Systems-Biology Study of RGD Mimetic Peptide in Breast Cancer

Systems-Biology Study of RGD Mimetic Peptide in Breast

Cancer

阮雪芬Nov 30, 2003

Yuki Juan’s Systems Biology Lab

SCIENCE, 2001, 294, 82-85


Outline

Introduction cDNA microarray Proteomics Bioinformatics


Introduction


Systems Biology Hiroaki Kitano

Science, 2002 Mar. Nature, 2002 Nov.


牛頓雜誌 2003 年 11 月號


Traditional Biology & Systems Biology Traditional biology :

Single genes or proteins Systems biology:

Simultaneously study the complex interaction of many levels of biological information to understand how they work together

Genomic DNAmRNA Proteins

Functional proteinsInformational pathways Informational networks


Systems Biology and Omics Data

Systems BiologySystems Biology

GenenomicGenenomic

ProteomicProteomic

TranscriptomicTranscriptomic

MetabonomicMetabonomic

Drug discovery Drug discovery Development processDevelopment process

Understanding drug toxicologyUnderstanding drug toxicology


The Structure of an Integrin Hynes in 1987 to emp

hasize the role of these RGD receptors in integrating the extracellular matrix outside the cell with the actin-containing cytoskeleton inside the cell.


The Interactions of Integrins with Other Proteins


Schematic Model of the Protein-protein Interactions

Signal are presumably transmitted into the nucleus, where they stimulate the transcription of gene involved in cell growth and proliferation


Integrin-activated Survival Signals

Integrins

Shc

FAK

Grb2/Sos

PI 3-kinase

Ras Raf MEK MAPK

Cell survival

Trends in Cell Biology, 1997, 7, 146-150


How RGD Trigger Apoptosis?

By integrin-mediated signal? Directly interact with the proteins in

cytosol?


How RGD Trigger Apoptosis?

b. RGD trigger apoptosis via integrin

Nature, 1999, 397, 534-539

a. Cell survive

c. Cell apoptosis by activating procaspase-3


Control Aggregation Cell Death

RGD(Arg-Gly-Asp) is the smallest motif that bind with the integrin receptor on the cell surface and play important role in cell cycle.

RGD and Cell Death


Our Study


Human breast cancer cell MCF-7

Cell Apoptosis

Genomic Study

Proteomics

Bioinformatics

Our Study


The Structures of RGD Mimetic Peptides

Asp

GlyArg NH

H2N O

O

N

O

HN

NH

O

O

OH

HN

O

HN

O

S

S

HN

O

NH

NH

H2N

ArgGly Asp

Trp

Pro

Cys

Tpa

Cyclic-RGD

RGD


RGD cRGDcontrol

1mM

5mM

0.5mM

1mM

control


Crystal Structure RGD and Integrin Crystal Structure of the

extracellular segment of integrin alpha Vbeta3 in complex with an RGD ligand

Science 296 pp. 151 (2002)


The 3D Structure of Cyclic-RGD

Cis form Trans form


Apoptosis Mechanism in MCF7

C-RGDCis or Trans?

C-RGDCis or Trans?


cDNA Microarray


Introduction to Microarray A powerful approach to

accurately measure changes in global mRNA expression levels.

Used to Discover novel genes Determine gene functions Evaluate drugs Dissect pathways Classify clinical samples.

Yuki Juan’s Systems Biology LabEur J Nucl Med 2002, 29, 115-32

Introduction to Microarray

mRNA

Data analysis

Control or treatment

Reverse transcriptase to generate Cy3/Cy5 cDNA probes

Hybridization to the gene chip


cDNA Microarray

C-RGD, 6hr C-RGD, 24hr

C-RGD, 48hr C-RGD, 72hr


Apoptosis Total 34 genes, but after

filtering there are only 19 genes

Total 11 genes have expression fold >2 (up or down changes)


Apoptosis Regulator

U60519

U97075

AF051941

U13738

AF005775

U60521

Z48810

AAF19819

U67319

U28976

AF015450


DescriptionGenebankaccession

No.

6 hFold Change

24 hFold Change

48 hFold Change

72 hFold Change

Group 1

caspase 10, apoptosis-related cysteine protease U60519 - - - 0.471

CASP8 and FADD-like apoptosis regulator U97075 - - - 0.355

nucleoside diphosphate kinase type 6 (inhibitorof p53-induced apoptosis-alpha) AF051941 - - - 0.376

Group 2

caspase 3, apoptosis-related cysteine protease U13738 - 2.301 - -

CASP8 and FADD-like apoptosis regulator AF005775 - 2.272 - -

Group 3

caspase 9, apoptosis-related cysteine protease U60521 - - 2.519 -

Group 4

caspase 4, apoptosis-related cysteine protease Z48810 2.615 - 2.796 2.819

Group 5

inhibitor of apoptosis protein AAF19819 - - - 5.249



Group 6

CASP8 and FADD-like apoptosis regulator AF015450 - - - 6.912

Apoptosis Regulator


6 7224 48

time (hour)0.01

0.1

1

10

Normalized Intensity(log scale)

p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1

6 7224 48

time (hour)0.01

0.1

1

10


p1


Using Linear Model to Construct Gene Network

Linear Model

ti;,btywΔt

tΔy

jiji,j

i

tBtyWt

ty

,

1~~~

YYYt

YW TT

D’haeseleer, 2000


Weights Matrix of Apoptosis Regulator

Weights Gene 1 Gene 2

2.670363 AF015450 U60521

2.068236 AAF19819 U60521

-1.889373 AF015450 AAF19819

-1.427408 AAF19819 AAF19819

-0.81632 AF005775 AF005775

-0.761848 U13738 AF005775

0.753277 AF015450 U60519

0.682257 U13738 AAF19819

0.646907 Z48810 AF005775

0.636552 AF015450 AF005775

0.632796 AF005775 Z48810

0.594627 AF005775 AAF19819

-0.55848 Z48810 AAF19819

0.543142 AAF19819 U60519

-0.527872 U60521 U60521

0.518056 U28976 U60521

0.508007 U60521 AF005775

0.499483 U13738 Z48810


Gene Network of Apoptosis Regulator

caspase 9, apoptosis-related cysteine protease

(U60521)

inhibitor of apoptosis protein (AAF19819)

CASP8 and FADD-like apoptosis regulator

(AF015450)

+2.068236

+2.670363

6

-1.889373


(U13738)

+0.682257

-0.761848

CASP8 and FADD-like apoptosis regulator

(AF005775)


(U60519)


(Z48810)

+0.646907

+0.636552

+0.753277


J03071 X15215 S75361 M37483 U14187 D12614 NM_005130 AF179274 AB017365 AF035835 D25328 NM_003242 S81439 AB017364 L13858 L27475 AF068868 M34480 NM_005928 AF005271 X53038 AI127370 AF002986

M37763 AF107885 AF081513 AJ000185 AF251118

D10202

U12535 AF026692

X14253

X51602 AF119815 U72338 AF041240 M37435

AB000509

AI634668 S77035 AF107885 U52112 AF056087

AF019634 X76079

BE336944 X52599 L24494 M64347 M12783 AF107885

U52112 M83575 L13857 AI692949 AW663903 AJ222700 M57399 U31176 X03438 AI885899 AB009249 U73737 U66406 AF266504 AW887370 AB039723 M77227 AF010312 M35878 NM_005429 AAC17439 U28054 L13858 L34641

X14253

NM_004791 AF035835 D63395 U94888 J03071 D87845 L13857 M21188 NM_004114 X14885 X70340

1 2

Signal Transducer


Weights Gene1 Gene2

-0.875598 NM_003242 L274750.834468 M37435 L27476-0.78655 L27475 L27477

-0.760695 M21188 L27478-0.728793 D63395 L274790.645775 NM_003242 NM_005429

0.608239 NM_003242 NM_005430

0.558504 L27475 NM_005431

0.538674 D63395 NM_005432

0.530764 U72338 L274750.455149 AF041240 L274760.453129 AI634668 L27477-0.452645 AB017364 L274780.449854 NM_003242 U12535

0.444301 M21188 AF251118

-0.442259 M37435 U52112

0.437687 M21188 U12535

0.429097 NM_003242 AF251118

Weights Matrix of Signal Transducer


transforming growth factor,

beta receptor II (70-80kD)

(NM_003242) Human interleukin-1 beta

converting enzyme gene, 5' flank.

(L27475)

colony stimulating

factor 1 (macrophage)

( M37435)

insulin-degrading enzyme

(M21188)

Notch (Drosophila)

homolog 4

(D63395)

-0.875598

+0.834468

vascular endothelial growth factor C

(NM_005429)

+0.645775

+0.608239

+0.558504

Human platelet activating factor

acetylhydrolase, brain isoform, 45

kDa subunit (LIS1) gene, exon 7.

(U72338)

hypocretin (orexin)

neuropeptide precursor

(AF041240)

+0.455149

msh (Drosophila) homeo box

homolog 1 (formerly homeo box 7)

(AI634668)

+0.453129 frizzled (Drosophila) homolog 2

(AB017364)

epidermal growth factor

receptor pathway substrate 8

(U12535)

+0.449854

Interleukin-1 Superfamily z

(AF251118)

+0.444301

+0.530764

-0.452645

-0.728793

+0.538674

-0.760695

Gene Network of Signal Transducer


Proteomics

Yuki Juan’s Systems Biology LabNature 2000, 405, 837-846

Two-dimensional Gel Approach


Control cRGD

97000

66000

45000

30000

14400

20100

4.0 5.0 6.0 7.0 10.08.0 9.03.2 5.5 4.0 5.0 6.0 7.0 10.08.0 9.03.2 5.5

1

Control vs c-RGD (6hr)

Yuki Juan’s Systems Biology Lab4.0 5.0 6.0 7.0 10.08.0 9.03.2 5.5

97000

66000

45000

30000

14400

20100

4.0 5.0 6.0 7.0 10.08.0 9.03.2 5.5

2

34

5 6 78

910

11 13

14

121516

17

18

Control c-RGD

Control vs c-RGD (24hr)


97000

66000

45000

30000

14400

20100

4.0 5.0 6.0 7.0 10.08.0 9.05.5

19 20

21 22

Control vs c-RGD (48hr)Control c-RGD


97000

66000

45000

30000

14400

20100

4.0 5.0 6.0 7.0 10.08.0 9.03.2 5.5

23

24 25

26

Control vs c-RGD (72hr)Control c-RGD


Proteomics Results 1. Semenogelin I protein precursor (S

GI) 2. Cell division protein kinase 6 3. Zinc finger protein 74 isoform 4 4. Keratin 5. Similar to presomitic mesoderm sp

ecific gene 6. Unnamed protein product 7. RNA-binding protein regulatory su

bunit 8. Similar to Claudin-6 (Skullin) 9. Unnamed protein product 10. Similar to Per-hexamer repeat pro

tein 5 11. Similar to L1 repetitive element O

RF 12. Hypothetical protein 13. Zinc finger protein 189

ISOFORM 2

14. Hypothetical protein 15. Similar to stretch response prote

in 553 16. Zinc finger protein 83 17. Immunoglobulin heavy chain var

iable region 18. Hypothetical protein 19. Cytokeratin 8 20. Cytokeratin 8 21. Zinc-alpha-2-glycoprotein precu

rsor 22. Keratin 18 23. Platelet-activating factor acetylh

ydrolase precursor 24. Topoisomerase II alpha 25. 13kD differentiation-associated

protein 26. Purified protein derivative-specif

ic T-cell receptor beta chain Red color: up-regulatedBlack color: down-regulated


Bioinformatics


RGD Peptides Can Be Used in Many Diseases

Thrombosis Osteoporosis Cancer

Any one else ??


Blood Clots Form Blood clots form wh

en platelets adhere to one another through fibrinogen bridges that bind to the platelet integrin


Clustering Analysis of Proteins

http://uranus.csie.ntu.edu.tw:9000/index.jsp


RGD-containing Proteins in Swiss-Prot Database In Swiss-Prot database, there are 738 h

uman RGD-containing proteins which containing 5 caspase proteins . Caspase 1, caspase 2, caspase 3 and caspa

se7, caspase 8.


RGD-containing Proteins in Swiss-Prot Database

Heat shock protein Dna Chaperone DnaJ Alzheimer's disease SM22　　　 Leiomyoma


Conclusion and Discussion



Cyclic RGD exerts more potency than that of liner RGD on the inhibiting cell growth.

The cyclic RGD exerts 8-10 times potency more than that of liner RGD peptide in inhibiting proliferation and inducing clustering of MCF-7 cells.

Cyclic RGD can induce the apoptosis of MCF7. We showed many caspases involved in this apoptosis and constructed the caspase pathway.



CASP8 and FADD-like apoptosis regulator, caspase 9 and inhibitor of apoptosis protein formed the positive and negative feedback control system.

Vascular endothelial growth factor C and human interleukin-1 beta converting enzyme gene have the important positions in the gene network because they will affected many other genes.

Clustering tool maybe could predict some novel functions in RGD-containing proteins.


Outlook

cDNA microarray

Drug discovery

ProteomicsApoptosis pathway

Cellular mechanism

Bioinformatics


Acknowledgement

中央研究院生化所陳水田黃宣誠徐駿森陳正良曾湘文吳嘉欽

台灣大學資工系歐陽彥正陳倩瑜蕭博仁台北科技大學化工系王怡琇


Thank you!

Documents

Systems-Biology Study of RGD Mimetic Peptide in Breast Cancer