TSLC(TUMOR SUPPRESSOR IN LUNG TSLC(TUMOR SUPPRESSOR IN LUNG CANCER)1 SUPPRESSES EPITHELIAL CELL CANCER)1 SUPPRESSES EPITHELIAL CELL

SCATTERING AND TUBULOGENESISSCATTERING AND TUBULOGENESISMari Masuda, et. al. (2005)Mari Masuda, et. al. (2005)

J. Biol. Chem. 280, 42164-42171J. Biol. Chem. 280, 42164-42171

銘傳大學生物科技學系銘傳大學生物科技學系姓名姓名 : : 尹馭群尹馭群學號學號 :91390576:91390576

IntroductionIntroduction TSLC1TSLC1 EMT EMT CystCyst Cell adhesion moleculesCell adhesion molecules Epithelial tissue and connective tissue Epithelial tissue and connective tissue ZO-1ZO-1 Tet-off SystemTet-off System Cell scattering and tubulogenesisCell scattering and tubulogenesis Rac, Rho, Rac, Rho, Actin cytoskeleton

TSLC1TSLC1 TSLC/IGSF4A is a tumor suppressor gene in nonsmall cTSLC/IGSF4A is a tumor suppressor gene in nonsmall c

ell lung cancer (NSCLC), which encodes a single membrell lung cancer (NSCLC), which encodes a single membrane-spanning glycoprotein belong to the family of immuane-spanning glycoprotein belong to the family of immunoglobulin superfamily cell adhesion molecules(IgCAMs)noglobulin superfamily cell adhesion molecules(IgCAMs)

The cytoplasmic tail of TSLC1 contains a juxtamembrane The cytoplasmic tail of TSLC1 contains a juxtamembrane sequence interacting with protein 4.1 and a class II PDZ sequence interacting with protein 4.1 and a class II PDZ binding motifbinding motif

In a primary NSCLC we have found a 2-bp deletion in thIn a primary NSCLC we have found a 2-bp deletion in the TSLC1 the cause replacement of the c-terminal 19 amie TSLC1 the cause replacement of the c-terminal 19 amino acid residues, indicating that the cytoplasmic domain no acid residues, indicating that the cytoplasmic domain of TSLC is crucial for tumor suppressionof TSLC is crucial for tumor suppression

Epithelial tissueEpithelial tissue Epithelial tissue covers the whole sEpithelial tissue covers the whole s

urface of the body. It is made up of urface of the body. It is made up of cells cells closely packedclosely packed and ranged in and ranged in one or more layers. one or more layers.

Epithelial tissue, regardless of the tEpithelial tissue, regardless of the type, is usually separated from the uype, is usually separated from the underlying tissue by a thin sheet of cnderlying tissue by a thin sheet of connective tissue; basement membronnective tissue; basement membrane. The basement membrane proane. The basement membrane provides structural support for the epitvides structural support for the epithelium and also binds it to neighbohelium and also binds it to neighbouring structures uring structures

Connective tissueConnective tissue

Connective tissue is any type of biological Connective tissue is any type of biological tissue with an extensive extracellular matritissue with an extensive extracellular matrix and often serves to support, bind togethex and often serves to support, bind together, and protect organsr, and protect organs

Connective tissue has abundant extracelluConnective tissue has abundant extracellular matrixlar matrix

Composed by smaller cells and low densitiComposed by smaller cells and low densitieses

EMT (epithelial mesenchymal transitions)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.

Cyst Cyst

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

Cell adhesion molecules (CAM)Cell adhesion molecules (CAM)

ZO - 1ZO - 1

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

Apical

Basal

Tet-off systemTet-off system

Cell scattering Cell scattering

Cell scattering is used to describe the dispersiCell scattering is used to describe the dispersion of compact colonies of epithelial cells inducon of compact colonies of epithelial cells induced by certain soluble factors such as growth faed by certain soluble factors such as growth factors, cytokines, and phorbol esters ctors, cytokines, and phorbol esters

Procedures:Procedures:

1.1. Cell spreadingCell spreading

2.2. Dissociation cells from each otherDissociation cells from each other

3.3. Migration as individual cellsMigration as individual cells

TubulogenesisTubulogenesis

Tubulogenesis processed during HGF-inTubulogenesis processed during HGF-induced EMT. Generally speaking, it is reladuced EMT. Generally speaking, it is related with tumor cells’ invasion and matastted with tumor cells’ invasion and matastasis asis

Procedures:Procedures:A.A. Formation of extensionsFormation of extensions

B.B. Formation of single file chainsFormation of single file chains

C.C. Conversion to multilayered cordsConversion to multilayered cords

D.D. Maturation of tubulesMaturation of tubules

*Actin cytoskeleton is regulated by small GTP-binding proteins

(Rho, Rac )

Previous studies fromothers have shown tPrevious studies fromothers have shown that a constitutively active Rac mutant abolihat a constitutively active Rac mutant abolished HGF-mediated scatteringshed HGF-mediated scattering

And high in Rac and low in Rho activities aAnd high in Rac and low in Rho activities are correlated with an epithelial phenotype re correlated with an epithelial phenotype by stabilizing E-cadherin cell-cell adhesionby stabilizing E-cadherin cell-cell adhesion

A transient decrease in Rac activity is essA transient decrease in Rac activity is essential to induce disassembly of cell-cell junential to induce disassembly of cell-cell junctionsctions

Material and MethodMaterial and Method Cells and proliferation AssayCells and proliferation Assay AntibodiesAntibodies Expression Vectors and TransfectionExpression Vectors and Transfection Protein AnalysisProtein Analysis Three-dimensional Culture in Collagen Gels and TubulogThree-dimensional Culture in Collagen Gels and Tubulog

enesis Assayenesis Assay Cell Scattering AssayCell Scattering Assay Immunostaining and Confocal MicroscopyImmunostaining and Confocal Microscopy Rac and Rho Activation Assay (GST pull-down assay)Rac and Rho Activation Assay (GST pull-down assay) StatisticsStatistics

Three-dimensional cultureThree-dimensional culture

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

ImmunostainingImmunostaining

Direct immunofluorescenceDirect immunofluorescence Indirect immunofluorescence Indirect immunofluorescence

ResultResult

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

MDCK(-)

TSLC1

ΔC-HA

TSLC1 TSLC1 ZO-1 Merged

TSLC1

TSLC1 TSLC1 ZO-1 Merged

Δ4.1-BM

ΔPDZ-BM

HGF(-)

DOX(-) DOX(-) DOX(+)

HGF(+)

MDCK(-)

GFP

TSLC1

ΔC-HA

HGF(-)

DOX(-) DOX(-) DOX(+)

HGF(+)

TSLC1

Δ4.1-BM

ΔPDZ-BM

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 < 0.0001), whereas other cell clones showed no statistically significant difference between the Dox-treated and untreated cysts (p>0.05)

HGF(-)

DOX(-) DOX(-) DOX(+)

HGF(+)

MDCK(-)

GFP

TSLC1

ΔC-HA

HGF(-)

DOX(-) DOX(-) DOX(+)

HGF(+)

TSLC1

ΔPDZ-BM

Δ4.1-BM

A quantitative analysis of cell scattering confirmed that the difference between the Dox-treated and untreated MDCK/TSLC1 cells was statistically significant with p <0.0001. In contrast, other cell clones showed no significant difference between the Dox-treated and untreated cells (p>0.05)

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.

ConclusionConclusion

Both the Protein 4.1-BM and the PDZ-BM are NBoth the Protein 4.1-BM and the PDZ-BM are Necessary for the lateral localization of TSLC1 in ecessary for the lateral localization of TSLC1 in cyst grown in a 3D culturecyst grown in a 3D culture

Expression of TSLC1 in MDCK cells suppressed Expression of TSLC1 in MDCK cells suppressed HGF-induced tubulogenesisHGF-induced tubulogenesis

TSLC1 inhibits HGF-induced cell scattering by sTSLC1 inhibits HGF-induced cell scattering by suppressing induction of EMTuppressing induction of EMT

MDCK/TSLC1 cells retained E-cadherin-based cMDCK/TSLC1 cells retained E-cadherin-based cell-cell adhesion even after treated with HGFell-cell adhesion even after treated with HGF

ConclusionConclusion

The expression of TSLC1 induces the prolonged The expression of TSLC1 induces the prolonged activation of Rac and the reduced activation of Ractivation of Rac and the reduced activation of Rho in HGF-stimulated MDCK cellsho 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

The End

ConclusionConclusion

Both the Protein 4.1-BM and the PDZ-BM are NBoth the Protein 4.1-BM and the PDZ-BM are Necessary for the lateral localization of TSLC1 in ecessary for the lateral localization of TSLC1 in cyst grown in a 3D culturecyst grown in a 3D culture

Expression of TSLC1 in MDCK cells suppressed Expression of TSLC1 in MDCK cells suppressed HGF-induced tubulogenesisHGF-induced tubulogenesis

TSLC1 inhibits HGF-induced cell scattering by sTSLC1 inhibits HGF-induced cell scattering by suppressing induction of EMTuppressing induction of EMT

MDCK/TSLC1 cells retained E-cadherin-based cMDCK/TSLC1 cells retained E-cadherin-based cell-cell adhesion even after treated with HGFell-cell adhesion even after treated with HGF