WS: Notch Signalling

Question 1

Outline the sequence of events in canonical notch signalling:

Answer 1

• S1 cleavage of receptor in golgi (Furin)
• -> receptor fragments remain associated and are transported to cell surface
• Notch ligand on adjacent cell interact -> S2 cleavage by members of ADAM family of enzymes (ADAM10 or ADAM17) leaving the NCTF ‘stump’ in the membrane -> subsequent S3/S4 cleavage by gamma secretase complex
• NICD (notch intracellular domain) translocates to nucleus, displace co-repressor from CSL TF
• CSL-NICD recruits MAML and a coactivator
• Gene repression (differentiation + proliferation)

Question 2

How are human notch receptor typically modified? Give examples of 3 of these enzymes

Answer 2

• Post translational modification by ‘Fringe’ glycosylases
• Determines which ligand the receptor interacts with
• Fringe glycosylases: Radical, manic, lunatic
• Takes place in golgi apparatus

Question 3

List the various mammalian notch ligands, with 2 key extracellular and 1 key intracellular domain:

Answer 3

• Jagged 1 and 2
• DII 1, 3 and 4
• Differ by domain structure
• I-C: PDZ (post synaptic density signalling)
• E-C: DSL, EGF repeats

Question 4

What is the active component of the gamma secretase complex?

Answer 4

• Aspartyl proteinase called Presenilin
• Require a water molecule -> issue in hydrophobic environment and area of doubt in field

Question 5

What hallmarks of cancer is Notch signalling involved in?

Answer 5

• Cell proliferation
• EMT
• CSC maintenance
• Angiogenesis
• Resistance to traditional chemo and radiotherapy

Question 6

What are ADAM proteins?

Answer 6

• A disintegrin and metalloproteinases
• Proteolytic enzymes involved in shedding of membrane bound proteins, GFs etc
• Generally activated by proprotein convertases which cleave the pro-domain
• ADAMs act as regulators of cell proliferation and may also be involved in cancers; potential prognostic markers in case of lung cancer

Question 7

Give the 4 components of the gamma-secretase complex:

Answer 7

• Presenilin (including catalytic site)
• Nicastrin (Gatekeeper which blocks access to substrates with larger ectodomains)
• Aph-1
• Pen-2

Question 8

Outline the role of Notch signalling:

Answer 8

• Cell fate determination
• Proliferation
• Apoptosis
• Adult tissue homeostasis
• Embryonic development

Question 9

By what mechanisms is S2 cleavage by ADAMs enacted?

Answer 9

• Notch has a NRR (negative regulatory region) which contains structural machinery for MMP resistance in absence of ligand
• When ligand binds, activation switch is exposed -> ADAM10/17 able to cleave
• This occurs due to mechanical pulling force from the ligand as it is internalised in endocytosis

Question 10

Structure of Notch receptors:

Answer 10

• I-C: PEST (degradation signals), TAD (only in certain members), ANK, NLS, RAM (associates with TFs in nucleus)
• E-C: NRR (HD, LNR), EGF-like repeats(ligand interaction), SP (signal peptide)

Question 11

What sets notch signalling apart from other pathways covered in this module?

Answer 11

• Relatively irreversible
• The receptor itself is irreversibly cleaved to enact transcriptional activation

Question 12

2 examples of Notch target genes:

Answer 12

• Hes family
• Hey family

Question 13

JAG1 in HCC:

Answer 13

• Found to be upregulated in majority of hepatocellular carcinoma cases
• Oncogenic role
• Notch signalling via JAG1 upregulate osteopontin (OPN) which is a key driver of extra-hepatic HCC metastasis
• RUNX3 is downregulated in many HCCs; usually opposes Notch signalling
• JAG1 is also linked with therapy resistance

Question 14

What pathways does JAG1/Notch signalling crosstalk with during oncogenic activities?

Answer 14

• ILs
• TGF-B
• Hippo
• Wnt/B-catenin
• All contributes to cancer development through regulation of behaviour of tumour cells and cells in the TME

Question 15

How can JAG1 be targeted as a therapeutic strategy against cancer? (4 novel and 1 historical including key issue)

Answer 15

• JAG1 gene silencing by lentivectors containing JAG1 siRNA or expression of certain miRNAs
• Interfering with signalling cascade (e.g. upregulating Neur1/inhibiting Mib1, treating cells with ‘dynasore’ which inhibits dynamin and thus promotes cell apoptosis, or use of antibodies against JAG1-Notch interactions)
• Most commonly used strategy is GSI (gamma secretase inhibitor) -> serious issues with toxicity

Question 16

What two E3 ligases are required for JAG1 endocytosis?

Answer 16

• Neuralized1 -> negatively affects Notch signalling
• Mindbomb-1 -> positively affects Notch signalling
• Dynamin is also important for JAG1 endocytosis and is an endocytic adaptor protein

Question 17

Notch signalling and apoptosis:

Answer 17

• Prevents apoptosis through p53 inhibition via the PI3K-Akt pathway
• Alt: NFkB activation
• -> Upregulation of anti-apoptotic proteins (survivin, Bcl2)
• Notch inhibition is shown to increase expression of pro-apoptotic proteins

Question 18

Notch signalling in angiogenesis:

Answer 18

• Notch signalling has been shown to stimulate angiogenesis (linked with DLL-4)
• e.g. Head and Neck SCC

Question 19

TSG activity of Notch in BCC:

Answer 19

• Notch signalling via Hh pathway results in enhanced Gli expression, which controls SC proliferation
• Inhibited by PTCH1
• Can also act as a TSG through Wnt signalling (inhibition or deletion associated with increased beta-catenin expression)

Question 20

Biphasic action of Notch signalling in small cell lung cancer:

Answer 20

• Downregulates Slug, Snail, Vimentin (EMT TFs) whilst upregulating E-cadherin -> TSG by preventing EMT
• Preventing Cdk-inhibition (decreased p21 expression)

Question 20

Biphasic action of Notch signalling in non-small cell lung cancer:

Answer 20

• Upregulation of survivin, IGF-1R -> tumour growth and survival
• However, DLL4 overexpression reduces survival rates

Question 20

List key therapies against Notch signalling:

Answer 20

• Thapsigargin (S1 inhibitor via malfunction of SERCA pump)
• Notch receptor antibodies
• Antibodies targeting JAG1, DLL3, DLL4
• GSI and GSM (against gamma secretase complex)
• S2 inhibitors

Question 21

NOTCH3 and ND disease:

Answer 21

• Notch signalling is known to impact development of neurodegenerative disease including alzheimer’s and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)
• NOTCH3 mutations are known to cause CADASIL
• Mutations in presenilin are common in AD
• Downs syndrome is a major risk factor for ND disease as amyloid precursor protein is upregulated -> drives competitive inhibition of Notch-1 cleavage by presenilin-1 enzyme