RIJKSUNIVERSITEIT GRONINGEN

Transcrição

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
7
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).
10
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
11
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
12
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 bestdescribed 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
13
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).
14
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.
15
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).
Hypermethylation
MLH1
PTEN
MSI
Mutations (e.g.
KRAS, BAX, MSH2)
Endometrial
hyperplasia
Type I endometrioid
adenocarcinoma
Endometrial
intraepithelial
carcinoma
Type II serous
adenocarcinoma
Normal
p53
HER2/NEU
P53, LOH,
HER2/NEU
Figure 1. Progression model of endometrial cancers type I and type II progression
adapted from Ryan et al. (2005).
18
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
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
1
Genetics,
2
Pathology,
3
Gastroenterology, University Medical Center
Groningen, University of Groningen, Groningen, The Netherlands.
4
Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Under review
25
ABSTRACT
A progressive accumulation of genetic alterations underlies the adenomacarcinoma 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 adenomacarcinoma 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 paraffinembedded 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 noncarriers; 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%
10% ( 2)
33% (4)
76
MSS
49% (17)
36% (65)
34% (11)
0%
33% (4)
97
( 3)
(0)
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 noncarriers 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)
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
D17S250
D5S346
D2S123
BAT40
BAT26
BAT25
NR24
NR21
NR27
MSI Status
MSI-L
MSIH
MSS
ND
LD
ND
LD
ND
HD
ND
HD
HD
HD
HD
ND
LD
LD
LD
HD
ND
ND
LD
ND
LD
LD
HD
HD
ND
LD
HD
HD
HD
ND
ND
ND
ND
ND
ND
MMR
mutation
Grade
Y313
Y239
Y72
Y65
Y71
Y190
Y264
Y200
Y81
Y210
Y234
Y89
Y123.1
Y192
Y123.2
Y79
Y301
Y176-2
Y13
Y39
Y56
Y167
Y170
Y225
Y257
Y267
Y202
Y223
Y268
Y309
Y311
Y317
Y327
Y331
Y294
Dinucleotide
markers
NON-CARRIERS
Patient ID
Mononucleotide
markers
B)
16T
HD
3T
HD
18T
ND
10T
LD
1
ND
5T
LD
29T
LD
21T
LD
25T
ND
5
ND
6
ND
14T
HD
6T
HD
Y21
ND
2
ND
3
ND
4
ND
7
ND
Y112
HD
MSI-H
MSI-L
MSS
MSI-H
LD
LD
MSS
Y241
HD
MSI-H
26T
HD
MSI-L
7T
LD
12T
LD
Y86
HD
1T
LD
MSH6
4T
20T
MSS
34
D17S250
D5S346
D2S123
BAT40
Dinucleotide
markers
BAT26
LD
BAT25
HD
13T
NR24
17T
NR21
LD
NR27
9T
MSI
Status
LD
MMR
mutation
LD
8T
MLH1
Grade
2T
MSH2
Patient
ID
Mononucleotide
markers
Table 3. MSI results for adenoma and carcinoma of the same patient.
1
2
3
4
5
6
7
AD
CRC
AD
CRC
AD
CRC
AD
CRC
AD
CRC
AD
CRC
AD
CRC
D17S250
D5S346
D2S123
BAT26
Dinucleotide
markers
BAT25
NR24
NR21
NR27
Mononucleotide
markers
MLH1
MLH1
MSH2
MSH2
MSH2
MSH2
MSH2
MSH2
MLH1
MLH1
MLH1
MLH1
MSH2
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 adenomacarcinoma sequence. We compared germline MMR mutation carriers and noncarriers, 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 noncarriers. 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 MMRproficient 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
LIFESCIHEALTH-5, proposal no. 018754).
38
European
Community
(FP6-2004-
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Boyer JC, Yamada NA, Roques CN, et al. Sequence dependent instability of mononucleotide
microsatellites in cultured mismatch repair proficient and deficient mammalian cells. Hum Mol Genet
2002;11(6):707-13.
Bronner CE, Baker SM, Morrison PT, et al. Mutation in the DNA mismatch repair gene homologue
hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 1994;368(6468):258-61.
De la Chapelle A. Testing tumors for microsatellite instability. Eur J Hum Genet 1999;7(4):407-8.
Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61(5):759-67.
Fishel R, Lescoe MK, Rao MR, et al. The human mutator gene homolog MSH2 and its association with
hereditary nonpolyposis colon cancer. Cell 1993;75(5):1027-38. Erratum in: Cell 1994;77(1):167.
Furlan D, Casati B, Cerutti R, et al. Genetic progression in sporadic endometrial and gastrointestinal
cancers with high microsatellite instability. J Pathol 2002;197(5):603-9.
39
2005;7(2):160-70.
Grady WM, Rajput A, Myeroff L, et al. Mutation of the type II transforming growth factor-beta receptor is
coincident with the transformation of human colon adenomas to malignant carcinomas. Cancer Res
1998;58(14):3101-4.
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):59.
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 nonpolyposis 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.
Shibata D, Peinado MA, Ionov Y, et al. Genomic instability in repeated sequences is an early somatic
event in colorectal tumorigenesis that persists after transformation. Nat Genet 1994;6(3):273-81.
2003;200(2):168-76.
1993;260(5109):816-9.
Young J, Leggett B, Gustafson C, et al. Genomic instability occurs in colorectal carcinomas but not in
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 OlderodeBerends1, 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 mononucleotide 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
CRC
Mutation carriers
EC
CRC
Total
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 noncarriers 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 markerdependent 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
LIFESCIHEALTH-5, proposal no. 018754).
54
European
Community
(FP6-2004-
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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.
5
Department of Medical Genetics, University of Helsinki, Helsinki, Finland.
6
Department of Patomorfology, Medical College, Jagiellonian University, Krakow, Poland.
7
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 MSIH 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). Freshfrozen 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, paraffinembedded 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
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
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
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
SEC16
SPG20
CAMSAP1L1
ZMIZ1
ZMIZ1
INTU
IGSF9
KIAA1370
JMJD1C
FAM135A
KIAA1797
MBD6
DACH1
POLE3
JAM3
HEXDC
CHD4
LYN
PPP1R10
SRPR
TTC3
NRIP1
AP3B1
INPPL1
FLNB
JAK1
BAT1
MTA1
FXR1
IFNGR2
RBM6
SF3B2
RABGAP1
TNPO3
TRPM5
ITM2B
SVIL
INTS12
C17orf63
KIAA1009
CPEB3
NOL7
ADD3
PHKB
TFPI
SEC16 homolog A (S. cerevisiae)
spastic paraplegia 20 (Troyer syndrome)
calmodulin regulated spectrin-associated protein 1-like 1
zinc finger, MIZ-type containing 1
zinc finger, MIZ-type containing 1
inturned planar cell polarity effector homolog (Drosophila)
immunoglobulin superfamily, member 9
KIAA1370
jumonji domain containing 1C
family with sequence similarity 135, member A
KIAA1797
methyl-CpG binding domain protein 6
dachshund homolog 1 (Drosophila)
polymerase (DNA directed), epsilon 3 (p17 subunit)
junctional adhesion molecule 3
hexosaminidase (glycosyl hydrolase family 20, catalytic domain) containing
chromodomain helicase DNA binding protein 4
v-yes-1 Yamaguchi sarcoma viral related oncogene homolog
protein phosphatase 1, regulatory (inhibitor) subunit 10
signal recognition particle receptor ('docking protein')
tetratricopeptide repeat domain 3
nuclear receptor interacting protein 1
adaptor-related protein complex 3, beta 1 subun
inositol polyphosphate phosphatase-like 1
filamin B, beta (actin binding protein 278)
Janus kinase 1 (a protein tyrosine kinase)
HLA-B associated transcript 1
metastasis associated 1
fragile X mental retardation, autosomal homolog 1
interferon gamma receptor 2 (interferon gamma transducer 1)
RNA binding motif protein 6
splicing factor 3b, subunit 2, 145kDa
RAB GTPase activating protein 1
transportin 3
transient receptor potential cation channel, subfamily M, member 5
integral membrane protein 2B
supervillin
integrator complex subunit 12
chromosome 17 open reading frame 63
KIAA1009
cytoplasmic polyadenylation element binding protein 3
nucleolar protein 7, 27kDa
adducin 3 (gamma)
phosphorylase kinase, beta
tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)
C7
T8
A8
C7
C7
A8
C7
A8
A8
A8
A7
3XC7
A7
A7
G7
C7 + G7
A7
A7
C7
A8
A8
A8
A8
C7
G7
A8
T8
G7
A8
T7
G7
A8
A8
C7
C7
C7
G7
T7
C7
T8 + A7
C7
A8
A8
A7
A7
Mutat Freq.
(%)
5,7 (2/35)
5,9 (2/34)
7,1 (2/28)
4,3 (1/23)
9,1 (2/22)
4,2 (1/24)
4,3 (1/23)
12,9 (4/31)
15,1 (5/33)
3,3 (1/30)
6,7 (1/15)
24,1 (7/29)
3,1 (1/32)
4,8 (1/21)
8,3 (3/36)
11,4 (4/35)
8,1 (3/37)
6,7 (2/30)
3,7 (1/27)
25,8 (8/31)
3,3 (1/30)
34,3 (12/35)
8,1 (3/37)
14,3 (5/35)
9,7 (3/31)
20 (7/35)
2,9 (1/35)
3,3 (1/30)
12,5 (2/16)
2,8 (1/36)
3,4 (1/29)
10,7 (3/28)
6,25 (2/32)
2,9 (1/34)
5,3 (1/19)
2,8 (1/36)
10,8 (4/37)
5,9 (2/34)
3,7 (1/27)
18,5 (5/27)
2,9 (1/34)
7,1 (2/28)
14,7 (5/34)
4,3 (1/23)
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
JMJD1C
9
A8
ADD3
14
INPPL1
26
SVIL
31
HEXDC
12
ND
7% (1/15)
19% (5/27)
3% (1/34)
0% (0/15)
15% (5/33)
A8
37% (11/30)
47% (7/15)
15% (5/34)
C7
10% (3/30)
20% (3/15)
14% (5/35)
G7
14% (4/29)
27% (4/15)
11% (4/37)
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 ligandactivated 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
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.
100
90
Mutation Frequency (%)
80
70
60
MSI-H EC
MSI-H CRC
50
MSI-H GC
40
30
20
10
JA
K
KI
AA 1
10
0
JM 9
JD
1C
AD
D3
IN
PP
L1
SV
IL
HE
XD
C
BD
6
M
NR
IP
1
SR
PR
0
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
LIFESCIHEALTH-5, proposal No 018754).
72
European
Community
(FP6-2004-
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76
CHAPTER 5
Estrogen, MSI and Lynch Syndrome-Associated
Tumors
Ana M. Ferreira1,2, Helga Westers1, André Albergaria2, Raquel Seruca2,3, Robert M.
W. Hofstra1
1
Department of Genetics, University Medical Centre Groningen, University of Groningen,
Groningen, the Netherlands.
2
3
Faculdade de Medicina da Universidade do Porto, Portugal.
Under review
77
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 syndromerelated 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 MMRrelated neoplasias.
78
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 ligandactivated 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 (KleinHitpass 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).
79
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.
Non-genomic
Membrane or
cytoplasmic
ERs
Activation of signaling routes such
as: MAPK, P13K, PKA,PKC /
binding of transcription factors to
the active ERs
Non ER-membrane bound
receptors/
adapters
Estrogens
(Nuclear) ERs
Genomic
Estrogen Response Elements (ERE)
dependent signaling
regulated by coactivators
and repressors
ERE
Binding of coactivators
and co-repressors
(e.g. NRIP1)
Proliferation
Growth
Differentation
Angiogenesis
Apoptosis
Target gene expression
(e.g. VEGF)
Estrogen response
elements
independent
signaling by binding
to DNA bound
transcription
factors directly
Transcription factors
(e.g. AP-1 / PS2)
Figure 1. Mechanisms of action of estrogens.
80
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
81
hypomethylated gene is PAX2 (Wu et al., 2005). As an example of estrogenrelated 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
82
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).
83
Cancer
progression
Estrogens and the MSI Model
Histological state
High Estrogen
Receptor activity
DNA damaging effect
by estrogen
metabolites
More estrogens>
more MMR activity
and vice versa
Normal endometrium
cell proliferation
apoptosis
Simple endometrium
hyperplasia
MSH2
Methylation aberrations
Mutations
in genes
e.g.
MLH1
Complex endometrium
hyperplasia
Hyper-methylation of
Estrogen receptor
Hypo-methylation
of PAX2
Other genes
Atypical
hyperplasia
Hyper-methylation
of MLH1
MSI
MSI
Type 1 endometrioid
adenocarcinoma
Mutations in target genes
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 upregulated 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
84
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).
85
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
Tamoxifen
Estrogen receptors
Estrogen receptors
ERα/ERβ
ERα
ERα
ERα/ERβ
ERβ
ERα
ERβ
ERα
ERβ
ERβ
receptor subtype
specific activation
by tamoxifen
receptor subtype
specific activation
by estrogen
Expression is
regulated
by co-activators
and
co-repressors
Tissue specific / Estrogen specific target genes Tissue specific / Tamoxifen specific target genes
Tissue specifc / Target genes activated by both Estrogen and Tamoxifen
Proliferation
processes
Expression is
regulated
by co-activators
and
co-repressors
Apoptosis
processes
Figure 3. Estrogen and tamoxifen signaling; different factors affecting the signaling pathways of both
ligands.
86
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 coactivators 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.
90
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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
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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 estrogenreceptor 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 corepressor protein of the estrogen-receptor (ER) pathway. The ER is an essential
pathway for endometrial tissue regulation; the endometrium is a hormonalresponsive 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.
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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 microdissected 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 MMRrelated 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 hormonerelated 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 MSIH). 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 nietcoderende 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%.
112
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.
113
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
114
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 naocodificantes
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.
115
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.
116
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
117
w
zakresie
markerów
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
118
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
zastosowaniu
analizowane
progu
15%,
dodatkowo,
jako
wyodrębniliśmy
geny
panel
‘towarzyszące’.
rzeczywistych
Po
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.
119
120
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.
121
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
122
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
124
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. 58
th
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”.
129

RIJKSUNIVERSITEIT GRONINGEN

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