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Sabine ZÖCHBAUER-MÜLLER, MD Medical University of Vienna Department of Medicine I

Life Sciences Day 2013. Mapping of CpG island methylation and its prognostic relevance in lung cancer patients. Sabine ZÖCHBAUER-MÜLLER, MD Medical University of Vienna Department of Medicine I Clinical Division of Oncology. OVERVIEW. Background of DNA methylation

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Sabine ZÖCHBAUER-MÜLLER, MD Medical University of Vienna Department of Medicine I

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  1. Life Sciences Day 2013 Mapping of CpG island methylation and its prognostic relevance in lung cancer patients Sabine ZÖCHBAUER-MÜLLER, MD Medical University of Vienna Department of Medicine I Clinical Division of Oncology

  2. OVERVIEW • Background of DNA methylation • Mapping of CpG island (CGI) methylation and its prognostic relevance in lung cancer patients • Genome-wide microRNA (miRNA) expression profiling identifies targets for DNA methylation in non-small cell lung cancers • Human ressources development • Publications • Research grants • Outlook • Acknowledgement

  3. EPIGENETIC MECHANISMS CONTRIBUTE TO TRANSCRIPTIONAL GENE SILENCING DNA methylation ….. • epigeneticchangeoccurringat CG dinucleotideswithin CGI located in 5` regionofmanycancer-related genes • affectstogetherwithotherepigeneticchangesbindingoftranscriptionfactorsto DNA leadingtogenesilencing • frequentlyoccuringchange in neoplasticcells • reversibel bydemethylatingdrugs

  4. DNA METHYLATION IN LUNG CANCER • Lung cancer leading cause of cancer deaths worldwide with patient`s 5-year overall survival rates of ~ 14% • Many protein-encoding genes identified to be frequently methylated in non-small cell lung cancers (NSCLC) • So far mainly analyses of single genes or small numbers of genes • Methylation of certain protein-encoding genes of potential clinical relevance in NSCLC patients • Mechanism for deregulation of microRNA expression in NSCLC?

  5. MAPPING OF CGI METHYLATION AND ITS PROGNOSTIC RELEVANCE IN LUNG CANCER PATIENTS Aims … • Genome-wide search for methylated CGIs in tumor (TU) and corresponding non-malignant lung tissue samples (NL) from a large number of NSCLC patients • Identification of tumor-specifically methylated genes • Confirmation of methylation by gene-specific analyses • Comparison of methylation with expression patterns • Investigation of effects of epigenetically active drugs on gene expression • Investigation of potential tumor suppressor gene function • Determination of potential clinical relevance of methylated genes in NSCLC patients Heller et al, Carcinogenesis 2013

  6. METHODS • Methylated DNA immunoprecipitation and microarray analysis using NimbleGen`s 385K Human CGI plus Promoter array (MeDIP-chip) including statistical analyses of primary TU and corresponding NL tissue samples of 101 stage I-IIIA NSCLC patients • Functional characterization using Ontologizer • Gene-specific methylation analyses using methylation-sensitive high resolution melting (MS-HRM) approach • Bisulfite genomic sequencing (BGS) • Microarray expression analyses of untreated/drug treated NSCLC cells • Immunohistochemistry (IHC) • Overexpression of genes in NSCLC cells followed by cell viability/proliferation assays • Comparison of methylation results with clinico-pathological characteristics of patients Heller et al, Carcinogenesis 2013

  7. CHROMOSOMAL DISTRIBUTION OF PROBES FOUND TO BE DIFFERENTIALLY METHYLATED • Identification of 2.414 probes differentially methylated between TU and corresponding NL samples • 97% of probes tumor-specifically methylated (dots above upper red line) • Tumor-specifically methylated probes at all chromosomes except chromosome 22 and Y chromosome • 3% of probes methylated at higher extent in NL samples (dots below lower red line) X-axis, probe position; y-axis, t-statistics; upper and lower red lines indicate significance levels (adjusted p-values for step-down multiple testing < 0.05) Heller et al, Carcinogenesis 2013

  8. FROM PROBES TO GENES • Annotation and dereplication of differentially methylated probes • Identification of 477 unique tumor-specifically methylated genes • 406 characterized protein encoding genes, 38 predicted genes, 33 non-coding RNA encoding genes • Association of 95% of protein encoding genes with a 5`CGI • Several tumor-specifically methylated genes located in gene clusters: HOXA, HOXB, HOXD, PCDHA, PCDHGA, PCDHGB • 149 tumor-specifically methylated genes involved in transcriptional gene expression and 61 in cell adhesion • From majority ofgenes methylation in NSCLCs unknown Heller et al, Carcinogenesis 2013

  9. COMPARISON OF PERCENT OF METHYLATION IN TU AND NL SAMPLES BY MS-HRM ANALYSES y-axis: % of methylation; each line represents an individual patient; * P ≤ 0.05; ** P ≤ 0.01; ***P ≤ 0.001 Heller et al, Carcinogenesis 2013

  10. TUMOR-SPECIFIC METHYLATION BY BGS 3 clones of TU and corresponding NL sequenced; relatively homogeneous methylation in TU; no or low methylation in corresponding NL; Heller et al, Carcinogenesis 2013

  11. EFFECT OF EPIGENETICALLY ACTIVE DRUGS ON EXPRESSION OF TUMOR-SPECIFICALLY METHYLATED GENES • MicroarrayexpressionanalysesofuntreatedandwithAza-dCorAza-dC/TSA treated NSCLC cells • Upregulationofexpressionof 31% of tumor-specificallymethylated genes* after drugtreatment in NSCLC celllines (foldchange ≥ 1.5, FDR ≤ 0.1); *identified by MeDIP- chip analyses of NSCLC cell lines A549, NCI-H1993 and NCI-H2073 Heller et al, Carcinogenesis 2013

  12. IHC STAINING OF TU AND NL SAMPLES Lack of expression in cancer cells Nuclear expression in normal bronchiolar epithelial cells Lack of expression in cancer cells Cytoplasmic expression in normal bronchiolar epithelial cells Lack of expression in cancer cells Cytoplasmic expression in alveolar epithelial cells and macrophages Weak expression in cancer cells Stronger cytoplasmic expression in normal bronchiolar epithelial cells and leucocytes TU samples methylated for individual gene; Heller et al, Carcinogenesis 2013

  13. REDUCED CELL VIABILITY/CELL PROLIFERATION AFTER L1TD1OVEREXPRESSION IN L1TD1 METHYLATED NSCLC CELLS Tumor-specific L1TD1 methylation determined by MS-HRM analysis Up-regulated L1TD1 expression after Aza-dC treatment Cell proliferation determined in real-time Cell viability normalized to GFP control Heller et al, in preparation

  14. REDUCED CELL VIABILITY/CELL PROLIFERATION AFTER ZNF677OVEREXPRESSION IN ZNF677 METHYLATED NSCLC CELLS Empty ZNF677 NCSLC cells transfected with empty control vector or ZNF677 expression vector Cell viability normalized to GFP control Cell proliferation determined in real-time Heller et al, in preparation

  15. STATISTICALLY SIGNIFICANT SURVIVAL DIFFERENCES* REGARDING METHYLATION Shorter DFSShorter OS Methylation of HOXA2§HOXA2 HOXA10§HOXA2 and/or HOXA10§ HOXA2 and/or HOXA10ZNF677§ Cumulative survival * Univariate analysis; § Independent prognostic parameters by multivariate analysis; HOXA2, HOXA10, HOXA2/HOXA10 : SCC patients; ZNF677: NSCLC patients; P = 0.013 Months Heller et al, Carcinogenesis 2013Heller et al, in preparation Kaplan-Meier plot of OS according to ZNF677 methylation.

  16. CONCLUSION • Identification of large number of tumor-specifically methylated genes in NSCLC patients • From many of them methylation in NSCLCs unknown so far • Involvement of about half of genes in regulation of gene expression or cell adhesion • Association of methylation of many genes with transcriptional regulation • Putative tumor suppressor gene function of L1TD1 and ZNF677 • Identification of HOXA2, HOXA10 and ZNF677 aspotential prognostic markers • Findings emphasize impact of methylation on pathogenesis of NSCLCs Heller et al, Carcinogenesis 2013 Heller et al, in preparation Heller et al, in preparation

  17. GENOME-WIDE miRNA EXPRESSION PROFILING IDENTIFIES TARGETS FOR DNA METHYLATION IN NON-SMALL CELL LUNG CANCER Aims … • Investigation of potential role of DNA methylation on miRNA silencing in NSCLCs • Investigation of miRNA methylation in TU and corresponding NL samples of NSCLC patients • Determination of potential clinical relevance of miRNA methylation in NSCLC patients Heller et al, Clin Cancer Res 2012

  18. METHODS • Determination of genome-wide miRNA expression of untreated and with epigentically activs drugs treated A549 cells by microarray analyses • miRNA target prediction and functional annotation • Gene-specific methylation analyses using MS-HRM approach in NSCLC cell lines, primary TU and corresponding NL tissue samples from 101 stage I-IIIA NSCLC patients • BGS • Comparison of methylation with gene expression patterns • Comparison of methylation results with clinico-pathological characteristics of patients Heller et al, Clin Cancer Res 2012

  19. MICROARRAY EXPRESSION ANALYSIS OF 856 miRNAs IN A549 CELLS Upregulationofexpressionof 66 miRNAs after drugtreatment (foldchange ≥ 1.5; P < 0.1) Associationof 33 miRNAswith CGI e.g. miR-7, miR-9-3, miR-29c, miR-34a, miR-125a, miR-193a, miR-200c, miR-375 GO analysis of predicted targets of 33 miRNAs Heller et al, Clin Cancer Res 2012

  20. TUMOR-SPECIFIC METHYLATION OF miR-9-3 AND miR-193a BY MS-HRMIN NSCLC PATIENTS Each circle represents individual sample. Heatmaps show comparison of percentage of methylation between TU and NL samples of each patient. Heller et al, Clin Cancer Res 2012

  21. miR-9-3 METHYLATION AND SURVIVAL OF LUNG SQUAMOUS CELL CARCINOMA PATIENTS Kaplan-Meier plots of disease-free and overall survival according to mi-9-3 methylation. Heller et al, Clin Cancer Res 2012

  22. CONCLUSION • Identification of 66 miRNAs with upregulated expression after drug treatment • Association of 33 miRNAs with a CGI • Identification of tumor-specific miR-9-3 and miR-193a methylation in NSCLC patients • Association of miR-9-3 methylation with shorter DFS and OS of SCC patients • Identification of methylation as mechanism for miRNA silencing in NSCLCs • Findings of potential clinical relevance Heller et al, Clin Cancer Res 2012

  23. HUMAN RESSOURCES DEVELOPMENT • Gerwin Heller*, PhD, post doc: permanent employment contract at the MUV, ready for “Venia docendi“ • Valerie Babinsky*, diploma student: finished thesis • Marlene Weinzierl, diploma student: finished thesis • Christian Noll*, diploma student: finished thesis • Corinna Altenberger*, diploma student: finished thesis, now PhD student • Bianca Schmid, diploma student: thesis in progress • Barbara Ziegler, technician: Nomination as lab manager *Financed by WWTF

  24. MAJOR PUBLICATIONS • Heller G et al, Anticancer Res 2009 IF 1.713 • Heller G et al, Cancer Metastasis Rev 2010 IF 7.787 • Kollmann K et al*, Blood, 2011 IF 9.060 • Kollmann K et al*, Oncotarget 2011 IF 6.636 • Heller G et al, ClinCancer Res 2012 IF 7.837 • Ghanim V et al*, Blood 2012 IF 9.060 • Brodowicz T et al*, Ann Oncol 2012 IF 4.120 • Heller G et al, Carcinogenesis 2013 IF 5.635 • Kollmann K et al*, CancerCell 2013 IF 24.755 • Preusser M et al*, Lung Cancer 2013 IF 3.392 • Höbaus J et al*, Int J Cancer 2013 IF 6.198 • Berghoff AS et al*, APMIS 2013 IF 2.068 • IF 88.261 Peer reviewed journals; * Co-Autorship; IF 2012

  25. RESEARCH GRANTS • Research grant from the FWF to Zöchbauer-Müller S: DNA methylation mediated microRNA gene silencing in non-small cell lung cancer patients, duration 3 years • Special research program (SFB) grant from the FWF to the SFB consortium (Valent P, Kralovics R, Lion T, Sexl V, Moriggl R, Zöchbauer-Müller S, Zuber J, Superti-Furga G, Mannhalter C, Nijman S): Myeloproliferative neoplasms - pathogenesis and development of new therapeutic strategies; subtheme: Epigenetic mechanisms involved in disease manifestation and progression in MPN, duration 4 years • Research grant from the Austrian Society of Hematology and Oncology to Heller G: Identification of novel epigenetically silenced tumor suppressor genes in NSCLCs, duration 1 year

  26. OUTLOOK • Characterization of additional tumor-specifically methylated genes regarding function and potential prognostic relevance in NSCLC patients • Identification of patients with “bad prognosis“ regarding pattern of tumor-specifically methylated genes • Identification of exclusively in NSCLCs tumor-specifically methylated genes • Investigation of methylation from panel of markers in serum samples to diagnose NSCLC • Relevance for more individualized treatment of patients and early detection of NSCLC ?

  27. THANK YOU FOR THE WWTF GRANT! Martin Posch, PhD, MUV Leonhard Müllauer, MD, MUV Walter Klepetko, MD, MUV Kwun Fong, MD, Prince Charles Hospital, Brisbane Balasz Döme, MD, PhD, Budapest Balasz Hegedüs, PhD, MUV Veronika Sexl, MD, Vetmeduni Peter Valent, MD, MUV Christoph Bock, PhD, CeMM Martin Bilban, PhD, MUV Christine Mannhalter, MD, MUV

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