4. Autophagy in cancer

Question 1

Which cell organelles perform degradation?

Answer 1

Degradation performed by:
- proteosomes
- lysosomes

Question 2

What needs to be degraded in cells? Why degradation mechanisms exist?

Answer 2

Need degradation for cellular homeostasis:
- toxic waste
- misfolded proteins
- regulate pathway activity (stop working when protein degraded)
- recycle molecules and cells for building blocks

Question 3

Why are there two organelles for degradation?

Answer 3

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

Question 4

What is needed for degradation in proteosomes to notice proteins meant for degradation?

Answer 4

Proteins for degradation are ubiquitinated by ubiguitin ligases -> noticed by proteosomes -> degraded

Question 5

What is autophagy?

Answer 5

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

Question 6

What are the types of autophagy?

Answer 6

Types of autophagy:
- Micro-autophagy
- Chaperone-mediated autophagy
- Macro-autophagy (=autophagy) - the mechanism discussed in this lecture

Question 7

Explain the mechanism of autopahgy (=macro-autophagy)

Answer 7

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

Question 8

Explain autophagosome structure

Answer 8

Pre- autophagosome vesicle:
- double membrane
- surface proteins

Autophagosome vesicle:
+ activated ATG8 proteins (ex LC3)

Question 9

What is an autophagosome

Answer 9

Autophagosome - double-membraned vesicle that plays a crucial role in the process of autophagy - fuses with lysosome for functional degradation

Question 10

Why is autophagosome fusion with lysosome needed for degradation?

Answer 10

Lysosomes contain digestive enzymes - inside low pH acidic - protection mechanism if aggressive degradation enzymes spilled - would not be functional in cytosol

Question 11

What was the first experiment done to investigate autophagy genes?

Answer 11

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

Question 12

Explain the ATG proteins

Answer 12

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

Question 13

What are the functions of ATG proteins?

Answer 13

ATG protein functions:
- autophagy cargo recruitment
- role in autophagosome growth and lysosome fusion

Question 14

Explain LC3 (ATG8) protein activation

Answer 14

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

Question 15

What is used to assess lipidation of LC3 (ATG8) as a readout for autophagy?

Answer 15

Fluorescence microscopy: change in fluorescence when LC3 tagged

SDS PAGE: aactive / inactive LC3 proteins show up differently by size - active run lower

Question 16

What are the two machinery types in involved autophagy?

Answer 16

Core autophagy mechinery: LC3 lipidation complex, ATG5 complex

Signaling autophagy machinery: Vps34 complex, ULK complex

Question 17

What are the signaling mechanisms involved in regulating ULK complex activity?

Answer 17

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

Question 18

Is autophogosome formation a dynamic or static process?

Answer 18

Dynamic - operates on levels - a balance must be kept in cells

Question 19

Summary of autophagy pathway

Answer 19

Question 20

What is selective autophagy?

Answer 20

Autophagy is selective - not all proteins degraded - proteins for degradation selected by receptor proteins - bring together autophagy substrates and autophagy machinery

Question 21

What are the types of substrates in autophagy?

Answer 21

Question 22

What happens to cells in autophagy absence?

Answer 22

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)

Question 23

Which diseases has autophagy been associated with?

Answer 23

Cancer

Question 24

What mice experiments are employed for studying autophagy in cancer?

Answer 24

Mice experiments used:
- genetically engineered mouse models - edit autophagy genes
- xenografts

Question 25

Explain the experiment of constitutive deletion in studying autophagy importance

Answer 25

**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

Question 26

Explain the experiment of inducible deletion in studying autophagy importance

Answer 26

**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

Question 27

Explain how it was tested that autophagy is essential in starvation survival

Answer 27

**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

Question 28

What is the underlying molecular cause of cancer?

Answer 28

Uncontrolled cell division: - **activation of oncogenes** - **mutated tumour suppressor genes**

Question 29

What are the factors influencing cancer formation?

Answer 29

Factors causing cancer: - **environmental** influences - **Natural** cell processes - **Inheritance** - **Viruses** => genomic **mutations in oncogenes** + **tumour suppressing genes**

Question 30

What is the role of autophagy in cancer?

Answer 30

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**

Question 31

Explain pancreatic cancer and its cause

Answer 31

**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

Question 32

What is the progression like of PDAC?

Answer 32

Morphologically - **loss of structure in ducts**

Question 33

How does autophagy impact the development of pancreatic cancer?

Answer 33

**Autophagy supports cancer development** - autophagy deletion prolonged life of cancer mice - autophagy helps cancer cells evade immune system

Question 34

What exactly is the role of autophagy in cancer maintenance?

Answer 34

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

Question 35

How can autophagy be targeted in anti-cancer therapy?

Answer 35

**Autophagy inhibition** - **immunity activation** can be used as anti-cancer therapy - shrinks tumours

Question 36

What is the current problem with targeting autophagy as anti-cancer therapy?

Answer 36

**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