Cell Cycle, Growth Inhibition and

Tumor Suppressor Genes

Cancer Biology, Week 3
 Cell Cycle
• Cancer is characterized by abnormal cell
proliferation. Cell proliferation involves the
reproduction of a cell to form two daughter
cells. Each daughter cell can reproduce to form
two daughter cells, and so on; thus cell
reproduction is cyclic – hence, cell cycle.
• The cycle is made up of four stages: G1, S
phase, G2, and M phase.
• G1, S, and G2 make up the part of the cycle
called interphase.
• The genetic material of a cell is replicated in S
phase (DNA synthesis).
• M phase involves the partitioning of the cell to
produce two daughter cells and includes mitosis
and cytokinesis.
• G1 and G2 are “gaps” preceding the S and M
phases, respectively, during which time the cell
prepares for the next phase.
 Cyclins and Cyclin-dependent Kinases
• The average length of the cell cycle is 16 h (15 h
for interphase and 1 h for mitosis)
• But this can vary depending on the cell type.
• Most cells in an adult are not in the process of
cell division. They are quiescent and enter an
inactive period called G0, a phase outside of the
cell cycle.
• Mitogens or growth factors can, however,
induce cells in G0 to re-enter the cell cycle and
pass a control point called the G1 restriction
point. Before the passage of the restriction
point, cell division is dependent on mitogens;
afterwards, cells are irreversibly committed to
progress through the cycle without the need for
growth factors.
• The passage of the cell through the different
phases of the cell cycle is coordinated and
regulated by a set of proteins called cyclins and
their associated cyclin-dependent kinases (cdks)
 Cyclins and Cyclin-dependent Kinases
• Cyclins were so named because of
the cyclical changes in their
concentrations that occur over a
series of cell divisions.
• The pairing of cyclins to the cdks is
highly specific.
• Cyclins are regulatory subunits of
their cdks. Upon binding of a cyclin to
its cdk partner, cyclin induces a
conformational change in the
catalytic subunit of the cdk, revealing
its active site. Note that the
concentration of cdks does not
fluctuate during the cell cycle.
 Cyclins and Cyclin-dependent Kinases
• Cyclin D is the first cyclin to be
synthesized and, together with cdks
4/6, drives progression through G1.
• Cyclin D plays a role in the regulation
of expression of the cyclin E gene,
whose product is important for the
G1 to S phase transition. Cyclin A–
cdk2 is important for S phase
progression. Cyclins A, B–cdk1 directs
G2 and the G2 to M phase transition
 Cyclins and Cyclin-dependent Kinases
• Cell cycle checkpoints a series of biochemical
signaling pathways that sense and induce a cellular
response to DNA damage, are important for
maintaining the integrity of the genome.
• The G1 checkpoint leads to the arrest of the cell
cycle in response to DNA damage, ensuring that
DNA damage is not replicated during S phase.
• The G2 checkpoint leads to the arrest of the cell
cycle in response to damaged and/or unreplicated
DNA to ensure proper completion of S phase.
• The M checkpoint leads to the arrest of
chromosomal segregation in response to
misalignment on the mitotic spindle.
• The components of the checkpoints are proteins
that act as DNA damage sensors, signal transducers,
or effectors.
• Disruption of checkpoint function leads to genomic
and chromosomal instability, leading to mutations
that can induce carcinogenesis.
 Cyclins and Cyclin-dependent Kinases
• The cyclin–cdk complexes exert their effect by
phosphorylating target proteins.
• The targets of cyclin– cdk complexes include a
diverse set of proteins:
• Transcriptional regulators,
• Cytoskeletal proteins,
• Nuclear pore and envelope proteins,
• Histones.
• Specific examples include
• condensins,
• nuclear lamins,
• GM130 of the Golgi apparatus,
• the famous transcriptional regulator retinoblastoma
protein (RB or pRB),
• transcription factors E2F and Smad 3.
• Consequently, essential events of the cell cycle,
including chromosomal condensation, nuclear
breakdown, fragmentation of the Golgi apparatus,
regulated gene expression, and mitotic spindle
assembly, are facilitated. Note that
dephosphorylation is an important mechanism for
resetting the cell for another round of the cell cycle.
 Cyclins and Cyclin-dependent Kinases
• The cyclin–cdk complexes exert their
effect by phosphorylating target
proteins.
• The targets of cyclin– cdk complexes
include a diverse set of proteins:
• Transcriptional regulators,
• Cytoskeletal proteins,
• Nuclear pore and envelope proteins,
• Histones.
• Specific examples include
• condensins,
• nuclear lamins,
• GM130 of the Golgi apparatus,
• the famous transcriptional regulator
retinoblastoma protein (RB or pRB),
• transcription factors E2F and Smad 3.
 M – Phase

• Consequently, essential events of the

cell cycle, including chromosomal
condensation, nuclear breakdown,
fragmentation of the Golgi apparatus,
regulated gene expression, and
mitotic spindle assembly, are
facilitated. Note that
dephosphorylation is an important
mechanism for resetting the cell for
another round of the cell cycle.
Growth Inhibition and Tumor
Suppressor Genes
 Growth Inhibition and Tumor Suppressor Genes
• The human body has mechanisms exerted by tumor suppressor genes that
normally “police” the processes that regulate cell numbers and ensure that
new cells receive DNA that has been precisely replicated.
• Many tumor suppressor gene products act as stop signs to uncontrolled
growth and therefore may inhibit the cell cycle, promote differentiation, or
trigger apoptosis.
• If both copies of a tumor suppressor gene become inactivated by mutation
or epigenetic changes, the inhibitory signal is lost, and the result may be
unregulated cell growth, a hallmark of cancer.
• Other tumor suppressor gene products are involved in DNA repair. If
inactivated, DNA repair may be defective, and failure to repair DNA may give
rise to mutations that lead to cancer.
• Two alleles of every gene are present in the human genome (except those on
sex chromosomes), and, in most cases, loss of tumor suppressor gene
function requires inactivation of both copies. This often happens by mutation
in one copy and loss of the remaining wild-type allele (loss of heterozygosity,
LOH).
 Definition of Tumor Suppressor Genes
• Inheritance of a germline mutation (passed on from egg/ sperm DNA and
thus present in all cells of an individual) in one tumor suppressor allele and
the acquisition of a somatic mutation or other inactivating alteration in the
second allele later in life. This was first proposed by Knudson and is known as
Knudson’s two-hit hypothesis. It states a strict definition of a tumor
suppressor gene: a gene in which a germline mutation predisposes an
individual to cancer.
• Breast and ovarian cancer susceptibility genes BRCA1 and BRCA2
• BRCA1 and BRCA2 germline inheritance leads to hereditary breast and
ovarian cancer syndrome and predisposes individuals to breast and ovarian
cancer. Individuals with this syndrome have a 50–80% lifetime risk of
developing breast cancer and a 30–50% lifetime risk of developing ovarian
cancer.
• Both BRCA proteins are involved in homologous recombination and double-
stranded break repair.
 Definition of Tumor Suppressor Genes
• Some genes that encode phosphatases which
antagonize kinase activity could act as tumor
suppressors as they remove the activating-
phosphate groups on proteins who are involved
in proliferation.
• One gene encoding a phosphatase that is
frequently mutated in many cancers is PTEN
(phosphatase and tensin homolog on
chromosome 10). PTEN codes for a phosphatase
with dual specificity—it can act as both a protein
and lipid phosphatase. Its role as a lipid
phosphatase in tumor suppression is best
known. PTEN dephosphorylates the membrane
lipid phosphatidyl-inositol-3 phosphate (PIP3) to
form PIP2. This antagonizes (shown by the
reversed red arrow) the PI3 kinase pathway
 Definition of Tumor Suppressor Genes
• Some genes that encode phosphatases which
antagonize kinase activity could act as tumor
suppressors as they remove the activating-
phosphate groups on proteins who are involved
in proliferation.
• One gene encoding a phosphatase that is
frequently mutated in many cancers is PTEN
(phosphatase and tensin homolog on
chromosome 10).
• PTEN codes for a phosphatase with dual
specificity—it can act as both a protein and lipid
phosphatase. Its role as a lipid phosphatase in
tumor suppression is best known.
• PTEN dephosphorylates the membrane lipid
phosphatidyl-inositol-3 phosphate (PIP3) to form
PIP2. This antagonizes (shown by the reversed
red arrow) the PI3 kinase pathway
 Definition of Tumor Suppressor Genes
• Loss of the inhibitory dephosphorylation
activity of PTEN in the PTEN mutant
phenotype may result in a constitutively
active PI3 kinase pathway, involving
activation of protein kinases Akt and m-TOR
(mammalian target of rapamycin). The net
result is the inhibition of apoptosis and
induction of cell proliferation. This favors
oncogenesis
 Retinoblastoma Gene
• Retinoblastoma is a rare childhood
cancer with a worldwide incidence of 1
in 20,000.
• There are two forms of the disease, a
familial (inherited) form and a sporadic
form.
• About 40% of all retinoblastoma cases
are familial, and about 60% are sporadic.
In the familial form of the disease, one
germline mutation in the Rb gene is
passed to the child and is present in all
cells. A second mutation is acquired in a
particular retinoblast that consequently
gives rise to a tumor in the retina.
• One inherited mutated gene results in a
sufficiently high probability that a second
mutation may occur. The second
mutation most often results from
somatic mitotic recombination, during
which the normal gene copy is replaced
with a mutant copy.
 Retinoblastoma Gene

• RB is a multi-functional protein that has over a hundred known

protein-binding partners. It is considered as a transcriptional co-factor
that can bind to transcription factors and either inhibit or induce
transcription factor activity. Its main role is to regulate the cell cycle
by inhibiting the G1 to S phase transition.
 Retinoblastoma Gene
• RB is an indirect regulator of
transcription for specific gene
expression that affects cell
proliferation and differentiation.
Protein–protein interactions
facilitate the function of RB as a
transcriptional regulator; RB
binds to, and modulates the
activity of, a critical transcription
factor (E2F) and chromatin
remodeling enzymes (Figure
5.4). Cell cycle arrest can also be
induced by RB via stabilization of
the Cdk inhibitor p27. RB acts as
a scaffold to recruit an inhibitor
of p27, S phase kinase-
associated protein 2 (SKP2), and
proteins that target SKP2 for
degradation; thus p27 remains
free.
 Retinoblastoma Gene

• Inactivation of RB promotes
chromosomal instability and
angiogenesis via increased
expression of E2F target genes
(e.g. MAD2, VEGF,
respectively). Some studies
suggest that RB may have a
role in differentiation, as well
as cell cycle progression, in the
developing retina
 Retinoblastoma Gene

• Although E2F1 inhibits angiogenesis in mouse models, growing evidence suggests a possible proangiogenic role for E2F1,
enhances angiogenesis by transcriptionally upregulating growth factors and cytokines like PDGF-B, IL8, and Groα, as well
as receptors like FGFR, FLT1, KDR, and PDGFRα.
• In Rb mutant mice, Id2 activated VEGF expression resulting in enhanced angiogenesis. Rb suppresses EMT by inducing
epithelial E-cadherin, in part through repression of ZEB1;
• Rb-inactive cancer show increased ZEB1 and decreased E-cadherin. E2F1 can induce the
mesenchymal fibronectin and vimentin promoters as well as induce β4 integrin and matrix-degrading enzymes MMPs
9,14,15, and 16. Cox2, which induces matrix-degrading enzymes including pro-UPA, MMP1, and MMP2, is repressed by
Rb; conversely, Rb inactivation leads to Cox2 expression resulting in increased expression of these enzymes. → Indicates
activation of expression, whereas ⊣ Indicates inhibition of expression.
 P53 Pathway
• The p53 gene was the first
tumor suppressor gene to
be identified, and, since its
discovery, scientists have
found that the p53 pathway
is altered in most human
cancers.
• Two p53 homologs, p73 and
p63, have also been
identified, but mutations in
cancer cells are rare. Its
protein product, p53, is at
the heart of the cell’s tumor
suppressive mechanism and
thus has been nicknamed
the “guardian of the
genome”.
 P53 Pathway
• In the absence of cellular stress,
low levels of p53 induce
antioxidant activity which
decreases the levels of reactive
oxygen species (ROS) and
subsequent DNA damage
• Normal cell metabolism produces
ROS that can react with DNA. It
has been estimated that
endogenous ROS modify
approximately 20,000 bases of
DNA per day in a single cell. p53
protects against ROS by
upregulating genes whose
products have antioxidant
functions such as glutathione
peroxidase 1 and sestrins—
proteins involved in hydrogen
peroxide metabolism. This
antioxidant activity guards against
mutation and may help prevent
cancer.
 P53 Pathway
• Many types of “danger signals,” such as
cell stress and DNA damage, can
activate p53 and trigger several crucial
cellular responses that suppress tumor
formation.
• Upstream stress activators include
radiation-, drug-, or carcinogen-induced
DNA damage, oncogenic activation,
hypoxia, and low ribonucleotide pools.
These conditions may nurture tumor
initiation. In response to these stress
signals, p53 can elicit downstream
cellular effects, including transient or
permanent cell cycle arrest, DNA repair,
apoptosis, and inhibition of
angiogenesis (see Chapter 10). The
ability to cause the cell cycle to pause
allows for the repair of mild DNA
 Structure of p53
• The p53 gene, located on
chromosome 17p13, contains 11
exons that encode a 53 kDa
phosphoprotein.
• The p53 protein is a transcription
factor containing four distinct
domains: the amino-terminal
transactivation domain, the DNA-
binding domain containing a Zn2+
ion, a tetramerization domain, and a
carboxy-terminal regulatory domain.
• The p53 protein binds as a tetramer
to a p53 response element
containing two direct repeats
 Regulation of p53 protein by MDM2
• Normally, the level of p53 protein in
a cell is low. The activity of p53 in a
cell is regulated at the level of
protein degradation, not at the level
of expression of the p53 gene.
• The MDM2 protein, a ubiquitin
ligase, is its main regulator.
Ubiquitin ligases are enzymes that
attach a small peptide called
ubiquitin to proteins, flagging it for
proteolysis (enzymatic protein
degradation involving cleavage of
peptide bonds) in proteosomes.
• In addition, MDM2 modifies the
activity of p53 as it binds to, and
inhibits, the p53 transactivation
domain at the amino-terminal and
transports the protein into the
cytoplasm, away from nuclear DNA
 Regulation of p53 protein by MDM2
• The binding of MDM2 to p53 is
part of an autoregulatory
feedback loop.
• Mdm2 gene is a transcriptional
target of p53. Therefore, p53
stimulates the production of its
negative regulator MDM2 that
causes the degradation of p53.
• Low amounts of p53 will reduce
transcription of the Mdm2 gene
and the amount of MDM2
protein, and this will result in an
increase of p53 activity, thus
completing the loop.
• MDM2 binds to N-terminal,
modifies C-terminal of p53.
 Upstream: molecular pathways of p53 activation
• The mechanism by which p53 becomes activated
depends on the nature of the stress signal. Stress is
“sensed” by cellular proteins, many of which are
kinases that convey the danger signals to p53 via DS Break
phosphorylation.
• Disruption of the p53–MDM2 interaction is
fundamental to the activation of p53 by its
upstream factors
• ATM and Chk2 phosphorylates N-terminus side of
p53, interfering with MDM2 binding (True for ATR
and Casein Kinase II, too).
• Activated oncogenes, such as Ras, induce the
activity of the protein p14arf, another modulator of
the p53–MDM2 complex. p14arf is one of two
translational products of the INK4a/CDKN2A gene.
• p14arf does not bind to the interface of p53–MDM2
but functions by sequestering MDM2 to the
nucleolus of the cell. All three pathways prevent
degradation of p53 by MDM2.
 Downstream: molecular mechanisms of p53 cellular
effects
• Cell cycle arrest or senescence: p21 inhibits
Cyclin/Cdk complexes
• DNA repair: XPC that is involved in nucleotide
excision repair is regulated by p53; PCNA is a
proliferation marker (proliferating nuclear antigen)
• Apoptosis: Genes encoding proteins that promote
apoptosis, pro-apoptotic proteins, are induced,
while genes encoding proteins that antagonize
apoptosis, anti-apoptotic proteins, are repressed.
The mitochondrial pro-apoptotic proteins NOXA,
PUMA, and p53AIP1, that cause the release of
cytochrome c and activate the apoptosome, are
induced. Also, p53 tips the balance regulated by the
Bcl-2 protein family towards apoptosis by inducing
gene expression of the pro-apoptotic protein Bax
and repressing the expression of the anti-apoptotic
protein Bcl-2.
• Inhibition of angiogenesis: Thrombospondin, an
inhibitor of angiogenesis, is also transcriptionally
regulated by p53.
 Factors that decide downstream outcome: cell cycle
arrest or apoptosis
• The regulation of a gene that codes for a
cyclin–cdk inhibitor, the p21 gene, plays a
pivotal role.
• Top: without competition from Myc, p53
and Miz-1 bind to the promoter of p21 and
induce transcription, resulting in cell cycle
inhibition.
• Bottom: upon oncogenic activation, Myc
competes with the binding of p53. Myc and
Miz-1 bind to the p21 promoter, inhibit
transcription, and block cell cycle inhibition.
ASPP (apoptosis-stimulating proteins of
p53) binds to p53 and facilitates activation
of apoptotic genes to induce apoptosis.
Interaction of DNA viral protein products
with RB and p53