Oncogenes Flashcards
1
Q
What is an oncogene
A
- A gene that has the potential to cause cancer when mutated or overexpressed.
- Several hundred oncogenes have been identififed
- It is derived from a normal cellular gene termed proto-oncogene.
2
Q
What are some characteristics of oncogenes
A
- Potentially cancer-causing gene
- Novel or unregulated activity
- High level of expression
3
Q
What is a proto-oncogene
A
- Proto-oncogene: control critical processes such as proliferation, survival and differentiation etc. in a regulated manner.
- **as a result of gene mutation → an altered version of the gene called an oncogene
4
Q
What are the functions of the gene products from proto-oncogenes
A
- function as growth factors, receptors, signal transduction molecules and transcription factors
- (all of these products control gene expression)
- Activated oncogenes over-express these normal proteins or produce altered proteins with altered (usually constitutive) activity that contribute to the hallmarks of cancer
5
Q
how does a proto-oncogene become an oncogene (name the mutation)
A
- Usually constitutively active
- Mutation of Proto-oncogene to become an oncogene is called a “gain of function mutation”
- E.g. Oncogene receptor may become constitutively active (does not require ligand interaction)
6
Q
are oncogenes dominant or recessive
A
- Dominant (opposite of tumour suppressor, you need both alleles to be mutated for the cell to become cancerous there)
7
Q
What are hallmarks of cancers?
A
- Hallmarks of Cancer: These are some of the processes that are degranulated in cancer cells compared to normal cells.
- Oncogenes are involved in many of these processes:
8
Q
Which mechanisms enable oncogene activation (4)
A
- Point mutation ⇒ protein with altered characteristics
- simple change in dna sequence, constitutively activated
- e.g. RAS, EGFR, BRAF
- Amplification of a genomic DNA region that includes the proto-oncogene ⇒ overexpression of the gene & increased amounts of protein
- proteins not mutated just over expressed leading to cancer because too many copies
- e.g. MYCN, EGFR
- Chromosome translocation that brings a proto-oncogene under the control of a different promoter ⇒ inappropriate gene and protein expression
- e.g. c-MYC, BCL-2
- Chromosome translocation that joins two genes together ⇒ creates a chimeric fusion gene and protein with novel characteristics
- e.g. BCR -ABL, EML4-ALK, NTRKCan have cancers with many different oncogenes that have different pathways of mutation
9
Q
What are some oncogenic single nucleotide variants (SNVs) seen in cancer
A
DIAGROM
10
Q
Describe receptor tyrosine kinases
A
- 58 human receptor tyrosine kinases (RTKs)
- Signalling domain is the functional bit of protein
- Examples include EGFR (ErbB1, Her1), ErbB2 (Her2), VEGFR, ROS, RET….
- Control cell growth, survival, motility, differentiation, metabolism….
- Share a similar protein structure of an extracellular ligand binding domain, a transmembrane helix, and an intracellular tyrosine kinase (TK) domain
- Dysregulation leads to many human diseases, especially cancer
11
Q
Describe EGFR signalling
A
- It is a monomer in the abscence of it’s normal ligand
- EGF binds to EGFR causing dimerisation and it autophosphorylates
- Once tyrosine residues phosphorylate, they act as docking stations for other cellular proteins
- Other cellular proteins transmit other signals and pathways down through the cell
- Signals through a range of proteins and eventually leads to a change in transcription factors in the nucleus which leads to a change in expression
- Signalling cascade that involves protein phosphorylation
- EGFR is frequently mutated in lots of cancers
12
Q
What are some common EGFR mutations
A
- See the below schematic of the EGFR (receptor) protein.
- The tyrosine kinase domain signals through to other molecules such as RAS and sets off the whole signalling cascade - there are common mutations in EGFR proteins in this part that cause cancer:
- small in Frame deletion of 6 aa at 747-752
- This results in changes protein conformation ⇒ prolongs active dimer configuration
- Missense mutation L858R (leucine ⇒ arginine) (amino acid substitution)
- Increases kinase activity 50-fold
- When the receptor has one of these 2 mutations it becomes constitutively activated and signals through the downstream pathway constantly.
- small in Frame deletion of 6 aa at 747-752
13
Q
What does mutant EGFR increase
A
- Increases RAS/MAPK activity
- You can mutate any proteins in this pathway and get the same effect where there are bigger changes in the gene expression
- eg: RAS
14
Q
What is RAS and what is its function
A
- RAS – a molecular switch involved in signal transduction
- One of the typical oncogenes is the RAS oncogene.
- RAS is a signalling molecule involved in a signalling cascade:
- On the cell membrane there is a receptor tyrosine kinase to which a growth factor binds to and activates its.
- The RTK then activates signals through a downstream signalling pathway which leads to changes in gene expression in the cell.
- RAS is one of the most important proteins involved in the signalling cascade.
- There are RAS genes in the human genome:
- KRAS
- NRAS
- HRAS
- And they are all basically the same.
15
Q
how is RAS different in cancer
A
- RAS is frequently mutated in cancers.
- Different RAS genes are associated with different cancers.
- Different cancers show variable patterns of KRAS, NRAS and HRAS mutations
- E.g. >95% of pancreatic cancer tumours have mutation in KRAS gene.
- In some cases the RAS genes are mutated in almost every case whilst in other RAS gene mutations are rare.
16
Q
What do oncogenic RAS mutations always involve and why
A
- involve missense mutations affecting one of 3 codons G12, G13 or Q61
- If we look at all the different mutations in the 3 RAS genes in these tumours, we find that the mutations occur at just 3 amino acids in the entire protein sequence
- This is because if we look at the 3D structure of RAS G12, G13, and Q61 are all part of the same pocket in the 3D structure and all surround the GTP binding site.
17
Q
How do point mutations of RAS affect its function
A
- RAS is involved in a signalling cascade from the RTK which ultimately results in changes in gene expression.
- RAS is switched between an on/off conformation depending on whether it is bound to GDP or GTP.
- When it is bound to GTP, RAS is active.
- When RAS hydrolyses GTP to GDP that switches off RAS activity and there is no signalling to the other proteins down streaming.
- Mutations at G12, G13, and Q61 at the GTP binding site, decrease GTP hydrolysis and so lock RAS in active GTP-bound state
- it is permanently switched on
- This leads to constitutive activation of RAS/MAPK pathway
18
Q
How are RAS and EGFR mutations typical to those seen in many oncogenes
A
- Mutations that activate RAS/EGFR genes are clustered in specific regions of the gene (called hotspots)
- These regions encode important functional domains in the proteins
- Most mutations are missense mutations (amino acid substitution) resulting in a gain of function.
- Mutations only need to happen in one of the 2 alleles.
19
Q
What are some oncogenes that are amplified in cancer
A
- MYCN
- c-MYC
- Cyclin D1
- EGFR
20
Q
Image showing in neuroblastoma MYCN amplification
A
- The image below was taken by FISH.
- Normally MYCN should only be on C2.
- Chromosomes are in blue and specific bits of the DNA are highlighted in green or red.
- C2 – green dots
- MYCN gene – red dots
- However, there are lots of red dots scattered over the image – these are additional copies of the MYCN gene that have arisen through gene duplication and gene amplification.
21
Q
Image showing N-myc protein expression increase in neuroblastomas
A
N-Myc protein expression is increased in neuroblastomas with MYCN gene amplification
- If you get a tumour sample and take to path lab they look for expression of the MYCN protein using immunohistochemistry.
- In E image you can see a normal section of cells where there no amplification of MYCN.
- On image on right you have MYCN amplification and brown staining is the MYCN protein.
22
Q
What is MCYN amplification associated with
A
- MYCN amplification frequently associated with aggressive disease, metastatic potential, therapeutic resistance and poor patient outcomes.
- See graph - MYCN amplification in children <1 year of age with metastatic neuroblastoma
- MYCN amplificationis used as a prognosis factor
23
Q
What gene amplification causes breast cancer, how do we treat this specific breast cancer
A
- ERBB2/Her2
- Another receptor tyrosine kinase that gets amplified in cancer
- Red dots = Her2 gene
- see over expression in the image
- Give an antibody which interferes with the protein and blocks its activity
- Emtansine
- Trastuzumab emtansine - google
24
Q
What are some examples of translocations leading to changes in oncogene expression and what do they do
A
SEE TRANSLOCATION TABLE
25
What is burkitt lymphoma?
- Highly aggressive **B cell** lymphoma derived from mature B cells
- Frequently presents as a fascial tumour
- One of the fasting growing tumours.
26
- What is one of the main hallmark of Burkett lymphoma, explain it further
- Translocations involving c-MYC are a hallmark of Burkitt lymphoma
- All BL tumour cells carry a chromosomal translocation involving c-MYC gene on chromosome 8 and one of the three immunoglobulin gene loci (most common involves heavy chain locus)
- You can have 3 immunoglobulin genes and the cmyc gene gets rearranged and joined onto one of these genes – the heavy chain and the kapa and lama light chains.
- t(8;14) MYC – IgH (**85%)**
- t(2;8) MYC – IgK
- t(8;22) MYC – IgL
27
How can translocation of chromosomes in cancer cells be detected
- Using Karyotyping
- Chromosomes stained with dye and matched up based on size and banding pattern.
- Chromosomes 8 – Where MYCN gene normally located.
- little but of the chromosome is missing
- Chromosome 14 – where normal immunoglobulin heavy chain is located.
- extra material on end of C14
- This is what is called a C8-C14 translocation and as a result we place the MYC gene next to the immunoglobulin gene on this chromosome.
28
What is the effect of translocation of chromosomes in Burkitt lymphoma on B cells
- t(8:14) translocation places IgH enhancer adjacent to c-MYC
- C-MYC is a very important transcriptional regulator and its activity is normally very tightly controlled by the cell.
- When you translocate onto C14 next to IgH enhancer you get over expression of MYC.
- This is because the IgH enhancer is very powerful in B cells as they need to produce a lot of antibodies.
- So, when you put MYC gene next to this enhancer and promoter you end up with super high level of expression of MYC gene.
29
What is a marker for cell proliferation to see the effect of the IGH enhancer
- Powerful IgH enhancer drives c-Myc protein overexpression
- Ki67 is a marker of cell proliferation – see cells are rapidly proliferating as there is high levels of Ki67 = cancer.
- MYC protein is stained – see tumour cells are all expressing very high levels.
- This drives then to proliferate very quickly.
30
Why does MYC give rise to cancer, what does MYC do
- C-myc is a transcription factor that regulates >600 cellular genes
- MYC is a very important TF that control many Genes required for:
- Cell growth
- Proliferation
- Ribosomal synthesis
- Protein synthesis
- Metabolism
- Energy generation
- These are all hall mark features of cancer.
- So if you deregulate MYC, because it controls many of the proteins involved in different features of cancer, then cell becomes cancerous.
31
What are some examples of translocations leading to fusion genes and their effects
SEE TRANSOCATION TABLE
32
Name a hallmark of chronic myeloid leukaemia (CML) and what is this new chromosome called
- BCR-ABL is a hallmark of chronic myeloid leukaemia (CML)
- All CML cells have this translocation in it.
- identify this chromosomal translocation by doing a cell karyotype.
- see this translocation between C9 & C22.
- you’ve fused 2 genes and created a new one
- new chromosome called Philadelphia chromosome.
- This results in a fusion between the BCR gene on C22 and the ABL gene on C9 creating a novel BCR-ABL fusion protein.
33
Describe the mechanism of how ABL and BCR-ABL causes cancer
- ABL and BCR-ABL are tyrosine kinases
- ABL activity usually supressed by p145 protein and this protein acts to control the activity of the tyrosine kinase domain
- WHen you fuse it with BCR you remove the inhibitory domains which is replaced by a part of the BCR sequence
- Tyrosine kinase domain no longer negatively controlled
- The brand new protein is formed and can now drive lots of intracellular pathways as there is no inhibition
34
What does genomic analysis of cancer cells enable
- Gives better understanding of the function of aberrant oncogenes
- Provides detailed information about mutational landscape of cancers
- Enables improved cancer diagnostic tests
- Can be linked to prognostic information
- Is important for designing new targeted cancer treatments
35
Why is understanding oncogenes important for clinical treatment
Understanding oncogenes is important for designing new targeted cancer treatments
36
What is precision cancer therapy and what are examples
- Increased genomics ⇒ Move away from ‘one size fits all’ approach to precision medicine
- Develop inhibitors of key oncogenes that drive tumour cell growth
- Improve patient outcomes and reduce side effects
- examples include
- BCR‐ABL – Imatanib
- EGFR – Gefetinib, Afatanib, Osimertinib
- HER2 – Trastuzumab
- RAS - Sotorasib
- -ib, small molecule inhibitor
- -ab, monoclonal antibody
37
How can BCR-ABL tyrosine kinase be inhibited and what are the clinical implications of this
- BCR-ABL: a paradigm for targeted therapy
- Imatinib (Gleevec) is a specific inhibitor of the BCR-ABL tyrosine kinase
- Competitive inhibitor of ATP binding to active site, therefore inhibiting tyrosine kinase activity
- This has improved outcomes and life expectancy of patients
- 83% 10-year survival
- This is an example of a targeted therapy: we known the mutation in this cancer, gone to lab and create a molecule that can inhibit the gene product.
38
What are some EGFR targeted therapies
- He didn’t go into that much detail however i’ve left it in there for the context and understanding. Know classes and examples and bining
- **First generation TKI**
- e.g. gefitinib, erlotinib
- Competitive ATP-mimics that binds to TK domain to prevent binding of ATP.
- Reversible binding
- Moderately increase life expectancy
- However Frequent drug resistance e.g. T790M mutation blocks binding of drug. (cancer cells are genetically unstable and mutate to become resistant to drug)
- **Second generation TKI**
- e.g. Afatinib , Dacomitinib
- Irreversible binding in the ATP pocket
- Binds to the mutated version which causes resistance
- So if we find patients given 1st gen drugs become resistant, we can give them new drugs which overcomes resistance mutation
- **Third generation TKI (he didn’t explain this in the lecture but it is in the diagram in the next toggle)**
- e.g. osimertinib
- Bind more avidly to EGFR T790M mutants than wild-type EGFR
39
Examples of precision targets for lung adenocarcinoma (don’t need to know)
- Proto-oncogenes encode growth factors, growth factor receptors, signal transduction molecules, second messengers, transcription factors etc.
- Oncogenes are abnormally expressed or mutated forms of the corresponding cellular proto-oncogenes
- Missense mutations often target specific regions of the gene (hotspots) and result in a gain of function
- Translocations can lead to gene overexpression or create fusion proteins with novel properties
- Only one copy of the gene needs to be mutated
- Some oncogenes are associated with specific tumours; others occur widely in many different cancers
- Proteins encoded by oncogenes can be targets for precision cancer therapy
