Cancer 11: Invasion Flashcards
What are the molecular mechanisms that regulate motility
microfilaments
regulation of actin dynamics
cytoskeletal proteins
signalling proteins
What are the steps of tumour progression
- Homeostasis
- Genetic alterations
- Hyperproliferation
- Dedifferentiation
- Invasion
What is involved in dedifferentiation
disassembly of cell-cell contacts
loss of polarity
What is inolved in invasion
increased motility
cleavage ECM proteins
Outline how cells change property during metastasis
Tightly bound together in priary tumours
Become mobile mesenchyme type cells and enter blood stream
Exit circulation and lose mesenchymal characteristics to form new tumour
Classify the types of tumour cell migratin
Amoeboid (small collection of cells) e.g. lymphoma
Mesenchymal (single cells or in chains) e.g. breast sarcoma
Cluster/cohorts e.g. epithelial cancer/melanoma, SCC
Multicecellular strands/sheets e.g. epithelial cancer/vascu tumours, SCC
Outline the the important proteins in each category of tumour cell migration
The individual (amoeboid and mesenchymal) and collective (cluster/cohort and multicellular strands) both require integrins and protease
The collective also require cadherins and gap junctions
Tumour cell metastasis is similar to what
Mimics morphogenetic events:
The cancer cell is de-differentiated and taken back to a stage of development in which cells would physiologically invade tissue
e. g.
- Branching morphogenesis
- Vascular sprouting
- Border cells (collective migration)
In these processes, there is a tip cell which is pushed up and degrades tissue (i.e. in lung development). SImilar to clusters and invasion
…
…
Differentiate the scratch wound assay with primary glial cells vs with glial tumour cell line
In primary glial cells, the cells can sense a gap, but they maintain cell-cell contacts and migrate coherently (collective migration)
In the cancer cell line, the cells can sense the gap, but do not migrate together and do not maintain any cell cell contacts
Compare the expression profile of invasive cells vs primary tumour cells
How was the experiment performed
Inject tumour cells into a mouse. Then insert a needle with EGF, which is chemotactic for the tumour cells (but only those which can METASTASISE, as we only want to look at the proteins in the cells that can invade… othe tumour cells that can’t migrate are left in the primary tumour)
There was upregulation (so more mRNA) of genes (in invasive compared to primary tumour) involved in:
- CYTOSKELETON REGULATION
- MOTILITY MACHINERY
Especially Arp2/3 and EGFR
What can stimualte cells to move
organogenesis and morphogenesis
wounding
growth factors/chemoattractants (i.e. like how EGF was injected into the mouse to attract cells from the primary tumour)
dedifferentiation (tumours)
What determines where the cells go
How do cells know when to stop
Where to go: directionality (polarity occurs so that the cells become the mesenchymal type in the slide, the thicker end is the direciton of movement)
When to stop: contact inhibition motility
How do cells move
specialized structures (focal adhesion, lamellae, filopodium)
How are cells atttached to ECM proteins
Via integrins, there is an intracellular plaque linking the cytoskelon to the ECM
What are focal adhesions
Focal adhesions are large, dynamic protein complexes through which the cytoskeleton of a cell connects to the ECM
Filamentous actin converges onto the plaque which, through which it links to integrin molecules (look at the image, where there are focal adhesions, there is convergence of filamentous actin )
What is vinculin
vinculin is a membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton
Differentiate filipodia and lamellipodia
Filopodia: Finger-like protrusions rich in actin filaments. Parallel filaments
Lamellipodia_ Sheet-like protrusions rich in actin filaments. Branched and crosslinked filaments.
Where can filipodia and where can lamallipodia be found
During extension of the cell in migration , there is the ‘leading edge of lamellipodia’ (see rock climbing image)
Filopodia (also microspikes) are slender cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating cells
Note, filopodia are not included on this diagram
What control is needed in cell movement
Control within a cell to coordinate what is happening in different parts
Control to regulate adhesion/release of cell-extracellular matrix receptors (i.e. to allow attachment to the ECM and then degrade these attachments at the back of the cell)
Control from outside to respond to external influences –
sensors
directionality
What allows for cell movement
Changing cell shape
Differentiate the types of motility in cell movement
Motility:
hapoptatic (cell moving in different direction)
chemotatic (all of the cell moving in one direction… required control)…
determined by external influences (sensors and directionality)
What are the stages of cell motility
Extension, adhesion, trnaslocation, deadhesion
Like rock climbing….. look at the diagram slide 15
What are the two types of actin
G actin and F actin
G is small soluble subinity
F is large filamentous polymer
What can hppen to actin in response to signal such as nutrient source
Disassembly of filments and rapid diffusion of subunits
Then reassemby of filaments at a new site (i.e. where the signal was detected)
Basically, filamentous actin in one part of the cell will break down into monomeric actin and then reassemble at the site that the migration is required
Explain the relationship between lamellipodia, filopodia, stressfibres and focal adhesions.
Ensure you compare the organisaiton of actin in each structure too
Lamellipodia= leading front of the migrating cell.
Filopodia= extensions of the lamellipodia branching out
Stress fibres=actin filaments which converge at focal adhesions with integrin molecules
Stress fibres have anti-parallel contractile strucutres.
These allow for the contraction at the back of the cell which drives the cell forward.
Relate slide 17 to slide 15
……
How can G-actin start being converted to F-actin
Nucleating (formation of a stable multimer of actin monomers)
F-actin can be modified. How?
After nucleation ,
severing, cross linking, capping, side binding, motor proteins and bundling
…
……..
Outline nucleation stage of actin polymerisation
Arp2 and Arp3 bind to other proteins and form an ARP compex
Then, at the MINUS end of the actin monomers which are forming into an actin filament, the ARP complex attaches
This leads to a NUCLEATED ACTIN FILMANET
Why is nucleation important in actin polymerisation
Limiting step in actin dynamics – formation of trimers to initiate polymerization
What happens in elongation stage of actin polymerisation
Addition and loss of G actin (dynamic) from the F actin chain.
Profilin binds to G- actin monomers and allows monomer binding to the forming actin filament
What is sequestering and what is it carried out by
Sequestering:
Think this refers to either the prevention of binding of G actin to the extending F-actin
Or the removal of G actin from the F actin chain
Look at which molecules
What can reduce the elongation process
SEQUESTERING:
- Profilin competes with thymosin for binding to actin monomers and promotes assembly
So thymosin binds to G-actin, but doesn’t allow monomers to be added to the forming actin filament.
Note that the Arp2/3 complex occurs at the negative end, but this means that the actin monomers are added on the positive end
They are reducing profilin action and thus reducing the polymerisation of monomers. They are a brake on polymerisation
- ADF/cofilin can also reduce elongation by severing monomers, therefore breaking down the actin filament
What occurs in capping, give examples of these proteins
The capping protein binds the end of the filament and prevent monomers from being added on.
The filaments are very dynamic. Once adding is blocked, there is a disassembly process that results that &shortening of the filament
Proteins:
+ve end capping: Cap Z, gelosin, fragmin/severin
-ve end: tropomodulin and Arp complex (technically caps in the nucleation stage)
What occurs in severing
The unsevered actin filament grows and shrinks, adding and removing monomers. Severing proteins chop the filament up
T/f severing actually speeds up the elongation process
T
In usevered popultin, actin filaments grow and shrink slowly
In severed population, they grow and shrink more rapidly
What is the function of gelsolin
Both caps at the +ve end and also severs
Give examples of severing proteins
gelsolin
ADF/cofilin
fragmin/severin
Elongation happens where on the filamentous actin
On the barbed end (requires energy, profilin-actin barbed end elongation)
Outline the fates of a cut actin filament`
- Barbed-end Capped (remains a shorter actin filament now)
- Elongation of the cut part of the filament
- Annealing to a new actin
Outline cross-linking and bundling
Note that everything up to this point has been involved in GENERATION OF THE ACTIN FILAMENT, whereas this stage involves ORGANISIATION of the generated ACTIN FILAMENTS
Some proteins can link separate actin polymers together in a parallel way, but at different distances apart
(fascin, fimbrin, a-actinin)
Spectrin links actin in a radiating structure
Filamin links actin in a non-parallel way
Dystrophin links actin to the membrane
Give examples of proteins involved in cross-linking and bundling
a-actinin fimbrin filamin spectrin villin vinculin
What occurs in buckling
When myosin acts between two actin polymers. It causes movements of the top strands together, allowing slack in the bottom strand
What is involved in branching
Arp complexes which allow for the polymerisation of actin (nucleation stage) attach to existing actin filaments always at 70 degree angle
in the lamellipod
(in fillipodia, which branch off of the leading edge of the lamellipodia, the actin filaments are now in parallel bundles, not branched/in this way, but there is still cross-linking between bundles)
What is the function of the arp complex
Nucleation of actin and also the branching
What is gel-sol trnastion
Conversion from a relatively rigid cytoplasm gel to flowing cytoplasm
Severing of actin is used (HENCE THE GELSOLIN PROTEIN! INVOLVED IN CAPPING AND SEVERING!) . The actin cross linking proteins (e.g. fascin) are still present, but the filaments are no longer forming a mesh, so it allows a sol that can flow
Allows cytoplasm to move into another area
Which of the following diseases is the actin cytoskeleton not involved in:
High blood pressure
Wiskott-Aldrich Syndrome – WAS (immunodeficiency, eczma, autoimmunity)
Duchenne Muscular Dystrophy (muscle wasting)
Bullous Pemphigoid (autoimmune disease)
Alzheimer (neurodegenerative)
Involved in all apart from alzheimers
Which cell activities need to occur during cell movement
The cell is adhered to the ECM through focal adhesion (involing integrin+actin filaments in the cell (stress fibres)
There is disassembly of actin then POLYERMISATION: nucleation, brancing, severing, capping, bundling etc. At the lamellipodium, the actin cytoskeleton will be branching, elongating, capping, severing etc. At the back of the cell, there will be severing and bundling etc
There is then gel/sol transition to allow the cytoplasm to flow into the lamellipodium
Then there must be a new attachment to the ECM at this point
Then contraction at the back of the cell
Then detachment fro the adhesion at the back of the cell
What will happen if there is no detachment of the focal adhesion at the back of the cell when the cell is moving forward
The cell will rip apart
Outline the process of lamellar protrusion
There is asembly of filaments due to brancing and capping at when the lamellipod protrudes in the direction of mocement
At the back of the lamellar there is severing, reasing G actin from the actin filaments which can move to the leading edge
NET FOLAMENT ASSEMBLY AT LEADING EDGE
NET FILAMENT DISSAMBLY BEHHIND THE LEADING EGE
Use of FILAMIN for cross linking (NOT parallel, but crosslinked)
What occurs in filipodia
There is actin polymerisation
There is lots of bundling and crosslinking of the actin to allow parallel actin strands with fascin. FAST ELONGATION
Then, upon removal of the stimulus, there is capping of the actin, and there is then retrograde flow and retraction as the base of the actin polymers are broken down
What are the important proteins in filipodia and in lamellipodia
Filipodia: fascin, a actinin
Lamellippdia: Arp2/3, fliamin (BRANCHED)
Which signallng mechanisms regulate the actin cytoskeleton
1 - ion flux changes (i.e. intracellular calcium)
2 – Phosphoinositide signalling (phospholipid binding)
3 – Kinases/phosphatases (phosphorylation cytoskeletal proteins)
4 - Signalling cascades via small GTPases
Think KIPS
How do small G proteins work
Once activated, small G proteins bind to specific proteins (known as effectors). These effectors are the messengers that carry out actions. Proteins are inactivated by hydrolysis of GTP –> GDP.
Give examples of the small G proteins
Rho subfamily of small GTPases belongs to the Ras super-family. Family members: Rac, Rho, Cdc42 best known
Which is the most important signalling mechanism in regulation the actin cytoskeleton
Small GTPases
What are small G proteins activated by
Proteins are activated by receptor tyrosine kinase, adhesion receptors & signal transduction pathways
T/F although some small G proteins are upregulated in some cancers, no mtations have been found yet
F:
Expression levels upregulated in different human tumours
Rho proteins are upregulated in tumours – mutations have been found that lead to hyper-activation
Give examples of how the Rho family of small GTPases can affect the cytoskeleton
Form cytoskeletal structures:
cdc42: induces filopodia
Rac; expansion and flattening of cell
Rho: induces stress fibres
Give an example of the mechanism by which a Rho family GTPase affects cytoskeleton
Rac protein activates WAVE and Arp-2/3, so Rac will induce polymerisation.
cdc42 will upregute WASP which increases Arp2/3 too
Actin binding proteins (arp) are regulated by which genes
Rac/Cdc42 GTPases
Outline the involvement of RAC, RHO and cdc42 at each stage of cell movement
Cdc42: filopodia, polarised motility and actin polierisation
RAC: forms lamellipodia. Focal adhesion assembly (with RHO)
RHO: contraction of stress fibres/tension at the end of the cell not moving. Also detaches the back of the cell from the old adhesion to allow forward movement
What could happen f you block Rho
Cell maybe ripped apart
Give 2 examples of molecules which are involved in the filopodia
Vinculin and actin
