Cancer 7: External factors controlling division and behaviour of normal and cancerous cells

Question 1

Define cell behaviour

Answer 1

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 is a lamellipod?

Answer 3

Cytoskeletal protein actin projection on the leading edge of the cell

Question 4

What is the basic behaviour of cells in culture?

Answer 4

Cell spreading - energy requiring process required to modulate cell adhesion and the cytoskeleton during spreading

Question 5

What is cell-ECM adhesion?

Answer 5

Cells need to be bound to the ECM to be fully competent for responding to soluble growth factors.

• They need to be attached to ECM to begin protein synthesis and proliferation.
• Attached to ECM for survival.

Anchorage dependence

Question 6

What external factors can influence cell division?

Answer 6

Growth factors
Cell-cell adhesion
Cell-ECM adhesion

Question 7

What can the cell phenotype be determined by?

Answer 7

The cell phenotype can be determined by the composition of the matrix.

Example: Cultured mammary epithelium

(A) in interstitial matrix (type 1 collagen), mammary epithelium does not differentiate to secretory cells;

(B) in basal lamina (basement membrane) matrix, mammary cells organise into “organoids” and produce milk proteins.

Question 8

How do cells receive information about its surrounding from its adhesion to ECM?

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

Question 9

What are integrins?

Answer 9

These are heterodimer complexes of alpha and beta subunits that 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

See slide for diagram

Question 10

How many different combinations of alpha and beta subunits are there?

Answer 10

More than 20. They bind specifically to short peptide sequences on ECM proteins.

For example alpha5beta1 fibronectin receptor binds arg-gly-asp (RGD)

Peptide sequences such as RGD are found in more than one ECM molecule.

Question 11

How do integrins link to the actin cytoskeleton?

Answer 11

Most integrins link to the actin cytoskeleton via actin-binding proteins.
Integrin complexes cluster to form focal adhesions (most) or hemidesmosomes.

These clusters are involved in signal transduction

Question 12

How does signalling happen from the the ECM to the inside of the cell?

Answer 12

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,
i. e. “outside-in” integrin signalling

Question 13

How do cell-ECM adhesion and signals be switched on and off?

Answer 13

Integrins complexes can adopt flexed and extended molecular confirmations.

Switching between these confirmations affects their ability to bind their ligands, and their signalling. In this way, cell-ECM adhesion, and signals, can be switched on and off.

Question 14

How can the the ECM alter the phenotype of the cell?

Answer 14

The composition of the ECM will determine which integrin complexes bind and which signals it receives.

Question 15

What do focal adhesions do?

Answer 15

They sense the mechanical properties of their surroundings - Cells can exert force on a environment causing it to stretch/deform.

The amount of force generated is dependent on the force generated by the cytoskeleton and the stiffness of the ECM

Question 16

What do integrins recruit?

Answer 16

Cytoplasmic proteins which promote both signalling and actin assembly.

Question 17

What is inside-out integrin signalling?

Answer 17

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 18

Summarise the conformation changes to the integrin complexes during activation and signalling

Answer 18

Low-affinity = bent confirmation, weak or no binding to ligand

High-affinity = extended confirmation, strong binding to ligand

Signal from inside the cell acts on the integrin complex to promote the switch to the extended high-affinity conformation: “inside-out” activation; switches adhesion on.

ECM ligand binds and causes the further opening of the legs. This exposes the binding sites for the recruitment of cytoplasmic signalling molecules:

Question 19

What is density-dependence of cell division

Answer 19

The higher the cell density the less cell proliferation - cells compete for growth factors

GF –> ERK MAP Kinase Cascade

Question 20

What signals control proliferation of tissue cells?

Answer 20

Growth factors (density dependence)
ECM (anchorage dependence)

Both signals needed for efficient stimulation of proliferation –> activate MAPK

Question 21

What are the different contact interactions between cells?

Answer 21

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 22

What happens when there is cell contact between non-epithelial cells?

Answer 22

When most non-epithelial cells “collide”, they do not form stable cell-cell contacts. They actually “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 23

Which cells form long terms contacts?

Answer 23

Epithelial cells and endothelial cells, which form layers, and neurones forming synapses.

strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions)

Question 24

How does cell-cell adhesion affect cell proliferation?

Answer 24

No cell-cell junctions, activated MAPK,
decreased p27KIP1, high proliferation

cell-cell junctions form, inactive MAPK,
increased p27KIP1, low proliferation

High calcium = no junctions produced. +ve Adhesion blocking
Low calcium = increased junctions formed. -ve adhesion blocking

Question 25

Describe the adherins junction?

Answer 25

Master junction - controls the formation of all the other junction types.

Molecule:
Cadherin (ca2+ depedent) - cell adhesion molecule
Beta-catenin
Alpha-catenin
Actin filament

Question 26

Describe the relevance of APC and a junction molecule.

Answer 26

APC gene-product is a protein involved in the degradation of the junction-associated molecule beta-catenin.

Question 27

beta-catenin dynamics

Answer 27

In the cytoplasma

APC complex active - rapid degradation of APC

APC complex inactive - LEF-1 –> nucleus - gene transcription leading to cell proliferation.

Question 28

What is the mechanism for contact inhibiton of proliferation?

Answer 28

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 29

What happens when cells lose their social skills?

Answer 29

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 

CANCER

Question 30

What does loss of contact inhibition cause?

Answer 30

Multilayering

Question 31

Other than promoting the formation of solid tumours, what is an important consequence of loss of contact inhibition of locomotion for the progression of cancer?

Answer 31

Metastasis - helps in the invasion properties of cells. They aren’t inhibited by other cells.

Question 32

How does a primary carcinoma cell metastasise?

Answer 32

• 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