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Id4 suppresses MMP2-mediated invasion of glioblastoma-derived cells by direct inactivation of Twist1 function

GJ Rahme and MA Israel

Oncogene, (IF=7.36 , 2013) Jan.13,2014

Doi: 10.1038/onc.2013.531

Presentation date : March 05,2014Advisor: Prof. Chi Shiun ChiangPresenter: Ahmad Usama Mohammad Mahmoud Student ID: 101012422

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The Outline

1- Introduction.

2- Materials & Methods.

3- Results.

4- Conclusion.

5- Comments.

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Introduction

Glioblastoma multiforme of the frontal lobe in a 60 year old woman. The tumor is multicolored (red and brown for recent and old hemorrhages, and yellow areas of necrosis).

Glioblastoma Multiforme (GBM):

- Glial Cells.- Primary brain tumor.- Common & Aggressive.- More common in males.- Age (over 50 years old).- Median survival : 4½ months.

http://www.uaz.edu.mx/histo/pathology/ed/ch_28/c28_s45.htm

http://library.med.utah.edu/WebPath/CNSHTML/CNS138.html

This glioblastoma has marked cellularity, with marked hyperchromatism and pleomorphism. Note the prominent vascularity as well as the area of necrosis at the left with neoplastic cells palisading around it.

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Matrix Metalloproteinases (MMPs) family :

- 23 members in human.- Zinc-dependent endopeptidases.- Digest Extracellular matrix (ECM).- Tumor invasion.- MMP2 & MMP9 for glioma invasion

http://en.wikipedia.org/wiki/File:Matrix_Metalloproteinases.png

Introduction

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introduction

Nature Review, Volume8, March 2007 , doi: 10.1038/nm2125

Frontiers in Bioscience 10, 353--366, January 1, 2005

MMP2 structure & expression control levels:

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Introduction

Inhibitor of DNA Binding/Differentiation Proteins (Id Proteins):

- 4 members (Id1 , Id2 , Id3 & Id4).- Ids – MMPs – Tumor invasiveness.- Id1 for MMP14.- Id1&Id3 for MMP2 &MMP9.- Id2 exception. - Id4 role in GBM invasion not well reported.- Twist1 (Basic Helix Loop Helix transcriptional factor).

Nature Review, Volume5, August2005, doi: 10.1038/nrc1673

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Materials & methods

1-Cell culture: U87, U251, A172, SW1088, SF767 and SNB44 adherent cell lines.

2-Invasion and motility assays

3-Kaplan–Meier analysis using TCGA(The Cancer Genome Atlas) data.

4-Retroviral infection.

5-Transfection:LipoD293 transfection reagent.

7-Chromatin Immunoprecipitation (ChIP).

8-RNA extraction and quantitative reverse transcription–PCR.

9-Western blot analysis.

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Materials & methods

10-Immunofluorescent staining.

11-Image deconvolution (Confocal microscopy).

12-Immunoprecipitation.

13-Conditioned media.

14-Zymography.

15-Function-blocking antibody experiment.

16-Luciferase reporter assays.

17-Site-directed mutagenesis.

18-Statistics.

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ResultsI-Id4 expression inversely correlates with invasiveness of glioma-derived cell lines

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ResultsII-Id4 attenuates glioma-tumor cell invasion without altering motility

- (V) Empty vector insertion.- (Id4) Id4 overexpression insertion- (Shld4 A&B ) infected cells express lower levels Id4 than others infected with (ShScram) insert.

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ResultsII-Id4 attenuates glioma-tumor cell invasion without altering motility

Cellular invasion through matrix is a complex process that involves several cellular processes including: 1-cellular motility.2-matrix degradation.

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ResultsIII- Id4 regulates expression of MMP2

A- Id4 overexpression (Intracellular level)

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ResultsIII- Id4 regulates expression of MMP2

B- Id4 knockdown (Intracellular level):

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ResultsId1 , Id2 , Id3 overexpression (Intracellular level) has no significant effect on expression levels of

MMP2 or MMP1, MMP3,MMP9 & MMP13

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ResultsIII- Id4 regulates expression of MMP2

Id4 overexpression (Extracellular level)

(1hr)

(6hrs)

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ResultsId4 regulates expression of MMP2

Id4 overexpression (Extracellular level)

cMMP2 levels can be regulated by inhibiting (MMP14/TIMP2) activation complex (no difference at this complex expression levels with Id4 overexpression)

These results indicate that Id4 downregulates MMP2 expression resulting in diminished extracellular levels of MMP2.

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ResultsIV- Id4 inhibits invasion of GBM cell lines by silencing MMP2 expression

- CM: Conditioned media from U87(v) & U251(v).- IgG: Control antibody.- IgG1αMMP2:Antibody known to block MMP2 function.- Sham Vector: Control empty vector.- pMMP2 Vector: MMP2 expression vector.

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ResultsV- Id4 interacts with Twist1 (Id4/Twist1 complex)

* Co-immunoprecipitation (Co-IP): (anti-FLAG tag Id4 & anti-Twist1 antibodies)

* Immunoprecipitation (IP): (Cell lysate for A172, SW1088, SF767 & anti-Twist1 antibody)

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ResultsV- Id4 interacts with Twist1 (Id4/Twist1 complex)

Id4 & Twist1 proteins expressed at similar subcellular localization that indicates these two proteins could interact

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ResultsAny Id family member interacts with Twist1 protein in U87 and U251 GBM cell lines ?

Id4 only the Id family member that interact with Twist1 protein in U87 & U251 GBM cell lines.

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ResultsWhether the Id4/Twist1 complex would alter mesenchymal makers expressed occasionally

in GBM ?

- Id4 can interact with Twist1 in glioma cells without influencing the expression of glioma mesenchymal markers.

Epithelial Mesenchymal Transition (EMT)

- TNC , Col5A1, CTGF, ACTA2, Fibronectin, N-cadherin & T-cadherin : are Mesenchymal Markers for EMT

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ResultsVI- Id4 inhibits Twist1 binding to the MMP2 promoter and Twist1-induced MMP2 expression

-pGL3 Basic vector: Luciferase reporter vector.- Two E-box sequences for Twist1 to bind with MMP2 promoter.

1.7 kb fragment lacks to Twist1 binding site but contains the other E-box sequence

Luciferase Assay

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ResultsVI- Id4 inhibits Twist1 binding to the MMP2 promoter and Twist1-induced MMP2 expression

- Twist1 binds to the MMP2 promoter and Id4 eliminates that association.- Overexpression of Twist1 overcomes the inhibitory action of Id4.

MM

P2

ChIP followed by RT-PCR

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ResultsVII- Id4 expression correlates with GBM patient overall survival and disease-free survival

, and inversely correlates with MMP2 expression

- Disease-Free Survival (DFS): The length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer.

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ResultsVII- Id4 expression correlates with GBM patient overall survival and disease-free survival

, and inversely correlates with MMP2 expression

- Overall Survival (OS): The length of time from either the date of diagnosis or the start of treatment for a cancer.

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ResultsVII- Id4 expression correlates with GBM patient overall survival and disease-free survival

, and inversely correlates with MMP2 expression

- MMP2 expression is significantly lower in patients with high Id4 than in patients with low Id4.

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conclusion

* Id4 expression inhibits glioma invasion in vitro by silencing MMP2 expression through a direct inhibitory interaction of Id4 with Twist1, a basic helix-loop-helix transcription factor highly expressed in GBM that mediates MMP2 expression and tumor invasion.

* Id proteins function to block the DNA binding activity of basic helix-loop-helix transcription factors. Twist1, a basic helixloop- helix transcription factor highly expressed in glioma, regulates EMT and upregulates MMP2 expression in many tumor types including GBM.

* Twist1 interacts directly with the MMP2 promoter to drive MMP2 expression.

* Upregulation of MMP2 resulting from decreased Id4 expression in GBM may contribute to the morbidity and mortality of GBM patients.

* Pharmacological inhibitors of Id1, Id2 and Id3 proteins proposed as therapeutics from other studies should be highly specific to avoid inhibition of Id4, as this would be expected to increase tumor invasion and enhance tumor associated morbidity.

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CommentsWhether the Id4/Twist1 complex would alter mesenchymal makers expressed occasionally

in GBM ?

- Id4 can interact with Twist1 in glioma cells without influencing the expression of glioma mesenchymal markers.

Epithelial Mesenchymal Transition (EMT)

- TNC , Col5A1, CTGF, ACTA2, Fibronectin, N-cadherin & T-cadherin : are Mesenchymal Markers for EMT

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Whether the Id4/Twist1 complex would alter mesenchymal makers expressed occasionally in GBM ?

- Observed a significant downregulation of EMT inducers Snail and Slug, which are downstream targets of Twist1, suggesting that

Id4/Twist1 complex is silencing other Twist1 downstream targets in addition to MMP2 and could regulate EMT in another cellular context.

Comments

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2) Recently, it has been shown in an evaluation of a brain tumor tissue array that Id4 expression is minimally detected in normal brain tissue, although it can be detected in glioma !!!

2) It may be that the tumors with elevated Id4 analyzed in the study by Zeng et al. have high expression of Twist1 and thus override the Id4/Twist1 inhibitory complex, or that Id4 has other functions in a subset of glioma.

1) why Id4 functions differently from Id1, Id2 and Id3. All Id proteins share a highly conserved HLH domain, yet Id1, Id2 and Id3 are associated with increased GBM invasion in contrast to our discovery that Id4 suppresses GBM invasion !!!

1) The amino-acid sequence of all Id proteins shows that Id4 has a unique polyalanine domain at the N terminus and a polyproline domain at the C terminus (data not shown). These domains that are unique to Id4 could convey specificity for the Id4/Twist1 interaction. Such an effect would be consistent with previous reports, suggesting that the N- and C-termini of Id proteins convey specificity for Id protein interactions.

Comments

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THANKS FOR YOR ATTENTION !

WELCOME BY YOUR QUESTIONS !