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Department of Gynecologic oncology
Identification of
hypermethylation based
markers
Frank Roossink
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• The second most common malignancy among women
world-wide.
 In the Netherlands 700 patients present with cervical cancer
each year.
 World-wide this number is 450,000 cases and 250,000 deaths.
• Disproportionally towards less developed countries.
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
2
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
Data from Globocan (2002)
23-5-2017
Identification of RT response methylation markers in cervical cancer
3
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• The second most common malignancy among women
world-wide.
 In the Netherlands 700 patients present with cervical cancer
each year.
 World-wide this number is 450,000 cases and 250,000 deaths.
• Disproportionally towards less developed countries.
• 99.7% of all cervical cancer patients are human
papilloma virus(HPV) positive. (Waalbomers, 1999)
 70% is due to infection with types HPV-16 and HPV-18.
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
4
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
HPV-82
0.3%
HPV-73
0.5%
HPV-68
0.6%
HPV-39
0.7%
HPV-51
1.0%
HPV-56
1.2%
HPV-59
1.3%
HPV-35
1.4%
HPV-58
2.2%
HPV-52
2.3%
HPV-33
2.6%
2.9%
HPV-31
6.7%
HPV-45
17.3%
HPV-18
53.5%
HPV-16
• Acknowledgements
0
10
20
30
40
50
60
70
80
90
100
Contribution to cervical cancer
Roden and Wu Nature Reviews (2006)
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Cancer is a disease of the genes.
• Changes in expression patterns of genes are thought to
be the cause for the development of cancer.
• In cervical cancer these changes in expression are
thought to initiate with the establishment HPV infection.
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
7
Normal cervix
ASCUS
Squamous Intraepithelial lesion (SIL)
Low grade
Invasive cancer
High grade
Infectious HPV particles
Superficial zone
Midzone
Basal layer
Basement membrane
Dermis
HPV infection
20%
Lesions
80%
clearance
Viral
integration
E6/E7
deregulation
Proliferation
Adapted from Snijders et al. J. of pathology (2006), Woodman et al. Nature reviews (2007).
Genetic instability
Normal cervix
ASCUS
Squamous Intraepithelial lesion (SIL)
Low grade
Invasive cancer
High grade
Superficial zone
Midzone
Basal layer
Basement membrane
Dermis
-Loss of Chr. 6p
-DR of MHC class I
-Loss of Chr. 3p,
4q, 6q and 10p
-UR telomerase
-DR GATA-3
-Loss of Chr. 1
-DR TSLC1
Immortalization
Adapted from Snijders et al. J. of pathology (2006), Woodman et al. Nature reviews (2007).
Invasion
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Cancer is a disease of the genes.
• Changes in expression patterns of genes are thought to
be the cause to the development of cancer.
• In cervical cancer these changes in expression are
thought to initiate with the establishment HPV infection.
• Changes in expression patterns can be due to
genetic or epigenetic alterations.
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
10
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
“The branch of biology which studies
the causal interactions between genes and
their products which bring the phenotype
into being”
C.H. Waddington, 1942
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
11
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
Waddington (1948)
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
“Heritable states of gene-expression that are
not due to alterations in the underlying DNA
sequence itself.”
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
13
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
Methylation
Methylation
Phosphorylation
Acetylation
miRNA
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
DNA packaging:
•
•
•
Every mitotic cell has 23 pair chromosomes.
• Approximately 30,000 genes.
• Approximately 2m DNA per cell.
Each individual consists of 10 billion cells.
That means 500,000 trips around the equator or
3.5 trips from the Sun to Pluto.
• Summary
• Acknowledgements
3.5x
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
Molecular biology of the cell, 4th edition
23-5-2017
Identification of RT response methylation markers in cervical cancer
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30nm fiber
DNA double helix
Molecular biology of the cell, 4th edition
Adapted from: http://www.newscientist.com/data/images/archive/2386/23865001.jpg
“Beads on a string”
Histone tail modifications
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
REF!!!
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
Histone modifications
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
Latham et al. Nature Structural & Molecular Biology, 2007
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
23-5-2017
• DNA methylation occurs at the 5th position of a cytosine.
• Cytosine that precede Guanine can become methylated
 So-called CpG dinucleotides.
• Methylation is mediated by DNA methyl transferases
(DNMTs)
 de novo methylation
• DNMT-3a and DNMT-3b
 Maintenance
• DNMT-1
Identification of RT response methylation markers in cervical cancer
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Side view
Adapted from O’Gara et al.
J Mol Biol. 1996 Sep 6;261(5):634-45
Frontal view
21
Frontal view
Adapted from O’Gara et al.
J Mol Biol. 1996 Sep 6;261(5):634-45
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• CpG dinucleotides are often found in clusters.
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• CpG-island
• Summary
 So-called CpG islands.




A region with at least 200bp
A G-C percentage of >50%
CpG-ratio Obs-Exp >0.6
Found in promoter regions of >50% of all genes.
• Promoter hypermethylation can silence genes.
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
23
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
Cancer
Normal
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
To identify genes, which methylation status
predicts the response to chemoradiation.
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
25
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
Different methylation pattern in responding
patients vs. non-responding patients.
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
26
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Originally our KWF-application listed 84 genes:
 52 genes from array data.
 •32
from literature.
Cellgenes
lines treated
with DAC (0, 0.2, 1 and 5µM) or 0.2µM DAC in
combination with 0.3µM TSA
• Expression
analysis
using
Affymetrix
Genechip®
• Together
with
OMS,
we
will analyze
our patient material
HG U133 plus 2.0 (>54,000 probes)
using a Methylation Specific PCR based screen.
• More than 3-fold UR after DAC(/TSA) treatment
 819 probes  546 genes
• Our new and improved list consists of 800+ genes.
• 85 genes that are in DNA-repair, cell cycle and
apoptosis pathways
• Summary
• Acknowledgements
• 52 genes that have a CpG island in their promoter
region
23-5-2017
Identification of RT response methylation markers in cervical cancer
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• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• MSP-based screening tool.
• Combines the large screen capabilities of microarray
with the semi-quantitative analysis of Q-PCR.
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
28
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Methylation specific PCR
 Bisulfite conversion.
Methylated DNA
Unmethylated DNA
A
C
G
C
G
C
G
C
C
A
C
G
C
G
C
G
C
C
A
C
G
C
G
C
G
U
U
A
U
G
U
G
U
G
U
U
Differential sequence
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
29
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
23-5-2017
• Nucleoside DNMT inhibitors:
 DAC (Decitabine), 5-aza-C, Zebularin.
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
DAPK
• Non-Nucleoside DNMT inhibitors:
 Hydralazine, EGCG, RG108, Procaine
 Procainamide
Identification of RT response methylation markers in cervical cancer
30
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
31
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
 Procainamide is a FDA approved drug which has demethylating
properties.
• However, it has severe side-effects.
 Also Zebularin has demethylating properties.
• Draw-back: it kills monkeys…
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
32
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Validation in a larger cohort of patient samples.
 Amsterdam series
 UMCG
• In vitro validation of our candidate genes.
 Over-expression of our candidate genes
• Clonogenic assay analysis.
 Knock-down of our candidate genes
• Clonogenic assay
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
33
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
Seeding of cells
Day 0
Irradiation
Day 1
0, 2, 4, 6, 8 and 10Gy
Read-out
Day 10
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
34
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
100
Surviving fraction (%)
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
Clonogenic Assay
10
1
SiHa
HeLa
CaSki
• Summary
• Acknowledgements
0.1
0
2
4
6
8
10
Dose (Gy)
23-5-2017
Identification of RT response methylation markers in cervical cancer
35
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
Seeding of cells
Day 0
Seeding of cells
Day 5
After 6h, irradiation.
0, 2, 4, 6, 8 & 10 Gy
Read-out
Day 15
DAC/TSA treatment
Day 2-4 200nM DAC
Day 4 300nM TSA
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
36
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
100
Surviving fraction (%)
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
SiHa Clonogenic Assay
10
1
Medium
DAC/TSA (200nM/300nM)
Zubularine (1mM)
• Summary
• Acknowledgements
23-5-2017
0.1
0
2
4
6
Dose (Gy)
Identification of RT response methylation markers in cervical cancer
8
10
37
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
23-5-2017
Identification of RT response methylation markers in cervical cancer
38
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• pcDNA3-HA-TAp73α
 Gift from G. Melino, Rome Italy
• Acknowledgements
• pIRES2-EGFP-TAp73α
23-5-2017
Identification of RT response methylation markers in cervical cancer
39
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
SiHa pcDNA3-HA-TAp73α αHA-tag (Y11) 1:50
Appr. 37% transfection efficiency
Marker
SiHa pcDNA3-HA-TAp73α
SiHa untransfected
Marker
SiHa medium control αHA-tag (Y11) 1:50
SiHa pcDNA3-HA-TAp73α
SiHa untransfected
Marker
SiHa pcDNA3-HA-TAp73α
SiHa pcDNA3-HA-TAp73α PBS control
SiHa untransfected
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
HA-TAp73α
• Acknowledgements
αHA (F7, monoclonal mouse) 1:200 1hour, 30s exposure
23-5-2017
Identification of RT response methylation markers in cervical cancer
40
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
CMV
G.O.I.
KanR pIRES2-EGFP
NeoR
IRES
• Summary
• Acknowledgements
23-5-2017
GFP
Identification of RT response methylation markers in cervical cancer
41
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• MSP analysis of the selected genes, to see what the
methylation status is of our 4 cell lines.
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• RT-PCR to see if the genes are transcribed.
• Summary
• Clone into pIRES2-EGFP.
• Develop RNAi constructs.
• Acknowledgements
• Clonogenic assay of transfected cells
with RNAi and plasmid constructs
23-5-2017
Identification of RT response methylation markers in cervical cancer
42
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• HeLa, SiHa are similar sensitive to RT in the clonogenic
setting
• CaSki is more resistant than SiHa and HeLa.
• After DAC/TSA and Zebularin treatment prior to RT, SiHa
cells are not changed in sensitivity to RT.
• Although procainamide is FDA approved (for arrythmia),
we are unlikely to use it due to side-effects.
• Summary
• Acknowledgements
• Cloning strategies in progress
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Identification of RT response methylation markers in cervical cancer
43
• Introduction
 Cervical cancer
 Epigenetics
• Chromatin
structure
• DNA methylation
• Our patient-material screen is starting shortly.
 Our candidate gene list has probably around 800 genes.
 Test samples already have been sent.
• Project
 Aim
 Hypothesis
 Gene Selection
 Biotrove
 MSP
 Demethylation
 Validation
 Clonogenic assay
 In vitro validation
 Plan of
investigation
• Summary
• Acknowledgements
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Identification of RT response methylation markers in cervical cancer
44
 Gynecologic oncology
 G. Bea A. Wisman
 Mirjam Kok
 Maartje G. Noordhuis
 Ate G.J. van der Zee
 Pathology
 Ed Schuuring
 Medical oncology
 Steven de Jong
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