Tumour supressor genes

Question 1

What is a tumour supressor gene?

Answer 1

‘Genes that sustain loss of function mutations in the development of cancer’

• • Encode for proteins whose absence, repression, inactivation or mutation promotes oncogenesis.
• • Both copies need to be mutated in order to have an effect – they are recessive.
• • Act as gatekeepers of cell cycle and act to maintain chromosomal integrity – inhibit proliferation, induce apoptosis, inhibit apoptosis, angiogenesis and induce cell adhesion.
• • One mutated TSG will not necessarily form a tumour, but if inherited there’s a higher chance of developing a tumour. They have different genetics from oncogenes.

Question 2

What is retinoblastoma? Explain the two-hit hypothesis?

Answer 2

• • Retinoblastoma is childhood cancer of retina. Two forms of cancer exist, familial (10-40%) and sporadic.
• • The RB gene is affected (either lost or mutated) in all cases of retinoblastoma.
• • Knudson’s Two-Hit Hypothesis: in the non-hereditary cancer occasionally one copy of the TSG is inactivated, but usually the other rarely won’t be inactivated leading to no loss of function. However in the inherited retinoblastoma, since there’s already an inherited inactivated Rb, it’s very likely the other will also be inactivated leading to formation of a retinoblastoma. ‘loss of heterozygosity’
• • RB is also knocked out in a substantial proportion of other human cancers.

Question 3

Explain Rb’s role in regulating the cell cycle.

Answer 3

• • pRb is a cell cycle regulator.
• • It regulates the cell cycle by inhibiting progression from G1 to S phase (at the R point) if the cell is not big enough and if there are no growth signals present.
• • It does this indirectly – by associating with the transcription factor E2F and histone deacetylases (HDAC).
• • pRb is pocket protein consisting of A and B domains. Normally the un-phosphorylated form binds to its two effector proteins E2F and HDAC simultaneously.
• • It sequesters and blocks E2F’s transcriptional activation of genes for progression of cell cycle and causes HDAC to coil up target genes.
• • Its interactions with these two proteins are regulated by serine and threonine proteases.

Question 4

What happens to pRb in response to a growth signal?

Answer 4

In response to a growth signal, cyclin D is expressed which phosphorylates and inactivate pRb so cell cycle progression can occur:

1. Cdk4/cyclin D (cyclin dependent kinases) phosphorylates the C terminal residues of pRb causing increase in negative charge, changing the lysine charges in its HDAC binding domain so HDAC is lost.
2. Then cyclin E is expressed and cyclin E/cdk2 will phosphorylate pRb at its E2F binding residues so E2F is lost.

– Once cells pass through the R point, the cells can complete the cell cycle without needing presence of further growth factor stimulation.

Question 5

What is p53? How does MDM-2 affect its levels?

Answer 5

• • It’s a TSG that is a transcription factor – binds to specific DNA sequences.
• • Unlike pRb, it is only produced in cells under stress - it’s activated by stress signals such as DNA damage, cell stress, hypoxia and radical species.
• • Under normal conditions p53 is very unstable and its levels are very low.
• • MDM-2, a ubiquitin ligase, ubiquitinates p53 so that it’s flagged for degradation by the proteasome in the cytoplasm. MDM-2 also binds to transactivation domain of p53 and transports it away from the nuclear DNA.
• • P53 stimulates the production of MDM-2 —> an auto-regulatory negative feedback loop.

Question 6

How is p53 activated?

Answer 6

– Cell stress, DNA damage and oncogenes will cause p53 levels to rise by acting on MDM-2:-

1. DSBs activates the Ataxia telangiectasia mutated (ATM) kinase that will phosphorylate the MDM-2 binding domain of p53, so that it’s levels rise.
2. Cell stress activates the two kinases ATR and casein kinase II. They will then phosphorylate p53 and disrupt its interactions with MDM2.
3. Activated oncogenes such as myc or Ras can induce P14arf which sequesters MDM2 to nucleolus so it can no longer bind to p53.

Question 7

Once activated,what does p53 act to do?

Answer 7

Once switched on, P53 will act as a TF to switch on genes that either:-

1. Cause cell cycle arrest - so that the damaged DNA is repaired before it can be replicated – including damaged DNA in daughter cells is dangerous.
   - - It induces transcription of p21, a cyclin dependent kinase inhibitor which inhibits formation of several cyclin-cdk complexes to stop the G1 –> S progression in cell cycle. It also acts on G2 –> M progression.
   - - P21 also induces proliferating cell nuclear antigen PCNA which mediates DNA repair.
2. Cause apoptosis -
   - - It induces pro-apoptotic factors (NOXA, PUMA, p53) while supressing expression of anti-apoptotic factors. It also regulates Bcl-2 family to induce apoptosis.
   - - It also induces Fas receptor that stimulates apoptosis via the extrinsic pathway.

Question 8

How is p53 function lost in many cancers?

Answer 8

P53 is mutated in up to 50% of cancers.

• • It has three main domains – transactivation domain, DNA binding domain and oligomerisation domain.
• • Most mutations occur in the DNA binding domain. Some mutations are more common than each other – usually in the amino acids that determine the sequence specific binding. Therefore the p53 will no more be able to bind DNA and arrest the cell cycle.
• • The mutations will also prevent it from binding to MDM-2 so its levels will rise – can be useful in detecting cancers.
• • Most P53 mutations are missense and are due to single amino acid substitutions.
• • P53 mutant cells will have genomic instability, as lack of checkpoints will cause high amount of DNA damage being present in cells.

Question 9

Explain how APC functions as a TSG and how its inactivation can lead to cancer.

Answer 9

APC normally functions in colonic epithelial cells which are very sensitive to proliferation.

• • It is part of a large complex of proteins. Regulates cell proliferation:
• • In most cells Wnt, a proto-oncogene is inactivated due to degradation of beta catenin. The TFs TCF/LEF bind to DNA and inhibit gene expression.

1. Activation in a normal cell —> Wnt signals through the Frizzled receptor and co-receptor LRP which dismantles the regulatory complex. Beta catenin is now free and its levels rise, so it can bind to TCF/LEF to activate gene expression.
   - — Genes expressed include c-myc and cyclin D1 transcription and hence causes cell proliferation.
2. In tumour cells —> the Wnt pathway is inactivated due to mutations in APC, so regulatory complex is dismantled and beta-catenin will increase causing unregulated gene expression.

Mutations in APC can cause familial adenomatous polyposis. Polyps – wart like growths – are formed due to loss of APC, can form adenocarcinomas. Found in 60-80% of FAP.

Question 10

Explain how VHL functions as a TSG and its relationship with HIF.

Answer 10

• • The von Hippel-Lindau (VHL) is a TSG that causes the rare genetic disease that can cause tumours to form in areas with a lot of blood vessels.
• • VHL is mutated in germ line cancers.
• • The VHL protein (pVHL) is part of a complex that destabilises hypoxia inducible factor - HIF (necessary for tumour growth) by interacting through a hydroxylated proline - this leads to ubiquitylation and proteasomal degradation.
• • When VHL is lost, HIF is activated and then promotes gene expression of genes that are involved in the proliferation of blood vessels such as VEGF.