, although one early
investigation of FoxO3 in clinical breast cancer suggested that
activation of FoxO3a was intriguingly associated with lymph nodal
metastasis and a poor prognosis [37].
1980 X. Zhang et al. / Biochimica et Biophysica Acta 1813 (2011) 19781986
3.2. FoxO-mediated suppression signaling in cell cycle
3.2.1. FoxO-mediated regulation of CIP/KIP family
Timely cell cycle regulation is accomplished by sequential activation
of a family of serine-threonine kinases called cyclin dependent kinases
(CDKs). Tight CDK regulation involves CDKIs which ensure the correct
timing of CDK activation in different phases of the cell cycle [38].
Disruption of cell cycle control is a hallmark of cancer [39]. In particular,
the reduced expression of the CDKI p27KIP1 has been extensively
observed in human cancers, and its low levels are often associated with a
worse prognosis [40,41]. In quiescent cells, FoxOs are located within the
nucleus in which they bind to the p27KIP1 promoter and initiate
transcription [25]. In response to growth factors or oncogenic signals,
FoxOs are phosphorylated by Akt, resulting in their nuclear export and
cytoplasmic sequestration that interferes with their nuclear transcrip-
tional functions including p27KIP1 transcription and thus promotes cell
proliferation [42]. As Akt is active in many tumors, this may represent an
additional mechanism to reduce p27KIP1 during tumor growth. A wealth
of studies to date have revealed that FoxOs target a range of genes
involved in multiple cellular processes including metabolism, cell cycle,
apoptosis, stress resistance, DNA repair, and immune system (Fig. 1).
One of the main effects of FoxOs is to promote cell cycle arrest at the G1/S
boundary by up-regulating multiple cell cycle suppressor genes such as
CDKI p27KIP1 [42]. In fact activation of FoxO3 is sufcient to elevate
p27KIP1 mRNA and protein levels, as well as to induce apoptosis [25].
FoxOs also induce cells to exit the cell cycle and enter a state of
quiescence in part by upregulating levels of the retinoblastoma family
member p130 [43]. Consistent with a role in driving or promoting cell
cycle arrest, FoxOs were found to be incapable of inducing G1 arrest in
p27KIP1/p130-decient broblasts, suggesting that p27KIP1 and p130 are
both critical mediators of FoxO-dependent G1 arrest. Thus, p27KIP1 plays
a crucial role in the control of cell proliferation by inhibiting the activities
of complexes of G1 cyclins and Cdks and, as such, is an important
candidate for therapeutic tumor suppression.
Another FoxO target gene is CDKI p21WAF1/CIP1. Seoane et al. [32]
demonstrated that the p21WAF1/CIP1 promoter contains both a Smad
binding region as well as a consensus forkhead binding element {FBHE;
T (G/A) TT (T/G) (G/A) (T/C)} immediately upstream of the rst Smad
Fig. 1. FoxO shuttling between nucleus and cytoplasm is phosphorylation-dependent. Boxes indicate select transcriptional targets of FoxOs within different pathways.
binding element (SBE) and that these features are conserved in the
human and mouse genes. Further experiments showed that in response
to TGF, FoxO1, FoxO3a, and FoxO4 bind specically and directly to
Smad3 and Smad4, and this complex targets the p21WAF1/CIP1 promoter
containing the FBHE and SBE to induce cell cycle arrest at the G1/S
transition [32,44]. Infact, FoxOs act as Smad partners to induce CDKIs
p21WAF1/CIP1 and p15 as part of the cytostatic response of epithelial cells
[45]. More recent studies also demonstrate that CDKI p21WAF1/CIP1 is an
important mediator of FoxO-induced G1 arrest and subsequent
quiescence. FoxO3a inhibited acute myeloid leukemia cell proliferation
in part by activating transcription of the Fas-L and p21WAF1/CIP1 genes,
whereas IKK activity maintains FoxO3a in the cytoplasm and thus
establish an important role of FoxO3a inactivation/nuclear exclusion in
the proliferation and survival of AML cells [46]. Furthermore, siRNA
knockdown of endogenous Sam68 (Src-associated in mitosis, 68 kDa), a
nuclear RNA-binding protein that plays a role in cell growth, inhibited
cell proliferation and tumorigenicity of breast cancer cells in vitro
through blocking the G1 to S phase transition. However, the anti-
proliferative effect of silencing Sam68 on breast cancer cells was
associated with up-regulation of CDKIs p21WAF1/CIP1 and p27KIP1,
enhanced transactivation of FoxOs, and attenuation of Akt/GSK-3
signaling [47]. Thus, FoxOs play a major role in G1 arrest by upregulating
cell cycle inhibitors (p21WAF1/CIP1 and p27KIP1) and consequent
attenuation of cell cycle promoting CDKs.
3.2.2. FoxO-mediated regulation of INK family and cyclins
FoxOs not only control cell cycle through transcription of p21WAF1/CIP1/
p27KIP1 genes, but also upregulate INK4 family of CDKIs such as p15 and
p19. The CDKIs of INK4 family (p16, p15, p18 and p19) specically bind to
and inhibit cyclin DCDK4/6 complexes, while the CIP/KIP family (p21,
p27 and p57) of CDKIs bind to and inhibit the cyclin ECDK2 as well as
other cyclinCDK complexes operating throughout the cell cycle [48].
Katayama et al. [49] have reported that p15 and p19 transcription was
associated with FoxO-mediated G1 cell-cycle arrest. Inhibition of Akt
signaling by PI3K inhibitors increased expression of CDKIs p15 and p19
but not p16 or p18. Conversely, Akt suppresses p15 and p19 expression by
Akt-dependent phosphorylation and inactivation of FoxOs. Intriguingly,
FoxOs can also promote cell cycle arrest by repressing cyclin D1 and D2,
 X. Zhang et al. / Biochimica et Biophysica Acta 1813 (2011) 19781986
the two positive regulators of cell cycle [50,51]. Additionally, forced
expression of FoxO1 causes a delay in the transition from G2 to M phase,
which is abrogated by overexpression of CDK1 and cyclin B1 [52]. shRNA-
mediated downregulation of cathepsin B and uPAR results in G0/G1 arrest,
prominent increase of p27KIP1 levels and inhibition of p-Rb. This increased
expression of CDKI p27KIP1 correlates with decreased expression of
p-PI3K, p-Akt, cyclin E, cyclin D1, cyclin D2 and increased expression of
FoxO3a protein [53]. Furthermore, FoxOs also regulate expression of
mitotic genes such as cyclin B and polo-like kinase (Plk) [54]. Since Plk
functions in mitosis and maintenance of DNA integrity, its regulation by
FoxO further highlights an important role of FoxOs in the control of
mammalian cell cycle. Cyclin G2 is a newly identied homologue of cyclin
G1, and in contrast to conventional cyclins, is known to induce cell cycle
arrest and is up-regulated when the cells undergo cell cycle arrest or
apoptosis. Cyclin G2 down-regulation has been reported in papillary
carcinomas of the thyroid [55], breast [56], oral [57], and gastric cancers
[58]. Modur et al. [59] found that FoxO overexpression stimulated
expression of several genes including cyclin G2. FoxO-dependent
upregulation of cyclin G2 is associated with PTEN-mediated transcription
in an Akt-dependent as well as an Akt-independent manner [60]. Taken
together, FoxOs mediate cell cycle arrest at the G1/S and G2/M transitions,
two checkpoints that are critical in the cellular response to stress, by
regulation of multiple cell cycle regulators [61]. FoxO-induced cell cycle
arrest may allow time for repair of damaged DNA as well as for
detoxication of cells.
3.3. FoxO/p53 crosstalk
As outlined here and in previous reviews [36,62], there are many
parallels in the functions and modes of regulation of FoxOs and p53 [36].
FoxOs mediate cell cycle arrest, cell death and cellular oxidative stress to
maintain metabolic stability by activating transcription of their target
genes. The tumor suppressor p53 performs similar functions to enable
DNA repair and to maintain genomic stability [36]. Cell-cycle arrest by
FoxO and p53 occurs through inducing their target genes such as p16,
p21WAF1/CIP1 and p27KIP1 [63]. FoxO and p53 orchestrate apoptosis
through multiple mechanisms but favor the upregulation of the pro-
apoptotic BH3-only proteins. FoxOs are known to upregulate Bim and
PUMA while PUMA is also transcriptionally upregulated by p53. The fact
that FOXOs target a number of genes including p21WAF1/CIP1, GADD45,
WIP1 and PA26 that are also regulated by p53, suggests that FoxOs and
p53 likely coordinate tumor suppression signaling [3]. Indeed available
data support that FoxO and p53 function together to mediate the effects
of cellular stress. In response to nutrient deprivation, FoxOs release p53-
dependent repression of the Sirt1gene, allowing the upregulation of
Sirt1 expression. This repression of Sirt1 seems to be mediated by direct
interaction between FoxO and p53 proteins and is independent of the
presence of FoxO binding site in the Sirt1 promoter [64]. FoxO3 has also
been found to interact with p53 in vitro in response to stress stimuli or to
nutrient deprivation [65]. Intriguing examples that further show the
overlapping functions of FoxO and p53 are derived from studies in
transgenic mice. Mice that lack p53 develop normally, but are
remarkably predisposed to developing broad spectrum of cancers
including thymic lymphoma and hemangioma, and usually die from
these malignancies within 36 months of birth [66,67]. Similarly,
conditional triple knockout mice lacking FOXOs (FoxO1, 3a, and 4) are
prone to develop hemangiomas and thymic lymphoma. On the other
hand, the individual or paired inactivation of FoxO1, FoxO3a or FoxO4
was found to cause a less severe phenotype [22]. These observations
suggest that FoxO and p53 play similar roles at least in suppressing
tumorigenesis by overlapping modes of target gene regulation. Overall,
FoxO3 together with partners including p53 provide a strong tumor
suppressor network to protect cells from damage to the DNA and thus
play an important role in maintaining genomic stability to prevent
clonal emergence and expansion.
1981
4. FoxO and apoptosis
According to Yin Yang theory, all matter consists of two sides. Yin is
the negative side, and Yang is the positive side. The coexistence of the
two sides in equilibrium and/or harmony is considered a key for the
overall health of a biological system. Consistent with this theory, gain-
and loss-of-function models of genes in the core apoptotic pathway
suggest that perturbation of cellular homeostasis can be a primary
pathogenic event that results in disease. Indeed, there is now
compelling evidence that insufcient apoptosis can manifest as
cancer or autoimmunity, whereas accelerated cell death contributes
to acute and chronic degenerative diseases [68].
Factors regulating the expression levels of survival proteins are likely
to play a crucial role in tumor formation. For example, aberrant
overactivation of PI3K/Akt signaling is a hallmark of many human
cancers. One major way by which PI3K/Akt promotes cell survival is
through sequestering FoxOs away from the promoters of apoptotic
genes. FoxOs therefore have emerged as an important effector arm
of PI3K/Akt signaling by driving multiple apoptotic gene expression [3].
4.1. FoxO-upregulated BH3-only proteins
Conventionally, apoptosis occurs via two main pathways: the
intrinsic pathway mediated by mitochondria, resulting in the activation
of caspase 9 while the extrinsic pathway is mediated by the activation of
death receptors (Fas and TNFR) and involves the activation of caspase
8 [69,70]. The BCL-2 family of proteins are well-characterized regulators
of apoptosis and are grouped into three subfamilies based on the
number of BH (BCL-2 homology) domains they share [71]. The BH3-only
group consists of pro-apoptotic proteins including BIK, EGl-1, Bim, BMF,
NOXA, BID, BAD, BNIP3, PUMA and Beclin-1. Initial investigations
indicate that the expression of constitutively nuclear forms of FoxOs
trigger cell death in different types of cells which involves transactiva-
tion of at least two members of pro-apoptotic BH3-only proteins (Bim
and BNIP3) [7275]. FoxOs induce Bim expression in hematopoietic
cells deprived of growth factors [74,75]. Overexpression of a FoxO3a
mutant that cannot be phosphorylated by Akt results in apoptosis of
BCR-ABL-transformed cells [76]. Treatment with proteasome inhibitor,
bortezomib, also reduces the burden of BCR-ABLinduced leukemic
disease by restoration of normal FoxO3a expression, and prolonged
survival of BCR-ABL-transduced mice [77]. SYUNZ-16, a new derivative
of alkannin that induces apoptosis is associated with an increase in the
nuclear accumulation of exogenous FoxO protein, and upregulation of
the mRNA expression of Bim and TRADD in liver cancer cells [78]. In
paclitaxel-sensitive breast cancer, upregulation of FoxO3a induced by
paclitaxel causes increased levels of Bim mRNA and protein, leading to
apoptosis and thus contribute to tumor suppression by paclitaxel [79].
PUMA is a potent regulator of mitochondrial outer membrane
permeabilization, attributing to two distinct mechanisms which both
rely on PUMA binding to the anti-apoptotic BCL-2 proteins: de-
repression and sensitization [80]. FOXO3a is responsible for the
transcriptional upregulation of Puma in response to cytokine or growth
factor deprivation [81], indicating that FoxO-induced transcription of
PUMA may play a role in regulation of apoptosis and stress response
[82]. Furthermore, FoxO4 indirectly suppresses the expression of the
pro-survival Bcl-2 family member Bcl-XL by regulating expression of the
transcriptional repressor Bcl-6 [83]. Thus, FoxOs can induce cell death
through the intrinsic apoptotic pathway mediated by mitochondria.
4.2. FoxO-mediated extrinsic apoptotic pathway
As described in Fig. 1, FoxO directly regulates the extrinsic
apoptotic pathway through enhancing the transcription of proapop-
totic factors such as FasL and TRAIL. The FasL promoter contains three
FBHEs, and Foxo3a-induced apoptosis of cerebellar neurons is
decreased when Fas/FasL interaction is blocked by the decoy fusion
1982 X. Zhang et al. / Biochimica et Biophysica Acta 1813 (2011) 19781986
protein Fas-Fc. Overexpression of a constitutively active form of
FoxO3 triggers apoptosis in cerebellar granule neurons through the
Fas signaling cascade [84]. IKK-insensitive FoxO3a protein mutated at
S644 translocated into the nucleus and activated the transcription of
the Fas-L and CDKI p21WAF1/CIP1 genes. This in turn inhibited leukemic
cell proliferation and induced apoptosis [46]. In addition, over-
expression of FoxO1 and FoxO3 in the prostate cancer cells results
in apoptosis and increased expression of TRAIL. FoxO3 DNA-binding
elements exist in the TRAIL promoter, indicating that TRAIL is a direct
target of FoxO3 [59]. Exposure of human hepatic stellate cells to TRAIL
led to dephosphorylation and nuclear accumulation of FoxO1 and
FoxO3a. Activation of FoxOs consequently promoted TRAIL-induced
apoptosis in LX-2 cells [85]. Active FoxO4 antagonized deregulated
proliferation and induced apoptosis in chronic myelogenous leuke-
mia-derived cell lines. Imatinib-resistant cells underwent apoptosis
after transfection with full-length TRAIL cDNA. These results suggest
that active FoxO4 can overcome imatinib resistance in CML cells via
TRAIL production [86]. It is worth noting that highly selective
expression of FoxO target genes has been observed in different cell
lines and treatment, suggesting that transcriptional targets of FoxOs
are tissue- and stimulus-specic. For example, HIV infection of CD4+ T
cells leads to induction of FoxO3a, which modulates multiple target
genes that are critical to the G0 cell cycle entry observed in the
infected cells and are active players in both the extrinsic and the
intrinsic pathways of apoptosis [87]. However, the precise mecha-
nisms are unclear. Taken together, FoxO proteins regulate cell death
by modulating the expression of BH3-only proteins (Bim and BNIP3)
and death receptor ligands (FasL and TRAIL) that function in autocrine
and paracrine pathways.
4.3. PTEN/FoxO axis in apoptosis
The PTEN tumor suppressor was discovered by its homozygous
deletion and other mutations following mapping of the human
chromosome 10q23 in cancer [88]. Sequencing of the PTEN gene in
tumors found it to be one of the most commonly mutated tumor
suppressors in human malignancies. Thus PTEN signaling is almost
universally altered in cancer [88]. Analysis of PTEN decient hematopoi-
etic system revealed that conditional ablation of PTEN drives exit from
quiescence through PI3K/Akt hyperactivation, and leads to exhaustion of
normal hematopoietic stem cells while at the same time initiating acute
leukemia [89]. Interestingly, mice with FoxO deletions never develop
AML, suggesting that the repression of FoxO activity by Akt is not
sufcient for leukemogenesis, although other types of malignancies
develop in these mice [22,90]. This suggests that alternative or additional
downstream targets of Akt are required for leukemic transformation.
PTEN is a major negative regulator of the PI3K-Akt pathway. In cells that
carry the PTEN mutation, the PI3K pathway is often constitutively active,
leading to inactivation of endogenous FoxOs and possible initiation and
progression of tumors [91]. Phosphorylation-dependent nuclear/cyto-
plasmic shuttling of FoxOs is mainly regulated by Akt and PTEN. FoxO
phosphorylation by Akt results in sequestration of FoxOs in the
cytoplasm and inhibition of target gene transcription. FoxO dephosphor-
ylation leads to nuclear translocation and target gene activation [92].
Since PTEN is a FoxO target gene, FoxOs can enhance transcription of
PTEN to positively reinforce their biological function of tumor suppres-
sion. Therefore, restoring nuclear expression of FoxOs and their target
genes may provide an important therapeutic approach for targeted
cancer therapy.
5. Targeting AKT-FoxO signaling axis
Based on tumor suppressor properties of FoxOs, their reactivation
is considered an attractive anti-cancer strategy. In contrast to other
tumor suppressors such as p53 or PTEN, the functions of which are
abrogated by genetic or epigenetic changes, inactivation of FoxOs
occurs mostly because of the overactivation of their inhibitory
signaling [36]. This offers a wide range of possibilities for rescuing
FoxO activity with two major strategies: 1) reactivation of FoxO and
its regulators; 2) targeting PI3K/ Akt/mTOR axis.
5.1. Reactivation of FoxO and its regulators
5.1.1. Restoration of FoxO gene expression
Since a hallmark of most cancers is the inactivation of FoxO by
PI3K-dependent phosphorylation and hyperactivation of PI3K/Akt
pathway that controls many biological functions such as cell
proliferation, survival, and insulin response, reactivation of FoxO
may be an invaluable therapeutic approach. Kau et al. [93] have
employed a cell-based, chemical genetics approach to search for
compounds that trap FoxO1 in the nucleus by modulating FoxO's
dynamic nucleocytoplasmic shuttling. Two classes of compounds that
inhibit FoxO1a nuclear export were identied: (1) compounds that
target the general nuclear transport machinery and (2) compounds
specic to targeting the PI3K/Akt/FoxO1a signaling pathway. They
have shown that 19 of the compounds that promoted retention of
FoxO1a in the nucleus were novel general protein export inhibitors.
All 19 compounds blocked the nuclear export of RevGFP and FoxO1a
by targeting CRM1, and not by inhibition of other factors in the
nuclear transport machinery [93]. There were 23 small molecule
inhibitors of FoxO1a that acted in the PI3K/Akt/FoxO1a signaling
pathway. Importantly, some of these inhibitors were able to suppress
cellular proliferation of PTEN-decient 786-0 cells, even though they
displayed IC50 values in the micromolar range [93]. Analyzing these
FoxO1 nuclear export inhibitors may not only yield a new class of anti-
cancer therapies, but may also provide novel regulators of the PI3-K,
Akt and FoxO pathways.
Phosphorylation of FoxO factors by Akt triggers the rapid
relocalization of FoxOs from the nucleus to the cytoplasm. Akt
phosphorylates FoxOs at three key regulatory sites (Thr32, Ser253,
and Ser315 in the FoxO3 sequence) that are conserved from
Caenorhabditis elegans to mammals and are part of a perfect consensus
sequence for Akt phosphorylation [91]. Based on this feature of FoxOs,
another strategy could be to mutate the three well-characterized Akt
phosphorylation sites on FoxOs to alanine. This AAA FoxO mutant can
no longer undergo phosphorylation and will accumulate in the
nucleus, allowing reconstitution of FoxO activity in the PTEN null cells.
Indeed, overexpression of Foxo1 or Foxo3a triple AAA mutant by
adenovirus-mediated gene transfer inhibited cell growth by inducing
cell cycle arrest in sub-G1 phase, and subsequently apoptotic cell
death, as evident by typical apoptotic morphologic changes, annexin
V-PE analysis, and caspase cascade activation in melanoma [94] and
endometrial cancer [95]. These data suggest that adenovirus expres-
sing a FoxO triple mutant could be a useful vector for gene therapy of
cancers resistant to chemotherapy and radiotherapy induced by
hyperactivity of PI3K/Akt.
It has been shown that the some cancer therapeutic drugs activate
FoxOs by attenuating Akt activity. In paclitaxel-sensitive MCF-7 breast
cancer cells, up-regulation of FoxO3a by paclitaxel can cause increased
levels of Bim mRNA and protein, resulting in apoptosis. Silencing of
FoxO3a by small interfering RNA greatly reduces the induction of
apoptosis caused by paclitaxel [79]. Furthermore, paclitaxel not only
induces FoxO3a expression but also enhances its nuclear relocation
through JNK-dependent inhibition of the PI3K/Akt signaling pathway,
suggesting a crosstalk between the PI3K-Akt-FoxO3a and JNK signaling
pathways [96]. Phosphorylation of FoxO3a by JNK reduces the interaction
between FoxO3a with 14-3-3 protein, leading to impaired nuclear export
of FoxO3a, and thereby stimulating FoxO3a activity [96]. KP372-1 (a
multiple kinase inhibitor) also inhibits pathways critical for acute myeloid
leukemia survival, including PDK1/Akt and FLT3 through activation of
FoxO activity [97]. In addition, selenium (a chemopreventive agent),
which signicantly reduces the incidence of prostate cancer, upregulates
 X. Zhang et al. / Biochimica et Biophysica Acta 1813 (2011) 19781986
FoxO1 expression when compared to gene expression prole of a control
group [98]. Further analysis demonstrates that FoxO1 activation is critical
for the anticancer effects of methylseleninic acid in prostate cancer cells
[99]. Since androgens provide an Akt-independent cell survival signal in
prostate cancer cells, FoxO transcription factors therefore are important
nuclear targets for both Akt-dependent and -independent survival signals
in prostate cancer cells [100]. Thus restoring FoxO activity by different
approaches can be benecial for cancer treatment and cancer prevention.
5.1.2. Restoration of PTEN gene expression by targeting miRNA
MicroRNAs (miRNAs) are short non-protein-coding RNAs (~22
nucleotide) that are known to alter gene expression at a post-
transcriptional level [101,102]. Over 1200 validated human miRNAs
have been discovered to date (http://www.mirbase.org), and they are
predicted to regulate one third of the human genome with involve-
ment in development and progression of many diseases [103105].
miRNA overexpression has been shown to be indicative of invasion
and chemoresistance through two mechanisms. The rst is by
increased cell proliferation and decreased expression of programmed
cell death 4 (PDCD4). The second mechanism is by repression of the
tumor suppressor PTEN in non-small cell lung cancer, which is
involved in regulation of cell growth and invasion [106]. Several
miRNAs have been shown to function as oncogenic miRNAs (onco-
Mirs) due to the fact that they target transcripts encoding key
regulators of cell proliferation and apoptosis such as PTEN and PDCD4
[107,108]. The PTEN mRNA has an unusual, about 3.3 kb long 3
untranslated region (UTR) that is suggestive of tight post-transcrip-
tional control of PTEN expression. Adding to the complexity of PTEN
regulation, recent evidence further indicated that the PTEN mRNA
undergoes post-transcriptional repression/degradation by specic
miRNAs. Within the PTEN 3UTR are several conserved miRNA target
sites, and PTEN has been shown to be repressed by a variety of miRNAs
derived from miRNA precursors containing a single hairpin structure
such as miR-19a, 21, 22, 26a, 214, 216a, 217, 221, 222, and 494, as well
as those with a polysictronic structure, such as mir-1792, mir-106b-
25, mir-367-302b, and mir-221-222 [107,109116]. Emerging evi-
dence also indicates that aberrant expression of miRNAs is highly
linked to chemoresistance to cancer therapeutic drugs such as Her2
monoclonal antibody trastuzumab and the anti-estrogen tamoxifen
[106]. Therefore, antisense oligonucleotides targeted to oncoMirs
represents a potential new approach for cancer therapy as well as a
solution for chemoresistance via restoration of depressed intracellular
PTEN levels [115,117120].
5.2. Targeting PI3K/Akt/mTOR axis
Given the central role of PI3K/Akt/mTOR pathway in tumorigenesis,
its inhibition seems a powerful approach to induce anti-tumor activity.
Akt is a core player in PI3K/Akt/mTOR axis activated through
phosphorylation of Ser-473/474 and Thr-308/309 [94]. Targeting the
upstream regulators or downstream effectors of Akt is one strategy to
identify new targets for drug discovery. More than 100 lead compounds
targeting multiple nodes of PI3K signaling, including PI3K, Akt, mTOR,
etc., are in the preclinical drug-development pipeline so far [121,122],
and a growing number of promising agents continually enter intensive
phase I, II, and III clinical trials (http://www.clinicaltrials.gov). PTEN is
the key antagonist of the PI3K/Akt pathway. Loss of PTEN in many types
of cancer is associated not only with tumor development but also with
clinical resistance to many anticancer drugs. Thus, targeting key
components of PI3K/Akt/mTOR axis is a desirable means to overcome
PTEN functional loss and consequent restoration of FoxO function.
Wortmannin and LY294002 are well-known PI3K inhibitors that have
been widely used as research tools to elucidate the role of PI3K/Akt/mTOR
signaling in various tumor cells. Unfortunately, high cytotoxicity of both
inhibitors precluded their clinical application. Nonetheless, several
inhibitors of PI3K have moved through preclinical studies and into
1983
phase I and II clinical trials [123].These range from inhibitors reported to
act on a single class I PI3K such CAL-101, to inhibitors of multiple class I
PI3K isoforms such as PX-866, XL-147, and GDC-0941, and to inhibitors
acting on multiple class I isoforms and other PIK family members such as
BEZ235 and XL765 [123]. Overall, there is limited clinical benet with
inhibitors of PI3K. The development of such inhibitors raises issues as to
what specic member or members in the PI3K family should be inhibited
to achieve maximal therapeutic benet, and can specic inhibitors be
developed with the necessary pharmacologic properties to allow them to
proceed to clinical trials? The question also remains to be answered
whether greater therapeutic efcacy will be obtained through the use of
inhibitors with increased specicity, or through inhibitors that act on a
spectrum of targets within the pathway [124].
To date, several types of Akt inhibitors have been investigated,
including phosphatidylinositol analog inhibitors, allosteric Akt kinase
inhibitors, ATP-competitive inhibitors, and alkylphospholipids
[125,126]. However, the use of these inhibitors is limited either by
high toxicity or by low bioavailability and stability in vivo, even
though several agents (e.g., perifosine, MK-2206, RX-0201, PBI-05204,
GSK2141795) have been in various stages of clinical testing [127].
Severe side effects of Akt inhibitors may be due to the critical role of
Akt in multiple intracellular processes [128].
The most studied target in PI3K/Akt/mTOR pathway is mTOR. Four
drugs that target mTOR have been explored for clinical use:
rapamycin, temsirolimus, everolimus and deforolimus [129,130]. In
patients with advanced breast cancer, these inhibitors have modest
anticancer activity in the range of around 10% because of collateral
effect of these inhibitors on feed-back activation of Akt and PI3K
[131,132]. To date, with the exception of kidney cancer, mTOR
inhibitors have failed to inhibit tumor progression in a variety of
cancers [133,134]. Since there is a crosstalk between the estrogen
receptor and the PI3K/Akt/mTOR pathway, clinical trials have
explored the combination of mTOR inhibitors and aromatase in-
hibitors. Recent data show that everolimus in combination of letrozole
proved to be superior over letrozole alone with a signicant response
rate (68% vs. 59%) in the patients with ER positive breast cancers
[130]. Although molecular targeting of tumor-specic signal trans-
duction pathways is a promising strategy for discovering and
developing novel and potent anticancer drugs, evolution of our
knowledge of the PI3K/Akt/FoxO pathway will serve as an important
guide in design of future anti-cancer agents with maximal efcacy and
minimal side effects.
6. Conclusions
FoxO transcription factors are emerging as key regulators of cell
growth. Their ability to limit cell proliferation by promoting
quiescence, apoptosis, or both plays a critical role in a variety of
physiological and pathological processes including tumor suppression.
FoxOs are one of the major targets of the PI3K/Akt signaling in the
presence of growth factors and nutrients. Activation of the PI3K
pathway promotes phosphorylation, and consequent nuclear exclu-
sion of the FoxOs results in blockage of their growth inhibitory
transcriptional functions. Since growth inhibitory functions of FoxOs
involve activation of several proteins that include inhibitors of cell
cycle, retinoblastoma protein, inducers of extrinsic as well as intrinsic
apoptosis, and interactions with tumor suppressors such as p53,
restoring activities of FoxOs therefore is an excellent anti-cancer
strategy. Indeed, over-expression of phosphorylation-decient FoxO
mutants has provided a proof-of-principle approach for suppression of
cell growth. Although FoxOs are known to function in a tissue and
context-dependent manner, and functional redundancy exists among
the FoxOs, identication of small molecule compounds that selectively
target one or multiple FoxOs and promote their nuclear retention or
inhibit their nuclear export nevertheless offer excellent opportunities
for cancer-related drug design. Furthermore, as Akt-dependent
1984 X. Zhang et al. / Biochimica et Biophysica Acta 1813 (2011) 19781986
phosphorylation promotes nuclear exclusion and degradation of
FoxOs, pharmacologic targeting of the PI3K-Akt pathway in combina-
tion with strategies that promote nuclear retention of FoxOs have the
potential to be superior in targeting a range of inammation-
associated pathologies including cancer.
Acknowledgements
Supported by the Department of Veterans Affairs Merit Review
grant to AKR.
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