M2L8: Cell death pathways

  • There are many different cell death pathways, but apoptosis was the first discovered

Apoptosis

  • Morphological changes

    • Separation from other cells

    • Membrane blebbing

    • Cell shrinkage

    • Nuclear/chromatin condensation

    • DNA fragmentation

  • Non-inflammatory, active process

  • Apoptosis vs necrosis

    • Necrosis: passive, cell swelling, membrane rupture, inflammation

    • Apoptosis: active, cell shrinkage, intact memnbrane, no inflammation, nuclear condensation, caspase-dependent

  • Intrinsic or extrinsic pathway

    • Extrinsic apoptosis is initiated by the extracellular microenvironment and driven by death receptors

      • Surface receptor interaction with death ligands leads to the assembly of the death-inducing signalling complex (DISC) which is a dynamic multiprotein complex at the intracellular tail of the death receptor (death domain)

        • Death receptors contain a cytoplasmic domain called the death domain (DD) which helps transmit death signals from the cell surface to intracellular signalling pathways

      • During FasL and FAS binding, Fas-associated proteins with death domains (FADD) are recruited and during TNF and TNFR binding, a TNFR1-associated death domain protein (TRADD) is recruited with FADD

      • Adaptor proteins exhibit appropriate death domains to bind to their corresponding receptor and they then recruit procaspase-8 by dimerising the death effector domain (DED)

        • Results in DISC formation and caspase-8 is thus cleaved and activated

      • Active caspase-8 initiates apoptosis by cleaving and activating executioner caspase-3

    • Intrinsic apoptosis (mitochondrial pathway) is initiated by microenvironment triggers like DNA damage or withdrawal of growth factors

      • Apoptotic cells retain their plasma membrane and metabolic activity during the apoptotic process due to the clearance by macrophages and other phagocytic cells via efferocytosis

      • At the end stage, apoptotic cells have a complete breakdown of the plasma membrane and acquire a necrotic morphotype (secondary necrosis)

      • Intrinsic apoptosis is irreversible and regulated by pro- and anti-apoptotic members of the Bcl2 family of apoptosis regulator proteins

      • The first step is widespread mitochondrial outer membrane permeabilisation (MOMP) which is mediated by Bcl2-associated X apoptosis regulator (BAX) and/or Bcl2 antagonist/killer (BAK)

      • BAX continuously travels between the outer mitochondrial membrane and the cytosol in an inactive form whereas BAK is located in the outer mitochondrial membrane

      • When apoptosis is triggered, BAX ceases to retranslocate, and BAX and BAK are directly or indirectly activated by pro-apoptotic proteins such as BID and BAD

      • The apoptogenic factor are released, including cytochrome C and second mitochondrial activator of caspases (SMAC)

      • Cytochrome C binds to apoptotic peptidase activating factor 1 (APAF1) and pro-caspse 9 to form the supramolecular complex apoptosome

      • The apoptosome activates caspase 9, which then catalyses the proteolytic activation of the executioner caspase 3

      • SMAC associates with the inhibitor of apoptosis (IAP) protein family to regulate apoptosis

      • Activation of executioner caspases triggers morphological and biochemical changes in the cell, including DNA fragmentation, phosphatidylserine (PS) exposure and formation of apoptotic bodies

      • Caspase 3 catalyses the proteiolytic inactivation of DNA fragmentation factor subunit alpha (aka. inhibitor of CAD, ICAD) and releases the catalytic activity of caspase-activated DNAse (CAD) to trigger DNA fragmentation

  • Bcl-2 family

    • Anti-apoptotic proteins (eg. Bcl-2, BcL-xl)

    • Pro-apoptotic proteins (eg. Bax, Bak)

    • BH3-only proteins (eg. Bid, Bad)

  • Caspase

    • Initiators - caspase 2, 8, 9, 10

      • Exist as inactive monomers which undergo proximity induced dimerisation

      • Dimers undergo autocatalytic proteolytic processing to form the active tetramer

    • Executioners (effectors) - caspase 3, 6, 7

      • Exist as inactive dimers which get cleaved by initiator caspases to form the active tetramer

    • Caspase 8 activation can link extrinsic and intrinsic pathways

  • Evasion of apoptosis in cancer

    • Upregulation of anti-apoptotic genes and downregulation of pro-apoptotic genes

    • p53 mutations can protect against apoptosis due to role of p53 in activating pro-apoptotic molecules

      • In the intrinsic pathway:

        • p53 activates BIM, PUMA, and NOXA (BH3 only proteins within Bcl-2 family) which inhibit anti-apoptotice Bcl-2 proteins including Bcl-XL, MCL-1 and Bcl-2

        • This disinhibits apoptosis effectors Bax and Bak to activate MOMP and apoptosome formation 

      • p53 also modifies the apoptotic response 

        • Activates other genes which fine-tune apoptosis, such as…

        • FAS/DR5 - death receptors (extrinsic pathway)

        • ZMAT3 - unclear role, modifies apoptotic signalling 

        • miR-34a - inhibits translation of anti-apoptotic Bcl-2

        • BTG2/PLK2 - stress response genes

  • Apoptosis inhibitors and apoptosis inducers 

    •     Venetoclax (venxlexta)

      • Bcl-2 inhibitor for chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL)

      • Can be in combination with azacytidine/decitabine/low-dose cytarabine for AML

Autophagy

  • Self devouring process initiated by starvation, ER stress, p53, energy deprivation

    • Provides nutrients to maintain cell function by degrading large molecules, organelles and proteins

    • Removes damaged organelles, misfolded proteins, lipid droplets —> cell homeostasis

  • Types of autophagy

    • Macroautophagy - characterised by formation of autophagosomes

    • Microautophagy - endosomes or lysosomes directly engulf and degrade autophagic cargo

    • Chaperone-mediated - cytosolic proteins are delivered to lysosomes for degradation by a process involving chaperones which bind proteins via specific targeting motifs

  • Macroautophagy - most common

    • Induction and nucleation - energy/nutrient stress activates AMPK/inhibits mTOR to form the ULK1 initiation complex and the formation of a membrane ‘seed’ (isolation membrane) at the ER or other organelles

    • Elongation - isolation membrane expands around cytoplasmic material and incorporates LC3 (cleaved from LC3-I to LC3-II) to capture cargo 

    • Closure and maturation - membrane edges fuse to form autophagosome

    • Fusion: mature autophagosome fuses with a lysosome to form and autolysosome

    • Degradation and recycling - lysosomal enzymes break down cargo and the resulting metabolites are recycled into the cytoplasm for energy and biosynthesis

  • Autophagy in cancer

    • Can be pro- or anti-cancer

    • Autophagy can sense genomic instability and help maintain genomic integrity

    • Autophagy may also help maintain dormancy

    • Autophagic MHC-I degradation for immune escape

    • Autophagic degradation of damaged organelles to provide nutrients and energy for the stress response

    • Can promote migration/invasion

    • Can promote resistance to anoikis to prevent cell death when metastasising cells detach from the primary tumour and migrate to distant sites

    • Can maintain dormancy, which may be harmful especially at distant metastatic sites

  • Autophagy inhibitors

    • Bafilomycin is a well known autophagy inhibitor

    • In clinic chloroquine and hydroxychloroquine (anti-malaria and anti-inflammatyory drugs) can inhibit fusion of autophagosomes with lysosomes and theri degradation

    • When combining chloroquine + radiation, overall survival does not notably change but helps brain metastases progression free survival (cure vs quality of life) 

Ferroptosis

  • A cell death pathway which requires iron, lipid peroxidation and defective/insufficient antioxidant response

    • Cells take up iron via TRF1 receptor which imports Fe3_ bound to transferrin

    • In endosomes, STEAP3 reduces Fe3+ to Fe2+

    • DMT1 moved Fe2+ into the cytosol, producing the labile iron pool

    • Some iron is stored as ferritin and excess Fe2+ participates in Fenton reactions which generates reactive hydroxyl radicals, initiating lipid peroxidation

    • Polyunsaturated fatty acids (PUFAs) are susceptible to oxidation and enzymes such as ACSL4 and LPCAT3 incorporporate PUFAs into phosphatidylethanolamine (PE) in the membrane to form PUFA-PE

    • PUFA-PE is oxidised by 15-LOX and hydroxyl radicals to form lipid hyperoxides (PUFA-PE-OOH)

    • PUFA-PE-OOH accumulation causes membrane damage and ferroptosis

    • Antioxidant response:

      • The system Xc- antiporter imports cystine in exchange for glutamate

      • Cystine is converted to cysteine and used to synthesise glutathione

      • GPX4 uses GSH to reduce PUFA-PE-PE-OOH to PUFA-PE-OH, preventing lipid damage

      • When System Xc- is inhibited or GPX4/GSH is low, lipid peroxides accumulate, causing ferroptosis 

  • Inducers - erastin, RSL3, ML-210, FIN56, sulfasalazine, BSO, inositol

    • Erastin and RSL3 were found to kill Ras mutated tumour cells through ferroptosis, which is how this pathway was discovered

    • Sulfasalazine - anti-inflammatory drug for bowel disease which can also cause ferroptosis

    • Some chemotherapy drugs are known to induce ferroptosis

  • Inhibitors - ferrostatin-1, liproxstatin-1, DFO

    • DFO - iron chelator to prevent induction of ferroptosis

  • There is significant cancer research focusing on ferroptosis is due to this pathway as it is very specific for aggressive and invasive tumour types

    • ZEB1 is an important transition factor for EMT induction which also upregulates PUFAs

    • GPX4 inactivation causes PUFA accumulation and lipid peroxidation leading to drug resistance

    • Drug resistant tumour cells often also have higher PUFA content and depend on GPX4 to manage redox stress

    • Inducers of ferroptosis which inhibit GPX4 can therefore cause ferroptosis in drug resistant cells

  • Ferroptosis sensitises invasive and drug resistant tumour cells*

    • Morphological features - mitochondrial condensation/shrinkage, membrane rupture

  • Ferroptosis chain reaction - death of one cell can be propagated to others 

  • Autophagy plays a significant role in ferroptosis

    • Autophagy mediated by NCOA4 can degrade ferritin and release iron into the cytoplasm to trigger ferroptosis

    • Lipid can be stored as lipid droplets within cells which can be broken down by autophagy mediated by TPD52, releasing free fatty acids including PUFAs which are substrates for lipid peroxidation

    • ARNTL is a part of the circadian clock which regulates antioxidant defence that can be degradaded by SQSTM1 mediated autophagy

    • HSPA8 in chaperone mediated autophagy can regrade GPX4

  • Ferroptosis lacks specific markers to measure it

    • Lipid peroxidation is the main indicator

      • 4-HNE is a widely used lipid peroxidation marker in tissue staining

    • Cell death is another indicator

      • When using ferroptosis inducers (molecule being tested for whether it causes ferroptosis) and inhibitors simultaneously, the amount of rescue can indicate how much ferroptosis was happening

  • MAFF is an important factor regulating ferroptosis via lipophagy reaction

  • Inducers in clinic - none, repurposing drugs to induce ferroptosis

  • Radiation can induce different types of cell death by causing DNA damage, including ferroptosis in cancer

    • Direct action (30% of action) - radiation directly targeting DNA to induce damage

    • Indirect action (70%) - radiation ionises other molecules, mainly water, creating free radicals that damage DNA

  • Ferroptosis is DNA damage independent

  • Ferroptosis inhibitor can also reduce lung fibrosis after radiation therapy and reduce cardiac toxicity after doxorubicin treatment

  • FLASH radiation (increasing dose rate, speed of radiation) - reduces normal tissue injury

    • One hypothesis for the effect of FLASH  is induction of lipid peroxidation and Fenton chemistry (possibly because tumour tissues have higher iron)