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Predicting the response of cervical cancer to radiotherapy

Predicting the response of cervical cancer to radiotherapy. Identification of hypermethylation based markers. Frank Roossink. Cervical cancer. Introduction Cervical cancer Epigenetics Chromatin structure DNA methylation Project Aim Hypothesis Gene Selection Biotrove MSP

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Predicting the response of cervical cancer to radiotherapy

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  1. Predicting the response of cervical cancer to radiotherapy Identification of hypermethylation based markers Frank Roossink

  2. Cervical cancer • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • The second most common malignancy among women world-wide. • In the Netherlands 700 patients present with cervical cancer each year. • World-wide this number is 450,000 cases and 250,000 deaths. • Disproportionally towards less developed countries. Identification of RT response methylation markers in cervical cancer

  3. Cervical cancer • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Data fromGlobocan (2002) Identification of RT response methylation markers in cervical cancer

  4. Cervical cancer • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • The second most common malignancy among women world-wide. • In the Netherlands 700 patients present with cervical cancer each year. • World-wide this number is 450,000 cases and 250,000 deaths. • Disproportionally towards less developed countries. • 99.7% of all cervical cancer patients are human papilloma virus(HPV) positive. (Waalbomers, 1999) • 70% is due to infection with types HPV-16 and HPV-18. Identification of RT response methylation markers in cervical cancer

  5. Cervical cancer • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements 0.3% 0.5% 0.6% 0.7% 1.0% 1.2% 1.3% 1.4% 2.2% 2.3% 2.6% 2.9% 6.7% 17.3% 53.5% Roden and Wu Nature Reviews(2006) Identification of RT response methylation markers in cervical cancer

  6. Cervical cancer • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Cancer is a disease of the genes. • Changes in expression patterns of genes are thought to be the cause for the development of cancer. • In cervical cancer these changes in expression are thought to initiate with the establishment HPV infection. Identification of RT response methylation markers in cervical cancer

  7. Normal cervix Invasive cancer ASCUS Squamous Intraepithelial lesion (SIL) High grade Low grade Infectious HPV particles Superficial zone Midzone Basallayer HPV infection Viral integration E6/E7 deregulation Proliferation Basement membrane Dermis 20% Lesions Genetic instability 80% clearance Adapted from Snijders et al. J. of pathology (2006), Woodman et al. Nature reviews (2007).

  8. Normal cervix Invasive cancer ASCUS Squamous Intraepithelial lesion (SIL) High grade Low grade Superficial zone Midzone Basallayer Basement membrane Dermis • Loss of Chr. 6p • DR of MHC class I • Loss of Chr. 3p,4q, 6q and 10p • UR telomerase • DR GATA-3 • Loss of Chr. 1 • DR TSLC1 Immortalization Invasion Adaptedfrom Snijders et al. J. of pathology(2006), Woodman et al. Nature reviews (2007).

  9. Cervical cancer • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Cancer is a disease of the genes. • Changes in expression patterns of genes are thought to be the cause to the development of cancer. • In cervical cancer these changes in expression are thought to initiate with the establishment HPV infection. • Changes in expression patterns can be due to genetic or epigenetic alterations. Identification of RT response methylation markers in cervical cancer

  10. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements “The branch of biology which studiesthe causal interactions between genes and their products which bring the phenotype into being” C.H. Waddington, 1942 Identification of RT response methylation markers in cervical cancer

  11. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Waddington (1948) Identification of RT response methylation markers in cervical cancer

  12. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements “Heritable states of gene-expression that are not due to alterations in the underlying DNA sequence itself.” Identification of RT response methylation markers in cervical cancer

  13. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Identification of RT response methylation markers in cervical cancer

  14. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements DNA packaging: • Every mitotic cell has 23 pair chromosomes. • Approximately 30,000 genes. • Approximately 2m DNA per cell. • Each individual consists of 10 billion cells. • That means 500,000 trips around the equator or 3.5 trips from the Sun to Pluto. 3.5x Identification of RT response methylation markers in cervical cancer

  15. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Molecular biology of the cell, 4th edition Identification of RT response methylation markers in cervical cancer

  16. “Beads on a string” 30nm fiber DNA double helix Histone tail modifications Molecular biology of the cell, 4th edition Adapted from: http://www.newscientist.com/data/images/archive/2386/23865001.jpg

  17. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements REF!!! Identification of RT response methylation markers in cervical cancer

  18. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Histone modifications Latham et al. Nature Structural & MolecularBiology, 2007 Identification of RT response methylation markers in cervical cancer

  19. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • DNA methylation occurs at the 5th position of a cytosine. • Cytosine that precede Guanine can become methylated • So-called CpG dinucleotides. • Methylation is mediated by DNA methyl transferases (DNMTs) • de novo methylation • DNMT-3a and DNMT-3b • Maintenance • DNMT-1 Identification of RT response methylation markers in cervical cancer

  20. Side view Frontal view Adapted from O’Gara et al. J Mol Biol. 1996 Sep 6;261(5):634-45

  21. Frontal view Adapted from O’Gara et al. J Mol Biol. 1996 Sep 6;261(5):634-45

  22. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • CpG dinucleotides are often found in clusters. • So-called CpG islands. • CpG-island • A region with at least 200bp • A G-C percentage of >50% • CpG-ratio Obs-Exp >0.6 • Found in promoter regions of >50% of all genes. • Promoter hypermethylation can silence genes. Identification of RT response methylation markers in cervical cancer

  23. Epigenetics • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Normal Cancer Identification of RT response methylation markers in cervical cancer

  24. Aim • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements To identify genes, which methylation status predicts the response to chemoradiation. Identification of RT response methylation markers in cervical cancer

  25. Hypothesis • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Different methylation pattern in responding patients vs. non-responding patients. The best feature of methylation is that it is reversible. Identification of RT response methylation markers in cervical cancer

  26. Gene selection • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Originally our KWF-application listed 84 genes: • 52 genes from array data. • 32 genes from literature. • Together with OMS, we will analyze our patient material using a Methylation Specific PCR based screen. • Our new and improved list consists of 800+ genes. Identification of RT response methylation markers in cervical cancer

  27. Biotrove • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • MSP-based screening tool. • Combines the large screen capabilities of microarray with the semi-quantitative analysis of Q-PCR. Identification of RT response methylation markers in cervical cancer

  28. MSP • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Methylation specific PCR • Bisulfite conversion. A A A C U C G G G U C C G G G U C C G G G U C U U U C A C G C G C G C C Methylated DNA Unmethylated DNA Differential sequence Identification of RT response methylation markers in cervical cancer

  29. Demethylation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Nucleoside DNMT inhibitors: • DAC (Decitabine), 5-aza-C, Zebularin. • Non-Nucleoside DNMT inhibitors: • Hydralazine, EGCG, RG108, Procaine • Procainamide 200nM DAC Combi DAC/TSA 1mM VPA 5µM DAC IV Medium control 10µM Hydralazine Leucocyte 300nM TSA Combi Hydr./VPA M U M U M U M U M U M U M U M U M U M U DAPK Identification of RT response methylation markers in cervical cancer

  30. Demethylation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements 5µM DAC 200nM DAC 40µM Hydralazine 1mM Hydralazine 1mM Zebularine reseed 10µM Procainamide 500µM Procainamide 120µM Zebularine 1mM Zebularine Medium reseed 200nM DAC + 300nM TSA Reseed Medium control M U M U M U M U M U M U M U M U M U M U M U M U Identification of RT response methylation markers in cervical cancer

  31. Demethylation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Summary • Procainamide is a FDA approved drug which has demethylating properties. • However, it has severe side-effects. • Also Zebularin has demethylating properties. • Draw-back: it kills monkeys… Identification of RT response methylation markers in cervical cancer

  32. Validation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Validation in a larger cohort of patient samples. • Amsterdam series • UMCG • In vitro validation of our candidate genes. • Over-expression of our candidate genes • Clonogenic assay analysis. • Knock-down of our candidate genes • Clonogenic assay Identification of RT response methylation markers in cervical cancer

  33. Clonogenicassay • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Seeding of cells Day 0 Irradiation Day 1 0, 2, 4, 6, 8 and 10Gy Read-out Day 10 Identification of RT response methylation markers in cervical cancer

  34. Clonogenicassay • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Identification of RT response methylation markers in cervical cancer

  35. Clonogenicassay • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Seeding of cells Seeding of cells Day 5 After 6h, irradiation. 0, 2, 4, 6, 8 & 10 Gy Day 0 Read-out Day 15 DAC/TSA treatment Day 2-4 200nM DAC Day 4 300nM TSA Identification of RT response methylation markers in cervical cancer

  36. Clonogenicassay • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Identification of RT response methylation markers in cervical cancer

  37. In vitro validation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements Identification of RT response methylation markers in cervical cancer

  38. In vitro validation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • pcDNA3-HA-TAp73α • Gift from G. Melino, Rome Italy • pIRES2-EGFP-TAp73α Identification of RT response methylation markers in cervical cancer

  39. In vitro validation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements SiHa pcDNA3-HA-TAp73α PBS control SiHa medium control αHA-tag (Y11) 1:50 SiHa pcDNA3-HA-TAp73ααHA-tag (Y11) 1:50 Appr. 37% transfection efficiency SiHa untransfected SiHa pcDNA3-HA-TAp73α SiHa untransfected SiHa pcDNA3-HA-TAp73α SiHa untransfected SiHa pcDNA3-HA-TAp73α Marker Marker Marker HA-TAp73α αHA (F7, monoclonal mouse) 1:200 1hour, 30s exposure Identification of RT response methylation markers in cervical cancer

  40. In vitro validation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements pIRES2-EGFP CMV G.O.I. KanR NeoR IRES GFP Identification of RT response methylation markers in cervical cancer

  41. Plan of investigation • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • MSP analysis of the selected genes, to see what the methylation status is of our 4 cell lines. • RT-PCR to see if the genes are transcribed. • Clone into pIRES2-EGFP. • Develop RNAi constructs. • Clonogenic assay of transfected cellswith RNAi and plasmid constructs Identification of RT response methylation markers in cervical cancer

  42. Summary • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • HeLa, SiHa are similar sensitive to RT in the clonogenic setting • CaSki is more resistant than SiHa and HeLa. • After DAC/TSA and Zebularin treatment prior to RT, SiHa cells are not changed in sensitivity to RT. • Although procainamide is FDA approved (for arrythmia), we are unlikely to use it due to side-effects. • Cloning strategies in progress Identification of RT response methylation markers in cervical cancer

  43. Summary • Introduction • Cervical cancer • Epigenetics • Chromatin structure • DNA methylation • Project • Aim • Hypothesis • Gene Selection • Biotrove • MSP • Demethylation • Validation • Clonogenic assay • In vitro validation • Plan of investigation • Summary • Acknowledgements • Our patient-material screen is starting shortly. • Our candidate gene list has probably around 800 genes. • Test samples already have been sent. Identification of RT response methylation markers in cervical cancer

  44. Acknowledgements • Gynecologic oncology • G. Bea A. Wisman • Mirjam Kok • Maartje G. Noordhuis • Ate G.J. van der Zee • Pathology • Ed Schuuring • Medical oncology • Steven de Jong

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