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Cancer mechanisms and therapeutics > p53 > Flashcards

p53 Flashcards

1
Q

What kinds of stresses might lead to the activation of p53?

A
  • DNA damage
  • oncogene activation
  • hypoxia
  • oxidative stress
  • increased E2F
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2
Q

What is the TP53 gene?

A
  • encodes p53
  • one of the most frequently mutated genes in cancer
  • involved in apoptosis, senesence, DNA metabolism etc
  • transcription, post transcriptional and post translational roles
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3
Q

How does p53 work in brief?

A
  • in response to damage, p53 is stabilised
  • can promote transcription of genes involved in cell cycle arrest + repair or apoptosis
  • maintains cellular and genetic stability
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4
Q

What is the structure of the p53 protein?

A
  • Transactivation domain binds MDM2 and other inhibitors and also DNA
  • co-activators bind at the N terminus
  • sequence-specific DNA binding domain is where 95% of cancer mutations occur
  • oligermirisaion domain allows tetramer formation
  • carboxy-regulatory domain is modified to activate p53
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5
Q

Under what conditions does p53 bind the DNA?

A
  • works best as a tetramer
  • specifically binds to consensus sequence
  • two copies of the sequence with a spacer region between them of 0-21 bp long
  • different orientations with different affinities
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6
Q

What kinds of post-translational modifications can occur to p53?

A
  • many, mostly at the c-terminus
  • phosphorylation and acetylation are key
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7
Q

What are the other p53 family members?

A
  • p63 and p73
  • some overlap in function and domains
  • p63 more important in development than apoptosis
  • p73 can induce cell cycle arrest and apoptosis AND important in development
  • p73 could potentially be upregulated in lieu of p53
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8
Q

How is p53 activity regulated in normal cells?

A
  • present in very low amounts due to its short half life
  • activation at the level of the protein rather than the gene
  • allows a rapid response by stopping p53 turnover and degradation and quickly turning it back on
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9
Q

How does Mdm2 regulate p53 levels in normal cells?

A
  • keeps it low in the absense of DNA damage
  • works in a heterodimer with mdm4
  • ubiquitin ligase that binds p53 transactivation domain, blocking its binding to DNA and causing its acetylation and ubiquitination
  • shuttles it to the cytoplasm where it is degraded
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10
Q

What else can Mdm2 do to p53?

A
  • can enhance p53 translation under genotoxic stress
  • context-dependent role
  • not fully understoof
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11
Q

How does p53 recognise DNA damage?

A
  • DNA damage activates ATM/ATR kinases
  • these phosphorylate p53
  • the activity of these kinases is directly dependent on the presence of DNA ends/damage
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12
Q

What happens when kinases phosphorylate p53?

A
  • at the N terminus in or near the Mdm2 binding region
  • drives off Mdm2 and stabilises p53
  • Mdm2 phosphorylation may also occur
  • phosphates can undo this sometime after the damage has occurred
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13
Q

What can phosphorylation of the c-terminus do to p53?

A
  • further activation
  • stimulates the sequence specific DNA binding activity of p53
  • enhances its transcriptional activity
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14
Q

How might the cell respond to huge damage?

A
  • phosphorylation on ser46 favours transactivation of pro-apoptotic genes like Noxa and PUMA
  • skips cell cycle arrest and repair and goes straight to apoptosis
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15
Q

How can p53 be regulated by acetylation?

A
  • co-activators such as p300 and CBP are acetyltransferases
  • chromatin remodelling enhances trnascriptional activity
  • acetylation of the C-terminus end increases stability and activates the protein
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16
Q

How can modifications play a role in p53 target preferences?

A
  • K120 acetylation leads to p53 accumulation at proapoptotic genes like Bax and PUMA
  • K320 acetylation favours apoptosis
  • ubiquitination of K320 favours growth arrest
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17
Q

In what two ways can p53 arrest the cell cycle?

A
  • directly by acting as a TF and swtitching on arrest genes
  • indirectly by regulating genes without a consensus sequecne
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18
Q

What are some examples of indirect gene regulation by p53?

A
  • repression of transcriptional activators
  • interfering with the assembly of transcriptional machinery
  • recruitment of repressors such as HDACs
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19
Q

What kinds of genes might p53 repress?

A
  • growth promoting or anti-apoptotic genes
  • c-myc, Bcl-2, cyclin A
20
Q

How does p21 inhibit the cell cycle?

A
  • p53 binds to p21 regulatory elements and increases its expression
  • p21 binds to and inhibits cyclin CDKs
  • prevents cell cycle progression
  • can also inhibit DNA synthesis to allow repair
21
Q

Other than p21, how else can p53 cause cell cycle arrest?

A
  • upregulates Siah-1 which degrades B-catenin and reduces cyclin D1, haulting the cell cycle
  • can also arrest cells in G2 by repressing cyclin B1 and stopping mitosis
  • loss of p53 - loss of G1 arrest + attenuation of G2 arrest
22
Q

What role does p53 play in DNA repair?

A
  • increases DNA polymerase B activtiy
  • cells without p53 are unable to efficiently repair DNA lesions
23
Q

Which function of p53 appears to be more important in tumour suppression?

A
  • mice with mutated p53 that can’t arrest the cell cycle but can induce apoptosis are abble to suppress cancers
  • pro-apoptotic role more important?
24
Q

What is the role of p53 in cellular senescense?

A
  • induces senescence and prevents lesions from developing into malignant tumours
  • inactivation of p53 prevents ras-induces senescence
  • sustained p53 + p16 expression can lead to senescence
25
Q

What is senescense?

A

permanent withdrawl from the cell cycle

26
Q

How do cells make the choice between repair or die? (3)

A
  • post-transcriptional modifications of p53 favour specific promoter binding
  • different affinities to different response elements
  • low p53 levels favour growth arrest and high or persisting levels override this and cause apoptosis
27
Q

WHat is the ASPP family of proteins?

A
  • apoptosis stimulating proteins of p53
  • direct p53 towards proapoptotic genes
  • upregulation of E2F or DNA damage activate ASPP 1+2
28
Q

How are ASPP proteins regulated?

A
  • turned on by DNA damage or E2F upregulation
  • iASPP prevents ASPPs from binding to p53 and allow growth and proliferation
  • ASPP 1+2 can also be silenced by methyatlion
29
Q

How might p53 be inactivated in cancer? (4)

A
  • missense p53 mutations seen in over hald of cancers
  • mutations in upstream activators such as ATM
  • increased levels of inhibitors such as MDM2 (sarcomas)
  • interaction with oncogenic viral proteins that bind and inactivate p53
30
Q

How might mutant p53 act? (3)

A
  • not binding its consensus sequence
  • not activating indirectly by promoters
  • not suppressing promoter activity
31
Q

What makes p53 mutations different to other oncogenes?

A

most oncogene mutations are frameshifts but not p53 are missense mutations

32
Q

What kinds of mutations occur in the DNA binding domain of p53?

A
  • contact mutations that prevent protein-DNA interaction
  • conformation mutations that destabilise the core structure (more common)
33
Q

Why does p53 usually have mutations that destabilise the structure?

A
  • has a selective advantage
  • only 1 copy needs to be mutated out of the 4 proteins in the tetramer to drive them all into mutant conformation and stop it binding DNA
34
Q

What are gain of function p53 mutations?

A
  • tumours may benefit from mutant p53 as they can do more
  • mutations can regulate transcription on promoters without p53 binding sites
  • tetramer may be locked in a mutant conformation
35
Q

What is Li Fraumeni syntdrome?

A

weakly penetrant p53 mutations are inherited and lead to increased cancer predisposition

36
Q

How does p14ARF stabilise p53 in response to stress?

A
  • binds Mdm2 and blocks its ubiquitin ligase activity
  • leads to stabilisation of p53
  • E2F can activate p14ARF
  • loss of ARF promoters or overexpression of repressors are associated with cancer development
37
Q

How do E2F and p53 work together?

A
  • E2F upregulation constitutes oncogenic stress
  • upregulates expression fo proteins that stabilise/activate p53 such as ATM, ARF and ASPPs
38
Q

How does PI3K signalling suppress p53 function?

A
  • Akt phosphorylates Mdm2 and triggers its translocation to the nucleus where it binds p53
  • PTEN inhibits PI3K signalling and restricts Mdm2 to the cytoplasm where it cant bind p53
39
Q

How can viral oncoproteins interact with p53?

A
  • have similar effects as mutations
  • complex with the WT protein in tetramers and inactivate them
  • adenovirus, HPV E6+E7
  • viral oncoproteins bind both Rb and p53 to block their pathways to allow viral infected cells to continue proliferating
40
Q

How are Rb and p53 important together in cancer?

A
  • loss of Rb must be combined with apoptosis resistance to promote tumour development
  • selective pressure to reduce the otherwise apoptotic consequences of regulated E2F
41
Q

What are the main goals in targeting p53 for cancer therapy?

A
  • reactivating WT functions
  • stabilising p53
42
Q

How might mutant WT functions be reactivated in p53?

A
  • small molecule drugs
  • can restore the WT conformation and sequence-specific binding
  • can restore protein folding of mutated p53 into a formation more similar to WT
43
Q

What is the target for increasing p53 stabilisation?

A
  • targeting Mdm2
  • Nutilin binds to Mdm2 and prevents its interaction with p53
  • can activate p53 pathways in cancer cells
  • small molecules can also inhibit Md2m
  • has side effects in normal tissue
44
Q

Why might it be appealing to inhibit p53 in cancer therapy?

A
  • most cancer therapy side effects are cause by p53-mediated apoptosis
  • could protect from lethal side effects
  • could allow the use of higher doses in those less responsive to treatment
45
Q

What is p53 gene therapy?

A
  • retroviral vector containing WT-p53 gene and an actin promoter
  • injected directly into tumours of lung cancer patients
  • not yet approved

Cancer mechanisms and therapeutics (12 decks)

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