Epigenetic in Cancer Development

Gara Samara Brajadenta, MD, PhD

Department of Genetics, Faculty of Medicine
Universitas Swadaya Gunung Jati
The Human Genome Project

3 billion bases 40,000 genes

http://www.genome.gov/
 Gregor Rosalind Watson & Crick HGP
Mendel Franklin
1952
1866 - 1950Mol. Biol 1980’s, Oregon
DNA Model
Genetics

DNA Forensic
Cloning – Dolly
Karry 1995
Gene Therapy Mullis 1997
AJ. Simpson
1990 PCR
1993

 Changing bad genes for good ones. We now have the tools to make the whole world better
through science ‚ the science of the human genome. HGP……
 For the sake of human being ………..
 Cancer
• Cellular level: over–proliferation of
the cell
• Tissue level: cells deviate from
their natural place in the tissue and
spread
• 3 main principles:
– Tumors are monoclonal
– DNA mutations (6-7 usually)
– Selection (from bad to worse)
 Cellular mechanisms in cancer
• Tumor cells uncontrolled
proliferation
• Growth factors constitutive
activity
• Signaling pathways
damage
• Constitutive up/down
regulation
• DNA repair problem
• Apoptosis mechanism not
active
• Cells acquire metastatic
potential
 Four
mechanisms
of oncogene
activity to
deregulate cell
division
> 120 genes

Fig. 19.13
 Oncogenesis
carcinogen
results in mutation increased GF

increased GF receptors
proto-oncogenes oncogenes
exaggerated response to GF

tumor
dysfunctional loss of ability to
suppressor
tumor suppressor repair damaged
genes
genes cells or induce
apoptosis

inherited
defect
Historically, Cancer Was Considered to be Driven Mostly
by Genetic Changes
GENETIC

Example:
Replication errors

X X

Altered
DNA sequence

Altered
Oncogenesis
DNA/mRNA/proteins

Tumor
 Recent Evidence : Epigenetic Changes are Also
Important in Causing Cancer
GENETIC EPIGENETIC

Example: Example:
Replication errors Chromatin modification errors

X X

Altered Altered
DNA sequence chromatin structure

Altered
Oncogenesis Altered levels of
DNA/mRNA/proteins mRNA/proteins

Tumor
 Cancer epigenetics
• Epigenetic alterations found in almost all types of
cancers
• 50% of genes that cause familial forms of cancer
are found silenced in sporadic forms of cancer
• Large number of epigenetic alterations found in
cancer cells due to:
– Stochastic occurrences that accumulate with age,
selected for during tumour formation
– Caused by defects in components of the
epigenetic machinery
– Repair gene (repair defects) alterations
 Cancer epigenetics
• Changes in 5-me-C distribution in DNA
– Hypermethylation of promoter CpG islands (found in
~50% genes) associated with TSGs
• Decreased expression levels/silencing of TSGs
• Increased mutations
– Global hypomethylation 
• Chromosome instability (particularly pericentromeric
repeats)
• Activation of viruses
• Activation of proto-oncogene
• Changes in chromatin structure
– Histone modifications:
• Histone deacetylation
• Histone methylation (H3-K9); demethylation (H3-K4)
• Histone sumoylation
– Heterochromatin-associated proteins
Microarray Technology
Biomarkers and gene expression

normal
malignant

Perou et al. Molecular Portraits of Breast Cancer, Nature, May 2000

Epigenetics & Genetic Mutations Can Cooperate to
Promote Oncogenesis
GENETIC EPIGENETIC

 Oncogene function  Oncogene levels

↓ Tumor suppressor function ↓ Tumor suppressor levels

Oncogenesis

Tumor
 Defining Epigenetics

Epigenetics is: Genome

• Reversible changes in gene DNA

expression
– Without changes in DNA
sequence
Chromatin
– Can be inherited from
precursor cells Epigenome

• Epigenetic information is
Gene Expression
included in the epigenome

Phenotype
 Epigenetics Play Important Roles in
Normal & Cancer Development
EPIGENETICS

Normal epigenetic Deregulated epigenetic

mechanisms mechanisms

Normal differentiated cells, e.g. embryonic Malignant

cells, hematopoetic cells progenitor cell Tumor

• Epigenetic mechanisms can regulate genes involved in differentiation, cell

cycle, and cell survival
• Deregulation of epigenetic mechanisms results in aberrant gene expression,
which can lead to cancer
• Reversal of deregulated epigenetic changes is a rational strategy for
targeting cancer
 Cancer Stem Cell Theory: the ‘Root’
of Cancer Growth

Tumor

Tumor
Developme
and
Growth

Epigenetically
altered, self-
renewing cancer
stem cells
Conventional Therapies May Target Tumor Cells,
Not Cancer Stem Cells
Conventional
therapy

Target tumor Cancer stem Tumor

cells survive regrowth
Epigenetic Therapy May Target Cancer Stem Cells and
Tumor Cells
Epigenetic
therapy

Target tumor Tumor and No

and cancer cancer stem regrowth
stem cells cell death
Section 1: Importance of Epigenetics in Cancer

Section 2: Mechanisms of Epigenetics: Focus on Deacetylation

Section 3: Therapeutic Targeting of Epigenetics

 Chromatin is a Key Component of Epigenetic
Mechanisms
Cellular DNA is packaged into a structure called
chromatin

The unit of chromatin is the nucleosome, a complex

of a histone tetramer with approx. 125 bp of DNA
wound around it

histone nucleosome
DNA

chromatin

• Chromatin organizes genes to be accessible for transcription,

replication, and repair
 Basic Epigenetic Mechanisms: Post Translational
Modifications to Histones and Base Changes in DNA

• Epigenetic modifications of histones and DNA include:

• Histone acetylation and methylation, and DNA methylation

Histone Histone
Acetylation Methylation

Ac
Me Me Me

DNA Methylation
Histone Proteins in Chromatin Can be Modified by
Acetylation

Histone Histone
Acetylation
Methylation

Me Me
Ac
Me

DNA Methylation
Epigenetic Changes can Alter Chromatin Structure
and Regulate Gene Expression

TF TF
Ac
Ac
Ac

Ac
Ac
Ac
Ac
Ac
Ac

Gene Gene
expression expression

• Gene expression (transcription) requires DNA to be physically accessible to

transcription factors (TF)
• Epigenetic changes alter the structure of the chromatin, which determines
whether DNA is accessible
– Open chromatin allows gene expression
– Closed chromatin prevents gene expression
 In Cancer, Changes in Chromatin Structure Can
Silence Tumor Suppressor Genes

Normal Cancer
Ac
cells Ac Ac cells
Ac
Ac Ac
Ac Ac Ac

Tumor suppressor gene Tumor suppressor

expression gene expression

• Silencing of tumor suppressor genes, a major process in

tumorigenesis, may result from epigenetic changes that
condense chromatin structure
Balance of Histone Acetylation is a Key Factor in
Transcriptional Regulation in Normal Cells

HAT
TF
Ac Ac Ac
HISTONE
Ac Ac Ac
ACETYLATION
TF Ac Ac Ac

HISTONE
DEACETYLATION
Deacetylated Acetylated Histones
Histones
HDAC Open chromatin
HDAC
Closed chromatin Transcription factors
Transcription factors can access DNA
cannot access DNA

Gene Gene
expression expression
 Imbalanced Levels of Histone Acetylation in
Cancer Deregulate Gene Expression
HAT

HISTONE TF
ACETYLATION Ac Ac Ac

Ac Ac Ac

Ac Ac Ac
TF HISTONE
DEACETYLATION

Deacetylated Acetylated Histones

Histones HDAC
Open chromatin
Closed chromatin Transcription factors
Transcription factors can access DNA
cannot access DNA

• Increased HDAC activity or decreased HAT activity may result in

aberrant gene expression, contributing to cancer
Other Epigenetic Modifications of Histones and
DNA

Histone
Histone Methylation
Acetylation
Me Me
Ac Me

DNA
Methylation
 Histone Methylation can Affect Chromatin
Structure
Me
HDAC Me

Me
HMT
Me
Ac
Ac
Me

Me
Me Me Ac

HMT Me
Me
HDAC

Gene Gene
expression expression

• Histone methylation by histone methyltransferases (HMTs) can

recruit HDACs, leading to:
– Closed chromatin structure
– Gene silencing
 DNA Methylation can Prevent Gene
Expression
TF
DNMT DNMT

TF DNMT DNMT
Ac Ac
Ac Ac Ac Ac
Me Me
Me Me Me Ac Me
Ac
Ac Ac Ac Me Ac
Ac Ac
Ac Ac
Ac Ac

Gene Gene
expression expression

• DNA methylation involves the transfer of methyl groups to cytosine

residues in DNA by DNA methyltransferases (DNMTs)
– May prevent transcription factors from binding to DNA
– May serve as binding site for methylated DNA-binding proteins,
such as MECP2, which then recruit HDACs
Epigenetic Modifications to Histones and DNA Can Cooperate to
Silence Gene Expression

DNMT DNMT HDAC

Ac Ac Ac
Ac
Ac

Ac Ac Ac Ac
Ac
Ac
Ac Ac Ac
Ac
Ac
HDAC

TF
Me
Me Me
Me

Me
Gene
Me
Me expression
• Epigenetic modifications can cooperate to silence gene expression
Section 1: Importance of Epigenetics in Cancer

Section 2: Mechanisms of Epigenetics: Focus on Deacetylation

Section 3: Therapeutic Targeting of Epigenetics

 Increased HDAC Activity Can Alter Gene Expression
and Result in Cancer

• Increased HDAC activity,

which has been associated
with certain tumors, can
alter expression of genes
HDAC involved in normal cell
TF development, resulting in:
– Loss of cell-cycle arrest
– Inhibition of
differentiation
– Cell growth and
Gene proliferation
expression – Evasion of apoptosis
– Migration and metastasis

Cell nucleus
 HDAC Inhibition Can Reverse Altered
Gene Expression
• Inhibition of HDAC activity
can restore the balance of
histone acetylation
DAC
Inhibitor • Targeting HDAC activity may
therefore allow
TF HDAC re-expression of silenced
genes to reverse oncogenesis
Ac Ac Ac

Cell-cycle arrest

Differentiation
Gene
Growth control
expression
Apoptosis
Adhesion

Cell nucleus
 Therapeutic Targeting of Both Histone and DNA
Modifications Can Synergize

DNMT
Inhibitor • Since DNA
DAC methylation and
Inhibitor
DNMT histone deacetylation
can co-operate to
HDAC
TF silence tumor
Ac Ac Ac
suppressors, 
Ac Ac Ac
inhibition of both
DNMT and HDAC
Cell-cycle arrest activities can
Differentiation
synergize to restore
Gene
expression of
expression
Growth control silenced genes
Apoptosis
Adhesion

Cell nucleus
Central Dogma Genotype

RNA function &

structure
DNA
Protein sequence

RNA Protein structure

Protein Protein Function

Phenotype
Genome

Expressome

Proteome

Metabolome

Functional
 Biomolecular  Diagnostics
Cancer : – Genomic : Diagnostic

Genetics • DNA
Epigenetics – Epigenomics Prognostic
Viral Infections : • HDAC
EBV
HPV
• DNMe Treatment
HBV – Transcriptomic Follow up
HIV • RNA
H Pylorii
– Proteomic: Prevention
• Protein
– Metabolomic : Discovery
• Enzym
• Biomarker
Example:
A closer
look at
p53
 17-gene metastatic signature

Upregulation: Protein translation apparatus

TP73 Inactivation of TSGs
MLH1 RASSF1
CDKN2A  p16INK4A + p14ARF

HIC1 TP53
BRCA2

cadherin BRCA1

Red = only hypermethylation identified HIC1 = hypermethylated in cancer 1

Green = only genetic mutations identified (frequent LOH)
Purple = both mechanisms found Nature Rev 3:415, 2002
 Inactivation of TSGs

E
Genetic Alteration
P
I
G
E
N
E
T
I
C

A (wt allele)
L
T
E
R

Cell Research, 15:237-246, 2005

 Inactivation of TSGs

(MMR gene) (DNA repair gene)

Nature Rev 3:415, 2002

DNA hyper-/hypomethylation

BORIS –
Reactivated in cancer: amplified
Overexpression  proliferation

CTCF –
In region of frequent LOH
Overexpression  inhibits
proliferation

Nature Rev 6:597, 2005

DNA hypermethylation
• Silencing may occur early, even in non- or pre-
cancerous cells
– E.g. Promoter methylation of
CDKN2A/p16INK4a is detectable in
preinvasive bronchial lesions, in pituitaries of
Cushing’s patients and in normal breast tissue
– Many other examples in other tissues
– Changes may be age-related or pre-
malignant, or may be associated with
environmental exposures and/or diet
Analysis of methylation status

• Gene expression microarray

analysis
– Identifies non-transcribed genes
as candidates for promoter
hypermethylation
– Re-expression of silenced genes
after HDAC/DMT blocks
 Analysis of methylation status
• Promoter DNA methylation status
1. Bisulfite genomic sequencing (known TSGs)
a. Converts unmethylated C to U
2. Bisulfite-based methylation-specific PCR (known TSGs)
3. Bisulfite-based methylation-specific microarray and probes
a. Two spots (sequence +/- bisulfite); bisulfite treated PCR probes
(multiple known TSGs)
4. Methylation-sensitive RE digests (RLGS, MS-RDA/PCR)
a. RLGS = restriction landmark genome scanning
– eg NotI for total genomic digestslook for missing spot on 2D gel
b. MS-RDA = methylation sensitive representational difference analysis
– Eg HpaII, SacII  ligate adaptors as primer sites; look for bands diffs
c. MS-PCR = methylation sensitive PCR
1. eg HpaII (sens) vs MseI (insens) PCR  look for bands or hybridize to
CpG island microarrays (random or known TSGs)
Hypermethylation of RASSF1A

Biochem (Moscow) 70:576, 2005

RASSF1A

Biochem (Moscow) 70:576, 2005

 Epigenetic analysis
• Heterochromatin analysis
– ChIP-PCR – using Ab’s to acetylated
H3/H4
• specific band or hybridize to CpG
microarray (random/known TSGs)
SNPs as Gateway to Genome-Wide
Association (GWA) Studies

 SNPs much more numerous than other

markers and easier to assay
 Genome-wide studies attempt to capture
majority of genomic variation (10M SNPs!)
 Variation inherited in groups, or blocks, so not
all 10 million points have to be tested
 Blocks are shorter (so need to test more
points) the less closely people are related
 SNP technology allows studies in unrelated
persons, assuming 5kb – 10kb lengths in
common (300,000 – 1,000,000 markers)
 Single Nucleotide Polymorphisms (SNPs)
GAAATAATTAATGTTTTCCTTCCTTCTCCTATTTTGTCCTTTACTTCAATTTATTTATTTATTATTAATATTATTATTTTTTGAGAC
GGAGTTTC/ACTCTTGTTGCCAACCTGGAGTGCAGTGGCGTGATCTCAGCTCACTGCACACTCCGCTTTCCTGGTTTCA
AGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGTCACACACCACCACGCCCGGCTAATTTTTGTATTTTT
AGTAGAGTTGGGGTTTCACCATGTTGGCCAGACTGGTCTCGAACTCCTGACCTTGTGATCCGCCAGCCTCTGCCTCCCA
AAGAGCTGGGATTACAGGCGTGAGCCACCGCGCTCGGCCCTTTGCATCAATTTCTACAGCTTGTTTTCTTTGCCTGGAC
TTTACAAGTCTTACCTTGTTCTGCC/TTCAGATATTTGTGTGGTCTCATTCTGGTGTGCCAGTAGCTAAAAATCCATGATTT
GCTCTCATCCCACTCCTGTTGTTCATCTCCTCTTATCTGGGGTCACA/CTATCTCTTCGTGATTGCATTCTGATCCCCAGT
ACTTAGCATGTGCGTAACAACTCTGCCTCTGCTTTCCCAGGCTGTTGATGGGGTGCTGTTCATGCCTCAGAAAAATGCAT
TGTAAGTTAAATTATTAAAGATTTTAAATATAGGAAAAAAGTAAGCAAACATAAGGAACAAAAAGGAAAGAACATGTATTCTA
ATCCATTATTTATTATACAATTAAGAAATTTGGAAACTTTAGATTACACTGCTTTTAGAGATGGAGATGTAGTAAGTCTTTTAC
TCTTTACAAAATACATGTGTTAGCAATTTTGGGAAGAATAGTAACTCACCCGAACAGT G/TAATGTGAATATGTCACTTACT
AGAGGAAAGAAGGCACTTGAAAAACATCTCTAAACCGTATAAAAACAATTACATCATAATGATGAAAACCCAAGGAATTTTT
TTAGAAAACATTACCAGGGCTAATAACAAAGTAGAGCCACATGTCATTTATCTTCCCTTTGTGTCTGTGTGAGAATTCTAGA
GTTATATTTGTACATAGCATGGAAAAATGAGAGGCTAGTTTATCAACTAGTTCATTTTTAAAAGTCTAACACATCCTAGGTAT
AGGTGAACTGTCCTCCTGCCAATGTATTGCACATTTGTGCCCAGATCCAGCATAGGGTATGTTTGCCATTTACAAACGTTT
ATGTCTTAAGAGAGGAAATATGAAGAGCAAAACAGTGCATGCTGGAGAGAGAAAGCTGATACAAATATAAAT/GAAACAA
TAATTGGAAAAATTGAGAAACTACTCATTTTCTAAATTACTCATGTATTTTCCTAGAATTTAAGTCTTTTAATTTTTGATAAATC
CCAATGTGAGACAAGATAAGTATTAGTGATGGTATGAGTAATTAATATCTGTTATATAATATTCATTTTCATAGTGGAAGAAAT
AAAATAAAGGTTGTGATGATTGTTGATTATTTTTTCTAGAGGGGTTGTCAGGGAAAGAAATTGCTTTTT

SNPs 1 / 300 bases

~ 10 million across genome
 One Tag SNP May Serve as Proxy for Many
Block 1 Block 2

SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
CAGATCGCTGGATGAATCGCATCTGTAAGCAT

CGGATTGCTGCATGGATCGCATCTGTAAGCAC

CAGATCGCTGGATGAATCGCATCTGTAAGCAT

CAGATCGCTGGATGAATCCCATCAGTACGCAT

CGGATTGCTGCATGGATCCCATCAGTACGCAT

CGGATTGCTGCATGGATCCCATCAGTACGCAC
 SNPs and Function:
We know so little…

• Majority are “silent”

– No known functional change
• Some alter gene expression/regulation
– Promoter/enhancer/silencer
– mRNA stability
– Small RNAs
• Some alter function of gene product
– Change sequence of protein

Courtesy S. Chanock, NCI

 SNPs within Genes
Coding SNPs (cSNPs)
• Synonymous: no change in amino acid
previously termed “silent” but…..
Can alter mRNA stability
DRD2 (Duan et al 2002)
Can alter speed of translation and protein folding
MDR1 (Gottesman et al 2007)
• Nonsynonymous: changes amino acid (codon)
conservative and radical
• Nonsense: insertion of stop codon
Frameshift (insertion/deletion): Disrupts codon
sequence, rare but disruptive

After S. Chanock, NCI

 SNPs Outside Genes

• Majority distributed throughout genome are “silent”

(excellent as markers)
• Alter transcription
– Promoter, enhancer, silencer
• Regulate expression
– Locus control region, mRNA stability
• Most are assumed to be ‘silent hitchhikers’
– No function by predictive models or analysis

Courtesy S. Chanock, NCI