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BMS336 - Modelling Human Disease and Dysfunction > 9. Cancer 3: Zebrafish as a Cancer Model > Flashcards

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

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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
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3
Q

What are the risk factors of melanoma?

A
  • Age
  • Gender
  • Skin tone
  • UV exposure/sunburn
  • Genetics
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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
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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
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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

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7
Q

How does UV exposure/sunburn affect melanoma risk?

A

Having >5 blistering sunburns increases risk of melanoma by 50%

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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
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9
Q

What is the main initiator event in melanoma?

A

Constitutive activation of the MAPK pathway (promotes proliferation)

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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
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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
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12
Q

What model is used to study core cell cycle machinery?

A

Yeast

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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

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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
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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
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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

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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
Q

What were the results from preclinical testing of PLX4032 in nude mice xenotransplants?

A
  • 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
Q

What were the results from clinical testing of PLX4032?

A
  • 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
Q

How have zebrafish been used in melanoma research?

A
  • 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
Q

What was the model used by White et al (2011) to identify additional melanoma treatments?

A

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
Q

What was significant about the expression of BRAF(V600E) by the mitfa promoter?

A

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
Q

How did they gain insight into these initiating events?

What did this show and suggest?

A
  • 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
Q

What screen did White et al (2011) perform?

How did they do this?

A
  • 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
Q

What were the results of White et al (2011) chemical screen?

A
  • 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
Q

What was significant about using zebrafish in White et al (2011) chemical screen?

A
  • High n numbers were required as 2000 chemicals were screened
  • This would not be possible in rodents due to costs
34
Q

What were the results of preclinical testing of leflunomide in cell lines?

A
  • 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
Q

What were the results of preclinical testing of leflunomide in nude mice xenotransplants?

A
  • 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
Q

If BRAF mutations are insufficient to form tumour alone, what must be occurring?

A
  • 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
Q

What was identified to promote progression of melanoma?

A
  • A large region of chromosome 1

- Very difficult to identify specific gene as region was large and contained many genes

38
Q

What model was used in Ceol et al (2011) candidate gene screening?
What was the problem with this model?
How was this fixed?

A
  • 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
Q

How did Ceol et al (2011) identify the gene in chromosome 1 causing melanoma progression?

A
  • 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
Q

What were the results of Ceol et al (2011) screen?

A
  • 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

BMS336 - Modelling Human Disease and Dysfunction (14 decks)

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