Deregulation of apoptosis in cancer

Question 1

What is apoptosis?

Answer 1

This is programmed cell death which functions in normal cell turnover, elimination cells that are superfluous or abnormals and is induced by death signals, absence of survival signals or abnormal conditions

Question 2

What are the types of caspases involved in apoptosis?

Answer 2

There are initiator caspases (2,8,9,10) which have long, regulatory prodomans and low constitutive enzymatic activity they are activated when adaptor protein (FADD, PIDD and Apaf-1) induce their oligomerisation
Effector caspases (3,6,7) have short prodomains, no constitutive activity and are activated by proteolytic cleavage by initiator caspases they will then cleave substrates leading to the dismantle of cells

Question 3

What are death receptors?

Answer 3

These are receptors which receive apoptotic signals from their ligands and include
TNFR-1
Fas
Death receptor-3
DR-4 aka TRAIL-R1
DR-5 aka TRAIL-R2

Question 4

What are decoy receptors?

Answer 4

These are receptors which bind death ligands but lack transmembrane and intracellular domains so they do not generate a death signal, this allows them to compete with and prevent the activation of death receptors
DcR4 which competes for Fas binding
TRAIL-R3 and 4 compete for TNF-like ligands

Question 5

What is the role of death receptors on both normal cells and tumour cells?

Answer 5

Normal cells may express death ligands to mediate tissue remodelling or to regulate immunity
They can also act with both anti and pro tumour functions

Question 6

How is Fas expression changed in cancers?

Answer 6

Fas, FADD and the initiator caspase may be down regulated epigenetically to suppress apoptosis it is also a p53 target so p53 knockouts will also reduce its expression
It may also undergo increased internalisation and degradation
Inhibitors of Fas such as DcR3 and FLIP (which competes with initiator caspases for binding to FADD) may be over expressed

Question 7

What is the Fas death pathway?

Answer 7

Fas engages its activating FasL ligand, this now apoptotic fas will then be palmitoylated, locating it to glycolipid-rich rafts on the plasma membrane this is followed by recruitment of the adaptor Fas-associated-death domain protein, FADD which recruits initiator caspases 8 and 10 through death effector domains these will then cleave and activate

Question 8

How can Fas have an anti-apoptotic effect?

Answer 8

FasL can signal to immune cells to stimulate anti-tumour immunity through inducing the maturation and function of dendritic cells, recruitment and activation of neutrophils and activation of both CD4 and CD8 T cells

Question 9

What are the tumour promoting features of Fas?

Answer 9

When FAS is found outside rafts it engages in various signalling pathways allowing it to be switched to an oncoegen by FLIP and mutant Ras these include
Signals via Jun-N-terminal kinase to induce proliferation
Activation of NFkappaB to promote survival
Signals in concert with tyrosine kinases and PI3K to induce MMps
Uses tyrosine kinases and phospholipase Cgamma to activate the actin severing protein cofilin allowing actin remodelling and extension of cytoplasmic protrusions and migrations

Question 10

How can FasL promote tumour growth?

Answer 10

This can recruit wound repair-type macrophages which release cytokines, growth and angiogneic factors and MMPs
It can kill activated T cells that are very sensitive to FasL induced apoptosis
Killing fo other cells such as hepatocytes which allows the spread of hepatic metastases

Question 11

How was BCL-2 discovered?

Answer 11

Follicular B cell lymphomas typically contain a translocation between chromosome 14 and 18 where the BCL-2 proto oncogene is translocated into and activated by the IGH locus
This gene was later found to extend cell survival and does not induce proliferation

Question 12

What are the anti-apoptotic members of the Bcl family?

Answer 12

Bcl-2 and Bcl-XL and other proteins have Bcl-2 homology domains 1 to 4
These proteins can heterodimerise with and inhibit the function of proapoptotic members of the family

Question 13

What are the pro-apoptotic members of the Bcl-2 family?

Answer 13

Bak and Bax which possess BH1 to BH3 domains in normal cells they exist as inactive monomers on mitochondria (Bak) or in the cytoplasm (Bax)
When activated by apoptotic stimuli they form multimers in mitochondrial membrane laeding to mitochondrial outer membrane permeabilisation

Question 14

What is the evidence showing the role of Bak and Bax acting as pro-apoptotic genes in cancer?

Answer 14

Cells from Bak/Bax-double knockout mice are resistant to many apoptotic stimuli
Bak is inactivated in 10% of colon and gastric cancers, and Bax in 50% of mismatch repair-deficient colon and gastric cancers
When MMR Bax +/- colon cancer cells were inoculated in mice the normal allele but never the mutant allele is lost during tumour growth indicating that there is selective pressure against Bax

Question 15

What are the pro-apoptotic BH3-only proteins?

Answer 15

These are a diverse group of proteins that share only a BH3 domain, this is a short amphipathic helix that engages a hydrophobic pocket (composed of BH3,1 and BH2 alpha-helical regions) in anti-apoptotic proteins

Question 16

What is the role of BH3-only proteins in stress?

Answer 16

These proteins sense many stresses
Bid is activated by caspase-8 cleavage and amplifies the death receptor pathway as well as being activated by DNA damage and caspase2
Bim induces apoptosis in response to the stress kinase JNK (which is induced by UV radiation), paclitaxel and integrin disengagement
PUMA is induced by glucocorticoids or p53 following genotoxic damage

Question 17

How do BH-3-only proteins function?

Answer 17

These can act as sensitisers engaging anti-apoptotic proteins displacing them from Bax and Bak or as activators where they directly activate proapoptosic proteins allowing Bax and Bak to oligomerise and induce MOMP

Question 18

How have BH3 only proteins lead to drug treatment options?

Answer 18

Proapoptotic low molecular weight drugs are being developed as BH3 only mimetics which will compete with pro-apoptotic members for binding to anti-apoptotic members of the BCL-2 family allowing the displaced pro-apoptotic members to induce cell death
Clinically this has been effective though does cause tumour lysis syndrome

Question 19

What is tumour lysis syndrome?

Answer 19

This is when so many tumour cells are killed that their released DNA can clog/kill and damage the kidneys

Question 20

What role does the mitochondria play in caspases activation and apoptosis?

Answer 20

The mitochondrial intermembrane space contains mediators of apoptosis and the permeability of mitochondrial membranes will then regulate apoptosis with pores being formed through two key mechanisms including formation of a megachannel or the permeability transition pore complex
Alternatively Bak or Bax homo-oligomers can directly form pores in the outer membrane

Question 21

How is the megachannel which plays a role in mitochondria driven apoptosis formed?

Answer 21

This opens in response to pro-apoptotic Bak and Bax which allows the influx of water and solutes, the loss of the mitochondrial electrochemical potential, swelling of the organelle, rupture of the outer membrane and release of apoptotic mediators
The PTPC may then be formed by the voltage dependent anion channel or adenine nucleotide transporter which is opened by atractylosiode and closed by bongkrekic acid

Question 22

What does MOMP lead to?

Answer 22

Irreversible dissipation of the mitochondria membrane potential which leads to cessation of the mitochondrial ATP synthesis
Uncoupling of the respiratory chain leading to generation of ROS which further promotes apoptosis
Release of apoptotic regulators such as cytochrome C which induces oligomerisation of the apoptotic protease-activating factor-1 (Apaf-1) with caspases-9 leading to formation of the apoptosome (caspases 9 was catalytically active without cleavage and activates caspase 3)
Release of apoptosis mediators such as nucleases that mediate large-scale cleavage of DNA