MICROSATELLITE INSTABILITY PROFILING OF LYNCH … · 9 Associated extra-colonic cancers: cancer of...

MICROSATELLITE INSTABILITY PROFILING OF LYNCH SYNDROME-ASSOCIATED CANCERS

The studies described in this thesis were supported by the “Fundação para a Ciência e a Tecnologia”

(SFRH/BD/18832/2004), Portugal, and by the European Community (FP6-2004-LIFESCIHEALTH-5,

proposal no. 018754).

The printing costs of this thesis were supported by: Stichting Nationaal Fonds tegen Kanker – voor

onderzoek naar reguliere en aanvullende therapieën te Amsterdam; Fundação para a Ciência e a

Tecnologia; University of Groningen; University Medical Center Groningen (UMCG); Graduate School

for Drug Exploration (GUIDE).

Printed by: Grafimedia Facilitair Bedrijf RUG Cover design by: Grafimedia Facilitair Bedrijf RUG

© 2009, A.M. Monteiro Ferreira. All rights are reserved. No part of this publication may be reproduced or

transmitted in any form or by any means without permission of the author.

ISBN: 978-90-367-3821-7

2

MICROSATELLITE INSTABILITY PROFILING OF LYNCH SYNDROME-ASSOCIATED CANCERS

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

woensdag 13 mei 2009 om 10.30 uur

door

Ana Maria Monteiro Ferreira

geboren op 4 november 1980 te Amarante, Portugal

3

Promotores: Prof. dr. R.M.W. Hofstra Prof. dr. R. Seruca Copromotores: Dr. H. Westers Dr. R.H. Sijmons Beoordelingscommissie: Prof. dr. M.J.E. Mourits Prof. dr. H. Morreau

Prof. dr. J. Lubiński

4

Recomeça… Se puderes

Sem angústia

E sem pressa.

E os passos que deres,

Nesse caminho duro

Do futuro

Dá-os em liberdade.

Enquanto não alcances

Não descanses.

De nenhum fruto queiras só metade.

E, nunca saciado,

Vai colhendo ilusões sucessivas no pomar.

Sempre a sonhar

E vendo,

Acordado

O logro da aventura.

És homem, não te esqueças!

Só é tua a loucura

Onde, com lucidez, te reconheças.

Miguel Torga

5

Paranimfen: Maria Alves

Mateusz Siedliński

6

CONTENTS

Chapter 1: Introduction 9

General background 10

Aim and outline of the thesis 19

Chapter 2: Mononucleotide precedes dinucleotide instability during

colorectal tumour development in Lynch syndrome patients 25

Chapter 3: Do microsatellite instability profiles really differ between

colorectal and endometrial tumours? 43

Chapter 4: The hunt for new target genes in endometrial tumors

reveals the involvement of the estrogen-receptor pathway in

microsatellite unstable cancers 57

Chapter 5: Estrogens, MSI and Lynch syndrome-associated tumors 77

Chapter 6: General discussion, conclusions and future perspectives 97

Chapter 7: Summary 107

Nederlandse samenvatting 111

Resumo 114

Streszczenie 117

Acknowledgments 121

Curriculum Vitae 125

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8

CHAPTER 1

Introduction

9

GENERAL BACKGROUND

1.1. Lynch syndrome

Clinical definition Lynch syndrome is an autosomal dominant inherited cancer-susceptibility

syndrome. It is named after Dr. Henry Lynch, whose role was crucial in the clinical

and scientific identification of the syndrome as an inherited and relatively frequent

cause of colorectal and extra-colonic cancer. Lynch syndrome is also known as

hereditary nonpolyposis colorectal cancer (HNPCC), a rather misleading name, as

several cancers other than colorectal also belong to the disease spectrum. Lynch

syndrome is the most common hereditary cause of colorectal cancer.

Long before the genetic mechanism underlying the disease was known,

several major clinical features were described, and a first attempt to define a

uniform set of minimal criteria for clinical diagnosis of Lynch syndrome based on

family history was made in 1990, in a meeting of the International Collaborative

Group on HNPCC (ICG-HNPCC), in Amsterdam (Vasen et al., 1991). These

became known as the Amsterdam criteria (I). With time, and according to the new

findings in the field, especially those related to the genetic basis of the disease,

several refinements to this set of criteria were suggested, such as the Japanese,

Mount Sinai, and Bethesda criteria (Fujita et al, 1996; Peltomaki et al., 2004; Umar

et al., 2004). In 1999, the ICG-HNPCC proposed a new definition for

HNPCC/Lynch syndrome and, with it, the revised Amsterdam criteria (II) (Vasen,

1999). (See Box.1)

Genetics of Lynch syndrome The start of unravelling the genetic cause of Lynch syndrome was in 1993, when

two major findings came together. One was the report of genetic instability

associated with replication errors in microsatellite sequences in a large percentage

of tumours from Lynch syndrome patients (Aaltonen et al., 1993; Ionov et al., 1993;

Peltomaki et al., 1993a; Thibodeau et al., 1993). The other was the identification of

two Lynch syndrome loci by linkage analysis, at chromosomes 2p and 3p

(Lindblom et al., 1993; Peltomaki et al., 1993b).

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Box 1. Details of the Amsterdam criteria for identifying Lynch syndrome families and the definition

of the syndrome, by the 1999 International Collaborative Group on HNPCC (ICG-HNPCC) (Vasen

et al., 1999).

Amsterdam criteria II

At least three relatives should have histologically verified colorectal cancer, cancer of the

endometrium, small bowel, ureter, or renal pelvis;

One of them should be a first-degree relative of the other two;

Familial adenomatous polyposis (FAP) should be excluded;

At least two successive generations should be affected;

In one of the relatives colorectal cancer should be diagnosed before 50 years of age.

ICG-HNPCC definition of HNPCC/Lynch syndrome Familial clustering of colorectal and/or endometrial cancer;

Associated extra-colonic cancers: cancer of the stomach, ovary, ureter/renal pelvis, brain,

small bowel, hepatobiliary tract, and skin (sebaceous tumours);

Development of cancer at an early age;

Development of multiple cancers;

Features of colorectal cancer: (1) predilection for proximal colon; (2) improved survival; (3)

multiple primary (synchronous/metachronous) colorectal cancers; (4) increased proportion of

mucinous tumours, poorly differentiated tumours, and tumours with marked host-lymphocytic

infiltration and lymphoid aggregation at the tumour margin;

Features of colorectal adenoma: (1) the numbers vary from one to a few; (2) increased

proportion of adenomas with a villous growth pattern and (3) probably rapid progression from

adenoma to carcinoma;

High frequency of MSI (MSI-H);

Immunohistochemistry: loss of hMLH1, hMSH2, or hMSH6 protein expression;

Germline mutation in MMR genes (hMSH2, hMLH1, hMSH3, hMSH6, hPMS1,hPMS2).

During 1994, the first germline mutations were found in two genes identified in

those loci (MSH2 and MLH1), both being human homologues of the well-known

mutS and mutL mismatch repair (MMR) genes of bacteria and yeast. Thus,

deficient DNA mismatch repair was identified as the cause of Lynch syndrome.

This functional inactivation of the DNA MMR genes is due to germline mutations as

the first hit (Fishel et al., 1993; Leach et al.,1993; Bronner et al., 1994;

Papadopoulos et al., 1994), followed by somatic inactivation of the second allele as

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the second hit (Hemminki et al., 1994; Lu et al., 1996). This second hit is usually a

somatic mutation or loss of heterozygosity (LOH).

Germline mutations in the MLH1 and MSH2 genes form the vast majority of

mutations found in Lynch syndrome cases (Peltomaki et al., 2004). Two other

MMR genes – MSH6 and PMS2 – were later reported as being involved in the

disease as well, since germline mutations are also found in a fraction of Lynch

syndrome families (Berends et al., 2002; Hendriks et al., 2006). Germline deletion

of the 3' exons of TACSTD1 can cause heritable somatic methylation and

inactivation of the neighbouring MSH2 gene and thus Lynch syndrome (Kovacs et

al., 2009; Ligtenberg et al., 2009). Also an interstitial deletion at 3p21.3 resulting in

the genetic fusion of MLH1 and ITGA9 has been recently reported in a Lynch

syndrome family, presumably defining a novel subclass of Lynch syndrome

patients (Meyer et al., 2009). Several other genes, such as MLH3 and EXO1, also

belong to the MMR pathway and these were therefore screened over the years as

well. Germline mutations in MLH3 and EXO1 have been found (Wu et al.,

2001a&b), but due to their low frequencies and type, mostly missense, they are not

considered to be major players in Lynch syndrome (Hienonen et al., 2003;

Jagmohan-Changur et al., 2003; Ou et al., 2008).

1.2. Mismatch repair and microsatellite instability

The MMR system is responsible for correcting errors that escape the activity of the

polymerases during DNA replication. The system is able to correct mispaired

nucleotides, as well as insertions and deletions loops (IDLs) that typically occur at

short DNA tandem repeats - microsatellites. Therefore, when an MMR protein is

inactivated, mutations will accumulate in those repeat sequences at a much higher

rate (100- to 1000-fold) than that of spontaneous mutations in normal cells (Shibata

et al., 1994). This phenomenon is referred to as “microsatellite instability” (MSI)

(Ionov et al., 1993). It is easily recognized by decreased or increased lengths of the

microsatellite, and therefore the detection of MSI became a key technique when

searching for MMR-deficient tumours.

MSI was reported in Lynch syndrome patients in 1993, occurring in over 90%

of tumours in those patients, and it was another important piece of the puzzle

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linking MMR deficiency and Lynch syndrome (Aaltonen et al., 1993; Ionov et al.,

1993; Peltomaki et al., 1993a; Thibodeau et al., 1993). However, it is also found in

a large proportion (15-25%) of sporadic tumours, not only of colorectal origin, but

also in gastric and endometrial carcinomas (Boland et al., 1998). The underlying

mechanism characterizing the sporadic forms of MSI tumours is also the functional

inactivation of an MMR gene, namely MLH1, but in this case the bi-allelic

inactivation of the gene is typically due to somatic promoter hypermethylation. MSI

is a very early event in the tumorigenic process of tumours with MMR problems, as

it has been detected in early lesions such as colorectal adenomas (Giuffrè et al.,

2005).

1.3. Adenoma-carcinoma sequence

The adenoma-carcinoma sequence of colorectal cancer represents one of the

best-known models of cancer development. Colorectal carcinomas arise through a

multistep process, starting from early to high-grade dysplastic adenomas to

carcinomas. This process of cancer development is basically caused by the

progressive accumulation of genetic alterations in genes involved in cell growth,

differentiation, proliferation, and apoptosis (Fearon & Vogelstein, 1990).

This accumulation of genetic alterations is thought to be due to genetic

instability, in which several distinct forms can be distinguished. Those that are best-

described are chromosomal instability (CIN) and microsatellite instability (MIN or

MSI) (Royrvik et al., 2007). CIN is characterized by widespread chromosomal

abnormalities such as aneuploidy and frequent loss of heterozygosity (LOH). MSI

is caused by defects in the DNA mismatch repair (MMR) pathway, and is

characterized by the accumulation of mutations in microsatellites (see above).

MSI is found in the very early stages of the adenoma carcinoma sequence,

although generally in lower frequencies than in carcinomas. It is reported in about

1-2% of sporadic adenomas (Young et al., 1993; Iino et al., 1999; Loukola et al.,

1999; Sugai et al., 2003) and in 10-90% of Lynch syndrome-associated adenomas

(Aaltonen et al., 1994; Iino et al., 2000; Giuffrè et al., 2005). This wide range of MSI

frequencies might be explained by the method of dissection used (laser

microdissection vs. manual dissection) and it is related to the multi-clonality of the

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tissue, i.e. different areas show different degrees of MSI and different degrees of

dysplasia (de Wind et al., 1998; Iino et al., 1999, 2000; Giuffrè et al. 2005;

Greenspan et al., 2007). In addition, there may be considerable variation in the

methods used to score MSI by different laboratories and between different

observers.

1.4. MSI detection

An international consensus panel of five microsatellite markers for detecting MSI

was proposed in 1997 (Boland et al., 1998) to facilitate the production of easily

comparable results, and this has become widely used. The panel includes two

mononucleotide markers (BAT-25 and BAT-26) and three dinucleotide markers

(D2S123, D5S346 and D17S250). Samples that are unstable for two or more of

these markers are designated MSI-high (MSI-H), while samples unstable for one

marker are MSI-low (MSI-L); samples that are stable for all the markers are

designated microsatellite stable (MSS). If it is necessary to distinguish between

MSI-L and MSS, then additional markers should be used (Boland et al., 1998). It is,

in fact, common that some labs use a different number of markers. In that case, a

sample is MSI-H if it is unstable for more than 30% of the markers used. More

recently, a pentaplex PCR assay for 5 mononucleotide markers was proposed

(Buhard et al., 2004). It includes the following markers: BAT-25, BAT-26, NR-21,

NR-22 and NR-24. The authors claim a sensitivity and specificity of 100%, and

suggest that the use of quasi-monomorphic mononucleotide repeats over

dinucleotide repeats is advantageous, as the latter are typically polymorphic and

more difficult to interpret. It is also believed that there is a greater sensitivity of

dinucleotides for MSI-L cases than for MSI-H cases (Hatch et al., 2005). In addition,

the use of mononucleotide markers might avoid needing normal tissue for

comparison in CRC cases. In fact, it has also been proposed that BAT-26 alone

and without normal matching mucosa might be sufficient for detecting MSI-H CRC

(Hoang et al., 1997; de la Chapelle, 1999). The above-mentioned pentaplex panel

has also been advised for endometrial carcinomas, although normal matching

mucosa DNA is in that case still recommended (Wong et al., 2006).

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1.5. Target genes and tissue selection

Microsatellites are short DNA tandem repeat sequences spread throughout the

genome, including non-coding and coding regions of genes. When MSI occurs in

high frequencies in a coding sequence of a gene with important regulatory

functions (involved in processes like apoptosis or proliferation for example), it is

believed that such a gene, when impaired, contributes to development of cancer.

These genes are generally called “target genes”. This is a rather simplistic

definition; however, it has been controversial to agree on the criteria to define a

real target gene (Woerner et al., 2001, 2003; Duval & Hamelin, 2002; Perucho,

2003). Due to the general lack of functional studies proving the true involvement of

target genes in tumour development, these genes are generally classified as such

based on a high mutation frequency. One major problem is the establishment of a

valid cut-off value for the mutation frequency to separate real target genes from

passengers or bystanders (those having the background mutations expected in an

MMR-deficient context but not related with the progression to cancer). In 2002,

Duval et al. proposed a cut-off frequency value of 10-15% and this has been used

by other groups (Vilkki et al., 2002).

A number of target genes have been identified in MMR-deficient tumours,

and these are thought to be key players in MSI-H tumorigenesis. Mutations were

mostly searched for in MSI-H colorectal tumours, although now many of the genes

have been screened in endometrial and gastric tumours as well (Duval et al., 1999;

Schwartz et al., 1999; Duval et al., 2001; Vilkki et al., 2002; Royrvik et al., 2007).

From several studies it became clear that there are target genes, such as BAX,

commonly involved in MSI-H tumours of diverse origin, whereas others show

considerable qualitative and quantitative differences between different tumour

types, probably due to tissue-specific selection (Myeroff et al., 1995; Duval et al.,

1999; Gurin et al., 1999; Schwartz et al., 1999; Semba et al., 2000; Duval et al.,

2002a). It is also clear from these studies that more important genes remain to be

found, especially in endometrial cancer. These tumours are subjected to less

screening than colorectal ones, and only a few genes with a high mutation

frequency have been found in them.

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1.6. Endometrium

1.6.1. Histology and functional changes The uterus is a hollow muscular, pear-shaped organ weighing 40-80 gram in a

nonpregnant woman. The size of the uterus is highly variable as is demonstrated

during pregnancy. There are two parts to the uterus: the main body, known as the

corpus, and the lower part, which opens into the vagina, called the cervix. The wall

of the uterus consists of three layers: different types of mucosa at the inner side; a

thick muscular, highly vascularised part; and a thin layer of serosa covering the

intraperitoneal part of the corpus. The cervical canal is covered by a single layer of

clindrical mucus secreting cells which extends into the underliying myocervix

forming endocervical crypts. The inner lining of the corpus is called endometrium.

The endometrium consists of a supportive stroma and an epithelial component the

endometrial glands. The thickness and differentiation of the functional layer of the

endometrium is highly regulated by the hormonal changes occurring during the

menstrual cycle. The endometrial mucosa can be sub-divided in two areas related

to those changes: a functional layer, adjacent to the cavity of the uterus, that is

sloughed during menstruation and built up afterwards, and a basal inert layer,

adjacent to the myometrium, that is not shed during the menstrual cycle and that

functions as a regenerative zone for the functional layer. After menopause, in a low

estrogenic situation, the endometrium consists of the basal layer only.

1.6.2. Endometrial cancer

Aetiology Endometrial cancer (EC) is one of the most common types of gynaecological

cancer in women worldwide. The highest incidence is found in North America,

although the highest levels of mortality are in Eastern Europe. The incidence of

endometrial cancer increases after menopause; approximately 75% of cases are

diagnosed in postmenopausal women (Cancer Research UK website).

The major risk factor for endometrial cancer is the high, unopposed exposure

to oestrogens (Sherman, 2000; Amant et al., 2005). Therefore, conditions

16

increasing the oestrogen levels are considered to increase the disease risk. These

include for instance: early menarche, late menopause, nulliparity or low parity, and

hormone replacement therapy (HRT) with exogenous oestrogen but without

progesterone. Long-term use of tamoxifen, a drug used to treat breast cancer, also

increases the risk for endometrial cancer (Polin & Ascher, 2008). Other risk factors

for the disease include: a high-fat diet, obesity, hypertension, diabetes, age (more

common after age of 50), personal history of breast, colorectal, or ovarian cancer

and a family history of endometrial cancer or colon cancer (Lynch syndrome).

Endometrial cancer risk has also been suggested to be increased in Cowden

syndrome, caused by germline PTEN mutations. The use of oral combined

contraceptives, on the other hand, is reported to reduce the risk of EC.

Histopathological and molecular types of endometrial carcinomas Endometrial carcinomas are usually divided into two major groups that have

different clinical and histological characteristics, as well as molecular differences

(Emons et al., 2000; Lax et al., 2004; Ryan et al., 2005).

Type I or oestrogen-dependent endometrioid carcinomas (EEC)

Representing 80% of sporadic cases, this is the group of oestrogen-related

tumours. They occur in both pre- and post-menopausal women, and their

architectural features resemble endometrial glands. Tumours of this type are

usually well differentiated (low grade) and confined to the uterus (low stage) and

therefore the patient generally has a good prognosis. They are frequently preceded

by endometrial hyperplasia.

Type II or non-oestrogen-dependent ECs

The tumours belonging to this group are unrelated to oestrogenic stimulation, and

mainly occur in post-menopausal women. They display a more aggressive

behaviour and poor prognosis. Frequently, by the time of diagnosis, the tumour has

already spread outside the uterus. They are not usually preceded by hyperplasia,

but originate from an atrophic endometrium instead. They are high-grade tumours

with serous or clear-cell morphology.

17

In addition to the histopathological differences referred to above, there are also

genetic differences between these two categories of endometrial carcinomas (Doll

et al., 2007). In type I EC we can basically find mutations in PTEN (35-50%), K-Ras

(15-30%) and β-catenin (25-40%) genes, and MMR defects, detected by high

levels of MSI (20-40%). These characteristics are rarely seen in type II EC, which

are characterized by high mutation frequencies on P53 gene (90%) and alterations

on HER2/NEU and CDH1. The main type of genetic instability in this group is

chromosomal instability (CIN), being aneuploidy and loss-of-heterozygosity (LOH)

typical of EC type II. MSI (MIN) is extremely rare in these tumours (Emons et al.,

2000; Lax et al., 2004; Ryan et al., 2005). Figure 1 shows the progression model of

endometrial cancer proposed by Ryan et al. (2005).

Normal

Endometrial hyperplasia

Endometrial intraepithelial

carcinoma

Type I endometrioid adenocarcinoma

Type II serous adenocarcinoma

p53

PTEN

MLH1

Hypermethylation

MSI Mutations (e.g. KRAS, BAX, MSH2)

P53, LOH, HER2/NEU

HER2/NEU

Figure 1. Progression model of endometrial cancers type I and type II progression

adapted from Ryan et al. (2005).

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AIM AND OUTLINE OF THE THESIS

The main focus of this thesis was to understand the development of tumours that

follow the MSI pathway. The study covered Lynch syndrome-associated tumours,

with particular emphasis on colorectal and endometrial carcinomas and their

sporadic counterparts.

Chapter 1 reviews the general background to Lynch syndrome, microsatellite

instability, and the hereditary and sporadic cancers associated with this pathway.

Chapter 2 addresses how instability evolves along the adenoma-carcinoma

sequence of colorectal cancer, and whether we are able to establish different

profiles of MSI for hereditary and sporadic adenomas and carcinomas. Knowledge

on this process might be helpful in understanding tumour development and in

identifying Lynch syndrome patients in an easier and more specific way.

In chapter 3, we report on our comparison of the frequencies of instability of

different types of microsatellites between colorectal and endometrial MSI-H

tumours. In addition, we analyze features such as type (deletions/insertions) and

size of microsatellite mutation for possible correlations with tissue specificity.

Chapter 4 describes our hunt for new genes involved in MSI-H endometrial

tumours. It addresses the instability of mononucleotide repeats occurring in coding

sequences. The aim of this work was to identify novel target genes that could

explain MSI-H endometrial tumour development, and to unravel molecular

pathways related to this type of cancer. We further wanted to determine whether

the identified genes were also involved in colorectal and gastric tumours, and we

speculate about the functional role of the proteins that are encoded by the genes

we found mutated.

In chapter 5 we review and try to clarify the mechanisms linking hormones to

cancer, and in particular how hormones can play a role in MSI tumorigenesis.

Finally, in chapter 6, the major findings of this project are discussed,

conclusions are drawn and future perspectives are formulated.

19

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24

http://info.cancerresearchuk.org/cancerstats/types/uterus/incidence/

CHAPTER 2

Mononucleotide Precedes Dinucleotide Instability during Colorectal Tumour Development in Lynch

Syndrome Patients

Ana M. Ferreira1, 4, Helga Westers1, Sónia Sousa4, Ying Wu1, Renée C. Niessen1,

Maran Olderode-Berends1, Tineke van der Sluis2, Peter T.W. Reuvekamp1, Raquel

Seruca4, Jan H. Kleibeuker3, Harry Hollema2, Rolf H. Sijmons1, Robert M.W.

Hofstra1

Departments of 1Genetics, 2Pathology, 3Gastroenterology, University Medical Center

Groningen, University of Groningen, Groningen, The Netherlands. 4Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.

Under review

25

ABSTRACT A progressive accumulation of genetic alterations underlies the adenoma-carcinoma sequence of colorectal cancer. This accumulation of mutations is driven by genetic instability, of which there are different types. Microsatellite instability (MSI) is the predominant type present in the tumours of Lynch syndrome patients and in a subset of sporadic tumours. It is generally accepted that MSI can be found in the early stages of tumour progression, such as adenomas; however, the frequencies reported vary widely among studies. Moreover, data on the qualitative differences between adenomas and carcinomas, or between tumours of hereditary and sporadic origin, are scarce. We compared MSI in colorectal adenoma- and colorectal carcinoma samples in order to identify possible differences along the adenoma-carcinoma sequence. We compared germline mismatch repair (MMR) gene mutation carriers and non-carriers, to address possible differences of instability patterns between Lynch syndrome patients and patients with sporadic tumours. We found a comparable relative frequency of mono- and dinucleotide instability in sporadic colorectal adenomas and carcinomas, dinucleotide instability being observed most frequently in these sporadic tumours. In MMR gene truncating mutation carriers, the profile is different: colorectal adenomas show predominantly mononucleotide instability and also in colorectal carcinomas more mononucleotide than dinucleotide instability was detected. We conclude that MSI profiles differ between sporadic and Lynch syndrome tumours, and that mononucleotide marker instability precedes dinucleotide marker instability during colorectal tumour development in Lynch syndrome patients. As mononucleotide MSI proves to be highly sensitive for detecting mutation carriers, we propose the use of mononucleotide markers for the identification of possible Lynch syndrome patients.

26

INTRODUCTION The adenoma-carcinoma sequence of colorectal cancer is the best-known model of

cancer development. Colorectal carcinomas arise through a multi-step process,

starting from early adenomas to high-grade dysplastic adenomas to carcinomas.

This process of cancer development is basically caused by the progressive

accumulation of genetic alterations in genes involved in cell growth, differentiation,

proliferation, and apoptosis (Fearon et al., 1990). This accumulation of genetic

alterations is thought to be driven by genetic instability, of which several distinct

forms can be distinguished. Those that are best-described are chromosomal

instability (CIN) and microsatellite instability (MIN) (Komarova et al., 2002). CIN is

characterized by widespread chromosomal abnormalities, such as aneuploidy and

frequent loss–of-heterozygosity (LOH). MIN is characterized by the accumulation of

mutations in short repetitive sequences, known as microsatellites. The underlying

mechanism of microsatellite instability is a defect in the DNA mismatch repair

(MMR) pathway. The MMR pathway corrects replication errors, such as mispaired

nucleotides, as well as small insertions and deletions resulting from slippage of the

polymerases during replication of microsatellites. MMR deficiency therefore leads

to an accumulation of mutations in microsatellites and it is recognized by

decreased or (less often) increased microsatellite lengths. This phenomenon is

referred to as microsatellite instability (MSI) (Ionov et al., 1993); it was first

described in Lynch syndrome patients (Aaltonen et al., 1993; Ionov et al., 1993;

Peltomaki et al., 1993; Thibodeau et al., 1993) and detected in over 90% of

tumours in those patients. It is also found in a large proportion (15-25%) of

sporadic colorectal (CRC) and endometrial (EC) carcinomas (Boland et al., 1998).

It was shown that the functional inactivation of the MMR pathway by a germline

mutation in one of the MMR genes and, in addition, somatic inactivation of the

corresponding wild-type allele causes Lynch syndrome (Fishel et al., 1993; Leach

et al., 1993; Bronner et al. 1994; Papadopoulos et al., 1994).

The occurrence of MSI is not only reported in colorectal carcinomas but also

in colorectal adenomas (AD) although generally in lower frequencies, in

approximately 1-2% of sporadic AD (Young et al., 1993; Iino et al., 1999; Loukola

27

et al., 1999; Sugai et al., 2003), and in 10-90% of Lynch syndrome-associated AD

(Aaltonen et al., 1994; Iino et al., 2000; Giuffrè et al., 2005).

Previous studies comparing patterns of MSI in different tumour types and

stages suggest that tumours with different tissue origin or in different stages of

tumorigenesis show different levels of instability (Furlan et al., 2002; Kuismanen et

al., 2002). However, these studies generally refer to quantitative differences in

(mononucleotide) instability between tumour types. Data on qualitative differences

are scarce. In this study we have analyzed mononucleotide and dinucleotide

markers to define specific qualitative profiles of MSI in colorectal adenomas and

carcinomas of Lynch syndrome patients and of sporadic cases.

MATERIALS AND METHODS

Samples The participants in this study came from two sources: either known MMR gene

mutation carriers under surveillance at our Hospital, or participants from previous

studies in our group who had been diagnosed with CRC under the age of 50 years

or had two or more Lynch syndrome-related cancers, including at least one CRC,

irrespective of age and family history. This study was approved by the local

medical ethical committee. All patients had been analyzed for germline mutations

in the MLH1, MSH2, and MSH6 genes (for details see Rijcken et al., 2002; Niessen

et al., 2006). In total we included paraffin-embedded tumour tissue sections and

the respective normal tissue/blood from 67 colon adenomas (AD) and 213 colon

carcinomas (CRC). Twenty-two of the AD were classified as low-grade dysplasia

and 20 as high-grade dysplasia; information on the grade of dysplasia was not

available for the other 25 AD. Thirty-two out of the 67 AD and 20 out of the 213

CRC were from patients who carried a pathogenic germline mutation in one of the

three MMR genes (MLH1, MSH2, and MSH6). These patients are referred to as

truncating mutation carriers. This group includes 7 pairs of adenoma and

carcinoma samples of the same patient. In the CRC group, in addition to the 20

pathogenic mutation carriers, 12 patients carry missense mutations, all of unknown

pathological significance.

28

MSI Analysis MSI analysis was performed using a panel of three mononucleotide markers

(BAT25, BAT26, BAT40) and three dinucleotide markers (D2S123, D5S346,

D17S250) as described previously (Berends et al., 2002). For the AD and the

AD/CRC pairs from the same patient, 3 additional mononucleotide markers were

analyzed (NR27, NR21, NR24). DNA was extracted from formalin-fixed paraffin-

embedded tumour sections and compared with DNA isolated from normal tissue

from paraffin-embedded sections (when available) or peripheral blood lymphocytes

from the same patient, as described previously (Berends et al., 2002). The samples

were classified as MSI-High when more than 30% of the markers analyzed were

unstable.

Statistical Analysis

The statistical analyses were performed using the 2 test or Fisher’s exact test. P

values <0.05 were considered to be significant.

RESULTS Frequencies of instability – truncating mutation carriers show the highest MSI frequency Frequencies of MSI-H, MSI-L and MSS samples distributed by tumour type and

presence/absence of MMR mutations are shown in Table 1. As expected, the

samples from the truncating mutation carriers show higher frequencies of MSI-H

and lower frequencies of MSS/MSI-L than the samples from the non-carriers.

Among the AD a significantly smaller proportion exhibit MSI-H (6% of the non-

carriers; 56% of the truncating mutation carriers) compared to the CRC (36% of the

non-carriers; 90% of the truncating mutation carriers) (P< 0.05). The missense

mutation carriers show instability frequencies very similar to the non-carriers.

29

Table 1. Distribution of the samples by presence or absence of germline MMR mutations, tumour tissue

type (adenoma/carcinoma) and MSI status.

Non-carriers Mutation carriers Total (N)

Truncating Missense

AD CRC AD CRC CRC

(35) (181) (32) (20) (12) (280)

MSI-H 6% (2) 36% (65) 56% (18) 90% (18) 33% (4) 107

MSI-L 46% (16) 28% (51) 9% ( 3) 10% ( 2) 33% (4) 76

MSS 49% (17) 36% (65) 34% (11) 0% (0) 33% (4) 97

AD colon adenomas; CRC colorectal cancer; MSI-H microsatellite instability high; MSI-L microsatellite

instability low; MSS microsatellite stable.

Figure 1. Frequencies of instable mono- and dinucleotide markers in MSI-L and MSI-H samples from

CA and CRC. * significant.

Higher mono- and dinucleotide instability in MSI-H tumours compared to MSI-L tumours

Figure 1 shows the observed frequencies of unstable mono- and dinucleotide

markers in AD and CRC, in both MSI-L and MSI-H cases, without stratification of

samples into mutation carriers or non-carriers. For the carcinomas, both mono- and

30

dinucleotide markers are more unstable in the MSI-H group than in the MSI-L

group, and instability at the dinucleotide level was significantly more frequently

observed than mononucleotide instability. In AD, in MSI-L samples dinucleotide

instability was also significantly higher than mononucleotide (36% vs. 5%, p<0.05),

however in the MSI-H group mononucleotide was markedly increased and

significantly more frequent than dinucleotide instability (85% vs. 43%, p value

<0.05).

Repeat instability depends on MMR mutations Stratifying the samples for the presence/absence of germline mutations in one of

the MMR genes MLH1, MSH2 and MSH6, the profiles of instability obtained were

in the CRC set quite different from those described above (compare Figure 1 and

2).

Figure 2. Frequencies of instable mono- and dinucleotide markers in MSI-L and MSI-H of non-carriers,

missense mutation carriers, and truncating mutation carriers, for colorectal carcinoma samples. NS not

significant; * significant.

When comparing truncating mutation carriers and non-carriers in the MSI-H

CRC group (Figure 2) several significant differences were observed: the non-

carriers show significantly more dinucleotide instability (66%) than mononucleotide

31

instability (49%); truncating mutation carriers had more mononucleotide instability

than non-carriers (78% vs. 49%, p<0.05), but similar frequencies of dinucleotide

instability (67% vs. 66%); CRC MSI-L samples from non-carriers showed

significantly more dinucleotide instability, while the MSI-L samples of truncating

mutation carriers had similar frequencies of mono- and dinucleotide instability.

The MSI-H cancers of carriers of MMR missense mutations retained

preferential dinucleotide instability over mononucleotide instability, resembling

more the pattern seen for the non-carriers, but the sample size was too small to

make a clear statement.

In AD, the following observations were made: dinucleotide instability is seen

in both non-carriers and mutation carriers groups at similar frequencies, whereas

mononucleotide instability was by far more frequently present in mutation carriers

(p<0.05) (Table 2).

When comparing low-grade (LD) with high-grade dysplastic (HD) adenomas

from mutations carriers, we detected MSI-H in 38% (5/13) of the LD adenomas and

in 67% (6/9) of the HD adenomas. In LD samples of mutation carriers, instability

was found only in adenomas from MLH1 mutation carriers, whereas in HD

samples, instability was observed in adenomas from all three types of mutation

carriers (MLH1, MSH2, MSH6). Moreover, LD adenomas from mutation carriers

showed mainly mononucleotide instability, whereas HD adenomas showed both

mono- and dinucleotide instability (Table 2).

Pairs adenoma and carcinoma from the same patient We also had 7 patients from whom we could obtain both an AD and a CRC. We

observed both mono- and dinucleotide instability at high frequency.

Mononucleotide instability was seen more frequent than dinucleotide instability (but

not significantly different) (Table 3).

32

Table 2. Instability results for colon adenomas.

A) Mononucleotide

markers Dinucleotide

markers

Patie

nt ID

Gra

de

MM

R

mut

atio

n

MSI

Sta

tus

NR

27

NR

21

NR

24

BA

T25

BA

T26

BA

T40

D2S

123

D5S

346

D17

S250

Y313 ND

Y239 LD MSI-

H

Y72 ND

Y65 LD

Y71 ND

Y190 HD

Y264 ND

Y200 HD

Y81 HD

Y210 HD

Y234 HD

Y89 ND

Y123.1 LD

Y192 LD Y123.2 LD

Y79 HD Y301 ND

Y176-2 ND

MSI

-L

Y13 LD

Y39 ND

Y56 LD

Y167 LD

Y170 HD

Y225 HD

Y257 ND

Y267 LD

Y202 HD

Y223 HD

Y268 HD

Y309 ND

Y311 ND

Y317 ND

Y327 ND

Y331 ND

Y294 ND

NO

N-C

AR

RIE

RS

MSS

A) adenomas from non-carriers; B) adenomas from mutation carriers

LD low-grade dysplasia; HD high-grade dysplasia; ND no data available.

Results in black mean unstable; grey mean stable; white no result available.

33

B)

Mononucleotide


markers

Patie

nt

ID

Gra

de

MM

R

mut

atio

n

MSI

St

atus

NR

27

NR

21

NR

24

BA

T25

BA

T26

BA

T40

D2S

123

D5S

346

D17

S250

2T LD

8T LD

9T LD

17T HD

13T LD

16T HD

3T HD

18T ND

10T LD

1 ND MSI-H

5T LD

29T LD MSI-L

21T LD

25T ND

5 ND

6 ND

ML

H1

MSS

14T HD

6T HD

Y21 ND

2 ND

3 ND

4 ND

7 ND MSI-H

Y112 HD

4T LD

20T LD

MSH

2

MSS

Y241 HD MSI-H

26T HD MSI-L

7T LD

12T LD

Y86 HD

1T LD

MSH

6

MSS

34

Table 3. MSI results for adenoma and carcinoma of the same patient.

Mononucleotide


markers

D17

S250

NR

27

NR

21

NR

24

BA

T25

BA

T26

D2S

123

D5S

346

1 AD MLH1 CRC MLH1 2 AD MSH2 CRC MSH2 3 AD MSH2 CRC MSH2 4 AD MSH2 CRC MSH2 5 AD MLH1 CRC MLH1 6 AD MLH1 CRC MLH1 7 AD MSH2 CRC MSH2

AD colorectal adenoma; CRC colorectal carcinoma; black means unstable; grey means stable; white no

result available.

DISCUSSION

In the present study, we compared MSI in colorectal adenomas (AD) and colorectal

carcinomas (CRC) in order to identify possible differences along the adenoma-

carcinoma sequence. We compared germline MMR mutation carriers and non-

carriers, to address possible differences of instability patterns between Lynch

syndrome patients and patients with sporadic tumours.

The CA in our study showed a significantly lower proportion of MSI-H cases

than CRC, both in non-carriers (6% vs. 36%, p<0.05) and in truncating mutation

carriers (56% vs. 90%, p<0.05) (no adenomas were available from missense

mutation carriers). Our study confirms the reported difference in MSI frequencies

during the transition from adenoma to carcinoma (Shibata et al., 1994; Grady et al.,

1998; Loukola et al., 1999; Iino et al., 2000; Sugai et al., 2003; Giuffrè et al., 2005).

We further analyzed how instability is distributed amongst mononucleotide

versus dinucleotide markers, in both MSI-L and MSI-H groups of AD and CRC

35

samples. When no distinction is made between mutation carriers and non-carriers,

the distribution of instability is similar between MSI-L and MSI-H samples, in CRC:

dinucleotide markers are more frequently unstable than mononucleotide markers.

In AD, unstable dinucleotide markers are also more frequent than mononucleotide

markers in MSI-L tumours, but in MSI-H the opposite situation is found.

The results are, however, different when we split the CRC samples into

different groups: truncating mutation carriers, missense mutation carriers and non-

carriers. We observed that the high frequency of dinucleotide instability in MSI-H

tumours is due to the inclusion of non-carriers and missense mutation carriers, and

that the mononucleotide instability is mainly due to the inclusion of mutation

carriers.

In AD from non-carriers mainly the dinucleotide markers were unstable; a

very low frequency of mononucleotide instability was seen, resembling the MSI-L

CRC of non-carriers. In the AD of truncating mutation carriers, mononucleotide

instability was generally predominant. As more mono- to dinucleotide instability is

observed, our data suggest that mononucleotide instability is a very early event in

the carcinogenic process of tumours having mismatch repair mutations, and that

mononucleotide instability precedes that of dinucleotide repeats. We also included

7 adenoma/carcinoma pair from the same patient. Again we observe a difference

between mononucleotide instability and dinucleotide instability however this was

not significant due to the small number of cases analysed.

This idea is further supported by our results in low- and high-grade dysplastic

adenomas from mutation carriers. Low-grade adenomas have less MSI and mainly

mononucleotide instability, whereas 67% of the high-grade adenomas were MSI-H,

with the instability found in both mono- and dinucleotide markers.

As far as the observed “preference” of dinucleotide instability in early lesions

(AD) of non-mutation carriers is concerned, we hypothesize that the dinucleotide

instability in these cases represents a kind of background as seen in MSI-L

tumours, which is not a sign of an underlying MMR deficiency. Part of the

dinucleotide instability seen in MSI-H CRC of carriers and non-carriers might

therefore likely also occur independently of MMR deficiency. In the case of Lynch

syndrome tumours, with proven MMR deficiency, mononucleotide instability can be

considered a true result of the underlying MMR deficiency.

36

Our hypothesis is in line with the finding of only dinucleotide instability in a

study of MSI-L CRC using a large number of MSI markers (Laiho et al., 2002). The

same authors suggest that all CRCs would display a MSI-L phenotype if a large

number of markers is used (Laiho et al., 2002).

A possible explanation for our findings might be that the normal MMR system

more easily corrects mismatches in mononucleotides than dinucleotides; this would

mean that part of the dinucleotide instability is ‘background noise’ in tumours.

However, to our knowledge, such a difference in repair outcome has not been

demonstrated. Interestingly, the number of mononucleotide repeats in the entire

genome is higher than the number of dinucleotide ones (Borstnik et al., 2004), and

as more instability is seen in these less frequent dinucleotide repeats in MMR-

proficient tumors, this suggests either a higher vulnerability of dinucleotide repeats

to the occurrence of mismatches and/or a lower capacity of the normal MMR

system to repair them. It is important to keep in mind that, although the number of

mononucleotide repeats in the genome is higher compared to the number of

dinucleotide repeats, they are not always expected to be more unstable than

dinucleotide repeats; the size and base composition of the repeat can have a

strong influence on the degree of instability (Boyer et al., 2002). In addition, one

can also speculate that, depending on the rate of replication of tumour cells,

dinucleotides might be more prone to acquire mutations than mononucleotides.

Another explanation why mononucleotide instability is seen earlier and more

frequently in AD compared to dinucleotide instability in mutation carriers might be

the fact that the instability seen in mononucleotide repeats is almost always due to

deletions of a certain length, and this has consequences for the chance of

detection. As new mutations in an MMR-deficient tumour happen often and in

multiple cells, the tumours should be considered multi-clonal. The different clones

will not obscure detection of mononucleotide instability because of their similar

mutation size. For dinucleotide repeats this is different. The mutations in these

repeats are often different and because of this, the different clones, with differently

sized alleles might make it less easy to detect instability in these repeats. This,

however, does not explain the finding of mostly dinucleotide instability in non

mutations carriers.

37

Implications for diagnostics of HNPCC colorectal adenomas Our results suggest an advantage to using mononucleotide markers for identifying

colorectal adenomas and carcinomas associated with Lynch syndrome, since we

observed that the MSI-H adenomas from mutation carriers predominantly show

mononucleotide instability. Moreover, assuming that mononucleotide instability

precedes dinucleotide instability in adenomas of MMR-truncating mutation carriers,

analyzing mononucleotide markers would make it possible to detect MSI in very

early lesions. To our knowledge, our data are the first to show that the use of a

panel of only mononucleotide markers, as previously recommended for the

detection of MSI-H hereditary CRC (Buhard et al., 2004), should also be used for

the identification of Lynch syndrome patients through the testing of colon

adenomas. Another practical advantage of using a mononucleotide marker panel is

the fact that DNA from corresponding normal tissue is not always necessary (de la

Chapelle, 1999; Buhard et al., 2004).

CONCLUSIONS

We show that mononucleotide instability is a very early event in the development of

MSI tumours with MMR truncating mutations and that in Lynch syndrome

associated tumours mononucleotide instability precedes dinucleotide instability. We

therefore recommend using mononucleotide markers to identify possible Lynch

syndrome patients.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. Hermien de Walle for assistance with the statistical

analyses, Dr. Richard Hamelin for helpful comments, and Jackie Senior for editing

the manuscript.

This work was supported by Fundação para a Ciência e a Tecnologia, Portugal

(SFRH/BD/18832/2004) and by the European Community (FP6-2004-

LIFESCIHEALTH-5, proposal no. 018754).

38

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Iino H, Jass JR, Simms LA, et al. DNA microsatellite instability in hyperplastic polyps, serrated

adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52(1):5-

9.





Komarova NL, Lengauer C, Vogelstein B, et al. Dynamics of genetic instability in sporadic and familial

colorectal cancer. Cancer Biol Ther 2002;1(6):685-92.

Kuismanen SA, Moisio AL, Schweizer P, et al. Endometrial and colorectal tumors from patients with

hereditary nonpolyposis colon cancer display different patterns of microsatellite instability. Am J Pathol

2002;160(6):1953-8.

Laiho P, Launonen V, Lahermo P, et al. Low-level microsatellite instability in most colorectal

carcinomas. Cancer Res 2002;62(4):1166-70.

Leach FS, Nicolaides NC, Papadopoulos N, et al. Mutations of a mutS homolog in hereditary

nonpolyposis colorectal cancer. Cell 1993;75(6):1215-25.



Niessen RC, Berends MJ, Wu Y, et al. Identification of mismatch repair gene mutations in young

patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-

polyposis colorectal cancer. Gut 2006;55(12):1781-8.

Papadopoulos N, Nicolaides NC, Wei YF, et al. Mutation of a mutL homolog in hereditary colon cancer.

Science 1994;263(5153):1625-9.

40


characterize the hereditary non-polyposis colorectal carcinoma syndrome. Cancer Res

1993;53(24):5853-5.

Rijcken FE, Hollema H, Kleibeuker JH. Proximal adenomas in hereditary non-polyposis colorectal

cancer are prone to rapid malignant transformation. Gut 2002;50(3):382-6.

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adenomas. Hum Mutat 1993;2(5):351-4.

41

42

CHAPTER 3

Do Microsatellite Instability Profiles Really Differ Between Colorectal and Endometrial Tumours?

Ana M. Ferreira1, Helga Westers1, Ying Wu1, Renée C. Niessen1, Maran Olderode-

Berends1, Tineke van der Sluis2, Ate G. van der Zee3, Harry Hollema2, Jan H.

Kleibeuker4, Rolf H. Sijmons1, Robert M.W. Hofstra1

Departments of 1Genetics, 2Pathology, 3Gynecology, and 4Gastroenterology, University

Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

Genes Chromosomes and Cancer, in press.

43

ABSTRACT

Microsatellite instability (MSI) occurs in more than 90% of the tumours of Lynch syndrome patients, and in 15-25% of sporadic colorectal (CRC) and endometrial carcinomas (EC). Previous studies comparing EC and CRC using BAT markers showed that the frequency of unstable markers is lower in EC, and that the size of the mutations is smaller in EC. In the present study we analyzed the type (insertions/deletions), size and frequency of mutations occurring at three BAT and three dinucleotide markers in CRC and EC, in order to elucidate whether it is possible to establish different MSI profiles in carcinomas of different tissue origin. We show that mononucleotide markers nearly always become shorter whereas dinucleotide markers can become shorter or longer, in both CRC and EC. We therefore conclude that the type of mutation is a marker-dependent feature rather than tissue-dependent. However, we observed that the size of the deletions/insertions differs between CRC and EC, with EC having shorter alterations. The frequency of mono- and dinucleotide instability found in both tissues is comparable, with mononucleotide and dinucleotide markers being affected at similar rates. We conclude that it is not possible to define clearly different MSI profiles that could distinguish MSI-H in CRC and EC. We propose that the differences observed might indicate different durations of tumour development and/or differences in tissue turnover between colorectal and endometrial epithelium, rather than reflecting truly different MSI profiles. We therefore suggest that the same MSI tests can be used for both tumour types.

44

INTRODUCTION

Microsatellite instability (MSI) is a form of genetic instability caused by defects in

the DNA mismatch repair (MMR) pathway. MSI was first associated with Lynch

syndrome patients in 1993 (Aaltonen et al., 1993; Ionov et al., 1993; Peltomäki et

al., 1993; Thibodeau et al., 1993) and occurs in over 90% of the tumours of these

patients. It is also found in a large proportion (15-25%) of sporadic colorectal

(CRC) and endometrial (EC) carcinomas (Boland et al., 1998).

The MMR pathway corrects mispaired nucleotides as well as small insertions

and deletions, this last group resulting from slippage of the polymerases during

replication of short DNA repeat sequences (microsatellites). When inactivating

mutations occur within the MMR genes, such as MLH1, MSH2, and MSH6, the

MMR pathway becomes deficient and an accumulation of insertions or deletions is

observed in microsatellite sequences (Ionov et al., 1993). This phenomenon is

referred to as microsatellite instability. MSI is therefore easily recognized by

increased or decreased microsatellite lengths. An international consensus panel of

five microsatellite markers was established to facilitate the detection and analysis

of MSI and this panel is widely used (Boland et al., 1998). Samples that are

unstable for two or more of these markers are considered MSI-high (MSI-H);

samples unstable for one marker are called MSI-low (MSI-L); samples stable for all

markers are called microsatellite stable (MSS). When a different number of

markers are used, a sample is MSI-H when it shows instability in more than 30% of

the markers used.

Microsatellite mutations occur both at coding and non-coding repeats. Genes

frequently found mutated in MSI-H tumours (also called target genes) play

important roles in tumour development pathways and show mutations in their

coding microsatellite sequences. The profile of target genes is thought to be

different in CRC and EC, both in quantitative and qualitative ways (Duval et al.,

2002). Previous studies also compared patterns of MSI in sporadic and Lynch

syndrome-associated CRC and EC at the non-coding level, in particular by

analyzing the mononucleotide BAT markers. It was shown that in both sporadic

and Lynch syndrome-associated tumours, the proportion of unstable markers is

45

lower in EC than in CRC, and that the size of the allelic variations is smaller in EC

than in CRC (Furlan et al., 2002; Kuismanen et al., 2002).

These studies thus show quantitative differences in non-coding

mononucleotide instability between the two types of tumours. Data on MSI of

different types of markers and on qualitative differences are, however, scarce. Do

CRC and EC show different ratios of insertions/deletions? Do EC display smaller

insertions/deletions than CRC also in dinucleotide markers? Do CRC and EC have

different “preferences” for specific types of MSI markers, as they have for target

genes? In this study we addressed these questions in order to elucidate whether it

is possible to define different profiles of MSI for tumours with different tissue origin,

such as colorectal and endometrial cancers.


Samples The patients participating in this study were all suspected of having Lynch

syndrome and were selected from other research studies being conducted by our

group (Berends et al., 2002; Niessen et al., 2006). All patients gave their informed

consent for the study. The patients had either been diagnosed with CRC or EC

under the age of 50 years, or had two or more Lynch syndrome-related cancers,

including at least one CRC, irrespective of age and family history. The cases had

been analyzed for germline mutations in the MLH1, MSH2, and MSH6 genes

(Berends et al., 2001; Berends et al., 2003; Niessen et al., 2006). In total we

included paraffin-embedded tumour tissue sections and normal tissue/blood

samples from 194 colon carcinomas (CRC) and 68 endometrial carcinomas (EC).

Thirteen out of the 194 CRC and 7 out of the 68 EC were from patients who carried

a pathogenic germline mutation in one of the three MMR genes (MLH1, MSH2, and

MSH6). These patients are referred to as mutation carriers. Average age of tumour

onset for the different groups of patients is presented in table 2.

46

MSI Analysis MSI analysis was performed by fragment analysis using a panel of three mono-

nucleotide markers (BAT25, BAT26, BAT40) and three dinucleotide markers

(D2S123, D5S346, D17S250) as described previously (Berends et al., 2002). DNA

was extracted from formalin-fixed, paraffin-embedded tumour sections and

compared with DNA isolated from normal tissue from paraffin-embedded sections

(when available) or peripheral blood lymphocytes from the same patient, as

described previously (Berends et al., 2002). The samples were classified as MSI-H

if more than 30% of the markers analyzed were unstable. Only samples with

informative results for four or more of the six MSI markers (independent of the type

of marker) were included in this study. For the MSI-H tumours, type of

microsatellite mutation (deletion/insertion) and the size of these mutations were

analyzed for every marker, and frequencies of instability were calculated. The size

of mutations was measured as the difference between the highest peak in the

normal tumour and the farthest peak in the unstable tumour.

Statistical Analysis For the differences in type of mutations and frequencies of instability, the 2 test or

Fisher`s Exact test were used. P values <0.05 were considered to be significant.

Two-way factorial ANOVA was used for the differences in mutation size between

markers and tumour tissues.

RESULTS Frequency of microsatellite instability is highest in mutation carriers One-hundred and five tumors (40%) were classified as MSI-H and selected for

further analysis. The frequencies of instability were overall as we expected from the

literature, with mutation carriers showing significantly higher frequencies of MSI-H

than non-carriers, both in colorectal tumors and in endometrial tumors (table 1).

MSI-L was found in mutation carriers in the CRC group only, in two cases, both

harboring an MSH6 mutation. In the EC mutation carriers, two MSS cases were

detected; one carried an MSH6 mutation and the other an MSH2 mutation.

47

Table 1. Distribution of the samples by MSI status, presence or absence of germline mismatch repair

mutations, and tumour tissue type

Non-carriers Mutation carriers Total

CRC EC CRC EC N

N 181 61 13 7 262

MSI-H 36% 40% 85% 71% 105

MSI-L 28% 29% 15% 0% 71

MSS 36% 31% 0% 29% 86

CRC colorectal carcinomas; EC endometrial carcinomas; MSI-H microsatellite instability high; MSI-L

microsatellite instability low; MSS microsatellite stable; N, absolute number.

Table 2. Average age of tumor onset of the different groups of patients

Non-carriers Mutation carriers CRC EC CRC EC

MSI-H 48.4 48.3 43.4 48.6 MSI-L+MSS 46.8 46.9 50.0 65.5

CRC colorectal carcinomas; EC endometrial carcinomas; MSI-H microsatellite instability high; MSI-L

microsatellite instability low; MSS microsatellite stable.

Type of microsatellite mutations (insertions/deletions) depends on type of repeat Frequencies of deletions and/or insertions occurring at each microsatellite marker

were calculated for the MSI-H CRC and EC samples. No association between the

prevalence of deletions or insertions and tumour type was found. A strong

correlation between type of markers (mononucleotide vs. dinucleotide markers)

and type of microsatellite mutation (insertion/deletion) was, however, observed.

Mononucleotide markers were almost exclusively targets of deletions (98% in CRC

and 100% in EC), whereas dinucleotide loci showed both deletions and insertions

(Fig. 1). Simultaneous insertions and deletions were also detected in all

dinucleotide markers (Fig. 1).

48

Figure 1. Frequencies of deletions, insertions, and simultaneous deletions and insertions in

mononucleotide (BAT25, BAT26, BAT40) and dinucleotide (D2S123, D5S346, D17S250) MSI markers

in MSI-H colorectal (upper panel) and endometrial (lower panel) carcinomas.

Size of mutations in EC is smaller than in CRC

The size of insertions/deletions was analyzed for each unstable locus of MSI-H

tumours. The difference in size as defined in this study corresponded to the allele

with the maximum length difference from the normal allele (observed in normal

tissue/blood from the same patient). The mutation sizes were determined for both

tumour types (Fig. 2). The mutations were significantly smaller in EC (average

6.02±0.45bp) than in CRC (average 7.67±0.34bp) (ANOVA: F(1,203)=11.25,

49

P<0.001). However, these differences are not statistically significant when

analyzing each marker separately, except for D2S123.

Different markers showed different mutation sizes (ANOVA: F(5,203)=14.67,

P<0.001), but the relative differences between them remained similar in both

tissues, as there is no interaction between the two variables (ANOVA: F(5,203)=1.3,

P>0.1).

Figure 2. Size of the insertions/deletions (in bp) for each MSI marker, in MSI-H colorectal (CRC) and

endometrial (EC) carcinomas. * Statistically significant differences between the CRC/EC pair for each

marker.

Distribution of microsatellite instability Frequencies of instability were calculated for each MSI marker in both types of

tumours. Neither of the two tissues showed a statistically significant preference for

a specific type of marker. Both mononucleotide and dinucleotide markers are

equally affected in the two tumour types and none of the markers was differently

affected when we compared colorectal and endometrial tumours (Fig. 3).

50

Figure 3. Frequencies of instability observed for the mononucleotide (BAT25, BAT26, BAT40) and

dinucleotide (D2S123, D5S346, D17S250) microsatellite markers, in MSI-H colorectal and endometrial

tumours. The upper panel shows the frequencies for each marker; the lower panel shows the total of

mononucleotide and dinucleotide instability. CRC, colorectal carcinomas; EC, endometrial carcinomas.

DISCUSSION

We report an analysis of mononucleotide and dinucleotide MSI markers, with

regard to type, size and frequency of the mutations in MSI-H tumours with different

tissue origins, namely colorectal and endometrial carcinomas. Looking at these

features we found no significant differences between the EC and CRC MSI profiles,

or at least not great enough to justify applying different MSI tests for the two tumour

types.

No statistically significant differences between mutation carriers and non-

carriers were found. For this reason we were able to group all MSI-H cases

51

together for the analyses of the different MSI features. Nevertheless, we should

keep in mind that the number of mutation carriers used in this study is much

smaller than the number of non-carriers. Considering the age of the patients,

mutation carriers developed CRC earlier than non-mutation carriers for MSI-H

cases (table 2). Overall the average age of onset did not differ significantly among

the mutation carriers and the non-mutation carriers. Our data show that the ratio of insertions and deletions is a marker-

dependent feature rather than a tissue-dependent one, as all the mononucleotide

markers we studied showed almost exclusively deletions, while dinucleotide

markers showed deletions, insertions, and simultaneous deletions and insertions,

both in CRC and EC.

The occurrence of mainly deletions in the mononucleotide markers was what

we expected from the literature, since mutations in these markers are commonly

referred to as shortenings. In the first reports on the involvement of microsatellite

mutations in colon carcinogenesis mediated by a mutation in the MMR system

(“mutator mutation”) (Ionov et al., 1993), a striking imbalance of deletions over

insertions in Poly (A) sequences in CRC cell lines, with various degrees of

microsatellite instability, was described. It was also known that, in Saccharomyces

cerevisae, frameshifts on single base pair tracts tend to be deletions (Kunkel et al.,

1989; Henderson and Petes, 1992). With respect to dinucleotide instability, if the

three dinucleotide markers are taken as a whole, a tendency for only insertions

over only deletions was observed in both CRC (44% vs. 37%) and EC (49% vs.

32%). Simultaneous deletions and insertions were found in 19% of dinucleotide loci

in CRC and in 20% in EC. These results are in agreement with previous studies

suggesting that insertions are more common than deletions among dinucleotide

repeats (Twerdi et al., 1999; Ellegren, 2000; Yamada et al., 2002).

This close association of the occurrence of insertions or deletions with the

type of MSI marker suggests that characteristics of the repeats, such as repeat

length, have more influence on the type of mutation occurring at a microsatellite

repeat than the tissue origin of the tumour in which those mutations arise. Repeat

length, together with base composition and number of repeat units per tract, are

some of the features known to influence the mechanism of “slipped-strand

mispairing” (Boyer et al, 2002), the main mechanism generating insertions or

52

deletions in microsatellites during DNA replication (Levinson and Gutman, 1987;

Henderson and Petes, 1992).

While analyzing the size of the deletions/insertions, we observed significant

differences between CRC and EC, with EC showing smaller mutations than CRC

for all markers, as previously described in the literature for the BAT markers. The

differences are, however, not statistically significant in our study when comparing

each marker alone. For instance Kuismanen et al. (2002) reported a mean

deviation (bp) of 6.7 in CRC and 4.1 in EC for BAT25; and a mean deviation (bp) of

13.5 in CRC, and 8.5 in EC for BAT26. For the same markers we observed the

same tendency to larger mutations in CRC: a mean deviation (bp) of 6.08 ± 0.41 in

CRC, and 5.25 ± 0.62 in EC for BAT25; and 7.82 ± 0.65 in CRC and 6.00 ± 0.89 in

EC for BAT26. The apparent differences found between our data and the

mentioned study (Kuismanen et al., 2002) might be explained by differences in the

classification of MSI by different observers. The number of samples included might

also play a role in these differences, namely the inclusion of tumours with MMR

mutations, typically more unstable than those not carrying MMR mutations. The

stage of the tumour – more specifically the rounds of replication that a given

tumour has undergone – might, in our opinion, also influence the size of

microsatellite mutations.

Furthermore, although the different markers showed different mutation sizes,

the relative differences between them remained similar in both tissue types, leading

to comparable patterns of instability, as observed for the type of mutation.

Analyzing each marker alone, the frequencies of instability were not

significantly different between CRC and EC for any of the markers. If we consider

two groups, one of the three mononucleotide markers and one of the three

dinucleotide markers, the pattern was again similar, with mono- and dinucleotide

markers being affected in equal amounts and both of them similarly affected in

CRC and EC.

In conclusion, our results suggest that it is not possible to define specific

profiles of MSI marker instability to distinguish tumours of different tissue origins.

The features analyzed in our study – type, size and frequency of instability of MSI

markers – seem to be representative of common patterns of MSI in CRC and EC.

53

A possible explanation for the quantitative differences described between

CRC and EC, with EC having usually less unstable markers and smaller

deletions/insertions, might be that they indicate different durations of tumour

development, rather than reflecting real differences in profiles of the two tumour

types. Tissue specificities, such as differences in tissue turnover between

colorectal and endometrial epithelium might lead to different timings of tumour

development and, in practice, result in different levels of instability. This would be in

agreement with the tumour clock model of Shibata et al. (1996), who proposed that

microsatellite alterations could be seen as a proxy for the number of cell divisions.

Mutations accumulate with the number of replications, serving as a molecular clock

to define the time of tumorigenesis and tracing the history of the tumour.

ACKNOWLEDGEMENTS

The authors thank Pedro Lourenço for the statistical analyses, and Jackie Senior

for editing the manuscript.

This work was supported by Fundação para a Ciência e a Tecnologia, Portugal

(SFRH/BD/18832/2004) and by the European Community (FP6-2004-

LIFESCIHEALTH-5, proposal no. 018754).

54

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marker in hereditary and non-hereditary endometrial hyperplasia and cancer. Int J Cancer 92:398-403.

Berends MJ, Wu Y, Sijmons RH, et al. 2002. Molecular and clinical characteristics of MSH6 variants: an

analysis of 25 index carriers of a germline variant. Am J Hum Genet 70:26-37.

Berends MJ, Wu Y, Sijmons RH, et al. 2003. Toward new strategies to select young endometrial cancer

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Boyer JC, Yamada NA, Roques CN, et al. 2002. Sequence dependent instability of mononucleotide

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Duval A, Reperant M, Compoint A, et al. 2002. Target gene mutation profile differs between

gastrointestinal and endometrial tumors with mismatch repair deficiency. Cancer Res 62:1609-12.

Ellegren H. 2000. Heterogeneous mutation processes in human microsatellite DNA sequences. Nat

Genet 24:400-2.

Furlan D, Casati B, Cerutti R, et al. 2002. Genetic progression in sporadic endometrial and

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Ionov Y, Peinado MA, Malkhosyan S, et al. 1993. Ubiquitous somatic mutations in simple repeated

sequences reveal a new mechanism for colonic carcinogenesis. Nature 363:558-61.

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Pathol 160:1953-8.

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Levinson G, Gutman GA. 1987. Slipped-strand mispairing: a major mechanism for DNA sequence

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Niessen RC, Berends MJ, Wu Y, et al. 2006. Identification of mismatch repair gene mutations in young

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polyposis colorectal cancer. Gut 55:1781-8.

Peltomäki P, Lothe RA, Aaltonen LA, et al. 1993. Microsatellite instability is associated with tumors that

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Shibata D, Navidi W, Salovaara R, et al.. 1996. Somatic microsatellite mutations as molecular tumor

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Thibodeau SN, Bren G, Schaid D. 1993. Microsatellite instability in cancer of the proximal colon.

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Twerdi CD, Boyer JC, Farber RA. 1999. Relative rates of insertion and deletion mutations in a

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56

CHAPTER 4

The Hunt for New Target Genes in Endometrial Tumors Reveals the Involvement of the Estrogen-Receptor

Pathway in Microsatellite Unstable Cancers

Ana M. Ferreira1,7, Iina Niittymäki5, Sónia Sousa7, Frans Gerbens1, Krista Bos1,

Krista A. Kooi1, Chris Esendam1, Peter Terpstra4, Menno Hardonk4, Tineke van der

Sluis2, Monika Zazula6, Jerzy Stachura6, Ate G. van der Zee3, Harry Hollema2, Rolf

H. Sijmons1, Lauri A. Aaltonen5, Helga Westers1, Raquel Seruca7, Robert M. W.

Hofstra1

Departments of 1Genetics, 2Pathology, 3Gynecology, 4Medical Biology, University Medical

Center Groningen, University of Groningen, Groningen, the Netherlands. 5Department of Medical Genetics, University of Helsinki, Helsinki, Finland.

6Department of Patomorfology, Medical College, Jagiellonian University, Krakow, Poland. 7Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.

Manuscript in preparation

57

ABSTRACT

Microsatellite instability (MSI) in tumors results among others in an accumulation of mutations in (target) genes. Previous studies suggest that the profile of highly mutated target genes differs by tumor-type and that in particular for endometrial tumors the frequently mutated target genes remain to be identified. In our search for such highly mutated target genes in mismatch repair deficient endometrial cancers we identified 44 possible target genes of which 7 are highly mutated (>15%). Besides a high mutation frequency, 5 of these 7 could, by function, be linked to cancer development. Two genes encode proteins involved in chromatin remodeling (MBD6 and JMJD1C), one protein (JAK1) is involved in the JAK/STAT pathway, a pathway known to be implicated in cancer, one protein (KIAA1009) is essential in chromosome segregation and mitotic spindle assembly, and finally the most frequently mutated gene, NRIP1, encodes a co-repressor of the estrogen receptor (ER) pathway. Furthermore, we analyzed colorectal and gastric MMR deficient (MSI-H) tumors for mutations in ten of the identified target genes. Our data show that some of these newly identified target genes are tissue specificity, while others seem to play a more common role in MSI-H tumors, independently of the origin of the tissue. We therefore present a new profile of target genes, genes likely involved in endometrial cancer development. The most promising one is NRIP1, a gene influencing the ER pathway, the pathway with proven association with endometrial cancer development. These findings might prove relevant to look for additional target genes and it might give new insights for the design of novel therapeutic treatments.

58

INTRODUCTION

Endometrial carcinoma (EC) is one of the most common forms of cancer among

women in Western countries. High exposure to estrogens, obesity and family

history are considered the main risk factors for the disease. Moreover, EC is the

most common extra-colonic cancer in Lynch syndrome patients. This syndrome,

also known as hereditary nonpolyposis colorectal cancer (HNPCC), is caused by

germline mutations in the DNA mismatch repair (MMR) genes (Aaltonen et al.,

1993; Ionov et al, 1993; Thibodeau et al., 1993). The MMR system repairs DNA

replication errors that are not immediately corrected by DNA polymerase. The

MMR system, therefore, plays a crucial role in DNA replication accuracy.

Functional inactivation of MMR genes by mutations or epigenetic changes leads,

among others, to the accumulation of insertions/deletions. These are easily

identified in short DNA tandem repeat sequences (microsatellites); this phenotype

is called microsatellite instability (MSI). MSI can be detected in tumors from Lynch

syndrome patients (Aaltonen et al., 1993; Ionov et al., 1993; Peltomäki et al., 1993;

Thibodeau et al., 1993), but is also present in a fraction (~15-25%) of sporadic

cases of endometrial, colorectal, and gastric cancer (Boland et al., 1998).

Genes containing repeat sequences are vulnerable to replication errors in

MMR-deficient tumors. MSI can occur at non-coding but also at coding repeat

sequences of regulatory genes which might play a role in tumor development. Such

genes are generally called target genes and thought to be the key players during

MSI-H carcinogenesis. Over 160 target genes have been identified to date in

MMR-deficient tumors (Vilkki et al., 2002; Røyrvik et al., 2007). Mutations were

mostly searched for in MSI-H colorectal tumors (CRC). Endometrial and gastric

(GC) tumors have been analyzed to a lesser extent and were mainly studied for

target genes previously reported in CRC. From previous studies it becomes

however clear that although there are target genes commonly involved in MSI-H

tumors of diverse origin, e.g. BAX, others show considerable tissue specificity

(Duval et al., 1999; Schwartz et al., 1999; Semba et al., 2000). It has been shown

that the profile of target genes differs between EC and gastrointestinal tumors with

MMR deficiency, in both qualitative and quantitative manners (Myeroff et al., 1995;

Gurin et al., 1999; Duval et al., 2002). In fact in EC a small number of (highly

59

mutated) target have been identified so far, suggesting that other not yet identified

target genes remain to be found. This study aims at identifying these target genes

for MSI-H EC.


Samples -Six fresh-frozen normal endometrial tissue samples were obtained from six

women undergoing surgery at the University Medical Center Groningen (UMCG,

Groningen, the Netherlands) for other reasons than uterine cancer. These samples

were used for the expression arrays experiments.

-Forty-two paraffin-embedded tissue sections from MSI-H endometrioid

endometrial carcinomas were obtained from the Department of Pathology,

University Medical Center Groningen (Groningen, the Netherlands) and from the

Department of Pathomorphology, Jagiellonian University (Cracow, Poland). Fresh-

frozen tumor tissues were available for 10 of these samples.

-Forty MSI-H colorectal tumors were obtained from the Department of Medical

Genetics, University of Helsinki (Helsinki, Finland), and from the Department of

Pathology, University Medical Center Groningen (Groningen, the Netherlands).

-Fifteen MSI-H gastric tumors were obtained from The Institute of Molecular

Pathology and Immunology of the University of Porto (Porto, Portugal)

All the patients participating on this study have given their written consent.

DNA isolation Genomic DNA was isolated from fresh-frozen tissue and formalin-fixed, paraffin-

embedded tumor tissue using the Qiagen DNA Mini Kit (Qiagen, Venlo, the

Netherlands), using a standard protocol (protocol available on request).

Micro-array experiments RNA isolation

RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, CA) and was

treated with RNase-free DNase I (Qiagen) as described by the manufacturer.

60

mRNA Amplification and Cy-dye coupling

Linear amplification of mRNA was performed essentially according to a protocol of

the Dutch Cancer Institute (www.nki.l.nl/nkidep/pa/microarray/protocols.html).

Briefly, amplification started with first strand cDNA synthesis from 2 µg of total

RNA, using Superscript II RT-polymerase (GIBCO - Invitrogen) and a specific

oligo(dT) primer containing a 17bp T7 polymerase recognition site (5'-

ggccagtgaattGtaatacgactcactatagggaggcggT-24-3') (Eurogentec, Seraing,

Belgium). After second strand synthesis, double-stranded cDNA was purified with

the Qiaquick PCR purification kit (Qiagen). In vitro transcription was performed with

the T7 Megascript kit (Ambion, Huntingdon-Cambridgeshire, UK) as described by

the manufacturer, but using instead of UTP, a 1:1 mixture of aminoallyl-UTP

(Ambion) and UTP with a final concentration of 7.5 mM for all NTPs ('t Hoen et al.,

2004). Amplified RNA (aRNA) was purified with the RNA clean up protocol

(Qiagen). Five µg of aRNA was labeled by coupling monoreactive Cyanine 3 (2.5

nmol per reaction) or Cyanine 5 (2.5 nmol per reaction) fluorophores (Amersham

Biosciences, Little Chalfont, Buckinghamshire, UK) to the aminoallyl-modified

nucleotides. Labelled aRNA was separated from unincorporated Cyanine 3 or

Cyanine 5 molecules with Microspin G50 columns (Millipore Corp, Amsterdam, The

Netherlands) following the recommendations of the manufacturer.

Experimental design

For the identification of genes expressed in normal endometrium a randomized

design was applied for micro-array hybridization. Each of all six normal cDNA

endometrium tissue samples was labeled with Cyanine 3 and Cyanine 5 separately

and subsequently assigned at random to a sample labeled with the opposite dye

for hybridization.

Micro-array hybridization

In-house manufactured human oligonucleotide arrays were used containing the

Qiagen/operon 21,329 70-mer human gene specific oligonucleotide set version 2.1

extended with 4,000 negative and positive control features. The oligonucleotides

were printed in a concentration of 10 pM on Ultra-GAPS amino-silane coated slides

(Corning BV. Life Sciences, New York, USA) using BioRobotics 10K quill pins with

61

http://www.nki.l.nl/nkidep/pa/microarray/protocols.htm

the MicroGrid spotter (Isogen). Blocking, prehybridization and hybridization were

performed as described by Hegde et al. (2000), with some modifications (detailed

protocol available on request). Hybridization was performed in hybridization

chambers (Telechem International Inc, Sunnyvale, CA, USA) in a water bath at

52°C in the dark for approximately 48 h. Subsequently, slides were washed, dried

by centrifugation at 800 rpm during 3 min and scanned with an Affymetrix

GMS428TM array scanner.

Micro-array data analysis

Fluorescent signal intensity data for each spot and for each fluorophore were

extracted from the scanned images of each micro-array slide using ImaGene

version 5.6 (BioDiscovery, El Segundo, California, USA). Signal intensity data were

log transformed and for each spot the Cyanine 5 signal intensity/Cyanine 3 signal

intensity ratio was determined and subjected to print-tip lowess intensity dependent

normalization using the Limma package from the Bioconductor project in R

(http://bioinf.wehi.edu.au/limma). Since no dependency exists between both

samples during hybridization ('t Hoen et al., 2004), normalized log-ratios were back

transformed to log intensities. Further data analysis was performed using BRB

ArrayTools v3.2 developed by Dr. Richard Simon and Amy Peng Lam

(http://linus.nci.nih.gov/~brb/download.html). Basically, data was vigorously filtered

to exclude control spots, empty spots, spots with high between-pixel-intensity

variability and spots designated as bad by eye. Genes that had more than 25%

missing data across all observations were excluded from the analysis. Genes with

expression 10 times higher than the background were identified.

Selection of mononucleotide repeats A computer program (Repeat Finder) was created for the purpose of finding

repetitive tracts in DNA sequences. A data file containing the coding sequences

(CDS) of all the genes selected by microarrays in normal endometrium was

uploaded to the program. Genes with mononucleotide tracts of (A)7, (T)7, (C)7,

(G)7, (A)8, and (T)8 in there coding sequence were identified and selected.

62

http://bioinf.wehi.edu.au/limma

Mutation screening The sequences encompassing the repeats of interest were extracted from the

Ensembl database (http://www.ensembl.org/). Primers were designed using the

Primer3 program (http://frodo.wi.mit.edu/). Amplicons were amplified and

subsequently PCR products purified using ExoSAP-IT enzymatic reagent (US

Biochemical Corporation) according to the manufacturer’s instructions. Mutation

analysis was performed by direct sequencing using big dye Terminator Kit version

3.1 (Applied Biosystems) and ABI 3730 Automatic DNA sequencer (Applied

Biosystems) following the recommendations of the manufacturer. The list of genes

screened and the primer sequences and PCR conditions are available on request.

The mutation analysis in the gastric tumor samples was performed by size

separation using multiplex PCR. The products were read in a ABI 3100 sequence

analyzer using Peack scanner Software v1,0 with a 500 liz size standard

electropherogram.

RESULTS

Expression profiling of normal human endometrium Six normal endometrial tissue samples were used for expression profiling with 21K

oligonucleotide micro-arrays. A total of 2338 genes showed expression values 10X

higher than the background signals. These genes were considered as being clearly

expressed in normal endometrial tissue and therefore selected for the next step of

finding repeat sequences.

Genes containing coding mononucleotide repeats (A/T)7, (C/G)7, (A/T)8 The analysis of the coding sequences of the 2338 selected genes with the

computer program Repeat Finder revealed 573 repeats of interest identified in 432

out of the 2338 genes. The number of repeats of each type was the following: 244

(A)7, 74 (T)7, 114 (C)7, 57 (G)7, 72 (A)8, and 12 (T)8 repeats. The program

Repeat Finder is home made.

63

http://www.ensembl.org/

Mutation screening of the mononucleotide repeats in MSI-H EC Four hundred and seventy six (476) primer sets were successfully designed (for

the others we did not succeeded designing good primer pairs) encompassing a

total of 496 repeats (in 382 genes): 214 A(7), 61 T(7), 104 C(7), 46 G(7), 62 A(8),

and 9 T(8) repeats. The primers were first used for mutation analysis in 10 MSI-H

EC for which frozen material was available. Heterozygous frameshift mutations

(figure 1) resulting either from insertions or deletions (all +/-1bp) were found in 49

repeats (for at least 1 tumor DNA sample), in 44 candidate genes (some genes

host more than one repeat of interest). The screening of those repeats was then

extended to 32 additional MSI-H EC samples (results are given in table 1). Seven

genes were found to be mutated in 15% or more of the samples: NRIP1, SRPR,

MBD6, JAK1, KIAA1009, JMJD1C, ADD3, with mutation frequencies of 34%, 26%,

24%, 20%, 19%, 15% and 15%, respectively (Table I). The other genes had

mutation frequencies below the cut-off of 15%.

Figure 1. Example of frameshift mutations at coding mononucleotide repeats. In the upper panel, the

normal (reverse) sequence of the (A)8 repeat in NRIP1 gene is depicted; the lower panel shows a

deletion of an (A) base found for that repeat in a tumor sample.

64

Table I. Results of the mutational screening for the forty-four candidate target genes for which a

mutation was found in at least one tumor.

Gene Description Repeat Mutat Freq. (%)

SEC16 SEC16 homolog A (S. cerevisiae) C7 5,7 (2/35) SPG20 spastic paraplegia 20 (Troyer syndrome) T8 5,9 (2/34) CAMSAP1L1 calmodulin regulated spectrin-associated protein 1-like 1 A8 7,1 (2/28) ZMIZ1 zinc finger, MIZ-type containing 1 C7 4,3 (1/23) ZMIZ1 zinc finger, MIZ-type containing 1 C7 9,1 (2/22) INTU inturned planar cell polarity effector homolog (Drosophila) A8 4,2 (1/24) IGSF9 immunoglobulin superfamily, member 9 C7 4,3 (1/23) KIAA1370 KIAA1370 A8 12,9 (4/31) JMJD1C jumonji domain containing 1C A8 15,1 (5/33) FAM135A family with sequence similarity 135, member A A8 3,3 (1/30) KIAA1797 KIAA1797 A7 6,7 (1/15) MBD6 methyl-CpG binding domain protein 6 3XC7 24,1 (7/29) DACH1 dachshund homolog 1 (Drosophila) A7 3,1 (1/32) POLE3 polymerase (DNA directed), epsilon 3 (p17 subunit) A7 4,8 (1/21) JAM3 junctional adhesion molecule 3 G7 8,3 (3/36) HEXDC hexosaminidase (glycosyl hydrolase family 20, catalytic domain) containing C7 + G7 11,4 (4/35) CHD4 chromodomain helicase DNA binding protein 4 A7 8,1 (3/37) LYN v-yes-1 Yamaguchi sarcoma viral related oncogene homolog A7 6,7 (2/30) PPP1R10 protein phosphatase 1, regulatory (inhibitor) subunit 10 C7 3,7 (1/27) SRPR signal recognition particle receptor ('docking protein') A8 25,8 (8/31) TTC3 tetratricopeptide repeat domain 3 A8 3,3 (1/30) NRIP1 nuclear receptor interacting protein 1 A8 34,3 (12/35) AP3B1 adaptor-related protein complex 3, beta 1 subun A8 8,1 (3/37) INPPL1 inositol polyphosphate phosphatase-like 1 C7 14,3 (5/35) FLNB filamin B, beta (actin binding protein 278) G7 9,7 (3/31) JAK1 Janus kinase 1 (a protein tyrosine kinase) A8 20 (7/35) BAT1 HLA-B associated transcript 1 T8 2,9 (1/35) MTA1 metastasis associated 1 G7 3,3 (1/30) FXR1 fragile X mental retardation, autosomal homolog 1 A8 12,5 (2/16) IFNGR2 interferon gamma receptor 2 (interferon gamma transducer 1) T7 2,8 (1/36) RBM6 RNA binding motif protein 6 G7 3,4 (1/29) SF3B2 splicing factor 3b, subunit 2, 145kDa A8 10,7 (3/28) RABGAP1 RAB GTPase activating protein 1 A8 6,25 (2/32) TNPO3 transportin 3 C7 2,9 (1/34) TRPM5 transient receptor potential cation channel, subfamily M, member 5 C7 5,3 (1/19) ITM2B integral membrane protein 2B C7 2,8 (1/36) SVIL supervillin G7 10,8 (4/37) INTS12 integrator complex subunit 12 T7 5,9 (2/34) C17orf63 chromosome 17 open reading frame 63 C7 3,7 (1/27) KIAA1009 KIAA1009 T8 + A7 18,5 (5/27) CPEB3 cytoplasmic polyadenylation element binding protein 3 C7 2,9 (1/34) NOL7 nucleolar protein 7, 27kDa A8 7,1 (2/28) ADD3 adducin 3 (gamma) A8 14,7 (5/34) PHKB phosphorylase kinase, beta A7 4,3 (1/23) TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) A7 3,1 (1/320

Mutation screening of 10 target genes in CRCs and GCs (NRIP1, SRPR, MBD6, JAK1, KIAA1009, JMJD1C, ADD3, INPPL1, SVIL, and

HEXDC)

The seven genes with mutation frequencies equal or higher than 15% and three

additional genes (INPPL1, SVIL, HEXDC) were then screened in CRC and GC

samples. Frameshift mutations were found in all the genes analyzed, in at least

65

one of the tumors. All mutations found were heterozygous and consisted of +1 or -

1 bp, as in figure 1, except for SRPR in a GC sample, where -2 bp mutations were

also found. Three CRCs and 2 GCs did not show mutations for any of the repeats.

For the CRC group, no associations were found between mutations and the

classification of the patients as sporadic/Lynch syndrome patients (data not

available for GCs or ECs). Also no correlation was found between the mutations

and the gender of the patients. Mutation frequencies higher than a cut-off value of

15% were found in SRPR, ADD3, MBD6 and NRIP1 genes (47%, 37%, 25% and

22%, respectively) in CRC samples; and in ADD3 (47%), SRPR (27%), SVIL (27%),

JAK1 (20%) and INPPL1 (20%) in GC samples (Table II). Figure 2 shows the

comparative profiles of the three types of tumors for the new target genes. After analyzing repeats in 10 genes, we found: 62 mutations (in 321 repeats) in EC

patients, giving 1.9 mutations per patient on average; 53 mutations (in 289 repeats)

in CRC giving 1.8 mutations per patient on average; 27 mutations (in 150 repeats)

in GC (1.8 mutations per patient).

Table II. Mononucleotide repeats analyzed and respective mutation frequencies found in the MSI-H

colorectal and gastric tumor samples used; the results obtained for the MSI-H endometrial tumors are

included for comparison. ND: not determined.

Gene Exon Repeat CRC GC EC

NRIP1 3 A8 22% (8/36) 13% (2/15) 34% (12/35) SRPR 4 A8 47% (14/30) 27% (4/15) 26% (8/31) MBD6 7/8 3XC7 25% (10/40) 13% (2/15) 24% (7/29) JAK1 5 A8 3% (1/30) 20% (3/15) 20% (7/35) KIAA1009 12 T8+A7 ND 7% (1/15) 19% (5/27) JMJD1C 9 A8 3% (1/34) 0% (0/15) 15% (5/33) ADD3 14 A8 37% (11/30) 47% (7/15) 15% (5/34) INPPL1 26 C7 10% (3/30) 20% (3/15) 14% (5/35) SVIL 31 G7 14% (4/29) 27% (4/15) 11% (4/37) HEXDC 12 C7 3% (1/30) 7% (1/15) 11% (4/35)

66

Box 1: Function of the highly mutated proteins in EC

NRIP1 (nuclear receptor-interacting protein 1) is a modulator of several, if not all,

nuclear receptors (e.g. retinoic acid receptor, thyroid receptor, androgen receptor). It

is also a known co-repressor of the estrogen-receptor (ER) pathway. Silencing of

NRIP1 leads to growth advantages in breast cancer derived cell lines.

SRPR encodes the signal recognition particle receptor subunit alpha (‘docking

protein’), which together with the SRP (signal recognition particle) ensures the correct

targeting of the nascent secretory proteins to the endoplasmic reticulum membrane

system (Janin et al., 1992). MBD6 (methyl-CpG binding domain protein 6) contains a methyl-CpG-binding

domain (MBD) and is possible involvement in DNA methylationas are other MBD

proteins. Another MBD protein, MBD4, is a known target gene in MSH-H tumors

(Røyrvik et al., 2007).

JAK1 (Janus kinase 1) is a protein-tyrosine kinase (PTK). It is a widely expressed

membrane-associated phosphoprotein. Deregulation of the JAK-STAT signaling

pathway has been described in a variety of cancers and immune disorders. Mutations

in JAK1 have been reported in human leukemias and in several solid cancers (Jeong

et al., 2008). Furthermore, the JAK/STAT3 pathway has been suggested as a new

potential target for therapy of CRC (Xiong et al., 2008).

KIAA1009 is a new microtubule-associated ATPase involved in cell division, a

protein with essential role on chromosome segregation and mitotic spindle assembly.

It is expressed throughout mitosis, and it is located at the pole of the mitotic spindle,

associated with microtubules, and in the centrosome. The cell death induced by

transfection with QN1/KIAA1009 siRNA suggests that QN1/KIAA1009 protein is a

potential target for novel antimitotic cancer therapies (Leon et al., 2006).

JMJD1C (jumonji domain containing 1C), formerly TRIP8 (thyroid hormone receptor

interactor 8) codes for a nuclear protein predicted to be a transcriptional regulator

associated with nuclear thyroid hormone receptors. JMJD1C is believed to be a

histone H3K9 demethylase, therefore playing a major role in histone code.

ADD3 (adducin 3) is a membrane-cytoskeleton-associated protein that is involved in

the assembly of the spectrin-actin network in erythrocytes and at sites of cell-cell

contact in epithelial tissues. Not much is know about this protein.

67

DISCUSSION

In the present study we report seven new target genes for MSI-H endometrial

cancer. Additionally, we show that most of those genes are also mutated in

colorectal and gastric tumors, although with different frequencies.

A mutational screening was performed on mononucleotide repeats in the

coding sequence of genes that are expressed in normal human endometrial tissue.

By using normal tissue, we aimed to include genes with a potential role in the

normal maintenance of the endometrium and therefore theoretically the ones to be

affected in disease context. More commonly, an approach of comparing tumor

versus normal tissue would have been followed and down-regulated genes would

have been selected; however, in that case, possibly the mutations reported in this

study would not be found, since they are heterozygous and the gene can thus still

be expressed.

For the mutational screening we selected the genes with expression signals

ten-fold higher than the background signal for further analysis. We are aware that a

large number of candidate genes will in this way be excluded because of their

expression low expression. Another reason that we have missed target genes is

the fact that we selected only for specific repeat length. Type and length of the

repeats are highly relevant for their mutation frequencies. Mononucleotide repeats

are commonly considered the most MSI-H specific type of repeats and tracts with

lengths between 6 and 10bp are usually taken. Considering the recent paper of

Sammalkorpi et al 2007, to our knowledge the first study on mutation frequencies

of intergenic repeats, we decided to not include (G)8 and C(8) repeats or longer, to

avoid high background mutation frequencies interfering with the results. Known

target genes like BAX and TGFβRII for instance have mononucleotide repeats

longer than 9 bases and are therefore by definition not included in our screen. We

have reasons to believe that this is a safe set-up of the experiment to avoid false

positive candidates. These reasons imply that we only found a subset of all genes

mutated in EC.

Are the target genes found really involved in endometrial cancer

development? To define a real target gene, criteria have been formulated (Duval

and Hamelin, 2002). They consist of: (1) a high mutation frequency; (2) biallelic

68

inactivation of the gene by simultaneous alteration of the second allele’s repeat

tract or by point mutation or allelic loss; (3) possible involvement of the encoded

protein in tumor development; (4) occurrence of mutations within the pathway in

MSI-negative tumors; (5) in vitro or in vivo functional suppressor studies. These

criteria are considered rather strict and some are controversial (Perucho, 2003).

Due to this controversy and to the general lack of functional evidence

proving the relevance of the candidate genes, usually a high mutation frequency

(above a cut-off value of 12-15%) is taken as major criteria to classify a gene as a

real target gene (Duval and Hamelin, 2002). When applying the 15% cut-off rule, 7

new target genes were identified: NRIP1, SRPR, MBD6, JAK1, KIAA1009,

JMJD1C, and ADD3. To our knowledge these genes have never been reported

before in MSI-H endometrial cancer.

Bi-allelic mutations, the second criteria, were never found. All the mutations

in this study were heterozygous. Whether however biallelic mutations are indeed

necessary can be debated. Haploinsufficiency, caused by the loss of only one

allele, is frequent finding in cancer. A good example is mono-allelic loss of PTEN,

the main mutated gene in endometrial cancer (Nardella et al., 2008).

The function of the encoded protein, and thereby its possible involvement in

tumor development, is also an inclusion requirement for a real target gene. In Box

1 a short description of the proteins encoded by the newly identified target genes is

given. Considering the mutation frequency and the function of the protein, NRIP1

came out of our study as the best candidate target gene for MSI-H EC. It was the

highest mutated gene (34% of EC tumors) and it is a known co-repressor of the

estrogen-receptor (ER) pathway. The ER is a very important pathway for

endometrial tissue regulation, as the endometrium is a sex hormone responsive

tissue, highly regulated by the concentrations of estrogens. The ER is a ligand-

activated transcription factor from the nuclear receptor superfamily. Several

estrogen-responsive genes have been described. Genetic alterations in ER and

ER-responsive genes are thought to be key players in the development of

hormone-dependent tumors (Notarnicola et al., 2001). Furthermore, the high

exposure to estrogens is currently considered the major risk factor for EC.

Moreover, approximately 80% of all sporadic EC tumors – the endometrioid

endometrial carcinomas - are estrogen-dependent carcinomas. In addition to this,

69

it has been reported that NRIP1 is essential for female fertility in mice (White et al.,

2000), and that mutations in NRIP1 may act as predisposing factor for human

endometriosis (Caballero et al., 2005). We believe that it is very likely that NRIP1

mutations might result in functional differences at the ER-pathway level. We expect

that inactivation of NRIP1 will interfere with the process of co-repression of the ER

complex and lead to differences in the expression of estrogen-dependent genes.

This could eventually be linked to tumors growth advantages.

Most of the other six genes found highly mutated can by function also be

coupled to the carcinogenic process. Two of the proteins are involved in chromatin

remodeling (MBD6 and JMJD1C), JAK1 is likely involved in a pathway often found

implicated in cancer in general, and KIAA1009 is essential in chromosome

segregation and mitotic spindle assembly, a process which, when disturbed, will

contribute to cancer development.

Taking the mutation frequencies and the (known) function of the newly

identified target genes we have reasons to suggest that for sure part of the seven

genes do play a role in MSI-H EC development.

Comparison of endometrial versus gastrointestinal tumors

All the genes showed high mutation frequencies in at least one of the tumor types,

except HEXDC, which reached the highest frequency of only 11%, in EC samples.

Comparing the mutation frequencies found in the CRC and GC samples with the

EC samples we observe some differences in the profile of target genes affected

(table II and figure 1). We conclude that the target genes included in this study are

involved both in EC and gastrointestinal carcinogenesis, although in a different

order of mutation frequencies and therefore giving a different profile dependent on

the tissue origin, as expected from previous studies on target gene profiles of MSI

tumors (Duval et al., 2002).

However, the differences found in the JAK1 repeat (3% in CRC; 20% in GC

and EC) is quite striking, especially because JAK1 mutations have been reported in

several solid cancers and the JAK/STAT3 pathway has even been suggested as a

new potential target for therapy of CRC (Xiong et al., 2008). As this screening only

looked for mutations in one mononucleotide repeat, which is only a very small part

of the coding sequence of the gene, it can not be excluded that other mutations are

70

present and that the gene plays a more important role in CRC as well. Of course

this holds true for all genes analyzed.

Finding mutations in NRIP1, a protein clearly connected to estrogens and

estrogen receptor signaling, in CRCs and GC seems surprising as the colon tissue

is not typically hormone responsive. However, NRIP1 mutations in CRC and GC

have been reported before, although at lower frequencies (under the threshold of

15%). Frameshift mutations were found in an A9 coding microsatellite, in 13% of

MSI-H GCs and 7% of MSI-H CRC (Røyrvik et al., 2007). Moreover several

findings in colorectal cancer support a hypothesis that high estrogen levels can

have a protective effect. These findings have been used as an explanation for the

gender bias observed on CRC incidence, with a lower incidence of the disease in

women than in men. In particular, hormonal changes associated with pregnancy

(McMichael and Potter, 1980), and hormone replacement therapy (HRT) have

been associated with lower risk of CRC (Potter, 1995; Peipins et al., 1997; Chen et

al., 1998; Kadiyska et al., 2007).

It is interesting to notice that two other genes of our list, JMJD1C and SVIL

encode proteins involved in the regulation of hormone receptors. JMJD1C is a

transcription regulator of nuclear thyroid hormone receptors; SVIL has been

described as an androgen-receptor (AR) co-regulator that can enhance AR

transactivation in muscle and other cells (Ting et al., 2002). SVIL has already been

linked to cancer, as it is underexpresssed in prostate cancer (Vanaja et al., 2006).

CONCLUSIONS

Future studies at the functional level are essential to elucidate how NRIP1 and the

other genes are implicated in carcinogenesis, since even when mutations are

found in genes with putative roles in tumor-related processes, the chance of having

a bystander gene instead of a real target gene can not be discarded. However, with

this study we propose 7 new genes, and in particular NRIP1 as novel target genes

for MSI-H endometrial cancer. Our results also support the idea that MSI

gastrointestinal and EC tumors present some differences in the profile of target

genes affected but that there are also some genes affected at similar frequencies

71

http://www.pnas.org/search?author1=Huei-Ju+Ting&sortspec=date&submit=Submit

among the different types of tumors. More importantly, the present study suggests

that there might exist a stronger link between hormones and MSI than thought so

far, and that genes of hormone-related pathways should be considered important

candidates when searching for new target genes of MSI tumors.

0

10

20

30

40

50

60

70

80

90

100

NRIP1SRPR

MBD6JA

K1

KIAA1009

JMJD

1CADD3

INPPL1

SVIL

HEXDC

Mut

atio

n Fr

eque

ncy

(%)

MSI-H ECMSI-H CRCMSI-H GC

Figure 2. Distribution of mutation frequencies found in MSI-H endometrial (EC), colorectal (CRC) and

gastric carcinomas (GC), for the 10 most mutated target genes.

ACKNOWLEDGEMENTS

This work was supported by the Portuguese Foundation for Science and

Technology (“Fundação para a Ciência e a Tecnologia”), Portugal (Grant

ref.:SFRH/BD/18832/2004) and by the European Community (FP6-2004-

LIFESCIHEALTH-5, proposal No 018754).

72

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CHAPTER 5

Estrogen, MSI and Lynch Syndrome-Associated Tumors

Ana M. Ferreira1,2, Helga Westers1, André Albergaria2, Raquel Seruca2,3, Robert M.

W. Hofstra1

1Department of Genetics, University Medical Centre Groningen, University of Groningen,

Groningen, the Netherlands. 2Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal. 3Faculdade de Medicina da Universidade do Porto, Portugal.

Under review

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ABSTRACT Estrogens play a major role in the biology of hormonally responsive tissues but also in the normal physiology of various non-typical hormone responsive tissues. In disease, estrogens have been associated with tumor development, in particular with tumors such as breast, endometrium, ovary and prostate. In this paper we will review the molecular mechanisms by which estrogens are involved in cancer development, with a special focus in Lynch syndrome- related tumors. Further, we discuss the role estrogens might have on cell proliferation and apoptosis, how estrogens metabolites can induce DNA damage, and we discuss a possible connection between estrogens and changes in DNA (hypo- and hyper-) methylation. In this review we will also address the protective effect that high levels of estrogen have in MMR-related neoplasias.

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INTRODUCTION The most common types of cancer worldwide occur in hormonally responsive

tissues, such as breast, endometrium, ovary and prostate. Tumors occurring in

these tissues show strong associations with the exposure to exogenous or

endogenous steroidal hormones.

Estrogens are a group of steroid compounds which are present in both men

and women; however, their levels are significantly higher in women of reproductive

age. There are three types of estrogens of which 17β-estradiol is the most potent

one as has the highest affinity for its receptors. It is produced in high amounts in

pre-menopausal women by the ovary. The second endogenous but less potent

estrogen is estrone. It is produced from androstenedione, the immediate precursor

of estrone. The third estrogen is estriol, a metabolite of estradiol. It is mainly

produced by the placenta during pregnancy and is found in lower concentrations

than estradiol and estrone in non-pregnant women (Chen et al., 2008).

Estrogens act through the estrogen receptors (ERs). ERs are ligand-

activated transcription factors that have several domains that can bind estrogens

and activate transcription of several estrogen-responsive genes (see Figure 1)

(Notarnicola et al., 2001). There are two receptor isoforms, ERα and ERβ (Tsai &

O’Malley, 1994; Hall et al, 2001). When estrogen binds to these receptors, the

receptors dimerize, go to the nucleus and bind to specific DNA sequences, the

consensus estrogen response elements (EREs) of ER-responsive genes (Klein-

Hitpass et al., 1989).

The receptors may form ERα (αα) or ERβ (ββ) homodimers or ERαβ (αβ)

heterodimers (Li X et al., 2004). The activation of ER is influenced by a set of

different co-activators, enzymes, and co-repressors. These factors influence the

assembly of the transcriptional complex and the subsequent transcription of the

ER-responsive genes. This is called ´the canonical pathway´ of ER.

A ´non-canonical’ pathway of ER has also been described, in which genes

are activated without having ERE-like sequences. This non-classical mechanism

accounts for the transcriptional activation of approximately one-third of all estrogen

responsive genes (Huang et al., 2004).

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Alternative mechanisms without DNA binding have also been described.

DNA binding proteins such as specificity protein 1 (SP1) are activated by the direct

binding of ER (Velarde et al., 2007), for a schematic representation see Figures 1

and 3.

Estrogens play a major role in controlling the menstrual cycle, pregnancy,

thus female reproduction. However, estrogens are not only important for the

biology of hormonally responsive tissues; they also play an important role in bone

strengthening and cholesterol metabolism, and have an influence on the central

nervous system and the gastrointestinal physiology (Roy & Liehr, 1999; Nilsson &

Gustafsson, 2001). On one hand ER signaling plays an important role in many

normal physiological processes, on the other hand several studies have shown that

estrogens and their metabolites are also involved in tumor development.

In this review we will address the different possible mechanisms by which

estrogens can be involved in tumor development and in particular, we will focus on

how the hormone can be involved in the development of Lynch syndrome-related

neoplasias showing microsatellite instability.

Figure 1. Mechanisms of action of estrogens.

Proliferation Growth Differentation Angiogenesis Apoptosis

Activation of signaling routes such as: MAPK, P13K, PKA,PKC / binding of transcription factors to the active ERs

Estrogens

Genomic

Non-genomic

(Nuclear) ERs

Non ER-membrane bound receptors/

Estrogen Response Elements (ERE) dependent signaling regulated by co- activators and repressors

Transcription factors (e.g. AP-1 / PS2)

Target gene expression (e.g. VEGF)

Binding of co-activators and co-repressors (e.g. NRIP1)

ERE

Estrogen response elements independent signaling by binding to DNA bound transcription factors directly

adapters

Membrane or cytoplasmic ERs

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ESTROGEN AS A CARCINOGEN

The International Agency for Research on Cancer (IARC) recognized in 1987, for

the first time, that elevated concentrations of estrogens lead to an increased risk of

breast and uterine cancers (IARC, 1987). However, only in 2006, Russo & Russo

described in vivo malignant transformation of human breast epithelial cells by

estrogens (Russo & Russo, 2006). Salama et al. (2008) showed that

catecholestrogens induce oxidative stress and malignant transformation of human

endometrial glandular cells.

These and many other studies point towards a direct link between cancer

initiation and estrogens. However, how estrogens contribute to cancer is still not

totally clear. Several possible mechanisms have been put forward (see Figure 2):

1) Estrogens promote cell proliferation via ER mediated signaling, both

through genomic and non genomic pathways, which promotes cell proliferation and

growth, and reduces sensitivity to apoptosis. For instance, estrogens stimulated

ERs which then can up-regulate Wnt11 expression, which causes the tumor to

resist going into apoptosis (Katoh, 2003).

2) Another possibility is that estrogens promote signaling via cell membrane-

related but ER-independent phosphorylation of target genes. Examples are the

phosphorylation of AKT and ERK by estrogens in ER negative cells (Bouskine et

al., 2008; Zhang et al., 2009).

3) Estrogens lead to the production of toxic species that are able to induce

tumor development. Estrogens are converted to catecholestrogens by cytochrome

P450-mediated hydroxylation. Catecholestrogens, specifically the 4-hydroxylated

steroids, and their semiquinone and quinone reactive intermediates are considered

carcinogenic (Liehr, 2000). Various types of damage are found associated with

either estrogen quinones binding covalently to DNA or by free radical action.

Aneuploidy, gene amplification, arrest of DNA replication as a result of estrogen–

DNA adduction, single strand breaks, microsatellite instability, small insertions, and

deletions are all examples of these types of DNA damage (Roy & Liehr, 1999; Liehr,

2001; Fernandez et al., 2006).

4) Estrogens are found associated with changes in the methylation status,

both hypo- and hyper–methylation of DNA. An example of an estrogen-related

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hypomethylated gene is PAX2 (Wu et al., 2005). As an example of estrogen-

related hypermethylation gene is the estrogen receptor gene and MLH1 (Slattery et

al., 2001; Campan et al., 2006). The last example is particularly interesting, since in

this review we focus on MMR related neoplasia.

What is also believed to have a significant effect on cell growth and tumor

formation is the balance between the ERα and ERβ isoforms (Matthews &

Gustafsson, 2003). The activation of ERα is associated with increasing cell

proliferation whereas ERβ promotes apoptosis. For instance in endometrial cells it

was shown that a down-regulation of ERα results in a reduction of cell proliferation,

an effect that was not seen when ERβ was blocked.

ASSOCIATION BETWEEN LOW LEVELS OF ESTROGEN AND MSI IN LYNCH SYNDROME-ASSOCIATED TUMORS

Lynch syndrome-associated MSI tumors Several studies have showed that there is an association between estrogen

exposure and the presence of microsatellite instability in tumors. Microsatellite

instability (MSI) is a hallmark of tumors from patients with Lynch syndrome, also

known as hereditary non-polyposis colorectal cancer (HNPCC). This inherited

cancer syndrome is characterized by the development of colorectal cancer (CRC),

endometrial cancer and various other cancers and is caused by a mutation in one

of the mismatch repair (MMR) genes MSH2, MLH1, MSH6 or PMS2. Colorectal

cancer is the most common cancer found in Lynch syndrome: almost all patients

develop colorectal cancer, followed by endometrium cancer which occurs in

approximately 30% of all female patients (for a review on this see Vasen et al.

2007). Although MSI is the major characteristic of Lynch syndrome patients, it is

also found in 15-25% of sporadic colorectal, endometrial and gastric (Boland et al.,

1998). MSI is defined by the accumulation of insertions and/or deletions at short

DNA repeats (microsatellites), leading to different lengths of the repeat.

Accumulation of such mutations in coding mononucleotide repeats of genes with

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important regulatory functions, e.g. tumor-suppressor genes, is thought to be a key

event in the development of MSI tumors.

Estrogens and MSI Notarnicola et al. (2001) described a significant association between MSI and ER

status in colorectal tumors. Interestingly, they verified that the MSI tumors showed

low levels of ER expression. Moreover, the withdrawal of estrogens also resulted in

an increasing risk of MSI CRC tumors. An interesting hypothesis was made by

Breivik et al. (1997) by linking estrogens to gender differences in CRC through a

mechanism involving MSI.

The presence of low levels of ERs can be linked to hypermethyaltion of the

estrogen receptors (Slattery et al., 2001). However, the mechanism linking

estrogens to ER methylation and to MSI is not yet known.

Since ER inactivation is due to hypermethylation in 90% of colon cancers

(Issa et al., 1994), it was hypothesized that estrogens affect DNA methylation in

general (Slattery et al., 2001). In fact, it was suggested that estrogens might be key

factors in the development of the CpG island methylator phenotype (the CIMP

phenotype) (Baylin & Herman, 2000; Newcomb et al., 2007), a phenotype

commonly present in many different types of tumor. In CIMP tumors,

hypermethylation of promoter regions of regulatory genes, such as tumor

suppressor genes, is a general feature. This CIMP phenotype might be the missing

link between estrogens and the hyper-methylation of the ERs and the MSI

phenotype, as MSI in the sporadic tumors is mostly caused by hypermethylation of

the MLH1 promoter.

Besides global hypermethylation (CIMP phenotype), global hypomethylation

of introns and coding sequences of genes is also observed in tumors. In CRC,

35%-60% of the cases show reduction of methylation (Shen et al., 2009). An

example of a gene that is hypomethylated in estrogen-responsive tumors is PAX2

(Wu et al., 2005). This is corroborated by in vitro and animal studies that showed

that estrogens lead to a lower DNA methylation of specific genes and that they are

able to restore protective methylation patterns (Newcomb et al., 2007).

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Cancer progression

Normal endometrium

Simple endometrium hyperplasia

Type 1 endometrioid adenocarcinoma

Atypical hyperplasia

Complex endometrium hyperplasia

Histological state

Mutations in target genes

Other genes

Estrogens and the MSI Model

cell proliferation apoptosis

Hyper-methylation of MLH1

High Estrogen Receptor activity

Mutations in genes e.g. MLH1

Hypo-methylation of PAX2

Methylation aberrations

DNA damaging effect by estrogen metabolites

Hyper-methylation of Estrogen receptor

MSH2

More estrogens> more MMR activity and vice versa

MSI MSI

Figure 2. Model for the role of estrogens in the progression of microsatellite unstable tumors.

Estrogens, Mismatch Repair and cancer Slattery et al. (2001) raised the idea that at least one of the major MMR genes is

estrogen-responsive and that loss of estrogen results in loss of DNA mismatch

repair capacity. Wada-Hiraike and colleagues (2005) reported a direct interaction

between ER and the MMR gene, MSH2, from immunoprecipitations and pull down

assays. In fact the authors suggested that MSH2 is a potent co-activator of ERα.

An interesting possibility arose from this study: common co-activators of ER and

even ER itself might have a functional role in DNA MMR (Wada-Hiraike et al.,

2005). Miyamoto et al. (2006) demonstrated that cells under a high-level estrogen

environment have increased levels of both MLH1 and MSH2 proteins. Moreover, it

was observed by the same authors that MLH1 and MSH2 expression is up-

regulated and activity MMR increased by estradiol treatment mediated by the ER

pathway (Miyamoto et al., 2006). The higher MMR activity would ideally

compensate the replication errors occurring at a highly proliferative stage. Lower

MMR activity would then also explain the occurrence of endometrial carcinogenesis

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in a less proliferative stage, when the estrogens levels are low, as in

postmenopausal women. These data support all the association between

estrogens and MMR, suggesting an estrogen-mediated transcriptional activation of

the MMR complex protein.

In summary, both studies show a positive correlation between estrogens and

MMR activity. So high levels of estrogen give rise to higher cell proliferation and the

system seems to protect itself against DNA damage by activating the MMR system.

Thus estrogens may initially protect against cancer by activating the MMR system.

However, when the MMR system is deregulated, for instance, by hypermethylation

of the MLH1 gene, this protective mechanism is lost. It is also interesting to note

that tumors of the endometrium are seen mostly in postmenopausal women,

women who have low levels of estrogens.

ESTROGEN AND ENDOMETRIAL CANCER

Endometrial cancers (EC) can be divided in two classes, an estrogen- associated

type and a non-estrogen-associated type. The first group, to which all MSI-high

endometrium tumors belong, is called type I EC. These tumors are found in women

with long-term unopposed exposure to estrogens, which might be caused by

nulliparity, early menarche, late menopause or the use of estrogen replacement

therapy. Obesity is also considered a major risk factor, as adipose tissue gives rise

to a higher estrogen concentration (Salama et al., 2008). About 80% of all

endometrial carcinomas are estrogen-associated carcinomas (Amant et al., 2005;

Sherman, 2000).

Interestingly, the profile of estrogens and metabolites present in these

tumors seems to play an important role in the mechanism leading to endometrial

cancer. Different rates of the different possible metabolites of estrogens are

associated with different effects on the endometrium, and thus carry different risks

of developing endometrial cancer (Takahashi et al., 2004).

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Tamoxifen, estrogen and endometrial cancer Besides estrogens, tamoxifen can also be associated with cancer development, in

particular endometrial cancer. Tamoxifen is a drug used as adjuvant treatment of

ER–positive breast cancer. It acts as an estrogen antagonist in those tumors,

reducing tumor growth. However, it was shown that it acts as an estrogen agonist

in other tissues such as bone, where it prevents osteoporosis (Howell et al., 2004;

Smith & O`Malley, 2004). In the endometrium it induces cell proliferation. An

increased risk of developing EC is reported for postmenopausal women

undergoing tamoxifen therapy (Polin & Ascher, 2008). The working mechanism of

tamoxifen is binding of the compound to ERs and inducing a tamoxifen-specific

signaling (see figure 3). This signaling probably depends on the concentration of

estrogen (e.g. menstrual status of the patient), the ratio between ERα and ERβ,

and on the expression of the co-activators and co-repressors, all of which might be

tissue-specific.

Estrogen

Estrogen receptors

Proliferation Apoptosis processes processes

ERα ERβ ERα/ERβ ERα

ERβ

Tamoxifen

Estrogen receptors

ERα

ERβ

ERα ERβ ERα/ERβ

Expression is regulated

by co-activators and

co-repressors

Tissue specific / Tamoxifen specific target genes

Tissue specifc / Target genes activated by both Estrogen and Tamoxifen

Tissue specific / Estrogen specific target genes

receptor subtype specific activation by estrogen

receptor subtype specific activation by tamoxifen

Expression is regulated by co-activators and co-repressors

Figure 3. Estrogen and tamoxifen signaling; different factors affecting the signaling pathways of both

ligands.

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ESTROGENS AND COLON CANCER Expression of ERs has also been demonstrated in non-hormonally responsive

tissues, such as the gastrointestinal mucosa and its associated tumors, suggesting

that estrogens also have a role in these tissues that were previously not thought of

as hormone-responsive tissues (Potter 1995; Grodstein et al., 1999; Slattery et al.,

2001; D’Errico and Moschetta, 2008).

All findings in colorectal cancer support the hypothesis that high estrogen

levels can have a protective effect in specific phases of life. These findings have

been used as an explanation for the gender bias observed in CRC incidence, with

women having a lower incidence of the disease than men. Hormonal changes

associated with pregnancy (McMichael and Potter, 1980), and hormone

replacement therapy (HRT) have been associated with lower risk of CRC (Potter,

1995; Peipins et al., 1997; Chen et al., 1998; Kadiyska et al., 2007).

Most likely this protective effect of estrogens in CRC largely depends on the

ratios of receptor α and β. ERβ is the predominant ER isoform in colon tissue and

probably the responsible isoform for estrogen transcriptional effects (Foley et al.,

2000; Jassam et al., 2005; Kennelly et al., 2008). This situation is quite different in

breast and endometrium, where ERα is the main isoform present. It has, however,

been suggested that ERβ modulates the function of ERα, and that an increased

ratio of ERα/ERβ is associated with a progression from a healthy to carcinoma

state in those tissues (Jazaeri et al., 2001).

ESTROGENS AND KNOWN CANCER-RELATED PATHWAYS

Estrogens can activate proteins other than the ERs. For instance they have been

reported to activate the protein kinase A pathway (Fu & Simoncini, 2008), by

binding to the G protein–coupled receptor GPR30. Moreover, both insulin-like

growth factor 1 (IGF-1) receptor signaling and EGF receptor signaling can be

activated by estrogens (Song et al., 2007). Binding of estrogen to these growth

receptors leads to dimerization of the receptor, and activation of their kinase

activity. Phosphorylation of these proteins leads to activation of downstream

87

signaling pathways, such as the MAPK and PI3K/Atk pathways. The activation of

PI3K/Akt pathway has been observed in ER-positive human breast cancer cells

(Castoria et al., 2001; Marquez & Pietras, 2001; Sun et al., 2001; Duan et al.,

2002; Razandi et al., 2004; Lee et al, 2005), in rat and mouse endometrial

epithelial cells (Dery et al., 2003; Chen et al., 2005), and in human endometrial

cells during the proliferative phase (Guzeloglu Kayisli et al., 2004). Activation of

PI3K/Akt has been associated with cell survival in a variety of cancers (Castoria et

al., 2001; Lee et al., 2005).

CO-ACTIVATORS AND CO-REPRESSORS OF ER PATHWAY ER-mediated transcriptional regulation depends on the recruitment of co-activators

and components of the RNA polymerase II transcription complex, which enhances

target gene transcription (Klinge, 2000). Thus, the cellular availability of co-

activators and co-repressors is an important determinant in the biological response

to both steroid hormone agonists and antagonists in ER responsive tissues

(Edwards, 2000).

Many such co-activators and repressors contribute to ER-mediated

transcription events. ER-dependent gene transcription frequently depends on the

presence of FOXA1. FOXA1 is expressed in the mammary gland, liver, pancreas,

bladder, prostate, lung, and colon. Recently, Carroll et al. demonstrated that

FOXA1 is required for optimal expression of nearly 50% of ERα-regulated genes

and estrogen-induced proliferation, by enhancing binding of ERα to its target genes

(Laganière et al., 2005; Carrol et al., 2005; Carrol & Brown, 2006).

In colon, binding of estrogens to ERα induces a cancer promoting response,

whereas binding to ERα seems to exert a protective action (Weyant et al., 2001).

Reasons for this can reside not only in the different expression patterns of ERα and

ERβ in vivo but also their need to interact with cellular transcription cofactors which

are not functionally equivalent and ubiquitously expressed in all cells (McDonnell &

Norris, 2002), highlighting the importance of the ER-co-activators in colon cancer.

In endometrium, it has been suggested that co-regulators of ER are involved

in tumor progression. It has been proposed that p160 steroid receptor cofactor

88

(SRC) can modulate ER activity and that overexpression of another member of

SRC family, AIB1 (Amplified in Breast Cancer 1) in endometrial carcinoma lead to

ER increased action and consequent progression to malignancy (Balmer et al.,

2006). Moreover, recently we identified mutations in NRIP1 in MSI-H endometrium

tumors in 35% of the investigated tumors (Ferreira et al., unpublished data). NRIP1

is a co-repressor of ER signaling. Our data convincingly show the importance of

ER cofactors in the development of the tumor type.

GENERAL CONCLUSIONS

Estrogens are essential for maintaining of several tissues in humans, but they also

play an important role in the carcinogenic process of many different tumor types.

Although we know fairly well how estrogen signals, it is still not totally clear how

estrogen exerts different effects in different tissues. The differences in the effects of

estrogen mentioned in this review in the endometrium and colon may be partly

explained by the different physiological roles these organs: the endometrium

belongs to the reproductive system, and is a main target of sex hormones, whereas

the colon belongs to the digestive system, which is less influenced by sex

hormones. Moreover, the regulation of estrogen-responsive genes is different

among tissues and is, for example, dependent on the mechanism of action of the

ligand or the distribution of ER isoforms alpha and beta and their dimerization

(Castiglione et al., 2008). Also the co-activator and co-repressor molecules might

exist in different combinations or concentrations among tissues and lead to

different results.

Furthermore, several ER and non-ER related pathways are now known to

activate or be activated by estrogens. These pathways might be affected differently

in distinct tissues and therefore have a broad spectrum of influence in tumor

formation. Therefore the networks they form with estrogen should not be forgotten,

even in tumors occurring in less hormonally-dependent organs.

There is still a lot to learn about the possible connection between

microsatellite instability (MSI) and estrogens. Estrogens may have a protective

effect, which is likely lost after the MMR system has somehow been modulated

89

(mutated). On the other hand estrogens can, by there carcinogenic effect, directly

(by mutations) or indirectly (by methylation) inactivate the MMR pathway which

results in MSI. MSI can subsequently result in mutations in cofactors or ER

signaling which will modulate ER regulated transcription. Clearly the MMR system

is a (direct or indirect) target of estrogens, making genes involved in the estrogen

pathway potential candidates to be studied in MMR-deficient tumors. These

findings might also prove useful in the design of novel therapies for such tumors.

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96

CHAPTER 6

General Discussion, Conclusions and Future

Perspectives

97

DISCUSSION

MSI profiles change during the adenoma to carcinoma sequence, but not between colorectal and endometrial carcinomas Microsatellite instability (MSI) is the predominant type of genetic instability present

in the tumors of Lynch syndrome patients and also in a subset of sporadic

colorectal and endometrial tumors (Boland et al., 1998). It is generally accepted

that MSI can be found in the early stages of tumor progression, such as at

adenoma level. Studies comparing patterns of MSI in different tumor types and

stages suggest that different levels of instability are observed in tumors originating

in different tissues or in different stages of tumorigenesis (Furlan et al., 2002;

Kuismanen et al., 2002). However, the frequencies reported by different studies

vary widely and data on the qualitative differences are scarce.

In chapter 2 of this thesis we analyzed mononucleotide and dinucleotide MSI

markers to define specific qualitative MSI profiles in colorectal adenomas and

colorectal carcinomas. We included tumors from Lynch syndrome patients and

from sporadic cases in order to elucidate possible differences between tumors of

hereditary and sporadic origin. In chapter 3 we focused on MSI profiling of

colorectal carcinomas versus endometrial carcinomas. We looked at features such

as instability frequencies, type of microsatellite mutations, and the size of these

mutations in order to evaluate whether these features are tissue-dependent and

thus might reveal distinct profiles of MSI between tumors of distinct origin.

We found differences in MSI frequencies during the transition from adenoma

to carcinoma, as expected from the literature (Shibata et al., 1994; Grady et al.,

1998; Loukola et al., 1999; Iino et al., 2000; Sugai et al., 2003; Giuffrè et al., 2005).

The adenomas in our study showed a significantly lower proportion of MSI-H cases

than the colorectal carcinomas, both in non-carriers and in truncating mutation

carriers. Considering the different distributions of instability, our results from the

adenomas were of particular interest. It was mainly the dinucleotide markers that

were unstable in colorectal adenomas from non-carriers, whereas a very low

frequency of mononucleotide instability was seen, resembling the MSI-L CRC of

non-carriers. In contrast, mononucleotide instability was generally predominant in

the adenomas of truncating mutation carriers. Based on these data we suggest

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that mononucleotide instability is a very early event in the carcinogenic process of

tumors with mismatch repair mutations, and that mononucleotide instability

precedes that of dinucleotide repeats in such tumors. We further hypothesize that

the dinucleotide instability in non-carriers represents a kind of background

instability, as seen in MSI-L CRC tumors, which is not a sign of an underlying MMR

deficiency, whereas in Lynch syndrome tumors, with proven MMR deficiency,

mononucleotide instability can be considered to truly result from the underlying

MMR deficiency.

A possible explanation for these findings might be that the normal MMR

system more easily corrects mismatches in mononucleotides than in dinucleotides.

We assume that if more instability is seen in dinucleotide repeats in MMR-proficient

tumors (which are less frequent repeats in the genome than mononucleotide

repeats) then this suggests either a higher vulnerability of dinucleotide repeats to

the occurrence of mismatches and/or a lower capacity of the normal MMR system

to repair them.

In chapter 3 we suggest that it is not possible to define clearly different MSI

profiles distinguishing MSI-H CRC and EC, as we observed that the MSI-related

features that we studied showed similar patterns in both types of carcinomas. The

frequency of mononucleotide and dinucleotide instability found in both types of

tissues is comparable, with mononucleotide and dinucleotide markers being

affected at similar levels. In terms of type of microsatellite mutation,

mononucleotide markers virtually always become shorter, whereas dinucleotide

markers can become shorter and/or longer, both in CRC and EC. This close

association of the occurrence of insertions or deletions with the type of MSI marker

suggests that characteristics of the repeats, such as repeat length, have a bigger

influence on the type of mutation occurring at a microsatellite repeat than the tissue

origin of the tumor in which those mutations arise. Differences between CRC and

EC could be seen in terms of the size of the deletion/insertions detected, with EC

having shorter alterations than CRC. However, the relative differences between the

markers remained similar in both tissue types, leading to comparable patterns of

instability. We propose that the differences observed might indicate different

durations of tumor development and/or differences in tissue turnover between

99

colorectal and endometrial epithelium, rather than reflecting different profiles of the

two tumor types.

Implications for diagnostics

Since we observed that the MSI-H adenomas from mutation carriers mainly

showed mononucleotide instability, our results have implications for the diagnosis

of Lynch syndrome colorectal adenomas. Our results indicated that analyses of

mononucleotide markers are advantageous for identifying colorectal adenomas

and carcinomas associated with Lynch syndrome. To our knowledge, our data are

the first to show that the use of a panel of only mononucleotide markers, as

previously recommended for the detection of MSI-H hereditary CRC (Buhard et al.,

2004), can also be used to advantage in the identification of Lynch syndrome

patients by testing colon adenomas.

With respect to the MSI detection in carcinomas, we suggest that the same

MSI tests can be used for both colorectal and endometrial tumors, as we found no

great differences between the EC and CRC MSI profiles; the differences were not

enough to justify using different MSI tests for the two tumor types.

Identification of new target genes for MSI tumors: genes in the estrogen-receptor pathway are good candidates Genes containing repeats are frequent targets of mutations in MMR-deficient

tumors. Particularly mutations accumulating at coding sequences of important

regulatory genes (target genes) have been implicated in the development of MSI

tumors. The profile of target genes affected in MSI-H CRC has been well

established, with several genes being highly mutated, such as TGFβ-RII which has

a mutation frequency of approximately 90% in these tumors. For MSI-H EC, the

profile is less well known, or at least genes having such a high mutation frequency

have not been identified so far. Previous studies suggest that the profile of target

genes differs between endometrial and colorectal carcinomas and that frequently

mutated target genes remain to be found in the EC (Duval et al., 2002).

In chapter 4 of this thesis we described our work on the identification of new

target genes for EC. We identified 44 genes that were mutated in the MSI-H EC we

examined, of which seven were mutated relatively frequently. We propose these

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seven genes – NRIP1, SRPR, MBD6, JAK1, KIAA1009, JMJD1C, and ADD3 – as

new target genes for MSI-H EC. They encode proteins with several functions, with

some already reported to play a role in cancer. Interestingly, the most frequently

mutated gene in EC in our study was NRIP1 (34%). This gene encodes a co-

repressor protein of the estrogen-receptor (ER) pathway. The ER is an essential

pathway for endometrial tissue regulation; the endometrium is a hormonal-

responsive tissue, highly regulated by the concentrations of estrogens. Several

estrogen-responsive genes have already been described, and genetic alterations

in ER and those ER-responsive genes are thought to be key players in the

development of hormone-associated tumors, such as endometrial carcinomas

(Notarnicola et al., 2001). High exposure to estrogens is currently considered the

major risk factor for developing EC. Approximately 80% of all sporadic EC tumors –

the endometrioid endometrial carcinomas – are estrogen-associated carcinomas

(Doll et al., 2008). NRIP1 has been described as essential for female fertility in

mice (White et al., 2000), and mutations in NRIP1 may act as a predisposing factor

for human endometriosis (Caballero et al., 2005). We believe that it is very likely

that the NRIP1 mutations we found in our MSI-H endometrial carcinomas might

result in functional differences at the ER-pathway level. We expect inactivation of

NRIP1 to interfere with the process of co-repression of the ER complex and lead to

differences in the expression of estrogen-responsive genes that could eventually

result in tumor growth advantages, as previously observed in breast cancer studies

(White et al., 2005).

We further showed that most of these genes are also mutated in colorectal

and gastric tumors, although in different frequencies. These results confirm that

some target genes show tissue specificity, while others seem to play a more

common role in MSI-H tumors, independently of the tissue origin. We were

surprised to find NRIP1 mutations in colorectal carcinomas. All the reasons

mentioned above make NRIP1 an obvious target gene for EC, but a less obvious

one for CRC. However, this gene has already been reported as a target gene for

gastrointestinal MSI tumors, despite their lower mutation frequencies in such

tumors. Frameshift mutations were found in an A9 coding microsatellite, in 13% of

MSI-H GCs and in 7% of MSI-H CRC (Røyrvik et al., 2007). Furthermore, although

not a typical hormone-associated cancer, CRC does have a hormone component.

101

The presence of estrogen receptors and products of estrogen-related genes in the

colon suggests that estrogens have a role in the organization and architectural

maintenance of the colon, and their down-regulation accompanies the progression

of CRC (Francesca et al., 2008). Moreover, some studies suggest that the

combined estrogen and progesterone hormone replacement therapy might be the

factor underlying the reduction of incidence of CRC in postmenopausal women

(Chlebowski et al., 2004). In addition, CRC incidence and mortality rates are lower

in females than in males. Some authors have therefore suggested that estrogens

have a protective effect against CRC (Wada-Hiraike et al., 2006). Slattery et al.

(2001) explored the contribution of several estrogen-related factors to the

differences in MSI tumor frequency observed in men and women, and also in

younger versus older women. They showed that withdrawal of estrogen may

increase the risk of MSI-positive CRC. In fact, our finding of NRIP1 mutations in the

CRC group reinforces the possible link between this particular gene (and also the

ER pathway) and MSI carcinogenesis.

This subject is more thoroughly addressed in chapter 5, in which we tried to

clarify the mechanisms linking hormones to Lynch syndrome-associated tumors

and, in particular, to discuss how hormones could play a role in MSI tumorigenesis.

Our data suggest that there might be a stronger link between hormones and MSI

than so far thought, and that genes of hormone-related pathways, such as the ER

pathway, might be good candidates for target genes in MSI-H tumors, not only for

estrogen-responsive tissues, such as the endometrium, but also for other tissues

such as the colon.

CONCLUSIONS

Our data have provided new insights into the process of MSI-H related tumor

development. Our results suggest that mononucleotide instability is a very early

event in the carcinogenic process of colorectal tumors with mismatch repair

mutations, and that mononucleotide instability precedes that of dinucleotide

repeats in such tumors. We therefore propose that analyses of mononucleotide

markers are advantageous for identifying colorectal adenomas and carcinomas

102

associated with Lynch syndrome. Furthermore, we showed that there were no

substantial differences of MSI profile between CRC and EC, thus we propose that

the same MSI tests can be used for both colorectal and endometrial tumors. We

also found mutations in coding microsatellite repeats likely to indirectly affect the

estrogen-receptor pathway in MSI-H tumors. Our data suggest that genes in the

ER pathway would be very good candidate genes for mutation analysis in MSI-H,

and possibly also in microsatellite-stable tumors. These findings could prove

interesting in the design of novel therapeutic treatments.

FUTURE PERSPECTIVES

Although the work presented in this thesis gave many new insights in MSI and in

the development of MMR-related tumors, it also raised many questions and it

opened avenues for further research in this field.

With respect to the new findings on microsatellite instability patterns of

colorectal adenomas and carcinomas, it would be very interesting to conduct

further (basic) experiments. It would for instance be of great interest to obtain a

large set of micro-dissected adenoma and carcinoma tissues from the colon of the

same Lynch syndrome patient to evaluate at a larger scale the differences in MSI

profile between adenomas and carcinomas. Moreover, analyzing several micro-

dissected samples of different areas of the same MMR-deficient adenoma would

give us great insight in this matter. Finding only mononucleotide instability or

simultaneous mono- and dinucleotide instability, and no areas with only

dinucleotide instability, would corroborate our hypothesis that mononucleotide

repeats are targeted first in adenomas with MMR mutations. Finding this in

colorectal adenomas raises the question whether this holds true for all MMR-

related cancers. Extending such studies to other types of Lynch syndrome cancers,

such as endometrial or gastric, would answer these questions.

The same applies to our project comparing MSI profiles in CRC and EC.

Although we are convinced that for these two tumor types the patterns of MSI are

comparable, we do not know whether the same holds true for all MMR-related

tumor types. Therefore, it would be good to compare colorectal tumors with all

103

other MMR-related tumors. Without these data we do not know whether the MSI

test generally used is good for all MMR-related tumor types.

Considering our project on target genes, studies at the functional level will be

of major relevance to show how the genes identified in this project are implicated in

tumor development and progression. Silencing of these genes in vitro and in vivo,

and analyzing the effects on tumor-associated processes, such as proliferation,

apoptosis or invasion, would be of great help to elucidate the effects of the

identified truncating mutations in those genes on cancer development. Studying

the highest mutated gene, NRIP1 gene, could not only prove the involvement of

this gene in cancer development, but also it could help us in understanding the

complicated field of hormone-responsive pathways and their involvement in

mismatch repair- deficient tumors. Finding one member of a pathway mutated

might indicate that other members of the same pathway might also play a role in

tumor development; in case of NRIP1, other members of the ER pathway, a

pathway frequently targeted in therapeutic procedures for estrogen-responsive

cancers, seem to be appealing targets to study in MMR-deficient tumors. Their

possible involvement and knowledge of their mechanism of action might lead to a

better understanding of tumor response or tumor resistance to some hormone-

related therapies.

104

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106

CHAPTER 7

SUMMARY

107

SUMMARY

Microsatellite instability (MSI) is the predominant type of genetic instability present

in tumors of Lynch syndrome patients and in a subset (15-25%) of sporadic

colorectal (CRC), endometrial (EC) and gastric tumors (GC). The underlying

mechanism of MSI is a defect in the DNA mismatch repair (MMR) pathway. MSI is

characterized by the accumulation of mutations in short, repetitive DNA sequences,

also called microsatellites. This type of mutation can be found in both non-coding

and coding microsatellites. This thesis presents a study on MSI in tumors from

Lynch syndrome patients, with particular emphasis on colorectal and endometrial

carcinomas and their sporadic counterparts.

In chapter 1 the general background to Lynch syndrome, microsatellite

instability, and to the cancers traditionally associated with these MMR defects is

presented. In chapter 2 we describe a study on how microsatellite instability

evolves along the adenoma-carcinoma sequence of colorectal cancer. We found

comparable MSI profiles, measured by the relative frequency of mono- and

dinucleotide unstable markers, in sporadic colorectal adenomas and carcinomas.

However, we found differences in MMR gene-truncating mutation carriers:

colorectal adenomas showed instability almost exclusively in mononucleotide

repeats whereas the frequency of dinucleotide marker instability was markedly

increased in colorectal carcinomas. We concluded that MSI profiles differ between

familial and sporadic cases, and that mononucleotide marker instability precedes

dinucleotide marker instability during colorectal tumor development in Lynch

syndrome patients. We therefore suggest that mononucleotide markers should be

the preferred markers for identifying possible Lynch syndrome patients.

In chapter 3, we address whether the MSI profiles of colorectal and

endometrial MSI-high (MSI-H) tumors differ. To answer this question we analyzed

the frequency of the MSI, the type of mutation (deletions/insertions), and the size of

the microsatellite mutation occurring at three mononucleotide repeat markers and

at three dinucleotide markers in both CRC and EC. The frequency of mono- and

dinucleotide instability found in both tissues was comparable, with mononucleotide

and dinucleotide markers being affected at similar levels. We show that the type of

mutation is a marker-dependent and not a tissue-dependent feature, since we

108

observed for both tissues almost exclusively deletions in mononucleotide markers,

and both deletions and insertions in dinucleotide markers. The size of the

deletions/insertions also differs between CRC and EC, with EC having shorter

alterations than CRC. We concluded that there was no substantial difference

between the MSI profiles of CRC and EC tumors. Furthermore, our data also

showed that the same MSI tests could be used for both tumor types.

Chapter 4 describes our hunt for new target genes for MSI-H endometrial

cancer. MSI is characterized by the accumulation of mutations in both non-coding

and coding microsatellites, and genes containing microsatellites are frequent

targets of mutations in MMR-deficient tumors. Particularly mutations in important

regulatory genes – which we call target genes – are thought to be key players in

the development of MSI-H related tumors. We set up an endometrium-specific

strategy to find new target genes for MSI-H endometrial tumors. We screened

genes that are expressed in normal endometrium tissue and that contain repetitive

sequences. From a list of 2,338 genes expressed in the normal endometrium, 382

genes were found to contain 496 repeats and these genes were therefore

sequenced. Mutations in these repeats were found in 44 genes, but whether all 44

genes really contribute to tumor development can be debated. A major criterion for

selecting target genes that really contribute to tumors is the mutation frequency.

Generally a cut-off of 15% is taken as revealing a real target gene. Genes mutated

in lower frequencies are considered bystanders. When we applied this 15% cut-off,

we found seven new, real, target genes. Subsequently we also analyzed 10 real

EC target genes in colorectal and gastric MSI-H carcinomas. Our study confirmed

that some target genes show tissue specificity, while others seem to play a more

common role in MSI-H tumors, independently of the origin of the tissue.

The gene most frequently mutated in EC was NRIP1 (34%). This gene

encodes a co-repressor protein of the estrogen-receptor (ER) pathway, one of the

main pathways known to play a role in endometrial carcinogenesis. Surprisingly

this gene was also highly mutated in CRC (24%). These results point towards an

important role for the ER pathway in the development of MSI CRC tumors as well.

Our data also suggest that genes of the ER pathway might be good candidates for

target genes in MSI-H tumors, not only for estrogen-responsive tissues, such as

the endometrium, but also for tissues of different origin such as the colon.

109

The findings described in chapter 4 led us to write a review in which we tried

to clarify the mechanisms linking hormones to Lynch syndrome-associated tumors

and, in particular, to discuss how hormones can play a role in MSI tumorigenesis.

In general our data has provided new insights into the process of MSI-H

related tumor development: we propose that in the colorectal adenoma-carcinoma

sequence of Lynch syndrome tumors mononucleotide instability precedes

dinucleotide instability; we showed that there is no substantial difference of MSI

profile between CRC and EC; and we found mutations that are likely to affect the

estrogen-receptor pathway. Our data suggest that genes in the ER pathway are

perfect candidate genes for mutation analysis in MSI-H, but possibly also in

microsatellite-stable tumors. These findings might prove interesting in designing

novel therapeutic treatments.

110

NEDERLANDSE SAMENVATTING

Microsatelliet instabiliteit (MSI) is de voornaamste vorm van genetische instabiliteit

in tumoren van Lynch syndroom patiënten en deze vorm van instabiliteit wordt ook

gevonden in 15 tot 25% van sporadische colorectale, endometrium en

maagtumoren. MSI ontstaat door een defect in de zogenaamde MisMatch Repair

(MMR) route. MSI karakteriseert zich onder andere door de accumulatie van

mutaties in korte repeterende DNA sequenties, zogenaamde microsatellieten.

Deze mutaties kunnen optreden in zowel niet-coderende als coderende

microsatellieten. Voor dit proefschrift is MSI bestudeerd in tumoren van patiënten

met erfelijke colorectale en endometrium carcinomas (Lynch syndroom) en hun

sporadische tegenhangers.

In hoofdstuk 1 wordt algemene achtergrondinformatie gegeven over het

Lynch syndroom, microsatelliet instabiliteit, en over kankers die van oudsher

geassocieerd zijn met MMR defecten. In hoofdstuk 2 beschrijven we een studie die

laat zien hoe microsatelliet instabiliteit zich ontwikkelt met betrekking tot de

adenoma-carcinoma sequence van colorectaal kanker. We hebben vergelijkbare

MSI profielen gevonden in sporadische adenomas en carcinomas. MSI profielen

zijn gemeten door de relatieve frequentie van instabiele mono- en dinucleotide

markers te bepalen. Voor patiënten met een truncerende mutatie in één van de

MMR genen hebben we echter wel verschillen gevonden: colorectale adenomas

hebben vrijwel alleen instabiliteit in mononucleotide repeterende sequenties, terwijl

in colorectale carcinomas de instabiliteit in dinucleotide markers aanzienlijk is

verhoogd. We concluderen dat MSI profielen verschillen tussen erfelijke en

sporadische kanker en dat mononucleotide marker instabiliteit voorafgaat aan

dinucleotide marker instabiliteit tijdens de ontwikkeling van een colorectale tumor in

Lynch syndroom patiënten. We stellen daarom voor dat met name mononucleotide

markers getest moeten worden om mogelijke Lynch syndroom patiënten te kunnen

identificeren.

In hoofdstuk 3 gaan we in op de vraag of de MSI profielen verschillen tussen

colorectale en endometrium tumoren die MSI zijn (dit noemen we MSI-High of MSI-

H). We hebben de MSI frequentie bepaald, het type mutatie (deleties of inserties)

en de grootte van de microsatelliet mutatie die optreedt in drie mononucleotide

111

repeterende sequentie markers en in drie dinucleotide markers zowel in colorectale

als endometium tumoren. De mononucleotide en dinucleotide mutatiefrequentie die

in beide weefsels is gevonden, was vergelijkbaar, evenals de mutatiefrequentie in

de mononucleotide en de dinucleotide markers. We laten zien dat het type mutatie

marker specifiek is en niet weefsel specifiek. In mononucleotide markers treden

vrijwel alleen deleties op, terwijl in dinucleotide markers deleties en inserties

voorkomen. De grootte van de deleties/inserties verschilt tussen colorectale en

endometrium tumoren, waarbij bij in endometrium tumoren de deleties en inserties

kleiner zijn dan bij colorectaal tumoren. We concluderen dat er geen substantieel

verschil is tussen het MSI profiel van colorectale en endometrium tumoren.

Bovendien laten onze data zien dat dezelfde MSI testen gebruikt kunnen worden

voor beide tumortypes.

Hoofdstuk 4 is gewijd aan een studie naar nieuwe ‘targetgenen’ voor MSI-H

endometrium kanker. Zoals hierboven vermeld, wordt MSI gekarakteriseerd door

de accumulatie van mutaties in korte herhalende DNA sequenties in zowel niet-

coderende als coderende DNA sequenties. Genen die herhalende sequenties

bevatten, zijn de beste ‘targets’ voor mutaties in MMR-deficiënte tumoren. Vooral

van mutaties die optreden in belangrijke regulatiegenen, die noemen we

‘targetgenen’ wordt aangenomen dat deze zeer belangrijk zijn voor de ontwikkeling

van MSI-H gerelateerde tumoren. Wij hebben een endometrium-specifieke

strategie gebruikt om nieuwe ‘targetgenen’ te identificeren. Mutatie analyse is

uitgevoerd op die genen die daadwerkelijk in normaal endometriumweefsel tot

expressie komen (10x hoger dan het achtergrond signaal) en die daarnaast

herhalende DNA sequenties bevatten. Uit een lijst van 2338 genen die tot

expressie komen in normaal endometriumweefsel, bleken 382 genen 496

herhalende sequenties te hebben. Van deze genen is de sequentie bepaald. In 44

genen hebben we mutaties gevonden. Het is discutabel of alle 44 genen ook

daadwerkelijk bijdragen aan de ontwikkeling van een tumor. Een belangrijk

criterium om genen aan te merken als ‘targetgenen’ die bijdragen aan

tumorontwikkeling is de mutatiefrequentie. Over het algemeen wordt een ‘cut off’

waarde van 15% genomen om een gen als target gen aan te merken. Genen met

een lagere mutatiefrequentie worden als ‘bystanders’ beschouwd. We hebben

zeven nieuwe targetgenen gevonden, gebaseerd op een cut off waarde van 15%.

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Daarnaast hebben we deze zeven target genen plus drie genen die bijna in 15%

van de tumoren gemuteerd zijn in MSI-H endometrium tumoren, in MSI-H

colorectale en maag carcinomas getest. Onze studie laat zien dat sommige

targetgenen specifiek voor een bepaald weefsel zijn, terwijl andere een meer

algemene rol lijken te spelen in de ontwikkeling van MSI-H tumoren onafhankelijk

van het type weefsel.

Het gen met de hoogste mutatiefrequentie in endometrium tumoren was

NRIP1 (34%). Dit gen codeert voor een co-repressor eiwit met een rol in de

oestrogeen-receptor (ER) route, een heel belangrijke signaal route in het goed

functioneren van het endometrium. Dit gen liet ook hoge mutatiefrequenties in

colorectaal tumoren zien (24 %), duidend op ook een mogelijke rol van de ER route

in de ontwikkeling van MSI colorectaal tumoren. Onze data suggereren daarnaast

dat de genen van de ER route goede kandidaat ‘targetgenen’ in MSI-H tumoren

kunnen zijn. Dit niet alleen voor weefsels die door oestrogenen worden

gereguleerd, zoals het endometrium, maar ook voor weefsels van een andere

oorsprong, zoals colonweefsel.

De resultaten die beschreven staan in hoofdstuk 4 hebben aanleiding

gegeven om een review artikel te schrijven waarin we de mechanismen

beschrijven die de rol van hormonen in de ontwikkeling van Lynch syndroom

geassocieerde tumoren verklaren, vooral de rol van hormonen in MSI

tumorontwikkeling.

Concluderend leiden onze data tot nieuwe inzichten in het proces van MSI-H

tumorontwikkeling. We hebben aangetoond dat er geen substantiële verschillen

bestaan tussen colorectale en endometrium tumoren. Daarnaast hebben we

mutaties gevonden in 7 genen die mogelijk allemaal op directe of indirecte wijze

invloed hebben op de ontwikkeling van de tumoren waarin ze zijn gemuteerd.

Belangrijk was de bevinding dat het hoogst gemuteerde gen dat werd gevonden

codeert voor een co-repressor van de oestrogeen-receptor route. Onze data

suggereren dat genen in de ER route de perfecte kandidaat-genen zijn voor

mutatie analyse in MSI-H tumoren en wellicht ook in tumoren die microsatelliet

stabiel zijn. De laatstgenoemde resultaten kunnen van belang zijn voor de

ontwikkeling van nieuwe therapeutische behandelingen.

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RESUMO

A instabilidade de microssatélites (MSI) é o tipo predominante de instabilidade

genética presente em tumores de doentes com Síndrome de Lynch e também

numa fracção (15-25%) de carcinomas esporádicos do cólon, endométrio e

estômago. Na origem da instabilidade de microssatélites estão deficiências no

sistema de mismatch repair (MMR) do DNA. A MSI caracteriza-se pela

acumulação de mutações em sequências repetitivas do genoma, também

chamadas microssatélites. Este tipo de mutação pode ser encontrado em

microssatélites codificantes e não-codificantes. Nesta tese, é apresentado um

estudo de MSI em tumores de pacientes com Síndrome de Lynch, com particular

enfâse nos carcinomas colorectais (CRC) e endometriais (EC), e respectivos

equivalentes esporádicos.

No capítulo 1 são apresentadas noções gerais sobre Síndrome de Lynch,

MSI, e sobre os cancros tradicionalmente associados com as deficiências de

MMR. No capítulo 2 descrevemos um estudo sobre como a instabilidade de

microssatélites evolui ao longo da sequência adenoma-carcinoma dos carcinomas

colorectais. Considerando a frequência relativa de instabilidade dos marcadores

de MSI compostos por mononucleotídeos e nos marcadores compostos por

dinucleotídeos, encontrámos perfis de MSI comparáveis nos adenomas e

carcinomas esporádicos. No entanto, foi possível observar diferenças nos

tumores portadores de mutações em genes do sistema de MMR - os adenomas

apresentam instabilidade quase exclusivamente nos marcadores com repetições

de mononucleotídeos, enquanto que a frequência de instabilidade dos

marcadores com repetições de dinucleotídeos é marcadamente aumentada nos

carcinomas. Assim, concluimos que os perfis de MSI diferem entre casos

esporádicos e casos com agregação familiar, e que a instabilidade de

mononucleotídeos precede a de dinucleotídeos durante o desenvolvimento dos

tumores em pacientes com Síndrome de Lynch. Por estas razões sugerimos o

uso preferencial de repetições de mononuleotídeos como marcadores de

instabilidade na identificação de possíveis pacientes com Síndrome de Lynch.

No capítulo 3, tentámos perceber se os perfis de MSI diferem entre tumores

instáveis (MSI-H) colorectais e endometriais. Para isso analisámos, para três

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marcadores de instabilidade de mononucleotídeos e três de dinucleotídeos, a

frequência de MSI, o tipo de mutação (delecção/inserção) e o tamanho das

mutações, em ambos os tipos de carcinoma: colorectal (CRC) e endometrial (EC).

A frequência de instabilidade de mono- e dinuleotídeos encontrada nos dois

tecidos é comparável, sendo os dois tipos de marcadores afectados em níveis

semelhantes. Mostrámos que o tipo de mutação é uma característica que

depende do tipo de marcador e não dependente do tecido, pois observámos para

ambos os tecidos quase exclusivamente delecções nos mononucleotídeos e

delecções e inserções nos dinucleotídeos. O tamanho destas deleções/inserções

difere entre CRC e EC, com EC apresentando alterações mais pequenas. Neste

capítulo concluimos que não existem diferenças substanciais nos perfis de MSI

entre os tumores colorectais e endometriais. Assim, os nossos resultados

mostram que os mesmos testes para MSI podem ser usados para ambos os tipos

de tumor.

O capítulo 4 descreve a nossa busca por novos genes-alvo (target genes)

envolvidos no cancro do endométrio com MSI (MSI-H EC). Como acima referido,

MSI caracteriza-se pela acumulação de mutações em microssatélites nao-

codificantes e/ou codificantes. Genes contendo microssatélites são

frequentemente alvos de mutação em tumores com deficiências de MMR. Em

particular, mutações em genes com importantes funções regulatórias – os

chamados target genes – são considerados genes-chave no desenvolvimento de

tumores MSI-H. Para encontrar esses novos target genes para tumores

endometriais com MSI-H, uma estratégia específica para o endométrio foi

estabelecida. Analisámos genes expressos no endométrio normal e que contêm

sequências repetitivas. De uma lista de 2338 genes expressos no endométrio

normal, 382 genes continham repetições (496) e, consequentemente, foram

sequenciados. Mutações nessas repetições foram detectadas em 44 genes. É, no

entanto, discutível se todos esses 44 genes contribuem realmente para o

desenvolvimento de tumores. Um dos pricipais critérios para a selecção de target

genes que realmente contribuem para os tumores é a alta frequência de

mutações. Geralmente um valor-referência de 15% é usado. Genes com

frequências inferiores a esse valor são considerados bystanders. Aplicando este

valor de 15%, encontrámos nos nosso estudo sete novos target genes.

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Adicionalmente, analisámos, em tumores MSI-H colorectais e gástricos, 10 dos

genes encontrados para os tumores do endométrio. O nosso estudo confirma que

alguns target genes revelam especificidade de tecido, enquanto outros parecem

exercer um papel comum em tumores MSI-H, independentemente da origem do

tecido.

O gene mais frequentemente mutado nos tumores do endométrio no nosso

estudo foi o NRIP1 (34%). Este gene codifica uma proteína co-repressora da via

do receptor de estrogénio (ER), uma das principais vias de sinalização na

carcinogénesis do endométrio. Surpreendentemente, este gene apareceu também

altamente mutado em carcinomas colorectais (24%). Estes resultados apontam

assim para um importante papel da via do ER também no desenvolvimento dos

tumores colorectais com instabilidade de microssatélites. Consequentemente,

sugerem que genes da via ER poderão constituir óptimos candidatos a target

gene nos tumores com MSI não só em órgãos dependentes de hormonas, mas

também em órgãos de outro tipo, tal como o cólon.

Os resultados encontrados no capítulo 4 levaram-nos a escrever um artigo

de revisão (capítulo 5), no qual tentámos clarificar os mecanismos que ligam

hormonas e tumores associados a Síndrome de Lynch, e particularmente discutir

como as hormonas podem desempenhar um papel importante no

desenvolvimento de tumores com MSI.

Em conclusão, os nossos estudos originaram novas ideias sobre o

processo de desenvolvimento de tumores MSI-H. Mostrámos que não existem

diferenças substanciais nos perfis de MSI entre tumores colorectais e

endometriais, e encontrámos mutações que provavelmente afectam

indirectamente a via do receptor de estrogénio, sugerindo que genes dessa via

serão óptimos candidatos para análise de mutações em tumores MSI-H, mas

possivelmente também em tumores estáveis. Este resultados poderão também

revelar-se interessantes do ponto de vista do desenvolvimento de novas terapias.

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STRESZCZENIE

Niestabilność mikrosatelitarnego DNA (MSI) jest głównym typem niestabilności

genomowej w guzach pochodzących od pacjentów z zespołem Lyncha oraz w

około 15-25% sporadycznych raków jelita grubego (CRC), raków endometrium

(EC) oraz raków żołądka (GC). Mechanizm leżący u podstaw MSI to zaburzenie

tzw „szlaku naprawy nieprawidłowego parowania zasad w DNA” – ang. „DNA

mismatch repair (MMR) pathway”. MSI to zjawisko charakteryzujące się

nagromadzeniem mutacji w krótkich, wysokopowtarzalnych sekwencjach DNA,

zwanych mikrosatelitami. Ten rodzaj mutacji może pojawiać się zarówno w

regionach kodujących jak i niekodujących zawierających sekwencje

mikrosatelitarne. Niniejsza praca prezentuje wyniki analizy MSI w guzach

pacjentów wykazujących zespół Lyncha, ze szczególnym uwzględnieniem raków

jelita grubego i endometrium oraz ich sporadycznych odpowiedników.

W rozdziale 1 został przedstawiony opis zespołu Lyncha, niestabilności

mikrosatelitarnej, oraz omówione zostały nowotwory wykazujące zaburzenia MMR.

W rozdziale drugim został zaprezentowany opis badań nad ewolucją zmian

fragmentów mikrosatelitarnych w sekwencji przemiany gruczolaka - poprzez

gruczolakoraka – w raka jelita grubego. W rozdziale tym przedstawiono

porównywalne profile MSI, oznaczane w oparciu o częstości krótkich fragmentów

repetytywnych - markerów mono- i dinukleotydowych w sporadycznych

gruczolakach i rakach jelita grubego. Opisane zostały różnice w rodzajach mutacji,

których skutkiem było skracanie genów MMR – w przypadku gruczolaków jelita

grubego wykazywano praktycznie wyłącznie niestabilność w zakresie powtórzeń

mononukleotydowych, gdy tymczasem częstość występowania markerów

dinukleotydowych była znacząco zwiększona w przypadku raków jelita grubego –

uważamy więc, że profile MSI różnią się pomiędzy rodzinnymi i sporadycznymi

rakami tego typu. Doszliśmy do wniosku, że profile MSI różnią się w przypadkach

rodzinnych i sporadycznych oraz, że niestabilność markerów mononukleotydowych

poprzedza niestabilność obserwowaną w zakresie markerów dinukleotydowych, w

czasie rozwoju guzów w obrębie jelita grubego u pacjentów z zespołem Lyncha.

Na tej podstawie proponujemy, że niestabilność w zakresie markerów

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mononukleotydowych powinna być preferowana w zakresie analizy markerów

mikrosatelitarnych w celu identyfikacji potencjalnych nosicieli zespołu Lyncha.

W rozdziale 3 zostało zadane pytanie, czy profile MSI raków jelita grubego i

raków endometrium o profilu MSI-high (MSI-H) różnią się między sobą. Żeby

odpowiedzieć na to pytanie przeanalizowaliśmy częstość występowania zjawiska

MSI, typy mutacji (delecje, insercje) oraz rozmiar mutacji mikrosatelitarnych

zachodzących w trzech markerach mononukleotydowych i trzech

dinukleotydowych, zarówno w rakach endometrium jak i rakach jelita grubego.

Częstość mutacji w obydwu typach tkanek była porównywalna, zarówno w

przypadku mono- jak i dinukleotydowych. Wykazaliśmy, że typ mutacji jest zależny

od rodzaju markera a nie od rodzaju badanej tkanki – zaobserwowaliśmy bowiem,

że w obu typach tkanki występowały prawie wyłącznie delecje w zakresie

markerów mononukleotydowych, oraz zarówno delecje jak i insercje w markerach

dinukleotydowych. Wykazano różnice pomiędzy CRC i EC w długościach

fragmentów podlegających delecjom/insercjom – gdzie raki endometrium

prezentowały zmiany o krótszej długości niż miało to miejsce w przypadku raków

jelita grubego. Stwierdziliśmy, że nie było znaczącej różnicy pomiędzy profilami

MSI w CRC i EC, co więcej – nasze wyniki wskazują, że ten sam panel badań MSI

może być zastosowany do analizy w przypadkach obu typów raków.

W rozdziale 4 opisujemy poszukiwania nowych genów targetowych dla

wysoce niestabilnych (MSI-H) raków endometrium. MSI to zjawisko

charakteryzujące się akumulacją mutacji zarówno w mikrosatelitarnych regionach

kodujących jak i niekodujących, a geny zawierające fragmenty mikrosatelitarne są

częstym celem mutacji w guzach wykazujących zaburzenia naprawcze.

Szczególnie mutacje w ważnych genach regulatorowych – które nazywamy genami

targetowymi – są uważane za główne czynniki w rozwoju guzów o wysokiej

niestabilności mikrosatelit. Zaproponowaliśmy strategię odpowiadającą rakom

endometrium, w celu znalezienia nowych genów targetowych w rakach

endometrium o fenotypie MSI-H. Przeprowadziliśmy analizę genów wykazujących

ekspresję w prawidłowym endometrium, zawierających sekwencje

wysokopowtarzalne. Z listy 2,338 genów wykazujących ekspresję w prawidłowym

endotelium, wybrano 382 geny zawierające 496 powtórzeń, geny te

sekwencjonowano. Mutacje w tych wysokopowtarzalnych fragmentach znaleziono

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w 44 genach. Jednak to, czy te mutacje (w 44 genach) rzeczywiście przyczyniają

się do rozwoju guza nadal pozostaje polem do spekulacji. Głównym kryterium do

oceny, czy geny targetowe rzeczywiście przyczyniają się do progresji nowotworu

jest częstość mutacji – jeżeli przekracza ona 15%, oznacza to, że gen można

określić mianem genu targetowego. Geny wykazujące częstość mutacji niższą niż

próg 15% były analizowane dodatkowo, jako geny ‘towarzyszące’. Po

zastosowaniu progu 15%, wyodrębniliśmy panel rzeczywistych genów

targetowych, których było siedem. Analogicznie zanalizowaliśmy panel 10

rzeczywistych genów targetowych w rakach jelita grubego o fenotypie MSI-H.

Nasze badania potwierdziły, że niektóre geny wykazują specyficzność tkankową,

gdy inne odgrywają rolę w guzach MSI-H, bez wzgledu na pochodzenie

analizowanej tkanki.

Najczęściej zmutowanym genem w rakach EC był NRIP1 (34%). Ten gen

koduje korepresor białka biorącego udział w szlaku receptora estrogenowego,

jednego z głównych szlaków związanych z karcynogenezą endometrium. Co

zaskakuąjce – ten gen jest również wysoce zmutowany w CRC (24%). Te rezultaty

wskazują na istotna rolę szlaku ER również w rozwoju niestabilnych

mikrosatelitarnie raków jelita grubego i może on się stać istotnym celem badań

również w innych rodzajach nowotworów wykazujących zjawisko MSI, a nie tylko

tkankach wykazujących odpowiedź na estrogeny.

Te obserwacje, opisane w rozdziale 4 skłoniły nas do opisania

mechanizmów hormonozależnych związanych z guzami związanymi z zespołem

Lyncha - i w szczególności - do przedstawienia dyskusji, w jaki sposób hormony

mogą odgrywać rolę w rozwoju guzów o fenotypie MSI.

Podsumowując, nasze wyniki pozwoliły spojrzeć na nowo na proces rozwoju

guzów o fenotypie MSI-H: wykazaliśmy, że nie ma właściwie różnicy pomiędzy

profilem MSI w CRC i EC oraz odkryliśmy, że mutacje w regionach

wysokopowtarzalnych mogą bezpośrednio wpływać na szlak receptora

estrogenowego. Nasze wyniki wskazują, że geny szlaku ER są nie tylko

doskonałymi celami badawczymi w analizie guzów MSI-H, ale również guzów

stabilnych mikrosatelitarnie. Te informacje mogą mieć w przyszłości konkretne

znaczenie przyczyniając się do rozwoju nowych strategii terapeutycznych.

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ACKNOWLEDGMENTS

First of all, I would like to thank my supervisor, Prof. Robert Hofstra. Robert, I am very

grateful for all the work and enthusiasm you put into this thesis, and for all the great

opportunities, trust and support that you have given me throughout these years. Thank you

also for so many crazy moments (on 3 different continents!), which made this journey a lot

more fun than just a normal job. Thank you so much for always being next door, ready to

share amazing laughs, problems, struggles and plans. It is so easy to be happy working with

you!

Prof. Raquel Seruca, I would like to thank you not only for being my second

supervisor, but also for introducing me to IPATIMUP when I had just finished studying

Biology. Raquel, thanks to you I had my first taste of research and start thinking of doing a

Ph.D. Thank you for the privilege and opportunity of working in such a great team in Portugal.

I am also very grateful that you encouraged me to go abroad and that you accompanied me

on this Dutch adventure.

Then I would like to thank Dr. Helga Westers and Dr. Rolf Sijmons. It is really a great

pleasure to have you both as my co-supervisors. Helga, thank you for dedicating so much of

your time to the papers in this thesis and to my other experiments. Besides enjoying your

company while travelling to meetings, I am personally very proud to be your first Ph.D.

student! Rolf, thank you for your help and always being so enthusiastic about the projects.

Your suggestions for the papers were very helpful, especially when we were struggling with

the adenomas story. And your good mood and funny stories are always helpful!

Three institutions have hosted my work as a Ph.D. student: the Department of Genetics of

the University Medical Centre Groningen, the Netherlands; the Institute of Molecular

Pathology and Immunology of the University of Porto, Portugal (3 months); and the

Department of Pathomorphology in the Medical College of Jagiellonian University, in Kraków,

Poland (6 months). Thank you also to Prof. Charles Buys, Prof. Cisca Wijmenga, Prof.

Sobrinho Simões and Prof. Jerzy Stachura for making me welcome at your institutions.

I am grateful to the Portuguese “Fundação para a Ciência e a Tecnologia” for financing my

project (grant ref.:SFRH/BD/18832/2004).

I am also grateful to the reading committee, formed by Prof. M.J.E. Mourits, Prof. H.

Morreau, and Prof. J. Lubiński, for their willingness to read my thesis and for giving their

approval for the defence.

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To all the co-authors of the papers, thank you for your collaboration. Jackie Senior, thank

you for your help in improving my manuscripts. Prof. Jan Kleibeuker and Prof. Harry Hollema,

thank for your input for my projects and for making me always feel very welcome at

meetings with you.

I would like to thank all the people in the three host institutions who helped me with this

thesis in some way or other. I apologize for not being able to mention all your names, but I

hope you can identify yourselves in the following references.

In Groningen, a special mention to the HNPCC/Oncogenetics group. Discussing with you

guys during the weekly work meeting and laughing a lot was the best therapy I could have

had at several moments. Thank you all also for your patience in speaking English at 9 am!

The group lost some elements, gained some new ones. I would like to mention a special

memory, of Frans Gerbens, whom we lost almost a year ago.

In the lab I cannot forget Krista B., who helped me a lot doing thousands of

sequences, and Chris, my first student, for his help when I had hundreds of primer sets to

design, to optimize and, of course, later on to use. Also thanks to Krista K., Bart, Paul,

Bahram, Jan O., Pieter and Yvonne.

I’m also grateful to the colleagues who made my lunch time in the first year a heel

gezellig tijd: Arja, Edwin, Bea, Jos, Chantal. Special thanks to Arja who literally opened the

doors of the department on my first day at work ☺

Very special thanks to all my room mates (I’ve had at least 13!) for the relaxed

atmosphere at work and sharing a lot more with me than only the room. My fellow Ph.D.

students thank you for the complicities proper of the Ph.D. life (and age). Thank you to all

the students with whom I got some educational experience. There’s always a lot to learn

with you. And of course everyone in the department with whom I have had contact and nice

talks, all those in the various sections who helped in different ways: labs, medical doctors,

secretariat, informatics, finances, etc.

With some of you I also had very good moments outside work, these usually included

food, or drinks, or trips, or a mixture of all three: Maria, Renée, Jihane, Marina, Greg,

Bahram, Jan Jongbloed, Yunia, Monique, Gosia, Agata, Mats, Gerben, Tjakko. Thank you

for that! Thank you Maria, Greg, Renée, and Jan for meeting up with me in Kraków! Renée,

we sure had a great time everywhere we went together, especially in Japan.

In Kraków, thank you Monika, Anastazja, Ania, Agnieszka, Rasa, Danuta, Piotr, Dr. Dąbroś,

the new students, and everybody around that received me with a smile while I spoke my first

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sentences in Polish. It was great to work and to live in that beautiful city with you. Monika,

thanks for welcoming me in your lab and to your family, and all the adventures you always

had for me. And, of course, for the Polish summary for this thesis!

In Porto, I am grateful to Cirnes and Sónia for the help with the MSI experiments, to the

friends I gained there, and to so many people that made IPATIMUP feel like home. Dear

Carla Oliveira and Prof. Fátima Carneiro, thank you for your recommendation letters to FCT

and everything you taught me.

I would also like to acknowledge the people, colleagues and friends, from recent and ancient

times, from my Portuguese, Polish and Dutch lives, who accompanied me through the years

in a more personal way.

Dear friends, thank you for the good times, the advices, the mails, the phone calls

at 4 am, the Euromeetings ☺ Basically, thank you for always being there for me at important

moments. Dear Paweł, thank you for all your efforts and everything that we shared during a

big part of this Ph.D. project. I lived and learned a lot with you and your family. To my closest

friends in Groningen, Gilda, Martin, Maria, Kaushal, Mateusz, Ana Duarte, Karla, Roberta,

Bispo, Susana, Vítor, thank you for being my “family” over here. I guess I don’t need to

describe how much I appreciate your company. Maria and Mateusz, my paranimfen, thank

you also for accepting this role.

Dear Pedro, thank you for being by my side during the joys and worries of the last year.

Thank you for always being interested and ready to help me with my work and for the

fantastic time we have been having together. First as a friend, then as vriend, you brought

new colours to “my” Holland and to my life! Thank you for all the trust and support in

important decisions. Life is so much easier and happier with you around.

To my family, especially to Chico, Pai, Sofia and Noémia (my brother, father, sister and

sister-in-law), I want to thank you all for always finding a way to be present in my daily life

and making me feel special, and to apologize for being absent from “home” for such long

periods. Thank you also for taking care of so many things for me. Above all, I am very

grateful to you for always believing in me and pushing me forward! To my mother and my

grandparents, I’m sure you are the stars behind all the luck I have had. I hope you can be

proud of my work and decisions. This thesis, and all it represents, is dedicated to you.

123

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Curriculum Vitae PERSONAL INFORMATION Name: Ana Maria Monteiro Ferreira Place and date of birth: Amarante, Portugal, 4th November 1980

Nationality: Portuguese

E-mail: [email protected]

ACADEMIC BACKGROUND

1998-2002: MSc in Biology, Scientific Technological Branch, specializing in “Applied Animal

Biology”, University of Porto, Portugal.

01/2005 - 01/2009: PhD in Medical Sciences, University of Groningen, the Netherlands.

Thesis to be defended on the 13th May 2009.

MAIN SCIENTIFIC AREAS OF INTEREST

Cancer genetics; Cancer biology; Mismatch repair related-tumours; Human Biology.

RESEARCH EXPERIENCE 11/2002 – 12/2004: Research grant as undergraduate student at Institute of Molecular

Pathology and Immunology of the University of Porto (IPATIMUP), Portugal, under the

supervision of Prof. Raquel Seruca, working on the project “Genetic and environmental

factors contributing to familial aggregation of gastric cancer”.

06/2004 – 09/2004: Short-term visiting researcher at Jagiellonian University Medical College,

Department of Pathomorphology, in Krakow, Poland, as participant in the Marie Curie

Research Training Network – Cellion Project – “Studies on cellular response to targeted

single ions using nanotechnology”.

01/2005 – 01/2009: PhD student in the Department of Medical Genetics, UMCG/ University

of Groningen, under the supervision of Prof. Robert Hofstra, working on the project

"Identification of microsatellite unstable (MSI-H) endometrial cancer associated target

genes".

02/2009 – 05/2009: Short-term researcher at Department of Medical Genetics, UMCG,

University of Groningen, within the HNPCC group.

125

PUBLICATIONS Oliveira C, Suriano G, Ferreira P, Canedo P, Kaurah P, Mateus A, Ferreira A, Ferreira AC,

Figueiredo C, Carneiro F, Keller G, Huntsman D, Machado JC, Seruca R. Genetic

screening for familial gastric cancer. Hereditary Cancer Clin Pract. 2004;2(2):51-64.

Oliveira C, Ferreira P, Nabais S, Campos L, Ferreira A, Cirnes L, Castro Alves C, Veiga I,

Fragoso M, Regateiro F, Moreira Dias L, Moreira H, Suriano G, Carlos Machado J, Lopes C,

Castedo S, Carneiro F, Seruca R. E-Cadherin (CDH1) and p53 rather than SMAD4 and

Caspase-10 germline mutations contribute to genetic predisposition in Portuguese gastric

cancer patients. Eur J Cancer. 2004;40(12):1897-903.

Oliveira C, Westra JL, Arango D, Ollikainen M, Domingo E, Ferreira A, Velho S, Niessen R,

Lagerstedt K, Alhopuro P, Laiho P, Veiga I, Teixeira MR, Ligtenberg M, Kleibeuker JH,

Sijmons RH, Plukker JT, Imai K, Lage P, Hamelin R, Albuquerque C, Schwartz S Jr,

Lindblom A, Peltomaki P, Yamamoto H, Aaltonen LA, Seruca R, Hofstra RM. Distinct

patterns of KRAS mutations in colorectal carcinomas according to germline Mismatch

Repair defects and hMLH1 methylation status. Hum Mol Genet. 2004;13(19):2303-11.

Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz S Jr, Duval A, Carneiro F,

Machado JC, Hamelin R, Seruca R. The prevalence of PIK3CA mutations in gastric and

colon cancer. Eur J Cancer. 2005;41(11):1649-54.

Zazula M, Ferreira AM, Czopek JP, Kolodziejczyk P, Sinczak-Kuta A, Klimkowska A, Wojcik

P, Okon K. Bialas M, Kulig J, Stachura J. CDH1 gene promoter hypermethylation in gastric

cancer: relationship to Goseki grading, microsatellite instability status and EBV invasion.

Diagn Mol Pathol. 2006;15(1):24-9.

Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval A,

Hamelin R, Machado JC, Schwartz S Jr, Carneiro F, Seruca R. KRAS and BRAF oncogenic

mutations in MSS colorectal carcinoma progression. Oncogene. 2007;26(1):158-63.

Davalos V, Dopeso H, Velho S, Ferreira AM, Cirnes L, Diaz-Chico N, Bilbao C, Ramirez R,

Rodriguez G, Falcon O, Leon L, Niessen RC, Keller G, Dallenbach-Hellweg G, Espin E,

Armengol M, Plaja A, Perucho M, Imai K, Yamamoto H, Gebert JF, Diaz-Chico JC, Hofstra

RM, Woerner SM, Seruca R, Schwartz S, Arango D. High EPHB2 mutation rate in gastric

but not endometrial tumors with microsatellite instability. Oncogene. 2007;26(2):308-11.

126

Ferreira AM, Westers H, Wu Y, Niessen RC, Olderode-Berends M, van der Sluis T, van der

Zee AG, Hollema H, Kleibeuker JH, Sijmons RH, Hofstra RMW. Do microsatellite instability

profiles really differ between colorectal and endometrial tumors? Genes Chromosomes and

Cancer, In press.

Ferreira AM, Westers H, Niessen RC, Wu Y, Olderode-Berends M, van der Sluis T,

Reuvekamp PTW, Seruca R, Hollema H, Kleibeuker JH, Sijmons RH, Hofstra RMW.

Mononucleotide precedes dinucleotide instability during colorectal tumour development in

Lynch syndrome patients. Under review

Ferreira AM, Westers H, Albergaria A, Seruca R, Hofstra RMW. Estrogens, MSI and Lynch

syndrome-associated tumors. Under review

SELECTED ORAL PRESENTATIONS

“Target genes profiling of microsatellite unstable endometrial tumours: finding needles in a

haystack”

• International Society for Gastrointestinal Hereditary Tumours (InSight) meeting, March

2007, Yokohama, Japan.

• Spring Meeting of the Dutch Society of Human Genetics, April 2007, Veldhoven, the

Netherlands.

“Identification of new target genes for MSI-H endometrial tumours”

• Genetica Retreat, March 2008, Rolduc, the Netherlands.

“Contribution to a new profile of target genes in microsatellite unstable tumours”

• Spring Meeting of the Dutch Society of Human Genetics, March 2008, Amsterdam, the

Netherlands.

POSTERS

“MSI profiles differ between Colon Adenomas and Carcinomas”. Ferreira AM, Niessen RC,

Hollema H, Wu Y, Kleibeuker JH, Sijmons RH, Hofstra RMW. GUIDE Early Summer

Meeting, June 2006, University Medical Center Groningen, University of Groningen,

Groningen, the Netherlands.

“Microsatellite Instability Profiles of Endometrial and Colorectal Tumors”. Ferreira AM,

Niessen RC, Hollema H, Wu Y, Kleibeuker JH, Sijmons RH, Seruca R, Hofstra RMW. 19th

127

Meeting of the European Association for Cancer Research (EACR), July 2006, Budapest,

Hungary.

“New target genes for microsatellite unstable endometrial tumors”. Ferreira AM, Gerbens F,

Westers H, Kooi KA, Bos K, Esendam C, van der Sluis T, Zazula M, Stachura J, van der

Zee AG, Seruca R, Hollema H, Hofstra RMW. 58th Meeting of the American Society of

Human Genetics (ASHG), November 2008, Philadelphia, USA.

“Mononucleotide precedes dinucleotide instability during colorectal tumour development in

Lynch syndrome patients”. Ferreira AM, Westers H, Niessen RC, Wu Y, Olderode-Berends

M, van der Sluis T, Reuvekamp PTW, Seruca R, Hollema H, Kleibeuker JH, Sijmons RH,

Hofstra RMW. Spring Meeting of the Dutch Society of Human Genetics, April 2009,

Veldhoven, the Netherlands.

“Estrogen-receptor pathway and microsatellite unstable tumors: a promissing link”. Ferreira

AM, Niittymäki I, Sousa S, Zazula M, Hollema H, Sijmons RH, Stachura J, Aaltonen LA,

Seruca R, Westers H, Hofstra RMW. International Society for Gastrointestinal Hereditary

Tumours (InSiGHT) meeting, June 2009, Düsseldorf, Germany.

PRIZES AND OTHER AWARDS RECEIVED

• Prize Rui Serpa Pinto for the best mark in Human Biology – Faculty of Sciences,

University of Porto, 2001.

• Short-term scholarship from the “Marie Curie Research Training Network – Cellion

Project”, 2004.

• 4-year PhD grant (ref.:SFRH/BD/18832/2004) from the Portuguese “Fundação Para a

Ciência e a Tecnologia” for the project on "Identification of MSI-H endometrial cancer

associated target genes", 2004.

• “Van Walree” travel award, for participation in the meeting of the International Society

for Gastrointestinal Hereditary Tumours (InSight) at Yokohama, in Japan, 2007.

• “Jan Kornelis de Cock-Stichting” funding for the project “Functional analysis of a newly

identified endometrial cancer related gene”, December 2008.

128

PRESENT POSITION From 06/2009: Post-doc position at Molecular Biology and Biochemistry Research Center

for Nanomedicine (CIBBIM-Nanomedicine), Vall d'Hebron University Hospital, Barcelona,

Spain, in the group of Dr. Simó Schwartz Jr., working on “Development and mechanistic

study of an animal model of human colorectal cancer metastases by KRAS and BRAF

expression”.

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