Lecture 21: Migration and invasion

Question 1

What are the different modes of cancer cell migration?

Answer 1

Amoeboid
Mesenchymal
Clusters
Multicellular strands/sheets

These can be switched between by cancer cells

Question 2

What is the role of Rho GTPases in mesenchymal migration?

Answer 2

1. Lamellipodium extension (Rac)
2. New adhesions o surrounding matrix proteins (Rac)
3. Cell body contracts (Rho)
4. Tail retraction

Activation: exchange of GDP for GTP (GEFs)
InactivationL hydrolysis of GTP to GDP (GAPs)

Question 3

What is the roles of Rho and Rac

Answer 3

Rho:
1. mDia: contructs actin bundles
2. Rock: increases myosin 2 activity
3. Stress fibre formation

Rac:
1. Regulate accumulation of actin at leading edhe
2. Binds to WASP protein family members activating Arp2/3 complex
3. Arp2/3 stimulates new actin filamets on side of existing filaments

Question 4

What are the two major groups of proteases in invasion>

Answer 4

1. Matric metalloproteinases (MMPs)
   a. family of 23 (secreted and membrane bound)
   b. degrade specific ECM components
   c. Release and activation of GFs
   d. Cleave cell surface adhesion molecules: integrins & cadherins
2. Serine proteases

Question 5

What is the strucuture of MMPs

Answer 5

1. Predomain: signal peptide - directs protein to the correct cellular location
2. Propeptide: synthesised as inactive pro-enzyme
3. Catalytic domain: Zn binding motif
4. Hemopexin domain: interactons with other MMPs
5. TM domain: membrane anchored MMPs

Question 6

How are MMPs activated and regulated?

Answer 6

they are secreted as pre-enzymes required proteolytic cleaveage for activation

Activation:
1. Serine proteinases (uPA / uPAR / plasminogen) - Pro-uPA binds
to uPAR and is activated, active uPA cleaves plasminogen to
plasmin (serine protease), Plasmin can cleave and activate a
number of pro-MMPs
2. Membrane bound MT1-MMP - this can activate MMP-2 & -13,
which can activate MMP-9
3. other secteted MMPs

Inhibition:
1. TIMPs (tissue inhibitors of MMPs)
2. Bind to MMPs and place them into inactive configuration

Question 7

What is the evidence for MMPs in metastasis?

Answer 7

In vitro
1. transwell assay
2. Highly invasive cells (HT1080)
3. MMP inhibition (active TIMP-2)
4. = reduction of HT1080 invasion (dose-dependent)

In vivo
1. expierimental metastasis assays
2. non-metastatic bladder cancer cell line (MYU3L) - over
expression of MMP-2 - activation of MMP-2 = increased
metastatic ability
3. Highly metastatic bladder carcinoma cell line (LMC19) = over
expression of TIMP-2
4. decreased metastatic ability

Clinical evidence
1. comparison of MMP-2 levels in normal and cacnerous breast tissue patients
2. increased production of MMP-2 associated with increasing breast cancer tumour severity

Question 8

How do MT1-MMP and MMP2 collaborate?

Answer 8

MT1-MMP expressed at leading edge of some cancer cells to breakdown basement membrane

Once in stroma, MT1-MMP activates MMP2 produced by stromal cells

These work together to degrade collagen matric in stroma, clearing a path for invasion

Question 9

Why have clinical trials to inhibit MMP activity failed?

Answer 9

1. clinical trials performed on late stage cancers
2. General MMP inhibitors affect almost all MMPs = too much ECM (side effect)
3. Some MMPs have anti-tumorigenic activity