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

j.nevins@duke.edu

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 22, 2002 as Manuscript R100063200 by guest on M

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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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