Cancer
Throughout the life of an individual, but particularly during development, every cell
constantly faces decisions.
Should it divide? Yes
No--> Should it differentiate? Yes
No-->Should it die? Yes-->Apoptosis
No
Proper development and tissue homeostasis rely on the correct balance between
division and apoptosis. Too much apoptosis leads to tissue atrophy such as in
Alzheimer’s disease. Too much proliferation or too little apoptosis leads to cancer.
cancer = unregulated cell proliferation
apoptosis = programmed cell death
necrosis = unprogrammed cell death
Cell Cycle
For recent reviews on this topic see:
Johnson and Walker (1999) Cyclins and cell cycle checkpoints. Annu. Rev. Pharmacol.
Toxicol. 39:295-312.
http://pharmtox.annualreviews.org/cgi/content/full/39/1/295
See also Gilbert’s website:
http://www.devbio.com/chap08/link0801.shtml
The cell cycle is at the center of the decisions a cell makes. Dividing cells go through a
cycle consisting of, G1 (growth or gap), S (DNA synthesis), G2 (growth) and M phase
(mitosis). Specific events must happen in a particular sequence for the cell to replicate.
During the G1 phase, the cell integrates mitogenic and growth inhibitory signals and
makes the decision to proceed, pause, or exit the cell cycle. S phase is defined as the
stage in which DNA synthesis occurs. G2 is the second gap phase during which the cell
prepares for the process of division. M stands for mitosis, the phase in which the
replicated chromosomes are segregated into separate nuclei and cytokinesis occurs to
form two daughter cells. In addition, the term G0 is used to describe cells that have
exited the cell cycle and become quiescent. When cells differentiate, they usually stop
dividing and therefore exit the cell cycle. Most cells that stop dividing to differentiate do
so in the G1 phase although some arrest in G2.
An important checkpoint in G1 is referred to as start in yeast and the restriction point
in mammalian cells. This is the point at which the cell becomes committed to DNA
replication and usually to completing a cell cycle. Before beginning the cell cycle, the
cell must assess whether sufficient growth has occurred (eg. are there enough
ribosomes? Other cell constituents?), whether there is cellular damage and whether
there is the proper complement of growth factor signaling. Another, checkpoint exists at
the G2 to M transition where cells become committed to divide. Again the cell must
assess whether the chromosomes are completely replicated and whether there is
cellular damage and proper growth factor signaling.
The events of the cell cycle are regulated by a protein complex consisting of a cyclin
dependent kinase (cdk) and a cyclin. Both the CDK and the cyclin are protein
kinases. Different CDK/cyclin complexes regulate different phases of the cell cycle. In
yeast, there is only one CDK that interacts with different phase-specific cyclins. In
mammals, there are different CDKs and different cyclins for different phases of the cell
cycle. Cyclins (and CDKs in mammals) are expressed cyclically; each cyclin is
expressed only in the appropriate phase of the cell cycle and then is rapidly degraded.
At each stage the CDK/cyclin complex performs 3 important functions:
1. They activate the cellular activities that are associated with that particular
phase in the cell cycle.
2. They activate the CDK/cyclin complex that controls the next stage in the cell
cycle.
3. They inactivate the CDK/cyclin complex from the previous stage in the cell
cycle. Often they trigger their own inactivation or degredation.
In this way, they control the forward progression of the cell cycle as well as the events
of that particular phase.
CDK/cyclin complexes are regulated in 4 major ways:
1. Association of the CDK and the cyclin (cyclin activates CDK)
2. Phosphorylation
3. Binding to cyclin dependent kinase inhibitors (CKIs)
4. Proteolytic degradation
Phosphorylation
Association of a CDK and cyclin is necessary but not sufficient for the formation of an
active complex. The proper phosphorylation status is also required for activity.
Phosphorylation of CDK/cyclin complexes can be either activating or inhibitory. As an
example we will consider the regulation of MPF activity. (Remember from cleavage that
MPF=maturation promoting factor, or mitosis promoting factor). MPF consists of a CDK
(cdc2) and cyclinB. After association of these 2 proteins, the CDK is phosphorylated on
a specific residue (tyrosine 15) by a kinase called wee1. This is an inhibitory
phosphorylation therefore wee1 ---| MPF. Then a second, activating phosphate is
added to another residue (threonine 161) by a kinase called CAK (CDK activating
kinase). However MPF is still not active because of the phosphorylated tyr15. MPF
finally becomes activated when the inhibitory phosphate added by wee1 is removed by
a phosphatase called cdc25 (or string). Then MPF is active and can trigger mitosis.
Cyclins can also be regulated by phosphorylation.
CDK inhibitors (CKIs)
There are 2 families of CKIs , the Cip/Kip family and the INK4 family.
• Members of the Cip/Kip family interact with all CDK/cyclin complexes. An
example of this family, p21, will be discussed below in relation to checkpoint
control.
• INK4 members interact with specific CDKs. For example, p16 interacts with the
G1 CDKs (cdc4 and cdc6 in mammals) and either prevent their association with
cyclinD or inhibit the preassembled complex. p16 is also important in checkpoint
control and will be further discussed below.
Ubiquitin dependent proteolysis
Ubiquitin dependent proteolysis is very important for rapidly degrading specific proteins.
Ubiquitin is a 76 amino acid peptide that is ligated (attached) to proteins targeted for
degradation. Additional ubiquitin peptides are sequentially added to the previous
subunit in a process called polyubiquitination. This is catalyzed by a protein complex
known as the ubiquitin ligase complex. Polyubiquitinated proteins are then
recognized and degraded by a proteolytic complex known as the 26S proteosome.
Ubiquitin dependent proteolysis is critical at 2 major steps in the cell cycle. Different
ubiquitin ligase complexes act at each step and the mode of regulation is different. The
progression from G1 to S-phase requires the action of a complex called SCF. SCF
recognizes proteins that are phosphorylated on PEST sequences, sites that are
common in proteins that are regulated by rapid turnover. Entry into S phase requires
the proteolytic degradation of an S phase inhibitor called Sic. Sic is phosphorylated on
it’s PEST motif by the G1 cyclinD. Thus Sic is targeted for polyubiquitination by SCF
and subsequent degradation by the 26S proteosome, removing the inhibitor and
allowing entry into S-phase. Ubiquitin dependent proteolysis is also important for
maintaining the forward progression of the cell cycle. SCF ubiquitinates cyclinD.
CyclinD is phosphorylated by the same CDK that it activates and this phosphorylation
targets it for ubiquitination by SCF and subsequent degredation.
Another ubiquitin ligase complex, APC (Anaphase Promoting Complex) is important for
the transition from metaphase to anaphase during mitosis. APC ubiquitinates M-phase
cyclins. The regulation of APC is very complex but at it is partly regulated by
phosphorylation of APC subunits. Phosphorylation of APC by the mitotic cyclin/CDK
activates it to ubiquitinate mitotic cyclins. It’s substrate specificity is controlled by
different subunits becoming part of the complex. For example, degradation of mitotic
cyclins is required for the onset of chromosome separation. Later, in telophase, APC
begins degrading proteins involved in anaphase. One subunit that provided specificity
for the mitotic cyclins is replaced by another subunit that confers substrate specificity
for the anaphase proteins. Thus, SCF activity is regulated by modification of the
substrates while APC activity is regulated by modification of the APC complex.
Checkpoint control
The most important checkpoint is called start in yeast or the restriction point in
mammalian cells. This is the point at which the cell commits to enter S phase. Since
most cells exit the cell cycle in G1 to differentiate, passing start generally commits to
undergoing an entire cell cycle. Cells assess whether adequate growth has occurred,
whether proper growth factor signaling is present and whether any cellular damage is
present before reaching the decision to pass start. Because defects in cell cycle
checkpoint control can lead to unregulated cell division, many of the factors involved in
checkpoint control were first identified as oncogenes.
One central protein in regulating start is the retinoblastoma protein (Rb). Rb binds and
inhibits a transcriptional activator called E2F. E2F activates the transcription of many
cell cycle genes, including those involved in DNA synthesis as well as S phase cyclins.
Rb dissociates from E2F when it is phosphorylated by cyclinD, thereby allowing E2F to
activate transcription and initiate S phase. Rb is also a negative transcriptional regulator
of the CKI p16. In cells lacking Rb, p16 is overexpressed. The inactivation of Rb by
cyclinD allows expression of p16 which then inhibits the activity of the G1 CDK. Thus
we see another example of how a cyclin promotes progression to the next phase while
inhibiting the current or previous phase.
CDK/cyclinD ——| Rb ——| E2F → S phase
Rb ——| p16 ——| G1 CDK
CyclinD does not oscillate with the cell cycle. Growth factors regulate the expression of
cyclinD. This is one of the ways that growth factors feed information into the cell cycle.
Thus cells cannot pass start without growth factor signals. Many growth factors signal to
the cell cycle through ras and the MAP kinase pathway. When quiescent cells re-enter
the cell cycle and divide (i.e. go from G0 to G1), cyclinD is the first cyclin to be
activated.
The myc transcription factor is also required for the G1 to S transition. myc activates
transcription of cdc25 which then activates the CDK complex, promoting cell cycle
progression to S phase.
Cells will not progress through the cell cycle if cellular damage is sensed. Cellular
damage induces the expression of a transcription factor called p53. p53 inhibits both
the G1 to S transition and the G2 to M transition. One of the genes whose transcription
is activated by p53 is the CKI p21. Thus:
Cellular damage → p53 → p21 ——| CDK/cyclin
Apoptosis
For recent reviews on this subject see the following:
King and Cidlowski (1998) Cell cycle regulation and apoptosis. Annu. Rev. Physiol. 60:
601-617.
http://physiol.AnnualReviews.org/cgi/content/full/60/1/601
Norbury and Hickson (2001) Cellular responses to DNA damage. Annu. Rev.
Pharmacol. Toxicol. 41:367-401.
http://pharmtox.annualreviews.org/cgi/content/full/41/1/367
Also the Aug 28, 1998 issue of Science was a special issue on the topic of apoptosis
and contains several informative reviews.
Apoptosis is generally considered as programmed cell death whereas necrosis signifies
unprogrammed cell death. The distinction between these was originally based on
morphological criteria; apoptotic cells shrink while necrotic cells swell. It is not clear
whether the distinction is quite so simple but there is a suite of morphological features
that characterize apoptosis. Apoptotic cells lose substrate attachment and become
rounded. Cells shrink, condense their chromatin, and display membrane blebbing.
Apoptotic cells fragment their DNA into approximately 200 base-pair fragments. At the
end of apoptosis, the cell is broken into multiple apoptotic bodies that are phagocytized
by neighboring cells. Thus these morphological changes are the outward manifestations
of the cell systematically dismantling itself and packaging itself up in membrane bound
vesicles to be absorbed by neighboring cells. Because cellular contents are not
released, this occurs with little inflammation.
Apoptosis is an essential component of normal development and homeostasis as well
as being critical for eliminating diseased or damaged cells. Approximately 50% of the
neurons undergo apoptosis during mammalian embryogenesis. Severe mental
retardation results if the extra neurons are not eliminated. In the immune system,
autoreactive lymphocytes are eliminated by apoptosis and failure in this system results
in autoimmune disease. Mutations in key components of apoptotic pathways are lethal.
As a safeguard against disease, every cell in our bodies expresses the components of
the apoptotic pathways and is ready for rapid self-destruction. In fact, it has recently
become clear that cells must receive the proper set of signals to prevent apoptosis or
they will self-destruct. Because of the importance of the correct balance between
division and apoptosis for proper development and tissue homeostasis and the dire
consequences of unregulated cell division, it is not surprising that the cell cycle and
apoptotic response are closely connected. This is one of the safeguards we have
against cancer (see below).
There are 2 major pathways that control the apoptotic response: the mitochondrial
pathway and the death receptor pathway. Both pathways utilize the same basic set of
proteases, called caspases.
Caspase Cascade
The central machinery of apoptosis consists of a cascade of cysteine proteases called
caspases. There are 2 major types of caspases, initiator caspases and effector
caspases.
Initiator caspases activate effector caspases by proteolytic cleavave of an effector
pro-caspase. The 2 most important initiator caspases are caspase-8 and caspase-9.
These are associated with the 2 major pathways for initiating apoptosis. Caspase-8 is
involved in receptor mediated apoptosis while caspase-9 is involved with the
mitocondrial pathway. Mice deficient for either of these caspases usually die before
birth and always within 3 days of birth. The mice show distinct defects for each
caspase, some of which include brain deformities, heart malformations and blood
overproduction.
Initiator caspases are regulated by association with cofactors. Available evidence
indicates that the cofactors facilitate caspase dimerization which leads to caspase
activation. One model proposes that dimerization results in the proteolytic cleavage of
each partner by the other, thereby activating the caspases.
Effector caspases are the enzymes responsible for disassembling the cells.
Substrates for effector caspases include:
1. apoptosis inhibitors (eg. Bcl2, Rb)
2. cell structures
3. other proteolytically activated enzymes
gelsolin—degrades cytoskeleton
CAD—caspase activated DNAse
Caspases are ubiquitously expressed, therefore every cell is poised for rapid self
destruction.
Mitochondrial pathway
The Bcl2 family of proteins are the central regulators of the mitochondrial pathway.
Bcl2 is an inhibitor of apoptosis. Bcl2 binds and inhibits a protein called Apaf. Apaf is an
activator of the initiator caspase-9. Therefore,
Bcl2 --| Apaf ! caspase-9 ! APOPTOSIS
Bcl2 is located on the cytosolic face of several membranes including the outer
mitochondrial membrane, the ER and the nuclear envelope. It is thought that Bcl2 may
somehow monitor damage in these compartments.
Bax is a protein,related to Bcl2, which inhibits Bcl2 thereby promoting apoptosis. It is
thought that binding of Bax to Bcl2 releases Apaf to then activate caspase9.
Thus, Bax --| Bcl2 --| APOPTOSIS, (net result, Bax ! APOPTOSIS)
CytochromeC is also an activator of Apaf and apoptosis. CytochromeC is released from
damaged mitochondria.
Death Receptor mediated pathways
A number of cell surface receptors can induce apoptosis when activated by a signal
ligand. One of the most well known of these is tumor necrosis factor (TNF) and the
receptor TNFR. Another important one is Fas. Death receptors are plasma membrane
spanning proteins that have a conserved motif in their cytoplasmic domain called the
Death Domain. The death domain mediates protein interactions with other death
domain proteins. A protein called FADD (Fas-associated protein with death domain)
associates with caspase-8 through the DED (death effector domain). This recruits
caspase-8 to the receptor complex leading to its activation (possibly as a result of 2
caspase-8 molecules being brought into proximity so they can cleave and activate each
other). Thus receptor mediated signaling can activate apoptosis independent of the
mitochondrial pathway.
TNF and other “death signals” can have very different effects on different cell types.
TNF can signal division in some cells, differentiation in other cells, and apoptosis in yet
other cell types. The response of a given cell depends on what other signals are being
perceived and what proteins are expressed by that cell. In the case of TNF signaling,
one protein of particular importance is a transcription factor called NF-ΚB. Cells that do
not express NF-ΚB are induced to die by TNF while those that do express NF-ΚB are
not.
Sensitization of cells
Inputs from different sources can act in combination to induce apoptosis. Cells that
receive stimuli that are insufficient to induce apoptosis become more sensitive to
induction by other stimuli. For example, mild DNA damage induces a low level of p53
expression. p53 inhibits the expression of Bcl2 and stimulates the expression of Fas.
Therefore, both the mitochondrial and receptor mediated pathways become more
sensitive to induction.
Cancer
Attributes of Cancerous Tumors
" Cell migration (metastasis): alterations in Cell-to-Cell Interactions Are Associated with
Malignancy. Metastatic cells break their contacts with other cells and the ECM in
their tissue of origin, and as a result, metastatic cells can invade adjoining tissue
or enter the circulation and establish themselves in a distant site.
" Angiogenesis (formation of new blood vessels): tumor growth requires formation of
new blood vessels to supply tumors with blood. Many tumors produce growth
factors that stimulate angiogenesis
" Unregulated cell division and growth (defects in cell cycle regulation).
" Failure to undergo apoptosis in response to inappropriate division
Cancer is an extraordinarily rare event
About 1 in 10 people will contract cancer at some point in their lives, and most of us
have some sort of personal experience with someone we know having cancer. As
such, we tend to think of cancer as common, and of course, it is a very serious
disease. However, when one considers it in a developmental context, cancer is
actually a very rare occurrence. First of all, cancer is rare before the age of forty. Then
when one considers all the billions of cell divisions that occur under proper regulation,
it is truly amazing how rarely a cell becomes malignant.
The reason for the rarity of cancer is the elaborate safeguards cells have against
inappropriate cell division. Normal cells require growth factor signals to instruct the
cells to divide and not to undergo apoptosis. Links between the cell cycle and
apoptotic machinery promote cell death for inappropriately dividing cells. For cancer to
occur requires the failure of the apoptotic safeguard mechanisms as well as the
deregulation of the cell cycle. Thus, most cancers require 2 or more mutations to
deregulate the cell cycle and to overcome the apoptotic safeguards against
inappropriate division.
Links between Apoptosis and the Cell Cycle
Because of the dire consequences of inappropriate cell proliferation, cells have evolved
failsafe mechanisms to eliminate cells that divide when they are not supposed to. We
tend to think of cancer as a common disease, but when one considers the millions of
cells and cell divisions in every individual, and that only about 10% of people develop
cancer (usually from a single defective cell and usually in older individuals), then it is
evident that cancer is in fact an extraordinarily rare event. This is because of the
failsafe mechanisms which allow cells to sense inappropriate proliferation and eliminate
themselves.
Several factors contribute to the rarity of cancers. One of these is the tight linkage
between the cell cycle and apoptosis. Factors that promote cell division also promote
apoptosis. Factors that inhibit apoptosis also inhibit cell division. There is a particularly
tight relationship between the control of cell cycle checkpoints and apoptosis.
p53 forms one of the key links between cell cycle regulation and apoptosis. Cell
damage induces p53. p53 inhibits cell division by activating transcription of the CKI p21.
p53 also induces apoptosis by inhibiting the expression of Bcl2 and activating the
expression of Bax and Fas.
P53! apoptosis
P53-- | cell cycle
p53 knockout mice are viable and cells can be induced to undergo apoptosis by other
means indicating that p53 does not have a direct role in apoptosis. However p53 mice
nearly always die of cancer indicating that p53 is a tumor suppressor.
Rb, the inhibitor of G1-S progression also inhibits apoptosis. Mice that are deficient in
Rb die because of widespread apoptosis. Lack of p53 suppresses the apoptosis in Rb
deficient cells indicating that Rb functions to suppress p53 dependent apoptosis. Rb is
one of the targets of degradation by caspases during apoptosis.
Rb --| cell cycle
Rb --| apoptosis
Myc is a transcription factor and proto-oncogene. Myc is required at the G1-S transition
and for quiescent cells to enter the cell cycle. Myc activates transcription of the cdc25
phosphatase which activates CDK. Thus Myc promotes cell division and in combination
with the appropriate growth factors is an important component of normal cell
proliferation. However, in the absence of growth factors, cells sense Myc induced cell
division as inappropriate and undergo apoptosis. This is because Myc also induces
p53.
Myc! cell cycle
Myc! apoptosis
The apoptotic regulator Bcl2 also feeds back to inhibit the cell cycle by an unknown
mechanism.
Bcl2 -- | apoptosis
Bcl2 ! exit to quiescence
Bcl2 --| re-entry to cell cycle
Genetic Basis of Cancer
There are 2 general classes of genes associated with cancers—oncogenes and tumor
suppressor genes. Gain-of-function mutations in proto-oncogenes convert them to
oncogenes (cancer causing). Loss-of-function mutations in tumor suppressor genes
can also cause cancer.
Oncogenes
Arise through gain-of-function mutations in Proto-oncogenes.
Proto-oncogenes generally encode factors that function to promote cell division or
inhibit apoptosis
Examples of Proto-oncogenes
" Growth factor signaling molecules (Growth factors, GF receptors, signal
transduction molecules like ras, MAPK, etc.)
" Transcription factors, such as myc
" Apoptosis inhibitors such as Bcl2
At least three mechanisms can produce gain-of-function mutations to generate
oncogenes from the corresponding proto-oncogenes.
" Point mutations in a proto-oncogene that result in a constitutively acting protein
product
" Localized reduplication (gene amplification) of a DNA segment that includes a
proto-oncogene, leading to overexpression of the encoded protein
" Chromosomal translocation that brings a growth-regulatory gene under the
control of a different promoter and that causes inappropriate expression of the
gene
In addition, oncogenes may be incorporated into viral genomes to generate tumor
viruses.
Tumor Suppressor Genes
Discovered because fusing cancer cells with some non-cancer cells inhibits tumor
growth unless a particular chromosome is lost. In general, they inhibit cell cycle
progression or promote apoptosis. Five broad classes of proteins are generally
recognized as products of tumor-suppressor genes:
•
•
•
•
•
Intracellular proteins that regulate or inhibit progression through a specific stage
of the cell cycle. Examples include:
o p16 cyclin-kinase inhibitor
o Retinoblastoma (Rb)
o p53
o p21
Receptors for paracrine factors (e.g. TGF-β) that function to inhibit cell
proliferation
Checkpoint-control proteins that arrest the cell cycle if DNA is damaged or
chromosomes are abnormal
Proteins that promote apoptosis
o Death receptors
o Bax
Enzymes that participate in DNA repair
Some Examples of Genetic Defects Common in Cancers
Many cancers involve defects in the factors that regulate the cell cycle or induce
apoptosis in response to inappropriate cell proliferation. Several examples follow:
Deregulated Myc expression is associated with a number of cancers, but Myc alone is
not sufficient to cause cancer. As mentioned, Myc alone induces apoptosis. However, if
Ras is also deregulated then cancer can occur. Ras is a signal transduction molecule
that acts downstream of growth factor receptors and deregulated Ras substitutes for
the growth factor requirement.
It is thought that deregulation of Rb is a ubiquitous feature of all cancers. This makes
sense since Rb blocks the cell cycle at start. For cancer to occur, cells must get past
this point somehow.
Overexpression of Bcl2 causes increased tumorogenesis by oversuppressing
apoptosis. Similarly, deficiencies in Bax are common in many types of cancer including
colon cancer because these cells are defective in apoptosis initiation.
Defects in death receptor signaling are associated with some cancers. For example,
mutations in the FasR gene for the Fas receptor increase the frequency of Hodgkin’s
lymphoma.
As mentioned, p53 is a tumor suppressor. Cells mutant for p53 no longer undergo
apoptosis or cell cycle arrest in response to cellular damage. This makes these cells
susceptible to accumulating genetic defects that could result in cancerous proliferation.
Mice lacking p53 are viable and healthy, except for a predisposition to develop multiple
types of tumors.
Oncogenic Viruses
Retroviruses
• Contain an RNA genome
• Reverse transcription produces a DNA copy that integrates into the host genome
• Host genes occasionally get incorporated into the viral genome
• Infection by a retrovirus containing an incorporated oncogene can cause cancer.
DNA viruses
• Do not integrate into the host genome
• Only a few examples of tumorogenic DNA viruses
•
•
•
human papillomavirus (HPV) is tumorogenic
encodes viral gene products that interfere with normal cellular regulation
o One HPV protein, E7, binds to and inhibits Rb
o another, E6, inhibits p53
o E5 protein causes sustained activation of the PDGF receptor
o Together these proteins induce transformation and proliferation of the
host cells
transformation occurs in the absence of mutations to cell regulatory proteins
Cancer Therapy
There are 4 major types of cancer therapy.
" Chemotherapy
" Radiation Therapy
" Angiogenesis suppression
" Gene therapy:
Many cancer therapies depend on induction of apoptosis by causing cellular damage.
For example, radiation therapy causes damage to cells and dividing cells are the most
sensitive. The idea is that actively dividing cancer cells will be induced to undergo
apoptosis. Many other therapeutic agents work by similar means. However, some
tumors become insensitive to therapy because of defects in the apoptotic machinery.
One of the most common examples is defects in p53. Since p53 is the central
regulator in damage induced apoptosis and cell cycle arrest, cells with defects in p53
no longer respond to agents such as radiation or certain drugs.
Since tumors require angiogenesis for a blood supply to support tumor growth, some
therapies target this process. The most common strategy is to develop and use
inhibitors of growth factors that promote blood vessel development.
The idea behind gene therapy is conceptually simple: replace a defective gene (such as
P53) with a functional copy. Of course technically this is a huge challenge. How to get
the functional gene into every cancer cell? Engineered viruses are the most promising
vector right now. Yes, some of the same families of viruses that can cause cancer
may be manipulated to deliver cancer suppressor genes to the defective cells.