Epithelial-Mesenchymal transition needed for organogenesis
Basics: cell contacts are lost, and as the primitive streak is formed during gastrulation, the transition occurs towards mesenchymal cells
TGF-B and Ras signaling required:
TGF-B: binding to its receptors leads to phosphorylation of SMAD2/3, and then SMAD4, then move to nucleus where SMAD complex can CONTROL Target gene expression.
A TF that aids in EMT during tumor development and progression
1. REPRESSES junction components E-cadherin, Claudin and Occludin, and desmoplakin.
2. NEGatively regulated by GSK-3B
Model of EMT induction used
In vitro. Looked at Snail and Smad involvement based on regulation of: CAR and E-cadherin
CAR: tumor suppressor, regulated by MEK-ERK pathway. DOWNregulated in TGF-B induced EMT
E-cadherin: established marker of E-cadherin
Fig 1 results
Main point: CAR and other junction components repressed with EMT, consistent with model. TGF-B treatment showed similar results to EMT positive model
EPH4 vs EPXT: Model to show EMT(XT) in mouse breast epithelial cells
XT showed: Western blot & mRNA detected decreases in Claudin, Occludin, and E-cadherin, and REPRESSION of CAR. Also Increase in Snail and Smad3&4
NMuMG: Shows EMT morphology
TGF-B induction: looked at NMuMG cells during timecourse w/TGF-B treatment and saw Decrease in same as above.
VIMENTIN (Marker for Mesen. Transition) was INCREASED after 72h!
Then looked at immunofluorescence: XT showed lacking of these junction proteins above, and same observed with TGF-B treated NMuMG cells.
Immunofluorescence for SNAIL & Smad3&4: In XT and TGF-B treated, saw LOCALIZATION to nucleus (consistent with model)
Fig 2 results:
Main point: Snail and SMAD (TFs) bind to regions of CAR, specifically the Snail binding elements (SBEs) and E-Boxes, respectively. These SNAIL and SMADS interact together
ChIP analysis showed SNAIL is involved with repression of CAR, by doing IP with different promoter regions and bringing Snail and SMADs. Then looked at binding to its promoter regions (SNAIL and SMADs are seen using antibodies against SMAD and SNAIL run on a gel). Seen with Both XT and NMuMG TGF-B treated cells.
Coimmunoprecipitation showed binding: With both XT and NMuMG TGF-B treated cells, Snail and SMAD3 and 4 were seen to interact together.
Fig 3 results
Main point: CAR does become repressed with SNAIL and SMAD 3 and 4 (TRANSCRIPTIONALLY REPRESSED), and repression seen with the other junction proteins claudin, occluding and E-cadherin.
Looked at promoter activity via luciferase reporter assay to see luciferase activity, which showed decrease as described above.
Also looked at mRNA and protein levels of CAR and e-cadherin with TGF-B treatment and GSK-3B inhibitor: saw decrease in mRNA levels, and only decreases in protein levels with TGF-B + , not GSK-3B. However, with both treatments, saw decreases and also INCREASES in SNAIL and SMAD3/4 protein.
Fig 4 results
Main point: Knockout of SNAIL and SMAD3/4 proteins with siRNA show that they act as transcriptional repressors to junctional components during EMT
siRNA showed a decrease in repression of CAR, occulin and E-cadherin at mRNA LEVELS
Fig 5 results
Immunofluorescence used to visualize mouse breast carcinomas: shows that SNAIL and SMADS colocalize together, and they are present in regions that are absent with CAR and e-cadherin. These numbers can be seen graphically
Vimentin used to show where mesenchymal area is…since is mesench. Marker
HER-2 used to show cells that are leaving towards EMT