Post on 29-May-2018
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
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- 152 + 160PIWIL2
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ocyte
s)
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- 159 + 162SAA1
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(fro
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ocyte
s)
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lative
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ressio
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s G
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)
0
200
400
600
800
1 2 3
Re
lative
exp
ressio
n
(SA
A1
vs G
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)
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50
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lative
exp
ressio
n
(PIW
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vs G
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)
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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
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Age (years)
ES
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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
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A
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M A A
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M A
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P M
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A
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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
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M
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A
M A
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M A
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149 154
P
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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
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A A
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A M
99 120 119
M U P
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P
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A
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A
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M
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A
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A
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A
116 120
U A
M A
U A
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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
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M A
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P
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P
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M A
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M
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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
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A
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A
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M
27 3 36 17
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…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
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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