4 Control of the Cell Cycle

Marcos Malumbres

S UMMARY OF K EY P OI N T S
• Most cells in postnatal tissues are intrinsic factors, such as growth cycle arrest, senescence, or death in
quiescent. Exceptions include factor or cytokine exposure, cell-to- response to cellular stress.
abundant cells of the hematopoietic cell contact, and metabolic • Checkpoints minimize replication
system, skin, and gastrointestinal constraints. and segregation of damaged DNA,
mucosa, as well as other minor • The internal cell cycle machinery is or the abnormal segregation of
progenitor populations in other controlled largely by oscillating levels chromosomes to daughter cells, thus
tissues. of cyclin proteins and by modulation protecting cells against genome
• Many quiescent cells can reenter of cyclin-dependent kinase (Cdk) instability.
into the cell cycle with the activity. One way in which growth • Disruption of cell cycle controls is a
appropriate stimuli, and the control factors regulate cell cycle hallmark of all malignant cells.
of this process is essential for tissue progression is by affecting the levels Frequent tumor-associated
homeostasis. of the D-type cyclins, Cdk activity, alterations include aberrations in
• The key challenges for proliferating and the function of the growth factor signaling pathways,
cells are to make an accurate copy retinoblastoma protein. dysregulation of the core cell cycle
of the 3 billion bases of DNA • Cell cycle checkpoints are machinery, and/or disruption of cell
(S phase) and to segregate the surveillance mechanisms that link the cycle checkpoint controls.
duplicated chromosomes equally rate of cell cycle transitions to the • Because cell cycle control is
into daughter cells (mitosis). timely and accurate completion of disrupted in virtually all tumor types,
• Progression through the cell cycle is prior dependent events; p53 is a the cell cycle machinery provides
dependent on both extrinsic and checkpoint protein that induces cell multiple therapeutic opportunities.

Most cells in the adult body are quiescent—that is, they are biochemi- exposed to external agents (e.g., reactive chemicals and ultraviolet
cally and functionally active but do not divide to generate daughter light) and to internal agents (e.g., byproducts of normal intracellular
cells. However, specific populations retain the ability to proliferate metabolism, such as reactive oxygen intermediates) that can induce
throughout the adult life span, which is essential for proper tissue DNA damage. A major goal of specific cell cycle checkpoints is to
homeostasis. For example, cells of the hematopoietic compartment detect DNA damage and activate cell cycle arrest and DNA repair
and the gut have a high rate of turnover, and active proliferation is mechanisms, thereby maintaining genomic integrity.
essential for the maintenance of these tissues. On average, about 2 Anything that disrupts proper cell cycle progression can lead to
trillion cell divisions occur in an adult human every 24 hours (about either the reduction or the expansion of a particular cell population.
25 million per second). The decision about whether to proliferate is It is now clear that such changes are a hallmark of tumor cells, which
tightly regulated.1,2 It is influenced by a variety of exogenous signals, carry mutations that impair signaling pathways that suppress prolifera-
including nutrients and growth factors, as well as inhibitory factors, tion and/or activate pathways that promote proliferation. In addition,
and the interaction of the cell with its neighbors and with the underlying most (if not all) human tumor cells have mutations within key
extracellular matrix. Each of these factors stimulates intracellular signal- components of both the cell cycle machinery and checkpoint path-
ing pathways that can either promote or suppress proliferation. The ways.1,5,6 This characteristic has important clinical implications, because
cell integrates all of these signals, and if the balance is favorable, the the presence of these defects can modulate cellular sensitivity to
cell will initiate a series of processes, collectively known as the cell chemotherapeutic regimens that induce DNA damage or mitotic
cycle, that lead to cell division into two daughter cells. catastrophe. This chapter focuses on the mechanics of the cell cycle
During the past four decades, extensive effort has been placed on and checkpoint signaling pathways and discusses how this knowledge
unraveling the basic molecular events that control the cell division can lead to the efficient use of current anticancer therapies and to the
cycle. Studies in a variety of organisms have identified evolutionarily development of novel agents.
conserved machinery that regulates eukaryotic cell cycle transitions
through the action of key enzymes, including cyclin-dependent kinases
(Cdks) and other kinases.3 It is essential that proliferating cells copy CELL DIVISION CYCLE
their genomes and segregate them to the daughter cells with high Overview of the Cell Cycle Machinery
fidelity. Eukaryotic cells therefore have evolved a series of surveillance
pathways, termed cell cycle checkpoints, that monitor for potential Cell division proceeds through a well-defined series of stages
problems during the cell cycle process.4 Human cells are continuously (Fig. 4.1). First, the cell moves from the nonproliferative, quiescent

56
 Control of the Cell Cycle • CHAPTER 4 57

G1/S Intra-S G2/M SAC

Microtubule nucleation
DNA replication & repair Cytokinesis
spindle

Cell cycle entry Centrosome duplication Chromosome Chromosome

G1 and separation condensation segregation

G0 G1 S G2 M M
Cyclin D
Cyclin E Cyclin A Cyclin B

Restriction point
Figure 4.1 • The cell division cycle. One round of cell division requires high-fidelity duplication of deoxyribonucleic acid during the S phase of the cell
cycle and proper segregation of duplicated chromosomes during mitosis, or the M phase. Before and after the S phase and M phase, the cell transits through
“gap” phases, termed G1 and G2. Quiescent (G0) cells require the appropriate mitogenic stimuli for reentry into the cell cycle by inducing D-type cyclins.
These stimuli are required up to a specific moment, known as the restriction point, at which time the cell cycle becomes independent of the mitogenic signals.
The cellular processes required for transition through the different cell cycle phases are controlled by the action of regulatory pathways and mostly are driven
by the expression of specific cyclins and the activation of cyclin-dependent kinases. These transitions are monitored by signaling pathways known as the cell
cycle checkpoints (represented by blue columns) that function to arrest the cell cycle at different phases (G1/S, intra–S phase, G2/M) in the presence of DNA
damage. In addition, the spindle assembly checkpoint (SAC) delays the exit from mitosis in the presence of defective chromosome alignment by inhibiting
the degradation of cyclin B1 and the subsequent inactivation of cyclin-dependent kinases.

(also known as G0) state into the first gap phase, or G1, in which (originally cdc2), was cloned by virtue of its ability to complement a
the cell essentially is readying itself for the cell division process. This mutant cdc2 yeast strain.12 Subsequent studies identified additional
process involves a dramatic upregulation of both transcriptional and human Cdks and determined that they regulate distinct cell cycle
translational programs, not only to yield the proteins required to stages; for example, Cdk4 and Cdk6 regulate cell cycle entry, whereas
regulate cell division but also to essentially double the complement Cdk2 may have specific roles during the G1-to-S transition and S
of macromolecules so that one cell can give rise to two cells without a phase. Cdk1 is essential in the control of G2 and mitosis and also
loss of cell size. Not surprisingly, this process takes a significant amount may play additional roles in earlier stages. The human genome encodes
of time (from 8 to 30 hours in cultured cells) and energy.7 Studies about 15 additional Cdks, although the functional relevance of many
with cultured cells show that mitogenic growth factors are essential for of them is still unknown.3,8–11
continued passage through the G1 phase. Specifically, if growth factors The activity of these kinases is controlled by multiple regulatory
are withdrawn at any point during this phase, the cell will not divide. mechanisms. Cdks act in association with a cyclin subunit that binds
However, as it nears the end of the G1 phase, the cell passes through a to the conserved PSTAIRE helix within the kinase.13 Cyclin binding
key transition point, called the restriction point, whereupon it becomes causes a reorientation of residues within the active sites that is essential
growth factor independent and is fully committed to undergoing cell for kinase activity.8,13 The associated cyclin also determines the substrate
division.1 The cell then enters the DNA synthesis phase, or S phase, specificity of the resulting cyclin/Cdk complex. The cyclins are quite
in which each of the chromosomes is replicated once and only once. divergent, especially in their N-terminal sequences, but they all share
This is followed by a second gap phase, called G2, which lasts 3 to a highly conserved 100–amino acid sequence, called the cyclin box,
5 hours, and the cell then initiates mitosis, or the M phase, a rapid that mediates Cdk binding and activation.9 As their name implies,
phase (lasting about 1 hour) in which the chromosomes are segregated. cyclins originally were identified as proteins whose expression was
On completion of mitosis, the daughter cells can enter quiescence restricted to a particular stage of the cell cycle14 because of cell
or initiate a second round of cell division, depending on the milieu. cycle–dependent regulation of both cyclin gene transcription and
Progression throughout the different phases of the cell cycle depends protein degradation. The human genome encodes more than 25
on the activity of key molecules that drive transcription, translation, cyclin-like proteins, yet only four distinct subclasses—D-, E-, A-, and
or the structural changes required for cell division (Table 4.1). A large B-type cyclins—are thought to play key roles in cell cycle regulation
number of these changes are modulated by protein phosphorylation (see Fig. 4.1).9 Each of these classes has a few paralogs (e.g., cyclin
and dephosphorylation, but other molecular processes such as D1, D2, and D3; cyclin E1 and E2; cyclin A1 and A2; and cyclin
SUMOylation, acetylation, or ubiquitin-dependent protein degradation B1, B2, and B3). The relative roles of these paralogs are not completely
are crucial for ordered cell cycle progression. Many of these cell cycle clear in most cases. Although some functional redundancy may exist,
regulators have been involved in tumor development or may be attractive published evidence suggests differences in regulation, expression pattern,
targets for cancer therapy and will be introduced in the following and substrate specificity.9,15
sections. The activation of cyclin/Cdk complexes requires considerable
posttranslational regulation.8,9,16 First, kinase activation is dependent
Cyclin-Dependent Kinases and Their Regulators on phosphorylation of a threonine residue that is adjacent to the
The Cdks constitute a large subfamily of highly conserved Ser/Thr active site (Thr160 in Cdk2). This phosphorylation is catalyzed by a
kinases that are defined by their dependence on a regulatory subunit, kinase, called Cdk-activating kinase (CAK).17,18 In mammalian cells,
called a cyclin.8–11 The first identified human Cdk, called Cdk1 phosphorylation occurs after cyclin binding. Although it appears that
58 Part I: Science and Clinical Oncology

Table 4.1 Representative Molecules Involved in Cell Cycle Regulation

Protein Family/
Complex Representative Members Function
KINASES
Cyclin-dependent Heterodimeric complexes formed of a cyclin (A, B, Phosphorylation of multiple proteins to drive progression
kinases D, and E types) and a Cdk (Cdk1, Cdk2, Cdk4, Cdk6); throughout the different phases of the cell cycle
Cdk7 functions as a Cdk-activating kinase
Wee1/Myt1 Wee1, Myt1 Inactivation of Cdks
Aurora-A holoenzyme Aurora-A and its nonkinase activator, Tpx2 Spindle dynamics, chromosome segregation, and cytokinesis
Chromosome passenger Aurora-B (Aurora-C?), Incenp, survivin, borealin Chromosome segregation
complex (CPC)
Polo-like kinases Plk1–Plk5 Centrosome function, chromosome segregation, and cytokinesis
NIMA-related kinases Nek1–Nek11 Centrosome function and mitosis
Haspin Haspin Phosphorylates histone H3 to recruit the CPC
Mastl Mastl Inhibition of PP2A phosphatases
CDK INHIBITORS
INK4 proteins p16INK4a, p15INK4b, p18INK4c, p19INK4d Inhibition of Cdk4 and Cdk6 during G1 progression
Cip/Kip inhibitors p21Cip1, p27Kip1, p57Kip2 Cdk inhibition and other roles in transcription or the cytoskeleton
TRANSCRIPTIONAL CONTROL
Retinoblastoma family pRB, p107, p130 Repression of the transcription of genes required for the cell cycle
E2F transcription factors E2F1–E2F8 Transcription of genes encoding S-phase and mitotic regulators
PHOSPHATASES
Cdc14 Cdc14a, Cdc14b Control of transcription and cell cycle progression
Cdc25 Cdc25a, Cdc25b, and Cdc25c Cdk activation and cell cycle progression
PP1 Multiple complexes with different regulatory Protein dephosphorylation
subunits
PP2A Multiple complexes with different regulatory Protein dephosphorylation; major Cdk-counteracting phosphatase
subunits
UBIQUITIN LIGASES
SCF E3 ubiquitin ligase formed of Rbx1, Cul1, Skp1, and Targets multiple cell cycle regulators (e.g., p27Kip1 or cyclin E) for
an F-box protein (e.g., Skp2 or βTrCP) ubiquitin-dependent degradation during interphase
APC/C E3 ubiquitin ligase composed for multiple subunits Targets multiple cell cycle regulators for ubiquitin-dependent
including Cdc20 or Cdh1 as coactivator molecules degradation during mitosis (cyclin B, securin) and G1 (e.g.,
Aurora-A, Plk1, or Tpx2)
OTHER FUNCTIONS
Kinesins More than 600 proteins including Eg5, CenpE, Microtubule-based motor proteins that hydrolyze ATP to generate
MCAK energy for movement along microtubule fibers
Cohesins and condensins Smc family (1–4), Rad21, Pds5, and SA (Stag) Structure and regulation of DNA
proteins, among others
Kinetochore proteins More than 100 proteins including the CENP Linking the chromosomes to microtubules and regulation of
(centromere-binding proteins) family, and the Knl1, microtubule dynamics
Mis12, Ndc80, and Dam1 complexes, among many
others
Mitotic checkpoint Mad2, Bub3, BubR1, and Cdc20 Inhibit the APC/C until complete bipolar attachment of
complex (MCC) chromosomes to the mitotic spindle

APC/C, Anaphase-promoting complex/cyclosome; ATP, adenosine triphosphate; Cdk, cyclin-dependent kinase; CPC, chromosomal passenger complex; NIMA, never in mitosis,
gene A; SCF, Skp1–Cullin1–F-box.

at least two mammalian CAKs exist, the major CAK is a trimolecular Activation of the cyclin/Cdk complex is then dependent on the action
complex composed of Cdk7, cyclin H, and Mat1. The Cdk7/cyclinH/ of a dual-specificity phosphatase called Cdc25. Mammalian cells have
Mat1 complex also is required for the control of basal transcription three different Cdc25 proteins, called Cdc25a, Cdc25b, and Cdc25c,
via regulation of RNA polymerase II function.11,18 Second, when it which show some specificity for different cyclin/Cdk complexes and
is first formed, the cyclin/Cdk complex frequently is subject to inhibi- cell cycle stages.20
tory phosphorylation of Thr14 and Tyr15 residues within the Cdk’s Cdks are modulated by a series of Cdk inhibitors (CKIs) that play
active site by the Wee1 (Tyr15) and Myt1 (Thr14 and Tyr15) kinases.9,19 a key role in restricting the activity of the cyclin/Cdk complexes in
 Control of the Cell Cycle • CHAPTER 4 59

response to either external signals or internal stresses.1,21 The CKIs regulates E2F through two distinct mechanisms. First, its association
can be divided into two distinct families based on their biological with E2F is sufficient to block the transcriptional activity of E2F.
properties. The first CKI family is named INK4, based on their roles Second, the pRB/E2F complex can recruit histone deacetylases to the
as INhibitors of Cdk4. The INK4 family has four members called promoters of E2F-responsive genes and thereby actively repress their
p16INK4a, p15INK4b, p18INK4c, and p19INK4d (encoded by the CDKN2A-D transcription. Cell cycle entry requires the phosphorylation of pRB
genes in humans). These INK4 proteins specifically prevent the binding by cyclin/Cdk complexes and the consequent dissociation of pRB
of cyclins to monomeric Cdk4 and Cdk6 but do not inhibit other from E2F.1,8,33
Cdks.13,21 The second CKI family is named Cip/Kip and includes Studies have identified eight E2F genes that encode nine different
three members: p21Cip1 (also called p21Waf1), p27Kip1, and p57Kip2 E2F proteins.34,35 Pocket proteins can regulate a subset of these factors:
(encoded by the CDKN1A-C genes in humans).21 These Cip/Kip E2F1, E2F2, E2F3a, E2F3b, E2F4, and E2F5. These E2F proteins
proteins have two major activities. First, they do not bind to monomeric associate with a dimerization partner, called DP, and the resulting
Cdks but associate with and inhibit the activity of cyclin/Cdk complexes complexes function primarily as either activators (E2F1, E2F2, and
already formed. Second, Cip/Kip proteins may promote the assembly E2F3a) or repressors (E2F4 and E2F5) of transcription under the
of cyclin D/Cdk4/6 complexes without dramatically perturbing its direction of the pocket proteins. Observations have suggested that
kinase activity.21,22 This activity is modulated by phosphorylation of several of these factors may act either as positive or negative regulators
Cip/Kip proteins by Src, Jak2, and Akt kinases,23–26 directly linking of transcription, depending on the cell type or the differentiation
Cdk regulation with the activity of these upstream mitogenic pathways. state.34,36–38 Most classic E2F target genes are regulated by the coor-
In addition to regulating the cell cycle, Cip/Kip proteins play important dinated action of these repressor and activator E2Fs.
roles in apoptosis, transcriptional regulation, cell fate determination,
cell migration, and cytoskeletal dynamics.27,28 Ubiquitin-Dependent Protein Degradation
The original observation that cyclin levels are tightly regulated during
Retinoblastoma Proteins and E2F Transcription Factors the cell cycle implies that these proteins are regulated not only at the
The retinoblastoma protein (pRB) was originally identified by virtue transcriptional level but also at the protein level. It is now evident
of its association with hereditary retinoblastoma.29 It behaves as a that ubiquitin-mediated protein degradation is a major regulatory
classic tumor suppressor: affected persons inherit a germline mutation mechanism to ensure ordered transition through the different phases
within one allele of the pRB-encoding gene, RB1, and loss of hetero- of the cell division cycle. Ubiquitylation depends on an enzymatic
zygosity is seen in all of the tumors. Subsequent studies showed that cascade, in which ubiquitin ligases recruit specific substrates for
the transforming ability of small DNA tumor viruses, including human modification. About 600 ubiquitin ligases are encoded by the human
papillomavirus, adenovirus, and simian virus, was dependent on the genome. Among them, the Skp1–Cullin1–F-box (SCF) and the
ability of virally encoded oncoproteins (E7, E1A, and SV40, respec- anaphase-promoting complex/cyclosome (APC/C) are known for
tively) to bind and inhibit pRB.30–32 Moreover, the RB1 gene is driving the degradation of cell cycle regulators to accomplish irreversible
inactivated in approximately one-third of all sporadic human tumors. cell cycle transitions.39–41 SCF has three core components: a RING
pRB and the pRB-related proteins p107 and p130, collectively finger protein, called Rbx1, which recruits the E2-ubiquitin conjugate;
known as the pocket proteins, are transcriptional repressors whose a cullin (Cul1); and Skp1 (Fig. 4.2). Skp1 acts to recruit a family of
major function is to inhibit the expression of cell cycle–related proteins proteins, called F-box proteins, which determine the target specificity
(see Table 4.1).33 This suppressive activity is largely dependent on the of the SCF complex. Once SCF binds its substrate, it transfers a
ability to prevent cell cycle entry through inhibition of the E2F ubiquitin molecule to lysine residues within the target protein to
transcription factors.33,34 The E2F proteins regulate the cell cycle– create a polyubiquitin chain, which targets the substrate to the protea-
dependent transcription of numerous targets, including core components some for degradation.42
of the cell cycle control (e.g., cyclin E and cyclin A) and DNA replica- The APC/C is a much larger complex, but it also contains a RING
tion (e.g., Cdc6, Cdt1, and the Mcm proteins) machineries.33–35 pRB finger protein, called Apc11, to recruit the E2-ubiquitin conjugate,

Apc11 E2
E2
Rbx1
Ubiquitin
Ubiquitin
Substrate
Substrate Apc2
P
Cul1 P
Activator
(Cdc20
F-box or Cdh1)
protein

Skp2

SCF ubiquitin ligase APC/C ubiquitin ligase

Figure 4.2 • Ubiquitin ligases. The Skp1–Cullin1–F-box (SCF) and anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligases play a key role in
enabling forward passage through key cell cycle transitions. These ligases are both large complexes that include three core components: a scaffolding protein
called a cullin in SCF, a protein that recruits the E2 and its associated ubiquitin molecule, and a specificity factor (called the F-box protein in SCF and the
activator in APC/C) that recruits the substrate. SCF and APC/C catalyze polyubiquitination of their substrates, which acts as a signal for substrate degradation
by the 26S proteasome. SCF has numerous substrates whose degradation promotes passage through the early stages of the cell cycle, including the cyclin-
dependent kinase inhibitor p27Kip1, cyclin E, E2F-1, and Cdt1. APC/C is essential for mitotic progression (by promoting degradation of securin and the
mitotic cyclins) and for cell cycle exit (by promoting degradation of multiple cell cycle regulators).
60 Part I: Science and Clinical Oncology

and a core cullin subunit (Apc2). In addition, APC/C is activated by centromeric region of chromosomes is determined by a complex
a cofactor that, in a manner comparable with that of the F-box proteins epigenetic, DNA sequence–independent mechanism.72–74 Kinetochores
of SCF, establishes substrate specificity (see Fig. 4.2).39 Cdc20 is the regulate the proper bipolar attachment between microtubules and
mitotic cofactor of APC/C, and it targets several cell cycle regulators chromosomes in order to distribute the replicated genome from a
(A- and B-type cyclins, Nek2, and securin) during mitotic entry and mother cell to its daughters.71,75 Given the relevance of the kinetochore
the metaphase-to-anaphase transition. Cdh1 (also known as FZR1 in in genome integrity, the composition of the kinetochore and the
mammals) replaces Cdc20 during mitotic exit and is the cofactor identification of various physical and functional modules within its
responsible for the elimination of many cell cycle regulators in the substructure is under deep investigation.76,77
G0 or G1 phase to prevent unscheduled DNA replication.43 Recent
studies have provided new insights into the intricate relationship Cell Cycle Phosphatases
between ubiquitylation and the cell division apparatus, including new Cdc14 phosphatases are the major Cdk-counteracting proteins in
roles for atypical ubiquitin chains, new mechanisms of regulation, yeast. Although two family members exist in mammals, their relevance
and extensive cross talk between ubiquitylation enzymes.44–49 in the cell cycle is not well understood.78,79 In eukaryotes, two major
complexes, PP1 and PP2A, account for more than 90% of protein
Mitotic Spindle and Mitotic Kinases and Kinesins phosphatase activity. In fact, these enzymes correspond to hundreds
In addition to the central role of Cdks in the cell cycle, many other of phosphatase complexes assembled from a few catalytic subunits
kinases play critical roles in cell cycle progression.3 Aurora and Polo (PP1α, PP1β/δ, and PP1γ1/2 for PP1, and the Cα and Cβ isoforms
kinases were first identified in genetic studies in flies as a result of for PP2A) and a diverse array of regulatory subunits.80 Recent evidence
their essential role in mitotic progression.50–52 Each of these families suggests that these protein families cooperate in the dephosphorylation
of kinases is represented by a single member in yeast, whereas three of most cell cycle kinase targets, including the retinoblastoma family
Aurora kinases (Aurora-A, Aurora-B, and Aurora-C) and five Polo-like or mitotic phosphoproteins.78,81–83 PP1 and PP2A are major phospha-
kinases (Plk1 through Plk5) exist in humans (see Table 4.1).52,53 tases responsible for pRB dephosphorylation during mitotic exit,
Aurora-A participates in several processes required for building a bipolar although the relative roles of these complexes or the particular
spindle, including centrosome separation and microtubule dynamics. holoenzymes involved are not clear.81,82 Similarly, both PP1 and PP2A
Aurora-A is activated by Tpx2, and the Aurora-A–Tpx2 holoenzyme are required for dephosphorylation of hundreds of mitotic proteins
may have critical implications in tumor development.51,52,54 Aurora-B, that are phosphorylated by Cdk1, as well as the other mitotic kinases.83,84
on the other hand, is part of the chromosome passenger complex Thus it has been suggested that the cell cycle ultimately is regulated
(CPC) that localizes to the kinetochores from prophase to metaphase by the dynamic equilibrium between Cdks (and partially by the other
and to the central spindle and midbody in cytokinesis. Other com- mitotic kinases) and PP1/PP2A activity. In the absence of Cdk activity,
ponents of the CPC include INCENP, survivin, and borealin, and the balance tilts in favor of the phosphatases. When Cdks are activated,
this complex regulates proper microtubule-kinetochore attachment phosphatase activity is overtaken.
and promotes biorientation during mitosis.51 Aurora-C may play similar Cdk1 is able to directly inhibit PP1 by direct phosphorylation of
roles to Aurora-B, although it is mostly expressed in germ cells and the catalytic subunit. The Cdk-dependent inhibition of PP2A, on the
may play a specific role in meiosis and during the first embryonic other hand, is not direct; rather, it is mediated by a new kinase known
cycles.55–57 Plk1 functions as a major regulator of centrosome matura- as Greatwall in flies and Xenopus or Mastl in mammals.85–87 Cdk1
tion, mitotic entry, and cytokinesis, whereas Plk4 is a critical regulator phosphorylates and activates Mastl, which in turn phosphorylates
of centriole duplication.50,58–60 The other members of the family, Plk2, Arpp-19 and Ensa, two highly related proteins that function as inhibitors
Plk3, and Plk5, are mostly involved in stress responses during interphase of a particular PP2A holoenzyme encompassing a regulatory subunit
or in neuron biology.3,53 of the B55 family.85–89 Reactivation of PP1 and PP2A phosphatases
A different group of additional cell cycle kinases with potential is a mandatory step for the exit from mitosis and the transition to
interest in tumor biology is the never in mitosis, gene A (NIMA)–related interphase.90–92
kinase family (Nek1 through Nek11). Four of these proteins, Nek2,
Nek6, Nek7, and Nek9, are involved in cell cycle progression, whereas Entry Into the Cell Cycle
all other family members are likely to play critical roles in cilia and
centrioles.3,61–63 Nek2, the closest relative to the Aspergillus NIMA Because most adult cells are quiescent, the mechanisms that determine
kinase, localizes to the centrosome and plays a role in establishing the their quiescent state and their reentry into the cell cycle with the
bipolar spindle by initiating the separation of centrosomes and appropriate stimuli are key determinants of tissue homeostasis. In
contributing to microtubule organization at the G2/M transition by quiescent cells, several DP/E2F complexes associate with the promoters
phosphorylating several centrosomal substrates. Nek2 also may play of E2F-responsive genes and recruit pRB-family members, along with
additional roles in chromosome condensation and the mitotic check- their associated histone deacetylases, to actively repress their transcrip-
point. Nek9, Nek6, and Nek7 function in a kinase cascade that tion.93,94 This repression machinery therefore prevents the expression
participates in centrosome separation and the formation and/or of proteins required for DNA synthesis and chromosome segregation.
maintenance of the mitotic spindle.61,63 In addition, CKIs normally are expressed in quiescent cells, preventing
A structurally different kinase, Haspin, is involved in the phos- the activation of Cdks.21 D-type cyclins are present at very low levels
phorylation of histone H3 on threonine 3 (H3T3).64,65 This kinase in most quiescent cells, in large part because they are phosphorylated
is activated by Cdk1 and Plk1,66 and phosphorylation of H3T3 is by an abundant kinase called Gsk3β and then exported to the cytoplasm
required for proper Aurora-B localization and activity and coupling for degradation.95
of mitotic chromosome structure with transcription.64,65,67 The transcription of D-type cyclins is induced in response to a
The activity of all these kinases is functionally linked to many wide variety of mitogenic stimuli.96,97 In addition, Gsk3β is inhibited
other proteins associated with chromosomes, microtubules, or different by mitogens, thus preventing the degradation of these cyclins. D-type
organelles, whose activity is essential for the structural changes associated cyclins then associate with Cdk4 and Cdk6, and the resulting complexes
with cell cycle progression and chromosome segregation.68 Among phosphorylate pRB proteins, partially inactivating their transcriptional
them, microtubule-associated proteins such as kinesin motor proteins suppressor function (Fig. 4.3).98–101 pRB inactivation causes the release
determine the dynamic behavior of the mitotic spindle required for of its associated DP-E2Fs. Repressive E2Fs such as E2F4 and E2F5
chromosome movement during mitosis.69,70 During mitosis, micro- dissociate from the DNA, and the free E2F complexes—DP-E2F1,
tubules associate with chromosomes through a large protein assembly DP-E2F2, and DP-E2F3—now occupy the promoters and activate
known as the kinetochore.71 The position of kinetochores in the their transcription. DP-E2F targets include many genes encoding
 Control of the Cell Cycle • CHAPTER 4 61

Inhibitory Mitogens
growth factors

P P
Cip/Kip p107
INK4 p130 P
P P P
pRb pRb
P
E2F P
1,2,3 E2F
Cdk4/6 Cdk2 4,5
CycD CycE
p107 HDAC
p130 DNA synthesis
E2F E2F and mitotic
4,5 1,2,3
regulators
G0 /G1 G1/S
Transcriptional Transcriptional
repression activation

Figure 4.3 • Entry into the cell cycle. The pocket proteins—pRB, p107, and p130—regulate a subset of the E2F family of proteins among many other
transcription factors. The pocket proteins bind to these E2Fs during the G0/early G1 phases, block their transcriptional activity, and recruit histone deacetylase
(HDAC), which comprises repressive complexes. Mitogenic signaling leads to the induction of D-type cyclins and the activation of interphase cyclin-dependent
kinase (Cdk) complexes, which phosphorylate the pocket proteins and release their associated E2Fs. This process allows the activating E2Fs to induce the
transcription of genes required for DNA synthesis and mitosis.

essential proteins required for DNA synthesis as well as mitosis.33–35

Through the control of pRB proteins, cyclin D-Cdk4/6 activity is a MCM MCM
central mediator of the reentry of cells into the cell cycle. Not surpris-
ingly, Cdk4/6 activity is frequently hyperactivated in cancer cells, as Cdc6 Cdt1
described later. Recent data suggest that Cdk6 may have kinase-
independent, transcriptional functions, although the exact mechanisms
behind these functions and their relevance in tumor development ORC
G1
deserve further investigation.102,103
Cyclin E is itself an E2F-responsive gene, and this regulation creates
a strong feed-forward loop. Cyclin E binds to Cdk2, and this complex
may promote further pRB inactivation.104,105 Cyclin E–Cdk2 also Cdc6
phosphorylates the CKI p27Kip1 on Thr187.106,107 This action creates MCM MCM
a high-affinity binding site for the SCF ubiquitin ligase bound to the Cdt1
F-box protein Skp2, leading to p27Kip1 degradation during the G1/S
transition.108 Finally, cyclin E-Cdk2 phosphorylates itself on multiple
DDK + Cdk
sites, creating a recognition site for SCF-Fbw7/Cdc4 and thereby dependent
ensuring its own destruction.109,110 The fact that lack of Cdk2 in the S
ORC
mouse does not result in defective mitotic cycles suggests that the
activity of this protein overlaps with other Cdks, with Cdk1 being
the best candidate.111–113 Indeed, Cdk1 is able to bind interphase
cyclins such as cyclin D and cyclin E, and it is sufficient for G1/S
C t
transition, at least in the absence of other interphase Cdks.114–115 Cdk2 1
is however an essential role in meiosis, although the molecular basis d
for this requirement is not fully understood.112,116
Figure 4.4 • Origin licensing and firing. The origin replication complex
(ORC) associates with replication origins. During the G1 phase, Cdc6 and
DNA Replication Cdt1 are loaded on chromatin, and they in turn load the mini chromosome
maintenance (MCM) complex on chromatin, at which point licensing is
The DNA replication machinery is optimized to ensure that the genome considered complete, and the multiprotein complex is called the prereplication
is copied once—and only once—in each cell cycle.117 This optimization complex (pre-RC). Once cells pass the G1-to-S transition, this complex is
is achieved through a two-step process that first establishes a prereplica- activated to form the preinitiation complex (pre-IC), and DNA replication
tion complex (pre-RC) at each origin of replication, a process that is is initiated. Activation requires both cyclin-dependent kinase (Cdk) and
frequently referred to as origin licensing, and subsequently transforms Ddf4-dependent kinase (DDK) activity. It results in recruitment of numerous
pre-RCs into the preinitiation complex (pre-IC) that activates DNA proteins and activation of the MCM complex, which unwinds the DNA.
Subsequently, core components of the replication machinery, including DNA
replication (Fig. 4.4). These two steps occur at distinct stages of the polymerase α and DNA polymerase ε, are recruited to initiation sites. The
cell cycle to ensure that origins are licensed only once per cell cycle transition from pre-RC to pre-IC results in inhibition of Cdt1 by ubiquitin-
and rereplication cannot occur. Pre-RC formation takes place in the mediated degradation and geminin binding. Origin licensing cannot occur
initial steps of the cell cycle. The first event in this process is the again until activation of anaphase-promoting complex/cyclosome at the end
recruitment of the multiprotein complex called the origin recognition of mitosis allows accumulation of Cdt1.
62 Part I: Science and Clinical Oncology

complex to the origin DNA.118 The origin recognition complex recruits Mitosis
additional proteins including Cdc6, Cdt1, and finally the mini
chromosome maintenance (MCM) complex, a helicase that is required The mitotic machinery is optimized to ensure that the replicated
to unwind the DNA strands to form the pre-RC. Once cells enter S chromosomes are faithfully segregated to the daughter cells. This
phase, the transformation of the pre-RC to the pre-IC requires the segregation is achieved through the use of a specialized microtubule-
activity of two kinases: a Cdk and the Ddf4-dependent kinase, which based structure, the mitotic spindle, on which the original chromosomes
is composed of the Dbf4 regulatory subunit and the Cdc7 kinase.119,120 and their newly replicated copies, called sister chromatids, align and
The action of these kinases allows numerous additional proteins to then are partitioned to opposite poles of the cell. The mitotic spindle
associate with the pre-RC and form the pre-IC.121 Assembly of the is a highly dynamic structure that is maintained by many protein
pre-IC is thought to trigger DNA unwinding by the MCM complex, families, including motor molecules and other microtubule-associated
recruitment of the DNA polymerases, and initiation of the replication proteins.69,70,136,137 The appropriate side-by-side alignment of the sister
process, frequently called “origin firing.” chromatids, termed biorientation, is facilitated by the physical tethering
The transformation of the pre-RC to the pre-IC can occur at of the sister chromatids to one another. This process, called cohesion,
different time points in S phase, depending on whether the origin actually occurs in S phase in a manner that is coordinated with the
fires early or late.117 The system can tolerate this heterogeneity because replication process.138–140 Cohesin is mediated by four proteins that
the pre-RC is disassembled after firing and cannot reform until the together make up the cohesin complex. Two of these proteins, Smc1
subsequent cell cycle. This process occurs through several mechanisms. and Smc3, have a long coiled structure with a dimerization domain
The MCM complex travels with the replication fork in its role as the at one end that allows them to heterodimerize to form a V-like structure.
DNA helicase. Some evidence also indicates that phosphorylation of Important to note, the remaining ends of Smc1 and Smc3 can associate
Orc1 reduces its ability to bind to origins. Finally, and most important, with each another to form a functional adenosine triphosphate (ATP)
Cdt1 is prevented from participating in pre-RC formation outside of domain. This domain acts in an ATP-dependent manner to recruit
the G1 phase in two distinct ways. First, Cdt1 is marked for destruction two additional proteins, Scc1 and Scc3, which form a closed-ring
by ubiquitination.122 This process is mediated by SCF-Skp2 and structure that most likely encircles the chromosomes.141–143 Cohesin
particularly by an E4 ubiquitin ligase that includes Rbx1 (to recruit loading onto chromosomes, catalyzed by a separate complex called
the E2-ubiquitin), a cullin (Cul4), Ddb1, and Dtl/Cdt2 (the substrate kollerin, is thought to be mediated by the entry of DNA into cohesin
specificity factor).123–125 Important to note, this Cul4-Ddb1-Dtl/Cdt2 rings, whereas dissociation, catalyzed by Wapl and several other cohesin
complex functions independently of Cdt1 phosphorylation. Instead, subunits, is mediated by the subsequent exit of DNA.144 Increasing
Cdt1 is targeted only when proliferative cell nuclear antigen is present evidence indicates that cohesin participates in other cellular processes
on the DNA, which occurs primarily as a consequence of the initiation that involve DNA looping such as transcriptional regulation. Interesting
of DNA replication.126 Second, cells possess a protein called geminin to note, mutations in genes encoding cohesin subunits and other
that sequesters Cdt1 and prevents it from participating in pre-RC regulators of the complex have been identified in several tumor types.145
formation. Geminin is present specifically in S-, G2-, and early M-phase The related family of chromosomal proteins, condensins (condensin
cells. The APC/C ubiquitinates geminin and thereby triggers its I and condensin II), are formed from a conserved pair of Smc proteins
destruction during mitotic exit. This action creates a window between (Smc2 and Smc4) and distinct sets of non-SMC regulatory subunits.146
late mitosis and the end of G1 phase (when APC/C-Cdh1 is inactivated) Condensins participate in a diverse array of chromosomal functions
in which geminin is absent, and therefore Cdt1 is free to participate including chromosomal organization in interphase or the assembly
in pre-RC formation.127 of mitotic chromosomes.
The A-type cyclins are first transcribed late during the G1 phase
under the control of the E2F transcription factors in a similar manner Mitotic Entry
to that of cyclin E. Cyclin A associates with both Cdk2 and Cdk1 In addition to its role in DNA replication as discussed earlier, cyclin A/
and acts both during S-phase and G2/M.128,129 At the start of S Cdk complexes are critical mediators of the changes that occur during
phase, cyclin A–Cdk2 enters the nucleus and is specifically localized the G2 phase in preparation for mitosis.128 Here they are thought to
at nuclear replication foci, where it is thought to be actively involved play a key role in initiating the condensation of chromatin and also
in the firing of replication origins.130 As was described previously, might participate in the activation of the cyclin B/Cdk1 complexes.
cyclin A–Cdk2 also is required to phosphorylate E2F-1 and mediate Data have suggested that cyclin A2 is also crucial for loading of kinesins
its degradation, which is required to prevent E2F1 from triggering to microtubules, thus contributing to the formation of a functional
apoptosis.131,132 spindle.134 Cyclin A2 is destroyed at nuclear envelop breakdown in
Given the redundancy between different cyclin and Cdk family an APC/C-Cdc20–dependent manner.147 A-type cyclins are then
members, the specific role of cyclin A in DNA synthesis is unclear. substituted with B-type cyclins. Cyclin B1 protein accumulates steadily
Studies in mouse models identified a partially overlapping role between through the G2 phase and associates mainly with Cdk1, although it also
E- and A-type cyclins in the control of DNA synthesis in a cell-type may associate with Cdk2.129 The resulting cyclin B1/Cdk1 complex is
specific manner.133 Fibroblasts lacking both cyclin A1 and cyclin A2 mostly sequestered in the cytoplasm, and it is retained in an inactive
are able to proceed to S phase, and this is abrogated if E-type cyclins form throughout the G2 phase via the inhibitory phosphorylation
are deleted. However, A-type cyclins are essential in hematopoietic of Thr14 and Tyr15 in Cdk1’s active site by the Myt1 and Wee1
stem cells, suggesting cell-type differences probably caused by the kinases.148–150
levels of expression of the encoding genes. Although these observations Activation of cyclin B1–Cdk1 occurs in a highly synchronous
suggest overlapping functions for E- and A-type cyclins in the activation manner during G2-prophase transition (see Fig. 4.1).151 This activation
of Cdks, a new kinase-independent role as an RNA-binding protein is mediated by two changes. First, the activities of Myt1 and Wee1
has been recently proposed for cyclin A2.134 Cyclin A2 binds directly are downregulated at the transition between the G2 and M phases.
to the 3′ UTR of Mre11 mRNA in a Cdk-independent manner to Second, a dramatic increase in the activity of the Cdc25 phosphatases
promote its translation. Mre11 is a component of the MRN complex, occurs that relieves the inhibitory phosphorylation of Thr14 and
composed of Mre11, Rad50, and Nbs1, which plays a central role in Tyr15. These activity changes are triggered by the phosphorylation
DSB repair and replication fork restart.135 Depletion of cyclin A2 of Myt1, Wee1, Cdc25a, and Cdc25c. Three different kinases are
results in a reduction in Mre11 levels and defective repair of replication thought to contribute to this phosphorylation: Polo-like kinase
errors. Thus cyclin A2 participates in DNA synthesis in a Cdk- (Plk1), cyclin A–Cdk1, and cyclin B1–Cdk1 itself. The involvement
dependent manner while reinforcing stabilization of the key machinery of cyclin B1–Cdk1 creates a powerful feed-forward loop; once a
responsible for repairing DNA lesions caused by replication errors.134 small amount of cyclin B1–Cdk1 is activated, it simultaneously
 Control of the Cell Cycle • CHAPTER 4 63

inactivates its own inhibitors and activates its activators, enabling the attachments of microtubules to the chromosomes, and possibly
a rapid transformation of the entire cyclin B1–Cdk1 pool from the the tension that is generated by microtubules on the kinetochores
inactive state to the active state. Once active, cyclin B1–Cdk1 phos- ensures that the sister chromatids are properly aligned at the metaphase
phorylates components of the centrosomes and initiates a process called plate.159,160 This process is the basis of one of several cell cycle check-
centrosome separation, in which the centrosomes move to opposing points, called the mitotic spindle assembly checkpoint (SAC), which
poles of the nascent spindle, an event essential for formation of the is described in more detail in the following sections.
mitotic spindle.68,152
The activation of mitotic kinases leads to dramatic changes in Anaphase
DNA structure. Largely on the basis of these morphologic changes, Anaphase is characterized by the segregation of the chromosomes.161
mitosis is divided into five different stages—prophase, prometaphase, This event is controlled by the mitotic ubiquitin ligase APC/C-Cdc20.
metaphase, anaphase, and telophase (Fig. 4.5)—before separation of APC/C-Cdc20 ubiquitinates, and thereby triggers the degradation of,
the daughter cells or cytokinesis. cyclin B1 and a protein called securin.39 Both securin and cyclin B1/
Cdk1 complexes are able to bind and inhibit a protease called sepa-
Prophase rase.162,163 APC/C-Cdc20 activity results in the degradation of cyclin
Prophase is characterized by condensation of sister chromatids— B and securin and the subsequent separase activation. Once released,
essentially packaging into a more compact chromatin structure. This separase cleaves the Scc1 component of the cohesin complex, which
process involves condensins I and II, which require phosphorylation opens the cohesin ring, unlinking the sister chromatids and allowing
by mitotic Cdks.145 During prophase, the nuclear envelope is still them to be pulled to opposite poles (see Fig. 4.5). The spindle poles
intact; consequently, differences in subcellular localization of the then move farther apart to ensure that the chromosomes are fully
condensin and Cdk complexes allow only condensin II and cyclin segregated. The separase-dependent cleavage of Scc1 also is essential
A-Cdk1 (nuclear), and not condensin I and cyclin B-Cdk1 (cytoplas- to link segregation of chromatids with the separation of centrioles
mic), to initiate condensation. In a parallel process called resolution, during mitotic exit.163 Cyclin B degradation results in the parallel
the sister chromatids are untangled via the action of topoisomerase inhibition of Cdk1 activity, thereby releasing the inhibitory mechanism
II.141,142 Resolution requires removal of the chromosome arm cohesin that limit PP1 and PP2A activity during the earlier phases of
through phosphorylation of Scc3 by Plk1 and histone H3 by the mitosis.84,90,92 The reactivation of these phosphatases results in the
Aurora-B kinase. Important to note, the cohesin complex at the massive dephosphorylation of mitotic phosphoproteins and results in
centromere is somehow protected from this modification by a protein the disassembly of the mitotic spindle, chromosome decondensation,
called shugosin (Sgo).153–155 and the reformation of the nuclear envelope.161
The second major event in prophase is the translocation of the During anaphase, Cdh1, which is inhibited by Cdk-dependent
cytoplasmic cyclin B1–Cdk1 to the nucleus, where it phosphorylates phosphorylation during mitosis, is dephosphorylated and replaces
components of the nuclear envelope and triggers its breakdown,68,156 Cdc20 as the main APC/C activator.39 APC/C-Cdh1 is responsible
which defines the transition from prophase to prometaphase. To prevent for the degradation of multiple cell cycle regulators, including Cdc20.
the activity of phosphatases induced by the mixture of cytoplasmic APC/C-Cdh1 also activates the ubiquitination and degradation of
and nuclear compartments, Mastl (which is mostly nuclear in prophase) geminin, allowing accumulation of Cdt1 for origin relicensing in the
is exported to the cytoplasm, thus inhibiting the Cdk-counteracting subsequent G1 phase, and the mitotic cyclins, allowing loss of Cdk
phosphatase PP2A-B55.68,157 Cdk1 phosphorylates more than 100 kinase activity. Loss of Cdh1 does not result in major abnormalities
proteins and participates in such activities as chromosome condensation, during mitotic exit but results in earlier entry into the following S
nuclear envelope breakdown, modifications in the Golgi apparatus, phase because of increased Cdk activity and DNA damage.164,165 Cdh1
and formation and dynamics of the mitotic spindle.9,68 Cdk1-deficient therefore is required to prevent unscheduled entry into S phase and
mouse embryos arrest during the first embryonic divisions, indicating genomic instability.43
that this kinase is absolutely required for mitosis and cannot be
compensated by other mammalian Cdks.115,158 Telophase
The reactivation of phosphatases during mitotic exit leads to the
Prometaphase dismantling of the mitotic spindle, leaving two discrete sets of
During prometaphase, the condensation process is accelerated because chromosomes with each nascent daughter cell.161 The elimination
condensin I and cyclin B-Cdk1 now have access to the DNA. The of mitotic phosphoresidues by these phosphatases also results in
sister chromatids become attached to spindle microtubules through DNA decondensation, and the nuclear envelope reforms around
the kinetochore, which is assembled onto centromeric DNA.71,72,74–76 the segregated chromosomes to create two new nuclei, an event that
Microtubules nucleated from the centrosomes attach to the kinetochore defines telophase.166
through a process called search and capture, in which individual
microtubules grow and shrink until they contact and bind the kin- Cytokinesis
teochore.73 Typically, one sister chromatid of the pair attaches first,
and this attachment is further stabilized through the recruitment of Finally, the cell undergoes cytokinesis, or cytoplasmic division. This
additional microtubules from the same pole of the mitotic spindle to process involves formation of a structure containing actin and myosin,
create a kinetochore fiber—that is, highly bundled microtubules bound called the contractile ring, on the inner face of the cell membrane.
to the kinetochore. The sister chromatids oscillate in the cell until The position of the contractile ring is carefully controlled. In cultured
the second sister chromatid is captured by microtubules emanating cells, the ring typically begins to form in anaphase, and its position
from the other pole. These oscillations continue until all of the is established by the position of the metaphase plate. As the membrane
chromosomes are properly aligned on the metaphase plate. grows, the contractile ring contracts steadily to form a constriction,
termed the “cleavage furrow,” which ultimately separates the two nuclei
Metaphase and forms the two daughter cells.167,168 This process partially depends
Metaphase is defined as the point at which all of the chromosome on several mitotic kinases such as Aurora-B and Plk1, which are
pairs are fully condensed, attached to the mitotic spindle, and aligned located at the midbody.51,52,58,169 Plk1 controls the activity of RhoA
at the center—termed the metaphase plate. The pulling of the kineto- guanosine triphosphatases, which are major regulators of the actomyosin
chore fibers toward the poles creates tension through the cohesin ring required for abscission.58,170 Aurora-B also ensures that abscission
complex at the kinetochores, which indicates that the sister chromatids does not occur in the presence of DNA bridges to avoid DNA damage
have achieved appropriate biorientation. The cell constantly monitors in a process known as the abscission checkpoint.168,171,172
64 Part I: Science and Clinical Oncology

Cyclin A/B-Cdk1 activity

Interphase

Chromatin begins to condense

Centrosomes move to poles
and mitotic spindle starts to form

NE Chromosomes attach to
breakdown microtubules of spindle
Prophase

Prometaphase

Chromosomes align at metaphase plate

Sister chromatids separate

Chromatin expands
Cytoplasm divides

APC
Metaphase

Cytokinesis
Anaphase

Telophase

Figure 4.5 • Key stages of mitosis. As the parent cell enters prophase, the chromosomes begin to condense, and multiple proteins associate to form the
kinetochores. The centrosomes segregate to the poles to begin formation of the mitotic spindle. Nuclear envelope (NE) breakdown denotes the start of pro-
metaphase. In this phase, the sister chromatids continue to condense, and they attach to spindle microtubules via their kinetochores. During metaphase, the
sister chromatids align at the metaphase plate and eventually achieve appropriate biorientation. At the onset of anaphase, the sister chromatids separate and
move toward the poles of the spindle. During telophase, the two daughter nuclei are reformed and the daughter cells are finally separated by cytokinesis.
APC/C, Anaphase-promoting complex/cyclosome.
 Control of the Cell Cycle • CHAPTER 4 65

CELL CYCLE CHECKPOINTS G1/S Checkpoint

At key transitions during eukaryotic cell cycle progression, signaling The molecular pathway that determines cell cycle entry with the
pathways monitor the successful completion of events in one phase appropriate mitogenic stimuli (previously described) is not considered
of the cell cycle before proceeding to the next phase. These regulatory a cell cycle checkpoint, strictly speaking. However, several of its
pathways are commonly referred to as cell cycle checkpoints.4 In a components also are used by a checkpoint that monitors DNA altera-
broader context, cell cycle checkpoints are signal transduction pathways tions before replication. In G1 cells, double-stranded DNA breaks
that link the rate of cell cycle phase transitions to the timely and (DSBs) are the most common and most deleterious type of DNA
accurate completion of prior dependent events. Checkpoint surveillance damage. The central components of the DNA damage response (DDR)
functions are not confined to monitoring normal cell cycle progression; are two members of the phosphoinositide 3-kinase–related kinase
they also are activated by both external and internal stress signals. family: ataxia telangiectasia mutated (ATM) and ATM- and rad3-related
The checkpoint pathways include sensor proteins that detect these (ATR).173 ATM originally was identified by virtue of its mutation in
lesions and simultaneously trigger two processes: they recruit additional a hereditary syndrome, ataxia-telangiectasia, which is associated with
effector complexes to correct the problems and activate signaling radiation hypersensitivity and cancer predisposition.174 ATR also is
pathways that induce a temporary cell cycle arrest. In certain situations, associated with a hereditary syndrome called Seckel syndrome.175 Early
which are determined by the cell type and the degree of damage, the studies suggested that ATM and ATR played distinct roles in the
checkpoint pathways eventually can induce permanent cell cycle arrest response to DSBs (ATM) versus replicative defects and single-stranded
(a process called senescence) or apoptosis. breaks (ATR). However, we now know that the regulation is more
To minimize the possibility of errors, checkpoints exist at the complex; considerable cross talk occurs between ATM and ATR, and
four different phases of the cell cycle: G1 (to prevent entry into S they share many mediators and effectors, but the precise composition
phase), intra S, G2 (to prevent mitosis), and M (to avoid mitotic and role of the DDR complexes vary depending on both the type of
exit with abnormal segregation of chromosomes) (see Fig. 4.1). In damage and the stage of the cell cycle (Fig. 4.6).176
general, these checkpoints monitor the status and structure of DNA These DSBs are recognized by the multifunctional Mre11-Rad50-
during cell cycle progression. In particular, cells scan the chromatin Nbs1 (MRN) complex.135,173 This complex recruits ATM to the site
for partially replicated DNA, as well as DNA strand breaks and other of damage. The active ATM then recruits proteins to modify the
DNA lesions that can result from both extrinsic (e.g., chemicals, chromatin at the region of the break and activate repair and signaling.
ionizing or ultraviolet radiation) and intrinsic (e.g., byproducts As a first step in this process, ATM phosphorylates histone H2AX to
of intracellular metabolism) DNA-damaging agents. In addition, form γH2AX, which helps hold the damaged ends together and acts
checkpoints also monitor the proper structure of chromosomes and as a binding platform for additional factors, including Mdc1, 53BP1,
their biorientation to ensure equal distribution between the two and Brca1, as well as more MRN and ATM. In contrast to the S and
daughter cells. G2 response, no recruitment of ATR to DSB in G1 cells occurs, and

G1 Intra S G2/M

DNA damage Replication- DNA damage

associated
error
DSB DSB

ssDNA
MRN RPA MRN

ATM ATR ATM

γH2AX ATM γH2AX ATR γH2AX

Mediators Mediators Mediators

repair repair repair
machinery machinery machinery

Chk2P Chk2P Chk1P Chk1P Chk2P

Figure 4.6 • Ataxia telangiectasia mutated/ATM- and rad3-related (ATM/ATR) signaling is activated by DNA damage and replication stress. The cell
constantly monitors the chromatin for lesions, using complex signal transduction pathways that center on the ATM and ATR kinases. The precise mechanism
of response varies according to the type of DNA damage and the cell cycle stage. Double-stranded breaks (DSBs) are the most deleterious form of DNA
damage. DSBs are recognized by the Mre11-Rad50-Nbs1 (MRN) complex that consists of Mre11, Rad50, and Nbs1. This complex recruits ATM to the site
of damage. ATM phosphorylates histone H2AX to form γH2AX, which creates a binding platform for additional proteins that propagate the DNA damage
response and activate repair. For S and G2 phase cells, but not G1 phase cells, ATR also is recruited to the damage site. ATR and/or ATM signal to their
effector kinases (Chk1 and Chk2, respectively) to influence cell cycle progression as described in Fig. 4.7. Errors in DNA replication also can activate the
DNA damage response machinery through the presence of single-stranded DNA (ssDNA), which is a hallmark of the replication fork. The ssDNA is coated
with replication protein A (RPA) and bound by ATR. Active ATR then recruits the DNA damage and repair machinery, including ATM, leading to the
sequential activation of Chk1 and then Chk2.
66 Part I: Science and Clinical Oncology

G1, Intra S, G2/M Intra S G1/S

DNA Replicative Oncogenic
damage stress stress

P P p14ARF
Chk1 and/or Chk1 and
P P
Chk2 Chk2

Phosphorylation of Hdm2
all three Cdc25 proteins
Ubiquitination
P P Degradation
Cdc25a
p53 p53

P Bound and
Cdc25b Oligomerizes to
P inhibited
Cdc25c form active
by 14-3-3
transcription factor

Cdk activation p21Cip1 Pro-apoptotic

genes
Cdk activity

Figure 4.7 • DNA damage, replicative stress, and oncogenic stress induce cell cycle arrest. DNA damage and replication stress lead to the rapid phosphorylation
and activation of the Chk1 and/or Chk2 kinases. These kinases enforce cell cycle arrest through two mechanisms. Chk1 and Chk2 both phosphorylate the
Cdc25 phosphatases, which triggers their ubiquitination and degradation (Cdc25a) or binding and inhibition by 14-3-3 (Cdc25b and Cdc25c), thereby preventing
activation of cyclin/Cdk kinase complexes. Chk1 and Chk2 also phosphorylate p53 and prevent it from being targeted by Hdm2 for ubiquitin-mediated degrada-
tion. As a result, p53 accumulates and activates transcription of p21Cip1, inhibiting Cdk2 and Cdk1 kinase complexes, or proapoptotic genes. Oncogenic stress
also leads to cell cycle arrest by activating replicative stress and/or inducing transcription or the p14ARF tumor suppressor and suppressing Hdm2-mediated
inhibition of p53. Cdk, Cyclin-dependent kinase.

thus ATM is solely responsible for checkpoint activation. The recruit- Important to note, p53 also is activated by other stress signals (see
ment of additional ATM amplifies the signal, and ATM acts via Fig. 4.7). In particular, it is now well established that numerous
phosphorylation and activation of the effector kinase Chk2.177,178 oncogenes trigger a stress response (called oncogene-induced stress)
Chk2 influences the G1 cell cycle arrest via two mechanisms (Fig. that leads to the activation of p53.188,189 The emerging view is that
4.7). First, it phosphorylates all three members of the Cdc25 family. this process occurs through two distinct mechanisms. First, oncogene
Phospho-Cdc25a is ubiquitinated by SCF-TrCPβ and degraded, whereas activation is thought to yield replicative stress that activates p53 via
phospho-Cdc25b and phospho-Cdc25c are bound and sequestered activation of Chk kinases and phosphorylation of Hdm2/Mdm2 as
by a cytoplasmic protein called 14-3-3.20,179 This process is a rapid just described.190,191 Second, many oncogenes activate transcription
response that can take effect within minutes after DNA damage, and of cell cycle inhibitors such as p16INK4a, p15INK4a, p21CIP1, or p19ARF
it has a widespread effect on cell cycle progression by preventing in a p53-independent manner.188,192–195 The protein p19ARF is encoded
activation of Cdks. Second, Chk2 phosphorylates p53, a critical regula- by the INK4A/ARF (CDKN2A) locus, and it actually shares two
tor of cell cycle checkpoints.180 In normal, nonstressed cells, p53 coding exons, which are read in alternate reading frames (hence the
protein is maintained at low steady state levels because it has a very name ARF) with the p16INKa tumor suppressor.194 The ARF protein
short half-life. This half-life is a result of rapid ubiquitination of p53 product, called p14ARF in humans and p19ARF in mice, binds to Hdm2/
by Hdm2 (the human ortholog of murine Mdm2 protein) and its Mdm2 and prevents it from regulating p53.196–199 As with the DDR,
consequent degradation.181,182 The importance of Mdm2 for mainte- this mechanism frees p53 to activate the transcription or proarrest or
nance of appropriate p53 levels in vivo is highlighted by the fact that proapoptotic targets.
absence of Mdm2 in knockout mice results in early embryonic lethality As an additional DDR in G1 cells, genotoxic agents also inhibit
that is rescued by a dual knockout of Mdm2 and p53.183,184 Phosphoryla- origin licensing by way of an ATM/ATR-independent process that is
tion of p53 by Chk2 is sufficient to prevent its association with Hdm2/ achieved through regulation of Cdt1.200 As described previously, Cdt1
Mdm2,183 which leads to an accumulation of p53, which functions is required for pre-RC formation. In an undamaged cell, Cdt1 is
as a transcriptional activator; p53 induces expression of many genes available during the G1 phase but is inhibited after origin firing by
involved in cell cycle arrest, including the CKI p21Cip1.185,186 This degradation (mediated by the SCF-Skp2 and Cul4-Ddb1-Dtl/Cdt2
p53-mediated arrest takes longer to develop than does the Cdc25 ubiquitin ligases) and geminin binding. As a key feature of this regula-
response (because it requires transcription and protein synthesis), but tory system, Cdt1 is completely resistant to Cul4-Ddb1-Dtl/Cdt2 in
it appears to be much more robust. Moreover, in addition to inducing the G1 phase. However, DNA damage allows the Cul4-Ddb1-Dtl/
cell cycle arrest, p53 has the capacity to induce apoptosis through the Cdt2 complex to ubiquitinate Cdt1 and induce its degradation.
transcriptional activation of proapoptotic regulators (e.g., the BH3-only Important to note, the degradation of Cdt1 is extremely rapid, occurring
proteins Puma and Noxa).187 within minutes of the DNA damage. Both Cdt1 and Cdt2 are
 Control of the Cell Cycle • CHAPTER 4 67

phosphorylated in response to DNA damage, which results in the localize to the outer kinetochore and, in the absence of biorientation,
ubiquitination of Cdt1. This process depends on the activity of the prevent the Cdc20 activator from binding to the APC/C.
p97 AAA+-ATPase and its cofactor Ufd1, a complex required for the The kinetochore is made of approximately 30 scaffold proteins
extraction of ubiquinated proteins from the chromatin.124,201,202 As a that link chromatin and the mitotic spindle.71,72,75–77 Additional regula-
result, origin licensing is completely blocked until the damage is repaired tory elements include sensors of microtubule-kinetochore attachment
and Cdt1 is resynthesized. and a complex signaling pathway that modulates APC/C-Cdc20 activity.
Lack of tension or lack of attachment at the kinetochore results in
Intra–S Phase Checkpoint stable Mad1/Mad2 complexes that convert an inactive open-Mad2
conformation into a closed-Mad2 conformation that is able to bind
One of the major goals of cell cycle checkpoints is to prevent the to Cdc20.159,213,214 The Mad2-Cdc20 association triggers the recruitment
deleterious consequences of replicating damaged DNA. Therefore of BubR1-Bub3 into an APC/C-inhibitory complex (the mitotic
S-phase cells must respond virtually instantaneously to DNA damage checkpoint complex [MCC]). Because the closed-Mad2 signal is
to halt initiation of new replication forks throughout the S phase.203 diffusible, a single unattached kinetochore is sufficient to form these
The most deleterious type of damage is DSBs. DSBs can occur through complexes and inhibit APC/C-Cdc20 activity. As a result, separase is
the action of DNA-damaging agents (from either extrinsic or intrinsic inhibited by the high levels of securin and cyclin B/Cdk1 complexes,
sources) or as a consequence of the replication process itself—for being unable to cleave the centromeric cohesin (see Fig. 4.8). Once
example, if the replication fork passes through nicked DNA or if all chromosomes are bipolarly attached to the mitotic spindle, the
replication stalls at sites of DNA damage.204 The cell senses the damage SAC is satisfied and the Mad1/Mad2 complex is removed from the
in different ways, depending on whether the lesion is associated with kinetochores.71,77 How the checkpoint signaling is inhibited currently
replication. Ultimately, both ATM and ATR are recruited to the site is unclear. Cdc20 is now released from the MCC complex and activates
of damage, but the order of binding is different.176,203 Replication-linked the APC/C, leading to the rapid ubiquitination and degradation of
DSBs are distinguished by the presence of single-stranded DNA cyclin B and securin. Inactivation of these two proteins results in two
(ssDNA), a hallmark of the replication process. The ssDNA is coated major processes. First, lack of securin and cyclin B results in the
by replication protein A (RPA) and bound by ATR and its regulator activation of separase, a caspase-like protease that cleaves cohesin, the
subunit ATRIP, even during the normal replication process. In response molecule that holds sister chromatids together at the centromere.
to DNA damage, the ATR kinase is activated, and it then recruits a Second, inhibition of Cdk1, due to the lack of cyclin B, leads to the
variety of complexes that mediate both repair and checkpoint activation, activation of mitotic phosphatases such as PP1 and PP2A, triggering
including ATM. In contrast, nonreplication-associated DSBs initially mitotic exit as described earlier.161
recruit and activate ATM through the MRN-dependent process
described previously for the G1/S checkpoint. However, in S-phase CELL CYCLE DEREGULATION IN
cells, DSB resection causes the formation of ssDNA (through the HUMAN CANCERS
action of the MRN endonuclease), which is then bound by RPA and
ATR/ATRIP.203 Thus in S-phase cells, ATR and ATM jointly orchestrate The central relevance of cell cycle regulation in cancer is underscored
the DDR. ATR contributes to the checkpoint response in a similar by the finding that virtually all human tumors carry mutations in (1)
manner to ATM: it activates Chk1, which also can phosphorylate the the basic cell cycle machinery that controls entry into the cell cycle
Cdc25 proteins and p53.205–208 and (2) checkpoint regulators such as the p53 pathway (Table 4.2)
and by the observation that most human tumors display aberrant
G2 Checkpoint chromosome numbers.1,5,6,173,215 Together, the pRB and p53 pathways
are critical gatekeepers of cell cycle progression and stress response,
Whereas the G1/S and intra–S phase checkpoints prevent cells from and their function has crucial implications in the maintenance of
unfaithful replication, the G2 checkpoint is required to prevent the genome integrity at the level of both structural and numeric aberrations
passage of DNA lesions to the two daughter cells during mitosis.173,209,210 in chromosomes.216,217
DSBs are detected exactly as described previously for the S-phase
nonreplication-associated DSBs. Similarly, the ATR/Chk1 and ATM/ Unscheduled Cell Cycle Entry in Cancer
Chk2 pathways enforce arrest through inhibition of G2 and mitotic
Cdk complexes via the rapid removal of the Cdc25 phosphates and The alterations in the cell cycle machinery that occur most frequently
the p53-dependent induction of p21CIP1 to inhibit mitotic Cdk include loss or mutation of the pRB tumor suppressor; overexpression
complexes (cyclin A/B in combination with Cdk1/2 kinases).208,210,211 of cyclins, Cdks, and Cdc25 phosphatases; and loss of expression of
Ubiquitin ligases also are involved in this process. SCF-βTrCP regulates CKIs.1 Mutations that affect the pRB pathway have been identified
the levels of Cdc25, Claspin, and Wee1, whereas APC/C-Cdh1 is in most human cancers.1,29,217 The RB1 gene originally was identified
critical for the elimination of Plk1, a kinase essential for checkpoint by virtue of its mutation in both familial and sporadic retinoblastoma,
recovery.211,212 but it is defective in many other tumor types, especially osteosarcoma
and lung cancer.
Spindle Assembly Checkpoint In tumors that lack RB1 mutations, alterations in other components
of the signaling pathways that regulate pRB frequently are found,
The SAC acts to ensure that appropriate partitioning of the chromo- including cyclin overexpression or loss of CKIs. Nearly 50% of inva-
somes occurs during mitosis.159,160 The concept that chromosome sive breast cancers have elevated cyclin D expression compared with
segregation is prevented until all condensed sister chromatid pairs are surrounding normal breast epithelium, and in transgenic mice with
aligned at the metaphase plate with the appropriate biorientation has overexpression of human cyclin D1 or cyclin E in mammary gland
already been introduced. This process actually is controlled by a signal- cells, mammary adenocarcinomas develop.218–220 Similarly, Cdk4 and
ing network that constitutes the SAC (Fig. 4.8). The core components Cdk6 gene amplification occurs in breast cancers, sarcomas, gliomas,
of this checkpoint—called Mad1, Mad2, BubR1, Bub1, and Bub3 and melanomas, and specific translocations lead to cyclin D or Cdk6
in humans—originally were identified through screens in yeast for overexpression in hematopoietic disorders.1,221 Modifications of CKIs
“mitotic arrest deficient” (MAD) and “budding uninhibited by that act upstream of pRB activity also commonly are found in human
benzimidazole” (BUB) mutants.159 Other components of the checkpoint tumors. The CKI p27Kip1 often is aberrantly expressed in human
include the Mps1 kinase and the three subunits of the Rod, Zwich, breast cancer, and reduced p27Kip1 protein levels are correlated with
and ZW10 (RZZ) complex. During prometaphase, these proteins more aggressive breast tumors.222–224 Likewise, decreased expression
68 Part I: Science and Clinical Oncology

Prometaphase

No attachment
Pole
“Wait” Cohesion
Unattached
kinetochore Spindle
Low checkpoint
Mad2 proteins Cdc20
Metaphase High
Mad2

Attachment
Active Inactive
APC/C APC/C
Securin
ubiquitination
+ degradation
Anaphase
Securin
Inactive
Active separase Separase

Cohesion
(by Scc1
cleavage)

Figure 4.8 • The spindle assembly checkpoint (SAC). Improper chromosome alignment on the mitotic spindle, disruption of microtubule dynamics, or
unattached kinetochores maintains the SAC in an active state. Checkpoint signaling is mediated by the Bub1, Bub3, BubR1, and Mad2 proteins, which
localize to kinetochores. These core spindle checkpoint regulators prevent Cdc20 from activating the anaphase-promoting complex/cyclosome (APC/C) and
therefore protect securin and cyclin B, two major APC/C-Cdc20 targets, from ubiquitin-mediated degradation. As a result, securin and cyclin B1-Cdk1 remain
bound to separase, which prevents cleavage of Scc1 and loss of centromeric cohesin. The “wait” signal is inhibited at the end of the metaphase by the appropriate
biorientation of the sister chromatids at the metaphase plate. The sensing mechanism involves detecting attachment or tension at the kinetochores that is
created by the pulling of the spindle fibers toward the poles. Mad2 then dissociates from the attached kinetochore, which allows Cdc20 to activate APC/C,
targeting securin and cyclin B for degradation. This process leads to the inactivation of Cdk1 and the activation of separase, triggering sister chromatid segregation
and mitotic exit.

of the CKI p57Kip2 is found in human bladder cancers. Although Mutations in p53 and Checkpoint Regulators
p21Cip1 is not commonly mutated in human cancer, its expression
is strongly reduced in multiple tumors as a consequence of defective The most frequently altered cell cycle checkpoint signaling molecule
p53 signaling.225 The CKI most frequently affected appears to be is the p53 tumor suppressor. The importance of p53-dependent signal-
p16INK4a; it was identified as a tumor suppressor that is associated ing in tumor suppression is underscored by the frequency of mutation
with familial melanoma, and it is inactivated by point mutation, (~50%) in sporadic tumors235 and the finding that germline mutations
deletion, and/or promoter methylation in approximately 30% of all of p53 result in Li-Fraumeni syndrome, a highly penetrant familial
human tumors.221,226 In contrast, point mutations in p15INK4b, p18INK4c, cancer syndrome that is associated with significantly increased rates
and p19INK4d are rare, but promoter methylation and reduced protein of brain tumors, breast cancers, and sarcomas.236 In human tumors
expression of some of these inhibitors have been seen in a variety that lack p53 gene mutation, p53 function may be disrupted by
of tumor types.1,221,227–229 Germline mutations in p16INK4a predispose alterations in cellular proteins that modulate the levels, localization,
individuals to melanoma, whereas deletion of p15INK4b and p16INK4a is and biochemical activity of p53. For example, in some tumors with
linked to the pathogenesis of lymphomas, mesotheliomas, and pancreatic wild-type p53 alleles, Mdm2 gene amplification occurs, resulting in
cancers.1,221 Although point mutations in Cdks are not common in Mdm2 protein overexpression and subsequent p53 inactivation.237 In
human tumors, a specific mutation of Cdk4 (R24C) that prevents its human papillomavirus–induced cervical carcinoma, p53 typically is
inhibition by INK4 proteins has been found in persons with familial not mutated; however, the human papillomavirus E6 protein binds
melanoma.230 Knockin mice harboring this mutation show not only p53 and targets it for degradation, abrogating p53-dependent
an increased susceptibility to melanoma but also the development signaling.238
of multiple tumor types, indicating the relevance of this regulatory Proteins that reside upstream of p53 (including ATM and Chk2)
circuit in human tumors.231,232 Both Cdc25a and Cdc25b phosphatases also are targeted for mutation in human tumors, and their discovery
also are overexpressed in more than 30% of primary breast tumors, and analysis have greatly deepened our insight into DDR signaling
40% to 60% of non–small cell lung cancers, 50% of head and neck pathways. ATM mutations occur in ataxia-telangiectasia, a disorder
tumors, and a significant fraction of non-Hodgkin lymphomas.20,233,234 in which patients have increased sensitivity to radiation and an
All these events can result in increased activation of Cdks, defec- elevated incidence of leukemias, lymphomas, and breast cancer.174,239
tive pRB signaling, unscheduled cell proliferation, and override of ATM-null mice exhibit growth retardation, neurologic dysfunction,
checkpoint arrest. infertility, defective lymphocyte maturation, and sensitivity to ionizing
 Control of the Cell Cycle • CHAPTER 4 69

Table 4.2 Alteration of Cell Cycle Regulators in Human Tumorsa

Tumors Associated With Mutations or Hereditary Syndromes Associated With
Protein (Gene) Altered Expression Germline Mutations
ATM Breast carcinomas, lymphomas, leukemias Ataxia-telangiectasia
Aurora-A (AURKA) Wide array of tumors NR
Bub1 Colon, lung, and pancreatic cancer NR
BubR1 (BUB1B) Wilms tumor, rhabdomyosarcoma, and acute leukemia Mosaic variegated aneuploidy and premature chromatid
separation syndrome
Brca1/2 Breast and ovarian carcinoma Familial breast and ovarian cancer
Cdc25a Carcinomas of the breast, lung, head, and neck and lymphoma NR
Cdc25b Carcinomas of the breast, lung, head, and neck and lymphoma NR
Cdk4 Wide array of cancers Familial melanoma
Cdk6 Wide array of cancers NR
Chk1 Colorectal and endometrial carcinomas NR
Chk2 Carcinomas of the breast, lung, colon, urogenital tract, and testis Li-Fraumeni syndrome
Cyclin D1 Wide array of cancers NR
(CCND1)
Cyclin D2 Lymphoma and carcinomas of the colon, testis, and ovary NR
(CCND2)
Cyclin D3 Lymphoma, pancreatic carcinoma NR
(CCND3)
Cyclin E Wide array of cancers NR
(CCNE1/2)
Mdm2 (HDM2) Soft tissue tumors, osteosarcomas, esophageal carcinomas NR
Mps1 (TTK) Lung, gastric and bladder cancer NR
Mre11 Lymphoma Ataxia-telangiectasia–like disorder
Nbs1 Lymphomas, leukemias Nijmegen breakage syndrome
p15INK4b (CDKN2B) Wide array of cancers NR
p16INK4a (CDKN2A) Wide array of cancers Familial melanoma
p27Kip1 (CDKN1B) Wide array of cancers NR
p53 (TP53) Wide array of cancers Li-Fraumeni syndrome
p57Kip2 (CDKN1C) Bladder carcinomas NR
p130 (RBL2) Wide array of cancers NR
pRB (RB1) Wide array of cancers Familial retinoblastoma
SA1 (STAG2) Bladder, colorectal, glioblastoma, melanoma and Ewing’s sarcoma NR
Tpx2 Lung, bone, and pancreatic cancer NR

a
Only genetic alterations or defects that are present in more than 10% of primary tumors are represented.
NR, Not reported.

radiation.240,241 The DNA double-strand break repair gene Mre11 also for Nijmegen breakage syndrome, a rare autosomal-recessive disorder
is mutated in persons with an ataxia-telangiectasia–like disorder.242 characterized by chromosome instability, hypersensitivity to ionizing
Mutations of Chk2 and Chk1 also arise in human cancers. Chk2 radiation, and high susceptibility to the development of tumors.135,247
mutations have been reported in several cancers, including lung cancer,
whereas Chk1 mutations have been observed in human colon and Aneuploidy and Chromosomal Instability
endometrial cancers.243,244 In addition, heterozygous alteration of Chk2
occurs in a subset of persons with Li-Fraumeni syndrome who lack Abnormal chromosome numbers, called aneuploidy, is a frequent feature
p53 gene mutations.245 These findings support the theory that in of cancer cells.6,215,248 In addition, many cancer cells also display
human tumors in which p53 is intact, the function of this signaling chromosomal instability or an aberrantly high change in the karyotype.
pathway might be disrupted by alterations in cellular proteins that Chromosomal instability may lead to aneuploidy, but not all aneuploidy
modulate the levels or activity of p53. In addition, the breast cancer cells display chromosomal instability, because many cancer cells exhibit
susceptibility tumor suppressors Brca1 and Brca2 are known to stable aneuploidy karyotypes. Aneuploidy has a high rate of frequency
participate in the DDR and repair.246 Similarly, Fanconi anemia proteins, (more than 75%) in human tumors, and about one-quarter of the
which originally were identified by virtue of their association with a genome is affected by whole-arm or whole-chromosome number
recessive development disorder called Fanconi anemia, which is associ- alterations.249 Yet it is not clear whether aneuploidy is a cause or
ated with increased cancer predisposition (particularly acute myeloid consequence of cancer, and the relative level of chromosomal instability
leukemia), also function in the DDR.246 Mutations in the Nbs1 gene, may have either oncogenic or tumor suppressor properties during
a component of the MRN complex that sensors damage, are responsible tumor development.6,250–252
70 Part I: Science and Clinical Oncology

In vitro estimates suggest that normal cells missegregate a chromo- THERAPEUTIC MANIPULATION OF CELL
some every hundred cell divisions.215 This rate is dramatically increased CYCLE CONTROLS
in cells that display chromosomal instability, which missegregate a
chromosome in every one to three cell divisions in vitro.253 These Research during the past several decades has shown that alterations
defects occur for multiple reasons, including aberrant centrosome in cell cycle machinery and checkpoint signaling lead to tumorigenesis.
numbers, abnormal microtubule-kinetochore attachments, and These findings have important implications for the optimization of
imperfect SAC. In particular, there has been considerable speculation current therapeutic regimens and for the selection of novel cell cycle
that disruption of the SAC could occur during tumor progres- targets for the future development of anticancer agents. A leading
sion.252,254,255 Notably, inactivating mutations in BUB1 have been goal in cancer research is to identify compounds that will target key
identified in human colon carcinoma cell lines, which are known to cell cycle controls in a tumor-specific manner.
have a high degree of aneuploidy.256 These mutations facilitate the
transformation of cells that lack the breast cancer susceptibility gene Targeting Cyclin-Dependent Kinase Activity
BRCA2.257 The gene encoding BubR1, BUB1B, is also mutated in
persons with mosaic variegated aneuploidy and the premature chromatid Considerable debate has occurred about whether inhibition of Cdk
separation syndrome. Both BUB1 and BUB1B also are silenced by activity is a rational strategy for anticancer therapies.267 Cdk activity
promoter hypermethylation in specific tumors.258 Additional mutations frequently is elevated in human tumors, but it also is required to
and epigenetic alterations have been found in the SAC components maintain specific cell populations in adults (e.g., the hematopoietic
Mad1 and Mad2.258 Most of these alterations are loss-of-function compartment and gut) that are essential for viability.3 Thus the key
mutations, suggesting that the SAC is not functional in a variety of issue is whether tumor cells may require different Cdks for proliferation
tumors. Moreover, haploinsufficiency of Mad2 has been shown to or whether sufficient difference in the Cdk activity exists to create a
cause elevated rates of lung tumor development in mice.259 Interesting therapeutic window. During the last few years, the analysis of Cdk
to note, overexpression of Mad2 also results in increased tumor and cyclin mouse models has yielded considerable insight into this
susceptibility and frequent relapses of chromosomally unstable tumors.260 question but also has raised additional questions.268 These mouse
In fact, Mad2 is also frequently overexpressed in human tumors. models show that the cell cycle machinery is extremely robust; it
Several other mitotic regulators such as separase, securin, condensins, adapts easily to the loss of Cdks or cyclins by using other Cdks or
Cdc20, or Aurora kinases, as well as the SAC component Mps1, are cyclins to substitute for the missing activity. For example, cells are
included in the overexpression signature that marks chromosomally able to proliferate without specific interphase Cdks because novel
unstable cancers.258,261 All these data, together with the recent evidence cyclin/Cdk complexes can be formed that allow the cell cycle to
gathered in mouse models, suggest that small aberrations, either progress.111–115 This ability raises the possibility that tumor cells will
overexpression or downregulation, in the levels of mitotic and SAC develop resistance to Cdk-inhibitory drugs rapidly simply by adapting
regulators may contribute to aneuploidy and/or chromosomal instability their cell cycle machinery. On the positive side, studies in cell lines
in human tumors.6,255 Because functional disruption of SAC proteins and mouse models clearly show that tumors can be more dependent
results in the abrogation of the checkpoint and a failure to arrest in on Cdk activity, or at least a specific Cdk activity, than are normal
mitosis in the presence of microtubule poisons such as taxol and tissues. In parallel to these biologic studies, numerous small-molecule
nocodazole, these aberrations also may play a role in the response to inhibitors have been developed with different specificity profiles against
mitotic poisons currently used in the clinic.262 Cdks (Table 4.3).268–270
Multiple cell cycle aberrations, including pRB or p53 inactivation, Which Cdk should be targeted in each tumor type? Pioneering
lead to aberrant levels of mitotic regulators resulting in “oncogene- studies in the mouse showed that loss of D-type Cdk had little or no
induced mitotic stress.”216 The consequences of this aberration in effect on the development and maintenance of many tissues but can
cancer cell proliferation are possibly limited by additional alterations greatly suppress the development of certain tumor types, depending
in other mitotic regulators. For instance, the cancer-associated upregula- on the tissue and the identity of the initiating oncogenic lesions.271–274
tion of the SAC component Mad2 can be balanced by upregulation Mammary gland tumor proliferation dramatically depends on cyclin
of its target Cdc20, thus maintaining the balance between SAC and D1/Cdk4 complexes, whereas the absence of these molecules does
APC/C activity required for genomic stability.216 In the same line, not alter normal mammary gland development. In a pioneering study
mutations in the APC/C component Cdc27 may slow down APC/C in mouse models, cyclin D1–Cdk4 was shown to be required for
activity, thereby facilitating the correction of chromosome segregation Her2 (ErbB2)- or HRas-driven tumors, whereas it was dispensable
errors in the presence of the weak SAC frequently found in tumor for Myc- or Wnt-induced neoplasias, suggesting that the efficacy of
cells.263 Tumor cells also accumulate mutations in other genes whose CKIs may have a strong dependence on the genetic background of
deregulation may induce chromosomal instability, such as cohesin target tumors.272,275,276 Similarly, Cdk4 is required for KRAS-mutant
subunits and their regulators. However, whether chromosome misseg- lung tumors, but it seems dispensable for similar lung tumors with
regation is the major contribution of cohesin mutations to cancer wild-type KRAS alleles.274 On the other hand, cyclin D3/Cdk6
progression is not clear at present.146 complexes are critical for Notch1-mutant T-cell leukemias.277 These
Chromosome missegregation not only results in whole-chromosome studies and parallel efforts in human cancer cell lines led to multiple
aneuploidy but also may generate chromosome breaks and rearrange- clinical trials to test the effect of specific Cdk4/6 inhibitors in a wide
ments. A possible mechanism for DNA damage caused by chromosome spectrum of tumors.278 The success of some of these trials resulted in
missegregation is based on the defective DNA replication of the the rapid approval of palbociclib for the treatment of hormone-positive
micronuclei present in cells affected by aneuploidy.264 These micronuclei breast cancer, and other Cdk4/6 inhibitors such as abemaciclib and
undergo asynchronous DNA replication, resulting in DNA damage ribociclib have showed significant efficacy in advanced clinical trials
and pulverization of chromosomes. This process may provide, at least in multiple tumor subtypes.279–281
partially, an explanation for “chromothripsis,” a situation in which
chromosomes undergo massive local DNA breakage and rearrange- Targeting DNA Damage Response Proteins
ments.264,265 In addition, chromosomes that missegregate may be
damaged during cytokinesis, resulting in DNA double-strand breaks In the past decade, there has been a growing appreciation that many
that can lead to unbalanced translocations in the daughter cells.266 tumor cells carry mutations that disrupt their DDR. This characteristic
All these processes provide additional mechanisms by which defective is a major factor in establishing the resistance of tumors to chemo-
chromosome segregation may induce genomic aberrations during therapeutic agents, many of which work by causing DNA damage
tumor development. and triggering apoptosis through induction of DNA damage pathways.
 Control of the Cell Cycle • CHAPTER 4 71

Table 4.3 Cell Cycle Regulators With Therapeutic Interest

Target Representative Small-Molecule Inhibitors Preclinical or Clinical Data
MITOTIC SPINDLE
Tubulin Stabilizing: taxanes, eleutherobins, epothilones, laulimalide, In clinical use for solid and hematopoietic tumors
sarcodictyins, and discodermolide; polymerization inhibitors:
vinca-alkaloids, cryptophycins, halichondrins, estramustine
Eg5 (KSP) ARRY-520, AZD4877, MK-0731, SB-715992 (ispinesib), Clinical trials in advanced solid tumors, lymphoma, and
SB-743921 taxane-resistant cancer
KINASES
ATR AZD6738, VX-970, ATR-101 Clinical trials in monotherapy or combination with
radiotherapy in solid tumors
Aurora kinases AT9283, CYC116, GSK1070916A, MLN8237, PF-03814735, VX-680 Clinical trials in solid tumors and hematopoietic malignancies
Cdks (1, 2, 4, 6) AG-024322, EM-1421, LEE-011 (ribociclib), LY2835219 Clinical trials in solid tumors and leukemias; Cdk4/6 inhibitors
(abemaciclib), P276-00, PD-0332991 (palbociclib) approved for hormone-positive breast cancer
Chk1 LY2606368, UCN01, CCT245737 Clinical trials in combination with DNA damaging agents
Plk1 BI2536, BI6727 (volasertib), GSK461364, NMS-1286937, Clinical trials in solid tumors and hematopoietic malignancies
TKM-080301
Mps1 (TTK) NMS-P153, BAY1161909, NTRC 0066-0 Clinical trials in metastatic solid tumors
PHOSPHATASES
Cdc25 ARQ-501, IRC 083864 Preclinical studies and clinical trials in advanced solid tumors
DNA REPAIR
PARP ABT-888, AZD-2281 (olaparib), AG014699, BMN-673, CEP 9722, Clinical trials in solid tumors and hematopoietic malignancies;
MK-4827 (niraparib) olaparib and rucaparib are approved for treating ovarian
cancer
UBIQUITIN LIGASES
APC/C TAME, APCIN Preclinical studies

APC/C, Anaphase-promoting complex/cyclosome; Cdk, cyclin-dependent kinase; ATR, ATM- and rad3-related; PARP, poly (ADP-ribose) polymerase.

Therefore considerable attention has focused on designing cancer Targeting the Mitotic Spindle
treatments that would be effective in cells with an impaired DDR.
Because it is difficult to restore the function of mutant or missing Microtubule poisons, such as taxol and vinblastine, kill cancer cells
proteins, the prevailing strategy is to identify drugs that would synergize at least partially by exploiting their effects on the mitotic spindle.292
with the defective DDR to selectively kill the tumor cells and not the Taxanes, such as docetaxel or paclitaxel, are microtubule-stabilizing
normal cells. For example, inhibitors of poly (adenosine diphosphate drugs widely used to treat breast and ovarian tumors, non–small cell
[ADP]-ribose) polymerase (PARP) selectively kill cells that lack either lung cancer, and Kaposi sarcoma. Vinca alkaloids, such as vinblastine
Brca1 or Brca2.282,283 The rationale for this action is that these proteins or vincristine, are microtubule-destabilizing compounds and have
provide two alternative repair mechanisms in response to DNA damage: shown clinical efficacy against a broad range of hematologic malignan-
homologous recombination (Brca1 and Brca2) and base excision repair cies. Both classes of drugs bind tubulin and inhibit microtubule
(PARP). Therefore loss of one but not both of these pathways can be dynamics, impairing the formation of a functional spindle. The SAC
tolerated. As a second example, inhibition of Chk1 sensitizes p53 senses lack of proper attachment of kinetochores and arrests cells in
mutant cells to DNA damage.284 Because p53 is mutated in approxi- prometaphase in the presence of these compounds. Thus inhibition
mately half of all human tumors and the absence of p53 is a major of spindle dynamics results in abnormal chromosome segregation that
predictor of poor response to classic chemotherapeutic agents, consider- frequently results in either aneuploidy or cell death. The low mitotic
able efforts are being made to develop small-molecular inhibitors of index in human tumors suggests that microtubule poisons may have
Chk1. Although the initial model proposed that this effect was due therapeutic potential in a mitotic-independent manner although this
to the simultaneous abrogation of the G2 (Chk1) and G1 (p53) deserves further exploration.293 About 30 microtubule poisons are
checkpoints, new evidence suggests that the toxicity of Chk1 inhibitors currently in clinical development.
may be due to the generation of replicative stress (RS) in cells, with The success of these molecules in the clinic led to the search for
the less restrictive S-phase entry due to the lack of p53.285 Chk1 additional compounds that target specific regulators of microtubule
inhibitors therefore might synergize with other mutations that promote dynamics rather than tubulin itself. In a pioneer screening, monastrol
a promiscuous S-phase entry. A similar rationale applies to other was identified as an inhibitor of the kinesin Eg5, a protein required
molecules that target the RS response, such as inhibitors of the Chk1- for centrosome separation and the formation of a bipolar spindle.294
activating kinase ATR. Indeed, ATR inhibitors display a selective New drugs have been characterized that result in similar defects by
effect in Myc-driven tumors that display high levels of RS as a con- inhibiting CenpE, another kinesin with critical roles in microtubule-
sequence of the overexpression of Myc.286 This strategy can be general- to-kinetochore attachment.295–297 Inhibition of Aurora-A or Plk1 also
ized to multiple oncogenic alterations that result in cell cycle defects, may be considered to be an antispindle strategy because these kinases,
causing an increased reliance on ATR checkpoint activity, as recently although involved in other mitotic processes, are essential for centrosome
shown for different tumor types.287–291 maturation and separation and the formation of a bipolar spindle.52,295
72 Part I: Science and Clinical Oncology

In general, the inhibition of all these molecules results in an SAC- prevents mitotic arrest in the presence of microtubule poisons and
dependent arrest that impairs proliferation or viability of targeted therefore generates aberrant cells.52,305,306 It has been shown that the
cells. The Plk1 inhibitor volasertib has shown considerable promise reduction in these checkpoint proteins makes tumor cells more sensitive
in clinical studies, having reached phase III trials and been granted than untransformed cells to low doses of spindle poisons.307 The efficacy
the breakthrough therapy designation for its effects in acute myeloid of several Aurora or Mps1 small-molecule inhibitors currently is being
leukemia.298 tested in preclinical or clinical assays (see Table 4.3).52,295,306,308
Aneuploidy is a hallmark of cancer and is now considered as a
Targeting Mitotic Entry and Exit highly attractive therapeutic target.215,309 Most tumor cells are aneuploid,
whereas this abnormality is infrequent (although this has not been
As indicated in the earlier sections of this chapter, multiple enzymatic precisely established) in wild-type cells. This specificity is especially
activities are required for mitosis. Cdk1 is the major engine in this interesting given the problems that most therapeutic strategies that
process, and its activity is essential for mitosis. However, it has not target mitosis have in specifically inhibiting tumor cells. Aneuploid
been considered as a major target because its inhibition may have cells display specific defects in cell cycle kinetics, growth rate, metabo-
strong undesired effects.270 Yet some evidence suggests that it may be lism, and the response to specific stresses.310,311 These defects can be
an interesting target in specific situations. Cdk1 is specifically required exploited by targeting specific cellular pathways that protect cells from
in cells transformed with Myc but not in cells transformed by other the deleterious effects of aneuploidy. For instance, inhibiting the
oncogenes. Inhibition of Cdk1 rapidly downregulates survivin expres- proteotoxic and metabolic stress pathways specifically reduces the
sion, a protein required to avoid apoptosis in the presence of an excess viability of aneuploidy cells because of the unbalanced load of proteins
of Myc. Cdk1 inhibition therefore results in Myc-dependent apoptosis in these cells.312 As previously described, targeting the mitotic checkpoint
and regression of Myc-dependent lymphoma and hepatoblastoma may increase the levels of aneuploidy in the tumor cells, which opens
tumors.299 Cdk1 also is implicated in DNA repair by homologous the opportunity to combine different types of antimitotic strategies
recombination, and its inhibition results in impaired Brca1 activity. to further induce chromosomal instability and aneuploidy and inhibit
Cdk1 inhibition synergizes with PARP inhibitors, and partial inhibition the pathways that cancer cells use to tolerate this abnormal state.309
of Cdk1 therefore may sensitize Brca1/2-proficient cancer cells to
inhibit PARP, suggesting specific applications of Cdk1-targeting SUMMARY
compounds in cancer cells.300 Cdk1 activators such as Cdc25 phos-
phatases are additional targets whose inhibition results in impaired During the past several decades, investigators have uncovered a wealth
Cdk activity and cell cycle arrest.20 The therapeutic usefulness of of information about the proteins that control the division of human
inhibition of additional mitotic kinases such as Mastl, Nek proteins, cells. A key finding is that deregulation of the cell cycle machinery and/
or Haspin, among others, is currently undergoing preclinical or its checkpoints is a universal alteration in human cancer.1,2 Because
evaluation.3,295,301 the basic machinery that controls the cell cycle is similar in all cell
Inhibiting mitotic entry or progression or preventing spindle types, the hope is that common strategies will be developed against
dynamics may result in different outcomes, including cell death or a wide variety of cancers. Even though several of the currently used
the exit from the cell cycle without chromosome segregation.295 In anticancer therapies target nonselective and non–mechanism-based
fact, a rapid exit from mitosis is a major resistance mechanism that targets, their effectiveness, albeit limited in many cases, is likely due
generates viable cells in the presence of mitotic poisons. This finding to the fact that they ultimately target cell cycle regulatory or DDR
led to the evaluation of mitotic exit pathways as new targets for signaling pathways, the status of which is different in normal cells
therapy.302 In fact, genetic ablation of APC/C-Cdc20 completely versus tumor cells. Identification of all the components of the cellular
prevents mitotic exit, and these mutant cells arrest in mitosis until machinery that control the cell cycle both positively and negatively
they die.90 This action results in complete tumor repression in mouse is vital to the continued development of anticancer agents that can
models, and a first generation of APC/C inhibitors is currently available preferentially eliminate cancer cells and minimize the toxicity to normal
to test this strategy in preclinical studies (see Table 4.3).90,303,304 tissues. The complexity of the human genome makes this task difficult
because many members of the major protein families that regulate the
Targeting the Spindle Assembly Checkpoint cell cycle have not been studied thus far. In addition, our knowledge of
and Aneuploidy the in vivo relevance of these proteins in different tissues is limited. It is
obvious from the mouse models studied that the efficacy of inhibiting
Similarly to what was discussed for the DNA damage checkpoint, a specific cell cycle targets depends on the tumor type and the oncogenic
different concept is provided by the use of SAC inhibitors (checkpoint environment in each specific tumor. As our understanding of cell cycle
abrogation) to increase instability in cancer cells. Checkpoint kinases regulation and checkpoint signaling improves, the goal is to use this
such as Mps1 (also known as TTK) and Aurora-B (not considered to knowledge in the design of mechanism-based therapeutics that will
be a “core” checkpoint protein but involved in an error-correction bring anticancer therapy to a new level. There can be little doubt of
mechanism) are required for the proper bipolar spindle attachment the value of targeting the cell cycle in drug discovery.
of chromosomes.3 Inhibition of these kinases results in rapid exit from
mitosis without chromosome segregation, generating tetraploid or The complete reference list is available online at
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