8th March - Endogenous inhibitors of angiogenesis Flashcards
What was the first endogenous negative regulator of angiogenesis to be discovered?
Angiostatin
What are the different effects of angiostatin at the primary tumour and at a distant site?
Angiostatin –> short half lives - bFGF and VEGF, long half lives - inhibitors
At primary tumour site - positive stimulus > negative stimulus
At secondary tumour site - negative stimulus > positive stimulus
What is the lewis lung tumour model?
that the primary subcutenous tumour –| lung metastases growth to less than 200µm diameter
Removal of the primary tumour led to rapid lung metastatic growth as they became angiogenic
–> Tumour is suppressing metastases
Serum/urine from tumour bearing mice –| metastatic growth therefore the inhibitor is present in either serum or urine of the tumour bearing mouse
Serum/urine of tumour bearing mouse –| capillary endothelial cell proliferation in culture
How was angiostatin purified?
18l urine from tumour bearing mice –> dialysis –> heparin-sepharose chromatography –> bio-gel A 0.5 m, agarose chromatography –> HPLC/C4 reverse phase chromatography –> 1327X purifcation
What is angiostatin?
A 38kDa fragment of plasminogen
How was the function of angiostatin verified?
It was injected into mice which had their initial tumour removed, found it specifically inhibits growing vascular endothelial cells
Inhibited primary tumour growth by up to 98%
Caused regression of large tumours
Is angiostatin secreted by the tumour?
No the tumour releases uPA and MMp which cleave plasminogen to form angiostatin
What is the structure of plasminogen?
5 kringle domains held together by disulphide bonds
K1-K3 is sufficient to provide the anti-angiogenic nature of angiostatin
K5 is the plasmin catalytic domain
What is angiostatins proposed mechanism of action?
Inhibits F1Fo ATPase activity which is required for endothelial cell survival under pH stress inherent int he tumour microenvironment
Binds to angiomotin which inhibits endothelial cell migration, invasion and morphogenesis
Binds to alphavbeta3 integrin which antagonizes the proliferative, migratory and pro-survival signals mediated by integrin-matrix interaction
Binds to c-Met decreases activation of PI3K/Akt leading to release of suppression of apoptosis and blocking of cell cycle progression into S-phase
What are the basement membrane derived inhibitors of angiogenesis?
Endostatin Restin Arrestin Canstatin Tumstatin alpha6 NCI domain Endorepellin
What are the receptors for endostatin?
alpha 5 beta 1 integrin, VEGFR2, glycipin
What is the receptor for arrestin?
alpha 1 beta1 integrin
What is the receptor for tumstatin?
alphavbeta3 integrin
What is the receptor for endorepellin?
alpha 2 beta 1 integrin
Who discovered endostatin in 1997?
O’reilly
What is endostatin?
A 20kDa fragment of collagen XVII which is a cryptic angiogenesis inhibitor
What happens when endostatin is applied to a tumour?
It increases the apoptosis rate 7x and has no effect on proliferation
What is the function of glypican?
It binds endostatin to the endothelial cell surface
What are the molecular responses to endostatin?
Inhibition of VEGF121, VEGF165, VEGFR2 –> reduced endothelial cell survival and proliferation
Inhibition of MMPs –> reduced endothelial cell invasion
Inhibition of VEGF driven relocalisation of Beta catenin from cell-cell junctions –> Maintains endothelial cell-cell adhesion
Binds alpha5 beta1 integrin preventing integrin dependent epithelial cell migration
Downregulates cyclin D2 –> G1 arrest in endothelial cells
Inhibits PDGF mediated recruitment of peri-vascular cells
Describe the use of endostatin in cancer therapy
Daily administration of endostatin suppressed lewis lung primary tumour growth
As endostatin is aimed at genomically stable endothelial cells they are less likely to develop resistance by mutation
What is thrombospondin?
A 140kDa fragment of TSp-1 which inhibits angiogenesis
How is thrombospondin expression related to angiogenic activity?
It is inversely proportional
How is thrombospondin expression regulated?
By WT p53 in fibroblasts and mammary epithelial cells
What effect does a loss of p53 have on thrombospondin levels?
Dramatic decrease in TSP-1
What is the function of type 1 repeats in thrombospondin?
Inhibits angiogenesis
Induces apoptosis
Binds CD36
ANTI-ANGIOGENIC ACTIVITY IS RESTRICTED TO TYPE I REPEATS
Describe the findings of Jiminez (2000)
Performed a corneal pocket assay in a mouse
Found that signalling molecules induces by TSP-1 in microvascular endothelial cells were essential for angiogenesis inhibition. This is dependent on CD36, p59 Fyn, caspase 3 and p38.
What is the mechanism of TSP1?
- TSP1 binds to CD36 receptor on neighbouring endothelial cell
- Activates p59 fyn
- Activates p38, JNK
- Activates transcription
- Transmitted through Fas receptor to neighbouring cell
- Activates caspases
- Triggers cell death
What are the advantages of targeting angiogenesis in cancer therapy?
Endothelial cells are genetically stable therefore less chance of resistance developing
Not restricted to certain histological tumours
Tumour microvasculature is easily accessible
In adult organisms angiogenesis is only induced under certain conditions
What are the processes of angiogenesis vulnerable to antagonists?
VEGF antagonists –> VEGFR
ECM degradation, adhesion antagonists –> alphavbeta3 and MMPs
Endothelial cell antagonists –> Angiostatin, endostatin, thromobospondin-1
What are the currently FDA approved drugs which target angiogenesis?
Avastin (Anti-VEGF Ab)
Sunitib (Blocks VEGFR signalling)
Pazopinib (Blocks VEGFR signalling)
Sorafenib (Blocks VEGFR signalling)
Outline the animal trial of Sunitib?
Sunitib (SU11248) in Rat C6 glioma cells implanted in the colon of nude mice. Animals were treated with SU5416 or DMSO for 16 days. Day 16 tumours were quantified. 73% decrease in tumour volume
Refined structure –> Sunitib (SU11248)
What is pazopanib used to treat?
metastatic renal cell carcinoma
What is sorafenib used to treat?
Hepatocellular cancer and metastaic renal cell carcinoma
What are the drugs in clinical trials that block activators of angiogenesis?
IgG fusion protein - VEGF-Trap
Small molecule inhibitors of VEGFR1 and VEGFR2
Small molecule inhibitors targeting Tie-2
What is Marimastat?
A synthetic inhibitor of MMPs
What is a drug in clinical trials that inhibits angiogenesis?
Endostatin
