Lecture 11 - Apoptosis and senescence

What biochemical changes occur during apoptosis?

• Blebbing 

• Cell shrinkage 

• Nuclear fragmentation 

  • Chromatin condensation 

  • Chromosomal DNA fragmentation  

How is apoptosis related to carcinogenesis?

• Unrepaired or excessive DNA damage leads to apoptosis 

• Inappropriate growth signals (e.g. due to oncogene activation) can lead to apoptosis 

• Therefore, ability to avoid apoptosis is a key hallmark of malignant tumours  

What can trigger apoptosis of a cell?

• Various stresses experienced by cells 

• Most notable: 

  • Hyperactive oncogenic signalling 

  • DNA Damage Response 

What are the two apoptotic pathways?

• Intrinsic

  • Response to DNA damage

• Extrinsic

  • Response to death receptor ligand binding

How does the intrinsic apoptotic pathway signal?

• In response to DNA damage, p53 causes activation of initiators of apoptosis NOXA and PUMA

• NOXA and PUMA then bind and inhibit members of the BCL-2 family

• This frees BAX and BAK which then form oligomeric pores in the mitochondrial outer membrane

• This causes release of cytochrome C from mitochondria

• Cytochrome C then binds APAF1 which then associates with Pro-caspase 9. This forms the apoptosome and causes activation of caspase 9

• This triggers activation of caspase signalling cascade which causes activation of caspase 3 and 7, triggering apoptosis

What are IAPs?

Inhibitors of Apoptosis (IAP)

• Family of proteins which inhibit caspases and prevent apoptosis

How are IAPs inhibited during apoptosis initiation?

• Following release of Cytochrome c, another protein called SMAC/DIABLO is released too

  • This protein inhibits IAPs by binding with them

  • This prevents inhibition of caspase proteins such as caspase 9

How does the extrinsic apoptotic pathway signal?

• Death receptor ligand binds with receptor

  • E.g. Fas ligand binding Fas receptor

• This results in formation of DISC complex (death-inducing signalling complex) which recruits FADD/caspase 8 complex

• Caspase 8 is then able to signal via the caspase cascade to activate caspase 3 and 7

• This then triggers apoptosis

How can the intrinsic and extrinsic apoptotic pathways cross-talk?

• Caspase 8 from the extrinsic pathway can activate BID by truncating it

• tBID can the interact with BAK/BAX to cause oligomerisation and formation of pores in the mitochondria membrane

• This then leads to release of cytochrome c

How can Mitochondrial Outer Membrane Permeabilization (MOMP) during apoptosis initiation lead to cancer development?

• Widespread MOMP

  • Leads to apoptosis and inhibition of cancer development

• Minority MOMP

  • Limited caspase activation

  • This can cleave DNA without fully engaging apoptosis

  • This is a form of genomic instability which can lead to cancer development

What is the BCL-2 family?

Key regulators of apoptosis 

Comprises both pro-apoptotic and anti-apoptotic members 

• Balance between pro and anti determines cell fate 

• Typically anti- members dominate to prevent apoptosis from occurring

Aberrant expression of BCL-2 is seen in cancer  

What are some examples of pro-apoptotic BCL-2 members?

• Initiators

BIM, PUMA, BAD and NOXA

Which domain is the only one all pro-apoptotic initiator members have?

• They have the BH3 domain only

• Unlike pro-survival guardians which are multi-domain

What are examples of anti-apoptotic BCL-2 members?

• Guardians

• BCL-2

• BCL-X

• MCL-1

What are examples of pro-apoptotic executioner BCL-2 members?

• BAX

• BAK

• BOK

All pro-apoptotic BCL-2 members contain the BH3 domain only.

True or False?

False.

• Initiator members only contain the BH3 domain

• Executioner members are multi-domain

Not all pro-apoptotic members can inhibit all pro-survival members. Why not?

Some members such as PUMA can inhibit all pro-survival members.

• However some like NOXA have specificity to certain members (e.g. MCL-1 and BCL2A1)

What is the importance of the BH3 domain?

Functions by binding to and inhibiting pro-survival BCL-2 members

• This releases the inhibition on pro-apoptotic executioner members, BAX and BAK, which induces mitochondrial pore formation

What are BH3 mimetics?

• Mimic the BH3 domain of pro-apoptotic proteins 

• Used in combination with other cytotoxic and targeted therapies 

What is an example of a BH3 mimetic?

Venetoclax  

• BCL-2 specific BH3 inhibitor  

• Reduced broad cytotoxicity compared to other broad BH3 inhibitors as it doesn’t target all BCL-2 family members

How can cancers develop resistance to BH3 mimetics?
(3 examples)

• Mutation of target

• Inactivation of pro-apoptotic members

• Upregulation of non-targeted pro-survival members

What is p53?

Tumour-suppressor gene

• Maintains genomic integrity and tumour surveillance 

• MDM2 is the critical regulator of p53 through inhibition

How is p53 activated and how does it exert its function?

• P53 is phosphorylated and activated by Chk1 and Chk2

• This releases it from MDM2 where it can then promote transcription of numerous gene targets

How does p53 generate a negative feedback loop?

• Upregulation of p53 causes upregulation of certain genes

• In particular, p53 causes upregulation of MDM2 which in turn inhibits p53

  • Regulates p53 tightly

• p53 also causes upregulation of Wip1 which in turn inhibits p53

What protein is able to inhibit MDM2 and cause an increase in p53 activity?

p14ARF

What is Wip1?

Inhibitor of p53 and p14

• This prevents activation of p53 signalling and therefore tightly regulates its activation

What are some examples of mechanisms to inhibit p53 signalling in cancer?

• TP53 mutations

• P14ARF deletion or promoter hypermethylation

• MDM2/MDMX amplifications/over-expression

• WIP1 overexpression

How does DNA damage cause activation of p53?

DNA damage activates ATR and ATM

• ATR phosphorylates CHK1 and p53

• ATM phosphorylates CHK2

• CHK1 and CHK2 both phosphorylate p53 resulting in its activation

How does Oncogene activation trigger p53 activation?

• Oncogene activation is recognised by ARF

• ARF then prevents MDM2-mediated degradation of p53

• This stabilizes and activates p53

P53 can initiate apoptosis through NOXA and PUMA. How else can it upregulate apoptosis?

• p53 can also upregulate the expression of BAX

• p53 can do protein-protein interactions with pro-apoptotic proteins to promote apoptosis

• p53 can upregulate apaf1 to help facilitate the formation of the apoptosome

• p53 can upregulate death receptor expression

What type of p53 mutations are the most common?

Most are missense mutations (~90%) - Single nucleotide change which causes a different amino acid to be incorporated

• Some are nonsense mutations (~10%) - Nucleotide change which causes introduction of a STOP codon, truncating the protein

Majority of these mutations occur in the DNA binding domain

What are the consequences of p53 mutations which result in cancer development?

• What do they cause?

• P53 mutations in cancer are complex

  • Loss of transcription of anti-tumour genes

  • Transcription of new targets

  • Disruption of tetramer formation

What is MDR-1 (Pgp) and what does p53 do to it?

Multi-drug resistance protein (MDR-1)

• p53 represses MDR-1 transcription via direct DNA binding

• This downregulation is mediated by microRNA-34a and LRPPRC

How does mutant p53 cause upregulation of MDR-1?

It was found that some mutant p53 can cooperate with ETS-1 to upregulate MDR-1 instead of downregulating it

• This contributes to multi-drug resistance in the cell

What is the p53 R72P single nucleotide polymorphism (SNP)?

Exon 4, in a proline rich region

• In the population there can be arginine or proline at position 72

• Proline more common in Africans

• Arginine more common in Caucasians

How does R72P SNP relate to cancer?

Conflicting reports of association with cancer susceptibility.

• p53R > p53P in induction of apoptosis

  • Transcriptional effects

  • Non-transcriptional effects

What is MDM2 T309G SNP?

• SNP in MDM2 gene promoter region

• SNP leads to increased Sp1 binding and increased expression of MDM2

What are examples of targets for p53 therapies?

Wt p53

• Activation of p53

Mutant p53

• Refolding of mutant p53

• Degradation of mutant p53

• Read-through of truncated mutant p53

  • Prevent production of truncated protein

How do growth factors lead to cell proliferation in normal cells?

• What gene is involved?

• In normal cells, mitogenic signals (growth factors) lead to upregulation of MYC 

  • This leads to cell proliferation 

How does MYC play a role in cancer?

• MYC is a proto-oncogene and is dysregulated in many cancers by amplification or constant activation of upstream factors 

How does high expression of MYC result in apoptosis?

• High expression of MYC for an extended time results in signalling to p14

  • This then leads to inactivation of MDM2 and subsequent activation of p53

  • This then triggers apoptosis

• High MYC expression will also lead to rapid cell proliferation which can lead to DNA damage

  • This will also result in p53 activation

How do healthy cells avoid apoptosis initiation during MYC signalling?

• Signalling will be below threshold so p14 wont be activated

• Growth factor receptor binding also causes PI3K signalling which causes AKT activation

  • AKT causes promotion of MDM2 and inhibition of apoptosis

What are some examples of genetic alterations cancer can have to resist cell death?

• PI3K mutation/activation

  • Causes activation of AKT and subsequent p53 inhibition

• P14 gene inactivation

  • Suppression of p53

• BAX mutations

  • Loss of pro-apoptotic protein

• CASP8 promoter methylation

  • Inactivation of extrinsic cascade by inhibiting CASP8 expression

• CASP3 repression

  • Inactivation of executioner caspase

• TNFR1 methylation

  • Repressed expression of death receptors

• APAF1 methylation

  • Loss of caspase 9 activation

• BCL-2 overexpression

  • Closes mitochondrial channels and prevents cytochrome c release

What is cellular senescence?

• State of permanent cell cycle exit

• Associated with morphological, metabolic, epigenetic and biochemical changes

  • E.g. shortening of telomeres

• Potent anti-cancer mechanism

What two signalling pathways are important for senescence?

• p14ARF activation

• p16INK4a activation

  • Both cause inhibition of Rb phosphorylation

  • Rb-E2F complex prevents expression of cell cycle genes

    • This then leads to senescence

How does p14 activation result in cellular senescence?

• p14ARF activation signalling p53 which then signals to p21

  • This inhibits CDK2-Cyclin E AND CDK4/6-Cyclin D

• Inhibition of these kinases results in inhibition of Rb phosphorylation

• Hypophosphorylated Rb then associates with E2F and prevents expression of cell cycle genes

  • This can then lead to senescence

How does p16 activation result in cellular senescence?

• p16INK4a activation

  • This inhibits CDK4/6-Cyclin D

• Inhibition of these kinases results in inhibition of Rb phosphorylation

• Hypophosphorylated Rb then associates with E2F and prevents expression of cell cycle genes

  • This can then lead to senescence

How can Oncogene Induced Senescence (OIS) occur?

Overexpression of MYC can activate p14ARF

• P53 can activate p21 which can inhibit Rb phosphorylation resulting in oncogene-induced senescence

Overexpression of RAS can activate p16INK4

• This inhibits Rb phosphorylation resulting in oncogene-induced senescence

What are the markers of cellular senescence?

• Enlarged cytoplasm, enlarged nuclei, flat, spreading  

• Changes in expression of components in key senescence pathways

  • E.g. p16

• Reduction/lack of cell proliferation markers 

• Increase in anti-apoptotic markers 

• Senescence Associated Secretory Phenotype (SASP) 

What is Senescence Associated Secretory Phenotype (SASP)?

Paracrine and autocrine effects

Cell type and stress context dependent 

• Can cause tumour suppression, immune cell attraction and prevention of fibrosis 

• However, can also promote cell proliferation, promotion of angiogenesis and chronic inflammation 

What is therapy induced senescence (TIS)?

• Cancer therapies can induce senescence in cancer and non-cancer cells 

• Contribute to treatment related adverse effects and relapse of cancer 

• Development and use of senescence targeted therapies could improve efficacy of therapies 

What are the two types of serotherapeutics?

• Senolytics

  • Eliminate the cells

• Senomorphics

  • Modulate the cells

What is the aim of senolytic therapies?

• Target anti-apoptotic pathways 

• Kills cells in senescence  

• ‘One Two Punch’ strategy  

  • Combination of drug to drive cells into senescence and then senolytic drug 

What is the aim of senomorphics?

• Inhibit or modulate SASP produced by senescence cells without causing cell death 

• Preserve the good and inhibits the bad 

• Target key pathways involved in SASP production 

  • Metformin and Rapamycin

What is a possible disadvantage of senolytic therapies?

• Older individuals will have a larger number of senescent cells

• Impact on their healthy cells could be very large

What is a possible disadvantage of senomorphic therapies?

• Treatment would likely have to used forever to prevent SASP production

• The moment therapy is withdrawn, SASP will be initiated