predicting the response of cervical cancer to radiotherapy

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


Data from Globocan (2002)Data from Globocan (2002)




• Summary


• 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



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)




• Summary


• 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


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.





• 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





• Summary



Waddington (1948)Waddington (1948)




• Summary


“Heritable states of gene-expression that are not due to

alterations in the underlying DNA sequence itself.”





• Summary






• Summary






• Summary


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.


Molecular biology of the cell, 4Molecular biology of the cell, 4thth edition edition




• Summary


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


REF!!!REF!!!




• Summary


Histone modifications


Latham et al. Latham et al. Nature Structural & Molecular Biology, 2007Nature Structural & Molecular Biology, 2007




• Summary


• 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





• Summary


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.





• Summary






• Summary


Normal Cancer

To identify genes, which methylation status predicts the

response to chemoradiation.





• Summary


Different methylation pattern in responding patients vs. non-

responding patients.





• Summary


• 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.





• Summary


• MSP-based screening tool.• Combines the large screen capabilities of microarray

with the semi-quantitative analysis of Q-PCR.





• Summary


• Methylation specific PCR Bisulfite conversion.


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




• Summary


• Nucleoside DNMT inhibitors: DAC (Decitabine), 5-aza-C, Zebularin.

• Non-Nucleoside DNMT inhibitors: Hydralazine, EGCG, RG108, Procaine Procainamide





• Summary


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





• Summary


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…





• Summary


• 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






• Summary


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 (%

)





• Summary


Clonogenic Assay

0 2 4 6 8 100.1

1

10

100

SiHaHeLaCaSki

Dose (Gy)

Surv

ivin

g fr

actio

n (%

)





• Summary


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





• Summary


SiHa Clonogenic Assay

0 2 4 6 8 100.1

1

10

100

Medium

Dose (Gy)

Surv

ivin

g fr

actio

n (%

)


0 2 4 6 8 100.1

1

10

100

MediumDAC/TSA (200nM/300nM)

Dose (Gy)

Surv

ivin

g fr

actio

n (%

)


0 2 4 6 8 100.1

1

10

100

MediumDAC/TSA (200nM/300nM)Zubularine (1mM)

Dose (Gy)

Surv

ivin

g fr

actio

n (%

)





• Summary


• pcDNA3-HA-TAp73α Gift from G. Melino, Rome Italy

• pIRES2-EGFP-TAp73α





• Summary






• Summary


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





• Summary


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




• Project Aim Hypothesis Gene Selection Biotrove MSP Demethylation Validation Clonogenic assay In vitro validation Plan of

investigation

• Summary


• 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





• Summary


• Our patient-material screen is starting shortly. Our candidate gene list has probably around 800 genes. Test samples already have been sent.





• Summary


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

predicting the response of cervical cancer to radiotherapy

Documents