L20: Tumour suppressor genes

Question 1

Normal role of TSG products:

Answer 1

• Inhibition of cell division
• Negative regulation of proliferative signalling
• Activating cell death

Question 2

Knudson’s 2 hit hypothesis

Answer 2

• One mutated copy of Rb not enough to cause disease; 2 muations required to occur
• ‘One mutation is inherited via the germinal cells ad the second occurs in the somatic cells. in the non-hereditary form, both mutations occur in somatic cells’

Question 3

Sporadic vs hereditary retinoblastoma

Answer 3

• Sporadic: no family history, unilateral, single tumours in one eye (60%)
• Familial: multiple tumours, bilateral (40%), disease appears dominant

Question 4

RB TSG (size, LOH)

Answer 4

• 180 Kb gene, 4.7 kb mRNA
• In familial retinoblastoma, tumours have lost wt TB allele; loss of heterozygosity
• Mitotic recombination between non-sister chromatids can lead to LOH

Question 5

Prevalence of p53 in sporadic cancers; Li-Fraumeni

Answer 5

• Approx. 50% of sporadic cancers have 053 mutation
• p53 protein: TF
• Li Fraumeni: syndrome associated with inheritance of one mutated p53 allele
  -> tumours develop when wt p53 lost

Question 6

p53 at DNA damage checkpoints

Answer 6

• p53 is an effector of DNA damage; DNA damage checkpoint can arrest cell cycle progression at restriction point or G2/M
• DNA replication checkpoint pathway can inhibit further DNA replication during S phase and delay entry into mitosis

Question 7

p53 strucutre

Answer 7

• Transcriptional activation domain (N-term); Ser15:ATR/ATM, Ser20:CHK2
• DNA-binding domain
• Nuclear export signal, tetramerization domain (active form is a heteromer)
• Ubiquitination, acetylation sites (C-term)

Question 8

p53 in presence vs absence of DNA damage

Answer 8

• No damage: MDM2 ubiquitinates lysines in p53; targeted for degradation, remains exported from nucleus
• Damage: kinase activity stimulated, phosphorylation of p53 and MDM2; no longer interact
  -> p53 tetramerises, blocking nuclear export. Interacts with transcription proteins, including p300, which acetylates histones (enhancing transcription pf proteins for cell cycle arrest and programmed cell death)

Question 9

Alternative p53 targets and their functions (x5)

Answer 9

• GADD45 - binds to PCNA, blocks it from functioning as a processivity factor (S-phase replication checkpoint)
• 14-3-3sigma - binds and sequesters CDC25 phosphatase (req. for activation of cyclinB-Cdk1) - arrests cells at G2/M boundary
• Bax - acts as a positive regulator of apoptosis (pro-apoptotic)
• FAS cell surface death receptors (extrinsic apoptosis pathway)
• APAF1 - activates caspases (intrinsic apoptosis pathway)

Question 10

p53 targeting of p21

Answer 10

• active, phosph. p53 binds to regulatory region of p21 gene, so p21 is transcribed and translated, which inhibits G1/S-Cdk and S-Cdk by comlexing with them

Question 11

Bax dimers under different conditions

Answer 11

• Normally, Bax and Bcl2 production balanced (*Bcl2: anti-apoptotic), so Bax-Bcl2 heterodimer present
• p53 activation of Bax leads to Bax-Bax homodimer
  -> apoptosis
• Activation of Bcl2 (e.g. through activation of oncogenes) blocks apoptosis
  -> cells immortalized

Question 12

Contribution of checkpoint failure to genetic instability (give examples of effects on genome)

Answer 12

• Increased genetic instability leads to increased chance of further mutations that may activate oncogenes or result in loss of TSGs
• Failure of spindle checkpoint -> aneuploid
• Failure of centrosome duplication checkpoint -> tetraploid
• Failure of DNA-damage checkpoint -> translocation, deletion, gene or chromosome amplification

Question 13

Cancers as a result of initiating mutations

Answer 13

• Skin cancer: basal cell carcinoma, squamous cell carcinoma (UV signature mutations, C->T or CC->TT transitions)
• Lung cancers (mutations in DNA bases subject to attack by polycyclic hydrocarbons in tobacco smoke

Question 14

p53 as a ‘poison subunit’

Answer 14

• Most p53 mutations genetically recessive
• Some p53 mutations have dominant negative effect (one subunit can disrupt whole tetramer, preventing activity)
  -> only 1/16 possible tetramers functional (50:50)

Question 15

Cancers as a result of p53 degradation

Answer 15

• Human Papillomaviruses (HPVs) are main cause of cervical cancer; mostly types 16 and 18
• HPV produce E6 and E7 oncoproteins
• E6 oncoprotein binds to p53, targeting it for destruction - prevents accumulation in response to DNA damage
• E7 oncoprotein sequesters Rb
  -> deregulates E2F activity

Question 16

Gatekeeper TSGs, example genes and example hereditary cancer syndromes

Answer 16

• Directly involved in restraining cell proliferation; loss of function leads directly to proliferation
• p53, p21, Rb, Bax

Hereditary Cancers
- Retinoblastoma (RB1)
- Li-Fraumeni (p53)
- Neurofibromatosis type 1 (NF1)
- Familial adenomatous polyposis coli (APC)
- Wilms tumour (WT)

Question 17

Caretaker TSGs

Answer 17

• Maintain the integrity of the genome
• Code for proteins mediating DNA repair; loss of function leads to genetic instability
• Increase chance of loss of further TSGs and mutation of proto-oncogenes to oncogenes
• Mutations associated w/ familial cancers

Question 18

Syndromes associated w/ caretaker TSG mutation

Answer 18

Lynch syndrome/HNHPCC
- Autosomal dominant (80% lifetime risk of colon cancer associated w/ inheritance of mutant allele of one of mismatch repair genes ; MSH2, MLH1, PMS1, PMS2)
- Patients developing hereditary non-polyposis colon cancer (HNPCC) are heterozygous, tumours show LOH
Xeroderma pigmentosum
- Autosomal recessive
- Skin cancer susceptibility associated w/ defective nucleotide excision repair capability
- Failure to repair UV inflicted DNA damage results in mutation accumulation
BRCA1, BRCA2
- 10% of all breast cancers
- Promote repair of DNA DSBs by high fidelity homologous recombination (mediated by RAD51, human homologue of RecA)
- Tumours show LOH (characterised by abnormal chromosomes)
Bloom
- recessive
Fanconi anaemia
- recessive