Tumour angiogenesis

Question 1

What are the characteristics of malignant tumours?

Answer 1

Growth

• Unlimited growth (not self-limited as in benign tumours) as long as an adequate blood supply is available

Invasiveness

• Migration of tumour cells into the surrounding stroma where they are free to disseminate via vascular or lymphatic channels to distant organs

Metastasis

• Spread of tumour cells from the primary site to form secondary tumours at other sites in the body

Question 2

What are the four key steps in cancer progression?

Answer 2

Transformation: extensive mutagenic and epigenetic changes followed by clonal selection - small tumour growing that has been transformed from a single cell

Angiogenesis: new blood vessel formation (overcomes limitations imposed by hypoxia and tumour receives nutrients) - starts to grow and expand further

Motility and invasion: epithelial to mesenchymal transition (invasive properties allowing intravasation into circulation and extravasation from circulation to tissues) -

Metastasis: colonisation of target organs (ability to expand from micrometastases)

Question 3

What is angiogenesis?

Answer 3

The formation of new blood vessels from pre-existing vessels

Vasculogenesis is where a primitive vessel of networks is forming from progenitor cells, that then form a capillary network.

Vasculogenesis is the formation of new blood vessels from progenitors.

Question 4

What are the three types of angiogenesis?

Answer 4

Developmental / vasculogenesis – organ growth, developing embryo has no existing network of vessels so vessels are derived from progenitors

Normal angiogenesis – wound repair, placenta during pregnancy, cycling ovary

Pathological angiogenesis - tumour angiogenesis, ocular disorders and inflammatory disorders

Question 5

What size do tumours grow up to before they need a blood supply?

Answer 5

Tumours don’t grow beyond 1-2mm^3 without their own blood supply, need blood supply to receive nutrients and oxygen.

Tumours initiate process to acquire new blood vessels from pre-existing capillaries in that tissue - neovascularisation.

Question 6

Describe the three stages of tumour angiogenesis

Answer 6

A. Small tumour risen from clonal cell and has proliferated. Oxygen and nutrients have become limited.

B.

Angiogenic switch within the tumour where a stimulus will allow the tumour to start upregulating angiogenic factors. These are cytokines and proteins that allow for new blood vessel growth. Growth factors acting on endothelial cells of blood vessel to allow vessels to form new sprout, sprouting angiogenesis.

Also chemotactic factors that allow for the cells within the blood vessels to grow towards the tumour.

Angiogenic and chemotactic factors

C.

New network starts to grow around the tumour, tumour receives more oxygen and nutrients and can start to grow and spread more.

Cells start to shed off, become more motile and invasive.

Vessels are also a root of metastatic spread through the new network of blood vessels and into the general circulatory/systemic circulatory system.

Question 7

What is hypoxia? What is hypoxia a stimulus for?

Answer 7

Hypoxia is low oxygen tension <1% in oxygen

Hypoxia is a strong stimulus for tumour angiogenesis, the switch that causes the increases in growth factors released from the tumour

Hypoxia increases with distance from capillaries

Question 8

What growth factors are released after hypoxia?

Answer 8

VEGF, GLUT-1, u-PAR, PAL-1

factors act as stimulus, endothelial cells of blood vessels begin to form new blood vessels, start sprouting from capillary.

Activates transcription of genes involved in angiogenesis, tumour cell migration and metastasis

GLUT-1 involved in glucose uptake.

If oxygen deprived you need to get energy from non-oxidative phosphorylation, here GLUT-1 is important.

Other genes that are switched on are involved in invasion and metastases.

Question 9

What angiogenic factors do tumour cells produce?

What enzyme releases these components from extracellular matrix?

Answer 9

1.

Vascular endothelial growth factors (VEGF)

Fibroblast growth factor 2 (FGF 2)

Placental growth factor (PIGF)

Angiopoietin 2 (Ang 2)

2.

Matrix metalloproteinase 2 (MMP-2)

Question 10

What biological processes are involvd in sprouting angiogenesis?

Answer 10

Proliferation, migration, invasion.

Endothelial cells lining the blood vessels need to be able to migrate into extracellular matrix.

Question 11

What is the process of vascular endothelial growth factor signalling?

Answer 11

VEGF binds to VEGF-R2 on endothelial cells

VEGF/VEGF R2 dimerises at the plasma membrane and recruits cofactors – cofactors involved in activating receptor tyrosine kinase

Subsequently activate 3 major signal transduction pathways that increase angiogenesis. Activated by phosphorylation.

Ultimately VEGF activates cell survival, vascular permeability, gene expression and cell proliferation

All of these pathways are essential for angiogenesis

Question 12

What are the three pathways VEGF signalling can activate?

Answer 12

1. Phospholipase C pathway with PIP2, activates diacylglycerol which activates PKC to increase cell proliferation and permeability.

IP3 increases calcium release, calcium activates nitric oxide synthase to induce nitric oxide to stimulate vasodilation and angiogenesis.

3. PI3 kinase pathway, forms PKB. Important for cell survival.

VEGF activates this pathway, decrease in cell apoptosis, there is cell survival instead, important for new blood vessel growth.

3. Ras Ref MEK pathway, MAP kinase pathway, substrates phosphorylated and substrate downstream mediators to increase gene expression and cell proliferation.

Question 13

What is epithelial-mesenchymal transition?

Answer 13

EMT, phenotypic switch of cells, of a normal epithelial cell acquiring mesenchymal type characterisitcs which are chracterised by invasive and motile properties

Question 14

What do epithelial cells lose in EMT?

Answer 14

Epithelial shape and cell polarity (due to downregulation of B-catenin, claudin-1 which maintain epithelial cell shape)

Cytokeratin intermediate filament expression, allows epithelial structure

Epithelial adherens junction protein (E-cadherin)

Question 15

What do epithelial acquire during EMT?

Answer 15

Fibroblast like shape and motility - elongated with cellular extensions that allow it to spread out, motile so move and invade.

Invasiveness

Vimentin intermediate filament expression

Mesenchymal gene expression ( proteins important for fibroblast motility fibronectin, PDGF receptor, avB6 integrin)

Protease secretion (enzymes that allow cells to move around and invade MMP-2, MMP-9)

Question 16

Where are E-cadherins expressed?

What are they?

Answer 16

In epithelial cells.

Homotypic adhesion molecule (adhesion of cells with same cadherin) - intra and extracellular domain, allow binding to actin and catenin

Calcium dependent

Inhibits invasiveness

Binds B-catenin

Question 17

What does E-cadherin do in normal cells?

Answer 17

Cells sititng in monolayer with high density, cell binds to another cell through adhesion molecule

signal for them to not migrate or invade - contact inhibition

at low density cells proliferate until they reach high density, contact inhibition prevents cells proliferating more

Question 18

What happens in cancer cell where there is loss in E-cadherin?

Answer 18

Loss of E-cadherin due to downregulation

contact inhibition deregulated, loss of cell to cell adhesion

no longer a monolayer, cells continue to proliferate and grow ontop of each other

forms tumour mass

cells more motile

Question 19

How do stromal cells contribute to tumour progression?

What factors do they release?

Answer 19

Factors released by stromal cells (macrophages, mast cells, fibroblasts) include angiogenic factors, growth factors, cytokines, proteases

E.g: urokinase-type plasminogen activator (uPA); activated by tumour cells – resulting in plasmin production.

Pro-uPA binds to uPA receptor on tumour cells, converted to the active form of uPA.

uPA can activate plasminogen in extracellular matrix to plasmin

Plasmin, a protease, activates other downstream enzymes.

activates matrix metalloproteinases (MMPs) (pro-MMP to MMP),

permit invasion by degrading ECM and releasing matrix-bound angiogenic factors such as transforming growth factor-B1 (TGF-B1)

Allows tumour cell to move into the environment.

Plasmin also activate growth factors that are latent, inactive in extracellular matrix, such as TGF-B1 activated to active form of it. Increases invasion of tumour cells.

Question 20

What are the 8 steps in cancer dissemination?

Answer 20

1. Primary tumour formation, angiogenesis occurring
2. Localised invasion into blood system
3. Intravasation – interaction with platelets, lymphocytes and other blood components
4. Transport through circulation
5. Arrest in micro vessels of various organs
6. Cell comes out of blood vessel, extravastion
7. Formation of micrometastasis
8. Colonisation – formation of macro metastasis

process inefficient, <0.02% cells actually for micrometastasis

Question 21

Where do common cancers metastasise?

Answer 21

Breast cancer metastasise to liver, lung, brain and bone.

Colorectal cancer metastasise to liver and lung

Stomach cancer to esophagus, liver and lung

Lung cancer goes to brain , liver and adrenal glands

Pancreatic to lung and liver

Prostate to bone

Question 22

What can be targeted to inhibit cancer?

Answer 22

Tumor angiogenesis, targeted therapy to angiogenic factors like VEGF.

targetting cell motility (cell-cell adhesion molecules) and invasion (matrix metalloproteinases) unsuccessful.

Question 23

What is the angiogenesis hypothesis, 1971?

Answer 23

Tumour growth dependent on new blood vessel growth.

If tumour could be held in non-vascularised dominant state it is possible that metastases won’t arise - target vasculature of tumour instead of tumour itself.

Both tumour and microvascular compartment valid therapeutic targets, work went into identifying GF involved and drugs to inhibit VEGF.

Question 24

What is avastin?

Answer 24

First specific anti-angiogenesis drug

approved for colorectal, lung, kidney and ovarian cancers and eye diseases

Question 25

What is the mechanism of avastin/bevacizumab?

Answer 25

An anti VEGF antibody.

Binds to VEGF secreted by tumour cell

mops it up, preventing the protein from binding to its ligand binding site on the receptor of endothelial cells.

no dimerization or downstream phosphorylation, and pathways linked to the receptor.

VEGF signal transmission is inhibited.

decrease in angiogenesis and progression of metastasis and survival of the tumour.

Drug reduces tumour burden.