L10: p53 tumour suppressor

Question 1

Which virus is associated with tumourigenesis in humans?

Answer 1

• There are a whole host of viruses that do this
• For example, cervical cancer famously associated with HPV, its viral oncoprotein E6 that binds p53

Question 2

What are the types of p53 mutations?

Answer 2

Point mutations – most common
- There are particular high frequencies of hotspot mutations
- Generally they are non-synonymous
- Not nonsense mutations, they don’t necessarily block the translation of p53
- Full end protein produced but possibly doesn’t have the same function

Deletions (and insertions) – rarer
- These are obvious deletions of p53
- Can be gross deletions (deletions of entire gene)
- Can be enormous deletions (loss of a copy of a chromosome)
- Or small deletions or insertions that lead to a frameshift in the p53 sequence, that usually results in a non-functional protein formation

Question 3

P53 and genomic integrity – so what does p53 actually do?

Answer 3

• Essentially prevents proliferation of cells with damaged DNA
• P53 controls both cell proliferation and genomic integrity – these functions are intertwined – hence such an important TSG
• P53 prevents DNA damage accumulating (particularly that consequent from single-strand (ss) and double-strand (ds) breaks)
• P53 sometimes referred to as the ‘guardian of the genome’
• P53 acts as a transcription factor
• P53 acts its function via DNA binding domain, which is where hotspot mutations occur

Question 4

How does the protein encoded by p53 work? How does it know it has to switch on transcription / block the cell cycle / engage repair?

Answer 4

• P53 is constantly transcribed in cells, however not much p53 is seen if you take a snapshot of cells, because p53 is degraded as fast as it’s made
• P53 is activated after stress through a mechanism called post-translational mechanism, by stabilizing the protein, suddenly the protein becomes very stable within seconds

Question 5

What is the function of Mdm2?

Answer 5

• Puts ubiquitin tags, that marks it for degradation
• P53 is constantly degraded by a protein called Mdm2, tags p53 for degradation by proteosome
• So this degradation is blocked upon stress
• P53 protein itself drives the transcription of Mdm2, acts as a negative feedback loop

Question 6

What happens during Mdm2 overexpression?

Answer 6

Mdm2 overexpression (with other oncogenes) can drive cancer
- So even if p53 is not mutated, p53 function is suppressed during tumourigenesis

Question 7

What are other potential regulatory roles of p53 PTMs (post-translational modifications)?

Answer 7

PTMs may also:
- Affect stability of p53 independent of Mdm2
- Directly affect transcriptional activation potential of p53 (including target selection)
- Affect DNA-binding of p53
- Sequester p53 from nucleus

Question 8

What is senescence?

Answer 8

Senescence – permanent exit from cell cycle (but cells alive)
- Often driven by oncogene activation (oncogene-induced senescence) via ARF

Question 9

What are the positive-dominant mutations of p53?

Answer 9

Positive-dominant mutations p53
- Subclass of point mutations that both inactive p53 tumour suppressor activities and cause it to acquire additional capabilities
- Sometimes called gain-of-function rather than positive-dominant, but this can be misleading
- Can act on many functions of p53

Question 10

How do we target a tumour suppressor with drugs?

Answer 10

• Chemo drugs that damage DNA: thus activate wild-type p53 to produce cell cycle arrest, senescence, apoptosis
• Restore function of p53 when it is suppressed?:
  o P53 deleted tumours – no
  o P53 mutant tumours – yes
  o P53 regulator defects – yes

Question 11

What are the important effects of many DNA binding domain mutations?

Answer 11

Two important effects of many DNA binding domain mutations
1. Disrupt folding but do not remove residues required for direct interaction with DNA or destabilise p53 – these mutations block contact with DNA they don’t cause protein to become catastrophically unfolded, they simply reduce affinity for binding the DNA
2. Mutant p53 hyperaccumulates! (no regulation by Mdm2 loop)

Question 12

Give examples of reactivating mutant p53

Answer 12

reactivate mutant p53
- Drugs have been assessed that can bind mutant p53, alter its confirmation so that it now can make productive contacts with DNA and therefore induce tumour suppressive effects

example 2: p53 regulator targeting agents
- In instances where negative regulator of p53 or Mdm2 is massively overexpressed and highly active
- Active area of research: developing drugs that could bind the Mdm2 and block its interaction sites with p53