CANCER 2 - Animal models & melanoma Flashcards
What are the most common mutations seen in melanoma?
Ptc inhibitor mutations
Risk factors to melanoma?
Age, gender (more common in males), skin tone and SUN BURN
What are some common mutations seen in melanoma?
- Mutations in DNA repair genes
- Melanocortin receptor - mutation = ginger hair
- CDKN2A gene (p16/p14 are the protein products which cause apoptosis of cells upon UV damage)
What is TERT and how is is relevant to melanoma?
TERT = gene which codes for protein which maintains the integrity of telomeres & cell survival
Mutations in this gene can occur and are rare.
Common genes with EARLY MUTATIONS
TERT, CDKN2A, SW1/SNF
Common genes with LATE MUTATIONS
p53, pTEN
How are the MAPK and Ras pathways relevant
Mutations in MAPK genes linked to initiation of melanoma
Ras and BRAF — why important?
75% of mutations seen in melanoma linked to the BRAF but this doesnt always lead to tumours
Advantages of using: yeast, drosophila/c.elegans, fish and mice
Yeast - good for observing cell cycle machinery
Mice - precise genetics used, closer to human as mammalian
FIsh - large scale, optic clarity, useful in drugs screens
Drosophila/c.elegans - look at signalling pathways such as Hh and Wnt, can look at upstream signalling pathways
THE NUDE MOUSE - what is it, advs and disadvs?
Mouse created with a defective immune system and mutations in the fox1 gene. Can use to create XENOTRANSPLANTS (transplant the mouse with human tumour
+ good model for human disease
- tumours not always transplanted and grown in the right location, the model is immunocompromised so transplants often don’t work
How are mouse genetically modified models useful? (Use of Ras in mouse models)
Can create tumours by knocking down CDK2NA, creating loss of PTEN (tumour suppressor) and also hitting the mouse with UV , which will lead to activated Ras in melanocyte cells.
This will be specific to MELANOCYTES ONLY
TyrCre-ERT2;Brafca+,PTEN(lox/lox)
What is this - explain the elements of the mouse model and outline what it allows us to do
PTEN is loxed so will be cut out upon injection of tamoxifen (tyr)
Braf = cre activated form of BRAF
ERT2 = melanocyte-specific promoter
Cre mouse!!!
When tamoxifen is injected, PTEN becomes inactivated and BRAF activated IN MELANOCYTES ONLY – this allows tumours to be created!
Example of research - BRAF inhibitors and RG7204
BRAF inhibitors mutated (BRAF6000E) – then treated with RG7204 - mutants are sensitive to RG7204 but only in the presence of BRAF6000E mutation
How was the nude mouse used in the testing of RG7204?
3 melanoma cell lines used from nude mice. 3 lines needed ars different mutations present in different lines.
RG7204 blocked tumour growth in the model very well
What problem was encountered with RG7204?
Cells became resistant to the inhibitor
What new method is causing zebrafish to become increasingly popular
CRISPR/Cas9 system
Why was a zebrafish screen carried out (mitfa:brafV6000E transgenic line)
- mitfa:braf6000E transgenic line created - mitfa is a melanoctye specific promoter and braf6000E is a melnoma-inducing oncogene
- melanomas induced in fish ONLY WHEN P53(-/-) PRESENT
- Melanomas identified to have NEURAL CREST PROGENITOR SIGNATURE
Outline procedure and outcome of the drugs screen
- 2,000 compounds tested which inhbit CRESTIN (a neural progenitor marker)
- DHODH identified to inhibit CRESTIN
- A771726 analogue of Lethlunamide tested futher - (similar compound to DHODH) at the same time as PLX4720 —»> Lethlunamide and PLX4720 in combination impacted tumour growth!!!!
Features of BRAF
cooperates with OTHER GENETIC PATHWAYS
Activated in 60% of melanomas
Gene found at Large region in chromosome 1
Features of mitfa - how is it used in investigation of cancers in fish - what was shown with regards to SETDB1?
- Expression plasmid encoding mitfa is injected into cells.
- There have been different expression plasmids created which target DIFFERENT CANDIDATE ENHANCERS FOR TUMOUR GROWTH and see which are most effected
- SETDB1 caused a decrease in melanoma free cell survival – could this be a potential therapeutic target?
What is SETDB1?
- histone methyl transferase enzyme
- amplified in melanoma
what is shown in the mitfa:braf(V6000E) which also had knocked down p53 (-/-)
When mitfa removed, FISH WERE NORMAL
