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EPIGENETICS OF CARCINOGENESIS
Olga Kovalchuk, MD/PhDOlga Kovalchuk, MD/PhDUniversity of Lethbridge, AB, CanadaUniversity of Lethbridge, AB, Canada
EPIGENETICS VERSUS GENETICS
EPIGENETICS
Alterations
Heritable transmission of information in the absence of changes in DNA
sequence
GENETICS
SNP
C/T
Heritable transmission of information based on differences in DNA sequence
MTHFR, C677TGCC→GTC
Allis CD et al., In: Epigenetics, 2007
Epigenetic alterations – changes induced in cells that alter expression of the information on transcriptional, translational, or post-translational levels without change in DNA sequence
EPIGENETIC CHANGES
Methylation of DNA
Modifications of histones
RNA-mediated modifications
• siRNA, miRNA, piRNA …
Control Treated-3.0 3.00
A
Me
P
U
- acetylation
- methylation
- phosphorylation
- ubiquitination
P UMe
A
METHYLATION LANDSCAPE OF THE HUMAN GENOME
LINE 22.9%
LTR 9.3%
SINE 10.1%
Other 38.7%
Low Complexity Repeats 1.3%
Other Repeats 0.15%
Alpha Satellite 2.07% Classical Satellite
2.1%
Simple Repeats 1.7%
CpG Island (Non-overlapping) 0.57%
Ensembl 1st Exons (Non-overlapping) 0.2%
CpG Island/1st Exons Overlap 0.11% Other Ensembl Exons 1.83%
DNA Transposons 3.6%
COMPOSITION OF GENOME
Rollins RA et al.,Genome Res, 2006
LINE 22.9%
LTR 9.3%
SINE 10.1%
Other 38.7%
Low Complexity Repeats 1.3%
Other Repeats 0.15%
Alpha Satellite 2.07% Classical Satellite
2.1%
Simple Repeats 1.7%
CpG Island (Non-overlapping) 0.57%
Ensembl 1st Exons (Non-overlapping) 0.2%
Promoters/1st Exons Overlap 0.11% Other Ensembl Exons 1.83%
DNA Transposons 3.6%
LINE 22.9%
LTR 9.3%
SINE 10.1%
Other 38.7%
DISTRIBUTION OF METHYLATED AND UNMETHYLATED DOMAINS
Rollins RA et al.,Genome Res, 2006
-Unmethylated CpG
- Methylated CpG
METHYLATION LANDSCAPE OF THE HUMAN GENOME
E1 E2 E3
E1 E2 E3
X
- Unmethylated CpG - Methylated CpG
Unmethylated domains (CpG islands at gene promoters)
Simple tandem repeat
DNA transposon
LTR – endogenous retrovirus
Methylated domains (repeated DNA sequences)
SR SR SR SR
IR IRTransposon
5’LTR 3’LTRgag envpol
TSDR TSDRMSC P ORF2ORF1A/T Rich
Site
TSDR TTTTDR DRPoly(A)SVA Element
Non-LTR autonomous retrotransposon: LINE
Non-LTR non-autonomous retrotransposon: SINE
Sequence compartment
CpG G + C (%)CpG
Obs/Exp (%)
Genome 29,848,753 41 24
Promoter 1,876,802 62 89
First Exon 508,553 56 65
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
Classical Satellite ~1,140,000 34 67
Other 8,358,888 42 15
Distribution of CpG sites in the human genome
Wilson AS et al., BBA, 2007Rollins RA et al., Genome Res, 2006
CpG island• G + C content > 0.55.
• Observed vs expected CpG densities > 0.5.• Lengh > 300 bp (500 bp).
POST-TRANSLATIONAL HISTONE MODIFICATIONS
H2AH3
H4H2B
CHROMATIN NUCLEOSOME
POST-TRANSLATIONAL HISTONE MODIFICATIONS
Histone modifications
Role in transcription
Histone-modified sites
Acetylation activation H3 (K9, K14, K18, K56)
H4 (K5, K8, K12, K16)
H2A
H2B (K6, K7, K16, K17)
Phosphorylation activation H3 (S10)
Methylation activation H3 (K4, K36, K79)
repression H3 (K9, K27)
H4 (K20)
Ubiquitination activation H2B (K123)
repression H2A (K119)
Sumoylation repression H3 (?)
H4 (K5, K8, K12, K16)
H2A (K126)
H2B (K6, K7, K16, K17)
TYPES AND ROLES OF HISTONE MODIFICATIONS
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 Astrand B
strand B
DNA replication
Maintenance DNA methylation
strand A
Maintenance of histone modifications
Felsenfeld G., In: Epigenetics, 2007
Initiation
Promotion
Progression
Normal cells
Single initiated cells
Focal proliferation
Single carcinoma cells
Carcinoma
STAGES OF CARCINOGENESIS
?
ENVIRONMENTAL EPIGENETICS – MECHANISMS OF
EPIGENETIC PROGRAMMING BY THE ENVIRONMENT
AND THEIR POSSIBLE IMPLICATIONS FOR TOXICOLOGY
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).
ESTROGENIC CHEMICAL BISPHENOL A
Dolinoy, 2007
SELECTED LIST OF ENVIRONMENTAL CHEMICAL AGENTS THAT
ALTER CELLULAR EPIGENETIC PATTERNS
Agent Effect Reference
Arsenic Global DNA hypomethylation
Hypomethylation of GC-rich sequencesGene-specific hypomethylation (Er-α, cyclin D1)Gene-specific hypermethylation (p53, p16INK4A, RASSF1A)
Inhibition of DNMT1 and DNMT3a expressionHistone acetylation
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, 2006Reichard JF et al., BBRC, 2007.Ramirez T et al., Chromosoma, 2007
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)
Takiguchi M et al., Exp Cell Res, 2003
Benbraim-Tallaa L et al., Environ Health Perspect, 2007
Hydrazine Global DNA hypomethylationGene-specific hypomethylation (p53, c-myc, HMG CoA reductase)
Fitzgerald BE, Shank RC, Carcinogenesis, 1996Zheng H, Shank RC, Carcinogenesis, 1996; Coni P et al., Carcinogenesis, 1992
Benzo(a)pyrene Global DNA hypomethylationGene-specific hypermethylation (CYP1A1) Promoter-specific histone H3 lysine 9 hypo- and hyperacetylationCpG-methylation-associated mutations (p53)
Wilson WL, Jones PA, Carcinogenesis, 1984Anttila S et al., Cancer Res, 2003Sadikovic B et al., J Biol Chem, 2008 Yoon JH et al., Cancer Res 2001
Aflatoxin B1 Gene-specific hypermethylation (GSTP, MGMT, RASSF1A, p16INK4A)
Zhang YJ et al., Mol Carcinog, 2002; Zhang YJ et al., Int J Cancer, 2003; Zhang YJ et al., Cancer Lett, 2005.
2-Acetylaminofluorene Gene-specific hypermethylation (p16INK4A)Loss of histone H4 lysine 20 trimethylationIncreased DNMT1 expression
Bagnyukova TV et al., Carcinogenesis, 2008
Peroxisome proliferators(WY-14643)
Global DNA hypomethylationHypomethylation of GC-rich sequencesLoss of histone H4 lysine 20 trimethylationGene-specific hypomethylation (c-myc)
Ge R et al., Toxicol Sci, 2001; Pogribny IP et al., Mutat Res, 2007.
Dibromoacetic acid Global DNA hypomethylation Tao L et al., Toxicol Sci, 2004
DO EPIGENETIC CHANGES PLAY A ROLE IN CARCINOGENESIS?
DNA METHYLATION CHANGES DURING SKIN CARCINOGENESIS
NS MCA3D PB MSCP6 PDV PAM212 MSCB119
MSC11A5
HaCa4 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
Status of global DNA methylation
CpG island methylation status of selected genes
Fraga MF et al., Cancer Res, 2004
SELECTED LIST OF GENES HYPERMETHYLATED IN HUMAN HEPATOCELLULAR CARCINOMA
Gene FunctionFrequency in HCC, %
Consequences
p16INK4A Cell cycle G1-to-S phase progression 32-65 Cell cycle alterations
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 Alteration in cytoskeletal organization, dissemination
CDH1 Cell adhesion 33-49 Dissemination
CDH15 Cell adhesion 55 Dissemination
SYK Immune and inflammatory responses, angiotensin II signaling pathway
27-77 Promotion of invasiveness and cell proliferation
GSTP1 Xenobiotic metabolism, conjugation of glutathione
54-65 Accumulation of carcinogens and their metabolites
NQO1 Xenobiotic metabolism 50 Accumulation of carcinogens and their metabolites
MGMT DNA repair 39 Increased mutation rates
PROX1 Homeobox gene 47 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 CARCINOGENESISBREAST CARCINOGENESIS
Estrogen Radiation
•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
•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
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
(i) Sham treated controls; (ii) Estrogen treated group; (iii) IR treated group; (iv)IR + Estrogen treated group.
COMBINED EFFECTS OF ESTROGEN AND IONIZING COMBINED EFFECTS OF ESTROGEN AND IONIZING RADIATION ON THE EPIGENETIC PROCESSES IN THE RAT RADIATION ON THE EPIGENETIC PROCESSES IN THE RAT
MAMMARY GLANDMAMMARY GLAND
LEVELS OF PROLIFERATION
EXPRESSION OF DNA METHYLTRANSFERASES IN THE MAMMARY GLANDS OF ESTROGEN- AND RADIATION-EXPOSED RATS
Kutanzi, EMM ,in revision
up-regulated down-regulated
IR+Estrogen
IR estrogen
30
8 31
32
IR+Estrogen
IR estrogen
27
4
1 6
3
34343535
1111 77 55 99
up-regulated down-regulated
IR+Estrogen
IR estrogen
30
8 31
32
IR+Estrogen
IR estrogen
30
8 31
32
IR+Estrogen
IR estrogen
27
4
1 6
3
IR+Estrogen
IR estrogen
27
4
1 6
3
34343535
1111 77 55 99
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 cancer progression Chemotherapy
Cancer cells
Carcinoma in-situ
Advanced cancer
Resistant relapse
? ?
Cancer progression- associated epigenetic changes
Novel epigenetic biomarkers of drug
resistance
Novel epigenetic
therapy
An
tica
nce
r d
rug
re
sist
ance
Chemotherapy-induced
epigenetic changes
CARCINOGENESIS
Cancer initiation- associated epigenetic changes
GENETIC AND EPIGENETIC MODELS OF THE CANCER INITIATION
Epigenetically reprogrammed cells
Mutator phenotype cells
En
dog
en
ou
sEn
dog
en
ou
s
En
vir
on
men
tal
En
vir
on
men
tal
ALTERATIONS IN CELLULAR EPIGENOME
Normal cells
Cancer cells
Clonal selection and expression of initiated cells
Mutator phenotype cells
En
dog
en
ou
sEn
dog
en
ou
s
En
vir
on
men
tal
En
vir
on
men
tal
ACQUISITION OF ADDITIONAL RANDOM MUTATIONS
Normal cells
Cancer cells
EPIGENETIC MODEL OF CARCINOGENESIS
Ant
ican
cer
drug
re
sist
ance
carcinogenesis cancer progression chemotherapy
Cancer cells
Carcinoma in-situ
Advanced cancer
Resistant relapse
ER cells
Screening for early-stage
disease
Detection and
localization
Disease stratification
and prognosis
Response to therapy
Risk assessment
Screening for disease recurrence
Cost and morbidity
Hartwell et al. Nat Biotechnol., 2006.
Hartwell et al. Nat Biotechnol., 2006
EPIGENETIC ALTERATIONS CAN PREDICT HUMAN HEPATOCARCINOGENESIS
Metabolic Liver Diseases
Hussain et al., Oncogene, 2007
Screening for early-stage disease
Detection and localization
Disease stratification and
prognosis
Response to therapy
Risk assessment
Screening for disease
recurrence
Cost and morbidity
CHEMICALAflatoxin B1
Ethanol/SmokingVinyl Chloride
LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODELMODEL
THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGESTHERAPEUTIC 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.
Results:• 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.
1 Gy
0.01 Gy
0 Gy (sham)
0.1 Gy
10 x 0.01 Gy
6 hours
4 weeks
96 hours
thymus
spleen
Global and locus-specif ic DNA methylation analysis
Global histone modif ication analysis
Analysis of microRNAome
DNA damage analysis by H2AX foci
Exposure Time points Organs Endpoints
Genome stability analysis 0.01Gy ‘prime’ followed by 1 Gy‘challenge’
Gene expression analysis
Koturbash et al., Cell Cycle, 2008Koturbash et al., Mutation Res., 2008
•Bystander effects occur Bystander effects occur in vivoin vivo
•Epigenetic changes are involved in generations and/or Epigenetic changes are involved in generations and/or
maintenance of bystander effectsmaintenance of bystander effects
•Bystander effects are tissue specificBystander effects are tissue specific
•Bystander effects are strain and species-independent, but Bystander effects are strain and species-independent, but
there are some mouse strain differencesthere are some mouse strain differences
•Bystander effects are persistentBystander effects are persistent
•Bystander effects are sex specificBystander effects are sex specific
•Bystander effects affect the germlineBystander effects affect the germline
Possible linkage between radiation-induced
bystander effects in vivo and carcinogenesis
•γH2AX foci accumulation
•DNA hypomethylation
•histone modifications
•gene expression changes
•altered proliferation and apoptosis
•microRNA changes
all are signs of carcinogenesis
mechanisms of IR and bystander-induced
carcinogenesis
means of prevention
RADIATION EFFECTS ON NEUROBLASTOMA AND GLIOBLASTOMA – AN EPIGENETIC CONNECTION
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.
INTRODUCTION
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.
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.
INRODUCTION
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.
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)
DNA methylation A-172 IMR-32 SK-B-NE 24 hours 19 3 17 72 hours 90 1358 9
Gene expression A-172 IMR-32 SK-B-NE 24 hours 113 225 2 72 hours 3 4 0
RESULTS
DNMT1, DNMT3a and MeCP2 in neuroblastoma and glioma cells
CT
24h
CT
72h
IR
24h
IR
72h
CT
24h
CT
72h
IR
24h
IR
72h
CT
24h
CT
72h
IR
24h
IR
72h
A 172 IMR-32 SK-N-BE
DNMT1
DNMT3a
MeCP2
loading
Low dose IR-induced changes in protein
expression in neuroblastoma and
glioma cells
CT
24h
CT
72h
IR
24h
IR
72h
CT
24h
CT
72h
IR
24h
IR
72h
CT
24h
CT
72h
IR
24h
IR
72h
A 172 IMR-32 SK-N-BE
γH2AX
p53
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
DNA methylation A-172 IMR-32 SK-B-NE 24 hours + + ++ 72 hours +++++++ +++++++++++++++++++++++ +
Gene expression A-172 IMR-32 SK-B-NE 24 hours ++++ +++++++++ + 72 hours + ++ -
Apoptosis A-172 IMR-32 SK-B-NE 24 hours + ++ - 72 hours ++ +++++ - -
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
Acknowledgements
Funding:Funding:
Collaborators:Bryan Kolb, CCBN, CanadaIgor Pogribny, NCTR, USA
Vasyl Chekhun, IEORB, Ukraine
CIHR Institute of Gender and Health – Chair Program
Kovalchuk groupBo Wang
Dongping Li
Anna Kovalchuk
Rocio Rodriguez-Juarez
Lidia Luzhna
Slava Ilnytsky
Alumni
Jody Filkowski
Natasha Singh
Julian St. Hilaire
Dmitry Litvinov
Kristy Kutanzi
Igor Koturbash
Jonathan Loree
James Meservy