9: External Factos Controlling Division and Behaviour of Normal and Cancerous Cells

Question 1

What is cell behaviour?

Answer 1

“Cell Behaviour” is the term used to describe the way cells interact with their external environment and their reactions to this, particularly proliferative and motile responses of cells.

Question 2

What external influences are detected by cells?

Answer 2

• Chemical:- hormones, growth factors, ion concs, ECM, molecules on other cells, nutrients and dissolved gas (O2/CO2) concs.
• Physical:- mechanical stresses, temperature, the topography or “layout” of the ECM and other cells

Question 3

What external factors can influence cell division?

Answer 3

• Although all external factors may influence cell proliferation, the ones to be considered
  here, in relation to cancer cell behaviour, are:-
• Growth factors
• Cell-cell adhesion
• Cell-ECM adhesion

Question 4

Steps in adhesion of tissue cells in culture

Answer 4

• cell settling on culture surface
• spreading
• acquiring motility (lamellipods are very important for this)

Question 5

Basic behaviour of cells in culture

Answer 5

• Cell-spreading is not a passive, gravity-dependent event. Energy is required to modulate cell adhesion and the cytoskeleton during spreading
• e.g.:
• A cell accidentally stuck on top of another.
• The top cell is blebbing as it has no contact with the ECM substratum.
• The bottom cell is beginning to spread, and it has less blebbing.

Question 6

How does Cell-ECM adhesion influence cell proliferation?

Answer 6

• Cells require to be binding to extracellular matrix to be fully competent for responding to soluble growth factors
• e.g. small adhesive patch: 30% chance of going into S-phase; bigger adhesive patch: 80% chance of going into S-phase.
• very small chance of entering S-phase in agar (without an adhesive patch)
• not only the amount but also the engagement if contact is important (e.g. one big piece of adhesive material cells die; same amount of adhesive material distributed and cells survive.

Question 7

Why is cell-ECM adhesion important?

Answer 7

• in suspension, cells do not significantly synthesise protein or DNA
• cells require to be attached to ECM (and a degree of spreading is required) to begin protein synthesis and proliferation (DNA synthesis)
• attachment to ECM may be required for cell survival
• cell phenotype can be determined by the matrix
• i.e. anchorage dependence
• Most cells need matrix

Question 8

A cell can receive information about its surroundings from its adhesion to ECM - how?

Answer 8

• Cells have receptors on their cell surface which bind specifically to ECM molecules
• these molecules are often linked, at their cytoplasmic domains, to the cytoskeleton
• this arrangement means that there is mechanical continuity between ECM and the cell interior
• INTEGRINS
• OUTSIDE IN SIGNALLING

Question 9

What are integrins?

Answer 9

• heterodimer complexes of alpha and beta subunits
• associate extracellularly by their “head” regions
• Each of the “leg” regions spans the plasma membrane.
• Ligand-binding occurs at the junction of the head regions
• more than 20 combinations of alpha and beta known -> each bind specifically to short peptide sequence on ECM proteins
• some can bind multiple molecules, others can only bind a specific molecule
• most link to the actin cytoskeleton via actin-binding proteins
• integrin complexes cluster to form focal adhesions (most) or hemidesmosomes (a6b4)
• these clusters are involved in signal transduction
• Some integrins also bind to specific adhesion molecules on other cells (e.g. avb3 binds to PECAM-1(CD31) and aIIbb2 to ICAM-1 on endothelial cells in inflammation)
• recruit cytoplasmic proteins which promote both signalling and actin assembly

=> important for blood clotting and immune function

Question 10

Which integrin does not bind to the actin cytoskeleton?

Answer 10

a6b4 integrin complex found in epithelial hemidesmosomes, linked to the cytokeratin (intermediate filament) network

Question 11

Focal adhesions

Answer 11

• formed by clusters of integrins

- actin filaments (ic) linked to the integrins and point to inside of the cell

Question 12

“outside-in” integrin signalling

Answer 12

• ECM receptors such as integrins can act to transduce signals
• e.g. ECM binding to an integrin complex can stimulate the complex to produce a signal inside the cell
• involves conformational changes in the complex
• the composition of the ECM will determine which integrin complexes bind and which signals it receives
• this can alter the phenotype of the cell
• recruit cytoplasmic proteins which promote both signalling and actin assembly

Question 13

What conformational changes occur in the complex in integrin signalling?

Answer 13

• Integrin complexes can adopt “flexed” and “extended” molecular confirmations
• Switching between these confirmations affects their ability to bind their ligands, and their signalling -> cell-ECM adhesion, and signals, can be switched on and off
• in the extended confirmation the legs are open

Question 14

How do focal adhesions sense the mechanical properties of their surroundings?

Answer 14

• The amount of force that is generated at a focal adhesion depends on both the force generated by the cytoskeleton (F cell) and the stiffness of the ECM

Question 15

Aspect of signalling to ECM receptors?

Answer 15

• signal generated inside the cell (e.g. as the result of hormone binding to receptor) can act on an integrin complex to alter the affinity of an integrin (i.e. alter its affinity for its ECM binding)
• this is “inside-out” intergin signalling (e.g. in inflammation or blood-clotting, switching on adhesion of circulating leukocytes)

Question 16

Outside-in vs. inside out signalling

Answer 16

Outside-in

• ECM receptors (e.g. integrins) can act to transduce signals
• e.g. ECM binding to an integrin complex can stimulate the complex to produce a signal inside the cell,

Inside-out

• a signal generated inside the cell (e.g. as the result of hormone binding to receptor) can act on an integrin complex to alter the affinity of an integrin (i.e. alter its affinity for its ECM binding)
• e.g. in inflammation or blood-clotting, switching on adhesion of circulating leukocytes

Question 17

Conformational changes ininteghrins at low and high affinity as well as when ECM binds.

Answer 17

• Low affinity: bent conformation, weak or no binding to ligand
• signal from inside the cell causes the change to high affinity conformation (inside-out signalling) -> switches on adhesion
• High affinity: extended conformation, strong binding to ligand
• ECM bound: further opening of the lies, the binding sites for recruitment of cytoplasmic signalling molecules.

Question 18

Cell populations in high density

Answer 18

• When cells in culture form a confluent monolayer, they cease proliferating and slow down many other metabolic activities.
• This used to be known as contact inhibition of cell division.
• Another set of experiments suggest that it is competition for external growth factors and not cell-cell contact responsible: Density-dependence of cell division.

Question 19

Is there cross talk between GF signalling and ECM?

Answer 19

• yes - SYNERGISTICALLY
• GF (density dependance) -> Ras -> MAPK -> Proliferation
• ECM binding via Integrins (anchorage dependence) -> ic signalling incl. MAPK -> Proliferation
• Both signals needed for efficient stimulation of proliferation (they activate the same pathway: MAPK); individually this activation is weak and/or transient.
  => the separate signalling pathways act synergistically (strong and sustained activation)

Question 20

What are the main types of contact interactions between cells?

Answer 20

• short-term: transient interactions between cells which do not form stable cell-cell junctions
• long term: stable interactions resulting in formation of cell-cell junctions

Question 21

What is the cell-cell contact in between non-epithelial cells like?

Answer 21

• when they collide they do not form stable cell-cell contacts.
• they “repel” one another by paralysing motility at the contact site, promoting the formation of a motile front at another site on the cell, and moving off in the opposite direction.
• This is contact inhibition of locomotion and is responsible for preventing multilayering of cells in culture and in vivo.

Question 22

Long-term cell-cell contacts

Answer 22

• Upon contact, some cell types strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions)
• This is true of epithelial cells and endothelial cells, which form layers, and neurones forming synapses.
• e.g. zonula (belts) and macula (spots)

Question 23

Cell-cell junctions in epithelia

Answer 23

Junctions are usually arranged as continuous belts (zonula) or as discrete spots (macula)

Question 24

Contact-induced spreading of epithelial cells

Answer 24

• Contact between epithelial cells leads to the mutual induction of spreading
  -> the total spread area of the contacted cells is
  greater than that of the sum of the two separated cells.
• This could result in a stable monolayer

The contact spreads along and actin is polymerised.

Question 25

How do cell-cell adhesions affect cell proliferation?

Answer 25

• No cell-cell junctions: activated MAPK,
  decreased p27KIP1, high proliferation
• Cell-cell junctions form, inactive MAPK,
  increased p27KIP1, low proliferation
• cell-cell junctions are calcium dependant so if you remove calcium, the junctions break down (but cells are still touching) so they go from low to high proliferation.
• if you add adhesion blocking antibodies (that bind to adhesion sites and block adhesion) you get the same effect with the increase in proliferation

Question 26

Molecular organisation of adherence junctions

Answer 26

• extracellularly there are cadherins that link the cells to identical molecules (cedherins) of other cells.
• cadherins are calcium dependent
• intracellularly there are catenins (beta and alpha) that connect this to actin filaments.
• beta-catenin may be the important link between cell adhesion and proliferation (not the only one but important)

Question 27

APC

Answer 27

• Adenomatous polyposis coli
• genetic, inherited colon cancer, thousands of polyps in the colon, high cancer risk
• the gene product in APC is a protein involved in degradation of the junction associated molecule beta-catenin (-> link between cell-cell adhesions and proliferation)

Question 28

Is there cross-talk between cell-cell adhesions and ECM adhesions?

Answer 28

• both have beta-catenin downstream
• APC is part of a complex that degrades beta catenin
• also Wnt pathway influences this

Question 29

What are the dynamics of beta-catenin in cells?

Answer 29

• beta-catenin does not last long in the cytoplasm (e.g. when the cell-cell junctions break down)
• if the APC complex is active there is rapid degradation
• if the APC complex is inactive, beta-catenin binds to Let-1 and acts in the nucleus, altering gene transcription leading to cell proliferation (e.g. in individuals with APC)

Question 30

Mechanism for contact inhibition of proliferation.

Answer 30

• when bound to cadherin at the membrane, b-catenin not available for LEF-1 binding and nuclear effects
• normally, cytoplasmic b-catenin rapidly degraded
• if b-catenin cytoplasmic levels rise as a result of inhibition of degradation or loss of cadherin-mediated adhesion, b-catenin/LEF-1 complex enters nucleus and influences gene expression, leading to proliferation.

Question 31

Other adhesion-associated signalling pathways are known to influence contact-induced inhibition of proliferation

Answer 31

• Clustering of cadherins after cell-cell contact is known to alter the activation of small GTPases
  e. g. Rac is activated, Rho is inhibited: this can influence proliferation.
• Some GFRs are associated with cell-cell junctions. This reduces their capacity to promote proliferation.

Question 32

What happens when cells lose their social skills?

Answer 32

• Under certain conditions, cells lose their behavioural restraints. As a result, they will:
  -proliferate uncontrollably (lose density dependence of proliferation)
  are less adherent and will multilayer (lose contact inhibition of locomotion and anchorage dependence)
  epithelia breakdown cell-cell contacts
  not Hayflick limited, express telomerase
  i.e. cancer
• there are consequences of loss of contact inhibition, e.g. these cells form solid tumours.

Question 33

What components in cell-signalling are photo-oncogenes?

Answer 33

many components of signal transduction pathways are proto-oncogenes:

• receptors
• signalling intermediates
• signalling targets (e.g. transcription factors)
• Mutations can make these pathways constitutively active
• uncontrolled proliferation as a result of loss of growth factor dependence etc. -> you don’t need the upstream signalling
• you need multiple mutations for cancer.

Question 34

Oncogenes vs. proto-oncogenes

Answer 34

• Oncogene: mutant gene which promotes uncontrolled cell proliferation
• Proto-oncogene: normal cellular gene corresponding to the oncogene

Question 35

In what proportion of cancers is Ras mutated?

Answer 35

Ras is mutated in ~30 % of all cancers

Question 36

Features of malignant cancers?

Answer 36

• in addition to deregulated proliferation, a major feature of cancerous tumours is their ability to spread
• most human cancers are carcinomas (i.e. of epithelial origin)
• in order to spread to other sites (metastasis), cells must break away from the primary tumour, travel to a blood or lymph vessel, enter the vessel, lodge at a distant site, leave the vessel, and ultimately establish a secondary tumour

Question 37

How does a primary tumour metastasise?

Answer 37

• cell-cell adhesion must be down-regulated (e.g. cadherin levels reduced)
• the cells must be motile
• degradation of ECM must take place; matrix metaloproteinase (MMP) levels increased in order to migrate through basal lamina and interstitial ECM
• the degree of carcinoma cell-cell adhesion is an indicator of how differentiated the primary tumour is, and indicates its invasiveness and the prognosis

Question 38

What fraction of cancer cells that enter the bloodstream survive to form a distant metastasis?

Answer 38

1 in 1000