7. Cancer 1: Developmental Biology Wnt Flashcards
What was found in inbred strains of mice?
Particular inbred strains of mice were highly susceptible to mammary tumours ie female mice developed mammary tumours with almost full penetrance
How were the mammary tumours said to form?
When an RNA retrovirus (Mouse Mammary Tumour Virus) integrates itself next to a proto-oncogene, resulting in abnormally high levels of expression of this gene
How do RNA retroviruses drive increased proto-oncogene expression?
- RNA retrovirus hijacks the host cell machinery to transform its RNA transcript into DNA which is then randomly inserted into the host’s genome
- Retrovirus contains strong, constitutively active promoters and if host gene is downstream, read-through can occur from the viral promoter turning on/enhancing expression of host gene
What is a proto-oncogene?
Gene that normally regulates cell cycle in controlled manner, but if gene is mutated or overexpressed it can become oncogenic
What was the proto-oncogene found to be unregulated by Mouse Mammary Tumour Virus?
Integration Site 1 (Int1)
What did the predicted protein of Int1 show?
What did this suggest?
- Int 1 protein starts with a signal sequence
- Protein likely to be secreted
- Protein may be a growth factor - gives growth advantage
How did developmental biology contribute to the understanding of Int1?
- Nusslein-Volhard and Wieschaus performed large-scale mutagenesis screen in Drosophila to identify patterning genes
- These included segment-polarity genes, 1 of which was Wingless
- Wingless showed homology with Int1, therefore was renamed to Wnt1
How was the Wnt signalling pathway elucidated?
What is the canonical Wnt signalling pathway?
- Wnt signalling pathway was elucidated in model organisms (Drosophila and Mice) and mammalian cell cultures
1. In absence of Wnt, B-catenin is degraded in cytoplasm
2. In presence of Wnt, Wnt binds to its receptor Frizzled resulting in the stabilisation of B-catenin
3. B-catenin enters nucleus and binds to co-activators and co-TFs to activate target gene expression
What are Wnt signalling target genes?
How do these lead to mammary tumours in mice?
- Genes directly governing cell proliferation (e.g. CmyC and Cyclin D1)
- In mice, abnormally high levels of Wnt1 in mammary cells leads to increased expression of CmyC and Cyclin D1 which increases cell proliferation
How were components of Wnt signalling identified?
How were these ordered into a pathway?
- In Drosophila, using KO or overexpression studies
- If KO/overexpress gene and get same phenotype as Wnt KO/overexpression then this tells you the gene is involved in WNt signalling
- Use epistatic studies to order components into a signalling pathway
Give an example of an epistatic study.
- If think B-catenin is downstream of Frizzled, KO Frizzled and then artificially put in stabilised B-catenin
- If B-catenin is downstream, this will rescue the effects of the Frizzled KO
What are the 2 ways of activating the Wnt signalling pathway?
- Increasing activators
2. Removing suppressors
What are oncogenes in the Wnt signalling pathway?
How could they lead to cancer?
- Activators - overexpression leads to increased Wnt signalling activation therefore increased expression of cell cycle regulator genes (CmyC, Cyclin D1)
- This leads to increased proliferation
What are tumour-suppressor genes in the Wnt signalling pathway?
How could they lead to cancer?
- Suppressors - normally act to suppress activation of Wnt signalling pathway (e.g. APC), but if BOTH copies are mutated/deleted this suppression is lost, leading to increased Wnt signalling activation
- This leads to increased proliferation
Why is cancer referred to as a multi-step disease?
Cancer can take many years to develop
- If 1 copy of tumour-suppressor gene is mutated/deleted then there is no effect as remaining copy is sufficient
- When both copies are mutated/deleted then suppressive effect is lost and cancer develops
- It can be many months, years or never before 2nd copy is mutated/deleted
Does overexpression of Wnt lead to human cancer?
- No evidence that overexpression of Wnt itself leads to human cancer
- But mutations in Wnt signalling pathway components leads to constitutive stabilisation of B-catenin which is clearly implicated in many human cancers
What are the 2 categories of mutations in Wnt signalling pathway?
- Activating mutations - B-catenin is always stabilised and in nucleus
- Loss of suppression - both copies of suppressor gene are lost so B-catenin is always stabilised and in nucleus
What is the most common cancer linked to mutations of Wnt signalling pathway components?
Colorectal cancer
What does the finding of signalling pathway components allow in cancer?
- Pathways have important predictive values in cancer
- By establishing how genes control signalling (e.g. act as activators or suppressors) can make educated guesses how these genes may contribute to human cancer
- Activator = oncogene
- Suppressor = Tumour-suppressor gene
How can signalling pathway components be used in assessing cancer risk?
- Signalling pathway components can be used as biomarkers for cancer risk
- E.g. if increased nuclear B-catenin causes increased proliferation can biopsy tissue and analyse levels of nuclear B-catenin to assess cancer risk
How can we see what cells in the body respond to Wnt?
Using Wnt-responsive transgenic reporter lines (e.g. take Axin promoter (Axin gene unregulated by Wnt signalling activation) and clone reporter protein (GFP) downstream of this promoter)
What did transgenic reporter lines show about Wnt-responsive cells?
In many parts of body, Wnt-responsive cells were tissue-specific stem cells
How might Wnt signalling activation lead to cancer?
Activation of Wnt signalling pathway in stem cells promotes self renewal therefore abnormal activation may cause uncontrolled self-renewal
Describe the gut crypt stem cell niche.
Gut crypt made up of columnar epithelial cells
- At base of crypt is the stem cell niche
- At top of crypt is differentiating/ed cells
- Stem cells at base of crypt are surrounded by supporting Paneth cells
- When stem cell divides give rise to progenitor which rapidly amplifies this cell type (Trans Amplifying cells)
- As TA cells migrate up crypt they begin to differentiate into villus cells
What signalling occurs in the gut crypt?
- At crypt base, high levels of Wnt and Notch promote stemness/proliferation in stem cells
- At top of crypt, high levels of BMP promotes differentiation
What is an adenoma?
- Pre-cancerous growth
- In colon adenomas, detect high levels of nuclear B-catenin in stem cells leading to increased proliferation
How are inbred/genetically engineered mice used in cancer study?
- Identify oncogenes/tumour-suppressor genes
- Identify signalling pathways that lead to specific cancer types
- Understand basic biology at a cellular level (e.g. uncontrolled stem cell self-renewal)
- Impact of diagnostics and treatment
What has been found about breast cancer treatment?
- In BC many cells respond to oestrogen and therefore treat with anti-oestrogen Herceptin to target HER2
- However, BC often returns due to breast cancer stem cells (BCSC) which are resistant to anti-oestrogens
- New rationale is to treat BC with a combination of anti-oestrogens and anti-Wnt/Notch/Shh to target BCSCs
What is linked to the progression of cancers?
Inflammatory pathways
How is tumorigenesis thought to occur following loss of APC?
- APC gene is mutated in majority of colorectal cancers and loss of APC leads to constitutively active Wnt signalling and activation of CmyC
- This leads to increased RAC1 expression which causes hyper-poliferation
- RAC1 mediates this effect by driving NFkB and ROS signalling - indicates role of inflammatory pathways in tumorigenesis
What are NFkB and ROS?
- NFkB is a pro-inflammatory factor that positively regulates the expression of pro-inflammatory genes e.g. cytokines
- Reactive Oxygen Species are key signalling molecules that are important in the progression of inflammatory disorders
What is the potential mechanism of action how inflammatory pathways promote cancer progression?
- As tumour grows, its need for oxygen and nutrients outstrips its supply
- Cancer cells secrete pro-inflammatory signals (e.g. cytokines)
- Macrophages invade tumour and secrete more cytokines to kick-start angiogenesis to increase nutrient and oxygen supply
- Inflammatory cells break down ECM to promote metastasis
