meng li school of informatics interdisciplinary program of biochemistry indiana university advisors:...
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Meng LiSchool of Informatics
Interdisciplinary Program of Biochemistry Indiana University
Advisors: Dr. Sun Kim, Dr. Kenneth Nephew4/25/2008
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OverviewBiology background and experimental modelObjectiveHigh-throughput data and data analysisCombinatorial study and data miningConclusions
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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
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DNA methylation
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CpG dinucleotides: 5’- AATACGCCACGA
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DNA methylation
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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
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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?
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In vitro drug resistance system
Drug-sensitiveparental cells
A2780 R-
Drug-resistant Cells
A2780 R+
Cisplatin
Rounds of Cisplatin treatment
IC5
0 (u
M)
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Cisplatin
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High-throughput data and data analysisGlobal promoter methylation dataGlobal gene expression data
8Louis Staudt, The nation’s investment in cancer research (NCI)
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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+
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Raw Data Normalized Data
Global promoter methylation profilingLoess normalization: correcting technical bias
Fold-change analysis: extracting gene methylation level
M = Green
Redlog2
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Global gene expression profilingAffymetrix U133 plus 2.0
microarray 54,675 probes
representing 20,606 genes
Single-color microarray analysisData processingEstimate gene expression
levels
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mRNA
cRNA
U133 plus 2.0 array
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Global gene expression profilingClustering
Cutoffs:
- p-value < 0.01
- fold change >= 1.5
A2780 R-A2780 R+
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Fold changeA2780R+ expressionA2780R- expression
Welch’s t-test p-values
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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?
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The number of hypermethylated genes positively correlated with the increase of IC50
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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
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Resistant cells re-establish cisplatin sensitivity after methylation inhibitor treatment
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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?
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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
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*S+
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*S-
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SCpG
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Methylation selectively occurs at certain TFBS
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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)
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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?
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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
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E2F
Hypomethylated and up-regulated pathwaysGenes involved for each pathway:
PIK3R3, PDGFRA, E2F1, TGFBR2
Smad2/3
Smad4
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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
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Hypermethylated and down-regulated pathwaysGenes involved for each pathway:
CAMs (ITGAV, CLDN11, NEO1, CDH2)Tight junction (CLDN11, PPP2R4, INADL)PPAR signaling (CPT1A, SLC27A6)
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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
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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
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AcknowledgementsAdvisors
Sun KimKenneth Nephew
CommitteeCurt BalchHaixu Tang
OSU ICBP centerDustin PotterPearlly YanTim H-M. Huang
IUPUI Lang LiJeanette
McClintick
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ColleaguesFang FangShu Zhang
Henry PaikJohn MontgomeryMikyoung JeongFuxiao XinNicolas BerryXinghua LongNicole NickersonXi RaoCori Hartiman-
FreyFunding Agencies NCI U54 CA11300
NCI R01 CA85289
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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.
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