CBIO 3.2: Apoptosis

Question 1

Define apoptosis

Answer 1

• apoptosis is programmed cell death where the cell is responding to defects that cannot be repaired, like extensive DNA damage
• preventing the damaged cell from dividing and replicating the error
• external signals can also trigger apoptosis

Question 2

What triggers apoptosis?

Answer 2

• apoptosis is triggered by the activation of a cascade of proteolytic enzymes, the caspases
• which results in the cell being progressively broken down and consumed by other cells

Question 3

Give an example of one of the most important apoptosis-triggering proteins

What happens when it is lost or defective?

Answer 3

• the p53 tumour suppressor
• it identifies irreparable DNA damage and signals to the cells to undergo apoptosis
• if p53 is lost or defective, apoptosis may be avoided by the cells
• when the cell subsequently replicates, not only will the original defect be replicated, the cells will be at increased risk of further DNA errors and will accumulate such damage

Question 4

What changes do cells undergo upon activation of apoptosis?

Answer 4

• cell shrinkage
• nuclear condensation and fragmentation
• surface expression of membrane phospholipid: phosphatidylserine
• membrane blebbing
• mitochondrial depolarisation
• DNA fragmentation
• formation of apoptotic bodies

Question 5

What are the two major apoptosis pathways?

Answer 5

• extrinsic apoptosis
• intrinsic apoptosis

Question 6

What initiates and drives extrinsic apoptosis?

Answer 6

• extrinsic apoptosis is initiated by the extracellular microenvironment
• and is driven by plasma membrane receptors: death receptors

Question 7

Describe the induction of extrinsic apoptosis

• more specifically use the FAS receptor pathway

Answer 7

Surface receptor interaction with the death ligand leads to an assembly of a dynamic multiprotein complex at the intracellular tail of the receptor, death-inducing signalling complex (DISC).

• The death receptors contain a cytoplasmic domain called “death domain” (DD).
• DD plays critical role in transmitting the death signal from the cell surface to the intracellular signalling pathways.
• During FasL and FAS binding, FADD adaptor proteins (Fas-associated protein with death domain) are recruited (see figure for consecutive steps of this process)
• and during TNF ligand and TNFR binding a TNFR1-associated death domain protein (TRADD) is recruited together with FADD.
• Adaptor proteins exhibit appropriate death domains to bind to their corresponding receptor, and they then recruit procaspase-8 via dimerisation of another domain, the the death effector domain (DED).
• Now, DISC is formed and caspase-8 is cleaved and activated.
• Active caspase-8 initiates apoptosis by cleaving and activating executioner caspase-3.

Question 8

Describe intrinsic apoptosis

• initiated by?
• cell morphology during apoptosis
• controlled by what regulator proteins?

Answer 8

• Intrinsic apoptosis (mitochondrial pathway) is initiated by microenvironmental triggers, like DNA damage or withdrawal of growth factors.
• The apoptotic cells retain their plasma membrane integrity and metabolic activity during the apoptotic process thanks to the clearance by macrophages and other phagocytic cells (in a process called efferocytosis).
• However, at the end stage, apoptotic cells can have a complete breakdown of the plasma membrane and acquire a necrotic morphotype (secondary necrosis).
• Intrinsic apoptosis is irreversible and is controlled by pro-apoptotic and anti-apoptotic members of the BCL2 family of apoptosis regulator proteins

Question 9

Outline the steps of intrinsic apoptosis

Answer 9

• The first step is a widespread mitochondrial outer membrane permeabilisation (MOMP) mediated by BCL2-associated X, apoptosis regulator (BAX) and/or BCL2 antagonist/killer (BAK).
• In cells, BAX continuously travels between the outer mitochondrial membrane and the cytosol in its inactive form.
• BAK is located within the outer mitochondrial membrane.
• During induction of apoptosis, BAX no longer retranslocates, and BAX and BAK are directly or indirectly activated by pro-apoptotic proteins, including BID and BAD.
• The apoptogenic factors are released, including cytochrome C (can you see it in the figure above?) and Second Mitochondrial Activator of Caspases (SMAC).
• Cytochrome C binds to Apoptotic Peptidase Activating Factor 1 (APAF1) and pro-caspase 9 to form the supramolecular complex apoptosome, which is responsible for activation of caspase 9.
• Activation of caspase 9 catalyses the proteolytic activation of the executioner caspase 3.
• SMAC regulates apoptosis by associating with the inhibitor of apoptosis (IAP) protein family.
• Once executioner caspases are active, the morphological and biochemical changes occur, including DNA fragmentation, phosphatidylserine (PS) exposure, and the formation of apoptotic bodies.
• DNA fragmentation occurs by caspase 3 catalysing the proteolytic inactivation of DNA fragmentation factor subunit alpha (also known as ICAD - Inhibitor of CAD) and releasing the catalytic activity of CAD (Caspase-Activated DNAse ).

Question 10

What is anoikis?

Answer 10

• a specific variant of intrinsic apoptosis
• is initiated by the loss of integrin-dependent attachment to the extracellular matrix

Question 11

Briefly define the other mechanisms of cell death

• apoptosis
• necrosis
• mitotic catastrophe
• senescence
• autophagy

Answer 11

• apoptosis: genetically programmed, caspase-dependent
• necrosis: not genetically determined, caspase-independent
• mitotic catastrophe: failure of cells to undergo mitosis due to chromosome damage
• senescence: metabolically active but unable to divide
• autophagy: genetically programmed self-digestion, caspase and p53 independent

Question 12

What is p53?

What is its role in apoptosis?

Answer 12

• p53 mediate apoptosis activation
• it is a transcription factor that governs anti-proliferative cell programmes
• cell-cycle arrest
• senescence
• apoptosis
• through these programmes, p53 facilitates repair and survival of damaged cells or eliminates severely injured cells

Question 13

What pathways do cells undergoing apoptosis via the p53-dependent mechanism follow?

Answer 13

• both the cytosolic and the mitochondrial pathway
• but mostly the mitochondrial pathway

Question 14

Describe the role of p53 in the cytosolic p53 pathway

Answer 14

• In the cytosolic p53 pathway, nuclear p53 induces activation of proapoptotic genes, including members of the Bcl-2 family, like Bax, NOXA, and PUMA.
• This releases cytosolic p53 from the inactive complex with Bcl-Xl, and allows it to induce Bax oligomerisation and mitochondrial translocation.
• Inside the mitochondria, p53 promotes Bax and Bak activation and blocks anti-apoptotic Bcl-2 and Bcl-XL. p53 also complexes with cyclophilin D, a protein responsible for mitochondrial permeability.
• The result is disruption of the mitochondrial membrane and release of cytochrome c (along with other apoptogenic factors), which consequently activates caspase 9.

Question 15

Describe the role of p53 in death receptor-mediated apoptosis

Answer 15

• p53 overexpression promoted Fas levels, activates DR5 (Death Receptor 5) and promotes cell death through caspase-8

Question 16

Describe mutant p53

• encoded by which gene
• tumour suppressor or oncogene
• effects on other genes

Answer 16

• p53 is encoded by TP53, which is the most frequently mutated gene in human cancers
• with over 50% of human cancers carrying loss of function mutations in TP53 genes
• While wild-type p53 is a classic tumour suppressor, mutant forms of p53 can act as oncogenes, by inhibiting wild-type p53 in a dominant-negative manner.
• Cancer-derived mutant forms of p53 can activate various growth-promoting and oncogenic genes, including
• c-MYC
• multiple drug resistance gene 1 (MDR1)
• epidermal growth factor receptor (EGFR)
• telomerase reverse transcriptase (TERT).

Question 17

What characteristics do cells with mutated p53 have?

Answer 17

• less sensitive to DNA damage-induced apoptosis
• chromosome damage
• low oxygen (hypoxia) protection
• anoikis protection

Question 18

Give examples of other anti-apoptotic alterations that can occur in cancers, other than p53 mutation

Answer 18

• P14ARF inactivation
• MDM2 overexpression
• BAX mutation
• BCL2 overexpression
• NFkB activation
• PTEN inactivation
• IGF-1/2 overexpression
• FLIP overexpression
• FAP-1 overexpression

Question 19

How do cancer cells evade anoikis?

Answer 19

• As you know from other e-modules, cancer cells have the ability to undergo epithelial to mesenchymal transition (EMT) and to travel to other organs to form metastases.
• During EMT, cells lose connection between each other and with the matrix, so are able to migrate as a single cell or cell cluster.
• In normal cells, loss of attachment to the extracellular matrix would result in anoikis, however cancer cells evade this death process
• there are several mechanism that allow cancer cells to evade anoikis which will be outlined later

Question 20

Describe the different mechanisms that cancer cells acquire to be able to evade anoikis

• note the key proteins responsible

Answer 20

1. Hyperactivation of receptor tyrosine kinases (RTK):
   - cancer cells secrete growth factors that constitutively activate pro-survival signals, including PI3K, Ras-Erk, or Rho GTPase
   - overexpression of RTK – ERBB2, which inhibits pro-apoptotic protein Bim, while maintaining EGFR expression
2. Apoptotic Signalling:
   - cancer cells activate anti-apoptotic Bcl-2 proteins
3. Cytoskeletal rearrangement sensing:
   - cancer cells undergo epigenetic silencing of adhesion-related genes
   - like the Src family member p66Shc, which results in Ras hyperactivation and RhoA inactivation
4. Oxidative stress:
   - Oncoproteins, like ErbB2, bypass the metabolic stress induced by production of reactive oxygen species (ROS) during cell detachment.
   - Increased antioxidant responses are further driven by upregulation of heme oxygenase 1 (HMOX1)
5. Autophagy:
   - Autophagy is a lysosomal self-digestion
   - Cancer cells initiate a protective autophagy, for example to allow them to become dormant during unfavourable conditions (like lack of nutrients)
   - During detachment, activation of autophagy in cancer cells contributes to glycolysis and cell proliferation
6. EMT:
   - activation of epithelial-to-mesenchymal transition (EMT) - will be discussed more in CBIO4
   - upregulation of matrix metallopeptidases (MMPs)

Question 21

List these for intrinsic apoptosis

1. trigger initiating the apoptosis
2. proteins driving the process
3. activated caspases

Answer 21

1. microenvironmental trigger, e.g. DNA damage or withdrawal of growth factors
2. controlled by pro-apoptotic (like BID, BAD, BAX) and anti-apoptotic (like Bcl-XL and BCL2 family) proteins, release of apoptogenic factors (like cytochrome 3), which bind to pro-caspase 9 and form the apoptosome
3. Caspase 9 and caspase 3

Question 22

List these for extrinsic apoptosis

1. trigger initiating the apoptosis
2. proteins driving the process
3. activated caspases

Answer 22

1. binding of death ligand to death receptor (e.g. FAS to FASL)
2. formation of death-inducing signalling complex (DISC) composed of FADD (binding of DD with DED) interacting with pro-caspase 8
3. Caspase 8 and caspase 3

Question 23

Describe some strategies to promote intrinsic apoptosis in cancer cells

Answer 23

Strategies to promote intrinsic apoptosis in cancer cells include interfering with the cyto-protective effects of antiapoptotic members of the Bcl-2 family via:

1. Interference with mRNA function:
   - To interfere with mRNA function antisense oligonucleotides (ASOs) are used.
   - ASOs are short synthetic sequences of single-stranded DNA that can bind to target mRNA.
   - They act by enhancing sensitivity to cytotoxic drugs.
   - One of the ASOs that is advanced in clinical settings is oblimersen sodium (G3139, Genasense), which is an anti-BCL-2 mRNA agent, and is currently used in clinical trials for lymphoma.
2. Development of small-molecule drugs to target specific proteins:
   - Small-molecule inhibitors of the Bcl-2 protein family include those targeting histone deacetylases (HDACs), which control gene expression.
   - Currently, there are two approved HDAC inhibitors: vorinostat and romidepsin, against refractory cutaneous T-cell lymphoma (CTCL), while over 350 clinical trials have been completed or are underway using HDAC inhibitors.
   - Other approaches include both natural and synthetic BH3 mimetics to induce apoptosis.
   - These mimic critical domains of pro-apoptotic members of the Bcl-2 family, which include Bax and PUMA.
   - A highly promising BH3 mimetics therapy was recently approved by FDA for the treatment of 17p‑deleted chronic lymphocytic leukaemia (CLL).
3. Shut down gene transcription:
   - Small-molecule inhibitors can also act by targeting Inhibitors of Apoptosis (IAPs) by mimicking SMAC or by antisense-mediated interference of XIAP mRNA and protein expression.

Question 24

Describe how the extrinsic pathway can be targeted for cancer therapy

Answer 24

• For the extrinsic pathway the early efforts in targeting apoptosis in cancer cells included purified TNF-α, to target one of the death receptors TNF, however it was too toxic for systemic therapy.
• The second approach was to activate the second death receptor FAS, however this approach was also too toxic, with excessive apoptosis of liver cells (hepatocytes).
• Targeting of another death ligand, TNFSF10 (also called Apo2L/TRAIL) resulted in more success, with the creation of TNFSF10 cognate proapoptotic death receptors DR4 and DR5 antibodies.
• These antibodies are better tolerated but resistance is common, limiting their efficacy in monotherapy or in a combined therapy with conventional treatments.

Question 25

Describe how Myc influences apoptosis

Answer 25

• Myc influences the expression and/or activity of Bcl-2 family members at several levels.
  1. Myc can induce the expression of the BH3-only factor Pumaeither directly or through p53.
• Increased levels of Puma disrupt the functions of Bcl-2 or Bcl-XL, leading to mitochondrial dysfunction, the release of proapoptotic factors such as cytochrome c, SMAC, endonuclease G and apoptosis-inducing factor (AIF).
  2. Myc can repress the transcription of bcl-2 or bcl-X.
  3. Myc may ‘activate’ Bax by other means, as Myc is essential, in at least some cells, for Bax activation.
  4. Also induces the association of the BH3-only factor Bid with mitochondria

Question 26

How does c-Myc affect apoptosis?

Answer 26

• Models show that crosstalk and feedback loops in the ARF-p53 and Bcl-2/Bcl-XI apoptotic pathways are provoked by c-Myc
• In Model I, c-Myc activates the two pathways independently and both contribute to cell death.
• In Model II, c-Myc downregulation of Bcl-2 and/or Bcl-XL serves as the ‘stress’ signal that triggers the induction of ARF and p53.
• In some cell types, loss of ARF or p53 results in upregulation of Bcl-2 levels, which might can explain why ARF- and/or p53-deficient cells are more resistant to Myc-induced apoptosis

Question 27

What is autophagy and how can it contribute to tumour survival?

Answer 27

• autophagy is essential for removing damaged intracellular components, by taking away toxic products to maintain homeostasis
• process used by normal cells
• cancer cells are able to survive prolonged starvation
• as it was found out that they had activated the autophagy pathway and were surviving through it
• Discovered that autophagy was often upregulated in hypoxic tumour regions—those are regions deprived of oxygen—and when you deleted autophagy in those tumour cells, then you end up with no tumour cells surviving hypoxia
• Suggested that even in vivo, autophagy was an important survival mechanism for cancer cells

Question 28

What roles do autophagy play in cancer and cancer therapy?

Answer 28

• both tumour suppressor and tumour promoter roles
• see image
• In cancer biology, autophagy plays dual roles in tumour promotion and suppression and contributes to cancer-cell development and proliferation.
• Some anticancer drugs can regulate autophagy.
• Therefore, autophagy-regulated chemotherapy can be involved in cancer-cell survival or death.
• Additionally, the regulation of autophagy contributes to the expression of tumour suppressor proteins or oncogenes.
• Tumour suppressor factors are negatively regulated by mTOR and AMPK, resulting in the induction of autophagy and suppression of the cancer initiation.
• In contrast, oncogenes may be activated by mTOR, class I PI3K, and AKT, resulting in the suppression of autophagy and enhancement of cancer formation.