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EPIGENETICS OF CARCINOGENESIS

EPIGENETICS OF CARCINOGENESIS. Olga Kovalchuk , MD/PhD University of Lethbridge, AB, Canada. GENETICS. EPIGENETICS. Heritable transmission of information based on differences in DNA sequence. Heritable transmission of information in the absence of changes in DNA sequence. Alterations. SNP.

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EPIGENETICS OF CARCINOGENESIS

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  1. EPIGENETICS OF CARCINOGENESIS Olga Kovalchuk, MD/PhD University of Lethbridge, AB, Canada

  2. GENETICS EPIGENETICS Heritable transmission of information based on differences in DNA sequence Heritable transmission of information in the absence of changes in DNA sequence Alterations SNP MTHFR, C677T GCC→GTC C/T EPIGENETICS VERSUS GENETICS Allis CD et al., In: Epigenetics, 2007

  3. DNMT1 P U DNMT3a Me DNMT3b -3.0 0 3.0 Treated Control SAM SAH A - acetylation A - methylation Me - phosphorylation P - ubiquitination U EPIGENETIC CHANGES Epigenetic alterations – changes induced in cells that alter expression of the information on transcriptional, translational, or post-translational levels without change in DNA sequence Modifications of histones RNA-mediated modifications Methylation of DNA • siRNA, miRNA, piRNA …

  4. DISTRIBUTION OF METHYLATED AND UNMETHYLATED DOMAINS COMPOSITION OF GENOME Promoters/1st Exons Overlap 0.11% Other Ensembl Exons 1.83% CpG Island/1st Exons Overlap 0.11% Other Ensembl Exons 1.83% Ensembl 1st Exons (Non-overlapping) 0.2% DNA Transposons 3.6% Ensembl 1st Exons (Non-overlapping) 0.2% DNA Transposons 3.6% CpG Island (Non-overlapping) 0.57% CpG Island (Non-overlapping) 0.57% • Unmethylated CpG - Methylated CpG Low Complexity Repeats 1.3% Low Complexity Repeats 1.3% Other Repeats 0.15% Other Repeats 0.15% LINE 22.9% LINE 22.9% LINE 22.9% Simple Repeats 1.7% Alpha Satellite 2.07% Rollins RA et al., Genome Res, 2006 Classical Satellite 2.1% Simple Repeats 1.7% Alpha Satellite 2.07% Rollins RA et al., Genome Res, 2006 Classical Satellite 2.1% Other 38.7% Other 38.7% Other 38.7% LTR 9.3% LTR 9.3% LTR 9.3% SINE 10.1% SINE 10.1% SINE 10.1% METHYLATION LANDSCAPE OF THE HUMAN GENOME

  5. E1 E1 E3 E3 E2 E2 SR SR SR SR IR IR Transposon X 5’LTR 3’LTR gag pol env TSDR TSDR MSC P ORF1 ORF2 A/T Rich Site TSDR TTTT DR DR SVA Element Poly(A) - Unmethylated CpG - Methylated CpG METHYLATION LANDSCAPE OF THE HUMAN GENOME Unmethylated domains (CpG islands at gene promoters) Distribution of CpG sites in the human genome CpG island • G + C content > 0.55. • Observed vs expected CpG densities > 0.5. • Lengh > 300 bp (500 bp). Methylated domains (repeated DNA sequences) Simple tandem repeat DNA transposon LTR – endogenous retrovirus Non-LTR autonomous retrotransposon: LINE Non-LTR non-autonomous retrotransposon: SINE Rollins RA et al., Genome Res, 2006 Wilson AS et al., BBA, 2007

  6. H2A H3 H2B H4 POST-TRANSLATIONAL HISTONE MODIFICATIONS CHROMATIN NUCLEOSOME TYPES AND ROLES OF HISTONE MODIFICATIONS POST-TRANSLATIONAL HISTONE MODIFICATIONS

  7. COORDINATED MODIFICATION OF CHROMATIN Allis CD et al., In: Epigenetics, 2007

  8. strand A strand B DNA replication strand B strand A Maintenance DNA methylation MAINTENANCE OF DNA METHYLATION AND HISTONE MODIFICATIONS DURING DNA REPLICATION Maintenance of DNA methylation Maintenance of histone modifications Felsenfeld G., In: Epigenetics, 2007

  9. Normal cells Initiation Single initiated cells Promotion Focal proliferation Single carcinoma cells Progression Carcinoma STAGES OF CARCINOGENESIS ?

  10. ENVIRONMENTAL EPIGENETICS – MECHANISMS OF EPIGENETIC PROGRAMMING BY THE ENVIRONMENT AND THEIR POSSIBLE IMPLICATIONS FOR TOXICOLOGY

  11. ESTROGENIC CHEMICAL BISPHENOL A Maternal BPA exposure shifts offspring coat color distribution toward yellow. (A) Genetically identical Avy/a offspring representing the five coat color phenotypes. (B) Coat color distribution of Avy/a offspring born to 16 control (n = 60) and 17 BPA-exposed (n = 73) litters (50-mg BPA/kg diet). Dolinoy, 2007

  12. SELECTED LIST OF ENVIRONMENTAL CHEMICAL AGENTS THAT ALTER CELLULAR EPIGENETIC PATTERNS

  13. DO EPIGENETIC CHANGES PLAY A ROLE IN CARCINOGENESIS?

  14. DNA METHYLATION CHANGES DURING SKIN CARCINOGENESIS Status of global DNA methylation CpG island methylation status of selected genes Fraga MF et al., Cancer Res, 2004

  15. SELECTED LIST OF GENES HYPERMETHYLATED IN HUMAN HEPATOCELLULAR CARCINOMA

  16. PREDICTIVE POWER OF GENE METHYLATION FOR EARLY DETECTION OF HEPATOCELLULAR CARCINOMA Rivenbark AG, Coleman WB., Clin Cancer Res, 2007

  17. BREAST CARCINOGENESIS

  18. Estrogen Radiation • Estrogen is a well-known breast carcinogen with both initiating and promoting properties • Estrogen is linked to the neoplastic transformation of normal breast cells in vitro and in rodent model • Women with elevated estrogen levels are considered to be a high-risk group for breast cancer development • IR is the only genotoxic agent generally accepted as a breast carcinogen • Promotes the neoplastic transformation of normal breast cells in vitro and in rodent model • Induces breast cancer in exposed humans (atomic bomb survivors and women exposed to diagnostic and therapeutic irradiation) • Average IR exposure doses linked to breast cancer development range widely between 0.02 and 20 Gy

  19. Estrogen-Induced Rat Breast Carcinogenesis is Characterized by Alterations in DNA Methylation, Histone Modifications and Aberrant MicroRNA Expression

  20. Level of DNA methylation in mammary glands of control rats and rats exposed to estrogen

  21. Histone modifications in rat mammary glands of rats exposed to estrogen

  22. Combined Effects of Estrogen and Ionizing Radiation on the Epigenetic Processes in the Rat Mammary Gland • Sham treated controls; • Estrogen treated group; • IR treated group; • IR + Estrogen treated group.

  23. Levels of proliferation

  24. EXPRESSION OF DNA METHYLTRANSFERASES IN THE MAMMARY GLANDS OF ESTROGEN- AND RADIATION-EXPOSED RATS Kutanzi, EMM ,in revision

  25. MicroRNAs up- and down-regulated in rat mammary gland tissue upon estrogen exposure, radiation exposure, and combined estrogen and radiation exposure as analyzed by microRNA microarray

  26. MicroRNAs up- and down-regulated in rat mammary gland tissue upon estrogen exposure, radiation exposure, and combined estrogen and radiation exposure as analyzed by microRNA microarray

  27. Cancer cells Carcinoma in-situ Advanced cancer Resistant relapse ? ? Anticancer drug resistance carcinogenesis cancer progression Chemotherapy Cancer progression- associated epigenetic changes Chemotherapy-induced epigenetic changes Novel epigenetic therapy Novel epigenetic biomarkers of drug resistance CARCINOGENESIS Cancer initiation- associated epigenetic changes

  28. GENETIC AND EPIGENETIC MODELS OF THE CANCER INITIATION Normal cells Normal cells Environmental Environmental Endogenous Endogenous ACQUISITION OF ADDITIONAL RANDOM MUTATIONS ALTERATIONS IN CELLULAR EPIGENOME Clonal selection and expression of initiated cells Epigenetically reprogrammed cells Mutator phenotype cells Mutator phenotype cells Cancer cells Cancer cells

  29. Cost and morbidity ER cells Cancer cells Carcinoma in-situ Advanced cancer Resistant relapse Anticancer drug resistance carcinogenesis cancer progression chemotherapy EPIGENETIC MODEL OF CARCINOGENESIS Risk assessment Screening for early-stage disease Detection and localization Disease stratification and prognosis Response to therapy Screening for disease recurrence Hartwell et al. Nat Biotechnol., 2006.

  30. Cost and morbidity Metabolic Liver Diseases Hussain et al., Oncogene, 2007 EPIGENETIC ALTERATIONS CAN PREDICT HUMAN HEPATOCARCINOGENESIS Risk assessment Screening for early-stage disease Detection and localization Disease stratification and prognosis Response to therapy Screening for disease recurrence Hartwell et al. Nat Biotechnol., 2006 CHEMICAL Aflatoxin B1 Ethanol/Smoking Vinyl Chloride

  31. Low dose radiation-induced epigenetic changes in an animal model Therapeutic and diagnostic exposure challenges

  32. Koturbash et al., Cell Cycle, 2008 Koturbash et al., Mutation Res., 2008

  33. Bystander effects occur in vivo • Epigenetic changes are involved in generations and/or maintenance of bystander effects • Bystander effects are tissue specific • Bystander effects are strain and species-independent, but there are some mouse strain differences • Bystander effects are persistent • Bystander effects are sex specific • Bystander effects affect the germline

  34. Possible linkage between radiation-induced bystander effects in vivo and carcinogenesis

  35. Radiation effects on neuroblastoma and glioblastoma – an epigenetic connection

  36. Introduction Neuroblastoma is a malignant tumor that develops from nerve tissue. It usually occurs in infants and children. It is a neuroendocrine tumor, arising from any neural crest element of the sympathetic nervous system. It most frequently originates in one of the adrenal glands, but can also develop in nerve tissues in the neck, chest, abdomen, or pelvis. Neuroblastoma is one of the few human malignancies known to demonstrate spontaneous regression from an undifferentiated state to a completely benign cellular appearance.

  37. Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans, involving glial cells and accounting for 52% of all functional tissue brain tumor cases and 20% of all intracranial tumors. Glioblastoma, the brain tumor that killed Senator Ted Kennedy, still mostly untreatable.

  38. Inroduction Recent studies report an increase in the risk of brain cancers arising from therapeutic and diagnostic exposure to ionizing radiation (IR). While high-dose IR is an established risk factor for glioma and neuroblastoma, but it remains unknown whether low-dose IR affects brain cancer cells. Such analysis is extremely important especially in the view of the recent debate about the benefits and risks of diagnostic low dose IR exposure. Tumors are diagnosed using CT scans and other types of IR-based diagnostics. ?Does this diagnostic exposure cause any effects on tumors? ?Is it harmless? By now effects of low dose exposure on tumors have been neglected.

  39. Model: IMR-32, A-172 (neuroblastoma) and SK-N-BE cells (glioblastoma) cells Exposure: Cells were exposed to 0.1 Gy of X-rays (30kVp; 5mA) and harvested 24 and 72 hours after exposure to see the persistence of IR-induced effects.

  40. RESULTS Summary of gene-specific DNA methylation and gene expression changes induced by low dose radiation in human neuroblastoma (A-172 and IMR-32) and glioma cells (SK-N-BE)

  41. DNMT1, DNMT3a and MeCP2 in neuroblastoma and glioma cells

  42. Low dose IR-induced changes in protein expression in neuroblastoma and glioma cells High H2AX, p53 – glioma cells repair damage really well!

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