Oncogenes and tumour suppressors Flashcards
How many mutations are found in human cancers?
Depends on the cancer type - loads in colorectal cancer as genome is unstable, few in others. A typical tumour contains 2-8 driver mutations, the rest are passengers that give no advantage
How are driver genes classified?
As mountains (often mutated) and hills (mutated less but still frequently). Using a 20/20 rule (in which more than 20 percent of mutations are truncating or inactivating suggest a tumour suppressor whilst more than 20 percent missense mutations in recurrent positions are oncogenes). Found 71 TSGs and 54 oncogenes in a high confidence study
What is haploinsufficiency?
Where one normal copy isn’t enough to sustain function. Don’t need to lose both copies. Most tumour suppressor genes are haploinsufficient.
What is MutSigCV?
A new approach to identify significantly mutated genes in cancer. Corrects for variation by using patient specific mutation frequency and spectrum as well as gene-specific background mutation rates incorporating expression level (if highly expressed more likely to be mutated) and replication time (when replicated in S phase can effect chance of mutation). Using this method significantly shrinks the list of mutated genes in lung cancer.
How can the Rb pathway be inactivated?
Direct: mutation of the Rb gene/binding and inhibition of Rb by proteins of small DNA tumour viruses.
Indirect: amplification of the gene encoding cyclin D1 (promotes Rb phosphorylation) or loss of the cyclin dependent kinase inhibitors p16 (INK4A)
How is the Rb pathway affected in human cancers?
Inactivated in most human cancers. Depending on the cancer, get different characteristic inactivations at different points (could be oncogenic or tumour suppressive) - this may be due to genetic background of the tissue
How can loss of a tumour suppressor gene occur?
Healthy cell with only one normal copy Nondisjunction causing chromosome loss Chromosome loss then chromosome duplication Mitotic recombination Gene conversion Deletion Point mutation
Describe the E2F family of proteins in humans
9 distinct proteins. 3 activators (1-3a) and 6 repressors (3b-8), though these aren’t strict (repressors do some activation etc). E2F1-6 dimerise with DP proteins (of which there are 4). E2F1-5 interact with Rb family and recruit chromatin modifiers and remodelling factors (each E2F has affinity for a different Rb family member). E2F6-8 repress transcription in an E2F independent way
How do different members of the E2F family interact in Drosophila
Only 2 members: E2F1 (activator) and E2F2 (repressor). Can rescue the phenotype of the knock out of one by knocking out the other - functional antagonism
How does E2F induce proliferation and apoptosis?
Through external signals. If there is an increase in E2F signalling, this drives apoptosis. Need the parallel mitogenic signals to turn off the apoptotic pathway so E2F only promotes proliferation. Apoptosis is suppressed by mitogenic signals
How does E2F1 regulate tumour development?
If there is proper growth signalling and no DNA damage, apoptotic potential of E2F1 is repressed. If cells experience DNA damage/lack of signalling, apoptotic activity is released. Knocking out E2F1 results in tumours in mice, a tumour suppressor like property
How does Rb have such a big effect on cellular fate?
Rb interacts directly with chromatin associated factors that regulate transcription, chromatin modification and remodelling. Inactivating Rb changes the transcriptional profile of many genes.
What is the effect on mitosis of loss of Rb?
Causes centromere dysfunction, aneuploidy and chromosomal instability. Could be due aberrant transcription of MAD2, a SAC protein and CENPA?
How does Rb loss cause DNA damage?
If E7 (small DNA virus protein) inactivates pocket proteins, this causes replication stress (E2F regulates expression of DNA replication proteins, nucleotide synthesis genes and DNA damage response and repair genes) and stalling of replication forks at repetitive regions of DNA. This increases DNA damage and loss of heterozygosity and CIN/anueploidy.
How does Rb affect chromosome condensation?
In Drosophila chromatin appears puffy if Rb is deleted. Found that Rb interacts directly with condensing II subunit of CAP-D3 in Drosophila and human cells and is required for condensin accumulation of chromosomes.
Describe the initial evidence surrounding p53
p53 expression levels directly correlated with T-antigen activity
p53 was found to associate with the E1B antigen of adenovirus
Many tumours but not normal tissue showed high p53 levels
Transfection of p53 clones from transformed cell lines could co-operate with HRAS (another oncogene) to transform primary cell cultures
p53 cloned from transformed cell lines could augment the transformed properties of established cell lines
Suggested that p53 was an oncogene
How has p53 shown to be a tumour suppressor?
50% of human tumour cells have p53 loss/mutation
Heritable p53 mutant allele predisposed to cancer
100% of p53-null mice got cancer
Describe the structure of p53
N terminus-TAD1-TAD2-PRD-DNAb–Tet-Basic-C terminus
TAD = transactivating domain
DNAb = DNA binding (cancer hotspot mutations)
Tet = tetramer, has a nuclear export signal
Basic = for non-specific DNA binding (scanning), has nuclear localisation signal
How does p53 bind DNA?
As a tetramer to bi-partite target response elements
What factors affect p53 DNA binding?
Trans: p53 activating stimuli, cellular environment, nuclear p53 levels, post-translational modifications, protein-protein interactions, cofactors and transcription factors, chromatin environment
Cis: non-canonical response elements (RE), canonical tetrameric RE, mismatches in RE number or position, spacer length, sequence context/nearby transcription factor REs, SNPs/CNVs. position of RE to transcription start site
What stimuli does p53 respond to?
Nutrient deprivation (through AMPK phosphorylation), telomere erosion, hypoxia, DNA damage (through ATM/ATR), Ribosomal stress (some ribosomal proteins bind MDM2 as stress indicators), oncogene activation (ARF)
What is the response of p53?
Can trigger apoptosis, cell-cycle arrest, senescence, DNA repair, survival and genomic stability. Can regulate metabolic pathways and autophagy. What it does depends on the modifications that activated it.
How is p53 activated?
Normally, p53 is ubiquitinated and degraded and exported from the nucleus by MDM2. Can be relieved of this and translocated to the nucleus through ARF, Mdmx and modifications of Mdm2 and p53. In the nucleus, modifications of p53 can de-repress it from Mdm2/Mdmx e.g. acetylation or phosphorylation can relieve MDM2/ MDMX inhibition of p53 bound to a response element. Then, p53 can recruit cofactors to aid expression of genes for its response.
How is p53 regulated by Mdm2 and Mdm4?
Mdm4 = MdmX. Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. Mdm2 and 4 bind p53 and inhibit its transactivation. Mdm4 binds Mdm2, stabilising it and boosting its ligase activity.
How can p53 regulate metabolic pathways?
Can channel glucose into the pentose phosphate pathway by activating TIGAR. This, along with p53 induction of sestrins leads to lower ROS levels and survival
Signalling through AMPK (which detects low glucose) to p53 causes it to activate p21 which induces survival.
How does p53 regulate apoptosis?
Upregulates PUMA which inhibits the binding of BCL-X to p53 in the cytoplasm. p53 then goes to recruit BAX to the mitochondrial membrane directly and inhibit anti-apoptotic proteins
How does p53 suppress tumour growth?
Multiple mechanisms! Includes senescence, apoptosis (both nuclear and cytoplasmic p53), autophagy, signalling through mTOR (down regulates growth, up regulates autophagy), decreasing ROS
What mechanisms exist for p53 gain of function mutations?
Prevent interaction with p53 look a likes
Novel protein interactions (e.g. a new protein or a transcription factor)
Bind different sites
Initiate different transcription
Describe the JAK/STAT signalling pathway
Cytokines bind cytokinR which has constituently associated tyrosine kinases (members of the JAK family such as Tyk2). These phosphorylate themselves and allow STATs to bind. Members of the STAT family bind to the phosphate and are phosphorylated. These then dimerise and are translocated to the nucleus where they up regulate interferon stimulated response element (ISRE) genes.
STATs can also be serine phosphorylated by MAPKs which can affect their localisation
How are STATs down regulated?
SOCs inhibit STATs. These are activated by STATs, so there is a burst of STAT activity which is then down regulated. In cancer, the down regulation doesn’t occur.
What are JAKs?
A family of Janus (JA) tyrosine kinases (K). There are 4 including Tyk2
What are STATs?
Signal Transducer and Activator of Transcription. Transcription factors. Are 6 STATs; 2 variants of STAT5 (encoded on different genes - gene duplication) = 7 overall. They can also have splicing variants.
What are SOCs?
Suppressors Of Cytokine signalling proteins. Activated by STATs.
What are PIAS?
Protein Inhibitor of Activated Stat
Describe they cytokine receptors involved in JAK/STAT signalling
Each cytokine binds a specific alpha chain which is associated with a common cytokine receptor gamma©. This then signals through either JAK1 or JAK3 to a different STAT.
Describe the receptor for IL-2 and IL-15
Has a common cytokine receptor gamma© and a specific-to-the-cytokine alpha chain. Also has an IL-2Rbeta chain to make a high affinity complex
Describe the structure of JAK
Has a C terminal JH1 domain which is the kinase domain
Then has a JH2 domain which is the pseudokinase domain (controls kinase activity) where many mutations are found
At the N terminal in domains JH3-7 there is a FERM domain which mediates receptor binding
Where are JAKs expressed?
Tyk2, Jak1 and Jak2 are expressed in all tissues; Jak3 is expressed in haematopoietic tissues
What JAK KOs are viable?
Neither JAK1 (perinatal) or JAK2 (embryonic). JAK3 is but has severe defects in lymphocyte development; Tyk2 is viable and fertile, has defects in cytokine signalling.
What is the structure of STATs?
N terminal can be acetylated and otherwise modified. Then have a CC (coiled coil) domain for nuclear receptor binding and a DNA binding domain. Then have a linker domain and an SH2 domain next to the phosphorylated tyrosine followed by a transactivating domain to bring in other factors, individual to the STAT (gives each STAT a variety of functions)
Where are STATs expressed?
Widely expressed apart from STAT4 which is restricted to mesodermal lineages and T cells where it commits to a Th1 response (Th2 is by STAT6)
How do STATs bind DNA?
As a dimer in a nutcracker formation. Go look at a picture.
