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Oncogene (1999) 18, 6094 ± 6103
1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc
Modulation of cellular apoptotic potential: contributions to oncogenesis
Vuk Stambolic1,2, Tak W Mak1,2 and James R Woodgett*,2
1
Amgen Institute, 620 University Avenue, Toronto, Ontario, Canada M5G 2C1; 2Ontario Cancer Institute, 610 University Avenue,
Toronto, Ontario, Canada M5G 2M9
The importance of apoptosis as a natural means to
eliminate unwanted or damaged cells has been realized
over the past decade. Many components required to
exercise programmed cell death have been identi®ed and
shown to pre-exist in most, if not all, cells. Such ubiquity
requires that apoptosis be tightly controlled and suggests
the propensity of cells to trigger the cellular death
machinery can be regulated. Recently, several signaling
pathways have been demonstrated to impact the
apoptotic potential of cells, most notably the phosphatidylinositol 3' kinase (PI3'K) pathway. The 3' phosphorylated lipid products generated by this enzyme
promote activation of a protein-serine kinase, PKB/
AKT, which is necessary and sucient to confer cell
PI3'K-dependent survival signals. The relevance of this
pathway to human cancer was revealed by the recent
®nding that the product of the PTEN tumor suppressor
gene acts to antagonize PI3'K. This review focuses on
the regulation and mechanisms by which PKB activation
protects cells and the oncologic consequences of
dysregulation of the pathway.
Keywords: apoptosis; cell survival; PI3'K; PTEN
Introduction
Despite its late recognition as a fundamental process in
multicellular organisms, apoptosis has rapidly become
integrated into the understanding of a wide variety of
biological events and has instituted appreciation of the
delicate balances within cells that dictate ongoing
viability or termination (Steller, 1995). It had long
been recognized that tumors are associated with
considerable cell death as well as proliferation.
However, the realization that the primary means by
which gamma irradiation and most chemotherapeutics
killed cells was by inducing a suicide response
revolutionized thinking about how such therapies
might be improved and how cells might evade their
action. A similar watershed in thinking was instigated
by the ®nding that the bcl2 oncogene acted not by
promoting growth, as was seemingly the case for
previously discovered oncogenes, but by suppressing
cell death (Korsmeyer, 1999). These discoveries pointed
to an equally important role for control of cell death as
for control of cell division and brought about a surge
in e€orts to identify genes involved in the process of
programmed cell death. These e€orts were accelerated
by the genetic characterization of apoptosis in the
nematode, largely through the work of Hengartner and
*Correspondence: JR Woodgett
Horvitz (1994) and also Metzstein et al. (1998), which
provided key insights into the nature of the positively
and negatively acting gene products.
With emphasis placed on the identities of the
`executioners' and the `appeal judges', there was rapid
progress in characterizing the caspase family of
proteases activated during apoptosis and additional
relatives and antagonists of Bcl2 such as Bcl-Xl, Bax,
Bag and Bad (reviewed in Li and Yuan, 1999; Reed,
1998). Simultaneously, work in a variety of biological
systems began to uncover di€erential sensitivities of
cells to survival in culture depending on the presence of
particular factors in the media (Dudek et al., 1997).
These experiments indicated that extracellular molecules could in¯uence cellular viability and provided
models to facilitate dissection of the pathways induced
by such factors.
Survival signaling
The viability of cultures of primary neurons has long
been known to be highly dependent upon particular
factors, such as neurotrophins (such as NGF, CNTF
and BDNF). Likewise, several hematopoietic cell lines
were known to be dependent upon certain growth
factors such as IL-3 and GM-CSF. Ectopic expression
of certain genes was found to prolong neuronal
survival (such as dominant negative c-Jun; Ham et
al., 1995) but most e€ort was focused on the
mechanisms by which these growth factors enhanced
survival. Yao and Cooper (1996) noted that inhibition
of phosphatidylinositol 3' kinase (PI3'K) induced
apoptosis. At the time, the e€ectors of this lipid
kinase were thought to be largely related to metabolic
controls such as protein synthesis, vesicular transport
and sugar transport (Vanhaesebroeck et al., 1997).
Furthermore, the only inhibitors of PI3'K (wortmannin
and LY294002) were known to e€ect other cellular
processes. However, there was a strong correlation
between activation of PI3'K and protection from cell
death.
This association was strengthened by a di€erent
avenue of work investigating the e€ects of the c-Myc
oncoprotein on apoptosis. Normally, overexpression of
c-Myc is associated with cell proliferation. In quiescent
cells, c-Myc levels are undetectable but are rapidly
elevated upon mitogenic stimulation. Serum-deprivation results in a decrease of Myc expression and is
accompanied by ordered exit of cells from the cell
cycle. However, forced expression of Myc induced
apoptosis rather than quiescence of cells. Thus, serum
deprivation of cells expressing a constant level of Myc
resulted not in exit from the cycle but apoptosis (Evan
et al., 1992). By screening for constituents in serum
Apoptosis and cell survival during oncogenesis
V Stambolic et al
capable of blocking this death, Evan's group identi®ed
factors such as IGF-1 and PDGF as being protective
(Harrington et al., 1994). These factors all induced
activation of PI3'K.
Progress in determining the molecular mechanism by
which PI3'K was able to transduce a survival signal
was signi®cantly enhanced by the discovery that a
previously identi®ed protein-serine kinase termed
protein kinase B (PKB or AKT) was activated by the
3' phosphorylated products of PI3'K (Burgering and
Co€er, 1995; Franke et al., 1995; Kohn et al., 1995).
An oncogenic variant of PKB had been identi®ed in
1991 as a retrovirally transduced fusion of PKB with
gag sequences resulting in a constitutively activated
enzyme (Bellacosa et al., 1991). Expression of gagPKB/v-Akt in cells was shown to confer a similar
degree of protection as the agonists of PI3'K. For
example, introduction of gag-PKB into cerebellar
neurons (Dudek et al., 1997) or ®broblasts (Kauffmann-Zeh et al., 1997) increased viability in the
absence of serum. Furthermore, expression of catalytically inactive PKB reduced the viability of the cells in
the presence of survival factors (Dudek et al., 1997;
Stambolic et al., 1998). In other cell types, expression
of activated PKB protected cells against UV irradiation
(Kulik et al., 1997) and IL-3 withdrawal-induced death
(Songyang et al., 1997).
negatively charged phosphates) results in a partially
activated enzyme that is neither further activatable by
agonists of PI3'K or inhibitable by wortmannin or
LY294002. Interestingly, following phosphorylation by
the PDKs, a signi®cant fraction of active PKB
molecules translocate to the nucleus (Andjelkovic et
al., 1997; Meier et al., 1997). The sequence of events
required for induction of PKB is thus complex (see
Figure 1). Following activation of PI3'K (for example,
by its own translocation to the plasma membrane via
recruitment of its regulatory subunit's SH2 domains to
phosphotyrosine docking sites on activated receptors or
receptor adaptors), a complex assembles, drawn to
newly generated 3' phospholipid microdomains. The
complex consists of PKB and its two processing
kinases, the PDKs. Following phosphorylation the
activated PKB is drawn into the cellular interior and
nucleus. Since aspartate mutants of the activation sites
are PI3'K independent and short circuit this process,
membrane binding of PKB acts simply to allow its colocalization with the processing enzymes, the PDKs.
Phosphorylation by these enzymes is thought to induce
a conformational change that both reduces anity for
3' phosphorylated lipids and opens up the catalytic
cleft, allowing access to substrates.
Regulation of PKB
PDK1 was puri®ed by anity chromatography, relying
on its anity for PKB (Alessi et al., 1997b; Stokoe et
al., 1997). The enzyme consists of a C-terminal PH
domain and a protein kinase catalytic domain (and
little else) (Alessi et al., 1997a). PDK1 appears to be
constitutively active in cells. When puri®ed, the protein
does not require phospholipids or any other co-factors
for expression of activity (Alessi et al., 1997b). Indeed,
PDK1 phosphorylates other protein kinases in addition
to PKB, including various PKCs (Dong et al., 1999; Le
Good et al., 1998), p70 S6 kinase (Alessi et al., 1998;
Pullen et al., 1998) and cAMP-dependent protein
kinase (PKA) (Cheng et al., 1998). In all cases, the
All PKB gene products encode a polypeptide of
approximately 55 ± 60 kDa containing an N-terminal
pleckstrin homology (PH) domain and a serine/
threonine kinase catalytic domain (reviewed in Co€er
et al., 1998). The PH domain exhibits anity for 3'
phosphorylated lipids although the exact preferences
are unclear (in part due to the importance of the
context of presentation of the lipids to the protein)
(James et al., 1996; Klippel et al., 1997; Takeuchi et al.,
1997). Activation of PKB requires an intact PH
domain but this is insucient for function. For
catalytic activation the enzyme must undergo a
conformational shift that occurs upon phosphorylation of two residues, a threonine in the catalytic loop
(T308 in PKBa) and a serine close to the carboxyl
terminus (S473 in PKBa) (Alessi et al., 1996). These
residues are conserved in all PKBs (including
Drosophila and C. elegans) except for a splice variant
of the PKBg gene (Konishi et al., 1995) which encodes
a truncated polypeptide that lacks the C-terminal
phosphorylation site (a longer splice variant encoded
by the same gene does contain a serine homologous to
S473) (Brodbeck et al., 1999; Nakatani et al., 1999).
Phosphorylation of the two activatory sites is
required for full activation of PKB (Alessi et al.,
1996). Mutation of either to a non-phosphorylatable
residue signi®cantly reduces activity and mutation of
both eliminates enzymatic activity. Phosphorylation of
the two residues is dependent upon PI3'K activity and
is inhibited by wortmannin. Two enzyme activities that
independently phosphorylate T308 and S473 of PKBa,
termed polyphosphatidylinositide-dependent protein
kinases 1 and 2 (PDK1 and PDK2), have been
characterized. Substitution of the activation site
residues with aspartic acid (with the aim of mimicking
PDKs have other roles
Figure 1 Components of the PI3'K signaling pathway in
mammals and C. elegans. Components of the PI3'K pathway
have been remarkably conserved between multicellular organisms
allowing genetic analysis and identi®cation of new components.
The worm genes and their corresponding mammalian counterparts are illustrated
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Apoptosis and cell survival during oncogenesis
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6096
Table 1 Tumors associated with dysregulation of the PI3'K pathway
Genetic
alteration
Tumor type
Reference
PI3'K
Hemangiosarcoma in
(Chang et al., 1997;
ampli®cation
chicken, ovarian
Shayesteh et al.,
cancer in humans
1999)
PKB/Akt
Thymic lymphoma in
(Bellacosa et al., 1995;
ampli®cation
mice, ovarian, prostate
Cheng et al., 1992;
and breast cancer in
Cheng et al, 1996)
humans
PTEN
Glioblastoma, endometrial, (Bostrom et al., 1998;
mutations
prostate, thyroid, breast, Cairns et al, 1997;
kidney, melanoma,
Dahia et al., 1997;
lymphoma; Cowden and Guldberg et al.,
Bannayan-Zonana
1997; Li et al., 1997;
syndrome
Liaw et al., 1997; Liu
et al., 1997; Marsh et
al., 1997; Podsypanina
et al., 1999; Rasheed
et al., 1997; Steck et
al., 1997; Suzuki et al.,
1998; Teng et al.,
1997; Wang et al.,
1997)
targeted phosphorylation site is deep within the kinase
domain and, in the case of PKA, plays a structural role
(Cheng et al., 1998). The PDK1 site on PKA appears
to be phosphorylated upon synthesis and initial folding
of the enzyme. These data support a tonic function for
PDK1 in the processing of certain other protein
kinases and may partly explain the unusual phenotypes associated with mutation of the PDK1 homologue in Drosophila (DSTPK61) (Belham et al., 1999).
The question remains, why is PDK1 phosphorylation
of PKB dependent upon 3' phosphorylated lipids. The
most likely explanation is that binding of 3'
phospholipids by the PH domain of PKB reveals the
PDK1 site. Thus, phosphorylation can only occur at
the membrane.
Elucidation of PDK2 has been more problematic.
Two distinct possibilities have been proposed. Alessi et
al. performed a two hybrid screen with PDK1 and
isolated a C-terminal 77 amino acid fragment of
PRK2, yet another protein-serine kinase. They found
that co-expression of PDK1 with the PRK2 fragment
modi®ed it's substrate speci®city, enabling it to
phosphorylate the PDK2 site on PKB (Balendran et
al., 1999). Although the PRK2 fragment does not exist
physiologically, the group hypothesized that another
molecule may act similarly and convert PDK1 into a
PDK2 like activity. This novel idea has yet to be
shown to have physiological signi®cance but, if true,
would remove the necessity for an independent PDK2
enzyme. A prediction of this model would be coregulation of both sites of phosphorylation on PKB
(T308 and S473). Phosphorylation of each is dependent
upon PI3'K. However, mutation of one site does not
a€ect phosphorylation of the other and certain kinase
inhibitors block T308 phosphorylation without a€ect
on S473 (B Hemmings, personal communication).
A distinct PDK2 candidate emerged from study of a
protein kinase isolated by virtue of its anity for the
intracellular domain of b-integrins. Integrin-linked
kinase (ILK) has an unusual catalytic domain located
C-terminally to ankyrin repeats (Hannigan et al.,
1996). Between these features lies a sequence with
homology to phospholipid binding domains. ILK
activity was found to be induced upon integrin
ligation and to be PI3'K-dependent (Delcommenne et
al., 1998). Furthermore, expression of ILK in cells
resulted in activation of PKB and its phosphorylation
on S473. In vitro ILK can incorporate phosphate into
PKB, speci®cally at this residue. Similar data have
been obtained using Drosophila ILK which phosphorylates Drosophila PKB at serine 505 (analogous to S473
in the mammalian protein) (A Ali and J Woodgett,
unpublished observations) These data raise the
possibility of a mechanism to couple PKB activation
with matrix attachment. When epithelial cells lose
contact with other cells, they undergo a form of
apoptosis termed `anoikis' (Frisch and Francis, 1994).
Expression of activated mutants of PKB suppresses
this death (Fujio and Walsh, 1999; Khwaja et al.,
1997). Given the key role of integrins in matrix
signaling, it is tempting to speculate that ILK provides
the integrin-mediated survival signal when cells are
anchorage-dependent.
Targets of PKB
Knowing that expression of an activated allele of PKB
is sucient to confer a high degree of protection from
apoptosis, there has been intensive e€ort to identify the
important substrates. One of the ®rst targets identi®ed
was glycogen synthase kinase-3 (GSK-3), which is
inactivated by PI3'K signaling and can be directly
phosphorylated by PKB in vitro (Cross et al., 1995).
Overexpression of GSK-3 can induce cell death (Pap
and Cooper, 1998). However, lithium inhibits this
enzyme without apparent e€ects on apoptotic propensity (Stambolic et al., 1996).
Several other gene products have been proposed that
play a direct role in promoting apoptosis. In particular,
the pro-apoptotic, Bcl-2 related protein, Bad, contains
a serine residue within a consensus sequence recognized
by PKB. Bad dimerizes with Bcl-Xl and smothers its
anti-apoptotic capacity. Co-expression of Bad and
PKB leads to the former protein becoming phosphorylated (at serine 136) which facilitates binding to 14-3-3
proteins (Datta et al., 1997; del Peso et al., 1997;
Gajewski and Thompson, 1996; Ya€e et al., 1997; Zha
et al., 1996). As a consequence of phosphorylation,
Bad dissociates from Bcl-Xl allowing that protein to
exert protective e€ects. In essence, phosphorylation of
Bad by PKB neutrilizes the suppressive e€ects of Bad
on Bcl-Xl.
The physiological signi®cance of this mechanism is
somewhat limited by the narrow expression pro®le of
Bad, which is largely con®ned to hematopoietic cells.
Due to the relatively low endogenous levels of Bad, it
has also been dicult to demonstrate the e€ect without
resorting to transient overexpression. Further, activation of PKB by cytokines and induction of Bad
phosphorylation can be uncoupled (Craddock et al.,
1999; Hinton and Welham, 1999; Scheid and Duronio,
1998).
The executioners of apoptosis include a family of
proteases that cleave after aspartate residues, the
caspases (Nunez et al., 1998). These largely fall into
one of two classes: upstream caspases that are coupled
to regulatory machinery (such as FLICE which
Apoptosis and cell survival during oncogenesis
V Stambolic et al
associates with the Fas receptor adaptor, FADD) and
those that are activated by proteolytic cleavages
catalysed by the activated upstream caspases (e€ector
caspases). Caspase 9 is an upstream caspase that is
activated upon stiimulation of cells by agents such as
TNF and other cellular stresses. Human caspase 9
contains a PKB consensus sequence and can be
phosphorylated by PKB in vitro, resulting in inactivation of its protease activity (Cardone et al., 1998).
Further, phosphorylation of caspase 9 at the PKB site
can be demonstrated in human cells. By phosphorylating caspase 9, PKB would therefore reduce the capacity
of a cell to induce the proteolytic activation of certain
downstream caspases. The signi®cance of this mechanism is unclear, however, since a major fraction of
caspase 9 molecules would have to be phosphorylated
to impact the caspase autolytic cascade. Further, the
site of phosphorylation in human caspase 9, which has
been mapped to Ser 196, lies within a V8 proteolytic
fragment KLRRRFSSLHFMVE (Ser 196 underlined)
(Cardone et al., 1998). However, the analogous peptide
from murine caspase 9, KLEHRFRWLRFMVE (R
Hakem, personal communication) does not contain a
phosphorylatable residue in the position corresponding
to Ser 196.
Perhaps the most compelling anti-apoptotic targets
identi®ed to date is the family of transcription factors
than include the Forkhead-like (FKHR)/Afx proteins
(Biggs et al., 1999; Brunet et al., 1999; Guo et al., 1999;
Kops et al., 1999; Nakae et al., 1999; Rena et al.,
1999). Among the genes induced by these factors are
various pro-death molecules including fas ligand. For
example,
PKB-dependent
phosphorylation
of
FKHRL1 at Thr 32 and Ser 253 promotes its
association with 14-3-3 proteins. This is associated
with nuclear export denying access of this transcription
factor to its DNA targets (Brunet et al., 1999). Thus,
activation of PKB e€ectively shuts down a genetic
program that includes genes that can trigger cellular
suicide. The ®rst clue that this family might be
regulated by PKB came from studies in the nematode
(see Figure 1). Genetic analysis of the daf2/insulin/
IGF1 receptor revealed a suppressor termed daf16,
which, when cloned, was found to encode a forkheadrelated transcription factor (Lin et al., 1997; Ogg et al.,
1997). Together, the biochemical and genetic data
suggest that several members of the forkhead family
are important targets for PI3'K mediated survival
signaling. Even so, there are likely many additional
PKB substrates that mediate its survival promoting
e€ects that remain to be identi®ed.
Importance of PI3'K signaling in cancer
The role of phosphatidylinositol metabolism in
tumorigenesis was ®rst implied a number of years
ago by the ®ndings that products of viral oncogenes
pp60 v-src and polyoma virus middle T antigen
associate with an intracellular phosphatidylinositol
kinase activity (Sugimoto et al., 1984; Whitman et
al., 1985). It was shown that the regulatory subunit of
phosphatidyinositol 3' kinase (PI3'K) was able to
directly interact with these oncogenes and was
responsible for the associated PI3' kinase activity
(Carpenter et al., 1990; Kaplan et al., 1987; Serunian
et al., 1990). In support of the role of PI3'K in cellular
transformation, an oncogenic form of the catalytic
subunit of PI3'K was cloned from a retrovirus that
causes hemangiosarcomas in chickens (Chang et al.,
1997) and shown to induce transformation of chicken
embryo ®broblasts. More recently, ampli®cation of the
human gene encoding the p110a catalytic subunit of
PI3'K in ovarian cancer tissue samples and cell lines
has been described (Shayesteh et al., 1999), as well as
the ability of the activated form of PI3'K to cause
transformation of 3T3 cells (Jimenez et al., 1998).
PI'3K functions in multiple cellular signaling pathways
and is implicated in regulation of cell proliferation,
survival and adhesion, organization of the cytoskeleton
and glucose metabolism (reviewed in Rameh and
Cantley, 1999; Leevers et al., 1999; Fruman et al.,
1998; Shepherd et al., 1998). A role for PI3'K in
tumorigenesis is underscored by the identi®cation of
activating mutations in both upstream and downstream
components of PI3'K signaling pathways in human
cancer. For example, ampli®cation of members of the
receptor tyrosine kinase family capable of activating
PI3'K such as platelet-derived growth factor receptor
(PDGFR) and epidermal growth factor receptor
(EGFR) genes have been demonstrated in glioblastoma (Cha€anet et al., 1992; Smits and Funa, 1998).
Identi®cation of PKB/Akt, a transforming oncogene
that causes thymic lymphomas in mice (see above), as a
major target of PI3'K signaling further supports the
importance of PI3'K/PKB in cancer. Moreover, in
humans, overexpression of PKB has been demonstrated in a proportion of ovarian (Cheng et al., 1992),
pancreatic (Cheng et al., 1996) and breast cancers
(Bellacosa et al., 1995). ILK has also been found to be
overexpressed in ovarian and breast cancer and is able
to transform epithelial cells (S Dedhar, personal
communication; Wu et al., 1998). Thus, changes in
the activity of PI3'K signaling pathway(s) due to
ampli®cation and/or upregulation of its components
could result in complex cellular outcomes resulting in
cellular transformation and development of cancer
(Table 1).
The recent identi®cation of the molecular mechanism of action of the tumor suppressor gene PTEN/
MMAC1/TEP1 (PTEN herein; see below) has o€ered
new insights into the involvement of PI3'K-regulated
signaling pathways in a large fraction of human
tumors. PTEN was originally identi®ed as a candidate
tumor suppressor gene frequently deleted at chromosome 10q23 in a number of advanced tumors such as
glioblastoma, prostate, kidney and breast carcinoma
(Li et al., 1997; Steck et al., 1997). PTEN was also
independently discovered in a search for novel tyrosine
phosphatases and named TEP1 (Li and Sun, 1997). A
systematic search for the involvement of PTEN
alterations in human cancer by a number of groups
demonstrated a signi®cant rate of PTEN mutations in
high-grade but not low-grade glioblastomas (Bostrom
et al., 1998; Liu et al., 1997; Rasheed et al., 1997;
Wang et al., 1997), prostate (Cairns et al., 1997), and
thyroid (Dahia et al., 1997) tumors, as well as in breast
(Li et al., 1997; Steck et al., 1997; Teng et al., 1997)
and melanoma (Guldberg et al., 1997) cell lines. In
contrast to other tumors where PTEN mutations are
frequent in the advanced phases of the disease, PTEN
mutations also occur at all stages of endometrial cancer
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Apoptosis and cell survival during oncogenesis
V Stambolic et al
6098
(Risinger et al., 1997, 1998; Tashiro et al., 1997),
suggesting a potential involvement of PTEN in the
process of tumor initiation in this organ.
In addition to frequent mutations in sporadic
tumors, germline mutations of PTEN are believed to
cause three related autosomal-dominant hamartoma
syndromes: Cowden syndrome (Liaw et al., 1997),
Bannayan ± Zonana syndrome (Liaw et al., 1997;
Marsh et al., 1997, 1998a,b) and Lhermitte ± Duclos
disease (Liaw et al., 1997). Although each of the three
conditions is characterized by distinct clinical symptoms, the a€ected patients share high susceptibility for
benign hamartomatous tumors throughout the body
early in life, as well as increased incidence of cancers of
the breast, thyroid and brain (Eng, 1998).
The human PTEN gene encodes a 403 amino acid
polypeptide with a high degree of homology to protein
phosphatases (Li et al., 1997; Steck et al., 1997) as well
as a protein associated with the actin cytoskeleton at
focal adhesions, termed tensin (Lo et al., 1994). The
importance of an intact PTEN phosphatase domain for
its tumor suppressor function was emphasized by the
®ndings that a majority of tumor-associated PTEN
mutations map to the region encoding the phosphatase
domain (Marsh et al., 1998a; Rasheed et al., 1997).
Furthermore, unlike wild-type PTEN, catalyticallyinactive PTEN is unable to suppress growth and
tumorigenicity of PTEN-de®cient glioblastoma cells
(Furnari et al., 1997). PTEN is capable of dephosphorylating both phosphotyrosine and phosphoserine/
threonine-containing arti®cial substrates in vitro (Myers
et al., 1997). However, the anity of PTEN for
proteinaceous substrates is relatively low and PTEN
exhibits preference for highly acidic substrates,
suggesting an unusual substrate speci®city. To that
end, it has been shown that PTEN dephosphorylates
the D3 position of phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3), the primary product of phosphatidylinositol 3' kinase activity (Maehama and Dixon,
1998). Thus, PTEN activity directly antagonizes PI3'K.
The relevance of PTEN PI(3,4,5)P3-phosphatase
activity for its tumor suppressor function was highlighted by the fact that mutant PTEN proteins found
in two unrelated patients with Cowden syndrome, as
well as some of the mutant PTEN proteins found in
sporadic tumors, have abrogated lipid phosphatase
activity yet retain the ability to dephosphorylate a
synthetic tyrosine-phosphorylated protein substrate
(Furnari et al., 1998; Myers et al., 1998). PTEN has
also been shown to interact directly with focal adhesion
kinase (FAK) and reduce its tyrosine phosphorylation.
Expression of PTEN was shown to inhibit cell
migration, integrin-mediated cell spreading, and formation of focal adhesions (Tamura et al., 1998).
Whether these e€ects are mediated via FAK or are a
consequence of modulation of ILK (which is PI3'Kdependent) remains to be established.
Further mechanistic insight into the physiological
function of PTEN was derived from investigations of
PTEN-de®cient cell lines. Immortalized mouse embryo
®broblasts (MEFs) generated from PTEN-mutant mice
exhibit signi®cantly lower sensitivity to cytotoxic
stresses known to induce apoptosis, such as osmotic
shock, UV-irradiation, heat treatment or stimulation
with tumor necrosis factor a (Stambolic et al., 1998).
Resistance to apoptotic stimuli is accompanied by
constitutively elevated activity and phosphorylation of
PKB, a crucial regulator of cell survival (see above).
Signi®cantly, both sensitivity to apoptotic stimuli and
hyperphosphorylation of PKB in PTEN-de®cient cells
could be restored to wild-type levels by expression of
exogenous PTEN. Examination of PI(3,4,5)P3 levels in
PTEN-mutant MEFs revealed elevated intracellular
levels of this lipid in comparison to that in their wildtype counterparts, in agreement with an active role of
PTEN in negative regulation of PI(3,4,5)P3 levels in
cells. Consistent with such a role, a number of PTENde®cient tumor cell lines also display increased
PI(3,4,5)P3 levels accompanied by hyperphosphorylation of PKB and elevated cellular survival (Dahia et
al., 1999; Davies et al., 1999; Haas-Kogan et al., 1998;
Li et al., 1998; Myers et al., 1998). Expression of high
levels of PTEN in certain cell lines leads to apoptosis, a
phenomenon that can be rescued by coexpression of
activated mutants of PKB (Stambolic et al., 1998). In
other cells overexpression of PTEN causes G1 arrest
(Furnari et al., 1998), a potential consequence of the
ability of PTEN to regulate the expression of the cell
cycle regulator p27KIP1 (Sun et al., 1999). It appears
that whether PTEN induces apoptosis or cell cycle
arrest depends on the type of investigated cells.
Resolution of this apparent discrepancy of the e€ect
of PTEN on di€erent cell types requires further
investigation.
Mice null for PTEN die during embryogenesis
between gestation day E6.5 and E9.5 (Di Cristofano
et al., 1998; Podsypanina et al., 1999; Suzuki et al.,
1998) from an apparent failure to form chorio-allantoic
fusion (Suzuki et al., 1998). The severity of PTEN
mutant phenotypes appears to depend on the genetic
background, as the animals generated by three
independent groups have slightly di€erent phenotypes
(Di Cristofano et al., 1998; Podsypanina et al., 1999;
Suzuki et al., 1998). Mutants from at least one group
gastrulate and form all three germ layers, even though
they are developmentally delayed (Suzuki et al., 1998).
PTEN-null embryos show abnormally patterned and
expanded cephalic and caudal regions (Stambolic et al.,
1998; Suzuki et al., 1998). BrdU staining has identi®ed
those regions as hyperproliferative, implicating PTEN
in control of proliferation during early mouse
embryogenesis (Stambolic et al., 1998). Disruption of
PTEN also interferes with di€erentiation of embryonic
stem (ES) cells into haematopoetic mesoderm (Di
Cristofano et al., 1998).
Mice heterozygous for PTEN are highly susceptible
to tumors. The predominant type of malignancies in
PTEN+/7 mice at a young age is of lymphoid origin.
15 ± 20% of all mice develop thymic and peripheral
lymphomas, predominantly of T-cell origin, with
in®ltration into multiple organs and tissues (Podsypanina et al., 1999; Suzuki et al., 1998). Moreover,
gamma-irradiation decreases the time of development
of thymic lymphomas in PTEN+/7 mice, which was
in each case accompanied by loss of heterozygosity at
the PTEN locus (Suzuki et al., 1998). These tumors
exhibit elevated phosphorylation of PKB in comparison to normal tissue, consistent with the notion that
PTEN negatively regulates PKB signaling (Suzuki et
al., 1998). Of note, v-akt, the oncogenic form of PKB,
also causes mouse T cell lymphoma (AKR) (Bellacosa
et al., 1991; Staal and Hartley, 1988). Lymph node
Apoptosis and cell survival during oncogenesis
V Stambolic et al
hyperplasia, dysplastic intestinal polyps, thyroid
neoplasms, atypical adenomatous hyperplasia in the
liver, and teratocarcinoma were also observed at an
increased frequency in PTEN heterozygous mice
(Podsypanina et al., 1999; Suzuki et al., 1998).
Interestingly, young PTEN+/7 mice fail to exhibit
characteristics of patients with Cowden's, BZ and L-D
syndromes. However, past six months of age, all
PTEN+/7 females present with atypical hyperplasia
of the endometrium which in a number of cases leads
to carcinoma (Podsypanina, 1999; V Stambolic et al.,
in preparation). Furthermore, almost all females
develop breast carcinoma in situ, whereas about half
of males show prostate malignancies (V Stambolic et
al., in preparation). Almost all of these tumors are
associated with LOH at the PTEN locus and manifest
hyperphosphorylation of PKB (V Stambolic et al., in
preparation), implicating PI3'K/PTEN/PKB regulated
pathway(s) in the development of these tumors in mice.
Thus, older PTEN heterozygous mice exhibit some of
the hallmarks of PTEN-associated hamartoma syndromes, and represent a model system for their
investigation in the laboratory.
The next major challenge in PTEN research is
identi®cation of modes of its regulation. It is reasonable to assume that stimuli that result in activation of
PI3'K and related pathways would result in inhibition
of PTEN activity. Alternatively, PTEN could be
constitutively active and signals resulting in activation
of PI3'K are able to transiently override the negative
regulation of PI3'K-mediated pathways by PTEN.
PTEN could also be regulated at the protein level by
control of its expression and/or degradation. Further
studies are also needed to delineate the role of PTEN
in control of cell proliferation and survival. It is
conceivable that the physiological function of PTEN is
neither to induce apoptosis nor cell cycle arrest, but to
negatively regulate the processes of cell survival and
proliferation. Thus, the results of PTEN overexpression
studies could represent an extreme outcome of the
suppressive e€ects of PTEN on these processes and not
a true representation of its physiological role. The
balance between PI'3K, PI(3,4,5)P3, PKB and their
downstream targets on one side, with PTEN on the
other, functions as a molecular indicator capable of
regulating the survival and/or proliferation potential of
individual cells. Any alterations of this balance due to
either ampli®cations of positive regulators of survival
and proliferation, or inactivating mutations of the
negative ones, can lead to tumorigenesis.
Genetics of PI3'K signaling in ¯ies and worms
The high degree of conservation of the PI3'K
pathway and its downstream mediators has allowed
genetic approaches for unravelling functions (Figure
1). There are two PKB genes in the worm, C. elegans
and only one in the fruit ¯y, D. melanogaster. In the
¯y, PKB is a maternal e€ect gene. In germ line
clones (which are mutant for both the zygotic and
maternal copies of the gene), loss of PKB is
associated with ectopic apoptosis (Staveley et al.,
1998). A similar phenotype is observed upon
expression of a dominant negative mutant of PI3'K
or an interfering mutant of PKB (A Manoukian, J
Jing and JR Woodge€ in preparation). During
normal development, certain cells undergo apoptosis.
Many of these apoptotic events can be suppressed by
expression of a dominantly activated PKB (PKBDD; A
Manoukian, J Jing and JR Woodge€ in preparation).
These data indicate that PKB activity is necessary to
suppress cell death during development and that it is
sucient to block certain forms of cell death.
Interestingly, the apoptotic phenotype is de-emphasized as development proceeds. Thus, interference of
the PI3'K pathway at the imaginal disc stage results
not in cell death but in reduction of cell size.
Genetic analysis of the PI3'K pathway is most
advanced in the nematode C. elegans. In this organism
the PI3'K homologue, age-1/daf-23, has been identi®ed
as one of the genes involved in regulation of overall life
span. Wild-type worms live for 2 weeks at 208C.
However, under stress conditions such as food shortage
or overcrowding, the animals enter a dauer stage,
characterized by slowed-down metabolism, storage of
fat and dormancy (reviewed in Hekimi et al., 1998;
Roush, 1997; Thomas and Inoue, 1998). In this state,
the worms can live up to two months. Mutations of a
number of genes in the worm result in `longevity' or
extension of the life span as a result of an extended
dauer stage. Worms mutant for daf-2 (insulin-receptor
homologue) (Kenyon et al., 1993; Kimura et al., 1997),
age-1/daf-23 (PI3'K homologue) (Morris et al., 1996),
pdk-1 (PDK1 homologue) (Paradis et al., 1999) and
AKT-1 and AKT-2 (PKB homologues) (Paradis and
Ruvkun, 1998) share a common, `longevity' phenotype,
resulting from the extension of the dauer stage. daf-18,
a mutant in the PTEN C. elegans homologue is able to
suppress the age-1 mutant phenotype and to a lesser
extent the daf-2 phenotype, providing genetic evidence
that PTEN acts as a negative regulator of PI3'Kregulated pathways (Gil et al., 1999; Mihaylova et al.,
1999; Ogg and Ruvkun, 1998; Rouault et al., 1999).
Mutations of another gene, daf-16 (Lin et al., 1997;
Ogg et al., 1997), are also able to suppress daf-2, age-1/
daf-23, pdk-1 and akt-1 and akt-2 phenotypes. As
mentioned above, daf-16 encodes a homologue of the
mammalian transcription factors FKHR, AFX and
FKHRL1, which have recently been shown to be
phosphorylated and negatively regulated by mammalian PKB (Figure 1). Thus, a pathway homologous to
the insulin signaling pathway in mammals, dates back
700 ± 800 million years, prior to divergence of
nematodes.
The nematode studies have already played a key role
in identifying pertinent PKB targets and it is likely that
further substrates will be revealed by suppressor and
enhancer screens in this and other organisms.
Pros and cons of apoptosis signaling
Since activation of PI3'K provides a survival signal, it
is a prime target for regulation by pro-apoptotic
pathways. Evidence for such cross-talk has come from
studies of the inhibitory e€ects of ceramides on PI3'K
and PKB activation (Summers et al., 1998; Zhou et al.,
1998; Zundel and Giaccia, 1998). Ceramides can be
produced by certain pro-in¯ammatory cytokines such
as TNF. In many cells, this cytokine induces divergent
signals which promote (via caspase induction) as well
6099
Apoptosis and cell survival during oncogenesis
V Stambolic et al
6100
as suppress (via NF-kB and SAPK/JNK activation)
cell death (Basu and Kolesnick, 1998). TNF has also
been reported to activate PKB in HeLa cells (Pastorino
et al., 1999). This cytokine thus acts as a double-edged
sword, sensitizing cells and forcing a decision on their
fate (Baker and Reddy, 1998). Interactions have also
been reported with other signaling pathways such as
the ERK and p38 MAPK systems, resulting in
modulation (Hayashi et al., 1999). The intimate
interaction between pro- and anti-apoptotic signals is
perhaps a re¯ection of the dangers associated with
suppression of cell death, as evidenced by PTEN
mutations.
Overview, potential therapies, new targets, etc.
Multiple components of PI3'K-regulated cellular pathways are present in most cells of the body and it is
reasonable to assume, given the multitude of ways in
which PI3'K is regulated, that their activation will
depend on cell type and its immediate microenvironment. The ability of PI3'K to regulate a variety of
cellular processes also suggests that the cellular context
of downstream targets of this pathway could represents
a determining factor in the interpretation of a PI3'Kgenerated signal. PI3'K pathway seems to be conserved
throughout eukaryotic evolution, judged by recent
discovery of C. elegans PKB, PDK1 and PTEN and
their interplay with previously characterized PI3'K and
insulin receptor tyrosine kinase homologues in this
organism.
Identi®cation of oncogenic forms of PI3'K and
PKB, together with a high rate of PTEN mutations
in a variety of malignancies, has established PI3'K
signaling as one of the most frequently deregulated
cellular pathways in human cancer. Antibodies speci®c
for the phosphorylated forms PKB have greatly
facilitated monitoring of the status of this enzyme
and the PI3'K pathway in general. Similar to phosphospeci®c MAP kinase antibodies, these reagents have
allowed facile determination of the ¯ux through the
PI3'K pathway in cells and tissue sections and will
undoubtedly aid in assessment of the status of this
anti-apoptotic cascade in various pathologies. In view
of its relevance to human cancer, the PI3'K pathway is
receiving much attention from researchers focused on
the development of novel anti-cancer therapies. Known
PI3'K inhibitors, such as wortmannin and LY294002,
are highly toxic and exemplify some of the problems
associated with therapies targeting this molecule.
However, therapies aimed at a target further downstream in this pathway, might alleviate some of the
broader side e€ects a compound targeting an upstream
component might have (indeed wortmannin and
LY294002 also inhibit protein kinases such as ATM
and DNA-PK; Sarkaria et al., 1998; Wymann et al.,
1996). Since activation of PI3'K helps tumor cells
tolerate the consequences of genomic instability, it is
possible that tumor cells will exhibit di€erential
sensitivity to inhibitors of PKB, its targets and its
regulators, providing a clinically useful therapeutic
index. Indeed, while tumor cell activation of the
pathway reduces the ecacy of conventional chemotherapeutics and irradiation treaments, dependence
upon chronic PI3'K signaling may prove to be an
achilles heel.
Acknowledgments
TW Mak and JR Woodgett are supported by grants from
the Medical Research Council and Terry Fox Foundation
for Cancer Research. JR Woodgett is additionally
supported by a Howard Hughes International Scholarship.
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