Chapter 10: Angiogenesis (Book, main)

Question 1

Why do tumor cells need angiogenesis?

Answer 1

All cells need to be within 100-200 µm of a blood vessel (this is the diffusion limit of oxygen) in order to receive essential oxygen and nutrients. Cells within the core of a tumor that do not receive sufficient oxygen and nutrients die by necrosis, which is why new blood vessels are needed

Question 2

The neovasculature that is formed is unlike that formed ‘naturally’ (e.g. in wound healing or female reproductive cycle). How is it different?

Answer 2

It is leaky and tortuous and provides direct entry, allowing cells easy access tot he circulation. The neovasculature is also different at the molecular level from resting endothelium

Question 3

The angiogenic switch is a dynamic balance of angiogenic inducers and inhibitors. Can you name a couple anti-antiogenic and pro-angiogenic factors?

Answer 3

Anti-angiogenic factors

• Angiostatin
• Endostatin
• Prolactin
• p53
• Thrombospondin-1 , -2

Pro-angiogenic factors

• Vascular endothelial growth factor (VEGF)
• Fibroblast growth factor (FGF)
• Hepatocyte-derived growth factor (HGF)
• Epidermal growth factor (EGF)
• Platelet-derived growth factor (PDGF)

Question 4

True/false: Growth factors can be both non-specific, as well as endothelial-specific

Answer 4

True. Non-specific are e.g. EGF, FGF, HGF, PDGF and endothelial specific is VEGF

Question 5

VEGF is the star player involved in the initiation of angiogenesis. The VEGF family consists of 5 family members and 3 VEGF tyrosine kinases. Which combination is responsible for the majority of antiogenic effects?

Answer 5

VEGF-A with its receptor VEGFR-2

Question 6

True/false: Tumor cells cannot affect the surrounding stromal cells to induce the VEGF-promoter

Answer 6

False, they can in fact do this.
E.g. a subset of tumor-infiltrating immune cell shave been shown to adopt a pro-angiogenic phenotype and produce high levels of VEGF

Question 7

What function does each VEGFR receptor have?

Answer 7

1. VEGFR-1: inhibitory effects of VEGF
2. VEGFR-2: mediates the endothelial effects of VEGF
3. VEGFR-3: vital for lymphatic vessels

Question 8

In what two ways is the angiogenic switch regulated during tumorigenesis?

Answer 8

• First, as the tumor grows, it creates conditions of hypoxia, which induces angiogenesis via the hypoxia-inducible factor-1a (HIF-1a) that is regulated by oxygen concentration. One target of HIF-1a is the VEGF gene.
• Secondly, the angiogenic swtich can be modified by oncogenic proteins and loss of tumor suppressors. 30 oncoproteins have been shown to tip the balance towards angiogenesis

Question 9

Explain the function of HIF-1a in Normoxic and Hypoxic conditions

HIF = hypoxia-inducible factor

Answer 9

• Under normoxic conditions (20% oxygen), HIF-1a is rapidly degraded. The von Hippel-Lindau (VHL) tumor suppressor protein is an important regulator of HIF-1a degradation. The first step in targeting hIF-1a for degradation under normoxic conditions is modification (hydroxylation) by the enzyme prolyl 4-hydroxylase. This enzyme binds direcly and links molecular oxygen to specific proline residues on HIF-1a, and thus acts as a direct oxygen sensor in this pathway. VHL binds to hydroxylated HIF-1a and activates a complex of proteins for ubiquitination (proteosomal degradation). HIF target genes cannot be transcriptionally activated, angiogenesis does not occus
• Under hypoxic conditions, the enzyme prolyl 4-hdyroxylase in inactivated, HIF-1a is not hydroxylated, and VHL cannot bind and target HIF-1a for proteosomal degradation. HIF-1a is rapidly satbilized and transported to the nucleus to target genes (most notable target is the VEGF gene)

Question 10

How can HIF-1 activity be increased?

Answer 10

By oncogene and tumor suppressor gene products

Question 11

What happens in response to angiogenic inducing signals (which cells/proteins are activated)?

Answer 11

Endothelial cells extend filopodia and migrate towards the signal. At the location of the highest concentration of VEGF-A, VEGFR-2 is activated. The signal is enhanced by co-receptor neurophilin-1 (Nrp1) and is transducted via the MAPK cascade. This stimulates the formation of a tip cell at the forefront of the sprout

Question 12

In response to angiogenic inducing signals, there is formation of a tip cell at the forefront of the sprout. What steps are then taken so that a new vessel is formed where blood can flow through?

Answer 12

• Behind the tip cell are proliferating stalk cells that extend the sprouting blood vessel. The phenotype of the two cell types is not fixed and depends on a competition for VEGFR-2 activation that is regulated by Notch.
• In brief, upon VEGFR-2 activation, tip cells induce the expression and release of the Notch ligand, Delta-like 4 (DLL4).
• DLL4 binds to the Notch receptor on neighboring cells. The Notch intracellular domain NICD is released and transported to the nucleus where it represses VEGFR-2 gene expression and induces VEGFR-1 expression (which acts as a VEGF trap and reduces VEGF concentration).
• The growing sprout moves along a VEGF gradient. When two tip cells meet, they fuse and allow for a connected lumen, allowing blood to flow through the new vessel.

Question 13

What else, besides angiogenesis, contributes to the formation of tumor vessels?

Answer 13

Vasculogenic mimcry and vasculogenesis

Question 14

What is vasculogenic mimicry?

Answer 14

Vasculogenic mimicry desribes the process whereby tumor cells (e.g. melanoma cells, see the gray circules) act as endothelial cells and form vascular-like structures (see red arrow in b)

Question 15

What is vasculogenesis?

Answer 15

Vasculogenesis involves the differentiation and proliferation of endothelial cells from endothelial progenitor cells

For those who are now confused, the difference between vasculogenesis and angiogenesis:

Vasculogenesis occurs during the very early developmental stages of an organism when the blood vessel pathways are created. Angiogenesis, while a similar process, does not depend on the same genes for activation as vasculogenesis and occurs instead in the presence of an injury to a blood vessel, such as a cut or the slight damage done to the ovary post-ovulation. Angiogenesis is a remodeling process only, while vasculogenesis creates the blood vessels themselves.

Question 16

Explain how vasculogenesis can result in tumor neovasculature by circulating endothelial progenitor (CEPs) cells.

Answer 16

Circulating endothelial progenitors have shown to be derived in bone marrow in 40% of tumor endothelial cells. Angiogeneic factors from the tumor, such as VEGF, are involved in the recruitment of these cells that express VEGFR-2. After reaching the tumor, CEPs differentiate and contribute to the tumor neovasculature. See figure c

It seems likely that different cancer may differ in thier requirement for CEP contributions to the new tumor vasculatore. It is known that they are necessary for lymphomas and colon cancer

Question 17

In what two ways are therapeutic strategies designed for angiogenesis?

Answer 17

Therapies aimed at the tumor vasculature designed either to halt the angiogenic process (anti-angiogenic drugs) or to destroy the existing new tumor vasculature (vascular targeting) have been developed.

Question 18

What is the aim of anti-angiogenic therapy and roughly explain how it works?

Answer 18

Anti-angiogenic therapy is designed to prevent the formation of new blood vessels. Rather than target the tumor cells directly, the aim of anti-angiogenic therapy is to interfere with the responsiveness of normal endothelial cells that is essential to the survival of the tumor

Drugs may be designed to prevent the cells from responding to pro-angiogenic signals or may be targeted to block the activity of the inducers.

Question 19

How do anti-angiogenic therapies (and vascular targeting, discussed later) differ from the therapies we have seen throughout this course?

Answer 19

First, as angiogenesis only occurs on occasion in the adult, drugs that inhibit it are predicted to cause minimal side effects. More importantly, the target endothelial cells recruited during angiogenesis are genetically stable, unlike the tumor cells that have accumulated mutations, and are therefore less likely to develop drug resistance rapidly

Question 20

Several strategies can be used to block VEGF signaling in order to prevent angiogenesis. What are these strageties?

Answer 20

Both ligands (VEGF) and receptors (VEGFR) may be good targets, and both antibodies and small-molecule inhibitors may be employed.

• Therapeutic agents are shown in red, cellular targets are indicated by the ‘dot’-symbol. The TKIs shown inhibit multiple targets, including VEGFRs and PDGFRs.*
• SUS416 is no longer in clinical use*

Question 21

True/false: Inhibition of the VEGF/VEGFR pathway causes a rapid and sustained response

Answer 21

False! This was what was expected since VEGF/VEGFR is the ‘star player’. However, although some improvements in prognosis is seen, responses are often transient, stop after treatment is stoped, and are not as effecting (prolonging survival only in the order of months) as was hoped. Broader approaches are needed

Question 22

As stated earlier, only inhibiting the VEGF/VEGFR pathway are not as effective as we thought it would be, and broader approaches are needed. Can you name some examples of strategies for other angiogenic regulators?

Answer 22

Similar strategies that are used to target the VEGF/VEGFR pathway can be used for other angiotenic regulators

• Trebanamib is similar to alflibercept, and binds to angiopoietin-1 and -2, ligands of the Tie2 receptor (and has entered phase III trials)
• Recombinant human endogenous inhibitor held potential promise, but did not deliver the expected results because there was no response
• Antagonists to integrins avß3 and avß5 would block endothelial integrin-ECM interactions and specifically induce apoptosis of angiogenic vessels with little effect on mature vessels. Two integrins were developed, but research has been stopped
• There are several approved HIF pathway inhibitors, but no direct HIF inhibitors

Don’t learn this by heart, try to understand roughly understand how it works

Question 23

Anti-angiogenic effects may be “side effects” of other cancer therapies. Can you further elaborate on this?

Answer 23

Cancer therapies targeted at oncogene products often affect angiogenesis.

E.g. Herceptin has shown to be anti-angiogenic by inhibiting the production of angiogenic inducers (TGF-a and angiopoietin-1) by tumor cells and upregulating angiogenic inhibitors (e.g. thrombospondin). These anti-angiogenic effects has also been shown in MTD

Question 24

Besides anti-angiogenic drugs, another way to target tumor vasculature was by destroying the existing new tumor vasculature (vascular targeting). Can you shortly explain this?

Answer 24

Vasculature targeting is a therapeutic approach designed to destroy the exisitng neovasculature in the tumor in order to starve it of oxygen and nutrients and lead to tumor regression. This approach is possible owing to the identification of molecular differences between tumor and normal vasculature

Question 25

What was the first vascular targeting drug and how does it work?

Answer 25

Combretastatin is selectively toxic to neovasculature, and combretastatin and recently developed derivatives are being tested in clinical trials. Combretastatin bind tubulin, cause depolymerization and disrupt the cytosceleton. Their effects have been explained by the hypohtesis that immature endothelium may have a more intrinsic need for a tubulin cytoskeleton to maintain its shape than stable mature vasculature, which is firmly supported by a basemement membrane. Loss of shape and rounding up of the endothelial cells in new blood vessels block blood flow and/or lead to vascular collapse, therby depriving the tumor of oxyten and nutrients. Necrosis occurs.