Signaling Networks That Link Cell Proliferation and Cell Fate ...
Transcript of Signaling Networks That Link Cell Proliferation and Cell Fate ...
Signaling Networks That Link Cell Proliferation
and Cell Fate
Rosalie C. Sears and Joseph R. Nevins *
Department of Genetics
Howard Hughes Medical Institute
Duke University Medical Center
Box 3054
Durham, NC 27710
* Corresponding author
(919) 684-2746
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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The maintenance of normal cell function and tissue homeostasis is dependent on the
precise regulation of multiple signaling pathways that must accurately control cellular decisions
to either proliferate, differentiate, arrest cell growth, or initiate programmed cell death
(apoptosis). Cancer arises when clones of mutated cells escape this balance and proliferate
inappropriately without compensatory apoptosis. Many studies have revealed that the disruption
of multiple pathways is required for the development of cancer. Thus, not only is it critical to
understand the normal function of specific cellular pathways, but equally important is an
understanding of how they interconnect to synchronously regulate cell growth versus apoptosis.
Studies of both oncogenic processes as well as normal cell growth control have revealed
the key role played by the pathway controlling the retinoblastoma tumor suppressor protein (Rb).
A number of other cell regulatory activities, including the c-Myc and Ras proto-oncoproteins,
have also been shown to control not only cell proliferation, but also pathways leading to
apoptosis. In this review, we will discuss our current understanding of the Rb/E2F pathway, the
c-Myc transcription factor, and the Ras signaling molecule, followed by recent work showing
interconnections between these pathways, leading to a more comprehensive picture of the
network controlling the balance between cellular proliferation and apoptosis.
The Rb/E2F pathway and cellular proliferation
The retinoblastoma (Rb) gene was the first identified tumor suppressor gene and is now
recognized to play a central role in the control of cell proliferation [for a recent review, see (1)].
A large body of research has shown that the E2F transcription factor is a key target for the growth
suppressing activity of Rb as well as two Rb family members, p130 and p107 [reviewed in (2-4)].
Additional work has demonstrated that Rb function, including its ability to interact with E2F, is
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regulated by phosphorylation mediated by specific G1 Cyclin-dependent kinases. In particular,
the D-type cyclins, together with their associated kinases Cdk4 and Cdk6, initiate the
phosphorylation of Rb and Rb family members, inactivating the capacity of these proteins to
interact with E2Fs (Figure 1). This phosphorylation allows the accumulation of E2F1, E2F2 and
E2F3a transcription factors that activate the transcription of a large number of genes essential for
DNA replication as well as further cell cycle progression (2;3). In addition, phosphorylation of
Rb and p130 also disrupts complexes with E2F3b, E2F4 and E2F5 found in quiescent cells that
function as transcriptional repressors of S phase genes as well as the genes encoding the E2F1,
E2F2, and E2F3a proteins (Figure 1).
Amongst the E2F targets are genes encoding a second class of G1 cyclins, cyclin E and
the associated kinase cdk2. E2F activation of Cyclin E/Cdk2 kinase activity leads to the further
phosphorylation and inactivation of Rb, thus further enhancing E2F activity and increasing the
accumulation of Cyclin E/Cdk2 (Figure 1). This feedback loop, leading to a continual
inactivation of Rb independent of the action of Cyclin D/Cdk4, may represent at least part of the
restriction point identified by Pardee and colleagues, defined as the juncture in the cell
proliferation response when passage through the cell cycle becomes growth factor independent
(5;6). Finally, the activity of the G1 Cdks is negatively regulated by a family of small protein
inhibitors referred to as CKIs, including p21, p27, and the p16INK4a family (7). Deregulation of
many of the proteins that participate in this regulatory pathway, such as loss of the tumor
suppressor protein Rb, overexpression of D-type cyclins, or loss of the CKI p16 is an essential
step in the development of the majority of human tumors (8).
E2F activity represents a series of heterodimers made up of six distinct E2F proteins
complexed with one of two DP proteins. Various properties of the individual E2F family
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members suggest distinct functional roles for the proteins. The E2Fs can be classified into three
or possibly four subgroups based on their sequence, structure, association with specific Rb family
members, expression pattern, and putative function [for review, see (2;3)]. The first group
consists of E2F1, E2F2 and E2F3a, whose expression is regulated by cell growth, with maximal
accumulation at the G1/S boundary. These three E2Fs associate exclusively with Rb and appear
to play a positive role in cell cycle progression. The next subgroup is composed of E2F4 and
E2F5, which bind all three Rb family members and appear to function in transcriptional
repression in combination with the p130 protein in G0 and early G1 phase. The E2F4 and E2F5
genes are not transcriptionally regulated in relation to cell growth. E2F3b, like E2F4 and E2F5 is
constitutively expressed, but appears to bind exclusively to Rb. Finally, E2F6 is in its own group
since it lacks the domains that are involved in transactivation and binding to Rb family members.
E2F6, like E2F4 and E2F5 functions as a repressor of E2F-dependent transcription.
The Rb/E2F pathway and apoptosis
The Rb/E2F pathway has also been shown to integrate with pathways that control
programmed cell death. Evidence for a role for the Rb/E2F pathway in apoptosis can be seen in
Rb-deficient embryos, which show defects in fetal liver hematopoiesis, neurogenesis, and lens
development, and in all three tissue types, ectopic S-phase entry and extensive programmed cell
death is observed (9;10). Moreover, E2F1 knockout mice crossed with Rb deficient mice
partially rescues the apoptotic phenotype (11), and ectopic E2F1 expression has been
demonstrated to induce apoptosis under conditions where serum growth factors, which normally
impart survival signals, are limiting.
The p53 protein plays a key role in cellular decisions to either arrest the cell cycle,
allowing the repair of damaged DNA, or to commit to cell death [reviewed in (12)]. p53
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accumulation is negatively regulated by Mdm2, which targets it for ubiquitin-mediated
proteasome degradation; Mdm2 is, in turn, negatively regulated by p19ARF [for review see
(13;14)]. E2F1 induces the expression of p19ARF (15), thus directly connecting the Rb/E2F
pathway to p53 accumulation and an apoptotic response (Figure 2). However, E2F1 can also
induce apoptosis in a p53 independent manner, which could be attributed, at least in part, to the
activation of a p53 family member p73. E2F1 was shown to activate transcription of the p73
gene and both E2F1 and p73 were required for the p53 independent apoptosis observed in
peripheral T cells (16;17). In addition, E2F1 has been shown to specifically induce expression of
Apafl (18), which in combination with cytosolic cytochrome C and the caspase 9 protease forms
the so-called apoptosome. This ternary complex then activates the downstream caspase proteases
that are the final effectors of cell death (Figure 2).
Further evidence for a unique role for E2F1 in an apoptotic response is seen from the
observation that E2F1 is specifically induced following DNA damage (19). p53 is also induced
upon DNA damage , and its induction involves the ATM and ATR protein kinases, which are
activated by DNA damage, and then target p53 directly or indirectly through the Chk2 kinase [for
review, see (20)]. The phosphorylation of p53 by ATM/ATRthen blocks the ability of Mdm2 to
target p53 destruction. The induction of E2F1 in response to DNA damage similarly involves the
ATM and related ATR protein kinases (21) (Figure 2). The ATM/ATR kinases phosphorylate
E2F1 and this phosphorylation also blocks the proteasome-mediated degradation of E2F1. The
specificity of ATM and ATR for E2F1, rather than other E2F proteins, reflects a unique
phosphorylation site within the N terminal domain of E2F1 that overlaps with sequence shown to
be important for degradation. Presumably, this induction of E2F1 in response to DNA damage
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provides for a synergistic activation of p53 through the activation of p19ARF, or contributes to
p53-independent apoptosis, possibly via activation of p73 (Figure 2).
The c-Myc transcription factor
A variety of studies demonstrate that tight regulation of Myc protein levels is essential for
normal cell function. Myc expression is regulated at multiple levels. Myc RNA expression is
controlled by both cell growth associated increases in myc gene transcription and an increase in
myc mRNA stability (22). Recent work has demonstrated that Myc protein expression is not only
regulated by new synthesis, dependent upon its mRNA levels, but also by cell growth related
changes in Myc protein half-life (23). Specifically, Myc protein is subjected to very rapid
degradation in quiescent fibroblasts with a half-life of approximately 10 minutes, but is
dramatically stabilized following serum stimulation and the initiation of cell cycle progression,
extending the half-life to approximately 60 minutes. The degradation and turnover of Myc
protein, as well as many other cell cycle regulatory proteins, including E2F1 and p53, has been
shown to occur via the ubiquitin/26S proteasome pathway. This pathway involves a specific
multi-step process that results in a poly-ubiquitinated target protein, which is then rapidly
destroyed by the 26S proteasome [reviewed in (24)]. In most cases, the multi-ubiquitination of a
target protein is a regulated event, often controlled by posttranslational modification of the target
protein.
Similar to the Rb/E2F pathway, Myc expression couples cellular proliferation with the
induction of apoptosis under specific growth conditions where survival growth factors are
limiting [reviewed in (25;26)]. It has been suggested that the ability of Myc to concomitantly
induce proliferation and apoptosis provides a mechanism to guard against a single proliferative
lesion leading to unrestrained cell growth. Thus, in order for cells to survive with deregulated
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Myc expression they would require either a continuous supply of survival factors or the
acquisition of additional anti-apoptotic mutations. Indeed, lesions in either the p53 pathway or
overexpression of Bcl-2 family members that are anti-apoptotic have both been shown to
collaborate with Myc for tumor formation in vivo (27;28). Regions of Myc required for the
induction of apoptosis coincide with those needed for cell proliferation and include all the
requisite motifs characteristic of a transcription factor (29). Nonetheless, substantial evidence
indicates that c-Myc-induced apoptosis and proliferation are discrete downstream programs, since
activation of the molecular machinery mediating cell cycle progression is not required for c-Myc
induced apoptosis (30)
Myc-induced apoptosis is largely dependent upon p53 signaling and, similar to E2F1,
involves the induction of p19ARF, inhibition of mdm2, and elevated p53 expression (31) (Figure
3). However, it is also evident that Myc functions as an initiator of apoptosis by sensitizing cells
to a wide variety of apoptotic stimuli, including serum/growth factor deprivation, p53-dependent
response to genotoxic damage, virus infection, tumor necrosis factor, and CD95/Fas signaling
[reviewed in (26)]. The fact that c-Myc can sensitize so many disparate triggers of apoptosis
suggests an action at some common point in the regulatory and effector machinery of apoptosis.
Recent experiments have demonstrated that Myc-induced sensitization to apoptotic stimuli is
mediated by changes in the mitochondrial membrane resulting in the release of cytochrome c into
the cytoplasm, and this process can be blocked by survival factors such as insulin-like growth
factor 1 (IGF-1) (32) (Figure 3).
Connecting Myc with the Rb/E2F pathway
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A number of target genes for Myc have been identified that could play a role in the action
of Myc in cell proliferation control, as reviewed in (33). Myc has been shown to induce G1
Cyclin-dependent kinase activity (Figure 3). In addition to the direct transcriptional activation of
Cyclin D1 and D2, the Cyclin D partner Cdk4, and the phosphatase Cdc25A that removes
negative regulatory phosphates from the Cdks, Myc expression in quiescent cells leads to the
rapid induction of Cyclin E/Cdk2 activity, which in most cases is essential for Myc-induced
cellular proliferation (30;34;35). Recent experiments have demonstrated that it is the induction of
cyclin D1 and D2 by Myc that results in sequestration of the p27 cyclin kinase inhibitor away
from Cyclin E, which leads to the inactivation of Cyclin E/Cdk2 complexes (36;37). Myc
expression also strongly down-regulates the p27 Cyclin kinase inhibitor (30).
Myc overexpression has also been reported to induce E2F DNA binding activity (38).
While this could result from the Myc-mediated induction of Cyclin D/Cdk4 and/or Cyclin
E/cdk2, leading to the phosphorylation and inactivation of Rb family members and the release of
free E2F transcription factor, recent work has also shown that Myc directly contributes to the
activation of the E2F1, E2F2 and E2F3 genes (Figure 3) (23;39;40). Specifically, ectopic Myc
expression in quiescent fibroblasts induces E2F1, E2F2 and E2F3 mRNA accumulation in the
absence of G1 Cdk activity and G1 to S phase progression.
Myc and E2F transcription factors share a number of functional properties including the
ability to induce quiescent cells to enter the cell cycle and progress into S phase and to control
cell fate by activating the p53-dependent apoptotic pathway. The fact that Myc and E2F share
these functional properties, coupled with the fact that Myc can induce E2F gene expression,
raises the possibility that Myc function might be mediated, at least in part, through the action of
the E2F transcription factors. This possibility has recently been addressed using a genetic
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approach (41). Primary mouse embryo fibroblases (MEFs) from embryos deleted for specific
E2F genes were used to evaluate the functional relationship between Myc and various E2F
proteins. Experiments using these E2F-deficient MEFs showed that the ability of Myc to induce
S phase in the absence of other mitogens is severely impaired in MEFs deleted for E2F2 or E2F3,
but not E2F1 or E2F4. In contrast, Myc induced apoptosis in primary serum-deprived MEFs was
dramatically reduced in cells deleted for E2F1, but not affected by E2F2 or E2F3 deletion. In
addition, the ability of Myc to induce p53 expression in the absence of survival factors is also
dependent upon the presence of functional E2F1, but not E2F2 or E2F3 (R. Sears, unpublished
data). Thus, the induction of specific E2F activities is an essential downstream event in the Myc
pathway that controls cell proliferation versus apoptosis, and some of the functions of Myc, such
as the induction of p19ARF and p53 could be explained, at least in part, with one pathway leading
through E2F activation (Figure 3).
Ras signaling pathways
The ras proto-oncogene plays a critical role in cell growth control as a central component
of mitogenic signal-transduction pathways [reviewed in (42;43)]. Studies on Ras signaling over
the past two decades have shown that this complex regulatory activity can stimulate very diverse
biological responses such as cell proliferation or growth arrest, senescence or differentiation, and
apoptosis or survival (44). Mitogen stimulation results in an increase in the active, GTP-bound
form of Ras. Oncogenic activation of Ras, due to point mutations that maintain Ras in the GTP-
bound form, occurs in a large number of human cancers (45).
Ras function is carried out by a family of Ras effector molecules, which specifically bind
to and are activated by Ras-GTP. Among the effector signaling pathways are the Raf/MEK/ERK
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kinase cascade, primarily involved in plasma membrane-to-nucleus signaling (46), the Ral
GTPase signaling pathway, also involved in G1 to S phase progression (47;48), and the PI3-
kinase/AKT pathway, which is involved in cell survival signaling (49) (Figure 4). Although these
effector pathways were originally thought to mediate discrete cell functions, it is now apparent
that there is extensive overlap in their function; and Ras-mediated phenotypic responses appears
to require the combination of multiple effector pathways [recently reviewed in (48;50)]. For
example, while the Raf/MEK/ERK pathway plays a major role in Ras-mediated cellular
proliferation, it also affects cell survival (51), and the PI3-K/AKT pathway, which plays a major
role in protecting cells from apoptosis, can also facilitate G1 to S phase progression [reviewed in
(47;48)].
A number of cell survival signals, generated in response to growth factor stimulation,
function through the Ras, PI3-K, and AKT pathway, and result in the inhibition of cytochrome C
release (52). This may in part be through AKT-mediated phosphorylation and functional
inactivation of BAD, a pro-apoptotic Bcl-2 family protein that promotes the release of
cytochrome C by interfering with the anti-apoptotic activity of Bcl-XL at the mitochondrial
membrane (53;54) (Figure 4). However, AKT also appears to have a post-mitochondrial function
in cell survival since even in the presence of released cytochrome C, AKT can inhibit cell death
(55). This appears to be a function of AKT-mediated inhibition of caspase-9 and –3 activation,
possibly by direct phosphorylation of caspase-9. A recent report also demonstrates that AKT
promotes the translocation of Mdm2 from the cytoplasm to the nucleus, facilitating the targeting
of p53 for destruction (56) (Figure 4).
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Connecting Ras with the Rb/E2F pathway
Activation of Ras signaling pathways has been shown to be essential for cells both to
leave a quiescent state and to progress through G1 phase of the cell cycle. Based on experiments
in cells expressing wild-type or mutant Rb, the main role for Ras in G1 progression is to
inactivate Rb through the activation of G1 Cdks (39;57). This has been shown to occur through
the stimulation of Cyclin D1 transcription as well as increases in the level of Cyclin D1/Cdk4
kinase activity (58;59). As depicted in Figure 4, three Ras effector pathways, the Raf/MEK/ERK
cascade, PI3-K signaling, and Ral activation are all involved in stimulating Cyclin D1 gene
transcription, with maximal stimulation requiring the co-operative action of several pathways
[reviewed in (47)]. In addition, PI3-K/AKT signaling, via inhibition of glycogen synthase kinase
(GSK-3), increases the stability of the Cyclin D1 protein (60).
Ras activity also stimulates transcription of the cyclin kinase inhibitor p21 and p16INK4a,
which could underlie the ability of Ras to induce cellular senescence (61). In contrast, Ras
activation has been shown to down-regulate the p27kip1 CKI, resulting in the activation of
Cyclin E/Cdk2 (62). The down-regulation of p27 involves both the ERK and PI3-K effector
signaling pathways, and it is associated with a decrease in the rate of p27 translation, stability and
association with Cyclin E/Cdk2, and this is essential for Ras-mediated entry into S phase (63).
Ras, via Raf, has also been reported to activate the Cdc25A phosphatase that removes inhibitory
phosphates from Cdk2 and Cdk4 contributing to their activation (64). Taken together, these data
place Ras upstream of the G1 Cdk/Rb/E2F pathway.
Connecting Ras with Myc
One of the classic paradigms of cellular transformation, and the original basis for the
multi-hit theory of cancer, is the collaborative effects of Myc and Ras coexpression in primary
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fibroblasts. While Myc expression or Ras expression alone can readily transform immortalized
cell lines, which have already escaped normal growth arrest check points, coexpression of both
Myc and activated Ras is necessary for the transformation of primary or early-passage cells as
well as some cell lines (65). However, with the complex and diverse signals emanating from Ras,
it is not surprising that the molecular mechanisms underlying Myc/Ras collaboration, both for
normal cell proliferation and oncogenesis, have remained elusive despite many years of intensive
research. One clear mechanism for Ras/Myc collaboration in oncogenesis is the fact that Ras
activation can provide a survival signal, via the PI3-K/AKT pathway, and prevent the
overexpression of Myc from inducing apoptosis (66). The ability of Ras to protect against Myc
induced apoptosis is key when one thinks about Myc and cancer, since Myc-induced apoptosis
can prevent the outgrowth of a cell population, even though Myc is stimulating cell cycle transit.
Myc and Ras collaboration can also be seen by the fact that while high-level expression of
c-Myc alone results in cellular proliferation, coupled with the induction of Cyclin E/Cdk2 kinase
activity and the inactivation of the p27 CKI (67;68), lower levels of Myc do not have this
function unless coexpressed with activated Ras (39). One molecular mechanism that is likely to
underlie this observation is based on recent experiments, which show that Ras signaling stabilizes
and increases the accumulation of functional Myc transcription factor (23). This finding also
provides an important mechanism that is likely to underlie Myc/Ras collaboration in oncogenic
cell transformation since Myc overexpression appears to be the key event in its oncogenic
activation. Indeed, Myc overexpression alone is sufficient for pre-malignant and malignant
transformation of some cell types in transgenic mouse models (69;70). As previously discussed,
Myc protein levels are controlled by ubiquitin-mediated proteolysis in a cell growth dependent
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manner. Further experiments have shown that the serum-induced increase in Myc protein half-life
is dependent upon activation of Ras signaling.
As diagramed in Figure 4, two Ras effector pathways contribute to the stabilization of
Myc - the Raf/MEK/ERK kinase cascade and the PI3-K/AKT signaling pathway. These Ras
effector pathways control the phosphorylation of two sites in the N-terminus of Myc, which are
conserved between all Myc family members, and have opposing effects on Myc stability (71).
Specifically, activation of ERK kinases results in the direct phosphorylation of Serine 62, which
stabilizes Myc protein, and activation of AKT phosphorylates and inactivates GSK-3 that is
responsible for phosphorylation of Threonine 58, which destabilizes Myc and targets it for
ubiquitin-mediated degredation. In addition, there is a hierarchical relationship between these
two phosphorylation sites where phosphorylation of Threonine 58 requires prior phosphorylation
of Serine 62. Thus, following serum stimulation and entry into the cell cycle, myc gene
transcription is induced, myc mRNA accumulates and Myc protein is synthesized. At the same
time Ras activation of ERKs leads to the phosphorylation of the newly synthesized Myc on
Serine 62 and activation of AKT down-regulates GSK-3 inhibiting the destabilizing
phosphorylation of Threonine 58, thus allowing rapid and high-level accumulation of Myc.
Then, as the cell cycle progresses and AKT activity falls, GSK-3 becomes active leading to the
phosphorylation of Threonine 58 and the increased degradation of Myc. As such, Myc protein
levels decline later in G1 and then persists at this level as a cell continues to grow. While this
regulation of Myc stability appears complex, it allows for precise timing and controlled levels of
Myc expression.
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Alterations in G1 signaling pathways in cancer
Lesions in the Rb/E2F, Myc, and Ras pathways occur in virtually all human tumors
described to date [for reviews, see (45;72;73)]. As discussed in this review, each of these
pathways plays an important role in the control of both cellular proliferation as well as apoptosis.
Moreover, recent evidence demonstrates that extensive cross-talk exists between these cell
regulatory pathways (see Figure 4). Specifically, Ras activation facilitates Myc function by
stabilizing Myc protein allowing high-level accumulation of functional Myc transcription factor;
and at the same time blocking Myc's pro-apoptotic effects. Ras activation also leads to the
activation of cyclin D/cdk4 and the Rb/E2F pathway. Myc activation can also lead to cyclin
D/cdk4 and Cyclin E/cdk2 activation. Finally, Myc activation can directly feed into the Rb/E2F
pathway by inducing E2F gene expression; and Myc function, both for proliferation and
apoptosis, is at least in part dependent upon activation of these specific E2F proteins. It is clear
from these recent experiments demonstrating that extensive networking exists between cellular
pathways controlling proliferation and apoptosis, that understanding how molecular pathways
interconnect is essential for our understanding of the cancer disease process, and for the
development of meaningful treatments.
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Figure Legends
Figure 1. The Rb/E2F pathway.
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Figure 2. Connecting the Rb/E2F pathway with apoptosis and the p53 response.
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Figure 3. Connecting Myc with the Rb/E2F pathway, S phase and apoptosis.
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Figure 4. Connecting multiple Ras effector pathways with the Rb/E2F pathway and the
control of Myc accumulation.
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Rosalie C. Sears and Joseph R. NevinsSignaling networks that link cell proliferation and cell fate
published online January 22, 2002J. Biol. Chem.
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