Transcriptional profiling with a pathway-oriented analysis Transcriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes in the identifies dysregulated molecular phenotypes in the

endometrium of patients with PCOSendometrium of patients with PCOS

Haengseok Song, Ph.D.

Laboratory of Molecular Developmental Genetics Department of

Biomedical Sciences, CHA University

• PolyCystic Ovary Syndrome (PolyCystic Ovary Syndrome ( 다낭성난소증후군다낭성난소증후군 ))

• Pathway-oriented Analysis for MicroarraysPathway-oriented Analysis for Microarrays

• Aberrant Signaling Pathway in the Endometrium Aberrant Signaling Pathway in the Endometrium of the Patients with PCOSof the Patients with PCOS

Transcriptional profiling with a pathway-oriented analysis Transcriptional profiling with a pathway-oriented analysis identifies dysregulated molecular phenotypes of identifies dysregulated molecular phenotypes of

endometrium in patients of PCOSendometrium in patients of PCOS

Polycystic Ovary Syndrome (PCOS)Polycystic Ovary Syndrome (PCOS)

HPO

Axis

Hypothalamus: GnRH

뇌하수체

자궁내막

FSH LH

난소

Estrogen Progesterone

황체난포Pituitary Gland

Ovary난소

Endometrium

Follicle Growth and OvulationFollicle Growth and Ovulation

Ovary w/ PCOS

Steroidogenesis of Follicles in the OvarySteroidogenesis of Follicles in the Ovary

Theca Cell

Granulosa Cell

insulin↑

• Chronic anovulation, hyperandrogenism, and Chronic anovulation, hyperandrogenism, and frequently frequently accompanying insulin resistance and hyperinsulinemiaaccompanying insulin resistance and hyperinsulinemia..

• In ovaries of patients with PCOS, growth of In ovaries of patients with PCOS, growth of early antral early antral follicles is typically arrested at 5-10 mm stagefollicles is typically arrested at 5-10 mm stage. .

• The theca cell layersThe theca cell layers are prominent in these arrested follicles are prominent in these arrested follicles and represent the major source of the and represent the major source of the increased circulating increased circulating androgensandrogens in women with PCOS. in women with PCOS.

Polycystic Ovary Syndrome Polycystic Ovary Syndrome (PCOS)(PCOS)

• A common endocrine & metabolic disorder A common endocrine & metabolic disorder in women of reproductive age (5-10%). in women of reproductive age (5-10%).

• Microarray analysis for cultured theca cells from patients with Microarray analysis for cultured theca cells from patients with PCOS has demonstrated distinct biochemical and molecular PCOS has demonstrated distinct biochemical and molecular phenotypes different from the cells of regularly cycling women. phenotypes different from the cells of regularly cycling women.

• Another expression profiling for PCOS showed considerable Another expression profiling for PCOS showed considerable overlap with those of ovaries from long-term androgen-treated overlap with those of ovaries from long-term androgen-treated female-to-male transsexuals. female-to-male transsexuals.

• Androgens play a key role in the pathogenesis of PCOSAndrogens play a key role in the pathogenesis of PCOS..

• Oocytes from PCOSOocytes from PCOS patients have patients have molecular abnormalitiesmolecular abnormalities even though they appear to be morphologically normal. even though they appear to be morphologically normal.

Polycystic Ovary Syndrome (PCOS)Polycystic Ovary Syndrome (PCOS)

Pathophysiological Characteristics of PCOSPathophysiological Characteristics of PCOS

Obesity Hirsutism, Acne, .…

Follicle Arrest

The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS

• Endometrial cells in women with PCOS (PCOSE) can be Endometrial cells in women with PCOS (PCOSE) can be aberrantly influenced by various factors, such as insulin, aberrantly influenced by various factors, such as insulin, androgens and unopposed estrogens. androgens and unopposed estrogens.

• Due to the absence of ovulation, Due to the absence of ovulation, continuous exposure tocontinuous exposure to the stimulatory and mitogenic effects of the stimulatory and mitogenic effects of estrogens in estrogens in PCOSE could result in endometrial overgrowthPCOSE could result in endometrial overgrowth, possibly , possibly leading to hyperplasia and cancer. leading to hyperplasia and cancer.

• Characteristics of PCOS may cause implantation failure, Characteristics of PCOS may cause implantation failure, miscarriage, and cancer in the endometrium. miscarriage, and cancer in the endometrium.

• However, mechanisms underlying the pathophysiology of However, mechanisms underlying the pathophysiology of PCOSE have not been thoroughly explored.PCOSE have not been thoroughly explored.

The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS

The Endometrium of Patients with PCOSThe Endometrium of Patients with PCOS

Clinical Features of the patients with PCOSClinical Features of the patients with PCOS

Hierachical Clustering & Heatmaps of Up- and Hierachical Clustering & Heatmaps of Up- and Down-Regulated Genes in PCOSEDown-Regulated Genes in PCOSE

• dCHIP: (Biosun1.harvard.edu/complab/dchip/)

• Gene Pattern Gene Pattern : Designed to identify stage-specific signatures in tumorigenesis

• Gene Set Enrichment Analysis (GSEA) :Gene Set Enrichment Analysis (GSEA) :

• Stanford University

Developed Algorithms for Bioinformatics Developed Algorithms for Bioinformatics

• Significant Analysis of Microarrays (SAM): 2001, PNAS

• Harvard School of Public Health

• Broad Institute (Harvard & MIT)

From Individual Genes to Whole Networks of Genetic InteractionsFrom Individual Genes to Whole Networks of Genetic Interactions

• Gladstone Institute (Univ of California at San Francisco) : GenMAPP

• NIAID (NIH) : DAVID

• Univ of Michigan : Oncomine

• Developed at Stanford University (2001, PNAS)Developed at Stanford University (2001, PNAS)

• Uses data permutations to Provides estimate of False Discovery Uses data permutations to Provides estimate of False Discovery Rate (FDR) for multiple testingRate (FDR) for multiple testing

• Convenient Excel Add-inConvenient Excel Add-in

• Available for both DNA and oligo microarraysAvailable for both DNA and oligo microarrays

• Adjustable threshold determines number of genes called significantAdjustable threshold determines number of genes called significant

• Gene lists in Excel worksheets, easily exportable into various toolsGene lists in Excel worksheets, easily exportable into various tools

• Genes are web-linked to Stanford Genes are web-linked to Stanford SOURCESOURCE database database

SAM (Significance Analysis of Microarrays)SAM (Significance Analysis of Microarrays)Supervised learning software for genomic expression data mining

• Able to be applied to protein expression data and SNP chip dataAble to be applied to protein expression data and SNP chip data

www~stat.stanford.edu/~tibs/SAM/

www~stat.stanford.edu/~tibs/SAM/

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.

.

.

www~stat.stanford.edu/~tibs/SAM/

www~stat.stanford.edu/~tibs/SAM/

www~stat.stanford.edu/~tibs/SAM/

www~stat.stanford.edu/~tibs/SAM/

Gene A

Gene B

Gene C

Gene D

….

RNA Isolation Microarrays

qRT-PCR

Next Step ???

Sample Biopsy

Purpose of Microarray AnalysisPurpose of Microarray Analysis

Discovering the association of gene expression w/ Discovering the association of gene expression w/ biological and/or clinical sample characteristicsbiological and/or clinical sample characteristics

• SAMSAM• dCHIPdCHIP• Gene PatternGene Pattern• ……… ………

Single Gene Oriented Analysis

Major Limitations of Single Gene-oriented Major Limitations of Single Gene-oriented Approaches for MicroarraysApproaches for Microarrays

• A long list of statistically significant genes A long list of statistically significant genes w/o any unifying w/o any unifying biological themebiological theme

• Very likely to miss important effects on pathwaysVery likely to miss important effects on pathways

30% in all genes of a metabolic pathway vs a gene w/ 20 fold30% in all genes of a metabolic pathway vs a gene w/ 20 fold

Which one is more important to follow up?Which one is more important to follow up?

• Distressingly little overlapDistressingly little overlap in the list of statistically significant in the list of statistically significant genes from the two studies on the same biological systemgenes from the two studies on the same biological system

Pathway-oriented Approaches ?Pathway-oriented Approaches ?

• Identify underlying genetic abnormalities or signal transduction Identify underlying genetic abnormalities or signal transduction networks driving disease pathologies networks driving disease pathologies

• Interpretation of Microarray Data Interpretation of Microarray Data at the Level of Gene Setsat the Level of Gene Sets

• Gene sets are defined based on prior biological knowledge, Gene sets are defined based on prior biological knowledge, e.g., published information about biochemical pathways or e.g., published information about biochemical pathways or coexpression in previous experimentscoexpression in previous experiments

• Effectively bridge microarray data with biological significanceEffectively bridge microarray data with biological significance

http://www.genmapp.org/default.html

http://david.abcc.ncifcrf.gov/home.jsp

Gene Set Enrichment Analysis (GSEA)Gene Set Enrichment Analysis (GSEA)

• The goal of GSEA is to determine whether the members of S are randomly distributed throughout L or primarily at the top or bottom.

• It is expected that sets related to the phenotypic distinction will tend to show the latter distribution.

Leading edge subset

GSEA for Expression Profiles of PCOSEGSEA for Expression Profiles of PCOSE

• 141/164 gene sets : Up 141/164 gene sets : Up

• 44 gene sets at FDR < 25%44 gene sets at FDR < 25%

• 23/164 gene sets : Up 23/164 gene sets : Up

• 2 gene sets at FDR < 25%2 gene sets at FDR < 25%

PCOSNOR

Enriched Gene sets in Normal EndometriumEnriched Gene sets in Normal Endometrium

• Cell Cycle

• Cancer_Cell Cycle

• Cell Proliferation

• Cell Cycle Checkpoint

• Proliferation_Genes

• Cell Cycle Regulator

• Glucose_Down

• Glycolysis & Gluconeogenesis

• Insulin_2 Fold Up• Glut_Down

• Leucine_Down

• Pyrimidine Metabolism

• Purine Metabolism

• Leucine_Up

• Pyruvate Metabolism

• Gene Sets Associated with Cell Cycle Are Collectively Gene Sets Associated with Cell Cycle Are Collectively Down-regulated in PCOSE. Down-regulated in PCOSE.

• Some Gene Sets Associated with Glucose Metabolism Some Gene Sets Associated with Glucose Metabolism Are Down-regulated in PCOSE.Are Down-regulated in PCOSE.

• Metabolic Pathways Are Systemically Down-regulated Metabolic Pathways Are Systemically Down-regulated in PCOSE.in PCOSE.

GSEA, A Pathway-oriented Analysis Method, GSEA, A Pathway-oriented Analysis Method, Provide Information That …….Provide Information That …….

Cell Cycle Gene SetCell Cycle Gene SetPCOSCON

TGFβ1

RB1

CDKN1A

PRKDC BUB3

CCND2

CDK4

CCNE2

CCNA2

CDC6

ORC MCMWEE1

CDC7

CDC2

CCNA2

CDC2

CCNB1

CCNB2

CDC25B

CDC25C

MAD2L1

BUB1B

BUB1

CHEK1

PCNA

MCM2

MCM4MCM6

MCM5ORC6L

SMC1L1

CDC20

PTTG1

ESPL1

Cell Cycle Genes Are Down-Regulated in PCOSE Cell Cycle Genes Are Down-Regulated in PCOSE

** ****

**; p<0.01

Cell proliferation in stroma but not in epithelial Cell proliferation in stroma but not in epithelial compartments is severely impaired in PCOSEcompartments is severely impaired in PCOSE

LCM-Realtime RT-PCR Validated Cell-Type Specific LCM-Realtime RT-PCR Validated Cell-Type Specific Aberration of Gene Expression in PCOSEAberration of Gene Expression in PCOSE

LCM Realtime RT-PCR

Cell(s) of interest

Transfer film

Tissue section

Glass slide

PixCell II LCM systemPixCell II LCM system

Spot size

< 7.5 m

~ 15 m

~ 30 m

40 mW

25 mW

20 mW

Power Duration

450 s

1.5 ms

5 ms

Activated transfer film

The cells of interest are positioned in the center of the field

Transfer film is applied to the tissue surface

A focused laser beam is pulsed to activated the transfer film

Laser Capture Microdissection (LCM)Laser Capture Microdissection (LCM)

Laser Capture Microdissection (LCM)Laser Capture Microdissection (LCM)

LCM with VeritasLCM with Veritas

Down-regulation of Down-regulation of Glycolysis in PCOSEGlycolysis in PCOSE

PCOSNOR

HK1

PGM1

ACYP1

PKM2

Glycolysis, but not gluconeogenesis, is cooperativelydown-regulated in PCOSE.

HK1 (Hexokinase 1)PGM1 (Phosphoglucomutase 1)ACYP1 (Acrylphosphatase 1)PKM2 (Pyruvate Kinase)

Glycolysis & Gluconeogenesis

GlycolysisGluconeogenesis

Glycolysis Enzymes Are Down-Regulated in PCOSEGlycolysis Enzymes Are Down-Regulated in PCOSE

RT-PCRRT-PCR

Realtime Realtime RT-PCRRT-PCR **

**

**

**

**; p<0.01

Is the PCOSE Insulin-resistant ?Is the PCOSE Insulin-resistant ?

(Diabetes, 2006)

(Metabolism, 2007)

Leading Edge AnalysisLeading Edge Analysis

1 1 2 2 3 3 44

1 1 2 2 3 3 44

11 22 33 44

1.1. Integrin PathwayIntegrin Pathway2. ST_Integrin Pathway2. ST_Integrin Pathway3. Rho Pathway3. Rho Pathway4. Actin_Cytoskeleton_by_Rho_GTPases4. Actin_Cytoskeleton_by_Rho_GTPases

Integrin-Rho-Cytoskeleton NetworksIntegrin-Rho-Cytoskeleton Networks

MAP2K1

MAP2K2

MAPK1

MAPK3

SOS1

CSK

PXN

VCL

ACTN1 PIP5K1B

PFN1

MYLK

VCL

CFL1

PAK2

Cell Proliferation

PFN1

Integrin-mediated cell adhesion

Integrin-mediated cell adhesion

CDC42

Integrin-mediated cell adhesion

Rho Pathway

Integrin-Rho-cytoskeleton networks

Integrin-Rho-cytoskeleton network is cooperatively Integrin-Rho-cytoskeleton network is cooperatively down-regulated in PCOSEdown-regulated in PCOSE

A

B

• A variety of signaling pathways such as the cell cycle, DNA A variety of signaling pathways such as the cell cycle, DNA replication, apoptosis, glycolysis & integrin-actin cytoskeleton-replication, apoptosis, glycolysis & integrin-actin cytoskeleton-Rho network are dysregulated in PCOSE.Rho network are dysregulated in PCOSE.

• Glucose metabolism is impaired in the endometrium as well Glucose metabolism is impaired in the endometrium as well as the muscle of patients with PCOS.as the muscle of patients with PCOS.

• Proliferation of endometrial stromal cells, but not epithelial Proliferation of endometrial stromal cells, but not epithelial cells is severely impaired in PCOSE.cells is severely impaired in PCOSE.

• Pathway oriented analyses are better approaches to translate Pathway oriented analyses are better approaches to translate profiles of genome-wide expression into biological significance. profiles of genome-wide expression into biological significance.

CONCLUSIONSCONCLUSIONS

• Jin Yeong Kim• Sung Ran Hong• Tae Jin Kim• 제일병원 아이소망센터

• Hyun Joo Kim• Jae Eun Lee• Ji Young Choi• Soo Jin Hwang• Chang Se Lee

제일병원 분자종양연구실제일병원 분자종양연구실