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Predicting the response of cervical cancer to radiotherapy. Identification of hypermethylation based markers. Frank Roossink. Cervical cancer. Introduction Cervical cancer Epigenetics Chromatin structure DNA methylation Project Aim Hypothesis Gene Selection Biotrove MSP - PowerPoint PPT Presentation
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Department of Gynecologic oncology
Frank Roossink
Identification of hypermethylation based
markers
• 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.
21-04-23 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
21-04-23 Identification of RT response methylation markers in cervical cancer 3
Data from Globocan (2002)Data from Globocan (2002)
• 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
• 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.
21-04-23 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
• Acknowledgements
21-04-23 Identification of RT response methylation markers in cervical cancer 5
0.3%
0.5%
0.6%
0.7%
1.0%
1.2%
1.3%
1.4%
2.2%
2.3%
2.6%
2.9%
17.3%
53.5%
6.7%
Roden and Wu Roden and Wu Nature ReviewsNature Reviews (2006) (2006)
• 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
• 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.
21-04-23 Identification of RT response methylation markers in cervical cancer 7
• 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
Dermis
Basal layerBasement membrane
Midzone
Superficial zone
Normal cervix Squamous Intraepithelial lesion (SIL)
Low grade High grade
Invasive cancerASCUS
HPV infection
Infectious HPV particles
80% clearance
Viral integration
E6/E7 deregulation
Proliferation
20% Lesions Genetic instability
Adapted from Snijders et alAdapted from Snijders et al. J. of pathology . J. of pathology (2006), Woodman et al. (2006), Woodman et al. Nature reviewsNature reviews (2007). (2007).
Dermis
Basal layerBasement membrane
Midzone
Superficial zone
Normal cervix Squamous Intraepithelial lesion (SIL)
Low grade High grade
Invasive cancerASCUS
-Loss of Chr. 6p-DR of MHC class I
Adapted from Snijders et alAdapted from Snijders et al. J. of pathology . J. of pathology (2006), Woodman et al. (2006), Woodman et al. Nature reviewsNature reviews (2007). (2007).
-Loss of Chr. 3p,4q, 6q and 10p-UR telomerase
-DR GATA-3
Immortalization
-Loss of Chr. 1-DR TSLC1
Invasion
• 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.
21-04-23 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
• Acknowledgements
“The branch of biology which studies
the causal interactions between genes and their products which bring the phenotype into being”
C.H. Waddington, 1942
21-04-23 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
21-04-23 Identification of RT response methylation markers in cervical cancer 12
Waddington (1948)Waddington (1948)
• 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
“Heritable states of gene-expression that are not due to
alterations in the underlying DNA sequence itself.”
21-04-23 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
• Summary
• Acknowledgements
21-04-23 Identification of RT response methylation markers in cervical cancer 14
• 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
21-04-23 Identification of RT response methylation markers in cervical cancer 15
• 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
3.5x
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.
21-04-23 Identification of RT response methylation markers in cervical cancer 16
Molecular biology of the cell, 4Molecular biology of the cell, 4thth edition edition
• 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
30nm fiber“Beads on a string”
Histone tail modificationsDNA double helix
Adapted from: http://www.newscientist.com/data/images/archive/2386/23865001.jpgAdapted from: http://www.newscientist.com/data/images/archive/2386/23865001.jpgMolecular biology of the cell, 4Molecular biology of the cell, 4thth edition edition
21-04-23 Identification of RT response methylation markers in cervical cancer 18
REF!!!REF!!!
• 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
Histone modifications
21-04-23 Identification of RT response methylation markers in cervical cancer 19
Latham et al. Latham et al. Nature Structural & Molecular Biology, 2007Nature Structural & Molecular Biology, 2007
• 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
• 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
21-04-23 Identification of RT response methylation markers in cervical cancer 20
• 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
21
Frontal viewSide view
Adapted from O’Gara et al.J Mol Biol. 1996 Sep 6;261(5):634-45
Frontal view
Adapted from O’Gara et al.J Mol Biol. 1996 Sep 6;261(5):634-45
• CpG dinucleotides are often found in clusters. So-called CpG islands.
• CpG-island 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.
21-04-23 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
• Summary
• Acknowledgements
21-04-23 Identification of RT response methylation markers in cervical cancer 24
• 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
Normal Cancer
To identify genes, which methylation status predicts the
response to chemoradiation.
21-04-23 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
• Summary
• Acknowledgements
Different methylation pattern in responding patients vs. non-
responding patients.
21-04-23 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
• Summary
• Acknowledgements
• Originally our KWF-application listed 84 genes: 52 genes from array data. 32 genes from literature.
• Together with OMS, we will analyze our patient material using a Methylation Specific PCR based screen.
• Our new and improved list consists of 800+ genes.
21-04-23 Identification of RT response methylation markers in cervical cancer 27
• 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
• MSP-based screening tool.• Combines the large screen capabilities of microarray
with the semi-quantitative analysis of Q-PCR.
21-04-23 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
• Summary
• Acknowledgements
• Methylation specific PCR Bisulfite conversion.
21-04-23 Identification of RT response methylation markers in cervical cancer 29
A C G C G C G C C
A C G C G C G U U
Methylated DNA Unmethylated DNA
Differential sequenceDifferential sequence
• 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
• Nucleoside DNMT inhibitors: DAC (Decitabine), 5-aza-C, Zebularin.
• Non-Nucleoside DNMT inhibitors: Hydralazine, EGCG, RG108, Procaine Procainamide
21-04-23 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
• Summary
• Acknowledgements
Medium co
ntrol
M U M U M U M U M U M U M U
200nM DAC
M U 300nM
TSA
Combi DAC/T
SA 10µM
Hydralazin
e 1mM
VPA
Combi Hyd
r./VPA
5µM DAC
DAPK
M U M U
Leuco
cyte
IV
21-04-23 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
• Acknowledgements
Medium co
ntrol
5µM DAC
200nM DAC
M U M U M U M U M U M U M U M U M U M U M U M U
40µM Hyd
ralazine
1mM Hyd
ralazine
10µM Pro
cainamide
500µM Pro
cainamide
120µM Ze
bularine
1mM Ze
bularine
Medium re
seed
200nM DAC + 300nM
TSA Rese
ed
1mM Ze
bularine re
seed
• 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…
21-04-23 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
• Summary
• Acknowledgements
• 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
21-04-23 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
• Summary
• Acknowledgements
21-04-23 Identification of RT response methylation markers in cervical cancer 34
• 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
Seeding of cells
IrradiationDay 10, 2, 4, 6, 8 and 10Gy
Read-outDay 10
Day 0
Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
SiHaHeLa
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
SiHa
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
21-04-23 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
• Acknowledgements
Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
SiHaHeLaCaSki
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
21-04-23 Identification of RT response methylation markers in cervical cancer 36
• 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
Seeding of cells
Read-outDay 15
Day 0
DAC/TSA treatment
Day 2-4 200nM DACDay 4 300nM TSA
Seeding of cellsDay 5
After 6h, irradiation.0, 2, 4, 6, 8 & 10 Gy
21-04-23 Identification of RT response methylation markers in cervical cancer 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
SiHa Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
Medium
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
SiHa Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
MediumDAC/TSA (200nM/300nM)
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
SiHa Clonogenic Assay
0 2 4 6 8 100.1
1
10
100
MediumDAC/TSA (200nM/300nM)Zubularine (1mM)
Dose (Gy)
Surv
ivin
g fr
actio
n (%
)
21-04-23 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
• Acknowledgements
• pcDNA3-HA-TAp73α Gift from G. Melino, Rome Italy
• pIRES2-EGFP-TAp73α
21-04-23 Identification of RT response methylation markers in cervical cancer 39
• 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
21-04-23 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
• Summary
• Acknowledgements
SiHa pcDNA3-HA-TAp73α PBS control SiHa medium control αHA-tag (Y11) 1:50 SiHa pcDNA3-HA-TAp73α αHA-tag (Y11) 1:50
Appr. 37% transfection efficiency
SiH
a un
tran
sfec
ted
SiH
a pc
DN
A3-H
A-TA
p73α
SiH
a un
tran
sfec
ted
SiH
a pc
DN
A3-H
A-TA
p73α
SiH
a un
tran
sfec
ted
SiH
a pc
DN
A3-H
A-TA
p73α
Mar
ker
Mar
ker
Mar
ker
HA-TAp73α
αHA (F7, monoclonal mouse) 1:200 1hour, 30s exposure
pIRES2-EGFP
21-04-23 Identification of RT response methylation markers in cervical cancer 41
• 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
CMV
G.O.I.
IRES
GFP
KanR
NeoR
• MSP analysis of the selected genes, to see what the methylation status is of our 4 cell lines.
• RT-PCR to see if the genes are transcribed.
• Clone into pIRES2-EGFP.
• Develop RNAi constructs.
• Clonogenic assay of transfected cellswith RNAi and plasmid constructs
21-04-23 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
• Summary
• Acknowledgements
• 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.
• Cloning strategies in progress
21-04-23 Identification of RT response methylation markers in cervical cancer 43
• 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
• Our patient-material screen is starting shortly. Our candidate gene list has probably around 800 genes. Test samples already have been sent.
21-04-23 Identification of RT response methylation markers in cervical cancer 44
• 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
21-04-2345
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