Evasion of apoptosis

Question 1

Explain the importance of apoptosis in cancer.

Answer 1

• • The balance between cell death, differentiation and apoptosis is what determines the net number of cells.
• • In cancer there is aberrant regulation of this balance.
• • Mutations in apoptosis can cause cancer as well as resistance to cancer chemotherapies.

Question 2

Define apoptosis. What induces it?

Answer 2

‘A complex program of cellular self-destruction triggered by a variety of stimuli and involving the activation of caspase enzymes that result in quick fragmentation and phagocytosis of a cell’.

Triggered by: DNA damage, oxidative stress and by some cytotoxic drugs.

Question 3

What are caspase enzymes?

Answer 3

• • Caspase enzymes – name is sort of an acronym - c stands for cysteine-rich, asp – aspartate and protease.
• • They are secreted as pro-caspases and need to cleaved at aspartate residues in order to be activated.
• • Involved in signalling – at least 12 are involved in apoptosis.
• • Caspases can also cleave each other and this leads to amplification cascade of caspases whereby one caspase is activated and then can go onto cleaving a lot of other pro-caspases.

Question 4

What are two apoptotic pathways?

Answer 4

1. Intrinsic - mediated by the mitochondria and induced by intracellular signals.
2. Extrinsic - mediated by the membrane death receptors.

Question 5

Describe the intrinsic pathway of apoptosis.

Answer 5

• • Mediated via the pro-apoptotic Bcl-2 members, e.g. Bax or Bak, are thought to initiate apoptosis by opening up channels in mitochondrial membrane.
• • Upon the release of an apoptotic signal, Bax undergoes conformational changes inserts itself into outer mitochondrial membrane.
• • This increases permeability of mitochondrial membrane leading to release of apoptotic mediators.
• • One of the mediators is cytochrome-c, which binds to Apaf-1 molecule in the cytoplasm and this complex forms apoptosome which will recruit and activate caspase 9.
• • Caspase 9 will then cleave caspase 3 and this triggers a caspase amplification cascade which continues until the final caspases cleave ‘death substrates’ which will cause cell degradation.
• • These death substrates include cytoskeleton proteins, nuclear laminins and DNAse inhibitors. They also cleave molecules in the mitochondria, causing release of ROS.

Question 6

Describe the Bcl-2 family of proteins.

Answer 6

• • Family of proteins that act at the outer mitochondrial membrane – they control flow of cytochrome-c.
• • Bcl-2 family consists of 20 members which have the Bcl-2 homology domain (BH) that mediates the protein-protein interactions. There are four BH domains.
• • Some Bcl-2 can promote apoptosis others will inhibit it – the outcome depends on ratio.

Question 7

Describe the extrinisic pathway of apoptosis.

Answer 7

• • Occurs due to extracellular signals on pro-apoptotic cell surface receptors.
• • Death receptor ligands are cytokines involved in immune system (such as activation of NK cells) and they will bind to the ‘death receptor’ – Fas on Fas receptor or TNF-alpha on TNF-alpha receptor.
• • This causes the receptor to undergo conformational changes exposing its cytoplasmic death domains called the Fas-associated death domain (FADD), which allows adaptor proteins to bind.
• • This complex is now called Death inducing signalling complex (DISC) and it recruits a lot of pro-caspase 8.
• • The DISC will trigger the pro-caspase 8 to cleave itself and start off the amplification cascade.
• • The caspases will then cleave target proteins to trigger the breakdown of the cell.
• • The extrinsic pathway can use elements of the intrinsic –> caspase 3 (activated by caspase 8) can cleave the Bcl-2 related protein Bid which will travel to mitochondria and induce cytochrome-c release.

Question 8

How does p53 induce apoptosis?

Answer 8

• • It’s a transcription factor so it will express pro-apoptotic ligands and receptors:
    1. Induces expression of the Fas receptor on the cell surface.
    2. Bax and Bak which is another Bcl-2 member – act to release cytochrome-c.
    3. PUMA, another Bcl-2 is essential for induction of apoptosis.
    4. Induces expression of IGF binding protein which will bind and sequester the pro-survival signals IGF1/2.
    5. It can also repress expression of anti-apoptic factors such as IAP.

Question 9

By what mechanisms do cancer cells evade apoptosis?

Answer 9

Cancer cells encounter a number of stressors which can induce apoptosis which is why the majority of them have to inactivate this pathway:

1. The most important is the inactivation of p53 pathway – altered in almost half all human cancers.
   a. ARF is either deleted or methylated in some cancers.
   b. Or there’s overexpression of MDM2.
   c. P53 can also be mislocalised – e.g. sequestered in cytoplasm so it cannot carry out its function as a transcription factor.
2. Suppression of pro-apoptotic proteins —>
   - - inactivation of PUMA, mutations of BAX – reported in colon cancers, loss of APAF-1.
   - - Also includes ubiquitin-mediated degradation and phosphorylation (inactivation) of these proteins.
3. Overexpression of anti-apoptotic proteins –> it has been reported in several cancers, e.g. Bcl-Xl in lung cancer and is related to poor prognosis.
4. Hyperactivation of the PI3K - Akt pathway – increases cell survival.
5. Cancer cells have upregulated IAPs – an apoptotic signal will induce release of more IAP in the cancer cell.
   a. IAP – inhibitor of apoptosis protein – can make cancerous cells if alone.
   b. Inhibit several caspases by binding to their active centres and preventing their proteolytic activity. Or they can mark them for degradation.
   c. Block both intrinsic and extrinsic pathways.
   d. But in normal cells it is affected by SMAC/Diablo – (second mitochondrial activator of caspases) released with cytochrome-c that will inhibit IAP and allows a cascade of caspases to become activated and this leads to apoptosis occurring.