3b. Apoptosis

Question 1

What is Anoikis?

Answer 1

Anoikis is a specific type of intrinsic apoptosis which is triggered by the loss of integrin-dependent attachment to the ECM.

Question 2

What are 4 other types of cell death?

Answer 2

· Necrosis – not genetically determined and caspase-independent

· Mitotic catastrophe – failure to divide by mitosis due to chromosome damage

· Senescence – metabolically active cells that do not divide

· Autophagy – genetically programmed self-digestion which is p53 and caspase-independent

Question 3

What is p53? Explain the role of p53 in extrinsic apoptosis?

Answer 3

The tumour suppressor, p53, can activate both intrinsic mitochondrial mediated apoptosis as well as extrinsic death receptor mediated apoptosis.

In death receptor mediated apoptosis AKA extrinsic, p53 overexpression promotes Fas levels which activates Death-receptor 5 (DR5) and promotes apoptosis via caspase 8.

Question 4

Explain the role of p53 in intrinsic apoptosis?

Answer 4

Nuclear p53 activates proapoptotic genes – BAX, NOXA and PUMA.

They dissociate cytosolic p53 from its complex with BCL-XL, allowing it to induce Bax oligomerisation and mitochondrial translocation.

Mitochondrial p53 activates Bax and Bak, inactivates BCL-2 and BCL-XL, and finally complexes with cyclophilin D to cause mitochondrial permeability. Cytochrome C can thereby leak, activating caspase 9 and causing apoptosis.

Question 5

What is the effect of mutant p53?

Answer 5

Mutant p53 can activate various oncogenic and growth-promoting genes such as c-myc, multiple drug resistance gene (MDR1), EGRF, and telomerase reverse transcriptase (TERT).

Cells with mutated p53 have:

Less sensitive to DNA damage-induced apoptosis
Chromosome damage
Low oxygen (hypoxia) protection
Anoikis protection

Question 6

Why are cancer cells susceptible to anoikis?

Answer 6

Cancer cells can metastasise by performing epithelial to mesenchymal transition (EMT) wherein cells lose connection with each other and the matrix, allowing them to migrate as a single cell or cell cluster. Usually, this would lead to anoikis, but cancer cells display protection against this.

Question 7

How do cancer cells evade anoikis?

Answer 7

There are 6 mechanisms by which cells are able to evade anoikis:

1. RTK hyperactivation – ERbB2 can inhibit pro-apoptotic proteins (Bim) while responding to growth factors secreted by cancer cells to activate pro-survival signals (PI3K, Ras-ERK, Rho-GTPase)
2. Inhibition of apoptotic signalling – anti-apoptotic BCL-2 activated
3. Cytoskeletal rearrangement not recognised – epigenetic silencing of adhesion-related genes such as p66Shc from the Src family which hyperactivates Ras and inactivates RhoA
4. Insensitivity to oxidative stress – ERbB2 allows the cell to bypass the stress induced by ROS and upregulation of haem oxygenase 1 (HMOX1) increases the antioxidant response.
5. Protective autophagy – cancer cells can initiate autophagy when in unfavourable conditions which allows cells to become dormant and promotes glycolysis and cell proliferation when cells detach.
6. Activation of EMT – matrix metallopeptidases are upregulated in order to activate EMT

Question 8

What is autophagy?

Answer 8

an organism’s mechanism for disassembling and recycling, through a regulated process, unnecessary or dysfunctional cellular components.

During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome.

The autophagosome then fuses with a lysosome and the contents are degraded and recycled.

In the context of disease, autophagy has been seen as an adaptive response to stress, which promotes survival; in other cases, autophagy appears to promote cell death and morbidity. In the extreme case of starvation, the breakdown of cellular components promotes cellular survival by maintaining cellular energy levels.

Question 9

How can apoptotic pathways be exploited in cancer?

Answer 9

The intrinsic apoptosis pathway can be interfered in the following ways:

1. Antisense Oligonucleotides (ASOs) – interfere with mRNA function by binding to target sequences and increasing sensitivity to cytotoxic drugs. An anti-BCL-2 mRNA agent known as oblimersen sodium (Genasense) are being trialled in lymphoma patients.
2. Small-molecule inhibitors of BCL-2 – HDAC inhibitors such as vorinostat and romidepsin can be used against refractive cutaneous T-cell lymphoma (CTCL). BH3 mimetics can also be used mimic important domains in Bax and PUMA which are useful for treating 17p-deleted chronic lymphocytic leukaemia (CLL)
3. Small-molecule inhibitors of IAP – these mimic SMAC or interfere with XIAP mRNA by antisense-RNA.

The extrinsic apoptotic pathway can be exploited by:

1. TNSFSF10 (TRAIL) – DR4 and DR5 antibodies can be created. Targeting TNF-a or FAS was too toxic as apoptosis in hepatocytes was excessive.