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Molecular Signatures of Cancer
Esther Edlundh-Rose
Royal Institute of Technology
School of Biotechnology
Stockholm 2005
© Esther Edlundh-Rose
School of Biotechnology
Department of Gene Technology
Royal Institute of Technology
AlbaNova University Centre
SE-106 91 Stockholm
Sweden
Printed at Universitetsservice US-AB
Box 700 14
SE-100 44 Stockholm
Sweden
ISBN 91-7178-348-2
Esther Edlundh-Rose (2006). Molecular Signatures of Cancer. School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden. ISBN 91-7178-348-2
ABSTRACT
Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations. Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15. These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment. Key words: BRAF, NRAS, MGMT, methylation, pyrosequencing, microarray technology, gene expression analysis
I feel so extraordinary
Something’s got a hold on me
I get this feeling I’m in motion
A sudden sense of liberty
True Faith, New Order
List of publications
This thesis is based on the following publications:
1. Edlundh-Rose E, Kupershmidt I, Gustafsson AC, Parasassi T, Serafino A, Bracci-
Laudiero L, Greco G, Krasnowska EK, Romano MC, Lundeberg T, Nilsson P and Lundeberg
J. Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine
treatment demonstrates cell cycle arrest and increased differentiation. Pathobiology.
2005;72(4):203-12.
2. Gustafsson AC, Kupershmidt I, Edlundh-Rose E, Greco G, Serafino A, Krasnowska EK,
Lundeberg T, Bracci-Laudiero L, Romano MC, Parassassi T and Lundeberg J. Global gene
expression analysis in time series following N-acetyl L-cysteine induced epithelial
differentiation of human normal and cancer cells in vitro. BMC Cancer. 2005 Jul 7;5:75.
3. Bresso F, Edlundh-Rose E, D’Amato M, Are A, Grecius G, Lidén A, Sjöqvist U, Löfberg
R, Arulampalam V, Lundeberg J and Pettersson S. Investigating the chemopreventive role of
ursodeoxycholic acid in colorectal cancer cells. Manuscript.
4. Edlundh-Rose E, Egyházi S, Omholt K, Månsson-Brahme E, Hansson J, and Lundeberg
J. Lower age at diagnosis in melanomas with BRAF as compared to NRAS mutations.
Submitted.
5. Edlundh-Rose E*, Ma S*, Hansson J, Lundeberg J, Egyházi S. Hypermethylation status of
the MGMT promoter in melanoma tumours in relation to clinical response to chemotherapy.
Manuscript. *These authors contributed equally to this work
Contents
INTRODUCTION .............................................................................. 5
1. From peas to -omics ............................................................................5
2. Cancer .................................................................................................7
2.1 Carcinogenesis ................................................................................. 7
2.2 Epigenetics of cancer ...................................................................... 10
2.3 DNA repair and drug resistance ........................................................ 13
3. Mutation analysis ..............................................................................14
3.1 Sanger sequencing ......................................................................... 14
3.2 Single-strand conformation polymorphism, SSCP ................................ 15
3.3 Pyrosequencing .............................................................................. 15
4. Methylation analysis..........................................................................18
4.1 Methylation-specific PCR (MSP)......................................................... 18
4.2 Pyrosequencing .............................................................................. 19
5. Microarray Technology ......................................................................20
5.1 Microarray platforms ....................................................................... 21 5.1.1 GeneChip® technology.......................................................................... 21 5.1.2 Spotted arrays .................................................................................... 21
6 Gene expression analysis ...................................................................24
6.1 Experimental design........................................................................ 24
6.2 Data analysis ................................................................................. 25
6.3 Validation of array data ................................................................... 28
PRESENT INVESTIGATION ...............................................................31
7. Gene expression analysis of epithelial cell differentiation .................31
7.1 Gene expression profiling of N-acetyl L-cysteine (NAC) induced
differentiation of epithelial cells (Papers 1 and 2)...................................... 31
7.2 Investigating the chemopreventive role of ursodeoxycholic acid in
colorectal cencer cells (Paper 3) ............................................................. 32
8. Cutaneous melanoma ........................................................................34
8.1 NRAS and BRAF mutations in melanoma tumours in relation to clinical
characteristics – a study based on mutation screening by pyrosequencing
(Paper 4) ............................................................................................ 36 8.1.2 RAS-RAF-MEK-ERK pathway .................................................................. 36
8.2 Hypermethylation status of the MGMT promoter in melanoma tumours in
relation to clinical response to chemotherapy (Paper 5) ............................. 39 8.2.1 O6-methylguanine DNA-methyltransferase, MGMT .................................... 39
9. Concluding Remarks..........................................................................43
Acknowledgements ...............................................................................45
References ............................................................................................49
5
INTRODUCTION
1. From peas to -omics
Human genetics dates back to Darwin’s theory of evolution in 1856 and Mendel’s study of
garden peas in 1865, where it was observed that certain traits are inherited. It was Avery,
however, who in 1944 discovered that DNA is the material behind which genes and
chromosomes are made [1]. This paved the way for Watson and Crick’s work, which
described the double-helical structure of the DNA molecule [2], for which they won the Nobel
Prize in 1962. Francis Crick also postulated the “central dogma”, which describes how the
genetic information found in the DNA molecule is replicated as cells divide and translated
into functional proteins via transcription to messenger RNA (figure 1) [3]. These
revolutionizing discoveries have created a platform on which modern molecular and genetic
science is based.
Figure 1. The Central Dogma was postulated by Crick in 1958.
6
2001 saw the completion of the first draft of the human genome [4, 5], with the number of
genes estimated today at around 20000-25000 (http://www.ensembl.org/). With this came the
concept of “genomics” and an increased potential to identify and gain an understanding of
genes involved in human physiology and disease. We have now entered the “post-genomic
era”, from which has sprung a plethora of new terms: proteome, transcriptome, epigenome,
metabolome etc., along with a barrage of new techniques to study these. Microarray
technology has made it possible to analyze thousands of samples in parallel and has a variety
of applications [6]; the human proteome atlas is well under way and will eventually produce
antibodies toward all human proteins [7] (http://www.proteinatlas.org/) and antisense
technology and micro RNAs have given us a method for studying gene function [8].
There is much still to learn before we can characterize and cure all human disease, however
the means for doing so are growing and the amount of knowledge gained is increasing
rapidly. We may no longer need only to dream of what the future holds in store.
7
2. Cancer
Cancer has been recognized for thousands of years, the first description being found on
Egyptian papyri written between 3000-1500 BC. Hippocrates was the first to differentiate
between benign and malignant tumours and it was he who gave origin to the medical terms
that are still used today: oncos, meaning swelling in greek and carcinos, which means crab
(the cross section of a malignant tumour somewhat resembling a crab). Today cancer is
considered one of the major human diseases, responsible for around 25% of deaths in the
western world [9]. Thus, cancer prevention and control are major health issues and one of the
largest fields of scientific research.
2.1 Carcinogenesis
Carcinogenesis means literally the creation of cancer and describes the process of
transformation from normal to cancer cells. Cancer arises when the homeostatic balance of a
cell is disrupted, leading to an increase in cell proliferation. There is much evidence
suggesting that carcinogenesis is a multistep process, where several events lead to the growth
of a malignant tumour. These steps reflect genetic or epigenetic alterations, where normal
cells go through several pre-malignant states before resulting in invasive cancer. Tumour
development can be compared to Darwinian evolution, where a mutation, or series of
mutations, gives a cell a selective advantage over surrounding cells [10, 11]. Several types of
genetic alterations affecting cell growth have been identified, including point mutations,
aneuploidy, chromosome translocations and gene amplification. These alterations can produce
oncogenes with a dominant gain of function, or tumour suppressor genes with a recessive loss
of function [12]. Epigenetic alterations can also lead to inhibition of transcription and gene
silencing (reviewed below). Cancer is a very heterogenous disease, making it difficult to study
and get a good understanding of. The determinants of cancer are many and varied including
8
genetic predisposition, environmental influences, infectious agents, nutritional factors and
radiation, to name a few [13]. There are some typical molecular, biochemical and cellular
characteristics that are common in most human cancers. One of the main theories of
carcinogenesis is that the following 6 alterations are essential for the onset of cancer [14]:
Self sufficiency in growth signals
Normal cells require mitogenic growth signals in order to progress from quiescence to
proliferation. These signals are transmitted into the cell by transmembrane receptors that bind
distinct classes of signalling molecules: diffusible growth factors, extracellular matrix
components and cell-cell adhesion/interaction molecules. Tumour cells are self sufficient and
are not dependent on exogenous growth signals. Oncogenes are generally constitutively
expressed and are most often found in pathways leading to activation of transcription, thus the
tumour cell creates its own growth signal [15]. Cancer cells can also switch expression of
extracellular matrix receptors (integrins) to favour those that transmit pro-growth signals [16,
17] . Both these types of growth signal can lead to activation of the Ras-Raf-MAPK pathway
(see below) [17, 18].
Insensitivity to growth-inhibitory signals
Antigrowth signals maintain cellular quiescence and tissue homeostasis either by forcing cells
out of the cell cycle into the G0 phase, or inducing cells to enter a post mitotic differentiation
state. Differentiation is characterized by expression of integrins and adhesion molecules and is
often switched off in tumour cells [19].
The tumour suppressor gene p53 plays a central role in tumorigenesis and is often mutated in
cancer cells [20]. Inactivation of p53 renders the cell insensitive to anti-growth signals, thus
stimulating proliferation.
Evasion of apoptosis
Apoptosis is an organized, genetically programmed cell death, by which multicellular
organisms specifically destroy, dismantle and dispose of cells [21]. It is essential in both
embryonic and adult tissues to eliminate unwanted or potentially harmful cells and maintain
cellular homeostasis. Apoptosis can be triggered by a variety of factors such as DNA damage,
cellular stress, hypoxia or cytotoxic drugs. The Bcl-2 protein family as well as p53 and pRb
are key regulators of this process and mutations in these have been shown to play a
fundamental role in the ability of cancer cells to evade apoptosis [20, 22, 23].
9
Limitless replicative potential
Telomeres cap the ends of chromosomes and are essential for maintaining genomic integrity
and stability [24]. Telomeres are shortened with every replication, although this process is
delayed by telomerase, a DNA polymerase that has the ability to elongate them [25].
Normally a cell has a limited copy number, as telomere length decreases from replication to
replication. Tumour cells, however, have been seen to have an upregulation of telomerase,
resulting in good telomere maintenance and a limitless replicative potential [26].
Sustained angiogenesis
Crucial to cell function and survival is the supply of oxygen and nutrients, thus requiring
vicinity to capillary blood vessels. For tumors to develop in size and metastatic potential they
must make an "angiogenic switch" through perturbing the local balance of proangiogenic and
antiangiogenic factors [27]. Here integrins and cell adhesion molecules mediating cell-matrix
and cell-cell association are again implicated. Vascular epithelial growth factor (VEGF) is an
example of one of these and is thought to play a central role in the angiogenic switch from
vascular quiescence [28, 29].
Tissue evasion and metastasis
A tumour will eventually begin to outgrow its surroundings and will send off a group of cells
to invade different sites and grow new colonies, or metastases. Metastases cause 90% of
human cancer deaths [30]. Yet again cell-cell adhesion molecules, calcium-dependent
cadherins and integrins are involved in this process by linking cells to the extracellular matrix
and allowing them to be transported to new locations [18]. E-cadherin is expressed in
epithelial cells and acts as a suppressor of invasion and metastasis and its functional
elimination results in an acquisition of metastatic ability [31].
The acquisition of these 6 capabilities depends on gene mutation, which is an inefficient
process as the cell has its own set of “caretakers”, or repair enzymes, which mend damaged
DNA, making it highly unlikely for a tumour genome to occur during a human life span [32,
33]. However, cancer does occur frequently and therefore there must be some additional
factors involved. Alterations of genes that encode DNA damage response proteins, such as
p53 or repair enzymes, can result in genomic instability causing a heightened predisposition
to cancer [34]. Inactivation of DNA mismatch repair genes (MMR), such as MSH2 or MLH1
can give rise to alterations at short DNA repeat sequences throughout the genome, resulting in
10
microsatellite instability (MIN) [35, 36]. Tumours with such profiles are referred to as
displaying a mutator phenotype [37]. In most cases genomic instability arises from larger
chromosomal changes, such as amplifications and translocations, which lead to chromosomal
instability (CIN) [33]. These observations suggest that genetic instability plays an important
role in tumourigenesis, although Darwinian selection is more likely to be the initiating and
main driving force for the on-set and progression of cancer [38].
2.2 Epigenetics of cancer
Epigenetics can be defined as alterations in gene expression that are not related to changes in
genotype. Epigenetic regulation of gene expression plays a critical role in development and
differentiation, X inactivation, genomic imprinting and several human diseases including
cancer [39].
DNA methylation is one of the most common and best studied epigenetic events taking place
in the mammalian genome and because it is reversible, it makes it a useful therapeutic target.
DNA methylation is a covalent chemical modification, resulting in the addition of a methyl
group (CH3) at the carbon 5 position of the cytosine ring and occurs predominantly at CpG
dinucleotides [40-43]. The human genome, however, is not uniformly methylated, but
contains regions of unmethylated DNA scattered with methylated regions [44]. Regions
containing short runs of cystosine-guanine repeats are known as CpG islands and are often
found in the promoter region of genes. In cancer cells, gene silencing has been associated with
aberrant hypermethylation at CpG islands in the promoter region, which are normally
unmethylated [45]. In mammals there are three biologically DNA active methyl transferases
(DNMTs): DNMT1, working mainly on maintenance of methylation and DNMT3a and
DNMT3b, whose main activity is de novo methylation [46-48].
The mechanisms of transcriptional repression by epigenetic regulation are at present not fully
understood, although several models have been proposed. Previously it was believed that
silencing through DNA methylation was caused by physical hindrance of binding of
transcription factors to binding sites in the promoter region [49]. It seems, however, that DNA
methylation in itself does not result in gene silencing; instead it serves as a target for
recruitment of other proteins, which are required for the formation of heterochromatin to
silence genes. Binding of methyl binding domain proteins (MBDs) to methylated CpG islands
recruits a corepressor complex containing histone deacetylases (HDACs), resulting in a
change in chromatin structure (figure 2) [50]. It is well established that there is a link between
11
DNA methylation, histone deacetylation and methylation at lysine 9 of H3, although the
precise string of events is not yet fully understood. One theory proposes that H3K9
methylation creates a foundation where an adaptor molecule e.g. HP1 can be recruited, which
in turn binds a DNMT, methylating local CpG dinucleotides. MBD can then bind to me-CpG,
recruiting HDAC complexes, which are required for H3K9 methylation [52]. H3K9
methyltransferases (HMTs) can then methylate lysine 9 of H3 and thus epigenetic information
can flow back and forth from histone to DNA [53].
DNA methylation and cancer has recently become focus of investigation. Tumour cells show
major disruptions in DNA methylation patterns as compared to normal cells and several genes
have been identified as being frequently hypermethylated in cancer. Groups of genes
particularly susceptible are cell cycle genes (p16INK4a, p15INK4a, Rb, p14ARF), genes associated
with DNA repair (MGMT, BRCA1), apoptosis, drug resistance, detoxification, differentiation,
angiogenesis and metastasis [54]. Many tumours show hypermethylation in several genes, an
example of this is a study on lung cancer, where more than 40 genes were found to have
alterations in DNA methylation patterns [55]. Methylation profiling may be a potential
clinical application in cancer diagnosis, prognosis or therapeutics. Development in
methylation detection techniques has led to an array of tools, the most common being
bisulphite sequencing, where bisulphite causes the deamination of unmethylated cytosines to
uridine, thus enabling discrimination between methylated and unmethylated cytosine residues
through sequence analysis. Methylation profiling may be useful as a prognostic factor for
chemotherapeutic response (see paper 5 in Present Investigation).
Epigenetic changes are reversible, which makes them an attractive target for novel therapeutic
agents. Clinical trials are presently in progress using HDAC inihibitors and DNA
demethylating drugs [56-59]. Perhaps the future will see an approach where agents inducing
reversal of epigenetic silencing in tumour cells are combined with conventional chemotherapy
and tailored to suit every individual tumour and patient.
12
Figure 2. Gene silencing by DNA methylation (figure modified from Molecular Biology of
the Cell 4th edition [51]).
13
2.3 DNA repair and drug resistance
Chemotherapy and radiation are currently the two main manners of cancer treatment.
However, resistance to these poses a considerable problem in clinical oncology, leading to the
death of a large number of patients. It is therefore of great importance to find tools to predict
response to chemotherapy and to find new therapeutic targets. Cytotoxic agents work by
inducing DNA damage in cells, activating several pathways starting with recognition of
damage and resulting in programmed cell death [60]. The human cell has developed an
efficient system for repair of DNA-lesions, which is crucial in the maintenance of genomic
integrity and cell survival. DNA repair is therefore an important mechanism for therapeutic
resistance and an understanding of these pathways may reveal new targets for treatment.
DNA repair mechanisms
The simplest mechanism of DNA-repair is direct reversal, which involves a single enzyme
reaction for the removal of certain types of damage directly from the DNA. Removal of alkyl
adducts by MGMT is the best described example of this (reviewed in more detail below) [61].
Base excision repair (BER) is a DNA-repair pathway for single-base abnormalities [61] and is
the main pathway dealing with damage induced by cellular metabolites [62].
Nucleotide excision repair (NER) is a DNA repair complex consisting of recognition, removal
and synthetic proteins that repair bulky adducts and kinks, e.g. as induced by cisplatin, in a
transcription-coupled manner [60, 61].
Mismatch repair (MMR) is a repair complex, containing mismatch recognition proteins that
detects and repairs incorrectly paired nucleotides that have been introduced during replication
[61]. Cisplatin adducts are recognized by MMR complexes and loss of MMR has been linked
to acquired tumour resistance due to failure to enter apoptosis in the presence of DNA damage
[63].
Double-strand break (DSB) repair functions through homologous or non-homologous
recombination [60]. Double-strand breaks are among the most fatal DNA lesions and if
unrepaired can result in chromosome rearrangement and nucleotide loss, which commonly
occur in cancer [64].
14
3. Mutation analysis
As discussed above, cancer is often a result of genetic alterations in a cell and can be large
chromosomal aberrations, such as amplifications, translocations or deletions, or subtle
changes in nucleotide sequence such as single base substitutions, insertions or deletions.
Although Sanger sequencing has been regarded as the “gold standard” for many years and has
been considerably improved, as the need for high-throughput, enhanced speed and low cost
increases, this technique faces many limitations. Several other methods have been developed
for the analysis of point mutations and can be classed into two categories: scanning methods,
which aim at finding unknown mutations in candidate genes; and screening methods, which
aim at finding known mutations, preferably with high-throughput. Scanning technologies
include: direct DNA sequencing, conformation-based techniques, cleavage-based techniques
and enzyme-based methods [65]. Screening technologies include: ligation-based methods,
hybridisation-based techniques and techniques based on polymerase extension [66]. Recently,
novel methods such as biosensors and array-based techniques have also been described for
these purposes [65]. A number of these methods are reviewed by Ahmadian and Syvanän
[67-69].
Here is a short description of some of the most popular methods of mutation analysis:
3.1 Sanger sequencing
The first DNA sequencing techniques were introduced in 1977: the Maxam and Gilbert
method of chain degradation [70] and the Sanger method of chain termination [71]. In Sanger
sequencing four primer extension reactions are performed in the presence of one of the four
dNTPs and a low concentration of dideoxy-dNTPs (ddNTP). ddNTPs have a missing OH-
group at the 3’-end preventing addition of another nucleotide and incorporation results in
15
chain termination. This produces a number of fragments of differing lengths, which are then
separated by electrophoresis and the sequence determined by reading the band pattern.
Originally, radioactively labelled nucleotides or primers were used for detection. Modern
methods, however, use dye-terminators, where each of the dNTPs is labelled with a different
fluorescent dye [72]. The major advantage of this is that the complete sequencing set can be
performed in a single reaction making it easily automated. This, together with the introduction
of capillary electrophoresis, has increased throughput considerably as well as reducing cost.
3.2 Single-strand conformation polymorphism, SSCP
SSCP is a simple and fast method for scanning many samples for DNA polymorphisms.
Single-stranded DNA molecules can assume varying secondary and tertiary structures,
depending on their nucleotide sequences. SSCP analysis detects point mutations based on
electrophoretic mobility differences that result from these conformational changes [73]. PCR
amplified DNA, covering the region of interest, is denatured using a denaturing agent, such as
formamide, and heat. The single-stranded molecules are then separated on a non-denaturing
polyacrylamide gel and compared to wild-type or known mutant DNA. Results are typically
visualized by autoradiography[74], silver staining or capillary electrophoresis based methods
[75]. Mutant samples are usually confirmed by DNA sequencing.
3.3 Pyrosequencing
A fairly novel method for performing sequence-based DNA analysis is the pyrosequencing
technique [76]. Due to the ability of automation, this system can easily be used for high-
throughput screening. Pyrosequencing is based on the “sequencing by synthesis” principle
[77], where nucleotides are sequentially added to a primed single-stranded DNA template of
which the sequence can be determined by the order of the incorporated nucleotides. Four
enzymes are involved: the Klenow fragment of E. coli DNA polymerase I, ATP sulphurylase,
luciferase and apyrase. If the added nucleotide is incorporated into the growing DNA strand,
pyrophosphate, PPi, will be released by the Klenow DNA polymerase. PPi serves as a
substrate for ATP sulphurylase, which produces ATP. Luciferase then converts ATP into
visible light, which can be detected by a CCD camera. Nucleotides are added to the reaction
one at a time and excess nucleotides are degraded by apyrase in between additions. If the
added nucleotide does not form a base-pair with the template DNA strand, it will not be
16
incorporated and no light will be detected. The overall reaction takes place within 3-4 seconds
at room temperature (figure 3) [76].
One of the major drawbacks of pyrosequencing has been its limited read-length ability, which
originally allowed read lengths of up to only 20-30 bp. However, many improvements have
been made to the technique since the earliest version. It was discovered that false signals were
obtained when dATP was added to the pyrosequencing reaction, due to dATP being a
substrate of luciferase. This problem was solved by the substitution to dATPαS, which is inert
to luciferase, in the polymerization reaction [76]. Secondary structured in the single-stranded
DNA template, as well as unspecific primer binding, can interfere with the pyrosequencing
reaction, also reducing read-length. The addition of ss-DNA binding protein (SSB) can reduce
this problem and has recently been introduced to commercial kits [78] (www.biotage.com).
Pyrosequencing has many other applications including: SNP genotyping [79], CpG
methylation analysis (see below) [80, 81], resequencing [82] and has recently been developed
into an ultra high-throughput method for whole genome sequencing by 454 Life Sciences
[83].
17
Figure 3. Schematic diagram of the priniciples of pyrosequencing.
18
4. Methylation analysis
In the recent years, it has become apparent that cancer is as much a disease of misdirected
epigenetics as it is a disease of genetic mutations [42]. Hypermethylation of promoter regions
is associated with gene silencing, through chromatin conformational changes and is a
common event in carcinogenesis (see section 2.3). Detection of these events is therefore of
great interest in both clinical diagnostics and therapeutics. Most methods for analysis of DNA
methylation patterns are based on bisulphite genomic sequencing of amplified products [84].
Sodium bisulphite converts unmethylated cytosines to uracil, leaving methylated cytosines
unchanged [85]. PCR amplification of treated template results in a C/T polymorphism, which
can be detected by various sequencing methods. Novel techniques include pyrosequencing
[80, 81] and array-based methods [86, 87]. The method of choice depends on the desired
application, whether it is to quantify overall methylation in genomic DNA, to map methylated
cytosines in specific DNA sequences or to find new methylation hot spots (several methods
are reviewed by Fraga and Esteller 2002 [88]).
Two methods are briefly described below:
4.1 Methylation-specific PCR (MSP)
Methylation-specific PCR is the most widely used technique for studying the methylation
profile of CpG islands. Primers are designed to anneal specifically to methylated or
unmethylated bisulphite-converted sequence. Because the strands are no longer
complementary, the primer-design must be customized for each chain [88]. MSP eliminates
the false positive results inherent to previous PCR-based approaches which relied on
differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA
19
[89]. This method has had a huge impact on the field of cancer epigenetics by making DNA
methylation analysis accessible to a wide number of laboratories [84].
4.2 Pyrosequencing
Pyrosequencing has recently been adapted for methylation analysis (figure 4) [80, 81, 90].
The main advantage of the pyrosequencing technology, compared to MSP, is the
quantification of the methylation status of each individual CpG in the sequence. This is of
particular interest as the degree of methylation can differ significantly at various CpGs within
a CpG island [90].
c/mcgcagactgcctcaggcccg
u/mcguagautguutuagguuug
bisulphite treatment
t/cgtagattgttttaggtttg
PCR amplification
1200
1300
1400
1500
E S A T C A G T C T A G A T G T T A G T C A G T C A G T C A G T C T G
T:52,6%C:47,4%
T:100,0%C:0,0%
T:74,0%C:26,0%
T:70,7%C:29,3%
T:70,4%C:29,6%
T:69,7%C:30,3%
5 10 15 20 25 30
Pyrosequencing analysis
c/mcgcagactgcctcaggcccg
u/mcguagautguutuagguuug
bisulphite treatment
u/mcguagautguutuagguuug
bisulphite treatment
t/cgtagattgttttaggtttg
PCR amplification
t/cgtagattgttttaggtttg
PCR amplification
1200
1300
1400
1500
E S A T C A G T C T A G A T G T T A G T C A G T C A G T C A G T C T G
T:52,6%C:47,4%
T:100,0%C:0,0%
T:74,0%C:26,0%
T:70,7%C:29,3%
T:70,4%C:29,6%
T:69,7%C:30,3%
5 10 15 20 25 30
Pyrosequencing analysis
1200
1300
1400
1500
E S A T C A G T C T A G A T G T T A G T C A G T C A G T C A G T C T G
T:52,6%C:47,4%
T:100,0%C:0,0%
T:74,0%C:26,0%
T:70,7%C:29,3%
T:70,4%C:29,6%
T:69,7%C:30,3%
5 10 15 20 25 30
Pyrosequencing analysis
Figure 4. CpG methylation analysis by pyrosequencing.
20
5. Microarray Technology
With the completion of the human genome project, comes the ability to identify genes of
interest and find a link to human disease. Along with this comes the need for an efficient high
throughput method of screening large numbers of genes at once. Previously, methods such as
Northern Blot Analysis and RT-PCR have been used, which allow expression analysis of a
small number of genes. Serial Analysis of Gene Expression (SAGE) was one of the first
methods of global gene expression analysis [91] and is a powerful method, although it has
several limitations. Microarray technology, however, allows for parallel and global analysis of
several thousands of genes and is one of the most widely used and versatile methods. The
earliest recorded study using this technology was published by Augenlicht et al. in 1987,
where 4000 cDNA clones were spotted on a nylon membrane to examine gene expression in
colon cancer [92]. During the past few years development of microarrays has bloomed and
progressed from obscurity to becoming routinely used in biological and medical research for
gene expression studies. Furthermore, the microarray platform can be used in a variety of
other applications and several new methods are emerging, such as protein interaction assays
e.g. CHiP-on-chip (on chip immunoprecipitation) [93], genome-wide epigenetic analysis [86,
87] and resequencing [94].
21
5.1 Microarray platforms
5.1.1 GeneChip® technology
Array manufacture
The first commercially available microarrays were high-density oligonucleotide chips,
reported by Affymetrix in 1995 [95, 96]. Oligonucleotide probes are chemically synthesized
on the surface of a chip, using lithographic masks in a similar technique to that used in the
production of computer chips [97, 98]. For each gene represented on the chip, 11-20 probes,
preferably chosen at unique regions, are used. The probes are approximately 25 nt in length
and are generated in pairs with a perfect match (PM) and a mismatch (MM), where one
nucleotide in the complementary sequence has been changed. The MM probes are used later
to correct for non-specific binding and background signals [99].
Sample preparation, labelling and hybridization
cDNA is synthesized from isolated mRNA using reverse transcriptase and a poly-T primer.
Samples are amplified using T7 RNA polymerase in the presence of biotin-labelled UTP and
CTP, yielding 50-100 copies of biotin-labelled cRNA. The cRNA is then fragmented into 25-
300 nt long fragments, to avoid formation of secondary structures, and hybridized to the chip.
After washing, biotin-labelled target is stained using an antibody amplification procedure
involving binding to streptavidin-phycoerythrin. Finally the chip is scanned in a confocal laser
scanner [100].
5.1.2 Spotted arrays
Spotted arrays are considerably cheaper to use than GeneChip® and have the advantage that
they can be manufactured in-house.
Array manufacture
The first step in the construction of a microarray is to identify which transcripts are to be
spotted. Spotted arrays can be designed to cover any combination of transcripts focusing on
the field of interest and the user is not limited to pre-designed arrays, which may be of a more
general nature. There are several publically available databases where genes of interest can be
identified e.g. UniGene (http://www.ncbi.nlm.nih.gov/UniGene/), where gene nucleotide
22
sequences can be found in a set of non-redundant clusters [101] and Ensembl
(http://www.ensembl.org/, [102]). Another public source of cDNA clones is the Integrated
Molecular Analysis of Genomics and their expression (IMAGE) Consortium [101]. Once a
suitable collection of genes and ESTs has been chosen, bacterial clones containing plasmids
with cDNAs of interest are grown. After extraction, the cDNAs are amplified by PCR using
vector sequences as priming sites. PCR-products are then purified and sequence-verified
before being spotted onto aminosilane or polysilane coated glass slides by a robotic
microarrayer. An alternative to cDNA clones is to spot conventionally synthesized long-mer
oligonucleotides [103], which have gained increased interest, due to higher specificity in
hybridization and simplicity in array manufacturing.
Sample preparation, labelling and hybridization
Total or mRNA is isolated from cells or tissues of interest, quantitated and checked for purity.
Labelling with fluorescent dyes, typically Cy3 and Cy5, is then performed either in a direct or
indirect reaction. In a direct labelling reaction dye-labelled nucleotides are incorporated
during cDNA synthesis and in an indirect reaction chemically modified nucleotides are
incorporated to which fluorescent dyes are subsequently coupled. The most common method
involves incorporation of amino-allyl modified nucleotides, which are coupled to Cy3- or
Cy5-esters [104, 105]. Although indirect labelling involves an extra step, it is often preferred
as it avoids bias due to uneven incorporation of the different fluorophores, often gives higher
signals and is usually cheaper.
Before hybridization, the microarray slide is subjected to a prehybridization bath, where
reactive groups on the slide surface are blocked by BSA, to avoid high background signals.
Labelled samples are mixed with hybridization buffer, generally containing denaturing agents
such as sodium dodecyl sulphate (SDS) and formamide, salts and Cot-1 and poly-A DNA to
block repetitive sequences. Hybridization is performed manually, by applying the mixture to
the slide and placing it in a hybridization chamber, or automatically in a hybridization station
for 16-24h. Slides are then washed and scanned in a confocal laser scanner (figure 5) [100].
23
Figure 5. Schematic diagram of the principles of cDNA microarray analysis.
24
6 Gene expression analysis
Gene expression analysis or transcript profiling, where simultaneous analysis of gene
expression levels in various tissues is performed, can give us novel information on genes and
pathways involved in various diseases or states. The process involves several steps and can be
performed with both GeneChip® and spotted arrays.
6.1 Experimental design
Before the experiment is performed, it is important to plan well. A poorly planned experiment
may result in a load of useless data, where time, money and perhaps valuable samples will
have gone to waste. The first thing to consider is the aim of the experiment, whether it is to
identify differentially expressed genes, to search for specific gene expression patterns or to
classify tumour subtypes. Once this has been established, one has to think about which
samples one wants to look at. The most common analyses are where two samples are
compared to each other e.g. normal and diseased tissues, or treated and untreated cell samples.
Here one has to take into account aspects such as, what is considered normal and the
heterogeneity of tissue samples, especially tumours. Time is also an aspect, where a disease or
a treatment may have very different effects on gene expression over time; in this case it might
be suitable to compare several samples taken over a period of time. Biological fluctuation is
one of the major sources of variation in a microarray experiment and it is therefore extremely
important to have several biological replicates, the minimum recommendation being five
[106, 107]. This, however, is not always accomplished, often due to lack of biological
material. Technical replicates, which allow only effects of measurement variability to be
reduced, are not often required, although they can be of use in quality control experiments or
cases where the number of samples is small [108].
25
In the case of spotted arrays, there are three main design choices: direct comparison, where
the two samples of interest are hybridized against each other on one slide; reference design,
where all samples are hybridized on separate slides against a common reference; or loop
design, where samples are hybridized to one another in a chain [109, 110]. Direct comparison
is the simplest choice and reduces technical variance, although dye swap experiments should
be done to avoid bias from either of the fluorophores. This method is, however, limited in its
uses as only the two samples hybridized together can be compared. If there is a desire to make
several comparisons with many different samples a reference design might be preferred. This
has become the most common design method as it allows any comparison to be made between
samples with equal efficiencies and it also allows additional samples to be added to the
experiment at a later date, providing availability of the reference sample [110]. An alternative
to reference design is the loop design, which involves fewer slides [111]. Efficiency may,
however, be lost if loops are large and many comparisons are made, or if an array falls out
[110].
6.2 Data analysis
Data preprocessing and normalisation
Microarray experiments produce an overwhelming amount of data and the handling of this is
a whole science of its own. Raw data is obtained by scanning the arrays with a confocal laser
scanner after which image processing done and foreground and background intensities of the
spots measured. In the case of spotted arrays, where samples are hybridized in two channels, a
ratio of the spot intensities is used for analysis. In order to present up- and downregulated
genes equally, these ratios are log-transformed, most commonly using a base-2 logarithm
[100]. The array fabrication and hybridization process, especially for spotted arrays,
introduces a lot of noise, which may interfere with results of downstream analyses. It is,
therefore, common to process the data through a series of filtering steps before proceeding
with the analysis. Examples of some filtering steps are removal of spots with intensities close
to background, differing mean-to-median pixel ratios, saturated intensities or deviating size
[100]. Filtering is, however, considered unnecessary by some, who suggest that aberrant spots
will display a high variance between slides and will therefore be removed in downstream
stastistical analysis.
26
Normalization is performed, to adjust for variability across a slide or between experiments.
There are several algorithms “on the market” and to date no consensus on which is the best.
Local weighted linear regression, or lowess, analysis is widely used and takes into account
intensity dependent effects. A local, or print-tip, lowess normalisation corrects for both
intensity and spatial variation between spotting tips [112]. Other normalisation methods
include total intensity normalization, which relies on the assumption that mRNA quantities
are equivalent for both samples hybridized and that equal amounts of genes are up- and
downregulated [113]. Housekeeping genes can be used for normalization, however, this
assumes that all housekeeping genes have constant levels of expression, which is rarely true.
The use of spike-in controls for normalization purposes is attractive, although it involves
more work in array design etc. [114].
Identifying differentially expressed genes
Once normalized expression ratios have been obtained, it is usually desirable to identify
which genes are most likely to be differentially expressed. Early microarray gene expression
analyses often used simply a fold change cut-off of 2 to define genes as differentially
expressed, which is now considered as an inadequate method [115, 116] as it does not take
into account variance and offers no associated level of confidence [115, 117]. Subtle,
although potentially important changes, may also be missed when using the fc cut-off method.
More recent methods involve ranking the genes in order of evidence for differential
expression and setting a suitable value of significance [118]. One choice of ranking genes is
to use the t statistic, where variability between replicates of each particular gene is taken into
account. However, some genes, especially those with low intensities, will have small sample
variance and result in large t-values even though they are not in fact differentially expressed
[118]. An alternative choice is to use a parametric empirical Bayes approach, where the B-
statistic is an estimate of the posterior log-odds that each gene is differentially expressed
[119]. It is not always obvious where to choose the cut-off value as both type I and type II
error needs to be kept low. It is generally preferable to allow a certain amount of false
positives in order not to miss the true positives. Other popular statistical methods for
identifying differentially expressed genes are Significance Analysis of Microarrays (SAM)
[120] and ANOVA-based approaches [111, 121].
As many thousands of genes are measured simultaneously, correction must be done for
multiple testing. Bonferroni correction is a family wise error rate control procedure and limits
the probability of making a type I error due to multiple testing [122]. However, as it is often
27
deemed acceptable to include a number of false positives, Bonferroni correction is usually
considered too stringent. Benjamini and Hochberg came up with a procedure called false
discovery rate (FDR) which is often considered more appropriate [123].
Data mining
Microarray analysis often gives rise to long lists of potentially differentially expressed genes
and poses the problem of how to interpret these from a biological point of view. A number of
tools have been developed to deal with this and the choice of which to use depends on the
questions being asked.
Genes involved in a common pathway, or that respond to a particular environmental factor are
expected to be co-regulated. It is, therefore, of interest to study genes which share similar
patterns of expression and this is done by clustering analysis. Clustering is based on distance
metrics, where a “distance” is calculated between each gene expression vector. Various
clustering algorithms are then used to group genes of similar expression pattern. Unsupervised
methods, which require no prior knowledge, such as hierarchical clustering [124], k-means
clustering [125], self-organizing maps (SOM) [126] and PCA [127] are commonly used.
Supervised methods are used for classification of e.g. cancer sub-types and require previous
information about which genes will cluster together. The data is trained on the classes of
genes already known, so as to distinguish between members and non-members, and is then
applied to the test data [128]. Examples of supervised clustering methods are: linear
discriminant analysis, nearest neighbour classifiers [129], and neural networks such as support
vector machine (SVM) [130].
It may also be of interest to find out more about the function of the genes and how they are
related to other biological pathways and processes. For this purpose, the Gene Ontology (GO)
Consortium has constructed a vocabulary to describe gene and gene product attributes to
biological processes, cellular components and molecular functions [131]. A number of open-
access tools are available, which can calculate overrepresentation statistics for all GO terms
with respect to a given data set, so called gene class testing (GCT) [108], e.g. EASE [132],
MAPPfinder [133] and Onto-Express [134].
Identification of metabolic pathways is also an important part of gene expression profiling, to
acquire a mechanistic understanding of disease or drug treatment etc. One of the biggest
efforts in this field is the Kyoto Encyclopedia of Genes and Genomes (KEGG) consisting of
28
graphical representations of most known biochemical pathways [135]. There are various tools
available, which allow the user to link pathway information to gene expression data or to
build new pathways, such as the open-source GenMAPP [136] or commercially available
PathwayStudio [137] (Ariadne Genomics, MD, USA) and GeneSpring (Agilent Technologies,
USA).
The management and analysis of microarray data has become more and more demanding,
making it quite a challenge. The requirement for comprehensive and flexible tools has led to
the development of a number of both commercial and open-source tools. The R open-source
language and environment for data analysis and visualization provides implementations for a
broad range of statistical and graphical techniques [138](http://www.r-project.org/). Together
with the Bioconductor (http://www.bioconductor.org/) open-source software package, R can
be used to cover most of the above mentioned data analysis steps and is applicable to all array
platforms.
In 2001, the Minimum Information About a Microarray Expermiment (MIAME) was
introduced by the MGED society [139] in an effort to standardize the presentation of
microarray data. These standards have been adopted by the microarray community and are the
basis of several array databases.
6.3 Validation of array data
Although microarray technology and data analysis is becoming more and more standardized
and thus more reliable, artifacts can still be introduced at any time during an experiment and it
is therefore necessary to validate the results. Once a number of target genes have been
identified by the microarray analysis, it is desirable to confirm RNA expression levels by an
independent method. The most common method is quantitative RT-PCR [140] because of its
speed and inexpensiveness. Northern blot has also been reported to give consistent results as
compared to microarray [141].
In addition to validating transcript levels, it is important to measure levels of the
corresponding proteins, as we know that they are not always correlated. This can be done by
various antibody based methods e.g.Western blot, immunohistochemistry or IHC via tissue
microarrays [142-144]. These methods are dependent on antibody availability, which can pose
a problem in the case of unknown transcripts. However, with the Human Proteome Atlas
(http://www.proteinatlas.org/) well on the way, this should soon be solved.
29
A key strategy in target validation is to determine the phenotypic effects when the activity of
a gene is blocked. RNA interference (RNAi) technology is a gene knockdown strategy, which
can be used for this purpose [145]. This method coupled with large scale cell based screening
can also be performed, perhaps leading to identification of novel therapeutic targets [146].
30
31
PRESENT INVESTIGATION
7. Gene expression analysis of epithelial cell
differentiation
Cancer arises when the balance between proliferation, differentiation and apoptosis is in some
way disturbed. Human cancers often arise in epithelial cells, highlighting the importance of
gaining knowledge about the underlying molecular mechanisms involved in differentiation
and proliferation in these cell types.
7.1 Gene expression profiling of N-acetyl L-cysteine (NAC) induced
differentiation of epithelial cells (Papers 1 and 2)
N-acetyl L-cysteine, a sulfhydryl reductant, has been shown to induce a 10-fold more rapid
differentiation in normal human epidermal keratinocytes (NHEK) and a reversion from
neoplastic proliferation to apical-basolateral differentiation in the colon carcinoma cell line
Caco-2. Decreased cell proliferation in these cell types occurred without compromising cell
viability and without induction of apoptosis [147]. Rearrangement of the cytoskeleton was
observed, demonstrated by an increase in cellular junctions and basal localization of actin, all
characteristics of differentiation.
To study the effects of NAC on gene expression, microarray studies were performed using
both the Affymetrix and cDNA platforms. The Affymetrix GeneChip Human U95Av2
containing 12000 transcripts was used to compare NAC treated to untreated NHEK and Caco-
2 in a time series. The KTH HUM30k cDNAarray containing 29760 transcripts was used to
compare NAC treated to untreated NHEK in a time series. Quantitative RT-PCR was
performed to verify the array results.
32
In the Affymetrix study NAC induced differentiation in Caco-2 and NHEK gave 253 and 414
definitely expressed targets respectively (according to our criteria) across the whole time
series. This corresponds to approximately 200 genes in both cell types after consideration to
multiple appearances.
In the cDNA microarray experiment 1007 genes were found to be differentially expressed in
NAC treated NHEK (according to our criteria).
Both experiments showed a limited early transient response at 30 min-3h, with an increasing
late response at 12-48h. A significant number of genes were classified as being involved in
differentiation/proliferation processes and reorganization of the cytoskeleton, which was in
concordance with previous morphological studies. Although both NHEK and Caco-2
displayed differential expression of genes involved in similar processes, the actual responses
seemed to be lineage specific, with little overlap between gene lists.
Interestingly, inhibitor of differentiation 1 (ID-1), which has previously been shown to be
required in G1 cell cycle progression, was found to be downregulated at 1h/30 min in both
NHEK and Caco-2, suggesting a common mechanism for NAC induced differentiation and
inhibition of proliferation.
7.2 Investigating the chemopreventive role of ursodeoxycholic acid in
colorectal cencer cells (Paper 3)
Colorectal cancer (CRC) is one of the leading causes of cancer death in the western world,
with numbers increasing every year [148]. CRC is believed to arise through a multistep
process characterized by histopathological precursor lesions and molecular genetic alterations
[149]. By interfering with these events, chemoprevention could inhibit or reverse the
development of adenomas or the progression from adenoma to cancer. Nonsteroidal anti-
inflammatory drugs (NSAIDs) are the best studied chemopreventive agents against CRC and
have proven efficiency, though they do have certain side-effects. Ursodeoxycholic acid
(UDCA) is a naturally occurring bile acid and is widely used in cholestatic liver disease with
minimal side effects [150]. Preclinical studies have demonstrated colon cancer
chemopreventive effects of UDCA [151-153]. Apoptotic and anti-proliferative properties of
UDCA derivatives have been described in colon cancer cells, with cell cycle arrest at the G1
phase [151, 154]. Hence, UDCA may be of use in the chemoprevention of colon cancer,
although the molecular mechanisms are yet to be elucidated.
33
Global gene expression analysis was performed using cDNA microarrays, in order to
characterize the molecular mechanisms of action and find marker genes for the
chemopreventive effect of UDCA.
Colonic epithelial SW480 adenocarcinoma cells treated with 500 µM UDCA were compared
to ethanol treated control cells in a time series including time points 2, 6, 10, 24 and 48h. A
list of 62 statistically significant differentially expressed genes was extracted from time point
10h and 17 of these were verified by quantitative RT-PCR. Many genes had fold changes of 2
or more at time points 24 and 48h, although not statistically significant. QRT-PCR performed
at time points 2h and 24h reflected previous morphological observations and microarray
results, which suggested increasing transcriptional regulation over time.
Among the genes found to be upregulated at 10h, was growth differentiation factor 15
(GDF15), a divergent member of the TGF-β superfamily, which has been seen to regulate
apoptosis [155] and to be involved in the differentiation of epithelium [156]. GDF15 is a
secreted protein [156], suggesting a role as a regulator of late/secondary transcriptional
response and making it an interesting candidate for further analysis.
Interestingly, a comparison of gene expression levels in SW480, by QRT-PCR, revealed that
UDCA also has a gene regulatory effect on hepatocarcinoma cell line HEPG2 and breast
cancer cell line MCF-7, suggesting a potential chemopreventive use of UDCA in other cancer
types.
Cell cycle analysis by FACS showed a G1 arrest in UDCA treated SW480 cells as compared
to control cells treated with ethanol or deoxycholic acid (DCA). These results support the
microarray data, where both GDF15 and growth arrest- and DNA damage- inducible gene
(GADD45), were upregulated.
In conclusion, our results demonstrate a clear antiproliferative effect of UDCA on colon
cancer cells. Together with previous studies, our results support UDCA as a potential
chemothpreventive agent in patients at risk for colon cancer and may even have its uses in
other cancer types. Although the molecular mechanisms are not entirely clear, a number of
target genes have been identified and follow up studies on these are in the pipeline.
34
8. Cutaneous melanoma
Malignant melanoma is a growing disease increasing at a rate of 2.5-4% every year [157].
Primary tumours are easily detected and can be cured in 95% of cases by surgery. However,
patients with disseminated melanoma are generally resistant to all current therapies and have a
poor prognosis with a one year survival rate of approximately 50% [158].
Clark suggested a model for the progression of melanoma in 1984, which is based on a series
of histopathological and clinical properties. The model is based on 5 stages of melanoma
progression (figure 6): 1. common nevus; 2. dysplastic nevus; 3. radial growth phase (RGP);
4. vertical growth phase (VGP); 5. metastatic melanoma [159]. Common nevi, or moles, may
be present at birth (congenital nevi) or may appear later. They are benign, but are considered a
potential precursor to melanoma. A nevus is defined as dysplastic when it begins to differ
from the normal, i.e. it becomes atypical, and is considered a high risk marker of melanoma.
At the RGP stage, the tumour has begun to spread horizontally, but is still confined to the
epidermis. These tumours are also known as cancer in situ and do not have the ability to
metastasize. In VGP, the tumour has become invasive and has spread to the papillary and
reticular dermis. At this point the tumour has acquired the ability to metastasize and can
continue to develop into metastatic melanoma [159].
Figure 6. Clark’s model for the progression of melanoma.
35
There are several histological subtypes of cutaneous melanoma: superficial spreading
melanoma (SSM) which is the most common type; nodular melanoma (NM) are ball shaped
and lack RGP, making them highly aggressive; lentigo maligna melanoma (LMM) are often
located on the face and acral lentiginous melanoma (ALM), which are commonly found on
the palms of the hand, soles of the feet and subungally and are sometimes difficult to diagnose
as they often look benign.
Melanocytes are epidermal cells derived from the neural crest [157]. Melanocyte precursors
migrate to the basal layer of the epidermis where they differentiate and acquire the ability to
synthesise melanin (figure 7). These cells do not divide rapidly, but are stimulated by
environmental factors such as UV-radiation, which induce division and production of
cytoprotective pigments [157].
Figure 7. The different layers of the skin (figure modified from www.infomedica.se).
Melanoma is a disease of homeostatic imbalance in the skin, where a number of components
influence tumour development: epidermal keratinocytes, dermal fibroblasts, endothelial and
inflammatory cells. In normal skin there is a fine balance of these, where keratincocytes keep
melanocytes under control from constant proliferation and a well-defined basement
membrane complex keeps melanocytes out of the dermis [157].
E –cadherin is normally expressed in melanocytes, allowing them to adhere and thus
preventing invasion [160]. In melanoma progression, the expression of E-cadherin is
36
progressively lost and completely absent in melanoma cells [157]. Instead they acquire the
expression of N-cadherin, which promotes interactions with fibroblasts and endothelial cells
allowing migration and invasion of the tumour [161].
There are a number of gene mutations associated with malignant melanoma and the risk of
developing melanoma is considerably higher if there is a family history [162, 163]. CDKN2A
and CDK4 are high penetrance genes, which give a high risk of developing melanoma if
mutated [164, 165]. The CDKN2A gene locus encodes the tumour suppressors p16INK4A and
p14ARF. p16INK4A controls proliferation by binding pRb and thus blocking E2F activated gene
transcription, leading to cell cycle arrest in the G1 phase. p14ARF binds mdm2, blocking
degradation of p53, which plays an important roll in apoptosis [166, 167]. Thus, a mutation in
this gene locus can lead to increased proliferation and/or dysregulation of apoptosis.
There is strong evidence that the Ras-Raf-MAPK signalling pathway is involved in cutaneous
melanoma [168, 169]. Studies have shown mutations in NRAS or BRAF in approximately 80%
of tumours. The most common BRAF mutation is a switch from valine to glutamic acid at
codon 600 in exon 15 leading to hyperactivation of BRAF and increased transcription through
MAPK activation [170] (see below for more details).
8.1 NRAS and BRAF mutations in melanoma tumours in relation to clinical
characteristics – a study based on mutation screening by pyrosequencing
(Paper 4)
8.1.2 RAS-RAF-MEK-ERK pathway
The MAPK pathway plays a central role in the control of proliferation, differentiation and cell
survival. Growth factors activate RAS through a G-protein coupled receptor, which in turn
recruits RAF to the cell membrane where it is activated by phosphorylation [171, 172]. RAF
is a serine/threonine kinase, which activates the protein kinase MEK by phosphorylation,
which in turn phosphorylates and activates ERK, also a protein kinase [173]. ERK
phosphorylates and activates cytosolic and membrane-localized cytoskeletal proteins,
regulating cell shape and migration [174]. ERK can also translocate to the nucleus and
activate transcription factors, regulating gene expression [173]. There are three mammalian
RAF proteins: Raf-1, ARAF and BRAF [175, 176]. Activation of Raf-1 and ARAF requires
signalling from both RAS and Src, whereas BRAF has a higher basal kinase activity and is
activated by RAS alone [175, 177]. Consequently, BRAF requires fewer steps to become
37
activated, which suggests that it will be activated under a greater variety of conditions and
may be the isoform that is primarily responsible for signalling between RAS and MEK in the
majority of cells [173]. This may also provide an explanation as to why BRAF is more
susceptible to mutation in cancer.
Activating mutations in NRAS are found in up to 30% of human melanomas [178-180] and
BRAF is mutated in 40-67% of cases [170, 181, 182].
This study
Screening and identification of gene mutations in tumours is generally performed by DNA
sequencing, which is high throughput and reliable, but very costly, or single strand
conformational polymorphism (SSCP), a cheap and simple pre-screening method, where
sensitivity and specificity has been questioned.
Pyrosequencing is a real time, sequence by synthesis method, which is high throughput, very
specific and reliable. Sivertsson et al. (2002) showed that mutations in NRAS could be
screened using pyrosequencing [183]. Here we expanded the study to include BRAF
mutations. Samples were amplified in an outer multiplex reaction and 2 separate inner PCR
reactions were done for each of the genes. Pyrosequencing was then performed on a PSQ HS
96A Pyrosequencer.
In our study we screened 219 tumours from 176 patients for mutations in NRAS codon 61 and
BRAF codon 600, using pyrosequencing. In addition, 75 tumours from 43 patients with
previously reported NRAS/BRAF mutation status [182] were added to the analysis. Thus,
data from 294 tumours from 219 patients were analysed. Mutations in NRAS were found in 86
(29%) of the tumours and 156 (53%) in BRAF. In total, 82% of the tumours analysed
displayed mutations in either BRAF or NRAS, and were mutually exclusive in all cases but 2,
which contained mutations in both genes. Multiple tumours were analysed in 57 of the 294
cases and NRAS/BRAF mutations were found in 47 of them, where all but 3 had the same
genotype, indicating that these mutations occur early, before metastasis takes place.
Significant differences with respect to association of a pre-existing nevus and to lymphocyte
infiltration were seen between the three genotype groups: NRAS mutants, BRAF mutants and
wt NRAS and BRAF. BRAF mutants were more commonly associated with a pre-existing
nevus and a higher infiltration of lymphocytes.
38
The two groups of patients defined with NRAS and BRAF mutations, respectively, were
compared to each other only, in a clinical analysis including parameters such as overall
survival. BRAF mutations were associated with an earlier onset than NRAS, with mean ages at
diagnosis of 58 and 64 years respectively. In addition, NRAS mutations were associated with a
higher Clark level of invasion than BRAF mutations. Ulceration, however, seemed to be more
common in tumours with mutations in BRAF, although the difference was not statistically
significant.
In our study, BRAF mutations were more commonly found in tumours on the trunk and lower
extremities, whereas NRAS mutations were found more frequently on the upper extremities,
with a more continual exposure to sun. This observation is not surprising, as the nature of the
mutations in NRAS and BRAF differ from each other. NRAS codon 61 mutations are classical
UVB related modifications with a formation of pyrimidine dimers, caused by UV-radiation,
resulting in a CC → TT or C → T switch, wheras the most common BRAF mutation is a T →
A at nucleotide 1799, where the mechanism is still unknown.
In conclusion, our results show that NRAS and BRAF mutations arise early and are preserved
from primary tumours to metastases. We have also shown that different genotype patterns are
associated with different clinical parameters. Perhaps the most interesting finding is that
tumours with BRAF mutations are more often associated with lymphocyte infiltration,
suggesting a potential target of immunotherapy.
39
8.2 Hypermethylation status of the MGMT promoter in melanoma tumours
in relation to clinical response to chemotherapy (Paper 5)
8.2.1 O6-methylguanine DNA-methyltransferase, MGMT
The gene O6-methylguanine DNA-methyltransferase (MGMT) encodes the repair protein
MGMT, which removes alkylating lesions at the O6 position of guanine. MGMT scans the
double-stranded DNA flipping each base into its active-site pocket, which contains a cysteine
to which the alkyl group is covalently transferred (figure 8) [184, 185]. After this, MGMT is
released from the DNA and subsequently ubiquitinylated and degraded [186]. If the lesion is
not repaired, a GC→AT mutation will occur in 90% of cases during replication due to
incorrect base-pairing by the DNA polymerase [187].
MGMT is interesting from several points of view: first of all it is important in the protection
of normal cells from exogenous carcinogens and endogenous methylating agents. Several
reports show that overexpression of MGMT suppresses tumour development in mouse models
[188-191] and that MGMT knockout mice are more sensitive to methylating agents than wt
[192].
Secondly it may increase drug sensitivity when used as a target of inhibition in the use of
alkylating chemotherapeutic agents. Alkylating agents are often used in chemotherapy and
work on the basis of their capacity to transfer alkyl groups such as methyl, ethyl or
chloroethyl to nucleophilic sites in DNA. Alkylation at the O6-position is one of the most
cytotoxic lesions and mainly responsible for the effects of chemotherapy [193]. Resistance to
these cytotoxic agents may be a result of efficient repair of O6-AG adducts by MGMT.
MGMT can be upregulated by response signals to DNA-damage. The promoter region
includes several transcription-factor recognition sites [194-196] and hypermethylation of CpG
islands in this region in tumour cells turns off MGMT expression (see below). Loss of
MGMT may promote mutagenic conversion in cells by allowing O6-mG lesions to remain
unrepaired, triggering activation of oncogenes or inhibition of tumour suppressor genes. This
may be a good early marker of carcinogenesis in the detection of tumours [61]. Demethylating
agents induce expression of MGMT by reducing promoter methylation [197] and may be of
potential clinical use.
40
Figure 8. MGMT repairs alkylating adducts at the O6 position of guanine in a suicide reaction.
Regarding expression of MGMT, there are two sides of the coin: tumours expressing low
levels of MGMT probably have a worse prognosis, due to an increased number of oncogenic
mutations; on the other hand these tumours may respond better to chemotherapy, although it
is still unclear whether these effects can be solely attributed to loss of MGMT [61].
This study
The purpose of this study was to evaluate the methylation status of the MGMT promoter
region in melanoma tumours and see if there is a correlation to clinical outcome of
chemotherapeutic response. We used methylation specific PCR (MSP) and pyrosequencing to
analyse 64 tumours from 64 patients with cutaneous melanoma, 29 responders and 45 non-
responders. Good concordance was seen between the two methods, in a comparison of
methylation status, with only 4 out of 64 samples showing contrasting results.
In a comparison to clinical response to DTIC/TMZ chemotherapy, tumours were defined as
having a methylated MGMT promoter if they: a. gave a PCR product in MSP analysis,
regardless of methylation level; or b. methylation index (MtI, the average methylation level
over all CpGs in the analysed region) was > 5% in the pyrosequencing analysis. In the MSP
analysis, methylation of the MGMT promoter was observed in 7 out of 19 (37%) responders
compared to 9 out of 45 (20%) non-responders, receiving DTIC/TMZ-based chemotherapy.
When DTIC or TMZ was administered as a single agent, 4 out of 6 (67%) responders
41
displayed tumours with MGMT promoter methylation compared to 6 out of 27 non-responders
(22%). Results were similar in the pyrosequencing analysis, with tumours from 7 out of 19
(37%) responders to DTIC/TMZ-based chemotherapy defined as methylated at the MGMT
promoter compared to 11 out of 45 (24%) non-responders. In a comparison to single agent
therapy tumours from 4 out of 6 (67%) responders displayed methylation of the MGMT
promoter compared to 7 out of 27 (26%) non-responders. Thus, our results show that tumours
from patients responding to DTIC/TMZ-based chemotherapy tend to have MGMT promoter
methylation more often than non-responders. These results are, however, not statistically
significant and the study needs to be expanded to include more patients.
To conclude this study, we have seen that MGMT promoter methylation may provide a
predictive role in the clinical response to DTIC/TMZ-based chemotherapy in patients with
cutaneous melanoma. Thus, analysis of the MGMT promoter methylation status could
potentially be used as a basis for individually tailored chemotherapy.
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43
9. Concluding Remarks
Cancer is an important public health concern in the western world, coming second only to
heart disease in causes of death. In the past few years progress has been made in the early
diagnosis and monitoring of cancer and many patients are successfully treated. However,
most current therapies have a limited efficacy in curing late-stage disease, thus underlining
the importance of finding improved methods of diagnosis, treatment and chemoprevention. It
is well established that cancer is not one disease, but a collective term describing a number of
diseases arising from an imbalance in cell growth and differentiation. Heredity, somatic
mutations and epigenetic events all play a role in the initiation and progression of cancer by
altering the pattern of gene and protein expression. Because of the heterogeneity of the
disease and its origin, an assortment of methods is needed to explain these genetic and
molecular changes.
In this thesis, a number of methods are described for the identification of the molecular
signatures of cancer at different genetic or transcriptional levels. Pyrosequencing has proved
to be a valuable, high throughput tool in the analysis of both point mutations and detection of
DNA methylation. Through this method we have managed to characterize several molecular
features of cutaneous melanoma, including the role of activation of proto-oncogenes and a
possible mechanism of drug resistance by the inactivation of a DNA repair gene. Microarray
technology has given us a method of determining the effects on global gene expression of
prospective anti-tumourigenic drugs. Our results suggest possible pathways of action and
offer a few genes that may be of potential therapeutic interest. Further investigations are
already in the pipeline as a follow up to these studies, to hopefully take us even closer to an
understanding of the underlying mechanisms of cancer and to find more efficient and specific
methods of therapy.
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45
Acknowledgements
Jaha, är du nu jag ska öva inför Oscarsgalan? Jag började här på labbet 2001 (samma år som humana genomet publicerades), så jag har ju varit här ett tag även om själva doktorandtiden varit kort. Det finns många personer som hjälpt mig på vägen och jag vill passa på här att tacka några stycken. Jag vill tacka min handledare Joakim Lundeberg för att du tog emot mig först som examensarbetare och sedan doktorand. Du lyckas alltid hitta något positivt i mina resultat när jag själv tycker att det ser hopplöst ut. Tack också för att du varit så uppmuntrande nu på slutet, jag har behövt det. Mattias Uhlén, en stor inspiration till oss alla. Tack för att du alltid är så glad och positiv och tar dig tid att lära känna oss. Tack alla PIs på DNA corner, ni bidrar alla till den sköna stämning här och jag tycker väldigt mycket om er allihopa. Tack till mina roomies gamla och nya, jag tycker om er allihopa: Cissi, tack för diskussioner om microarray och en massa annat... och för alla konferenser vi varit på tillsammans (särskilt Bergen!). Emma, ett fräscht och mycket välkommet nytillskott. Fortsätt att vara sådär smart o cool så kommer du hur långt som helst. Karin L, tack för en del intressant skvaller och för att du också fryser ibland. Tack också för att du är så himla trevlig. Mats, den sötaste guldmunken på DNA corner. Jag har saknat dina spännande telefonsamtal sen du lämnade oss. Det brukar vara rätt kul på dina fester också! Olle-snut, vårt rum har aldrig riktigt hämtat sig doftmässigt sen du lämnade oss. Tack för att alltid dansar tryckare med mig, I’m never gonna dance again... Ronny, det har varit skönt att ha någon att dela skrivarglädjen med, även om det varit lite kyligt ibland, tack för att du låtit mig höja. Tove, min ”partner in crime”, tack för alla samtal om precis allt möjligt och för att du är världens bästa partybrud och kompis. Vi har inte alltid varit överens om temperaturen, men det var värt alla diskussioner för att få sitta bredvid dig! Puss. Flera på DNA corner: Malin A, tack för att du är en bra kompis och för alla långa samtal om mat och rumpor. Det är härligt att du äntligen fattat var man ska bo nånstans.
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Torun, Emilie, Mikaela och Carro, tack för allt vi gjort tillsammans och för att ni är mina vänner. Erik F, tack för att vi blev vänner när jag var ny och för alla långa samtal och roliga fester, du är saknad. Maria S, du var bästa ex-jobbshandledaren och du inspirerade mig att börja doktorera. Du är också en av de mest sympatiska människorna jag känner.Vi ses på Dello. Anna G, min mentor och partypolare. Tack för alla samtal om vetenskap och annat och för allt roligt vi haft på konferenser och hemma. Du har verkligen varit en inspiration. Tack hela microarraygruppen. Johan L, min teddy-bä. Tack för all hjälp med data analys och för att du haft sån tålamod med mig. Valtteri och Marcus, tack för hjälp med array-stuff och annat skvaller. Nipe, tack för att du alltid är glad och trevlig. Jag känner att jag kan alltid fråga dig om jag behöver hjälp. Kicki, tack för att du alltid tar dig tid att hjälpa mig med pyro trots att du jämt har fullt upp själv. Det är trevligt att ha någon att skvallra med också! Jacob O, tack för intressanta samtal (även vetenskapliga!) och för att jag kan fråga dig om precis vad som helst och du vet svaret. Anna W, tack för alla microarray hybbar du gjort åt mig och för schysst skvaller, du är cool. Annika Å, tack för hjälp med pyro och för alla bra samtal, både roliga och allvarliga, du är också cool. Tack alla samarbetspartners: Suzanne, Shuhua, Johan, Katarina, Marianne och Doris på CCK tack för ett gott samarbete. Det har varit ett nöje att jobba med er. Tack särskilt Suzanne och Shuhua som jobbat jättehårt för att få ihop manuset till avhandlingen. Francesca, tack för att du är så skön och italiensk! Det har varit kul att jobba ihop med dig, särkilt när jag fick vara hos er o köra FACS. Gesan, tack för att du och Francesca har jobbat så hårt för att få ihop manuset till avhandlingen. Tack även Sven och Mauro för ett trevligt samarbete. Uppsalagänget: Figge, Anna, Helena och Sara m.fl. Tack för roliga middagar och hudcancerkonferensen i Sevilla där alla låg vid poolen o solade. Just det, tack även för intressanta vetenskapliga cancermöten.
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Thank you Tiziana, Ewa, Eugenia and everyone else at the CNER in Rome for our collaboration and for welcoming me to the lab last October. Tack alla vänner: KMSSK: Jånni, Lenny och Hanni-Banni, utan er hade jag aldrig uthärdat KC i fyra år. Vi hade fantastiskt kul ihop i Lund och den tiden kommer jag alltid att minnas. Sara & David, tack för alla roliga fester med mycket intriger och för att jag alltid får tea and biscuits hos er. Mattias E, tack för att vi alltid har kul ihop och för att ditt missförstånd blev världens godaste drink! Mattias J, tack för hjälpen med bilden och för sköna efterfesther. Kakan, Lisa, Anna o Holmen, tack för alla telefonjourer på helgmorgnar med summering av förra kvällens händelser... Ni är mina bästisar och jag älskar er. Kakan, min lilla kanariefågel, tack för alla samtal om livet, labbet och killar och för att du alltid vill dricka vin. Jag har ju följt i dina fotspår: doktorera, singelliv (med allt vad det innebär), horan, disputera och snart fyller jag även 30. Tack för du visar mig vägen, jag är nyfiken på vad som kommer härnäst. Lisa (aka Big mouth strikes again), va mycket kul vi haft tillsammans i Rom, på Street mm. Tack för att du alltid ställer upp när jag behöver dig, du är fin. Anna Maria, tack för att du alltid (med ytterst få undantag) vill ta en öl med mig, du har varit min räddning många gånger (inte minst i sommar). Tack för ”brandy”, snurra flaskan och allt annat kul vi hittat på! Holmen, tack för att du inte försvann i bodelningen. Se nu till att inte försvinna i parträsket! Toffe, tack för allt du gjort för mig och stått ut med, du är fantastisk. Mummy and Daddy, thank you for all your support and encouragement through my whole life. You both know that I often need a little extra “shove” to get things done and if you hadn’t been there to help me, I probably wouldn’t be where I am today. Carl, my baby brother, you’re the only one who really understands my silly sense of humour and by the way “look out for mice, in Swedish!” I love you all.
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