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EPIGENETICS OF CARCINOGENESIS Olga Kovalchuk, MD/PhD University of Lethbridge, AB, Canada EPIGENETICS VERSUS GENETICS EPIGENETICS Heritable transmission of information in the absence of changes in DNA sequence Alterations GENETICS Heritable transmission of information based on differences in DNA sequence SNP MTHFR, C677T GCC→GTC C/T Allis CD et al., In: Epigenetics, 2007 EPIGENETIC CHANGES Epigenetic alterations – changes induced in cells that alter expression of the information on transcriptional, translational, or posttranslational levels without change in DNA sequence Modifications of histones Methylation of DNA DNMT1 DNMT3a DNMT3b SAM P Me U RNA-mediated modifications • siRNA, miRNA, piRNA … -3.0 SAH A A - acetylation Me - methylation P - phosphorylation U - ubiquitination Control 0 Treated 3.0 METHYLATION LANDSCAPE OF THE HUMAN GENOME COMPOSITION OF GENOME CpG Island/1st Exons Overlap 0.11% Other Ensembl Exons 1.83% Ensembl 1st Exons (Non-overlapping) DNA Transposons 3.6% 0.2% CpG Island (Non-overlapping) 0.57% LINE 22.9% Other 38.7% LTR 9.3% SINE 10.1% Low Complexity Repeats 1.3% Other Repeats 0.15% Simple Repeats Alpha Satellite Classical Satellite 2.07% Rollins RA et al., 1.7% 2.1% Genome Res, 2006 DISTRIBUTION OF METHYLATED AND UNMETHYLATED DOMAINS Promoters/1st Exons Overlap 0.11% Other Ensembl Exons 1.83% Ensembl 1st Exons (Non-overlapping) DNA Transposons 3.6% 0.2% CpG Island (Non-overlapping) 0.57% LINE LINE 22.9% 22.9% -Unmethylated CpG - Methylated CpG Other Other 38.7% 38.7% LTR LTR 9.3% 9.3% SINE SINE 10.1% 10.1% Low Complexity Repeats 1.3% Other Repeats 0.15% Simple Repeats Alpha Satellite Classical Satellite 2.07% Rollins RA et al., 1.7% 2.1% Genome Res, 2006 METHYLATION LANDSCAPE OF THE HUMAN GENOME Unmethylated domains (CpG islands at gene promoters) Distribution of CpG sites in the human genome Sequence compartment Genome CpG 29,848,753 G + C (%) CpG Obs/Exp (%) 41 24 CpG island Promoter 62 89 • G + C1,876,802 content > 0.55. • Observed vs expected densities >650.5. First Exon 508,553 CpG 56 • Lengh > 300 bp (500 bp). Other Exons 1,337,271 48 40 DNA Transposons 565,601 29 23 Line Transposons 3,242,225 32 18 LTR Transposons 1,958,798 37 19 SINE Transposons 7,479,682 38 41 Alpha Satellite ~766,000 38 33 ~1,140,000 34 67 8,358,888 42 15 Classical Satellite Other Rollins RA et al., Genome Res, 2006 E1 X E2 E3 E1 E2 E3 - Unmethylated CpG - Methylated CpG Methylated domains (repeated DNA sequences) Simple tandem repeat SR DNA transposon IR LTR – endogenous retrovirus Non-LTR autonomous retrotransposon: LINE Non-LTR non-autonomous retrotransposon: SINE Wilson AS et al., BBA, 2007 SR TSDR SR Transposon 5’LTR TSDR SR gag MSC P DR ORF1 SVA Element pol IR env ORF2 Poly(A) 3’LTR A/T Rich Site TSDR DR TTTT POST-TRANSLATIONAL HISTONE MODIFICATIONS CHROMATIN NUCLEOSOME H2A H3 H2B H4 TYPES AND ROLES OF HISTONE MODIFICATIONS Histone modifications Acetylation Role in transcription activation Histonemodified sites H3 (K9, K14, K18, K56) H4 (K5, K8, K12, K16) H2A POST-TRANSLATIONAL HISTONE MODIFICATIONS H2B (K6, K7, K16, K17) Phosphorylation activation H3 (S10) Methylation activation H3 (K4, K36, K79) repression H3 (K9, K27) H4 (K20) Ubiquitination Sumoylation activation H2B (K123) repression H2A (K119) repression H3 (?) H4 (K5, K8, K12, K16) H2A (K126) H2B (K6, K7, K16, K17) COORDINATED MODIFICATION OF CHROMATIN Allis CD et al., In: Epigenetics, 2007 MAINTENANCE OF DNA METHYLATION AND HISTONE MODIFICATIONS DURING DNA REPLICATION Maintenance of DNA methylation strand A strand B DNA replication strand A strand B Maintenance DNA methylation Felsenfeld G., In: Epigenetics, 2007 Maintenance of histone modifications STAGES OF CARCINOGENESIS Normal cells ? Initiation Single initiated cells Focal proliferation Promotion Single carcinoma cells Carcinoma Progression ENVIRONMENTAL EPIGENETICS – MECHANISMS OF EPIGENETIC PROGRAMMING BY THE ENVIRONMENT AND THEIR POSSIBLE IMPLICATIONS FOR TOXICOLOGY 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 SELECTED LIST OF ENVIRONMENTAL CHEMICAL AGENTS THAT ALTER CELLULAR EPIGENETIC PATTERNS Agent Arsenic Effect Global DNA hypomethylation Hypomethylation of GC-rich sequences Gene-specific hypomethylation (Er-α, cyclin D1) Gene-specific hypermethylation (p53, p16INK4A, RASSF1A) Inhibition of DNMT1 and DNMT3a expression Histone acetylation Cadmium Global DNA hypomethylation (short-term exposure) Inhibition of DNMT activity (short-term exposure) Global DNA hypermethylation (long-term exposure) Increased DNMT activity (long-exposure) Gene-specific hypermethylation (p16INK4A, RASSF1A) Hydrazine Global DNA hypomethylation Gene-specific hypomethylation (p53, c-myc, HMG CoA reductase) Benzo(a)pyrene Global DNA hypomethylation Gene-specific hypermethylation (CYP1A1) Promoter-specific histone H3 lysine 9 hypo- and hyperacetylation CpG-methylation-associated mutations (p53) Aflatoxin B1 Gene-specific hypermethylation (GSTP, MGMT, RASSF1A, p16INK4A) 2-Acetylaminofluorene Gene-specific hypermethylation (p16INK4A) Loss of histone H4 lysine 20 trimethylation Increased DNMT1 expression Peroxisome Global DNA hypomethylation proliferators Hypomethylation of GC-rich sequences (WY-14643) Loss of histone H4 lysine 20 trimethylation Gene-specific hypomethylation (c-myc) Dibromoacetic acid Global DNA hypomethylation Reference Zhao CQ et al., PNAS, 1997; Chen H et al., Carcinogenesis, 2004; Sciandrello G et al., Carcinogenesis, 2004; Reichard JF et al., BBRC, 2007 Xie Y et al., Toxicology, 2007. Chen H et al., Carcinogenesis, 2004. Chandra S et al., Toxicol Sci, 2006; Cui X et al., Toxicol Sci, 2006 Reichard JF et al., BBRC, 2007. Ramirez T et al., Chromosoma, 2007 Takiguchi M et al., Exp Cell Res, 2003 Benbraim-Tallaa L et al., Environ Health Perspect, 2007 Fitzgerald BE, Shank RC, Carcinogenesis, 1996 Zheng H, Shank RC, Carcinogenesis, 1996; Coni P et al., Carcinogenesis, 1992 Wilson WL, Jones PA, Carcinogenesis, 1984 Anttila S et al., Cancer Res, 2003 Sadikovic B et al., J Biol Chem, 2008 Yoon JH et al., Cancer Res 2001 Zhang YJ et al., Mol Carcinog, 2002; Zhang YJ et al., Int J Cancer, 2003; Zhang YJ et al., Cancer Lett, 2005. Bagnyukova TV et al., Carcinogenesis, 2008 Ge R et al., Toxicol Sci, 2001; Pogribny IP et al., Mutat Res, 2007. Tao L et al., Toxicol Sci, 2004 DO EPIGENETIC CHANGES PLAY A ROLE IN CARCINOGENESIS? DNA METHYLATION CHANGES DURING SKIN CARCINOGENESIS Status of global DNA methylation CpG island methylation status of selected genes NS MCA3D PB MSCP6 PDV PAM212 MSCB1 MSC11 HaCa4 19 A5 CarB CarC BRCA1 U U U U U U U U U U U MLH1 U U U U U U U U U U U MGMT U M M M M M M M M M M CDH1 U U U U U U U M M M M Snail U M M M M M M U U U U MLT1 U M M M M M M M M M M Abbreviations: NS - normal skin; M - methylated; U - unmethylated Fraga MF et al., Cancer Res, 2004 SELECTED LIST OF GENES HYPERMETHYLATED IN HUMAN HEPATOCELLULAR CARCINOMA p16INK4A Cell cycle G1-to-S phase progression Frequency in HCC, % 32-65 p15INK4B Cell cycle G1-to-S phase progression 16-49 Cell cycle alterations CyclinD2 Cell cycle G1-to-S phase progression 45-68 Cell cycle alterations RB1 Cell cycle G1-to-S phase progression 33 Cell cycle alterations SOCS1 Inhibitor of JAK/STAT pathway 60 Activation of JAK/STAT pathway SOCS3 Inhibitor of JAK/STAT pathway 30 Activation of JAK/STAT pathway APC Inhibitor of β-catenin 53-71 Activation of β-catenin pathway RASSF1A Ras effector homologue 95-100 Inhibition of cell cycle arrest NORE1A/B Ras effector homologue 62 Inhibition of cell cycle arrest TIMP-3 Inhibition of matrix metalloproteinases 42 CDH1 Cell adhesion 33-49 Alteration in cytoskeletal organization, dissemination Dissemination CDH15 Cell adhesion 55 Dissemination SYK 27-77 Promotion of invasiveness and cell proliferation 54-65 NQO1 Immune and inflammatory responses, angiotensin II signaling pathway Xenobiotic metabolism, conjugation of glutathione Xenobiotic metabolism MGMT DNA repair 39 Accumulation of carcinogens and their metabolites Accumulation of carcinogens and their metabolites Increased mutation rates PROX1 Homeobox gene 47 Gene GSTP1 Function 50 Consequences Cell cycle alterations Misregulation of differentiation and cell proliferation PREDICTIVE POWER OF GENE METHYLATION FOR EARLY DETECTION OF HEPATOCELLULAR CARCINOMA Rivenbark AG, Coleman WB., Clin Cancer Res, 2007 BREAST CARCINOGENESIS Estrogen Radiation •Estrogen is a well-known breast carcinogen with both initiating and promoting properties •IR is the only genotoxic agent generally accepted as a breast carcinogen •Estrogen is linked to the neoplastic transformation of normal breast cells in vitro and in rodent model •Promotes the neoplastic transformation of normal breast cells in vitro and in rodent model •Women with elevated estrogen levels are considered to be a highrisk group for breast cancer development •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 Estrogen-Induced Rat Breast Carcinogenesis is Characterized by Alterations in DNA Methylation, Histone Modifications and Aberrant MicroRNA Expression Level of DNA methylation in mammary glands of control rats and rats exposed to estrogen Histone modifications in rat mammary glands of rats exposed to estrogen COMBINED EFFECTS OF ESTROGEN AND IONIZING RADIATION ON THE EPIGENETIC PROCESSES IN THE RAT MAMMARY GLAND (i) Sham treated controls; (ii) Estrogen treated group; (iii) IR treated group; (iv)IR + Estrogen treated group. LEVELS OF PROLIFERATION EXPRESSION OF DNA METHYLTRANSFERASES IN THE MAMMARY GLANDS OF ESTROGEN- AND RADIATION-EXPOSED RATS Kutanzi, EMM ,in revision 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 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 CARCINOGENESIS ? carcinogenesis Cancer initiationassociated epigenetic changes cancer progression Cancer progressionassociated epigenetic changes Advanced cancer Resistant relapse ? Chemotherapy Anticancer drug resistance Cancer Carcinoma incells situ Chemotherapyinduced epigenetic changes Novel epigenetic biomarkers of drug resistance Novel epigenetic therapy ACQUISITION OF ADDITIONAL RANDOM MUTATIONS Clonal selection and expression of initiated cells Mutator phenotype cells Cancer cells Environmental Normal cells ALTERATIONS IN CELLULAR EPIGENOME Epigenetically reprogrammed cells Mutator phenotype cells Cancer cells Endogenous Normal cells Endogenous Environmental GENETIC AND EPIGENETIC MODELS OF THE CANCER INITIATION 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 Cost and morbidity Hartwell et al. Nat Biotechnol., 2006. Cancer cells Carcinoma insitu Advanced cancer Resistant relapse Anticancer drug resistance ER cells carcinogenesis cancer progression chemotherapy 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 Cost and morbidity Hartwell et al. Nat Biotechnol., 2006 CHEMICAL Aflatoxin B1 Ethanol/Smoking Vinyl Chloride Metabolic Liver Diseases Hussain et al., Oncogene, 2007 LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODEL THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES Low dose radiation-induced epigenetic changes in an animal model Objective: to dissect the epigenetic basis of : induction of the low dose radiation- induced genome instability and adaptive response and the specific fundamental roles of epigenetic changes (i.e. DNA methylation, histone modifications and miRNAs) in their generation. Approach: we utilize an in vivo murine model to study epigenetic alterations in the radiation-target organs – thymus and spleen in context of low dose radiation effects and adaptive responses. We also archive and analyze other tissues – gonades, brain and liver. Exposure 0 Gy (sham) 0.01 Gy 0.1 Gy 1 Gy 10 x 0.01 Gy Results: • • • • • 0.01Gy ‘prime’ f ollowed by 1 Gy ‘challenge’ Time points Organs 6 hours thymus Global histone modif ication analysis Analysis of microRNAome 96 hours spleen 4 weeks Endpoints Global and locus-specif ic DNA methylation analysis DNA damage analysis by H2AX f oci Genome stability analysis Gene expression analysis In this study, we for the first time found that low dose radiation (LDR) exposure causes profound and tissue-specific epigenetic changes in the exposed tissues We established that LDR exposure affects methylation of repetitive elements in the genome, causes changes in histone methylation, acethylation and phosphorylation Importantly, LDR causes profound and persistent effects on small RNAs profiles. MicroRNAs are excellent biomarkers of LDR exposure. LDR exposure causes tissue-specific changes in gene expression. We identified several novel biomarkers of LDR exposure. Koturbash et al., Cell Cycle, 2008 Koturbash et al., Mutation Res., 2008 •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 Possible linkage between radiation-induced bystander effects in vivo and carcinogenesis •γH2AX foci accumulation •DNA hypomethylation all are signs of carcinogenesis •histone modifications •gene expression changes •altered proliferation and apoptosis •microRNA changes mechanisms of IR and bystander-induced carcinogenesis means of prevention RADIATION EFFECTS ON NEUROBLASTOMA AND GLIOBLASTOMA – AN EPIGENETIC CONNECTION 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. 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. 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. 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. 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) 24 hours 72 hours 24 hours 72 hours DNA methylation A-172 IMR-32 19 3 90 1358 Gene expression A-172 IMR-32 113 225 3 4 SK-B-NE 17 9 SK-B-NE 2 0 DNMT1, DNMT3a and MeCP2 in neuroblastoma and glioma cells A 172 CT CT IR IMR-32 IR CT CT IR SK-N-BE IR CT CT IR IR 24h 72h 24h 72h 24h 72h 24h 72h 24h 72h 24h 72h DNMT1 DNMT3a MeCP2 loading A 172 CT CT IR IMR-32 IR CT CT IR SK-N-BE IR CT CT IR IR 24h 72h 24h 72h 24h 72h 24h 72h 24h 72h 24h 72h γH2AX p53 Low dose IR-induced changes in protein expression in neuroblastoma and glioma cells PCNA cyclin D1 CREB1 cyclin E loading High H2AX, p53 – glioma cells repair damage really well! Correlation between the levels of gene expression, methylation and apoptosis in the studied neuroblastoma and glioma cells 24 hours 72 hours 24 hours 72 hours 24 hours 72 hours DNA methylation IMR-32 A-172 + + +++++++++++++++++++++++ +++++++ Gene expression IMR-32 A-172 +++++++++ ++++ ++ + Apoptosis IMR-32 A-172 ++ + +++++ ++ SK-B-NE ++ + SK-B-NE + SK-B-NE -- KEY CONCLUSIONS: •Low dose IR exposure affects gene expression and methylome of the studied cell lines •Gene expression changes were most pronounced in neuroblastoma cells •Gene expression changes in glioma cells were the least pronounced •IR induced apoptosis in neuroblastoma •IR blocked apoptosis in glioma THUS: Analysis of DNA methylation, gene expression and apoptosis in brain cancer cells lines reveals a potential anti-tumor effect of low dose radiation in neuroblastoma and an opposite tumor-promoting effect in malignant glioma Kovalchuk group Bo Wang Dongping Li Anna Kovalchuk Rocio Rodriguez-Juarez Lidia Luzhna Slava Ilnytsky Acknowledgements Collaborators: Bryan Kolb, CCBN, Canada Igor Pogribny, NCTR, USA Vasyl Chekhun, IEORB, Ukraine Funding: Alumni Jody Filkowski Natasha Singh Julian St. Hilaire Dmitry Litvinov Kristy Kutanzi Igor Koturbash Jonathan Loree James Meservy CIHR Institute of Gender and Health – Chair Program