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

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men

tal

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ALTERATIONS IN CELLULAR EPIGENOME

Normal cells

Cancer cells

Clonal selection and expression of initiated cells

Mutator phenotype cells

En

dog

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sEn

dog

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ou

s

En

vir

on

men

tal

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men

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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