Cancer Cell Signalling

Question 1

Drug development to target signalling molecules

Answer 1

Monoclonal antibodies to inhibit GFRs (Herceptin in breast cancer) or TKR inhibitors (Gefitinib for lung cancer)
Tumours can develop resistance through mutating to therapies.

Question 2

General domain structure of transmembrane receptors

Answer 2

Discriminator - binds a specific ligand
TMD - anchors in the membrane
Effector - directly / indirectly linked to intracellular enzyme activities.

Question 3

RTKs (EGFR) activation, mutations and therapies

Answer 3

Activation mechanism - ligand binding causes dimerisation, conformational change in cytoplasmic domain enables auto-phosphorylation and recruitment of signalling proteins or scaffold proteins (for signalling complexes)

Oncogenic mutations increase signalling - ligand independent activation (when no ligand or no extracellular domain), over expression of receptors or copy number amplification to increase receptor firing.

Therapies - Gefitinib targets the ATP binding site of RTK domains in EGFR, leading to no activation of the receptor. Tumours can develop resistance via ATP binding site mutations. Usually within 8-10 months.

Question 4

Mechanism of phosphorylation and its functional consequences

Answer 4

Addition of a phosphate group (Ser>Thr>Tyr residues). Kinases add, phosphatases remove.
30% of human proteins can be phosphorylated.
- conformational changes
- activation/inactivation
- alter binding partners
- change subcellular localisation
- degradation by the proteasome

Question 5

Mechanism of degradation by ubiquitination

Answer 5

PTM adding a Ubq group for degradation via the proteasome. Turns off signalling.
- E1 recruits the ubiquitin protein
- Activated Ub is transferred to E2 conjugating enzyme.
- E3 transfers Ub to Lys of target protein
- Deubiquitination occurs via DUB proteases
EX - B-catenin in Wnt

Question 6

How can mass spec be used to identify the ubiquitinated proteome?

Answer 6

Digest ubiquitnated proteins to leave a di-Gly tag.

This tag can be targeted by antibodies, measure abundance and identify the protein by mass spec.

Question 7

What are the PTMs possible and how do they interact?

Answer 7

Phosphorylation, ubiquitination, acetylation and methylation.
They may promote or antagonise each other.
Can affect the same amino acid residue.
One PTM may be required for a subsequent modification.

Question 8

What do signalling pathways lead to?

Example

Answer 8

Leads to transcriptional regulation
Wnt -> B-Catenin/TCF -> Cyclin D1 (g1-s phase), Myc (cell cycle and apoptosis), fibronectin, MMP7 - Overall increase in cell proliferation

Question 9

Functions of the Wnt pathway
Pathway basics
Defects

Answer 9

Functions - body axis determination, cell fate decisions, cell proliferation and cell migration.

Wnt ligand binds Frizzled and LRP5/6 co-receptor, activated Dishevelled and phosphorylates Axin so it cannot form destruction complex and cause ubiquitin degradation of B-catenin. B-catenin levels rise, translocates to the nucleus, binds TCF and activates transcription of target genes by displacing Groucho co-repressor

Under or over activity of the pathway can cause disease.
Cause cancers, neural tube defects, bone density defects, tooth agenesis and diabetes.

Question 10

Difference between canonical and non-canonical Wnt signalling pathways

Answer 10

Canonical pathway - B-catenin dependent. Causes differentiation and proliferation. Leads to colorectal cancers.

Non-Canonical - Planar cell polarity pathway. Activates JNK for tissue patterning and cell polarisation. Causes neural tube defects. Slightly involved in migration.

Question 11

What is the Wnt ligand?

Answer 11

Small secreted glycoprotein that targets the plasma membrane. 19 different Wnt ligands in the human genome and are all highly conserved, cannot tell which pathway they target.

Question 12

What are Dickkopf proteins?

Answer 12

Wnt antagonists that bind to the LRP5/6 co-receptor. Turned on by canonical Wnt signalling and provides negative feedback mechanism.
Dkk’s are tumour suppressors and turned off in colorectal cancers.
Dkk1 & Sclerostin inhibit Wnt signalling to reduce bone formation. Anti-sclerostin antibodies can treat osteoporosis.

Question 13

What is Tankyrase?

Answer 13

Tankyrase normally degrades Axin and can be inhibited to prevent B-catenin accumulation.

Question 14

How to measure Wnt signalling?

Answer 14

The Topflash system - connect TCF binding regions to reporter luciferase and introduced into cell as a plasmid.
Fopflash (just Luc) gives background radiation, so Top:Fop ratio is measured for Wnt output.

Question 15

Events in the formation of colorectal cancer.

Answer 15

Cells in the crypt become transiently amplifying cells, moving up the crypt walls into the villus. Wnt signalling is turned off, the cells stop proliferating and differentiate into the cells of the small intestinal lining. They continue moving to the tip of the villus where they are shed and die as a cancer defence mechanism.

When Wnt signalling is mutated (through APC or B-catenin mutations) it blocks the transiently amplifying cells from moving and stops the differentiation process. Cells accumulate, mutate further, form polyps and eventually carcinomas.

Question 16

What specific mutations lead to the overactivity of Wnt signalling?

Answer 16

B-catenin - deletion of codon encoding the phosphorylation site so it can escape GSK3B and the destruction complex.
APC mutations - Truncation mutation to remove the B-catenin binding site. APC is a tumour suppressor, one mutation in germline, one somatically (2HH)

Both mutations are not needed for colorectal cancer, independent.

Question 17

What are the experimental systems in cancer signalling?

How can these be perturbed? - EX (Wnt)

Answer 17

• Clinical samples - tumour biopsy samples and human bio fluid samples
• Model organisms - genetic knockout mice to generate a cancer-like environment or xenografts.
• Cultured human cells - immortalised cells from tumour to create mini organs in culture.

Adding ligands, genetic deletion, pharmacological inhibitors/activators, mutant kinases.

Add recombinant and purified Wnt ligand, inhibition of GSK3B or B-catenin genetic knockouts.

Question 18

What are genome wide RNAi screens and how do they work?

Answer 18

siRNA inhibits expression of certain genes. RNAi screens use siRNA to observe the effect on a phenotype of interest.
No mechanism, but shows P-P interactions and which are important for signalling.

Question 19

What is yeast-two hybrid?

Answer 19

Yeast Two-Hybrid is an assay performed to see if two proteins interact. It can be scaled up to test 1000s of proteins at a time using libraries.
Separates the activation and DNA binding domains that activate in close proximity.
See if the reporter gene is transcribed and the yeast cell is able to grow.

Question 20

Affinity-purification mass-spectrometry

Example

Answer 20

Insert an antibody against a certain protein of interest.
Semi-purify it and identify the attached interacting partners obtained from the sample by mass spectrometry.
Can identify complexes, which Y2H cannot do.

DNMT1 example - Discovered B-catenin as an interacting partner for DNMT1 using epitope tag. Cross-talk in Wnt pathway and DNA methylation. Regulates DNMT1 stability and can alter methylation patterns to silence Wnt antagonists.