Lecture 20 - Tumour suppressor genes Flashcards
What is the function of tumour supressor genes.
The normal role of TSG products is ‘inhibition’ of cell division:
* Negative regulation of proliferative signalling
* Inhibition of cell division
* Activating cell death
Mutations that lead to a decrease in the normal activity of a tumour suppressor protein contribute to cancer - lead to loss of inhibition of cell division.
Point mutations, chromosome mutations - any mutation that decreases expression or stability oof tumour suppressor protein, or inactivates it.
Mutations in classical tumour suppressor genes genetically recessive
What is retinoblastoma?
Retinoblastoma - most common eye tumour in children from birth to 4 years - arises in immature retinal cells. Affects 1 in 20,000 children
* With early detection and treatment there is a good prognosis
* All children are routinely screened in the UK - simple procedure.
○ White light shone in eye
○ Reflection from normal retina
○ Reflection appears white may be indicative of retinoblastoma
* 87% of children worldwide with retinoblastoma dies - almost exclusively in countries lacking robust screening systems.
* Sporadic - no family history - unilateral, single tumours in one eye (60% cases)
* Hereditary - multiple tumours, bilateral - both eyes (40% cases) - disease appears dominant
What is Knudson’s two-hit hupothesis.
Knudson’s two-hit mutation hypothesis (1970)
Retinoblastoma is a cancer caused by two mutational events. One mutation is inherited via the geminal cells and the second occurs in somatic cells. In the non-hereditary form, both mutations occur in somatic cells.
Describe the genetics of retinoblastoma.
Genetics of retinoblastoma
RB tumour suppressor gene - 180kb gene - 4.7kb mRNA
In familial retinoblastoma, tumours have lost wild type RB allele - loss of heterozygosity (LOH)
Normally occurs on the chromosomal level
* Chromosome loss
* Mitotic recombination
* Chromosome loss and reduplication
At gene level
* Gene inactivation
* Epigenetic splicing
Mitotic recombination between non-sister chromatids can lead to a loss of heterozygosity
If both parents are carriers during mitotic recombination the chromosomes can be rearranged to be homozygous for either disease or normal.
What is the normal role of pRB in regulating the passage of cells from G1 to S.
Normal role of pRB in regulating the passage of cells from G1 to S - sequestering E2F
Regulation of progression through restriction point is pivotal to tumourigenesis
rb/rb cells - pRB is often truncated, unstable, fails to bind E2F
Consequently E2F is free to activate genes required for G1/S transition and S phase
The consequence of loss of pRB - sustained proliferative signalling.
What is the function of the p53 gene.
The p53 tumour suppressor gene
P53 most commonly mutated gene in human cancers - approximately 50% of all sporadic cancers have p53 mutation
* P53 protein - transcription factor sometimes called the guardian of the genome
* Li-Fraumeni syndrome is associated with inheritance of one mutated p53 allele - 90% risk of developing wide range of cancers
* Tumours develop wen wt p53 gene is lost
P53 is an effector of DNA damage checkpoints
The DNA damage checkpoint can arrest cell cycle progression at the restriction point or G2/M
DNA replication checkpoint pathway can inhibit further DNA replication during S phase and delay entry into mitosis
How does p53 accumulate in response to damage.
When no DNA damage is present MDM2 ubiquitinates lysine’s in the p53 C-terminal domain, targeting it for degradation.
Any remaining p53 is exported from the nucleus
When DNA damage is present, kinase activity is stimulated and results in phosphorylation of p53 and MDM2, so they no longer interact
P53 tetramerises, blocking nuclear export
P53 interacts with transcription proteins, including p300, which acetylates histones (enhancing transcription) and p53.
MDM2 an-p53 association is disrupted by phosphorylation
Tetramerization of p53 blocks nuclear export increasing nuclear concentration
Phosphorylated p53 interacts with histone acetylase p300 enhancing transcription
P53 accumulation in response to DNA damage leads to arrest at G1/S
Major roles for p53
RB1 and accessory tumour suppressor proteins act as breaks on cell division
How does p53 activate transcription of multiple genes.
P53 activates transcription of multiple genes (each of which are also TSGs)
e.g.
* P21 - inhibits cyclin dependent kinases - arrests cells at G1/S boundary
* GADD45 - binds to PCNA - blocks PCNA functioning as processivity factor - S-phase DNA replication checkpoint
* 14-3-3σ binds and sequesters CDC25 phosphatase required for activation of cyclinB-Cdk1 - arrests cells at G2/M boundary. (CDC25 is required to remove inactivating phosphates introduced by Wee1 kinase at positions 14 and 15)
* Bax - acts as a positive regulator of apoptosis - pro-apoptotic * FAS cell surface death receptors - extrinsic apoptosis pathway APAF1 - activates caspases - intrinsic apoptosis pathway
P53 activation results in increased pro-apoptotic Bax
Increased Bax due to p53 activation results in Bax homodimers and initiation of apoptosis
Describe the downstream events triggered by p53
Following loss of wt p53 in tumour cells:
* There is no activation of the DNA damage checkpoint
* No cell cycle arrest
* Apoptosis is inhibited
Normally in response to DNA damage levels of p53 increase. Phosphorylation and acetylation of p53 leads to transcriptional activation of genes through either the arrest/repair pathway or apoptosis pathway
Cell death through synthesis of Bax
Arrest through synthesis of p21, GADD45, 14-3-3σ
Repair through synthesis of GADD45
Describe the contribution of checkpoint faiures to genetic instability
Increased genetic instability leads to increased chance of further mutations that may activate oncogenes, or result in the loss of tumour suppressor genes.
* Failure of spindle checkpoints (aneuploid)
* Failure of centrosome duplication checkpoint (tetraploid)
* Failure of DNA damage checkpoint (translocation, deletion, amplification)
Loss of p53 activity via inactivating mutation
Initiating mutation may be caused by specific environmental exposure:
* e.g. skin cancers- basal cell carcinomas and squamous cell carcinomas - UV signature mutations C->T or CC->TT transitions
* e.g. lung cancers - mutations in DNA bases subject to attack by polycyclic hydrocarbons in tobacco smoke
Most p53 mutations are genetically recessive - one mutated copy cant cause tumour.
Some p53 mutations have dominant negative effect - mutation of one copy is sufficient to disrupt p53 protein function - bad subunits still tetramerise but can then disrupt their tetramer - prevents activity.
If mutants and wild-type subunits are present in equal amounts and specific p53 mutations can block tetramer activity- only 1/16 will be functional tetramers.
Loss of p53 activity via degradation
* In the UK ~3000 women are diagnosed annually with cervical cancer. Human papillomaviruses are the main case of cervical cancer with types 16 and 18 accounting for 70% of cases
* HPV produce E6 and E7 oncoproteins.
* E6 oncoprotein bins to p53, targeting it for destruction - this prevents accumulation in response to DNA damage
E7 oncoprotein sequesters Rb - deregulates E2F activity.
What are gatekeepers and caretakers?
There are different classes of tumour suppressor genes
1. Gatekeeper TSGs - are directly involved in restraining cell proliferation
When lose function it leads directly to proliferation
○ P53 - Loss of G1/S and G2/M checkpoints
○ P21 - Loss of G1/S and S checkpoints
○ RB - Promotes proliferation, E2F is uninhibited
○ Bax - Failure to promote apoptosis of damaged cells.
Hereditary cancer syndromes resulting for loss of TSG - gatekeeper genes
○ Retinoblastoma - RB1
○ Li-Fraumeni syndrome - p53
○ Neurofibromatosis type - NF1
○ Familial Melanoma - CDKN2A
○ Familial adenomatous polyposis coli - APC
○ Wilms Tumour - WT
2. Caretaker TSGs - maintain the integrity of the genome Genes coding for proteins mediating DNA repair. Their loss of function leads to genetic instability. It increases the chances of loss of further TSGs and mutation of proto-oncogenes to oncogenes Mutations in caretaker genes are associated with familial cancer and are rarely seen in sporadic cancers
How are germline mutations in caretaker TSGs are associated with increased cancer suceptibility
Germline mutations in caretaker TSGs are associated with increased cancer susceptibility of variable penetrance
Lynch syndrome/HNPCC
It is an autosomal dominant disease with an 80% life time risk of colon cancer associated with inheritance of a mutant allele of one of the mismatch repair genes MSH2, MLH1 and PMS2
Patients developing hereditary non-polyposis colon cancer (HNPCC) are heterozygous, tumours show LOH.
Xeroderma pigmentosum
Autosomal recessive - skin cancer susceptibility associated with defective nucleotide excision repair capability. Failure to repair UV inflicted DNA damage results in mutation accumulation.
Name breast cancer caretaker genes.
Breast cancer and caretaker genes BRCA1 and BRCA2
10% of all breast cancer associated with inherited mutation in BRCA1 or BRCA2 . BRCA1/2 are large unrelated nuclear proteins.
In normal cells they promote repair of DN DSBs by high-fidelity homologous recombination mediated by RAD51, human homologue of RecA
Tumours show LOH - cells are characterised by abnormal chromosomes- resulting from repair of SBs by non-homologous end-joining.
Name some hereditary cancer syndromes resulting from loss of TSG - caretaker genes.
- Ataxia telangiectasia - recessive - ATM
- Xeroderma pigmentosum - recessive -
- Hereditary non-polyposis colon cancer - dominant
- Fanconi anaemia - recessive
- Familial breast cancer - dominant
Bloom syndrome - recessive
Describe some cancer therapy.
Cancer therapy
Intervention in cancer
Target therapies towards cancer cells. Target properties that cancer cells may use to survive.
* Telomerase inhibitors
* EGFR inhibitors
* Pro-apoptotic BH3 mimetics
Immune activating anti-CTLA4 mAb
