Transcript p53 and apoptosis

Apoptosis
Tumor-suppressor genes
Tumor-suppressor genes, function like brakes, keep
cell numbers down, either by inhibiting progress
through the cell cycle and thereby preventing cell
birth, or by promoting programmed cell death
(also called apoptosis). When cellular tumor
suppressor genes are rendered non-functional
through mutation, the cell becomes malignant.
Examples are the gene encoding the
retinoblastoma protein (Rb), inactivated in
retinoblastomas, p53, and p16INK4a, which inhibits
cyclin-dependent kinases and is inactivated in
many different tumors.
Oncogenes
Oncogenes stimulate appropriate cell growth
under normal conditions, as required for the
continued turnover and replenishment of the skin,
gastrointestinal tract and blood, for example. Cells
with mutant oncogenes continue to grow (or
refuse to die) even when they are receiving no
growth signals. Examples are Ras, activated in
pancreatic and colon cancers, and Bcl-2, activated
in lymphoid tumours. Amplification of oncogenes
(more than their normal gene copy number) is also
found in cancer: MDM2 is amplified in
liposarcomas.
Roles of p53 in apoptosis
1. p53 induces apoptosis through
transcriptional activation of proapoptotic
genes, such as Puma, Noxa, p53AIP1, Bax,
Apaf-1 etc.
2. It can also directly induce apoptosis by
localizing to mitochondria via interaction
with Bcl-2 family protein Bcl-xL and
facilitating Bax oligomerization
Reading:Vousden and Lu: Nature Reviews
Cancer, 2002, 2:594-604.
p53 and of
apoptosis
Ref: Mol. Cell, 2003, 11(3):552-4
p53 and apoptosis
Ref: Cell, 2002, 108:153-164
Tumor Suppressor p53
• First identified as a protein associated with
viral oncogenes
• Mutated/inactivated in a majority of human
cancers
• Integrates numerous signals that control
cell life and death
• A common denominator in human cancer
• Understanding functions and regulation of
p53 is of great importance in cancer biology
and cancer therapy
The p53 pathways
The p53 network
p53 is a sequence-specific DNA
binding protein
1. p53 central core-domain interacts directly with DNA
2. p53 binding sites consist of four copies of the pentamer
consensus sequence PuPuPuC(A/T). The pentamers are
oriented in alternating directions. A short stretch of
sequence up to 13 bp may be inserted between the pairs
of pentamers. The p53 target genes in the human genome
usually carry the consensus sequence.
3. Amino acid residues in the core-domain that are critical for
DNA-binding are among the “hot-spots” of tumor-derived
p53 mutations, attesting to the importance of DNAbinding for p53’s tumor suppression function.
Structure of p53 core-domain
Ref: Science, 265:346-355, 1994
p53 and apoptosis
Apaf-1
p53AIP1
p53 in apoptosis
1. p53 mediates apoptosis in response to DNA
damage, oncogene expression (adenovirus
E1A, myc etc.), or withdrawal of growth
factors
2. Overexpression wild-type of p53 leads to
apoptosis
3. p53 can induce the expression of proapoptotic
genes, such as Bax (ref Cell, 80:293) and
p53AIP1 (ref: Cell, 102:849)
p53 in apoptosis
4. p53 can also repress transcription of
certain genes, and it has been proposed
that the repression function may also be
required for apoptosis (ref: Genes Dev
13:2490-501)
5. In vivo, p53 transactivation mutant is
defective in inducing apoptosis, at least
for some cell types (ref: EMBO J.
19:4967-4975)
Physiological relevance of
p53-induced apoptosis
• Suppress oncogene-induced transformation
• Inhibit tumor growth and progression
• Remove cells with severe DNA damage
• Effectiveness of cancer chemotheraphy
correlates with the ability to induce p53dependent apoptotic response
Tumor-derived mutations
affecting apoptosis
Protein
Role in apoptosis
Ref
p53
Mutated or altered expression in many
cancers. Initiates the intrinsic apoptotic
pathway. p53-/- cells are resistant to drug
induced apoptosis.
Vogelstein et al.,
2000
p19ARF
Mutated or altered expression in many
cancers. Blocks MDM2 inhibition of p53.
Enhances drug-induced apoptosis by p53.
Sherr and Weber,
2000
Rb
Mutated in some cancers, and functionally
disrupted in many cancers. Inhibits E2Fmedidated transcription. Loss of Rb function
induces p53-dependent and independent
apoptosis.
Harbour and Dean,
2000
Chk2
Mutated in Li-Fraumeni syndrome. Senses
DNA double strand breaks and
phosphorylates and stabilizes p53.
Khanna and
Jackson, 2001
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations
affecting apoptosis
Protein
Role in apoptosis
Ref
ATM
Mutated in ataxia-talangiectasia syndrome. Senses
DNA double strand breaks and stabilizes p53.
Deficiencies increase risk of developing
haematological malignancies and breast cancer
Khanna and
Jackson, 2001
Bax (p53
Mutated or decreased expression in some tumors.
Mediates mitochondrial membrane damage.
Sufficient but not necessary for drug-induced
apoptosis.
Rampino et al.,
1997
Bak
Mutated or decreased expression in some tumors.
Mediates mitochondrial membrane damage.
Sufficient but not necessary for drug-induced
apoptosis.
Kondo et al.,
2000
PTEN (p53
Mutated or altered expression in cancers.
Regulates Akt activation and subsequent
phosphorylation of Bad. Loss of PTEN results in
resistance to many apoptotic stimuli.
Di Cristofano and
Pandolfi, 2000
target gene)
target gene)
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations
affecting apoptosis
Protein
Role in apoptosis
Ref
Apaf-1 (p53
target gene)
Mutated and transcriptionally silenced in
Soengas et al.,
melanoma and leukemia cell lines. Necessary for 2001
activation of caspase-9 following cytochrome c
release. Apaf-1-/- cells are chemoresistant.
CD95/Fas
Mutated and down-regulated in lymphoid and
solid tumors. Initiates the extrinsic apoptotic
pathway. Loss of function is associated with
resistance to drug-induced cell death.
Muschen et al.,
2000
TRAILR1/R2
Mutated in metastatic breast cancers. Initiate the
extrinsic apoptotic pathway. Mutations lead to
suppression of death receptor-mediated
apoptosis.
Shin et al., 2001
Caspase-8
Gene silenced in neuroblastomas. Activates both
extrinsic and intrinsic apoptotic pathways.
Silencing results in resistance to drug-induced
apoptosis.
Teitz et al., 2000
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations
affecting apoptosis
Protein
Role in apoptosis
Ref
Bcl2
Frequently overexpressed in many tumors.
Antagonises Bax and/or Bak and inhibits
mitochondrial membrane disruption. Inhibits
drug-induced apoptosis.
Reed, 1999
MDM2
Overexpressed in some tumors. Negative
regulator of p53. Inhibits drug-induced p53
activation.
Sherr and Weber,
2000
IAPs
Frequently overexpressed in cancer. Down
regulation of XIAP induces apoptosis in
chemoresistant tumors.
Deveraux and
Reed, 1999
NFkB
Deregulated activity in many cancers.
Baldwin, 2001
Transcriptionally activates expression of antiapoptotic members of the Bcl-2 and IAP
families. Can inhibit both the extrinsic and
intrinsic death pathways and induce drugresistance.
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations
affecting apoptosis
Protein
Role in apoptosis
Ref
Myc
Deregulated expression in many cancers. Induces
proliferation in the presence of survival factors, such
as Bcl-2, and apoptosis in the absence of survival
factors. Can sensitise cells to drug-induced apoptosis.
Evan and
Vousden, 2001
Akt
Frequently amplified in solid tumors. Phosphorylates
Bad. Hyperactivation induces resistance to a range of
apoptotic stimuli including drugs.
Datta et al., 1999
PI3K
Overexpressed or deregulated in some cancers.
Responsible for activation of Akt and downstream
phosphorylation of Bad. Inhibition of PI3K enhances
chemotherapeutic drug-induced apoptosis.
Roymans and
Slegers, 2001
Ras
Mutated or deregulated in many cancers. Activates
PI3K and downstream pathways. Induces
proliferation and inhibits c-myc and drug-induced
apoptosis.
el-Deiry, 1997
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations
affecting apoptosis
Protein
Role in apoptosis
Ref
FLIP
Overexpressed in some cancers. Prevents
activation of caspase-8 and apoptosis induced by
some chemotherapeutic drugs.
Tepper and Seldin,
1999
Ref: Cell, 2002, 108:153-164
p53 directly mediates
mitochondrial mechanism of
apoptosis
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Early studies indicate that transactivation-defective mutants of p53
are capable of inducing apoptosis, implying a transcriptionindependent role for p53 in apoptosis.
DNA-damage leads to mitochondrial translocation of p53.
p53 binds to Bcl-2 family protein Bcl-xL to influence cytochrome c
release.
p53 directly activates the proapoptotic Bcl-2 protein Bax in the
absence of other proteins to permeabilize mitochondria and
engage the apoptotic program.
p53 can release both proapoptotic multidomain proteins and BH3only proteins [Proapoptotic Bcl-2 family proteins that share only
the third Bcl-2 homology domain (BH3)] that are sequestered by
Bcl-xL.
Ref: Mol. Cell, 2003, 11:577-90; Science, 2004, 303:1010-4.
How p53 functions in mitochondria
to induce cell death
1. p53 or Bax alone does not permeabilize
membrane, but they together can do so.
2. p53 facilitates Bax oligomerization.
3. p53 binds to Bcl-xL, but not to Bax.
4. p53-Bcl-xL interaction releases Bax.
5. Released Bax forms oligomers in mitochondrial
membrane, leading cytochrome c release and
apoptosis.
6. The proline-rich domain (aa 62-91 in mouse) of
p53 is required for this effect.
Ref: Science, 2004, 303:1010-4.
Viral oncogenes inhibit p53
functions
1. p53 was first identified as binding protein
of SV40 large T antigen in 1979, which
interacts with p53 core domain
2. Adenovirus oncoprotein E1B 55-kDa binds
to p53 and inhibits p53 transactivation
activity
3. HPV E6 binds to p53 and targets it for
ubiquitin-mediated degradation.
Using p53 to kill cancer cells
The p53 protein is a tumor suppressor — it keeps cell
numbers down by stopping cells from multiplying or
by promoting cell death. Loss of p53 occurs in most
human cancers, so it would be useful to be able to
restore its function. Several innovative strategies have
been suggested:
• Introduce normal p53 genes into a cancer cell with
mutant p53.
• Introduce a small compound that converts mutant
p53 proteins from an abnormal to a normal shape.
• Add a protein that attaches itself to mutant p53 and
kills cells.
• Stimulate the host's immune response to mutant p53
peptides.
Using p53 to kill cancer cells
• Introduce drugs that disrupt the interaction
between the MDM2 or E6 proteins and p53. (MDM2
and E6 negatively regulate p53; they are present at
abnormally high levels in some cancer cells, so
'quench' any normal p53.)
• Introduce the adeno-associated virus, which mimics
damaged DNA. Cells with mutant p53 cannot activate
the usual p53-dependent 'checkpoint' that is induced
by DNA damage, and eventually die.
• Infect cells with viruses that can replicate only in
cells without normal p53; the viruses kill these cells.
p53 in DNA repair and apoptosis
ROS: reactive oxygen species
Ref: Bensaad &
Vousden, Nature
Med, 2005
Summary
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p53 is a tumor-suppressor protein that induces apoptotic cell death
in response to oncogenic stress. Malignant progression is dependent
on loss of p53 function, either through mutation in the TP53 gene
(which encodes p53) itself or by defects in the signaling pathways
that are upstream or downstream of p53.
Mutations in TP53 occur in about half of all human cancers, almost
always resulting in the expression of a mutant p53 protein that has
acquired transforming activity.
p53-induced apoptosis depends on the ability of p53 to activate gene
expression.
p53 can also directly trigger the apoptotic response, by interacting
with Bcl-2 family protein.
The apoptotic and cell-cycle arrest activities of p53 can be separated,
and apoptotic cofactors that play a specific role in allowing p53induced death are being identified.
Ref: Nature Reviews Cancer, 2, 594 - 604 (2002)
Summary
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Phosphorylation of p53 regulates its ability to activate the
expression of apoptotic target genes, and other post-translational
modifications such as acetylation might also have a role.
In tumors that retain wild-type p53, the apoptotic response might
be hindered by defects in the apoptotic cofactors. These, therefore,
represent additional targets for the design of therapeutics that are
aimed at reactivating p53-mediated apoptosis in cancer cells.
Ref: Nature Reviews Cancer, 2, 594 - 604 (2002)