Human Cancer Epigenetics

Dr. Manel Esteller mesteller@idibell.cat

Director

Cancer Epigenetics and Biology Program (PEBC),

Bellvitge Biomedical Research Institute (IDIBELL)

ICREA Research Professor

Genetics Professor, School of Medicine, University of Barcelona

DNA methylation plasticity of human adipose-derived stem cells in lineage commitment

- 293 + 92PTPRS

Re

gre

sse

d

Ste

mC

ells

(fro

mm

yo

cyte

s)

- 152 + 160PIWIL2

Re

gre

sse

d

Ste

mC

ells

(fro

mo

ste

ocyte

s)

Re

gre

sse

d

Ste

mC

ells

(fro

mm

yo

cyte

s)

- 159 + 162SAA1

Re

gre

sse

d

Ste

mC

ells

(fro

mo

ste

ocyte

s)

Re

lative

exp

ressio

n

(PT

PR

S v

s G

AP

DH

)

0

200

400

600

800

1 2 3

Re

lative

exp

ressio

n

(SA

A1

vs G

AP

DH

)

0

50

100

150

1 2 3

Re

lative

exp

ressio

n

(PIW

IL2

vs G

AP

DH

)

0

50

100

150

1 2 3 4 5

A

B C

OSTEOGENIC

Differentiation

medium

Suppression of

the inductorDifferentiation

medium

Suppression of

the inductor

MYOGENIC

- 293 + 92PTPRS

Adult

Stem Cells_1

Adult

Stem Cells_2

In vitro

myocytes_1

In vitro

myocytes_2

- 152 + 160PIWIL2

Adult

Stem Cells_1

Adult

Stem Cells_2

In vitro

myocytes_1

In vitro

osteocytes_2

Adult

Stem Cells_1

Adult

Stem Cells_2

In vitro

Osteocytes_1

In vitro

Osteocytes_2

- 159 + 162SAA1

Berdasco et al., American Journal of Pathology 2012

The Epigenetic Dice:

-DNA Methylation

-Histone Modifications

-ncRNAs

-….

Cover by Sanchez-Mut and Esteller, Dev Cell, November 15th 2010

Towards the Human Epigenome

50-year-old

A

C

B

D

3-year-old twins 50-year-old twins3-year-old twins 50-year-old twins

0,00

0,40

0,80

1,20

1,60

2,00

0 15 30 45 60 75

Age (years)

ES

D 5

mC

0,00

0,40

0,80

1,20

1,60

2,00

0 15 30 45 60 75

Age (years)

ES

D A

c H

4

0,00

0,40

0,80

1,20

1,60

2,00

0 15 30 45 60 75

Age (years)

ES

D A

c H

3Francis Collins, NIH director, said:

"Here's something where Mendel, Watson and Crick all seem to have missed some crucial goodies"

Heyn et al., Carcinogenesis 2012

Discordant Twins for Breast Cancer

HOUSEKEEPING GENES

TISSUE -SPECIFIC GENES

TISSUE -SPECIFIC GENES

CpG –rich genes

Normal Cells Cancer Cells

RB, P16 MGMT GSTP1 BRCA1MLH1

Gain

HOXA5 HOXA9 MASPIN

MCJ

Gain

Loss

MAGECAGE BAGE LAGE

CpG –poor genes

E3

PERICENTROMERIC SEQUENCES CODING REGION

REP REP

CpG RICH

E1 E2

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG RICH

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG RICH

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG RICH

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG RICH

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG RICH

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG POOR Loss

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG POOR Gain

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG POOR

PERICENTROMERIC SEQUENCES CODING REGION

E1 E2 E3REP REP

CpG POOR

?

?

Berdasco and Esteller, Developmental Cell, 2010

Esteller, New England Journal of Medicine, 2008

Roriguez-Paredes and Esteller, Nature Medicine, 2011

N-S R G K Q G G K A R A K A K S… …L R K G N… …L G K V T… …L P K K T E S H…-C 9 15 1

P A

5

A

13

A A

36

A M

99 120 119

M U P

H2A

14

P

N-…P A K S A... …K G S K K A V T K… …V Y K V L… …Y N K R S… …L A K H A… …K A V T K…-C 5

A

12

A

15 20

M

43

A

85

A

108

A

116 120

U A

M A

U A

H2B

M M M M

N-A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S A P A T G G V K K P H R Y R P G T V… 2 3

P

4 8 9

M A

10

P

11

P

17

A

18 23

M A

26 27

M A

28

P

36

M

37

A

14

M A

M A

41

P

…Y Q K S T… …D F K T D…-C 56

M

79

45

P

M A H3.1

N-S G R G K G G K G L G K G G A K R H R K... …R I S G L… …V L K V F… …K R K… …L K R Q…-C 1

P M

3

A

5

A

8 12

M A A

16 20

M A

47

P M

59

A

77

A

79

A

91

M

92 H4

H1.4

N-S E T A P… …A E K T P V… …K S A G A A K R K A S… …K A V A A S K E R… …A L K K A L... 26

M

27 3 36 17

A

34 18 46 52

…K S L V S K G T L V Q T K… …S F K L N… …K S A K K T… …K K A K S… …P K S P A… -C

P P P A P U A A

64

M

85

A

90

A

M A

97

M A

106

M M

149 154

P

63

168

M A

172

P

186

P

Portela and Esteller Nature Biotechnology, 2010

Histone Modification Scenarios

N-S R G K Q G G K A R A K A K S… …L R K G N… …L G K V T… …L P K K T E S H…-C 9 15 1

P A

5

A

13

A A

36

A M

99 120 119

M U P

H2A

14

P

N-…P A K S A... …K G S K K A V T K… …V Y K V L… …Y N K R S… …L A K H A… …K A V T K…-C 5

A

12

A

15 20

M

43

A

85

A

108

A

116 120

U A

M A

U A

H2B

M M M M

N-A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S A P A T G G V K K P H R Y R P G T V… 2 3

P

4 8 9

M A

10

P

11

P

17

A

18 23

M A

26 27

M A

28

P

36

M

37

A

14

M A

M A

41

P

…Y Q K S T… …D F K T D…-C 56

M

79

45

P

M A H3.1

N-S G R G K G G K G L G K G G A K R H R K... …R I S G L… …V L K V F… …K R K… …L K R Q…-C 1

P M

3

A

5

A

8 12

M A A

16 20

M A

47

P M

59

A

77

A

79

A

91

M

92 H4

H1.4

N-S E T A P… …A E K T P V… …K S A G A A K R K A S… …K A V A A S K E R… …A L K K A L... 26

M

27 3 36 17

A

34 18 46 52

…K S L V S K G T L V Q T K… …S F K L N… …K S A K K T… …K K A K S… …P K S P A… -C

P P P A P U A A

64

M

85

A

90

A

M A

97

M A

106

M M

149 154

P

63

168

M A

172

P

186

P

Histone Modification Scenarios

Portela and Esteller Nature Biotechnology, 2010

Melo and Esteller, FEBS Letters, 2010

Melo and Esteller, FEBS Letters, 2010

Nature Genetics, 2009

Science, 2009

Cancer Cell, 2010

Cancer Res, 2007

PNAS, 2008

Oncogene, 2011

NEJM, 2011

Esteller, Nature Reviews Genetics 2011

Esteller, Nature Reviews Genetics 2011

Cancer Res 2007

PNAS 2008

Oncogene 2011

Oncogene 2012

Esteller, Nature Reviews Genetics 2011

Cancer Res 2007

PNAS 2008

Oncogene 2011

Oncogene 2012

Oncogene 2010

Esteller, Nature Reviews Genetics 2011

Cancer Res 2007

PNAS 2008

Oncogene 2011

Oncogene 2012

Oncogene 2010

RNA Biology 2012

June 2012

Guil and Esteller, Nature Structural & Molecular Biology, Nov 2012

I see mutated epigenetic genes…

Rodriguez-Paredes and Esteller, Nature Medicine, 2011

Epigenetic drugs

DNA Demethylating Agents

Histone Deacetylase Inhibitors

microRNA Related Compounds

Histone Methyltransferase Inhibitors

Histone Demethylase Inhibitors

Histone Kinase Inhibitors

Histone Acetyltransferase Inhibitors

Bromodomain Inhibitors

Sirtuin Inhibitors

Preclinical

stage

Clinical

stage

Rodriguez-Paredes and Esteller, Cancer Discovery 2011

From Single Epigenetic

Biomarkers to Epigenomics

Esteller M et al., Cancer Res. 59:793-7, 1999.

Normal brain GBM patients GBM cell lines

Meth

ylatio

n le

vel

(BeadA

rray B

eta

valu

es)

1.0 0.0

Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 Epigenetics, Vol 4, May 15, 2009

A microarray-based DNA methylation study of glioblastoma multiforme (GBM)

BRCA1

-Breast cancer is one of the most common diseases, affecting one in

8 women during their life time in the Western world.

-BRCA1, a breast cancer susceptibility gene was mapped to

chromosome 17q21 by linkage studies. Defects in this gene are

predicted to be responsible for 45% of inherited breast cancer and >80%

of inherited breast and ovarian cancer.

-However, despite the high incidence of loss of heterozygosity of the

BRCA1 region in sporadic breast and ovarian cancers, BRCA1 somatic

mutations have only been found in a few cases of sporadic ovarian and

breast cancers.

-This suggests that an alternative mechanism for BRCA1-inactivation or

other genes in the vicinity of the BRCA1 gene may play an important role

in the development of sporadic breast and ovarian cancer.

U U U U U U U U U M M M M M M M M M

NB BC NB BC BC NB IVD NL H2O

Patient 1 Patient 2 Patient 3 A

U U U U U U U U M M M M M M M M

Ov93 Ov88 Ov51 Ov30 Ov27 Ov47 IVD NL H2O

U U U U U U U U U M M M M M M M M M

CRC1 CRC2 Leu1 Leu2 LivC1 LivC2 NL IVD H2O

U M

B

C

BRCA1 Promoter Hypermethylation in Breast,

Ovarian and Other Tumor Types

J Natl Cancer Inst, 2000

Exploting BRCA1 and BRCA2 defects in therapy

BRCA1 CpG Island Hypermethylation Predicts Sensitivity to PARP Inhibitors

Veeck et al. Journal of Clinical Oncology, 2010

Per

cen

t S

urv

ival

Per

cen

t S

urv

ival

Time (Days) Time (Days)

Cell Line BRCA1 status IC50 (+/- SD)

MDA-MB-231 (wild-type) 17.5 (+/-3.0)

MDA-MB-436 (mutated) 0.9 (+/-0.4)

UACC-3199 (methylated) 3.1 (+/-1.1)

HCC-38 (methylated) 4.7 (+/- 1.8)

B

D

0

MDA-MB-231

(wild-type)

MDA-MB-436

(mutated)

UACC-3199

(methylated)

HCC-38

(methylated)

A

C

P = 0.0087 P = 6.4 x 10-7

BRCA1 Epigenetic Inactivation Predicts Sensitivity to Platinum-Based Chemotherapy

Stefansson et al. Epigenetics, 2012

Esteller, 2011

A DNA Methylation Fingerprint

Heyn and Esteller, Nature Reviews Genetics, 2012

A DNA Methylation Fingerprint of 1,628 Human Samples

Fernandez et al., Genome Research, June 1, 2011

A DNA Methylation Fingerprint of 1,628 Human Samples

Fernandez et al., Genome Research, June 1, 2011

DNA methylation profiling in Leukemia

Wiliop et al., British Journal of Haematology, 2011

Adult Acute Myeloid Leukemia (AML) Childhood Acute Lymphoblastic Leukemia (ALL)

Sandoval et al., British Journal of Haematology, 2012

Epigenetics, June 1, 2011

A DNA methylation microarray for 450,000 CpG sites in the human genome

Group 1 A.

Group 2

0 0.5 1

Adeno

Squamous

Large

0 20 5 10 15

0

0.2

0.4

0.6

0.8

group2 (n=174)

group1 (n=24)

1.0

Pro

po

rtio

n w

ith

rec

urr

ence

-fre

e su

rvia

l

Years after surgery

p: 0.002

Hazard ratio, 2.30 (95% CI, 1.36-3.89)

B.

Group 1 C. Group 2

0 0.5 1

group2 (n=114)

group1 (n=33)

p: 0.005

Hazard ratio, 2.46 (95% CI, 1.32-4.59)

D. Adeno

Squamous

Large

0 20 5 10 15

0

0.2

0.4

0.6

0.8

1.0

Pro

po

rtio

n w

ith

rec

urr

ence

-fre

e su

rvia

l

Years after surgery

A 450,000 CpG Site Prognostic Classifier of 500 Non-Small Cell Lung Cancer Patients

Dynamic and Heterogeneous

Representative scatter plots reporting CpG methylation levels

between different individuals EC01 and EC10. (A) swim-up (Up)

sperm samples; (B) swim-down (Dn) sperm samples; (C) whole

sperm population at 1 h (Ws 1 h) samples; (D) Box plot representing

the inter-individual variability of DNA methylation levels in total CpGs

from the swim-up, swim-down and whole sperm population at1h

samples. The median value is shown. * corresponds to p value =

0.0213; R2 = Pearson coefficient. The boxes describe the lower

quartile (Q1, 25%), median (Q2, 50%) and the upper quartile (Q3,

75%); the whiskers extend 1.5 times the IQR from the box.

Intra- and Inter-Individual Variability of Sperm DNA Methylation

Krausz et al. PLOS ONE, October 2, 2012

Viral DNA Methylomes: Epstein-Barr Virus (EBV)

Fernandez et al, Genome Research 2010

ZEB1 ZEB2

E-cadherin CRB3 PALS1 PATJ LGL2

miR-200 Epigenetic regulation ?

Chr 1

b a 429 c 141

Chr 12

AAUACUG

AACACUG

miR-200 FAMILY

x MCF7 MDAMB231 HCT116 RKO H441 H23 H157

BR

EAST

C

OLO

N

LUN

G

-121

miR-200ba429 promoter

+210

miR-200c141 promoter -564

-170

miR-200ba429 miR-200c141

I. CpG methylation in miR-200 promoters

Master regulator of epithelial phenotype

Davalos et al., Oncogene 2011

BREAST

COLON

0

5

10

15

20

Log2 F

old

change

(Refe

rence: M

DA

MB

231)

MDAMB231*

MCF7

0

5

10

15

Log2 F

old

change

(Refe

rence: R

KO

)

RKO*

Co115

HCT116

DLD1

-10

-5

0

5

10

15

20

200b 200a 429 200c 141

Log2 F

old

change

(Refe

rence: H

157)

H157* H1264*

H23* H441

EBC1 Calu3

LUNG

II. Epigenetic silencing of miR-200 family III. Correlation between epigenetic inactivation of miR-200 and expression of their targets

Epithelial-related genes

ZEB1/2

miR-200

x

Epithelial-related genes

ZEB1/2

miR-200

x

Unmethylated miR-200 Methylated miR-200

* Methylated cell lines

* Methylated cell lines

0

5

10

15

20

25

ZEB1 ZEB2 CDH1 CRB3 LGL2 PARD6B

Fold

change (

Log2)

DLD1

MCF7

EBC1

H441

RKO*

MDAMB231*

MDAMB436*

H157*

Davalos et al., Oncogene 2011

468 PT

468 LN

miR-200ba429 miR-200c141

miR-200 expression

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

miR200b miR200a miR429 miR200c miR141

Fo

ld c

han

ge (

Lo

g2)

(Refe

rence 4

68_P

T)

468_PT 468_LN

IV. Dynamic epigenetic regulation of miR-200 expression during EMT. MDAMB468 model

Davalos et al., Oncogene 2011

miR-200ba419 miR-200c141

0

10

20

30 TGFB 30 days

TGFB 30 days + AZA

TGFB 30 days + AZA TSA

200b 200a 200c 141

Fold

ch

ange

TGFβ 30 days TGFβ 30 days + aza TGFβ 30 days + aza/TSA

-15

-10

-5

0

5

con

tro

l 1

0 d

ays

20

day

s 3

0 d

ays

TGFβ

200b 200a 200c 141

Fold

ch

ange

(Lo

g2)

-5

0

5

10

Epit

hel

ial

TGFβ withdrawal

Mes

ench

ymal

TGFβ

30

day

s

Fold

ch

ange

(Lo

g2)

Co

ntr

ol

10

day

s

TGFβ

tre

atm

en

t

20

day

s 3

0 d

ays

TGFβ

wit

hd

raw

al

EMT

MET

Un

tre

ate

d

10

day

s Epit

hel

ial

Mes

ench

ymal

IV. Dynamic epigenetic regulation of miR-200 expression during EMT. MDCK model

Carmona and Esteller, Cancer Research 2011

The Entangled Web of Cancer Metastasis and Angiogenesis

Metastasis-specific CpG Island Hypermethylation: Cadherin-11

Carmona et al., The Journal of Pathology 2012

Transfection

shRNA

Carmona et al., The Journal of Pathology 2012

Whole Genome

Bisulfite Sequencing

Distance correlation to closest CpG site

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

Promoter Exonic Intronic Intergenic

Mean

meth

yla

tio

n level

NB

Y103

**

** ** **

68%

70%

72%

74%

76%

78%

80%

82%

Cp

G m

eth

yla

tio

n

lev

el (%

)

NB Y103 Y26 Corr

ela

tio

n

Distance

0.1

1.0

10 100 1 1000

16,000,000

16,200,000

16,400,000

16,600,000

16,800,000

Me

thyla

ted

Cp

G s

ite

s

NB Y103 Y26

Chromosome 8

0.8

0.6

0.4

Meth

. le

vel

Chromosome 2

0.8

0.6

0.4

Meth

. le

vel

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

CG poor promoters

CGI promoters

Me

an

me

thyla

tio

n le

ve

l

NB

Y103

**

**

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

Promoter Exonic Intronic Intergenic

DM

R n

um

ber

CpG poor

promoters CpG island

promoters

671; 83%

140; 17%

585; 55%

479; 45%

15669;

87%

2261; 13%

Hypomethylated

in Y103Hypermethylated

in Y103

DMRs

Housekeeping genes Tissue-specific genes

56; 87%

8; 13%

1200; 66%

611; 34%

The Distinct DNA Methylomes of Newborns and Centenarians

Proc Natl Acad Sci USA, 2012

R² = 0.6456

-1

-0.5

0

0.5

1

-2.5 -1.5 -0.5 0.5 1.5 2.5

Dif.methylation

level (WGBS) D

if.m

eth

yla

tio

n

leve

l (4

50

K)

Corr

ela

tio

n

0

.

1

1.0

1 1000

Distance

100 10

1

10

100

1000

AIM2 miR-21 TNFRSF9 IGSF9B PTPRE

Rela

tive e

xpre

ssio

n v

alu

es (%

)

NB

Y103

0

0

C +13 -567

-482 +77

+733 +278

+5905 +5366

0 0

+829 +1436

The Distinct DNA Methylomes of Newborns and Centenarians

Proc Natl Acad Sci USA, 2012

0

500

1000

1500

2000

2500

3000

3500

4000

Promoter Exonic Intronic Intergenic

DM

R n

um

ber

1812;

93%

132; 7%

5073; 88%

701; 12%

Hypomethylated

in Y103

Hypermethylated

in Y103

81; 42%

113;

58%

E

B

G

Distance

0.1

1.0

10 100 1 1000

D

CpG island

promoters

CpG poor

promoters

Corr

ela

tio

n

1.0

0.5

0.0

1.0

0.5

0.0

1.0

0.5

0.0

Nonagenarians Newborn

Nonagenarians Newborn

Nonagenarians Newborn

Nonagenarians Newborn

F

WGBS-DMRs

(17,930)

Present on 450K array

(3,205 CpGs)

450K validation (NB/Y103)

(1,149 CpGs)

450K validation (Newborns/Nonagenarians)

(214 CpGs)

Additional dmCpGs (Newborns/Nonagenarians)

(5,774 CpGs)

C

A

The Distinct DNA Methylomes of Newborns and Centenarians

Proc Natl Acad Sci USA, 2012

Nonagenarians Newborn HG/W

Diseases Associated with Premature Aging:

Hutchinson-Gilford Progeria and Werner syndrome

Prediction of Biological Age Using DNA Methylation

Aging / Cancer / Neurodegenerative Disorders / Cardiovascular Disease

Thanks to our collaborators and

funding agencies