Epigenetics in Cancer

DNMT1
Histones

DNA

Epigenetics in Cancer

Sushant Dhar
PhD Microbiology
L-2013-BS-82-D

Epigenetics
 Conrad Waddington introduced the term “epigenetics” in the 1940s.
 'Epigenetics' refers to heritable changes in gene
expression that do not result from alterations in the
gene nucleotide sequence.
 Changes in gene expression independent of DNA sequence
alterations.
 DNA methylation and histone modification are considered the
two main epigenetic mechanisms.

Epigenetics
 The NIH "Roadmap Epigenomics Project," ongoing as of 2014, uses
the following definition: Epigenetics refers to both heritable
changes in gene activity and expression (in the progeny of
cells or of individuals) and also stable, long-term alterations in
the transcriptional potential of a cell that are not necessarily
heritable.“
 Cold Spring Harbor meeting, 2008: “Stably heritable phenotype
resulting from changes in a chromosome without alterations in
the DNA sequence"

Epigenetics

The structural organization of DNA into chromatin creates an
environment that is generally repressive for gene transcription.
However, chromatin is a highly dynamic structure that must be
modified to accommodate the transcriptional machinery when
gene expression is required, to facilitate DNA repair mechanisms,
or to allow DNA replication, DNA methylation and histone
modification, as epigenetic mechanisms, regulate the access of the
transcription machinery to their target genes, modulating transitions
from condensed heterochromatin to relaxed euchromatin and
vice versa.

Euchromatin




A lightly packed form of chromatin;
Gene-rich;
At chromosome arms;
Associated with active transcription.

Heterochromatin





A tightly packed form of chromatin;
At centromeres and telomeres;
Contains repetitious sequences;
Gene-poor;
Associated with repressed transcription.

 Epigenetics, in a broad sense, is a bridge between genotype and

phenotype—a phenomenon that changes the final outcome of a locus
or chromosome without changing the underlying DNA sequence. For
example, even though the vast majority of cells in a multicellular
organism share an identical genotype, organismal development
generates a diversity of cell types with disparate, yet stable, profiles of
gene expression and distinct cellular functions.
 Thus, cellular differentiation may be considered an epigenetic
phenomenon, largely governed by changes in what Waddington
described as the “epigenetic landscape” rather than alterations in
genetic inheritance.

Cellular Functions regulated by epigenetic mechanisms

Correct organization of chromatin
Genomic imprinting
Silencing of repetitive elements
X chromosome inactivation

Inactive Histone tail
modifications

Inactive DNA
Methylation

Structural Protein

Epigenetic Modifications
 DNA Methylation
 Post-translational histone modifications
 Non-coding RNA’s
 Histone variants
 Chromatin remodeling
Aberrations in Epigenetics

 Tumor suppressor DNA hypermethylation
 Genome wide DNA Hypomethylation

Stephen B. Baylin Review Paper

Acet. Histone
Deacet. Histone

Activator
s

Transcr. Ready
Gene

CpG
Unmet
h
Repress
ors

Unmethylated Promoter
Activators and repressors in steady state

CpG
Heavily
meth

Transcriptional Activators
dispersed
Steady sate
silencing
Repressor
tightly
tethered to
DNA
Aberrant Hypermethylation

Methylation at different stages

The extent of DNA methylation changes in an orchestrated way during

mammalian development, starting with a wave of demethylation during
cleavage, followed by genome wide de novo methylation after implantation.
Demethylation is an active process that strips the male genome of methylation
within hours of fertilization; by contrast the maternal genome is only passively
demethylated during subsequent cleavage divisions.
 The extent of methylation in the genome of the gastrulating embryo is
high owing to de novo methylation, but it tends to decrease in specific tissues
during differentiation. De novo methylation occurs rarely during normal
postgastrulation development but is seen frequently during the establishment of
cell lines in vitro and in cancer.

Genetic analysis of various DNMT’s has established that DNA

methylation is essential for vertebrate development. Loss of
methylation causes apoptosis in embryos and fibroblasts and leads
to widespread derepression of ectopic gene expression and
transcriptional activation of transposable elements. Deletion of Dnmt1
during brain development results in perinatal respiratory distress.
 Potential explanations for the evolution of DNA methylation invoke its
ability to silence transposable elements or its function as a mediator of
developmental gene regulation.
Interrupting this featureless sea of genomic methylation are CpG
islands.

How CpG islands remain methylation free in a globally
metylated genome is an open question???????

DNA Methylation

Role of DNA Methylation Regulation of Gene Expression
DNA methylation is also the principal epigenetic factor governing allelic

imprinting. Imprinting is the process by which only one allele of certain
genes is expressed depending on the parental origin. In other words, a gene
'remembers' whether it was inherited from the father or the mother and is
expressed accordingly.
 DNA methylation is part of this 'memory' process and loss of imprinting (LOI)
has been tightly linked to cancer susceptibility. For example, the insulin-like
growth factor -2 (IGF2) gene is an imprinted gene that is expressed only
from paternally-inherited alleles.
 However, aberrant methylation of the IGF2 imprinting control region (ICR)
on the maternal allele leads to additional expression of the maternal copy.
Thus, the levels of IGF2 rise approximately two-fold and this elevated level is
associated with cancer susceptibility.

CpG island
 CpG islands located in areas downstream of promoters have been found to
be methylated in some normal somatic tissues whereas the 5´ CpG islands of
most widely expressed housekeeping genes are usually unmethylated.
29,000 CpG islands in human genome (~60% of all genes are associated with
CpG islands)
 Genes with 5´ CpG islands do not normally rely on DNA methylation for the
control of their gene expression because the islands are unmethylated
regardless of expression level.

There are several situations in which 5´ CpG islands become de novo

methylated in normal development, thereby silencing the expression of the
associated gene. Examples of genes silenced by 5´ CpG island methylation
include imprinted genes and genes on the inactive X chromosome in female
mammals.

In humans, methylation occurs in carbon 5 of cytosine which is

positioned before guanine. In normal cells, the CpG methylation is mainly
done in the repetitive regions of genome including satellite regions.
 CpGs do not exist randomly in the genome, but they are located in GC
rich regions referred to as CpG islands. These islands are usually found at the
end of 5’ regulatory regions of many genes . In normal cells, a great part of
CpG islands (about 94%) is nonmethylated.

Epigenetics in Cancer
 Distinct methylation patterns are established during embryogenesis
and are mitotically heritable.

 The maintenance of normal DNA methylation patterns is
disrupted in cancer, where CpG islands become susceptible to
methyltransferase activity and CpG poor regions undergo
hypomethylation during transformation. Global hypomethylation of
cellular DNA might result in genomic and chromosomal instability,
including the possible activation of oncogenes.

Epigenetics in Cancer

Change of DNA methylation pattern in CpG islands was the first and most
significant abnormal epigenetic change identified in cancerous cells.

 Low level methylation in cancers is substantially due to the loss of methylation
at repetitive sequences as well as the introns demethylation. During the
development of cancer, hypomethylation degree may increase in the DNA and the
progressive lesion could participate in the reproduction and metastasis of cancer cells.

 The following 3 mechanisms have been suggested for DNA hypomethylation: (a) the
increased instability of the genome, (b) the reactivation of factors that are capable
of movement on DNA, and (c) the functional defect in elements related to genome.

Aberrant promoter hypermethylation is due to the infidelity of
DNMT1. The substrate for this DNA methylase is hemimethylated DNA.
Methylation occurs immediately after DNA replication with the primary
function to ensure that the identical methylation pattern of the parental cell
is passed on to each daughter cell.

 DNMT1 is part of a multiprotein DNA replication complex (Vertino et

al., 2002), which may be prone to methylation errors. It is known that
the human DNA polymerases replicate genomic DNA with very high
fidelity, but do commit errors at a very low frequency (Kunkel and
Bebenek, 2000).
 Does this also occur for DNMT1? In accordance with this
hypothesis is the steady increase in aberrant DNA methylation that
occurs with age (reviewed by Issa, 1999) and the progressive increase
in the number of methylated CpGs that occurs for some cancer-related
genes (Graff et al., 2000).

Cancer in context to Hypomethylation and Hypermethylation

Hypermethylation of tumor suppressor genes in CpG islands

leads

to

inactivation

of

tumor

suppressor

genes.

The

hypermethylation of CpG islands in promoter region is a crucial
incident in the initiation of carcinogenesis process.
 For instance, hypermethylation of CpG islands within the promoter
of tumor suppressor genes is associated with various cancers such as Ecadherin, MLH1, and CDKN2A.

DNA methylation may affect the transcription of genes in two ways. First,

the methylation of DNA itself may physically impede the binding
of transcriptional proteins to the gene and second, and likely more important,
methylated DNA may be bound by proteins known as methyl-CpG-binding
domain proteins (MBDs).
 MBD proteins then recruit additional proteins to the locus, such as histone
deacetylases and

other chromatin

remodeling proteins

that

can

modify histones, thereby forming compact, inactive chromatin, termed
heterochromatin.

 The link between DNA methylation and chromatin structure is very
important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has
been implicated in Rett syndrome; and methyl-CpG-binding domain protein 2
(MBD2) mediates the transcriptional silencing of hypermethylated genes in
cancer.
 At least three of the five known members of this family (MeCP2, MBD2 and
MBD3) have been shown to be associated with large protein complexes
containing histone deacetylase (HDAC1 and HDAC2) and chromatinremodelling (Sin3a and mi-2) activities. The action of these histone
deacetylase and chromatin-remodelling activities is thought to result in the
production of compacted chromatin that is refractory to transcription.

CpG Methylation Cancer Type Associated
 CpG island promoter hypermethylation and silencing of other
tumor suppressor genes such as VHL (von Hippel–Lindau) in
renal cancer, the cell cycle regulator CDKN2 A/p16 in bladder
cancer , the mismatch repair gene hMLH1 in colon cancer.

 Although CIMP has been associated with environmental and lifestyle factors,
the molecular basis for CIMP is only beginning to be explored. The first clues
came from two studies showing that glioblastomas with a hypermethylator
phenotype

are

associated

with

somatic

mutations

in

isocitrate

dehydrogenase-1 (IDH1), and that somatic mutations in IDH1, IDH2, as
well as loss-of-function mutations in ten-eleven translocation (TET)methylcytosine dioxygenase-2 (TET2) establish a hypermethylation
phenotype in leukemia. These are the first indications for a molecular basis of
CIMP, and provide an explanation for a very distinct set of tumors with
increased levels of hypermethylated DNA.

 In contrast with hypomethylation of intergenic CpG sites in cancer that
lead to genomic instability, hypermethylation of CpG islands promotes the
progression of tumorigenesis by silencing tumor-suppressor genes. For
example, PTEN, a protein that prevents rapid proliferation, is commonly
hypermethylated in brain and thyroid cancers, whereas APC, a protein
involved in cell-cycle regulation, cell–cell adhesion, and cell mobility, is
inactivated by hypermethylation in many lung, breast, and colorectal cancers
(Bird 2009).
 Suppression of p16, a cell-cycle regulator, occurs in essentially all common
human cancers (Ligget and Sidransky 1998).
 Inactivating these tumor suppressors directly promotes tumorigenesis
due to lack of control over cellular processes.

 In addition to tumor-suppressor genes, hypermethylation of

other classes of genes such as DNA repair genes and
transcription factors can indirectly lead to tumorigenesis through
silencing of further downstream targets or accumulation of genetic
errors. For example, GATA-4 and GATA-5 are transcription factors
silenced in colorectal and gastric cancers (Alvarez-Nunez et al.
2006).
 Inactivation of DNA repair genes, such as O-6-methylguanineDNMT, is commonly found in primary neoplasias (Esteller et al.
2000). Therefore, hypermethylation of CpG islands in cancers
can affect multiple pathways to promote carcinogenesis.

Which genes are hypermethylated depends on tumor type

 RB in Retinoblastoma
 MLH1 in colorectal cancer
 BRCA1 in Breast Cancer
 RUNX3 in Oesophageal Cancer
 GATA4 and GATA5 in Gastric Cancer

DNA hypermethylation markers of discrete genes in human cancers
 Hypermethylation in tumor suppressor genes thereby silencing the
genes by either physically inhibiting the binding of transcription factors,
or by recruiting proteins that have transcription repressive properties has
been reported in various human cancers.
 MutL homolog 1 (MLH1), breast cancer 1 and 2 ( BRCA1 and BRCA2 ),
death associated protein kinase 1 (DAPK1), retinoic acid receptor β
(RARβ), E-CADHERIN, CYCLIN A1, p14, p15, p73, RAS association
domain family (RASSF1A) and APC genes are hypermethylated in
cancers.
 A broad set of carcinogenic exposures have been found associated with
hypermethylation of these genes. Cigarette smoking has been identified as a
risk factor associated with hypermethylation of p16, RASSF1A, RARβ, CDH13,
MGMT and GSTP1, APC, and DNMT1.

Role of DNA Methylation is context dependent
Genetic depletion of DNA Methylation by deletion of Dnmt1 can
either enhance or suppress tumorogenesis:

 Driven by tumor suppressor hypermethylation, then depletion of
DNA methylation suppresses tumorogenesis.
 Driven by chromosomal instability, then depletion of DNA
methylation enhance tumorogenesis.

CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4

Carcinoma

5

Normal
Epithelia

Dysplasia

Carcinoma
in situ
Metastasis

CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4

Carcinoma

5

Normal
Epithelia

Dysplasia

Carcinoma
in situ
Metastasis

CpG Island Methylation: A Stable, Heritable and
Positively Detectable Signal
1
2
3
4

Carcinoma

5

Normal
Epithelia

Dysplasia

Carcinoma
in situ
Metastasis

Genome wide Hypomethylation
One of the earliest observed features of cancer cells observed in
many/ all forms of tumors. Occurs at repeats, CpG poor promoters,
introns. Hypomethylation contributes to tumorogenesis by:
 Increasing genomic instability- Chromosomal rearrangements
and mobilization of retrotransposons

Activating a restricted number of tissue specific genes and

imprinted genes. e.g R-RAS activation in gastric cancer, IGF2
displays loss of imprinting (LOI )in Wilm’s tumor.

In context of Hypomethylation, Few examples
Repetitive Elements
 Repetitive elements make up about half of the genome and are normally
heavily methylated.
 Centromeric tandem repeats, adjacent-centromeric (juxtacentromeric)
tandem repeats, and short- (Alu) and long interspersed elements (LINE-1)
are the most frequently studied repetitive elements in cancer that are found to
be hypomethylated.

Tandem repeats at and near the centromere
play a role in keeping the DNA packaged into
heterochromatin at the point of sister
chromatid
association,
allowing
for
chromosome stability.

Hypomethylation of these regions can lead to chromatin
decondensation and chromosome rearrangements through
unstable translocations, leading to widespread genomic
instability (Eden et al. 2003).
For example, in vitro experiments conducted to knock out
DnmtI, a DNA methyltransferase (DNMT1), in murine embryonic
stem cells showed an increase in chromosomal translocations.

CpG Island Methylation and Genome wide Hypomethylation
CpG island
CGCG CG

Normal

CG

1

2

CpG island
Hypermethylation
M

Cancer

CG

3

M

CG

M

CG

4

Genome Wide
Hypomethylation

CGMCG MCG

M

CG

1

2

X

CG

CG

3

4
C: cytosine
m
C: methylcytosine

CG

Progressive Alterations in DNA
Methylation in Cancer
Global
Hypomethylation

Normal

+

Region-Specific
Hypermethylation

Cancer
Accumulation of
alities
m
r
o
n
b
A
ic
t
e
n
e
Epig

Role of Histone modification in Regulation of Gene
Expression
 Histone methyltransferases; catalyze transfer of a methyl group onto
a

lysine

or

arginine

residue

on

histone

tails.

Histone

acetyltransferases (HATs), a group of transcription regulators,
covalently modify the epsilon amino group of lysines found in the Nterminal tails of histones, or other proteins, by the addition of an acetyl
group from acetyl coenzyme A.
 Acetylation of lysines inside histone tails generally leads to
transcriptional activation and deacetylation results in silencing.
.

Contd.

Histone Modifications

Acetylation
Ac

Me
Ub

Methylation

Ubiquitination

Su
P

Sumoylation
Phosphorylation

Me

‘Histone Code’

 The positive charge on unacetylated lysines in the histones is
attracted to the negatively charged DNA producing a compact
chromatin state that is repressive for transcription. On the other
hand, acetylation of the lysines by histone acetylase removes their
positive charge and results in an open chromatin structure, which
facilitates gene transcription.

Since a more open chromatin architecture enables the

recruitment of transcription factors and polymerases, histone
acetylation results in the promotion of gene expression

Histone deacetylase (HDAC) removes the acetyl groups from

lysine, which reverses this process and silences gene expression.

Acetylation of Lysines
Acetylation of the lysines
at the N terminus of
histones removes positive
charges, thereby reducing
the affinity between
histones and DNA.
This makes RNA
polymerase and
transcription factors easier
to access the promoter
region.
Histone acetylation
enhances transcription
while histone deacetylation
represses transcription.

Aberrations in Histone Modification
Now What If

Cancer
Histone hypoacetylation

Overexpression of HDACs

 Acetylation of histones is required to maintain chromatin in an open
and transcriptionally active state. This permits binding of transcriptional
factors, histone acetylases, and other regulatory coactivators that promote
gene expression.

 Conversely, HDACs act to keep these residues deacetylated and thus
maintain transcriptional silencing. Binding of HDACs to hypermethylated
chromatin is directed by DNMTs as well as methyl cytosine-binding
proteins, which form a complex with other regulatory proteins to block
access of the transcriptional machinery to the promoter.

Aberrations in Histone Modification

Histone deacetylases (HDACs), more properly termed lysine

deacetylases, reverse the action of histone acetyltransferases (HATs) by
catalysing the removal of acetyl groups from the lysine ε-amino group of
histones and other non-histone substrates. HDACs can be divided into four
classes: class I, including HDAC1, 2, 3 and 8; class II, including HDAC4, 5, 6,
7, 9 and 10; class IV (HDAC11) and class III, the NAD+- dependent Sir2-like
deacetylases or sirtuins.

HDACs play roles in the regulation of
gene transcription, cell growth, survival
and proliferation. Dysfunction of
these enzymes is often associated
with
disease,
ranging
from
neurodegenerative disorders to
cancer.

 Aberrant deacetylation of histones in nucleosomes is probably
due to dysregulation of the specificity of HDAC and may be
associated with neoplastic transformation. For example, gene
translocations in some types of leukemia can generate fusion
proteins that recruit HDAC and bind to promoters to silence genes
involved in differentiation (reviewed by Johnstone, 2002).
 The importance of histone modifications in cancer is illustrated by
the marked antitumor activity of different inhibitors of HDAC, both in
animal models and in preliminary clinical trials (Marks et al., 2001;
Johnstone, 2002).

Important
 Modifications that represent an active transcription include acetylation of
H3 and H4, and di- or tri-methylation of H3 at lysine 4 (H3K4me2 or me3).
On the contrary, methylation at H3K9 and H3K27 represents an
inactivation of transcription

 Global loss of acetylation of histone H3 at lysine 9 (H3K9ac), H3K18ac,
H4K12ac, H4K16ac, along with loss of trimethylation of histone H4 at lysine
20 (H4K20me3) and H3K4me2/me3 has been observed in various primary
tumors and has been linked with tumor progression.

Important
Histone methylation, which can be associated with transcriptional
activation or repression based on the specific residue methylated.

Inactive Transcription
Active Transcription

Aberrations in Histone Modification

 Retinoblastoma is a pediatric tumor of the developing retina from which the
genetic basis for cancer development was first described. Inactivation of both
copies of the RB1 gene is the predominant initiating genetic lesion in
retinoblastoma.

 RB1 gene was cloned and found to undergo biallelic inactivation in virtually
all retinoblastoma tumors (Friend SH, Dunn JM 1988)

Function of Rb (Retinoblastoma
protein) is to prevent excessive cell
growth by inhibiting cell cycle
progression

Continue

 Since then, hundreds of genetic lesions in human cancer have been
identified. These genetic lesions can be grouped into pathways with direct or
indirect mechanistic links to many of the common cellular properties, or
hallmarks of cancer, including activation of growth-signaling pathways,
evasion of cell death and senescence, acquisition of limitless replicative
potential, sustained angiogenesis, local tissue invasion, and metastasis.

Thus, the rate of cancer progression is related to the kinetics of

acquisition of multiple genetic lesions that ultimately confer the essential
cellular properties of cancer.
 In the retina, RB1 inactivation confers limitless replicative potential
to retinoblasts,

Continued

Chromothripsis- thousands of clustered chromosomal
rearrangements occur involved in both cancer and congenital

RETINOBLASTOMA AND CHROMOSOMAL INSTABILITY
 RB1 protein is required for maintaining chromosomal stability. Therefore,
RB1 inactivation, which leads to chromosome instability (CIN) in cultured
cells, could allow the secondary and tertiary mutations in key cancer pathways to
be rapidly acquired.
 Studies have suggested that loss of RB1 results in mitotic defects that can
lead to aneuploidy, which in turn can contribute to CIN. Indeed, most cancers
have suppressed RB pathways, and CIN is an important hallmark of many of the
most aggressive forms of sporadic human cancers

LOH is a gross chromosomal event
that results in loss of the entire gene
and the surrounding chromosomal
region.

Aneuploidy- Chromosome
number is abnormal

Fig. RB1 has been implicated in regulating most major epigenetic processes.
Loss of RB1 leads to chromatin remodeling, in which oncogenes that are normally
repressed switch to active chromatin, tumor suppressors that are normally
actively transcribed are repressed, or both. Abbreviation: meDNA, methylated
DNA.

Epigenetics and Retinoblastoma

RB1 has been implicated in regulating most major epigenetic processes,

including

microRNA (miRNA) regulation,

DNA methylation, histone

modification, and ATP-dependent chromatin reorganization.
MicroRNAs in retinoblastoma

miRNAs encode small noncoding RNA molecules that function in

transcriptional and posttranscriptional regulation of gene expression.
miRNAs function by base-pairing with complementary sequences within mRNA
molecules, usually resulting in gene silencing via translational repression or
target degradation.

MicroRNAs in Cancer

Members of the let-7 family are among the most widely studied tumor

suppressor miRNAs. Functionally, let-7 is involved in repressing members of
the Ras family, HMGA2, and c-Myc oncogenes. As for several other cancers,
reduced expression of let-7 in retinoblastoma has been reported.

Tumor suppressor miRNA, miR-34a, was identified as differentially

expressed

in

retinoblastoma.

Studies

have

demonstrated

that

p53

transcriptionally activates the miR-34 family. Loss of miR-34a silencing
functions has been identified in several human cancers, making miR-34a
an attractive miRNA for therapeutic development

DNA methylation in retinoblastoma
 Studies on retinoblastoma have also looked at the methylation status of
other genes beyond RB1. One of the first studies to look at promoter
methylation of tumor suppressor genes in retinoblastoma examined the
methylation

status

of

nine

genes:

p16INK4A,

MGMT,

GSTP1,

RASSF1A,APC, DAPK, RARβ , CDH11, and CDH13. Of these, RASSF1A
(RAS-associated domain family 1A) was hypermethylated in 59% of tumors
analyzed and APC in 6%.

Promoter hypermethylation of MGMT was found in 15% of the

retinoblastoma tumors analyzed. In addition, MGMT hypermethylation was
associated with advanced-stage retinoblastoma

MGMT- Key Gene for DNA
Repair

IMPORTANT
 A promoter methylation analysis of the tumor suppressor p16INK4A
showed hypermethylation in most retinoblastoma tumors that also exhibited
p16INK4A protein downregulation. Furthermore, in over half of these cases,
the same alteration in p16INK4A expression was observed in the parents
of these patients, suggesting that this alteration could be a novel inheritable
susceptibility marker for retinoblastoma.

Toyota and colleagues first identified CIMP in colorectal cancer

 In this study of silencing in colorectal cancer, it was found that there is
common repression of the entire 4-Mb band of chromosome 2q.14.2,
associated

with

global

methylation

of

histone

H3

Lys9.

DNA

hypermethylation within the repressed genomic neighborhood was localized
to three separate enriched CpG island ‘suburbs’, with the largest
hypermethylated suburb spanning 1 Mb.

?????????????
 What determines which CpG island–associated genes are
susceptible to hypermethylation and histone modification in
different cancer types?
 What dictates epigenetic silencing and what protects some
islands from methylation?

New insights into the mechanisms and the role of CpG island

hypermethylation in cancer have emerged from recent studies using integrated
analyses of the two types of epigenetic modifications. Various research groups
have reported that genes that are targeted by Polycomb group (PcG) proteins
in embryonic stem (ES) cells are susceptible to cancer-specific DNA
hypermethylation (Ohm et al. 2007)
 PcG target genes are characterized by trimethylation of histone H3 lysine
27 (H3K27me3), are maintained in a low expression state, and are poised to
be activated during development (Bernstein et al. 2007). More recently, it has
been found that genes targeted by H3K27me3 in normal tissues acquire DNA
methylation and lose the H3K27me3 mark in cancer (Gal-Yam et al. 2008;
Rodriguez et al. 2008). Importantly, epigenetic switching of H3K27me3 and DNA
methylation mainly occurs at genes that are not expressed in normal tissues.

Polycomb Group Proteins

The polycomb group (PcG) proteins are a family of proteins
responsible

for

cellular

differentiation

during

development

via

transcriptional repression. The polycomb group was first described in
Drosophila as

a

locus

involved

in

the

silencing

of Hox gene

expression (Lewis, 1978). Many PcG proteins achieve this control of gene
expression via catalyzing H3K27 methylation. These proteins have been
the subject of intense study as it is clear that they are vital for maintenance
of cell-type identity, differentiation, and disease by creating and maintaining
repressive chromatin environments. Most PcG proteins form two major
polycomb repressive complexes (PRC): PRC1, and PRC2.

 Recent studies from several groups indicated that colorectal tumors
with KRAS mutations may also be associated with a unique DNA
methylation profile.
 Shen et al. (2007) described the CIMP2 subgroup, which also showed
DNA hypermethylation of CIMP-associated loci, but was highly correlated
(92%) to KRAS mutations and not associated with MSI (Microsatellite
instability).

KRAS?????
 GTPase KRas also known as V-Ki-ras2 Kirsten rat sarcoma viral
oncogene homolog and KRAS, is a protein that in humans is encoded by
the KRAS gene. The protein product of the normal KRAS gene performs an
essential function in normal tissue signaling, and the mutation of a KRAS
gene is an essential step in the development of many cancers.

MSI???????
Microsatellite instability (MSI) is the condition of genetic hypermutability
that results from impaired DNA Mismatch Repair (MMR). In other words,
MSI is the phenotypic evidence that MMR is not functioning normally.
Types of genes targeted for DNA methylation in each subgroup and the effects
of DNA hypermethylation on gene
expression

???????????????????

To better characterize DNA methylation subgroups in CRC, genome-scale DNA
methylation profiling of 125 primary colorectal tumors and 29 adjacent nontumor colonic mucosa samples was done using the array-based Illumina
Infinium HumanMethylation27 (HM27) Platform.

Interestingly, it

was

noticed

that

112

genes

exhibited

DNA

hypermethylation and reduced gene expression in CIMP-H tumors, 48
were also silenced in non-CIMP tumors, but without substantial increases in
DNA methylation. CIMP status in CRC has been found to be inversely
correlated with the occurrence of chromosomal instability (CIN), which is
characterized by aneuploidy, gain and loss of subchromosomal genomic
regions, and high frequencies of loss of heterozygosity (LOH) (Goel et al.
2007).
 Recently, Chan et al. (2008) identified genes that are inactivated by both
genetic mechanisms (mutation or deletion) and DNA hypermethylation
in breast and colorectal cancer. They observed that these genetic and
epigenetic changes are generally mutually exclusive in a given tumor, and
that silencing of these genes was associated with poor clinical outcome.

IMPORTANT

Cigarette smoking was found to be associated with
increased risk of developing CIMP CRC in a report
(Limsui et al. 2010).

 Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes

are characterized by distinct genetic and epigenetic alterations. Comprehensive
genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent
normal tissues was done. Different DNA methylation–based subgroups of CRC using
model-based cluster analyses were obtained. Each subtype showed characteristic
genetic and clinical features, indicating that they represent biologically distinct
subgroups.
 There is a tight association between BRAF mutation and CIMP in colorectal
cancer.

B-Raf proto-oncogene- controls cell functions such as growth and division.

 A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally
high frequency of cancer-specific DNA hypermethylation, is strongly
associated with MLH1 DNA hypermethylation and the BRAFV600E
mutation. A CIMP low (CIMP-L) subgroup is enriched for KRAS
mutations and characterized by DNA hypermethylation of a subset of
CIMPH- associated markers rather than a unique group of CpG islands.
 Non-CIMP tumors are separated into two distinct clusters. One nonCIMP subgroup is distinguished by a significantly higher frequency of
TP53 mutations and frequent occurrence in the distal colon, while the
tumors that belong to the fourth group exhibit a low frequency of both
cancer-specific DNA hypermethylation and gene mutations and are
significantly enriched for rectal tumors.

Review
 The cell-type specific organization of heterochromatin, established upon cell
differentiation, is responsible for maintaining much of the genome in a
repressed state, within a highly compartmentalized nucleus.

 Recent evidence that in cancer the normal packaging and higher
organization of heterochromatin is often compromised

Lessons from X chromosome inactivation:
heterochromatin is controlled at multiple levels
 X-inactivation is the pre-eminent model for the formation and maintenance
of facultative heterochromatin. Failure to properly silence one X
chromosome results in embryonic lethality as this event is required to
compensate imbalanced gene expression between males (XY) and females
(XX).
 Random inactivation of one X chromosome in female cells initiates
early in development when the non-coding RNA XIST is expressed from
and coats one X chromosome (Xi) in cis.

 Euchromatic histone marks are quickly replaced by marks associated
with inactive heterochromatin including H3K27me3, H3K9me2, and UbH2A,
and individual gene promoters become methylated.

This is followed by a transformation in the chromosome’s overall

architecture to condensed, tightly packaged chromatin which moves into
the

peripheral

heterochromatin

compartment,

excluding

access

to

transcriptional machinery within the nuclear interior.
 This dense heterochromatin structure, termed the Barr Body, is easily
visualized by light microscopy in normal cells, and is frequently lost in cancer.

 Several studies have found that certain primary breast and ovarian cancer
cells which lack an inactivated X chromosome (Xi) retain at least 2 active X
(Xa) chromosomes.
 Two plausible mechanisms for loss of the Barr body have been proposed:

First involves decondensation of the heterochromatic Barr body

associated with loss of the XIST transcript, followed by gene activation.
Evidence to support the mechanism that XIST RNA mislocalizes in cancer comes
from in situ studies in breast carcinoma cells.

One study

implicated that loss of the Barr body in cancer was

attributed to loss of BRCA1, a tumor suppressor linked to several
fundamental

cell

regulatory

processes

and

widely

implicated

in

development of breast and ovarian cancers. The apparent cytological
overlap reported in this study between BRCA1 and XIST RNA across the
Xi raised the important possibility of a direct role of BRCA1 in localizing
XIST.
 However, further study revealed that BRCA1 does not structurally
paint the Xi or XIST territory, as do markers of Xi heterochromatin, nor
does BRCA1 status correlate with XIST localization. Instead, BRCA1
overexpression

correlates

with

enhanced

XIST

expression,

suggesting BRCA1 may impact XIST transcription which may be due,
directly or indirectly, to BRCA1’s function as a transcription factor
BRCA1 repairs damaged DNA, repair of chromosomal damage with an important role
in the error-free repair of DNA double-strand breaks.

Highlight
The

broad

epigenetic

heterochromatin

and

domains

genetic

is

an

impact

of

understudied,

instability
yet

within

widespread

phenomenon in cancer. We suggest that heterochromatic domains are
integral to the proper positioning of sequences within the nuclear
environment

and

highlight

that

loss

of

compartmentalization

of

heterochromatin is likely to destabilize the epigenome more generally.
Genomic instability is important in cancer, but defects in the maintenance
of heterochromatin, including loss of Xi, peri/centric heterochromatin and
the peripheral heterochromatic compartment, could result in an analogous
broad epigenetic instability.

HOW EPIGENETIC CONTROL DEFECTS??????

Stochastic occurrences that accumulate with age, and are

selected for during malignant outgrowth, or whether the large number
of changes is caused by a defect in one of the components of the
epigenetic machinery.

By analogy, both stochastic mutations and mutator phenotypes

are involved in producing the genetic alterations found in cancer.
Likewise, it is anticipated that both stochastic errors and defects in
epigenetic control participate in cancer epigenetics..

 Genetic predisposition and/or environmental exposures could
lead to systemic epigenetic control defects. One such systemic
epigenetic control defect appears to be loss of imprinting of the IGF2
locus, which occurs in the colorectal tumors, and in the normal colonic
mucosa and white blood cells of individuals with colorectal cancer.

Genome wide DNA Methylation analysis

Profiling DNA methylation across the genome is vital to
understanding the influence of epigenetics. There has been a
revolution in DNA methylation analysis technology over the past
decade: analyses that previously were restricted to specific loci can
now be performed on a genome-scale and entire methylomes can
be characterized at single-base-pair resolution.

 List Techniques

Detecting Methylation Pattern ??????

To determine the exact DNA methylation status ???????

 Represents an attractive diagnostic and therapeutic target

The Answer is Bisulphite Genome sequencing
 Gold-standard technology for detection of DNA methylation because it
provides a qualitative, quantitative and efficient approach to identify 5-methylcytosine
at single base-pair resolution.

First introduced by Frommer et al and it is based on the finding that the

amination reactions of cytosine and 5-methylcytosine (5mC) proceed with very
different consequences after the treatment of sodium bisulfite

 DNA methylation status can be interpreted by comparing the sequencing
results and the original DNA sequence. Basically, all unmethylated
cytosines (C) convert to thymine (T) and the presence of a C-peak indicates
the presence of 5-methylcytosine (5mC) in the genome.

If both C-and T-peaks appear, this indicates partial methylation or

potentially incomplete bisulfite conversion has occurred. The proportion
of 5mC to C can be interpreted by analyzing the relative square area of these
two bands.

 Cytosines in single-stranded DNA will be converted into uracil residues and
recognized as thymine in subsequent PCR amplification and sequencing,
however, 5mCs are immune to this conversion and remain as cytosines
allowing 5mCs to be distinguished from unmethylated cytosines. A subsequent
PCR process is necessary to determine the methylation status in the loci of
interest by using specific methylation primers after the bisulfite treatment.

 The actual methylation status can be determined either through direct PCR
product sequencing (detection of average methylation status) or sub-cloning
sequencing (detection of single molecules distribution of methylation patterns) .
Moreover, bisulfite sequencing analysis can not only indentify DNA methylation
status along the DNA single strand, but also detect the DNA methylation patterns
of DNA double strands since the converted DNA strands are no longer selfcomplementary and the amplification products can be measured individually.

MUTATIONS IN EPIGENETIC REGULATOR PROTEINS AS DRIVERS OF
ABERRANT DNA METHYLATION
 Recurrent mutations in epigenetic regulator proteins, including IDH1/2, TET2 and
DNMT3A were described by several groups.
 Functional studies have linked IDH, TET2 and DNMT3A mutations with distinct
DNA methylation phenotypes.

IDH and TET2 mutations cause a DNA hypermethylation phenotype in AML
 IDH1 and IDH2 are members of the IDH family of enzymes which bidirectionally
catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (a-KG).
Both IDH1 and IDH2 mutations occur frequently in AML.
 Mutant IDH enzymes synthesize the ‘oncometabolite’ 2-hydroxyglutarate (2-HG).
Expression of IDH-mutants in murine bone marrow cells results in both an increase
in the oncometabolite 2-HG (2-hydroxyglutarate, competitor of dioxygenases
including histone demethylases) and concurrent DNA hypermethylation.The
mechanism by which 2-HG results in genome-wide DNA hypermethylation is
thought to involve inhibition of a group of enzymes called ‘a-ketoglutaratedependent dioxygenases’. These include enzymes involved in histone
demethylation and 5-mC hydroxylation.
 TET2 (dioxygenase) is one of the a-ketoglutarate-dependent dioxygenases that
is inhibited by 2-HG. As a member of the TET protein family, TET2 catalyzes the
conversion of 5-mC to 5-hydroxymethylcytosine (5-hmC). Although the exact
function of 5-hmC is not clear, it is most likely an intermediate of DNA
demethylation. Indeed, TET2 has been suggested to be involved in both passive
and active DNA demethylation mechanism during development.

Transcrip.
Factor
binding lost

Deacet.
Via PML

Continued in Text

Emerging concepts of DNA methylation aberrant changes.

 (a) A conceivable model behind aberrant DNA methylation in AML with inactivating
mutations. Recent studies suggest that transcription factors protect their binding sites
from DNA hypermethylation. Once transcription factor binding is lost, DNA
methylation occurs.

Potentially, this mechanism drives aberrant DNA hypermethylation in AML with

inactivating mutations such as CEPBa-mutated AMLs. CEPBa mutations either result in
loss of DNA-binding capacity or inhibit the wild-type enzyme—in both cases probably
leading to a loss of protective binding and consequent hypermethylation. Further
experiments will be needed to validate this mechanism.

c) IDH mutations drive genome-wide DNA hypermethylation via

inhibition of ‘a-ketoglutarate-dependent dioxygenases’. IDH mutations
are gain-of-function mutations which result in the production of the
oncometabolite 2--HG. 2-HG, in turn, inhibits a group of enzymes called
‘a-ketoglutarate-dependent dioxygenases’ leading to genome-wide DNA
hypermethylation. Inhibition of the enzyme TET2 (demethylation) has
been suggested to be involved in this process.
 Whether or not DNA hypermethylated genes are TET2 target genes is
currently, however, still unclear. Further investigation will be needed to
delineate the specifics of this mechanism, for example, also which
enzymes mediate this DNA hypermethylation.

TET2 ????????????

 The ten-eleven translocation (TET) gene family, initially found as a
chromosomal translocation partner in leukemia, turned out to be a key
enzyme for DNA demethylation. TET genes hydroxylate 5-methylcytosine
to 5-hydroxymethylcytosine, which is then converted to unmodified cytosine
through multiple mechanisms.

 TET2 is critical for the function of hematopoietic stem cells, and
disruption of TET2 results in the expansion of multipotent as well as
myeloid progenitors, leading to the accumulation of premalignant clones.
In addition to cytosine demethylation, TET proteins are involved in
chromatin modifications and other cellular processes through the
interaction with O-linked b-N-acetylglucosamine transferase.

Recent Publications on Cancer Epigenetics

EZH2 is a transcriptional repressor

Neural crest stem cells (NCSC)

Epigenetic effects in context to
bacterial infection

miR-210- microRNA
regulates
gene expression

H3K27 demethylase Jmjd3 (also known as KDM6B) was found to
catalyse the demethylation of H3K27me2/3 in vitro.

Are epigenetic changes hereditary?
Researchers used to think that when a sperm and egg combined, all their
epigenetic tags were erased, leaving the resulting embryo with a clean slate.
Now they know that about 1 percent of our epigenetic tags escape erasure
and pass directly to our offspring — and potentially their offspring and
beyond.
That suggests that what men and women eat and smoke, and what toxins and
traumas they're exposed to, can affect their children and even
grandchildren.
University of Texas zoologist David Crews has done multigenerational
studies with rats that led him to speculate that soaring obesity and autism rates
could be due to our grandparents' exposure to "the chemical revolution of
the 1940s," including the introduction of new plastics, fertilizers, detergents, and
pesticides.

A PARADIGM SHIFT: NUTRITION & EPIGENETICS
Epigenetics, Nutrition and the Development of Cancer

Are these insights yielding medical therapies?
Over the past five years, evidence that epigenetics plays a major role in cancer
has become "absolutely rock solid," says Robert A. Weinberg, a biologist
at the Whitehead Institute in Cambridge. Andrew Feinberg, director of Johns
Hopkins University's Epigenetics Center, thinks it's a factor in autism and
diabetes as well. Drugs are in the works aimed at undoing cancerous
epigenetic changes. Even eating foods rich in gene-altering methyl groups
— such as soybeans, red grapes, and green tea — might protect against
disease by silencing detrimental genes. In one famous experiment,
researchers fed a methyl-rich diet to pregnant female mice that carried a
gene that made them fat, yellow, and prone to cancer and diabetes.
Though their offspring carried the same gene, they were born slim, brown, and
disease-free.

More on Epigenetic inheritance

We’ve known for some time that certain environmental factors

experienced by adult mice can be passed on to their offspring via
epigenetic mechanisms. The best example is a gene called agouti,
which is methylated in normal brown mice. However, mice with
an unmethylated agouti gene are yellow and obese, despite
being genetically essentially identical to their skinny brown relatives.
Altering the pregnant mother’s diet can modify the ratio of
brown to yellow offspring: folic acid results in more brown
pups, while BPA results in more yellow pups.

Drugs reversing epigenetic mechanisms????????
Are there any?????????????????????
Most of them are under clinical trials, being tested on animal models

Decitabine and Azacitidine only approved hypomethylating drugs for
the treatment of cancer.
Used for the treatment of Myelodysplastic syndrome, ineffective
hematopoiesis resulting in anemia
Decitabine, a hypomethylating agent that allows for the re-expression
of tumor suppressor genes
Decitabine (5-aza-2’-deoxycytidine) is a cytosine analogue modified in
position 5 of the pyrimidine ring

I offer my sincere and profound gratitude
to:

Dr. Peter Laird
Dr. Shiv Grewal
Director, USC Epigenome Center
Chief, Laboratory of
Biochemistry
and Molecular Biology