Transcript The abrogation of beta1 integrin function can generate single

Alpha5 integrin dependent 3D attachment, appearance, and migration
(Not 2D)
(pliable)
(cell-derived)
Some early studies in 3-Dimensional
Substrates: Bissell lab
• HMT-3522 Breast Epithelial cells
– S-1: nonmalignant
– T4-2: tumorigenic
• Grown on plastic: 2 cell lines appear similar
• Grown in reconstituted basement membrane
(rBM):
– S-1 cells form polarized, acinar structures in growth
arrest
– T4-2 cells form nonpolarized, proliferating amorphous
structures
S-1 +
anti-beta4
T4-2 +
anti-beta1
Physiological relevance of blocking beta1 function:
Cells in suspension, treated with anti-B1 antibody, injected SQ:
Reduced tumor number and size
AIIB2 = anti B1
Tyrphosin = anti EGFR
mAB225 = anti EGFR
•Reduced b1 levels
•Reduced EGFR
levels and activity
Grown in Basement Membrane
Active FAK, ILK and MAP Kinase downstream of beta1 and EGFR
Studies with HMT-3522 cell lines grown in a reconstituted BM link
Integrin signalling, adherens junction assembly, growth factor
Signalling, and tissue structure differentiation
Bissell & Radisky 2001
Drug Induced Apoptosis
ECM/Microenvironment
(Laminin)
Alpha6Beta4 integrin
(Hemidesmosome targeting domain)
•Beta4 integrin
deletion (HTD)
Polarity/ Basement Membrane
•Tailless beta4 integrin
•Blocking Abs to alpha6
NF-kappaB translocation
or beta4 integrin
•Mutant IkappaBalpha
To nucleus
•Proteosome inhibitor
Apoptosis
Survival
Membrane Type I Matrix Metalloproteinase Usurps Tumor Growth
Control Imposed by the Three-Dimensional Extracellular Matrix
Works in Collagen and Fibrin gels, not Matrigel; also in vivo
(uncleavable)
The Five Stages of Cell Migration
On 2D substrates: cells have fully mature focal contacts.
In 3D substrates: clustered integrins couple to less-completely assembled focal
interations and a predominantly cortical actin cytoskeleton; stress fiber formation is rare.
x
x
x
Lymphocytes
Neutrophils
The abrogation of beta1 integrin function can generate single-cell dissemination.
Insert movie with melanoma
explants
HT-1080 and MDA-MB-231 cells remain migratory after
pericellular proteolysis: compensation by mesenchymal-amoeboid
transition.
First insert videos from this paper
Now insert dermis/ SQ video
Green = not treated
Red = pretreated with PIs
•Elongated, spindle-shaped
•Beta1 integrin dependent
•ECM-degrading enzymes
colocalize with integrins
•Reduced elongation
•Increased morphodynamic flexibility
•Loss of focal beta1 integrin and MMP
clustering at interactions with ECM
•Less adhesion to collagens (lower B1 & B3
•More diffuse cortical actin distribution
Lymphocytes
Neutrophils
Role of Rho family of GTPases
In Mesenchymal-Amoeboid Transition
Effect of Rho and ROCK inhibitors on cells with a rounded vs. elongated
Phenotype - invasion into matrigel
Only rounded cells show a decrease in migration when treated.
3D in vitro matrigel
Tumor Xenograft
Effect of Rho levels on cellular phenotype
When grown in 2D, activation of Rho signalling completely inhibits
Motility of BE cells.
Comparison of A375M2 motility in 3D vs. 2D
•Motility on 2D substrate is not blocked by Y27632 treatment
Here insert movies 3 & 4
Green= beta1 integrin
BE
ezrin
A375M2
BE
M2
M2+ C3
M2+
Y27…
-/+ Ezrin Dominant Neg.
Rounded motility driven by Rho or ROCK does not require
pericellular proteolysis
WM266.4 Melanoma (mixed morphology)
??