Meng LiSchool of Informatics

Interdisciplinary Program of Biochemistry Indiana University

Advisors: Dr. Sun Kim, Dr. Kenneth Nephew4/25/2008

1

OverviewBiology background and experimental modelObjectiveHigh-throughput data and data analysisCombinatorial study and data miningConclusions

2

Ovarian cancer and drug resistanceOvarian cancer

Most deadly gynecological malignancyCisplatin

Widely used chemotherapeutic drugDNA intercalating agent

Cisplatin resistance70% to 80% of patients develop resistance

after 2-year treatment

3

DNA methylation

4

CpG dinucleotides: 5’- AATACGCCACGA

DNA methylation

5

C: cytosinemC: methylcytosine

CG MCG MCG

Normal

CpG island

CG CG CG CG

X

CG CG

Cancer

CGMCGMCG MCG MCG

De novo methylation: acquired methylation

ObjectiveDoes de novo DNA methylation plays a

role in the development of chemotherapeutic drug resistance in ovarian cancer cells?

How does de novo DNA methylation affect drug resistance development?

6

In vitro drug resistance system

Drug-sensitiveparental cells

A2780 R-

Drug-resistant Cells

A2780 R+

Cisplatin

Rounds of Cisplatin treatment

IC5

0 (u

M)

7

Cisplatin

High-throughput data and data analysisGlobal promoter methylation dataGlobal gene expression data

8Louis Staudt, The nation’s investment in cancer research (NCI)

Global promoter methylation profiling

Differential Methylation Hybridization (DMH)44,000 probes

representing 10,000 genes

Two-color microarray analysisData processingEstimate methylation level

9CpG Island Microarray (44K)

A2780 R- A2780 R+

Raw Data Normalized Data

Global promoter methylation profilingLoess normalization: correcting technical bias

Fold-change analysis: extracting gene methylation level

M = Green

Redlog2

10

Global gene expression profilingAffymetrix U133 plus 2.0

microarray 54,675 probes

representing 20,606 genes

Single-color microarray analysisData processingEstimate gene expression

levels

11

mRNA

cRNA

U133 plus 2.0 array

Global gene expression profilingClustering

Cutoffs:

- p-value < 0.01

- fold change >= 1.5

A2780 R-A2780 R+

12

Fold changeA2780R+ expressionA2780R- expression

Welch’s t-test p-values

Combinatorial study and data mining

Does de novo DNA methylation plays a role in the development of chemotherapeutic drug resistance in ovarian cancer cells?

How does de novo DNA methylation affect drug resistance development?

13

The number of hypermethylated genes positively correlated with the increase of IC50

14

DNA methyl-transferases (DNMT) are up_regulated in resistant cells

Welch's t-test Fold change Gene title

DNMT1 0.0011 1.63 DNA (cytosine-5-)-methyltransferase 1DNMT2 0.3673 1.17 DNA (cytosine-5-)-methyltransferase 2

DNMT3A 0.1009 1.20DNA (cytosine-5-)-methyltransferase 3 alpha

DNMT3B 0.0004 1.80DNA (cytosine-5-)-methyltransferase 3 beta

DNMT1: maintain genomic DNA methylationDNMT3B: de novo methylation

15

Resistant cells re-establish cisplatin sensitivity after methylation inhibitor treatment

16

x

Key questionsDoes de novo DNA methylation plays a

role in the development of chemotherapeutic drug resistance?Yes

How does de novo DNA methylation affect drug resistance development?Does de novo methylation selectively blocking

transcription factor binding?

17

Surveying Transcription Factor Binding Sites (TFBS) on differentially methylated regionsScan TFBS on hypermethylation (S+),

hypomethylation (S-), or hypermethylated CGI (SCpG) regions with match program against TRANSFAC database

.

.

.

.

.

*S+

.

.

.

.

.

*

*S-

.

.

.

.

.

*

**

SCpG

18

Methylation selectively occurs at certain TFBS

19

Statistical scoring

TFBS S+ vs. S- S+ vs. Sr S+ vs. SCpG

NCX 0.000698 5.49E-10 0.0007561HMGIY 0.00113 6.14E-16 2.88E-05CEBP 0.008758 1.53E-09 0.0001531BRCA 0.01259 1.67E-06 0.001406

• Fisher exact test• Multiple test correction – False discovery

rate (FDR)

20

Key questionsDoes de novo DNA methylation plays a

role in the development of chemotherapeutic drug resistance?Yes

How does de novo DNA methylation affect drug resistance development?Does de novo methylation selectively blocking

transcription factor binding? YesDoes de novo methylation selectively regulates certain

pathways?

21

Methylation regulated pathways

UpRegulated Genes fc > 1.5 (1037) HypoMethyl fc <-1.5 and UpReg genes p<0.01 fc >1.5 (55)

Impact Input genes in pathway

Corrected p-value Impact Input genes in

pathwayCorrected

p-valuefisher.test

p-value

Glioma 6.09 6 0.016 11.909 3 8.69E-5 0.013

Melanoma 4.516 5 0.060 10.268 3 3.91E-4 0.009

Pancreatic cancer 5.237 8 0.033 9.09 3 0.0011 0.023

Prostate cancer 5.064 9 0.038 8.991 3 0.0012 0.029

Colorectal cancer 3.108 6 0.184 8.758 3 0.0015 0.012

Chronic myeloid leukemia 3.169 6 0.175 8.221 3 0.0025 0.012

Non-small cell lung cancer 1.376 2 0.600 5.59 2 0.0246 0.044

• Hypomethylation up-regulated pathways

All are human cancer related pathways

22

E2F

Hypomethylated and up-regulated pathwaysGenes involved for each pathway:

PIK3R3, PDGFRA, E2F1, TGFBR2

Smad2/3

Smad4

23

Methylation regulated pathways

DownReg Genes fc < -1.5 (1286) HyperMethyl fc >1.5 and DownReg genes p<0.01 fc <-1.5 (55)

ImpactInput

genes in pathway

Corrected p-value Impact Input genes

in pathwayCorrected

p-valuefisher.test

p-value

Cell adhesion molecules (CAMs) 384.485 14 1.95E-164 897.568 4 0 0.005052

Tight junction 8.868 13 0.0014 17.229 3 9.93E-7 0.02503

PPAR signaling pathway 3.68 6 0.1172 7.377 2 0.0052 0.03946

• Hypermethylation down-regulated pathways

Cell adhesion molecules (CAMs): Environmental information processing

Tight junction: Experimental processes -> Cell communication

PPAR signaling pathway: Experimental processes -> Endocrine system

24

Hypermethylated and down-regulated pathwaysGenes involved for each pathway:

CAMs (ITGAV, CLDN11, NEO1, CDH2)Tight junction (CLDN11, PPP2R4, INADL)PPAR signaling (CPT1A, SLC27A6)

25

Key questionsDoes de novo DNA methylation plays a

role in the development of chemotherapeutic drug resistance?Yes

How does de novo DNA methylation affect drug resistance development?Does de novo methylation selectively blocking

transcription factor binding? YesDoes de novo methylation selectively regulates certain

pathways? Yes

26

ConclusionPromoter CpG island methylation

Participates in the development of drug resistance of ovarian cancer cells

Regulates gene expression alteration through drug resistance development by selectively occurring at certain TFBS

Regulates cellular functions by methylating key players in certain pathways

27

AcknowledgementsAdvisors

Sun KimKenneth Nephew

CommitteeCurt BalchHaixu Tang

OSU ICBP centerDustin PotterPearlly YanTim H-M. Huang

IUPUI Lang LiJeanette

McClintick

28

ColleaguesFang FangShu Zhang

Henry PaikJohn MontgomeryMikyoung JeongFuxiao XinNicolas BerryXinghua LongNicole NickersonXi RaoCori Hartiman-

FreyFunding Agencies NCI U54 CA11300

NCI R01 CA85289

F. Coste, J. M. Malinge, L. Serre, W. Shepard, M. Roth, M. Leng and C. Zelwer, "Crystal structure of a double-stranded DNA containing a cisplatin interstrand cross-link at 1.63 A resolution: hydration at the platinated site", Nucleic Acids Res, 1999, 27, 1837.

29