Transcript epigenetics of carcinogenesis

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