9. Cancer 3: Zebrafish as a Cancer Model Flashcards
1
Q
Why use animal models for cancer?
A
- Help understand the mechanism of how cancer develops
- Use in pre-clinical therapeutic development
2
Q
What does melanoma develop from?
How can it be treated?
A
- Melanoma develops from melanocytes (cells forming moles)
- When discovered early they can be excised without chance of recurrence
- If left, melanoma breaks into dermis and lymph vessels to metastasise and becomes deadly aggressive
3
Q
What are the risk factors of melanoma?
A
- Age
- Gender
- Skin tone
- UV exposure/sunburn
- Genetics
4
Q
How does age affect melanoma risk?
A
- Through life somatic mutations are accumulated
- As age increases, the number of mutations acquired increases, which increases risk of acquiring a mutation in gene causal to melanoma
5
Q
How does gender affect melanoma risk?
A
- Males are at increased risk for unclear reason
- Males may have higher somatic mutation rate
- May be due to hormonal differences
- May be due to psychosocial factors e.g. increased exposure to carcinogens, increased alcohol intake and not using suncream
6
Q
How does skin tone affect melanoma risk?
A
Individuals with pale skin a have a 24fold higher risk of developing melanoma than dark-skinned individuals
7
Q
How does UV exposure/sunburn affect melanoma risk?
A
Having >5 blistering sunburns increases risk of melanoma by 50%
8
Q
How does genetics affect melanoma risk?
A
- Mutations in DNA repair genes - people with mutations in nuclear excision repair enzymes (repairs UV-induced DNA damage) have Xeroderma Pigmentosum and develop many melanomas
- Mutations in melanocortin receptor causes people to have red hair and very pale skin
- Rare mutations in CDKN2A gene - encodes 2 proteins involved in cell cycle control and prevent cells with DNA damage from entering cell cycle and send them for apoptosis. Loss of function mutations result in these cells surviving
- Rare mutations in TERT gene - encodes telomerase which maintains telomeres. Activating mutations in TERT promoter enhances expression and promotes cell survival
9
Q
What is the main initiator event in melanoma?
A
Constitutive activation of the MAPK pathway (promotes proliferation)
10
Q
Give examples of:
- Initiator mutations
- Progression stage mutations
- Late stage mutations
A
- BRAF(V600E)
- N-RAS
- These are both components of MAPK pathway, therefore activating mutations cause constitutive activation of MAPK pathway
- TERT
- CDKN2A
- p53
- PTEN
11
Q
What is the main initiator mutation?
A
- 60% of nevi and melanomas have activating BRAF mutations (majority are BRAF(V600E) mutation)
- As cancer progresses more mutations are acquired
12
Q
What model is used to study core cell cycle machinery?
A
Yeast
13
Q
What model is used to study upstream regulators of cell division/differentiation/death?
Why is this?
A
- Drosophila
- Growth factor signalling pathways (MAPK, Hh, Wnt) are much easier to study in Drosophila
14
Q
What model is used to study tumour growth and metastasis?
Why is this?
A
- Vertebrate
- Tumour growth and metastasis require blood vessels, lymph vessels and an elaborate immune system which Drosophila do not have therefore vertebrate model must be used
15
Q
What are the advantages of using mice as a melanoma model?
A
- High gene conservation with humans
- Can generate KO animals with high precision
- Can use xenotransplants
16
Q
What are the advantages of using zebrafish as a melanoma model?
A
- Large numbers can be used in screens
- Optic clarity allows single cell tracing in tumours
17
Q
Why can xenotransplants be performed in nude mice?
A
They have a mutation in foxn1 gene which causes a defective immune system so can host human melanoma tumours
18
Q
What are the pro’s and con’s of xenotransplant models?
A
+ Can use human melanoma cell lines
- Can go to wrong location
- Mic have defective immune system , immune response likely to be important in tumour biology
19
Q
Is melanocyte expression of N-RAS sufficient to form a melanoma?
A
No, UV irradiation or CDKN2A co-inactivation is required to form melanoma
20
Q
Is melanocyte expression of BRAF(V600E) sufficient to form a melanoma?
A
No, get more moles but insufficient to form melanoma. In combination with loss of PTEN form melanoma
21
Q
What is the BRAF(V600E) mouse melanoma model?
Describe how it works.
A
Tyr: Cre-ER(T2); BRAFCA/+; PTENlox/lox
- Tyrosinase is a melanocyte-specific promoter that drives expression of Cre-ER(T2) only in melanocytes
- Cre recombinase is fused to a mutant estrogen ligand-binding domain that requires tamoxifen for activation
- BRAFCA/+ - Cre-activatable constitutively active BRAF
- WT BRAF is flanked by lox sites and when tamoxifen is injected, Cre recombinase recombines WT BRAF out, activating expression of BRAF(V600E) mutant
- PTENlox/lox - PTEN gene flanked by lox sites and when tamoxifen injected, Cre recombinase recombines PTEN out, resulting in loss of PTEN
- As Cre-ER(T2) is only expressed in melanocytes, these effects are only seen in melanocytes
- After tamoxifen is injected mice develop melanomas and die within 80 days
22
Q
What was discovered in a cancer genome project?
A
Many melanomas were sequence analysed and BRAF(V600E) mutation was shown to be a major driver of melanoma
23
Q
What is PLX4032?
A
A selective BRAF(V600E) inhibitor
24
Q
What were the results from preclinical testing of PLX4032 in cell lines?
A
- PLX causes growth inhibition in cell lines expressing BRAF(V600E) mutation
- PLX is not effective in cell lines expressing N- or K-RAS mutations
- Therefore PLX inhibits growth of melanoma cells expressing BRAF(V600E)
25
What were the results from preclinical testing of PLX4032 in nude mice xenotransplants?
- Used 3 different melanoma cell lines, important because different cell lines express different mutations, therefore if PLX is effective in one cell line cannot say it will be effective in all
- Control mice receiving vehicle showed rapid melanoma growth
- PLX-treated mice showed inhibition of melanoma growth
PLX also shown to extend survival
- Control mice receiving vehicle quickly died following tumour implant
- PLX-treated mice survived >600 days
26
What were the results from clinical testing of PLX4032?
- PLX was very effective against melanomas with BRAF(V600E) mutation (not others)
- However effect is temporary as tumours quickly return and are resistant to further treatment
- PLX extends survival nevertheless
27
How have zebrafish been used in melanoma research?
- Find additional treatments that can be combined with PLX4032 to extend time before relapse
- Large scale candidate gene screening to identify genes that promote tumour progression
28
What was the model used by White et al (2011) to identify additional melanoma treatments?
mitfa: BRAF(V600E); p53 -/-
- mitfa is a melanocyte-specific promoter therefore BRAF(V600E) mutant only expressed in melanocytes
- When crossed with p53 -/- fish, these fish develops melanomas rapidly and reliably
29
What was significant about the expression of BRAF(V600E) by the mitfa promoter?
mitfa promoter drives expression of BRAF(V600E) at a time that overlaps with with expression of embryonic neural crest markers (e.g. sox10), therefore events that occur early in embryogenesis are analogous to events occurring in tumour initiation
30
How did they gain insight into these initiating events?
| What did this show and suggest?
- They compared gene expression profiles of mitfa:BRAF(V600E); p53 -/- embryos with those of mitfa:BRAF(V600E); p53 -/- melanomas
- Identified a signature of 123 overlapping genes for melanoma cells, which is similar to the signature of neural crest progenitor cells
- Suggest melanoma cells adopt a neural crest progenitor cell fate
31
What screen did White et al (2011) perform?
| How did they do this?
- An in vivo chemical screen to identify compounds that inhibit formation of neural crest progenitors
- Zebrafish embryos were incubated with 1 of 2000 chemicals and then in situ hybridisation was performed for cresting gene (neural crest progenitor marker)
32
What were the results of White et al (2011) chemical screen?
- 1 compound significantly reduced crestin expression and was found to inhibit DHODH
- Leflunomide is an existing approved drug that inhibits DHODH and also inhibited neural crest progenitor formation
33
What was significant about using zebrafish in White et al (2011) chemical screen?
- High n numbers were required as 2000 chemicals were screened
- This would not be possible in rodents due to costs
34
What were the results of preclinical testing of leflunomide in cell lines?
- Number of cell lines were sensitive to leflunomide but not extremely sensitive
- When leflunomide was combined with PLX, it was clear this combination was more potent in killing cells
35
What were the results of preclinical testing of leflunomide in nude mice xenotransplants?
- Combination of leflunomide and PLX was more effective in inhibiting tumour growth than PLX alone
- As both drugs are approved, will be easier to move to clinical trials
36
If BRAF mutations are insufficient to form tumour alone, what must be occurring?
- BRAF must cooperate with further genetic/epigenetic lesions in melanoma formation and progression
- Oncogenes are well studied but these are initiating events
- Further genetic changes are required in form of deletions or implications of chromosomal regions
- Which genes cause progression all less understood
37
What was identified to promote progression of melanoma?
- A large region of chromosome 1
| - Very difficult to identify specific gene as region was large and contained many genes
38
What model was used in Ceol et al (2011) candidate gene screening?
What was the problem with this model?
How was this fixed?
- mitfa: BRAF(V600E) fish crossed with p53 -/- fish, fish form melanomas rapidly and reliably
- This led to problems maintaining the line as fish die before breeding
- Removed mitfa gene itself causing melanocytes to die, and fish are now healthy, therefore model fish were mitfa:BRAF(V600E); mitfa -/-; p53 -/-
- If inject plasmid containing mitfa gene expressed under control of its own promoter into these fish embryos, create a mosaic fish and some melanocytes are rescued which form melanomas
39
How did Ceol et al (2011) identify the gene in chromosome 1 causing melanoma progression?
- Made many plasmids containing mitfa gene and 1 gene from candidate genes in region of chromosome 1
- Injected plasmids into fish embryos and looked for gene that enhanced formation of melanoma
40
What were the results of Ceol et al (2011) screen?
- Found 1 plasmid significantly enhanced the formation of melanomas compared to mitfa gene only plasmid and reduced survival
- This plasmid contained the gene SETDB1, a methyl transferase which may now be a therapeutic target
