Chapter 6: Growth inhibition and tumor suppressor genes (lecture 2)

Question 1

What three functions does p53 have when induced by stress and/or dysregulation

Answer 1

1. Cell cycle arrest
2. DNA repair
3. Apoptosis

Question 2

What factors determine P53 target gene selectivity to drive cell cycle arrest or apoptosis?

Answer 2

• Amount
• Modification (phosphorylation, acetylation)
• Co-factors present at the promoter
• Co-activators binding to p53

Question 3

P53 is regulated downstream via the promoter it binds to. There are several mechanisms that change the promoter, which affects p53. In what four ways can this be done?

Answer 3

• Difference in promoter binding affinity
• Co-factors determine affinity of p53 for effector promoters;
  
  • MIZ-1
  • ASPP
• Competition with other proteins
• Myc Post-translational modification

(we will discuss this step-by-step)

Question 4

Explain how the difference in promoter binding affinity works for p53.

Answer 4

p53 can shift its affinity from cell cycle arrest to apoptosis (indicated by the gray arrow). The binding affinity can be increased for specifically e.g. p21 or e.g. Bax, as can be seen in the picture

Question 5

Explain how the co-factors determine affinity of p53 for effector promotors for MIZ-1.

Answer 5

MIZ-1 can attach to the promoter of a high-affinity, thereby ‘blocking it’, so that p53 accumulates and has no other choice than to affect to a low-affinity promoter

Question 6

Another co-factor that can determine the affinity of p53 for effector promoters is ASPP. How does this work?

Answer 6

ASPP can directly bind to p53 resulting in an increased affinity of a gene that might be pro-apoptotic

Question 7

In what way does Myc play a role in in the affinity of p53?

Answer 7

In a normal situation, p53 binds to MIZ-1 that can e.g. stimulate p21, but Myc can act as a competitor so that p53 has no other choice than to bind with promoters it has less affinity for (thus stimulating pro-apoptotic genes)

Question 8

Lastly, it was noted that p53 can be post-translationally modified by many different factors

Answer 8

Just know that this is an option, but you do not have to know the individual pathways/mechanisms for this

Question 9

In what two ways can p53 function be altered due to a mutation?

Answer 9

1. Mutation in the p53 gene
   
   1. no expression (loss of the gene or promoter defect)
   2. missense mutation in the coding sequence (this happens in >75% of the cases)
2. Inhibition of p53 function by a mutation in a gene that regulates it (e.g. MDM2, Parc, COP1, Pirh2, viruses)

Question 10

Earlier we described the Knudson’s two-hit hypothesis (two genes have to be hit for cell alteration to occur). This is true to most genes, but there is one exception to the rule. What gene is this?

Answer 10

p53

Question 11

In what two types of mutations can p53 be altered that does not fit with the Knudson’s two-hit hypothesis?

Answer 11

• Dominant negative mutation
• Gain of function mutation (will be discussed shortly)

Question 12

What happens when there is a dominant negative mutation in the p53 gene? How can a dominant negative mutation still be very harmful?

Answer 12

One gene (50%) of p53 is transcribed, can even bind to a promoter, but is not functional (loss of function). However, p53 is a tetramer, so even though only 50% is not functional, because it binds together with functional p53, eventually about only 20% of p53 is functional and 80% has lost its function. On top of that, the p53 that lost its function can still compete for the promoter site

Question 13

What happens when there is a gain-of-function mutation in the p53 gene?

Answer 13

When there is a gain-of-function mutation, the p53 formed are still functional and can still activate the ‘good’ genes such as effectors and MDM2. However, they also activate genes they’re not supposed to, that leads to carcinogenesis

Question 14

Explain (or even better: draw) how the Rb mechanism/cycle works

Answer 14

Rb suppresses E2F. When E2F is active, cell cycle induction is directly activated. Notice how a counteractive mechanism is in play, via p53. P14 is activated by E2F, which inhibits MDM2. Normally, MDM2 inhibits p53, but because it is suppressed, p53 is active, so there is a cell cycle arrest. This cell cycle arrest has a suppressing effect on the S-phase.

Overal, however, E2F leads to the activation of the S-phase (so DNA synthesis occurs).

Note: So if Rb is active, there is no DNA synthesis!

Question 15

DNA viral protein (VP) products can alter the Rb mechanism/cycle. How does it do that?

Answer 15

Since it is a DNA viral protein, it needs DNA synthesis in order to replicate/survive. Therefore, the goal is to induce the S-phase. The VP does this by inactivating Rb, so there is no suppression of E2F, leading to DNA synthesis. Because VP is a very smart protein, and it does not want p53 to arrest the cell cycle, p53 is inhibited. Now, there is only induction of the cell cycle and no cell cycle arrest, and the virus can benefit of the DNA synthesis that occurs.

Question 16

What is an example of a virus that inhibits Rb and p53 that might induce cancer?

Answer 16

SV40, adenovirus and HPV leading to cervical cancer (you don’t need to remember te viruses, just remember that this occurs via the inhibition of Rb and p53)

Question 17

We already know that >90% of tumors have a p53 activation that leads to carcinogenesis. However, a study by Xue at al in mice showed an interesting aspect of p53 in tumors. What was that?

Answer 17

That sustained p53 inactivation (or p53 defect) is required for tumor maintenance (and not only development!)

Question 18

What does the finding that sustained p53 inactivation is required for tumor maintenance suggest?

Answer 18

That restoration of p53 activity has potential as anti-cancer treatment.

Question 19

What are the potential p53 restoration anti-cancer treatments?

Answer 19

1. p53 gene therapy
2. restore aberrant protein conformation
3. oncolytic adenovirus

Note: in the book, more are discussed, but only these are highlighted in the lecture

Question 20

What is the mechanism/goal for p53 gene therapy? What is a ‘complication’ of this therapy?

Answer 20

A functional p53 is induced that can take over the function of a mutant/absent p53. Because of it, cell cycle arrest, apoptosis and DNA repair can occur. Unfortunately, you cannot target all the mutated cancer cells, and always some cells will remain. Moreover, a long-term treatment always has a risk for resistance.

Question 21

How is the restoration of aberrant protein conformation achieved?

Answer 21

The p53 protein structure will be modified with a small molecule, e.g. PRIMA-1 or APR-246. This shows results in mutations in the DNA binding domain (90% of all cancers)

Question 22

Before we delve into the strategies of oncolytic adenovirus. Two remarks/corrections have to be made where the book is wrong:

Answer 22

• This does not correct a p53 mutation, but kills cancer cells with defective p53
• It was later found that this virus does not work entirely according to its design

Question 23

How are oncolytic adenoviruses used?

Answer 23

NOTE: I did not fully understand what was explained. However, the main idea is: You exploit the idea that cancer cells have a mutation, in order to kill them. They remove the part of the virus that blocks p53, but the part that blocks Rb remains. Only in cancer cells will the VP replicate, because there is no functional p53. Later was found that this strategy is not fully functional, because it also attacks other (parts of?) the cell, because it lacks the functions that have been removed. It is not developed anymore.

Question 24

Lastly, there are strategies that aim to activate endogenous (=wild type) p53. When/why is this used?

Answer 24

For example, in sarcomas, where MDM2 is replicated, so although p53 is functional, it is heavily suppressed

Question 25

How does the activation of endogenous (=wild type) p53 work?

Answer 25

For example, the MDM2 is heavily expressed and needs to be blocked. A small molecule, Nutlin-3, can bind to the binding site of MDM2 where p53 would normally bind. Because it is occupied, the p53 is ‘reactivated’ (there are many more examples, but thus far there are not more molecules produced that work)

Question 26

What drug can be administered for a pre-mature stop in the p53 coding sequence? How does it work?

Answer 26

Atularen. This stimulates the transcription machinery to ignore this ‘stop’, and completes the protein production. The missense mutation is very rare, and atularen is very toxic, so not used nowadays.

Question 27

NOTE: the lecturer does not fully understand this, but mentions this topic because it is mentioned in the book. When/how is Pifithrin-a used?

Answer 27

This is used on healthy cells during chemo-/radiationtherapy. The idea is that during this therapy, DNA is damaged and p53 is activated, which might lead to apoptosis (in healthy cells). When tumor cells have a p53-defect, the chemo-/radiation therapy will not work due to the mutation. Therefore, a p53 transcription inhibitor (=pifithrin-a) is used so that healthy cells are not affected. The timing is very important. The lecturer has many doubts about if this is going to work.

Question 28

What is the effect of a p53 dominant negative mutation on the levels of p53 protein in a cell?

a. No p53 protein expression anymore
b. Decreased p53 protein expression
c. Increased p53 protein expression
d. No clear change in p53 protein expression

Answer 28

c. Increased p53 protein expression Because there is less (functional) p53 present, less effectors are transcribed. One of these effectors is MDM2, that degrades p53. Therefore, more p53 will accumulate

Question 29

A clinical scientist wishes to test an experimental anti-cancer therapy based on restoration of p53 function on a group of cancer patients. The scientist considers to do this with one of the treatments discussed today. What should the scientist know about the cancer cells in the patients to choose the most relevant approach?

Answer 29

The scientist should know what is the molecular defect in the cancer cells of the patients that causes dysfunction of p53. (e.g. there is no use in treating a patient who has mutant-p53 with an inhibitor of MDM2)