4. Autophagy in cancer Flashcards
Which cell organelles perform degradation?
Degradation performed by:
- proteosomes
- lysosomes
What needs to be degraded in cells? Why degradation mechanisms exist?
Need degradation for cellular homeostasis:
- toxic waste
- misfolded proteins
- regulate pathway activity (stop working when protein degraded)
- recycle molecules and cells for building blocks
Why are there two organelles for degradation?
They degrade different size proteins:
- proteosomes - barrel shaped - can’t fit large proteins in - such as protein aggregates and organelles - only degrade smaller
- lysosomes - can degrade large proteins
What is needed for degradation in proteosomes to notice proteins meant for degradation?
Proteins for degradation are ubiquitinated by ubiguitin ligases -> noticed by proteosomes -> degraded
What is autophagy?
Autophagy - self-degradation - cellular process in which a cell breaks down and recycles its own components, such as damaged organelles, misfolded proteins, or other intracellular debris
What are the types of autophagy?
Types of autophagy:
- Micro-autophagy
- Chaperone-mediated autophagy
- Macro-autophagy (=autophagy) - the mechanism discussed in this lecture
Explain the mechanism of autopahgy (=macro-autophagy)
Autophagy (=macro-autophagy):
1) Upstream signal for autophagy induction
2) Pre-autophagosome engulfs degradation target
3) ATG8 (ex LC3) activation by fusion with a lipid
4) LC3 integration into pre-autophagosome membrane
5) Autophagosome fusion with lysosome - contains degrading enzymes
6) Degradation + recycling of nutrients
Explain autophagosome structure
Pre- autophagosome vesicle:
- double membrane
- surface proteins
Autophagosome vesicle:
+ activated ATG8 proteins (ex LC3)
What is an autophagosome
Autophagosome - double-membraned vesicle that plays a crucial role in the process of autophagy - fuses with lysosome for functional degradation
Why is autophagosome fusion with lysosome needed for degradation?
Lysosomes contain digestive enzymes - inside low pH acidic - protection mechanism if aggressive degradation enzymes spilled - would not be functional in cytosol
What was the first experiment done to investigate autophagy genes?
Random mutagenesis induced in yeast cells -> mutants with active / inactive autophagy -> grown in starvation media (hypothesised that autophagy is needed to survive in starvation) -> some yeats survived, others not -> first autophagy genes defined - autophagy related genes (ATG) - the ones missing in dead mutants with inactive autophagy
Explain the ATG proteins
Autophagy related genes (ATG) - genes needed to perform autophagy - 2 subfamilies of ATG8 proteins:
- LC3: LC3A, LC3B, LC3C (the ones focuse din this lecture)
- GABARAP
-> these are ubiquitin like molecules
What are the functions of ATG proteins?
ATG protein functions:
- autophagy cargo recruitment
- role in autophagosome growth and lysosome fusion
Explain LC3 (ATG8) protein activation
LC3 (ATG8) protein activation - ubiquitin-like conjugation:
1) LC3 inactive - protease cleaves off an end -> exposes glycine
2) cleaved LC3 modified by ubiquitin-like enzymes
3) PE lipid conjugated to LC3 - lipidation => LC3 activated - can integrated into pre-autophagosome membrane
What is used to assess lipidation of LC3 (ATG8) as a readout for autophagy?
Fluorescence microscopy: change in fluorescence when LC3 tagged
SDS PAGE: aactive / inactive LC3 proteins show up differently by size - active run lower
What are the two machinery types in involved autophagy?
Core autophagy mechinery: LC3 lipidation complex, ATG5 complex
Signaling autophagy machinery: Vps34 complex, ULK complex
What are the signaling mechanisms involved in regulating ULK complex activity?
ULK complex - signaling autophagy machinery:
- Inhibits: mTORC1 inhibits ULK -> inhibits autophagy but mTORC1 is inhibited by am. a. starvation + anti-cancer therapy -> then ULK complex active
- Activates: AMPK activates ULK -> low energy activates AMPK -> activates ULK complex and upregulates autophagy
Is autophogosome formation a dynamic or static process?
Dynamic - operates on levels - a balance must be kept in cells
Summary of autophagy pathway
What is selective autophagy?
Autophagy is selective - not all proteins degraded - proteins for degradation selected by receptor proteins - bring together autophagy substrates and autophagy machinery
What are the types of substrates in autophagy?
What happens to cells in autophagy absence?
Autophagy absence - higher cellular damage:
- ROS
- protein aggregates
- DNA damage
- inflammation
-> increased cellular damage - activation of growth suppression pathways (ex p53 tumour suppression pathways) => cell cycle arrest (senescence) / apoptosis => organ malfunction, neurodegenerative disease (brain very sensitive to autophagy absence)
Which diseases has autophagy been associated with?
Cancer
What mice experiments are employed for studying autophagy in cancer?
Mice experiments used:
- genetically engineered mouse models - edit autophagy genes
- xenografts
Explain the experiment of constitutive deletion in studying autophagy importance
Constitutive deletion of autophagy improtant genes from the start of life
ATG7 gene deleted -> autophagy inactivation -> mice born but die 2 days after because can’t breastfeed - don’t have a milk pouch
Explain the experiment of inducible deletion in studying autophagy importance
Inducible deletion of autophagy - not from beginning - deleted upon tamoxifen addition
Died without autophagy pathway from neurodegenerative disease - protein accumulation - no degradation in the brain damages the brain
Explain how it was tested that autophagy is essential in starvation survival
Autophagy is essential in starvation survival - WT can survive a day without food only water - autophagy mutants die - can’t maintain glucose levels - hypoglycemia - if given external glucose without food - survive
What is the underlying molecular cause of cancer?
Uncontrolled cell division:
- activation of oncogenes
- mutated tumour suppressor genes
What are the factors influencing cancer formation?
Factors causing cancer:
- environmental influences
- Natural cell processes
- Inheritance
- Viruses
=> genomic mutations in oncogenes + tumour suppressing genes
What is the role of autophagy in cancer?
Autophagy both prevents and supports cancer - double-edged sword:
- Autophagy inhibits cancer - tumour suppression: in normal cells autophagy maintains cellular homeostasis - clears away waste, ROS, recycles nutrients -> absence of autophagy can induce cancer
-
Autophagy supports cancer - tumour maintenance: in cancer cells autophagy helps avoid the immune system by hiding MHC-I proteins - without autophagy in cancer cells -> suppression of growth, cell death
=> potential anti-cancer therapy
Explain pancreatic cancer and its cause
Pancreatic cancer:
- most common type - pancreatic ductal adenocarcinoma (PDAC)
- treatment involved surgical resection + chemotherapy, radiotherapy
- likely caused by acinar cells - produce enzymes - acquire mutations -> become cancerous
What is the progression like of PDAC?
Morphologically - loss of structure in ducts
How does autophagy impact the development of pancreatic cancer?
Autophagy supports cancer development - autophagy deletion prolonged life of cancer mice - autophagy helps cancer cells evade immune system
What exactly is the role of autophagy in cancer maintenance?
Autophagy promotes immune evasion by degrading MHC-I antigens on cancer cell surface - targets MHCI for lysososmal degradation - immunity can’t recognise and clear the cancer - autophagy inhibition restores MHCI levels - better cancer clearance - extends lifespan
How can autophagy be targeted in anti-cancer therapy?
Autophagy inhibition - immunity activation can be used as anti-cancer therapy - shrinks tumours
What is the current problem with targeting autophagy as anti-cancer therapy?
Current available autophagy inhibitors are not specific enough to only target autophagy in cancer cells - when administered also target normal cells -> problems - toxic
Current autophagy inhibitors:
- mTOR activators / inhibtors or mTOR inhibitors
- PI3K inhibitors
- lysosome inhibitors
=> all affect normal cells too - autophagy required for tissue homeostasis - further treatment development needs to make them more specific - not to have off-target effects on healthy cells
