16. angiogenesis Flashcards
what makes 99.9% of cancers fatal and what is this process facilitated by?
metastasis is facilitated by angiogenesis
what can be done medically to metastatic tumours?
very little
what is angiogenesis and why is it required?
the formation of new blood vessels
- it is essential for normal tissue and tumours to have good supply of oxygen and nutrients so that biosynthesis can occur
what happens as you move away from the capillary?
oxygen availability decreases very rapidly (it follows an inverse square law)
how does the blood supply in a tumour look different from that of healthy tissue?
it is disorganised and random as a pose to ordered and regular
why do tumours induce vascularisation of their environment?
so it can get good access to oxygen and nutrients
what does good vasculature allow in terms of tumour growth?
allows tumour to grow to large sizes
if we artificially induce angiogenesis in a microtumour, what is observed? what is then observed if this is then removed?
the tumour can expand
when the vasculature is removed the tumour regresses due to apoptosis and necrosis and you end up with a tumour similar in size to the initial microtumour that was limited in size
what might being able to control the vasculature of a tumour allows?
being able to control the size of the tumour and stop tumour cells getting access to factors that they need in order to proliferate
in addition to oxygen and nutrients, what does angiogenesis also provide?
an escape route for cells to migrate from
when oxygen is limited in a tissue, what occurs?
cells release factors which stimulate capillaries to sprout towards them, this is a normal physiological process
new vasculature allows the tumour to expand, what then occurs?
expansion means there are now new areas of low oxygen, this stimulates the production of further vasculature
where is Epo secreted?
from the kidneys
describe the appearance of Epo and what does it do?
its helical bundle which binds receptors on the surface of cells in BM and promotes red blood cell production
people that train and live at altitude are likely to have increased Epo production, why is this?
so that they produce more red blood cells to provide oxygen to their tissue
what happened with Epo in relation to the Tour de France?
some people had injected Epo to increase oxygen carrying capacity of the blood
it was hard to detect as injected proteins are similar to endogenous proteins
what does HIF stand for? and what is it?
Hypoxia Inducible factor
it is a heterodimeric transcription factor
HIF-1α and HIF-2α or HIF-3α and HIF-1β
what is hypoxia?
a deficiency in the amount of oxygen reaching the tissues
what is HIF sensitive to? and what does it do?
hypoxia
HIF is active at low levels of oxygen and switches on a cascade that allows angiogenesis to take place
what target sequences does HIF bind? and give an example of a gene that has one of these in their promoter
HRE target sequence
Epo
how is HIF regulated?
in normoxia, HIF is rapidly degraded
in hypoxia, HIF is stabilised
give two example experimental example that show that HIF degradation is reliant on Ubiquitin-mediated proteasomal degradation?
- proteasome inhibition stabilises HIF
- mutations in E1 stabilise HIF
what is the cancer prone disease that is characterised by highly vascularised tumours in the retina, brain and spinal chord called? and what gene has been seen to be upregulated in this disease?
Von Hippel-Lindau Syndrome
some components of HIF are upregulated
a second gene is associated with Von Hippel-Lindau Syndrome, what is it and how is it mutated in this syndrome?
VHL
disease mutations arise in the beta domain which disrupts normal interacts with HIF
VHL is an important regulator of HIF, which domain bind HIF and which domain regulates HIF?
beta domain bind HIF while the alpha domain regulates HIF
what is seen when the alpha domain is truncated from VHL?
VHL still binds HIF but does not regulate HIF in any way
which protein does the alpha domain show homology to? and what does this suggest?
skp2
this suggests that the alpha domain might mediate recruitment of ubiquitination machinery and cause HIF destruction
there is a second protein protein interaction motif in the alpha domain, name three proteins that interact with this,
eloB, EloC and cullin2
what are elongins?
there are big proteins that mediate other protein protein interactions
which domain of VHL does HIF bind?
beta domain
what is Cullen2?
E3 ubiquitin-protein ligase complexes
if mutation in VHL arise what does this lead to? and why must this mutation follow the two hit hypothesis?
HIF cannot be degraded
VHL is a classic tumour suppresser
what is the oxygen sensing enzyme that modifies HIF and what modification does it make? and what is this enzyme dependent on?
Prolyl-4-Hydrolase hydroxylates the 4 position of proline 402 and 564 in HIF1α
this enzyme depends on oxygen and α-ketoglutarate
what needs to have occurred for HIF to make interactions with VHL?
the formation of 4-hydroxyproline at two locations of HIF1α
in addition to Epo, what are some key target genes for HIF? and what links them?
bonus point for an additional unliked gene
hexokinase, phosphofructokinase and pyruvate kinase are all rate limiting enzymes in glycolysis and so their upregulation impoves the glyoltic flux
vascular endothelial growth factor (VEGF)
why is glycolysis essential in low oxygen conditions?
although the TCA cycle generate the most ATP, it is dependent on oxygen
why is improving the glycoltic flux in tumour cells good?
so that they can generate enough ATP from glucose when oxygen levels are low
apart from reduced ATP, what is a problem of relying on glycolysis?
pyruvate will build up and NADH will be depleted
how does the tumour cells prevent pyruvate build up?
it converts pyruvate to lactate by lactate dehydrogenase (another target of HIF) and secretes lactate into the environment
name the gene that is very sensitive to HIF levels
vascular endothelial growth factor (VEGF)
what are VEGFs two main functions?
- causing increased vascularisation when present in the environment
- causing increase vascular permeability
what two things does increased vascular permeability allow?
- more oxygen and nutrients can get out the vasculature
2. things like tumour cells can more easily enter the vasculature
what cells line the circulatory system and what are they sensitive to? and what function does it have?
endothelial cells are sensitive to vascular endothelial growth factor (VEGF)
this stimulated endothelial cells to proliferate and organise new vasculature
how was VEGF first purified?
it was identified as a heparin binding protein
describe the structure of VEGF
it is a homodimeric glycoprotein
name one thing that can elevate expression of VEGF?
receptor tyrosine kinases
VEGFR bind transmembrane VEGFR on cells that are sensitive to VEGF, describe VEGFR structure
- VEGFR has two immunogolobulin like domains on extracellular side and a tyrosine kinase domain on the intracellular side
- extracellular domain of VEGFR forms a dimer with a second receptor
what happens when VEGFR binds extracellular VEGFR? and what does this cause?
tyrosine kinase trans-auto-phosphorylate and this leads to activation of classical signalling pathways
this causes endothelial cells to proliferate and migrate to generate new capillaries
there are three types of VEGF with slightly different functions, name them and define their functions
VEGF-1 vascular permeability
VEGF-2 vascularisation
VEGF-3 increase lymphatic system
what is often found contaminated will cancer cells? and what is this an indication of?
the lymphatic system
this is an indication that there is cancer in the lymph nodes
describe HIF during hypoxia
HIF is not hydroxylated in hypoxia and so VHL culling complex does not bind. this leads to high levels of HIF
what can HIF bind in a low affinity interaction and how is this affected by hypoxia? and what does this induce?
HIF can bind p53 in a low affinity interaction, when HIF is stabilised during hypoxia HIF competes with MDM2 for binding of p53 at the same site
this induces an apoptotic response
what limits HIF signalling? and does this normally happen?
- the fact that if there is too high levels of HIF it will start binging p53 and induce an apoptotic response
- this does not normally happen in a hypoxia response as levels of HIF don’t build up to sufficient amounts for HIF to start binding better than MDM2
what does this limitation on HIF signalling do? but how is this overcome in some tumours?
it limits the size of the tumour
if p53 is compromised by mutations then this safe-guarding mechanism will not kick in
what happens when the vasculature in a tumour is limited? in this case what is proliferation balanced with?
they do not grow pass a certain size and so proliferation is balanced with apoptosis
what three things does HIF do?
- increases angiogenesis
- makes tumour cells fitter to survive in low oxygen environment by improving metabolic process
- in high levels HIF can stabilise p53, p53-depednent HIF degradation and growth arrest/apoptosis
what happens if p53 is mutated?
this leads to more aggressive tumour formation as HIF signalling is not limited by p53
what is avastin and what does this do?
an anti-VEGF that knocked out the extracellular function of HIF by blocking endothelial cells from responding to VEGF
how was avastin developed?
- it was initially a mouse monoclonal antibody
- antigen binding part was grafted onto human antibody
- bivalent version with an altered Fc domain
what was seen when avastin was injected into cancerous mice?
a massive suppression of tumour growth
what is seen when humans are given Avastin?
it increases lifespan on average by about 2 months with chemotherapy
why were not very impressive results seen with avastin in humans?
Avastin does not cause regression of the vasculature that has already formed, but it makes it more leaky and increases
tumours cell can enter the vasculature, and although when they reach their secondary location they cannot form new vasculature, enough cells in circulation leads to mortality
what is endostatin?
- a proteosomal protolytic product of collagen XVIII
- it circulates the body and regulates the amount of de novo angiogenesis
why endostatin better than Avastin?
it is cheaper and easier to generate than Avastin and it has a similar effects
what does endostatin do?
it inhibits endothelial cells from forming capillary structures
what is a draw back of endostatin?
it is a small peptide and so does not last very long in circulation - it is rapidly degraded in less than 6 hours
what four considerations need to me made in Pharmacokinetics?
- absorption - how it gets into body, injection/ingestion?
- distribution - where does it localise/even localisation?
- metabolism - does metabolism change its properties/deactivate it?
- excretion - is it readily excreted from the body?
how was this downside to endostatin overcome?
endostatin is conjugated with an antibody and so can be stability maintained in circulation
describe how does of endostatin affects tumour growth
- low dose of endostatin can inhibit tumour growth substantially
- doses above optimum have reduced affects
how does the vasculature of a patients tumour affect their prognosis?
patients with more highly vascularise tumours have a better prognosis. these tumour cells are less stressed and there is less selective pressure for them to escape and metastasize which leads to a worse prognosis
vasculature of tumours affects prognosis of patient, what implication does this have on anti-angiogenesis therapy?
therapies need to be quick and effective so that they kill the tumour and prevent metastasis
increased malignancy seems to be coupled with what?
increased adaption to hypoxia
