network signalling (mTOR)

Question 1

mTOR complexes function and general info

Answer 1

• mTORC1 consists of mTOR, GBL and Raptor
  • Raptor can be inhibited by rapalogs (like rapamycin)
• The protein kinase activity of mTORC1 increases in response to:
  • The presence of amino acids
  • Levels of ATP sufficient for cell growth
  • Oxygen
  • Signalling from growth-factor receptors (IGF1, EGF and insulin)
• mTORC1 is inhibited by various types of cellular stress, including hypoxia and low levels of ATP and nutrients. It is also the mTOR complex inhibited by rapamycin.
• Active mTORC1 regulates cellular metabolism to promote cell growth and stimulates ribosome synthesis and translation. It also inhibits autophagy.
• mTORC2 consists of mTOR, GBL and Rictor
  - activates Akt and thus mTORC1 –> survival and apoptosis inhibition
  - insensitive to AA, oxygen, ATP, only activated by growth factors via PI3K

Question 2

Unfolded protein respsonse (negative feedbackloop)

Answer 2

1. Proteins in the ER are unfolded (often HS-SH bridges do not form correctly due to lack of oxygen)
2. elF2 is phosphorylated by PERK (ER resident kinase)
3. elF2 cannot initiate new mRNA
4. Protein synthesis stops to prevent further accumulation of unfolded proteins in the ER
5. ATF4 is activated (during ER stress – build-up of unfolded proteins) which causes transcription of chaperones to refold the proteins or causes breakdown of the unfolded proteins
6. GADD34 is then activated via ATF4(phosphatase complex)
7. elF2 is dephosphorylated
   - prolonged activation of this signalling pathway can induce apoptosis

Question 3

cMyc and eIF4 feedforward loop

Answer 3

• c-Myc is a protooncogene and is frequently deregulated in cancer
• c-Myc is a transcription factor that drives expression of many genes involved in tumorigenesis
• c-Myc increases protein synthesis machinery (specifically 3 elFs)
• Increase protein synthesis includes an increased amount of c-Myc itself and thus stimulates tumorigenesis  positive feedforward loop

Question 4

cellular faith and feedback loops

Answer 4

• NGF and EGF have a similar signalling cascade and act on the same cell only a different receptor (and ligand)
• EGF binds to EGFR and causes MAPK siganalling: RAS–>RAF–>MEK–>ERK
  - ERK inhibits RAF: negative feedback loop
  - The EGFR is captured in endosomes after stimulation and degraded
  - ERK causes transcription of DUSP which causes deactivation of ERK
  - ERK causes FOS transcription in a temporal fashion which leaves FOS very
  unstable
• NGF binds to NGFR which is not degraded but recycled
  - the recycled receptor causes a constant signal making causing the stabilisation of FOS since ERK will be present in large amounts and will phosphorylate FOS
• NFG causes neurite outgrowth while EGF causes proliferation

Question 5

epithelial to mesenchymal transition - EGF

Answer 5

• Epidermal Growth Factor (EGF) can induce transition of epithelial cell type to mesenchymal cell type
• Via the MAP kinases signalling SLUG and SNAIL are transcribed which will repress CDH1 (E-cadherin) and RKIP (RKIP inhibits RAF, so inhibition of RKIP leads to prolonged MAP kinase signalling)  leads to loss of adherens junctions
• Very important during development (i.e. neural crest cells) and during metastasis

Question 6

EMT - ZEB1 and miRNA200

Answer 6

• ZEB1 and miRNA200 reciprocal negative regulation of each other –> they both inhibit each other meaning that the one of which there is more will win and the other will be inhibited
• In epithelial cells there is very little ZEB1 meaning that there is a lot of active miRNA200
• Inhibition of ZEB1 leads to E-cadherin expression and thus to epithelial cell state
• Mesenchymal cells have low levels of miRNA200 and thus high levels of ZEB1 –> ZEB1 then represses the expression of E-cadherin leaving the cell in a mobile/mesenchymal state
• TGF-beta or FGF can induce ZEB1 which causes miRNA200 repression and thus epithelial –> mesenchymal transition
• Hypoxia represses the miRNA200 leading to increased EMT (and thus metastasis in cancer)

Question 7

network-mediated drug resistance

Answer 7

• Inhibition of mTORC1 can be used as anti cancer treatment to prevent further proliferation
• After a while the rapalogs lose their function since the inhibition of mTORC1 also leads to the removal of the negative feedback that mTORC1 has via S6K on IRS1 (EGFR receptor) leading to increased AKT and ERK activation and to proliferation and survival of the cancer cells via other pathways downstream of Akt and ERK
• In melanoma BRAF and KRAS are frequently mutated  oncogenes (gain of function)
• BRAF or MEK inhibitors are perfect to target these mutated oncogenes and stop proliferation
• Prolonged treatment induces increased EGFR expression since ERK could no long serve as the negative feedback on the receptor

Question 8

mTOR activation

Answer 8

• mTORC1 is directly inhibited by AMPK (low ATP and DNA damage:p53)
• mTORC1 is activated by Rheb-GTP and inhibited by Rheb-GDP
  
  • this GTP is hydrolysed by TSC1/2 (Rheb/GAP) to inactivate mTORC1
  • TSC1/2 activity is increased by: REDD1 (hypoxia), AMPK (low ATP, DNA damage), p53,
    GSK3beta
  • TSC1/2 is inhibited by Akt (via mTORC2, growth factor signalling through PI3K and
    PDK1), ERK and Rsk (growth factor signalling through MAPKs)
• mTORC1 is activated by RagA/C
  
  • AA stimulate RagA/C
  • leucine and arginine lift the inhibition from GATOR2 which then lifts inhibition of
    GATOR2 on RAGA/C –> mTORC1 active
  • leucine in lysosomes will stimulate ragulator to stimulate RagA/C

Question 9

mTOR downstream (3)

Answer 9

1. protein synthesis (S6K and eIF4)
   
   • elF4 is regulated by 4E-BP which can be directly inactivated by mTORC1
   • S6 kinase (S6K), that phosphorylates the small ribosomal subunit protein S6 and additional substrates, leading to a further increase in the rate of protein synthesis.
2. metabolism (HIF1afla, ATF4, SREBP)
   
   • HIF: glycolytic enzymes
   • ATF4: nucelotide synthesis
   • SREBP: lipid synthesis
3. protein turnover (inhibition ULK1 and UPS)
   
   • autophagy and UPS inhibition

Question 10

insulin resistance

Answer 10

• mTOR plays a major role in nutrient sensing within the body.
• Under normal physiological state:
  1. Increase in glucose and amino acid levels following a meal stimulates the secretion of insulin by the pancreatic β cells.
  2. Insulin binds it receptor.
  3. Insulin receptor phosphorylates insulin receptor substrates 1 and 2 (IRS1 and IRS2) at its tyrosine residues, allowing them to associate with PI3K
  4. Leading to the activation of downstream effectors such as AKT and protein kinase C
  • AKT drives the metabolic action of insulin where it inactivates glycogen synthase 3 to mediate glycogen synthesis as well as the translocation of glucose transporter 4 (GLUT4) to the plasma membrane for glucose uptake into myocytes
  5. Additionally, AKT activates mTORC1 which in turn stimulates S6K1 to phosphorylate serine residues in IRS.
  6. IRS can now no longer bind to the insulin receptor –> negative feedback mechanism thus desensitizes and protects the cell to further insulin simulation
• Chronic overstimulation of mTOR makes the cell insulin resistant –> diabetes

Question 11

cancer

Answer 11

• Genes encoding components of the mTORC1 pathway are mutated in many human cancers, resulting in cell growth in the absence of normal growth signals
• Mutations in components of cell-surface receptor signal transduction pathways that lead to inhibition of TSC1/ TSC2 Rheb-GAP activity are also common in human tumours and contribute to cell growth and replication in the absence of normal signals for growth and proliferation.
  • Mutations in genes like Ras, Raf, Akt, PI3K will cause overstimulation of mTOR and tumorigenesis –> oncogenes
  • Also loss of TSG like p53, of the TSC1 or TSC2 genes will lead to increased mTOR signalling

Question 12

ageing

Answer 12

• Loss of proteostasis
 mTOR upregulates transcription and downregulates autophagy and UPS giving rise to more protein production and thus higher chance of faulty protein production and accumulation

• Mitochondrial dysfunction
 mTORC1 stimulates mitochondrial biogenesis and fission and fusion but decreases mitophagy (lower pink and parkin) –> more mitochondria but leads to more ROS and thus ageing

• Senescence
 mTOR signalling leads to inhibition of SASP protein breakdown leading to longer lasting senescence (also the other hallmarks will lead to senescence)

• loss of stem cell function