Evading growth suppressors Lecture 8

Question 1

What is the significance of the retinoblastoma protein (RB1)?

Answer 1

Classical example for a genetic predisposition to cancer. Recessive at the cellular level

Almost inevitable that after all the necessary cell divisions that one retinal cell with acquire the mutation to inactivate the remaining RB1 gene in the heterozygote- therefore the cancer shows dominant inheritance

Also shows increased incidence in other cancers- loss of heterozygosity RB is a master controller of the cell cycle controlling S-phase entry- when hyper-phosphorylated dissociates from E2F at R-point allowing the transcription of genes for S-phase

Question 2

What is Knudson’s 2-hit hypothesis?

Answer 2

Explains relationship between familial and sporadic cancers

-Retinoblastoma is a cancer caused by two mutational events

Non-hereditary

• Occasional cell inactivates 1 of its 2 good Rb genes
• Second copy very rarely inactivated in same line of cells leading to retinoblastoma

Hereditary

• Inherited mutant Rb genes
• Occasional cell inactivates its only good Rb gene copy and this directly leads to excessive proliferation and retinoblastoma

Question 3

Descrbie characteristcs of tumour suppressor genes mutations

Answer 3

mutaiton of both alleles is usuallyrequired

Mutatations aredistributed throughout the gene and are not confined to hotspot locations

Mutations result in loss of fucntions of the encoded proteins (complete protein loss or dysfunctinal loss)

Mutation in both copies

First hit must be nonsense, frameshift, splicing etc

Second hit can be same as first or due to loss of heterozygosity

Question 4

What is a cryptic splice site?

Answer 4

• Mutation leads to different splice site being used
• Results in deletion of AAs

Question 5

What types of mutation are there and how big are their impacts?

Answer 5

High impact: Truncated/no protein

• Nonsense - gives rise to stop codon
• Frameshift
• Deletion

Moderate impact: Protein with partial function

• Missense
• Splicing

Question 6

What is loss of heterozygosity?

Answer 6

Single mutation in one allele causes the cell to become heterozygous

• During mitosis the mutation will be copied
• In mitotic recombination it is possible for the mutation to be swapped onto another chromosome. When these chromatids separate there is a 1/4 chance of producing a cell with two mutations

Question 7

Descrbie RB1 within the cell cycle and its inactivation

Answer 7

RB1 can be phosphorylated by clyclin D and CDK4/6 which started phosphorylating RB1. This process continues with cyclin E and CDK2 to phosphorylate RB1.

WHen RB1 is fully phosphorylated, E2F can break away and can activate gene transcrption

To progress through G1 phase, E2F factors need to upregulate genes important for the cell cycle progression. But E2F is bound to RB1 which blocks their function so E2F cant upregulate other genes.

Mutations in Cyclin D1 in which cause an increase in cyclin D which cause more RB1/E2F to form, moe E2F means more transcrpition og genes.

Can also have mutations in CDKN2A, they are CDK4 cyclin dependant kinase inhibitors which block activity of CDK4/6. Blocking of the inhibor will allow for nomral fucntion.

RB1 inactivation causes cell cycle deregulation

Question 8

What is the significance of p53?

Answer 8

It is the ‘Guardian of the Genome’ Regulates cell cycle arrest, DNA repair, block of angiogenesis and apoptosis in response to lack of nucleotides, UV, IR, oncogenic signalling, hypoxia, blockage of transcription

Unlike most tumour suppressor a p53 mutations are predominately Mis-sense and a single allelic loss confers predisposition to cancer alone

DNA damage stabilises p53 via phosphorylation so it is not ubiquitinated by MDM2 Mutations in the DNA binding domains prevent the transcription of cell cycle arrest proteins like p21

Question 9

What different types of mutations can occur in p53?

Answer 9

Loss of function

• Due to nonsense, frameshift or LOH
• Encode a stable mutant protein that interferes with function of the WT

Gain of function

• Mutant p53 proteins often become stable and accumulate to high levels in tumours
• Alters their DNA binding, conferring the ability to interact with other TFs and modulate expression of their target genes

Question 10

What is Li Fraumeni syndrome?

Answer 10

Germline p53 mutation predisposes to multiple tumour types.

Familial clustering of

Adrenocortical carcinoma (ACC), Soft tissue sarcoma (STS), central nervous system tumours, breast cancer.

Question 11

What are miRNAs and IncRNAs and give an example?

Answer 11

miRNAs

Mediate post transcrpiotinal gene silcening by degrading the target RNAs, supress translation. Degrades mRNA and blocks protien translation (binds to 3 prime region of coding RNA which causes degradation of complete RNA or black protein translation). Used within breat cancer

IncRNAs

Regulate transcription of coding and non-coding RNA through:

• Chromatin interaction (stabilize chromatin loops)
• Sequester regulatory factors - transpcriotn factors
  i. e. WRAP53alpha
• Transcribed from bottom strand (overlaps with p53 coding on top strand)
• Because they overlap, their RNAs can interact and protect p53 mRNA from degradation
• In WRAP53 knockdown p53 mRNA is lost -> can contribute to development of cancer