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Meng Li School of Informatics Interdisciplinary Program of Biochemistry Indiana University Advisors: Dr. Sun Kim, Dr. Kenneth Nephew 4/25/2008 1 Overview Biology background and experimental model Objective High-throughput data and data analysis Combinatorial study and data mining Conclusions 2 Ovarian cancer and drug resistance Ovarian cancer Most deadly gynecological malignancy Cisplatin Widely used chemotherapeutic drug DNA intercalating agent Cisplatin resistance 70% to 80% of patients develop resistance after 2year treatment 3 DNA methylation CpG dinucleotides: 5’- AATACGCCACGA 4 DNA methylation CpG island CG CG MCG MCG MCGMCG MCG MCG CG CG CG CG CG CG Normal Cancer X C: cytosine mC: De novo methylation: acquired methylation methylcytosine 5 Objective 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? 6 In vitro drug resistance system Cisplatin Cisplatin Drug-sensitive parental cells A2780 R- IC50 (uM) Drug-resistant Cells A2780 R+ Rounds of Cisplatin treatment 7 High-throughput data and data analysis Global promoter methylation data Global gene expression data Louis Staudt, The nation’s investment in cancer research (NCI) 8 Global promoter methylation profiling A2780 R- A2780 R+ Differential Methylation Hybridization (DMH) 44,000 probes representing 10,000 genes Two-color microarray analysis Data processing Estimate methylation level CpG Island Microarray (44K) 9 Global promoter methylation profiling Loess normalization: correcting technical bias Raw Data Normalized Data Fold-change analysis: extracting gene methylation level M = Red log 2 Green 10 Global gene expression profiling Affymetrix U133 plus 2.0 microarray 54,675 probes representing 20,606 genes mRNA Single-color microarray cRNA analysis Data processing Estimate gene expression levels U133 plus 2.0 array 11 Global gene expression profiling Clustering Fold change A2780R+ expression A2780R- expression Welch’s t-test p-values A2780 R+ A2780 RCutoffs: - p-value < 0.01 - fold change >= 1.5 12 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 1 DNMT2 0.3673 1.17 DNA (cytosine-5-)-methyltransferase 2 DNMT3A 0.1009 1.20 DNA (cytosine-5-)-methyltransferase 3 alpha DNMT3B 0.0004 1.80 DNA (cytosine-5-)-methyltransferase 3 beta DNMT1: maintain genomic DNA methylation DNMT3B: de novo methylation 15 Resistant cells re-establish cisplatin sensitivity after methylation inhibitor treatment 16 Key questions Does 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? x 17 Surveying Transcription Factor Binding Sites (TFBS) on differentially methylated regions Scan 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 32.5 Occurrence percentage 30 Hypermethylated 27.5 25 Hypomethylated 22.5 20 17.5 15 12.5 10 7.5 5 2.5 0 19 Statistical scoring • Fisher exact test • Multiple test correction – False discovery rate (FDR) TFBS S+ vs. S- S+ vs. Sr S+ vs. SCpG NCX HMGIY CEBP BRCA 0.000698 0.00113 0.008758 0.01259 5.49E-10 6.14E-16 1.53E-09 1.67E-06 0.0007561 2.88E-05 0.0001531 0.001406 20 Key questions Does 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? Yes Does de novo methylation selectively regulates certain pathways? 21 Methylation regulated pathways • Hypomethylation up-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 pathway Corrected p-value fisher.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 All are human cancer related pathways 22 Hypomethylated and up-regulated pathways Genes involved for each pathway: PIK3R3, PDGFRA, E2F1, TGFBR2 E2F Smad2/3 Smad4 23 Methylation regulated pathways • Hypermethylation down-regulated pathways DownReg Genes fc < -1.5 (1286) HyperMethyl fc >1.5 and DownReg genes p<0.01 fc <-1.5 (55) Input genes in pathway Corrected p-value Impact Cell adhesion molecules 384.485 (CAMs) 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 Impact Input genes Corrected in pathway p-value fisher.test p-value 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 pathways Genes involved for each pathway: CAMs (ITGAV, CLDN11, NEO1, CDH2) Tight junction (CLDN11, PPP2R4, INADL) PPAR signaling (CPT1A, SLC27A6) 25 Key questions Does 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? Yes Does de novo methylation selectively regulates certain pathways? Yes 26 Conclusion Promoter 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 Acknowledgements Advisors Sun Kim Kenneth Nephew Committee Curt Balch Haixu Tang OSU ICBP center Dustin Potter Pearlly Yan Tim H-M. Huang IUPUI Lang Li Jeanette McClintick Colleagues Fang Fang Shu Zhang Henry Paik John Montgomery Mikyoung Jeong Fuxiao Xin Nicolas Berry Xinghua Long Nicole Nickerson Xi Rao Cori Hartiman-Frey Funding Agencies NCI U54 CA11300 NCI R01 CA85289 28 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