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TUMOUR SUPPRESSOR GENES II


Cellular mechanisms regulating Rb activation and the induction of growth arrest:
1
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2
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3
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This involves the accumulation of
proteins that effectively prevent Rb phosphorylation, and therefore S phase entry
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Therefore, even when the cells are presented with mitogen-induced signalling, these
cells cannot progress into S phase of the cell cycle
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The Cyclin-dependent Kinase Inhibitors (CKIs) promote cell cycle arrest by blocking
Rb phosphorylation
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The CKIs, p21 and p27 promote the assembly of cyclinD-CDK4/6 complexes but
inhibit the activity of cyclinE-CDK2
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As cyclinD-CDK4/6 complexes accumulate in response to growth factor signalling, i
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EGF, they provide a sink for CKI separation
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This effectively reduces the concentration of free p21/p27 available to inhibit newly
forming cyclinE-Cdk2 complexes, as they are bound to cyclinD/Cdk4 complexes
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Active cyclinE-Cdk2 complexes can then phosphorylate p27 targeting it for
degradation, further promoting cyclinE-Ckd2 activation
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Induction of tumour suppressor p53 results in transcriptional activation and rapid
accumulation of p21, and consequently the inhibition of cyclinE-Cdk2 complexes
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This is due to the lack of a “sink” that would have sequestered the CKIs
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The tumour suppressor p16 disrupts cyclinD-Cdk4/6 complexes, inactivating the
kinase and preventing the phosphorylation-dependent inhibition of Rb
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Elevated in p16 expression is associated with cellular senescence (irreversible
growth arrest) and correlates with the hypophosphorylation of Rb
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P16 expression is induced in response to oncogenic Ras signalling to promote a
senescence-like growth arrest
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• This is an inbuilt response mechanism to any abnormal signalling
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Germline mutations in the gene CDKN2A (which encodes both p16 and p14 tumour
suppressor genes with alternate reading frames) is associated with familial
melanoma
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In addition to disrupting cyclinD-CDK4/6 complexes, p16 also liberates sequestered
p21/p27
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These liberated CKIs can bind and inhibit cyclinE-CDK2
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Rb phosphorylation is thus prevented, and E2F-dependent transcription and cell
cycle progression is therefore inhibited
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CKIs antagonise cell growth signals by binding to and inhibiting the kinase activity of
cyclin-CDK complexes
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Their expression is elevated in quiescent (p27) and senescent (p21 and p16) cells and
this correlates with the hypophosphorylation of Rb (suppression of transcription)
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As cyclinD-CDK4/6 complexes accumulate during G1, they provide a “sink” for
p21/p27 sequestration (which also functions to promote cyclinD-CDK4/6 assembly),
reducing the concentration of free CKI available to inhibit newly forming cyclinECdk2 complexes
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P16 inhibits cyclinD-Cdk4/6 activities and liberates sequestered CKIs, which can then
inhibit cyclinE-Cdk2
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Because of its ability to suppress proliferative signalling, p16 represents important
tumour suppressor proteins
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P21 can overcome sequestration by cyclinD-Cdk4/6 following its elevated
expression in response to the tumour suppressor p53
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In addition to inhibiting E2F, RB also positively regulates differentiation-specific
gene expression
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Differentiation of retinoblasts (and possibly therefore their exit from the cell cycle)
may be acutely dependent on RB
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Additionally, RB loss is not just associated with retinoblastomas, i
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RB1 gene
deletion is also found in sarcomas, lung cancers, bladder tumours, mammary
tumours and prostate cancers
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Significantly, tumours that retain normal RB often exhibit other alterations in the Rb
pathway, i
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elevated activity of the RB kinases (Cdk4,6/CyclinD)
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Rb mutations are the rate limiting factors of retinoblastoma but are in no way
indication that they are the only changes needed
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p53 is a 53Kda transcription factor encoded by the TP53 gene
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p53 monomers associate to form a 177 Kda tetramer, which is recruited to p53specific response elements in the promoter of target genes
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WT p53 can actively suppress oncogene-mediated cellular transformation
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p53 is a key node in signalling networks involved in sensing cellular stress, in that, it
integrated different cell signals to produce appropriate responses that will benefit
the organism
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The tumour suppressive functions of p53 have largely been attributed to its roles in
the DNA damage response and suppression of oncogene activation
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p53 must ensure that the cellular response matches the severity of the insult
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• Transient cell cycle arrest response would be an output to low level cellular
stress, requiring low but sufficient levels of p53
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• Low levels of p53 would lead to cell cycle arrest in order to allow DNA repair
to occur and a continuation of the cell cycle
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The biological functions of p53 are mostly due to the activities of p53-regulated gene
products
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In normal cells, p53 levels are low due to the short half-life of the protein (6-20 min),
due to the constant degradation of p53 by MDM2
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p53 tetramers lead to the transcription of MDM2 genes, causing translation of
MDM2 mRna
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MDM2 is ubiquitin ligase, which is exported into the cytoplasm of the cell, where it
is phosphorylated by Akt
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Akt-dependent phosphorylation allows the nuclear importation of MDM2 and
binding to p53 tetramer, tagging it for ubiquitination
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This tagging allows this complex to be exported out of the nucleus and undergo
proteasome mediated degradation
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This negative feedback leads to out-of-phase oscillations in the levels of MDM2 and
p53
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Activation of p53 by DNA Damage:
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2
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4
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6
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8
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These residues lie within the N-terminal transactivation domain of p53 and
regulate MDM2 binding
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Ser15/Ser20 phosphorylation of p53 inhibits MDM2 binding, resulting in p53
stabilization and accumulation
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In addition to targeting p53, the Chk1/2 kinases also phosphorylate MDM2 (at a
residue distinct from that targeted by Akt), reducing its affinity for p53
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Therefore, Akt phosphorylation of MDM2 promotes nuclear entry and p53
association, while Chk1/2 phosphorylation of MDM2 promotes p53 dissociation
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Rising p53 levels induce cell cycle arrest through the transcriptional activation of a
CKI that targets and inhibits the cyclinE/Cdk2 complex, the activity of which is
required for G1/S transition
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Once the damaged DNA has been repaired, and DNA damage signals dissipate, p53
levels (and therefore p21 levels – the CKI) decline, removing the barrier to cell cycle
progression
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If DNA damage is so extensive that it is beyond repair, p53 can induce apoptosis,
preventing the transmission of potentially dangerous genetic errors to progeny cells
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The mechanism for the stabilization of p53 in response to oncogenic stress differs to
that employed by the DNA damage response pathway
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Oncogenes such as myc and ras promote the hyperactivation of mitogenic signalling
pathways (i
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by upregulating cyclin D expression) resulting in the
hyperphosphorylation and inhibition of Rb
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This in turn drives E2F-mediated gene transcription
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Deregulated E2F activity promotes the transcriptional activation of the Arf protein
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The Arf protein is encoded by the CDKN2A gene (which also encodes the tumour
suppressor p16, however, these two genes structurally unrelated)
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Once exprsses, Arf is recruited to the nucleus where it binds and sequesters MDM2
to the nucleolus, preventing its interaction with p53
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Arf diminishes the pool of MDM2 available to inhibit p53, and so p53 levels
accumulate, driving the transcription of p53-responsive genes
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Transcriptional activation and accumulation of p21 results in cell cycle arrest through
p21-dependent inhibition of cyclinE/Cdk2
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CyclinE/Cdk2 inhibition results in reactivation of Rb (due to a fall in Rb
phosphorylation), enabling Rb-dependent inhibition of E2F and therefore the
suppression of cell-cycle gene transcription
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Prolonged accumulation of p21 may ultimately induce premature cell senescence as
a mechanism of preventing uncontrolled cell proliferation and consequently tumour
growth
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Alternatively, sustained p53 activation may result in the induction of apoptosis
through the transcriptional activation of pro-apoptotic genes such as PUMA, Noxa
etc
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Transcriptional targets of p53 include genes involved in the inhibition of
angiogenesis (formation of new blood vessels), providing other mechanisms for
tumour suppression
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Oncogenic viruses evolve two mechanisms in order to successfully inhibit the
functions of both Rb and p53 to ensure apoptosis is not induced following potent
activation of cell proliferation
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For example, HPV has two mechanisms:
• E7 and E6 proteins binds to and degrades Rb and p53, respectively
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Evidence of co-operation between the Rb and p53 pathways:
• In the absence of Rb, lens fibre cells fail to withdraw from the cell cycle and
fail to differentiate, and is counterbalanced by a high rate of apoptosis
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• In the absence of both Rb and p53, lens tumours do develop due to the antiapoptotic signals and continuous cell proliferation
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This also demonstrates that p53 is a downstream target of Rb
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Majority of mutations in the TP53 gene are missense mutations that impair p53-DNA
binding, and in tumours that retain a functioning p53 gene, other elements of the
p53 are often defective
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For example, MDM2 or MDM4 overexpression in retinoblastoma leads to MDM2/4
levels that exceed the sequestering capacity of Arf, therefore even in extensive DNA
damage, p53 levels are too low to activate apoptotic genes
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Some tumours express p53 mutants that actively antagonise the function of the
wild-type protein
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These mutations interfere with the function of normal p53 by forming inactive
tetramers
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Therefore, a similar result is obtained from the mutation of one allele as for deleting
both
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There is an equal amount of WT and DN p53 produced: 50:50 chance of either
genotype
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Therefore, ½ x ½ x ½ x ½ = 1/16, in that, only one in sixteen tetramers will be fully
wildtype
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This is because each monomer within the tetramer has a 50% chance of being a
mutant
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Breast and colon cancer patients whose tumours carry certain missense p53
mutations tend to have a poorer prognosis than patients with p53-null tumours
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These mutations enhance the tumorigenic potential of the cell’s, leading to much
more detrimental effects and giving p53 an oncogenic trait
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Mutations that result in such pro-oncogenic functions unto p53 include:
• Altered target gene specificity:
o The inappropriate transcriptional activation of genes associated with
cell growth/survival
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• A switch from transcriptional repression to activation:
o The transcriptional activation of genes usually suppressed by p53
function, i
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c-myc
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• Selective loss of specific p53 transcriptional targets:
o The p53 mutant is defective in its ability to upregulate cell cycle arrest
proteins and pro-apoptotic factors, but is highly effective at activating
MDM2
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• Direct inhibition of signalling pathways through altered protein-protein
interactions:
o The mutant binds and inhibits the MRN complex, preventing its
recruitment to DNA double-stranded breaks
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A Problem Confronting p53 signalling:
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e
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- These covalent modifications may alter p53 transcriptional potency and
target gene specificity
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Phosphorylation:
• Low levels of p53 phosphorylation are sufficient to displace MDM2
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• Because the affinity of p53 for the promoters of target genes varies (i
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high
MDM2 & p21 vs
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• That is, transcriptional potency varies in respect to p53 phosphorylation
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Acetylation:
• Acetylation of p53 is required for the transcriptional activation of most p53
target genes but not MDM2
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