TSLC(TUMOR SUPPRESSOR IN LUNG CANCER)1 SUPPRESSES EPITHELIAL CELL SCATTERING AND TUBULOGENESIS Mari Masuda, et. al. (2005) J. Biol. Chem. 280, 42164-42171

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TSLC(TUMOR SUPPRESSOR IN LUNG CANCER)1 SUPPRESSES
EPITHELIAL CELL SCATTERING AND TUBULOGENESISMari
Masuda, et. al. (2005)J. Biol. Chem. 280,
42164-42171

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Introduction

• TSLC1
• EMT
• Cyst
• Cell adhesion molecules
• Epithelial tissue and connective tissue
• ZO-1
• Tet-off System
• Cell scattering and tubulogenesis
• Rac, Rho, Actin cytoskeleton

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TSLC1

• TSLC/IGSF4A is a tumor suppressor gene in
  nonsmall cell lung cancer (NSCLC), which encodes
  a single membrane-spanning glycoprotein belong
  to the family of immunoglobulin superfamily cell
  adhesion molecules(IgCAMs)
• The cytoplasmic tail of TSLC1 contains a
  juxtamembrane sequence interacting with protein
  4.1 and a class II PDZ binding motif
• In a primary NSCLC we have found a 2-bp deletion
  in the TSLC1 the cause replacement of the
  c-terminal 19 amino acid residues, indicating
  that the cytoplasmic domain of TSLC is crucial
  for tumor suppression

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Epithelial tissue

• Epithelial tissue covers the whole surface of the
  body. It is made up of cells closely packed and
  ranged in one or more layers.
• Epithelial tissue, regardless of the type, is
  usually separated from the underlying tissue by a
  thin sheet of connective tissue basement
  membrane. The basement membrane provides
  structural support for the epithelium and also
  binds it to neighbouring structures

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Connective tissue

• Connective tissue is any type of biological
  tissue with an extensive extracellular matrix and
  often serves to support, bind together, and
  protect organs
• Connective tissue has abundant extracellular
  matrix
• Composed by smaller cells and low densities

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EMT (epithelial mesenchymal transitions)
Epithelial cells are the cells that line
virtually every organ in your body and 85 of
cancers derive from epithelial cells. During
embryonic development epithelial cells sometimes
dissolve their junctions with their neighbors and
become mesenchymal. Mesenchymal cells have a less
rigid shape and are more likely to be motile.
Epithelial to mesenchymal transitions, as well as
the reverse process, are extremely important for
normal development. In addition, these
transitions are important in wound healing, and
tumor cells that develop from epithelial cells
must transform into motile cells in order to
metastasize.
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Cyst

• The cyst is a spherical monolayer of polarized
  cells that encloses a central lummen

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Cell adhesion molecules (CAM)
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ZO - 1

• A marker of tight junctions, which is normally
  detected on the apical surfaces facing inwards of
  parental MDCK cyst

Apical
Basal
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Tet-off system
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Cell scattering

• Cell scattering is used to describe the
  dispersion of compact colonies of epithelial
  cells induced by certain soluble factors such as
  growth factors, cytokines, and phorbol esters
• Procedures
• Cell spreading
• Dissociation cells from each other
• Migration as individual cells

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Tubulogenesis

• Tubulogenesis processed during HGF-induced EMT.
  Generally speaking, it is related with tumor
  cells invasion and matastasis
• Procedures
• Formation of extensions
• Formation of single file chains
• Conversion to multilayered cords
• Maturation of tubules

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• Previous studies fromothers have shown that a
  constitutively active Rac mutant abolished
  HGF-mediated scattering
• And high in Rac and low in Rho activities are
  correlated with an epithelial phenotype by
  stabilizing E-cadherin cell-cell adhesion
• A transient decrease in Rac activity is
  essential to induce disassembly of cell-cell
  junctions

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Material and Method

• Cells and proliferation Assay
• Antibodies
• Expression Vectors and Transfection
• Protein Analysis
• Three-dimensional Culture in Collagen Gels and
  Tubulogenesis Assay
• Cell Scattering Assay
• Immunostaining and Confocal Microscopy
• Rac and Rho Activation Assay (GST pull-down
  assay)
• Statistics

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Three-dimensional culture

• Three-dimensional culture is a powerful tool for
  investigating the molecular signals, that specify
  epithelial architecture

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Immunostaining

• Direct immunofluorescence
• Indirect immunofluorescence

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Result
1.The obtained clones express the transgenes
at a nearly equivalent level 2. MDCK Tet-off
cells express a low level of endogenous TSLC
detected as faint bands
Overexpression of TSLC or any of the truncation
mutants does not alter cell growth in MDCK cell
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TSLC1
TSLC1
ZO-1
Merged
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TSLC1
TSLC1
ZO-1
Merged
TSLC1
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HGF(-) DOX(-)
HGF()
DOX(-)
DOX()
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HGF(-) DOX(-)
HGF()
DOX(-)
DOX()
TSLC1
?4.1-BM
?PDZ-BM
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• Quantitativeanalysis of 30 cysts for each cell
  clone demonstrated that the differencein
  tubulogenesis between the Dox-treated (TSLC1 off)
  and untreated(TSLC1 on) MDCK/TSLC1 cells was
  statistically significant (p lt 0.0001), whereas
  other cell clones showed no statistically
  significant difference between the Dox-treated
  and untreated cysts (pgt0.05)

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HGF(-) DOX(-)
HGF()
DOX(-)
DOX()
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HGF(-) DOX(-)
HGF()
DOX(-)
DOX()
TSLC1
?4.1-BM
?PDZ-BM
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A quantitative analysis of cell scattering
confirmed that the difference between the
Dox-treated and untreated MDCK/TSLC1 cells was
statistically significant with p lt0.0001. In
contrast, other cell clones showed no significant
difference between the Dox-treated and untreated
cells (pgt0.05)
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(No Transcript)
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• In response to HGF, the parental cells showed
  immediate early increase in Rac activity,which
  returned to basal levels within 15 min as well,
  as sustained Rho activation

• the HGF treatedMDCK/TSLC1 cells exhibited
  prolonged Rac activation, whichwas still observed
  4 h after stimulation, but there was no increase
  in Rho activity.

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Conclusion

• Both the Protein 4.1-BM and the PDZ-BM are
  Necessary for the lateral localization of TSLC1
  in cyst grown in a 3D culture
• Expression of TSLC1 in MDCK cells suppressed
  HGF-induced tubulogenesis
• TSLC1 inhibits HGF-induced cell scattering by
  suppressing induction of EMT
• MDCK/TSLC1 cells retained E-cadherin-based
  cell-cell adhesion even after treated with HGF

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Conclusion

• The expression of TSLC1 induces the prolonged
  activation of Rac and the reduced activation of
  Rho in HGF-stimulated MDCK cells
• The regulatory effect of TSLC1 on Rac activity
  appeared to depend upon its cytoplasmic domain
  because MDCK/C-HA cells showed a profile of Rac
  activation panel) similar to that in parental
  MDCK cells
• TSLC1 suppresses induction of EMT by modulating
  the activities of Rac and Rho

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The End
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Conclusion

• Both the Protein 4.1-BM and the PDZ-BM are
  Necessary for the lateral localization of TSLC1
  in cyst grown in a 3D culture
• Expression of TSLC1 in MDCK cells suppressed
  HGF-induced tubulogenesis
• TSLC1 inhibits HGF-induced cell scattering by
  suppressing induction of EMT
• MDCK/TSLC1 cells retained E-cadherin-based
  cell-cell adhesion even after treated with HGF