Download DNA repair pathways and the effect of radiotherapy in breast cancer

Linköping University Medical Dissertations
No. 1112
DNA repair pathways and the effect of
radiotherapy in breast cancer
Karin Söderlund Leifler
Linköping University
Department of Clinical and Experimental Medicine
Division of Surgery and Clinical Oncology
SE-581 85 Linköping, Sweden
Linköping 2009
c Karin Söderlund Leifler, 2009
ISBN 978-91-7393-668-2
ISSN 0345-0082
Published articles have been reprinted with permission from the
respective copyright holder.
c Spandidos Publications
Paper I c Elsevier
Paper II c Elsevier
Paper III Illustrations made by the author using OmniGraffle, unless otherwise
specified
Cover illustration: ‘Feather & drop´, courtesy of tanakawho
Typeset using LATEX
Printed by LiU-Tryck, Linköping 2009
Till Ola luften i mina lungor
Little by little, one travels far
J.R.R. Tolkien
Abstract
A large proportion of breast cancer patients are treated with radiotherapy.
Ionising radiation induces different DNA damages, of which double-strand
breaks are the most severe. They are mainly repaired by homologous recombination or non-homologous end-joining. Different protein complexes
have central roles in these repair processes. In addition to the ability to
repair DNA damage, cellular radiosensitivity is also affected by mitogenic
signals that stimulate survival and inhibit apoptosis. The phosphatidylinositol 3-kinase (PI3-K)/AKT pathway controls cell proliferation, invasiveness and cell survival. AKT is regulated by upstream growth factor receptors, one of them being HER2 (also called ErbB2). HER2 is overexpressed
in 15-30% of all breast cancers and associated with poor prognosis.
In this thesis, we have studied factors that affect tumour cell resistance
to ionising radiation. In Paper I, the role of HER2/PI3-K/AKT signalling
in radiation resistance was investigated in two breast cancer cell lines. The
results support the hypothesis that the HER2/PI3-K/AKT pathway is involved in resistance to radiation-induced apoptosis in breast cancer cells in
which this signalling pathway is overstimulated.
We also investigated if the protein expression of several DNA repairassociated proteins influence the prognosis and treatment response in early
breast cancer. Moderate/strong expression of the MRE11/RAD50/NBS1
(MRN) complex predicted good response to radiotherapy, whereas patients
with negative/weak MRN had no benefit from radiotherapy as compared
to chemotherapy (Paper II). These results suggest that an intact MRN complex is important for the tumour-eradicating effect of radiotherapy. In Pav
per III, low expression of the BRCA1/BRCA2/RAD51 complex was associated with an aggressive phenotype, an increased risk of local recurrence
and good response to radiotherapy.
In Paper IV, we studied if a single nucleotide polymorphism, RAD51
135G/C, was related to RAD51 protein expression, prognosis and therapy
resistance. We found that genotype was not correlated to neither protein
expression nor prognosis. Patients who were G/G homozygotes had a
significant benefit from radiotherapy. The results also suggested that the
RAD51 135G/C polymorphism predicts the effect of chemotherapy in early
breast cancer.
In conclusion, DNA repair proteins are potential prognostic and predictive markers. The results indicate that proteins in different repair pathways may contribute differently to the effect of radiotherapy. Also, the
HER2/PI3-K/AKT signalling pathway protects cells from radiation-induced
apoptosis. In the future, it might be possible to target some of these proteins with inhibitory drugs to sensitise tumours to radiotherapy.
Contents
Abstract
v
Contents
vii
List of Figures
x
Populärvetenskaplig sammanfattning
1
Abbreviations
3
Original publications
5
Introduction
Importance . . . . . . . . . . . . . . . . . . . . . . . . . .
Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . .
Risk factors . . . . . . . . . . . . . . . . . . . . . . .
Prognosis versus prediction of treatment outcome
Breast cancer tumour biology . . . . . . . . . . . . . . .
Tumour suppressors versus oncogenes . . . . . . .
Tumour heterogeneity . . . . . . . . . . . . . . . .
Breast cancer treatment . . . . . . . . . . . . . . . . . . .
Surgery, chemotherapy and endocrine treatment .
Radiotherapy . . . . . . . . . . . . . . . . . . . . .
Biological effects of ionising radiation . . . . . . . . . .
Radiation-induced DNA damage . . . . . . . . . .
vii
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Cellular response to ionising radiation . . . . . . . . .
Cell cycle arrest . . . . . . . . . . . . . . . . . . .
Cell death . . . . . . . . . . . . . . . . . . . . . .
Repair of radiation-induced damages . . . . . .
Tumour cell resistance to ionising radiation . . . . . .
HER2 and AKT signalling in breast cancer . . . . . . .
The HER2 receptor . . . . . . . . . . . . . . . . .
Role in breast cancer . . . . . . . . . . . . . . . .
The PI3-K/AKT signalling pathway . . . . . . .
Connections between AKT signalling and DNA repair
DNA repair pathways as targets for cancer therapy .
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Aims of the thesis
General aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Specific aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Comments on materials and methods
Cell culture . . . . . . . . . . . . . . .
Patients and tumour material . . . .
Western blot . . . . . . . . . . . . . .
Cell cycle analysis by flow cytometry
Apoptosis detection . . . . . . . . . .
Immunohistochemistry . . . . . . . .
TMA . . . . . . . . . . . . . . .
Antibodies . . . . . . . . . . . .
Fixation and antigen retrieval .
Advantages and disadvantages
PCR-RFLP . . . . . . . . . . . . . . .
Statistical analysis . . . . . . . . . . .
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Results & Discussion
47
HER2/PI3-K/AKT signalling is important for resistance to radiationinduced apoptosis (Paper I) . . . . . . . . . . . . . . . . . . . 47
Cell cycle regulation after γ-radiation . . . . . . . . . . . . . 48
Radiation-induced apoptosis . . . . . . . . . . . . . . . . . . 48
DNA repair-associated proteins in breast cancer (Paper II and III)
50
Reduced or elevated expression of DNA repair proteins in
breast tumours? . . . . . . . . . . . . . . . . . . . . . .
Significance of the subcellular localisation . . . . . . . . . . .
Expression patterns of proteins that form a complex together
Reduced expression of complexes is associated with clinicopathological variables . . . . . . . . . . . . . . . . . .
Relation to recurrence-free survival . . . . . . . . . . . . . . .
Can the expression of DNA repair proteins predict the effect
of radiotherapy? . . . . . . . . . . . . . . . . . . . . .
Does a polymorphism in RAD51 influence protein expression, prognosis and the effect of therapy? (Paper IV) . . . . . . . . . . .
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Conclusions
Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements
63
Bibliography
67
Paper I
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Paper II
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Paper III
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Paper IV
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List of Figures
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Breast cancer incidence in the world . . . . . . . . . . . . . . . .
8
Illustration of the ionisation tracks from low LET radiation . . . 17
Illustration of local multiply damaged site in the DNA . . . . . 18
Activation of ATM and downstream networks by radiation-induced
DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Non-homologous end-joining . . . . . . . . . . . . . . . . . . . . 23
Homologous recombination . . . . . . . . . . . . . . . . . . . . . 25
Role of HER2 and PI3-K/AKT signalling in tumourigenesis . . 29
Connections between AKT signalling and DNA repair . . . . . . 31
Western blot of NBS1 expression . . . . . . . . . . .
Visualisation of apoptotic cells with M30 staining .
Immunohistochemical staining of NBS1 in normal
breast tumour . . . . . . . . . . . . . . . . . . . . . .
Restriction site of the MvaI enzyme . . . . . . . . . .
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and
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44
Populärvetenskaplig
sammanfattning
Bröstcancer är den vanligaste cancerformen som drabbar svenska kvinnor,
med ungefär 7000 nya fall per år. En stor del av patienterna med bröstcancer får strålbehandling som en del av sin behandling. Strålningen dödar
celler genom att orsaka olika sorters skador på DNA, varav dubbelsträngsbrotten är de mest allvarliga och svårast för cellen att reparera.
När en cell utsätts för strålning startar en signalkaskad som kan aktivera DNA-reparation, hämma celldelning och påverka cellens benägenhet att dö. En sådan signalväg är HER2/fosfatidylinositol 3-kinas (PI3K)/AKT, som bland annat reglerar celltillväxt, celldelning och en form av
celldöd som kallas apoptos. HER2 är uttryckt i onormalt höga nivåer i 1530% av alla brösttumörer och är relaterat till sämre prognos.
I den här avhandlingen studeras faktorer som påverkar tumörcellers
känslighet för strålbehandling. PI3-K/AKT-signalering undersöktes i två
olika bröstcancercellinjer, som skiljer sig åt när det gäller HER2-uttryck
(Paper I). När PI3-K hämmades blev celler som hade högt uttryck av HER2
mer känsliga för strålningsinducerad apoptos. Celler som hade normalt uttryck av HER2 blev mer motståndskraftiga mot strålning när PI3-K/AKTsignalvägen stimulerades. Sammantaget stödjer resultaten hypotesen att
denna signalväg bidrar till strålningsresistens i bröstcancerceller med högt
uttryck av HER2.
1
P OPULÄRVETENSKAPLIG SAMMANFATTNING
I andra och tredje arbetet studerades uttrycket av flera proteiner som
är inblandade i reparation av dubbelsträngsbrott på DNA. Syftet var att
undersöka om proteinuttrycket påverkar prognosen och effekten av strålbehandling vid bröstcancer, samt om dessa proteiner har något värde som
prediktiva markörer som kan förutsäga vilka patienter som har bäst nytta
av en viss sorts behandling. Ett av DNA-reparationskomplexen kallas
MRE11/RAD50/NBS1 (MRN). Resultaten visade att högt uttryck av MRN
i brösttumörer predikterade god nytta av strålbehandling, vilket tyder på
att intakt MRN-komplex är viktigt för den tumörcellsdödande effekten av
strålning (Paper II).
I det tredje arbetet undersöktes ett annat proteinkomplex som består
av BRCA1, BRCA2 och RAD51. Lågt uttryck av komplexet var relaterat
till aggressivare tumöregenskaper och ökad risk att få lokalt återfall i eller
nära det drabbade bröstet (Paper III). Samtidigt predikterade lågt uttryck
av dessa proteiner god effekt av strålbehandling jämfört med cytostatika.
Det fjärde arbetet bygger vidare på resultaten från föregående arbete. Vi
undersökte om en variation, en så kallad polymorfi, i genen som kodar för
RAD51-proteinet hade någon inverkan på proteinuttrycksnivån, patienternas prognos eller utfallet av behandling. Resultaten visade att polymorfin
inte kunde kopplas till uttrycket av RAD51-protein. Förekomst av polymorfin var inte heller relaterat till prognos. Däremot tycktes patienter som
inte hade polymorfin svara bra på strålbehandling. Resultaten antydde
också att förekomst av polymorfin predikterade god nytta av en viss sorts
cytostatikabehandling.
Sammanfattningvis pekar resultaten mot att DNA-reparationsproteiner
är potentiella prognostiska och prediktiva markörer. Eftersom högt uttryck av MRN-komplexet, men lågt uttryck av BRCA1/BRCA2/RAD51komplexet, predikterade god nytta av strålbehandling verkar det som att
proteinerna i olika reparationssystem bidrar på olika sätt till effekten av
strålning. Resultaten visar också att HER2/PI3-K/AKT-signalering skyddade bröstcancerceller från strålningsinducerad celldöd. I framtiden kan
det bli möjligt att hämma en eller flera av de studerade proteinerna för att
göra tumörer mer känsliga för strålbehandling.
2
Abbreviations
ATLD
ATM
BARD
BASC
BER
BMI
BRCA1/2
BSA
CI
CMF
CT
DAB
DSB
ECM
EGFR
ER
HER2
HNSCC
HR
HRP
HRT
IGF-I
IHC
ataxia-telangiectasia-like disorder
ataxia-telangiectasia mutated
BRCA1-associated RING domain
BRCA1-associated genome surveillance complex
base excision repair
body mass index
breast cancer (susceptibility genes) 1/2
bovine serum albumin
confidence interval
cyclophosphamide, methotrexate, 5-fluorouracil
chemotherapy
3,3’-diamino benzidine tetrahydrochloride
double-strand break
extracellular matrix
epidermal growth factor receptor
oestrogen receptor
human epidermal growth factor receptor 2
head and neck squamous cell carcinoma
homologous recombination
horseradish peroxidase
hormone-replacement therapy
insulin-like growth factor I
immunohistochemistry
3
A BBREVIATIONS
IR
LET
LOH
MDS
NER
NHEJ
PARP
PCR
PI
PI3-K
PS
PTEN
RFLP
RT
SCID
SSA
TGF-β
TMA
TNM
UTR
ionising radiation
linear energy transfer
loss of heterozygosity
multiply damaged sites
nucleotide excision repair
non-homologous end-joining
poly(ADP)-ribose polymerase
polymerase chain reaction
propidium iodide
phosphatidyl inositol 3-kinase
phosphatidylserine
phosphatase and tensin homologue
restriction fragment length polymorphism
radiotherapy
severe combined immunodeficiency
single-strand annealing
transforming growth factor-β
tissue microarray
tumour (size), node (nodal involvement), metastasis
untranslated region
4
Original publications
This thesis is based on the following original papers, which will be referred
to in the text by their Roman numerals (I-IV).
I
Karin Söderlund, Gizeh Pérez-Tenorio and Olle Stål.
Activation of the phosphatidylinositol 3-kinase/Akt pathway prevents radiation-induced apoptosis in breast cancer
cells. International Journal of Oncology 2005; 26:25-32
II
Karin Söderlund, Olle Stål, Lambert Skoog, Lars Erik
Rutqvist, Bo Nordenskjöld and Marie Stenmark Askmalm.
Intact Mre11/Rad50/Nbs1 complex predicts good response to radiotherapy in early breast cancer. International Journal of Radiation Oncology Biology & Physics 2007;
68(1):50-58
III
Karin Söderlund, Lambert Skoog, Tommy Fornander and
Marie Stenmark Askmalm.
The BRCA1/BRCA2/Rad51 complex is a prognostic and
predictive factor in early breast cancer. Radiotherapy and Oncology 2007; 84:242-251
IV
Karin Söderlund Leifler, Anna Asklid, Tommy Fornander
and Marie Askmalm Stenmark.
The RAD51 135G/C polymorphism is related to the effect
of adjuvant therapy in early breast cancer. Manuscript
5
Introduction
Importance
Over recent decades, the number of available therapeutic options for the
treatment of breast cancer has grown. As a result, it has become increasingly important to know whether or not a patient will respond to a specific treatment, so that the optimal therapy combination can be chosen for
each individual patient. One important treatment modality is radiotherapy,
which is given to a majority of patients with breast cancer.
The aim of the research presented in this thesis has been to increase
our understanding of the complex mechanisms that govern the cellular
response to treatment-induced damages. Cellular responses to radiation
have been studied extensively in vitro, but relatively little is known about
how these mechanisms impact the outcome of cancer treatment. Therefore,
it would be valuable to see if knowledge acquired from experimental studies is applicable on patient material.
A second goal of these studies has been to identify markers that can
predict the clinical response of breast tumours to radiotherapy. In the extension, knowledge of predictive markers could be of help in the search for
suitable targets of treatments that aim to increase the sensitivity of tumour
cells to radiation. A more immediate use of these markers is to use them as
a guide in the choice of treatment, in order to avoid overtreating patients
that have a good prognosis and, conversely, to prevent the recurrence of
more aggressive tumours. Validated markers can indicate which patients
7
I NTRODUCTION
Figure 1: The age-standardised incidence rate of breast cancer is typically high in
the Western world and lower in Asia and Africa.
GLOBOCAN 2002, IARC.
are predicted to benefit from radiotherapy, and which patients will probably need an additional, or different, therapy regimen.
Breast cancer
It is a well known fact that breast cancer is the most common malignancy
among Swedish women today. In 2007, the incidence of breast cancer in
Sweden (women only) was 7049 cases [1]. Although generally thought of
as a disease of the female breast, approximately 40 Swedish men are diagnosed with breast cancer each year. Sweden belongs to a group of countries
that have the highest breast cancer risk in the world (Fig. 1) [2]. More than
82,000 people live with the disease, and approximately 1,500 die of breast
cancer each year in Sweden [3]. For a Swedish woman the risk of developing breast cancer before the age of 75 is 9.7% [1].
Despite the high incidence, breast cancer mortality has decreased in
Western countries due to mammography mass screenings that find the tumours at an early stage and improvements in adjuvant therapy as a complement to surgery.
8
Breast cancer
Risk factors
The etiology of breast cancer is surrounded by uncertainty. Still, some risk
factors have been identified. A major risk factor is the cumulative exposure of breast tissue to ovarian hormones such as oestrogens. Early age
at menarche, high age at menopause, and hormone-replacement therapy
(HRT) in postmenopausal women, all increase the risk of breast cancer by
prolonging the duration of exposure to female sex hormones [4, 5].
Risk is also associated with reproductive patterns, since nulliparity (having no children), as well as a high age at first birth, is related to a higher risk
[4]. The influence of pregnancy on breast cancer risk is complex. The high
levels of oestrogens early during a pregnancy might stimulate latent cancerous cells in the breast, leading to a transient increase in risk for about
a decade after the pregnancy [5]. On the other hand, sex hormones also
induce differentiation of breast epithelial cells which make them less susceptible to mutations over the long term.
It has also been suggested that the risk of breast cancer could be affected
by lifestyle factors. Studies on migrants who moved from countries with
low breast cancer incidence (e.g. Japan) to countries with a high incidence
(e.g. the United States) have shown that the immigrants adopt the higher
risk of their new country, suggesting that environmental and lifestyle factors play a significant role in breast cancer development.
There are many studies on the possible relation between what people eat and their risk of developing cancer. A comprehensive review of
prospective studies conducted on diet and breast cancer incidence concluded that there is no consistent association, with the exception of alcohol
intake, high weight and weight gain [6]. Epidemiological studies consistently demonstrate that a high body mass index (BMI) increases the incidence of breast cancer in postmenopausal women [7]. A combined analysis of six prospective studies from North America and Europe shows that
alcohol consumption is associated with a linear increase in breast cancer
incidence in women [8].
Other factors that have been studied are fat intake, fruit and vegetable
intake, dietary intake and serum levels of antioxidants, carbohydrate intake, consumption of dairy products (which increases endogenous insulinlike growth factor I (IGF-I) levels), soy products and isoflavones (with a
9
I NTRODUCTION
chemical structure that resembles oestrogen) and green tea, but the results
are inconclusive [6].
In addition to environmental factors, the genetic setup also plays a role
in breast cancer etiology. It is well known that having a family history of
breast cancer increases a woman´s own risk. Approximately 5-10% of all
cases are hereditary [9]. Mutations in the breast cancer susceptibility genes
BRCA1 and BRCA2 account for 20-40% of the familial cases [10, 11].
Mutations in P53, CHEK2 and PTEN are also found in a small portion
of patients with hereditary breast cancer, but not all cases that seem to have
a familial component can be explained by mutations in these genes. Thus,
it is possible that other genes associated with a more modest influence on
breast cancer risk are also involved, perhaps by modifying the effects of
other risk factors. It might be that mutations or polymorphisms in several
low risk genes act together to increase the risk of breast cancer [12].
Prognosis versus prediction of treatment outcome
When talking about survival rates, it is most common to use 5-year survival
rates. The use of 1-year survival gives a very short term estimation of the
prognosis, while 10-year survival rates introduce possible sources of errors
since they refer to patients that were diagnosed long ago and treatments
may have improved since then.
One common misconception is that survival rate is the same as cure
rate. Unfortunately, the survival rates for breast cancer patients continue to
fall beyond the first five years after diagnosis. It is thus valuable to have a
longer follow up period in studies on patient material.
At present, the most important prognostic indicator is the TNM system,
that classifies tumours into five different stages (0, I, II, III and IV) according to their size, spread to the nearest lymph nodes (nodal involvement)
and spread to other organs (metastases). The higher stages have poorer
prognosis (Table 2) [13].
The prognosis is also affected by the molecular profile of the tumour.
Some biomarkers, such as HER2 overexpression and oestrogen receptor
(ER) expression, are associated with clinical outcome. In the treatment situation, it is therefore important to divide patients into different risk groups
to avoid recurrences among patients with high risk by combining several
10
Breast cancer
STAGE
I
IIA
IIB
III
IV
5-YEAR SURVIVAL
96%
85%
78%
55%
16%
Table 2: The approximate 5-year breast cancer-specific survival rate of Swedish
breast cancer patients by combined TNM stages [14].
treatment modalities. A second benefit is avoidance of overtreatment of
patients that have a good prognosis with low risk of recurrence of the disease.
When evaluating biomarkers it is important to distinguish between the
biological aggressiveness of a tumour versus response to therapy. Both
affect the clinical outcome, but in different ways. A prognostic factor indicates the inherent aggressiveness of a tumour. Nodal status and histologic
grade are examples of prognostic factors. Preferably, a prognostic factor
is assessed in patients that are treated by surgery only, without systemic
treatment, since it should reflect the natural history of the disease. However, nowadays it is difficult to establish new prognostic markers. Due
to the aggressive nature of breast cancer, and considering the abundance
of treatment options available today, withholding adjuvant treatment from
patients would be unreasonable for ethical reasons.
A predictive factor, on the other hand, is indicative of how well the
tumour will respond to a certain treatment. Oestrogen receptor status is an
example of a predictive factor, since it indicates the likelihood of tumour
response to endocrine treatment with anti-oestrogens. These prognostic
and predictive properties of biomarkers are not mutually exclusive, and a
factor can have both prognostic and predictive value (e.g. the oestrogen
receptor).
11
I NTRODUCTION
Breast cancer tumour biology
The human body can be thought of as a multicellular organism, consisting
of more than 1014 cells. Unlike unicellular organisms, e.g. bacteria, the cells
in a body collaborate for the good of the organism. In cancer, however,
a clone of mutant cells gains properties that allow them to prosper at the
expense of neighbouring cells. Cancer cells have several characteristics in
common; they divide uncontrollably, are often relatively resistant to apoptosis, are genetically unstable (with a high mutation rate), and cells in an
advanced tumour have the ability to invade other tissues and proliferate
in foreign sites. It is believed that the genesis of a cancer is a multi-step
process, that requires the accumulation of several independent mutations
in cancer critical genes.
Cancer cells circumvent many of the constraints that normal cells adhere to, but they are not independent of the surrounding tissue. During tumour formation the tissue microenvironment changes, resulting in
increased number of fibroblasts, formation of new blood vessels and remodelling of the extracellular matrix (ECM) [15]. A gene expression study
on the different cell types in breast tissue revealed that extensive gene expression changes occur in all cell types during cancer progression and that
many of the altered genes encode secreted proteins and receptors [16]. The
interactions and paracrine signalling between the microenvironment and
tumour epithelial cells are important in the regulation of the proliferative,
angiogenic, invasive and metastatic behaviour of cancer cells [15, 17].
Tumour suppressors versus oncogenes
Genes that are critical for the initiation or development of cancer can be divided into two broad groups, depending on whether the cancer risk arises
from too much activity of the gene product, or too little. Genes that are
associated with cancer due to gain-of-function mutations are called protooncogenes, and their mutant, over active forms are labelled oncogenes.
The other type of cancer critical genes are the tumour suppressors. Loss
of function of tumour suppressor genes, due to point mutations, chromosomal aberrations or epigenetic events, abolish part of the control systems
that regulate cellular behaviour. Many of the genes that are important in
tumour biology are involved in the regulation of cell division (e.g. cell cy12
Breast cancer tumour biology
cle checkpoints), cell death (e.g. apoptosis), transduction of extracellular
survival signals, DNA damage detection, repair and telomere stability.
Of the proteins studied in this thesis, HER2 and AKT are well established as having oncogenic properties. BRCA1 and BRCA2, on the other
hand, are well-known tumour suppressors, whereas there is some controversy surrounding the roles of RAD51 and NBS1 in tumour development.
Tumour heterogeneity
An important concept in cancer biology is the understanding of tumour
heterogeneity. A single tumour contains several subpopulations of tumour
cells, with different genetic and phenotypic characteristics. Two different
models to explain the evolution of heterogeneity within a tumour have
been proposed. The clonal evolution model states that tumourigenesis begins in a normal, differentiated cell due to genetic or epigenetic changes.
Clonal expansion of this abnormal cell leads to a population of identical tumour cells. Some of these cells acquire additional mutations in a multi-step
fashion, independently of each other, and this would explain the heterogeneity seen in clinically detectable tumours.
The other model, the cancer stem cell theory, hypotheses that the
changes that initiate tumour formation occur in a stem cell. The resulting
tumour initiating cell has high self renewal capacity, but it can also mature
or differentiate into breast cancer cells that comprise the bulk of the tumour
[18]. Such tumour initiating cells have been identified in breast cancer [19].
It has been proposed that the tumour initiating cells constitute only a small
subpopulation of cells in a tumour, but that they nevertheless drive breast
cancer initiation, progression and recurrence, and that they are more resistant to cancer therapy than the bulk of the tumour [20].
Since subpopulations in a tumour can differ in their expression of genes
and proteins, they can have markedly different phenotypes. These differences could influence both the aggressiveness of the tumour cells and their
sensitivity to anti-cancer agents, thereby affecting both prognosis and treatment outcome. Tumour heterogeneity adds a layer of complexity to studies of prognostic and predictive markers in clinical material, since it is not
known which subpopulations in a heterogeneous tumour that will have the
largest impact on the outcome.
13
I NTRODUCTION
Breast cancer treatment
Surgery, chemotherapy and endocrine treatment
The primary treatment of breast cancer is surgical removal of the tumour.
Breast conserving surgery in combination with post-operative radiotherapy is comparable to removal of the whole breast (mastectomy) when it
comes to prevent local recurrence and improve breast cancer specific patient survival, and it is therefore preferred over mastectomy for smaller
tumours [21].
The basis of all cancer therapies is that they kill tumour cells more
efficiently than normal cells. Chemotherapeutic drugs (also called cytostatic or cytotoxic drugs, depending on their effect on cells) are divided
into different groups depending on the mechanism by which they kill cells.
Chemotherapies commonly used to treat breast cancer include alkylating
agents, antimetabolites, topoisomerase inhibitors, plant alkaloids and terpenoids (e.g. taxanes and vinca alkaloids). Other anti-tumour agents that
are widely used include monoclonal antibodies (e.g. trastuzumab) and hormonal/endocrine therapies (e.g. tamoxifen and aromatase inhibitors).
Without going into detail on all therapies mentioned above, the drugs
that have been used in the studies in this thesis (Paper II, III and IV) will
be described briefly. Cyclophosphamide is an alkylating agent that binds
covalently to the DNA, thereby crosslinking the DNA strands. These base
modifications interfere with DNA synthesis, and cyclophosphamide has
also been shown to induce strand breaks [22].
Methotrexate and 5-fluorouracil (5-FU) both belong to the antimetabolite group of chemotherapeutics, and they both target the synthesis of thymidine, one of the bases in the DNA. Methotrexate is a folic acid analogue
that inhibits dihydrofolate reductase, which leads to depletion of reduced
folates that are needed for the production of thymidylate [23]. Similarly,
5-FU is a pyrimidine analogue of deoxyuridine monophosphate (dUMP),
another substrate needed for the generation of thymidylate [24]. Depletion
of thymidylate results in inhibition of RNA and DNA synthesis, and slows
the growth of rapidly dividing cells. Methotrexate enhances the cytotoxic
effects of 5-FU [24].
14
Breast cancer treatment
Radiotherapy
A brief history of radiotherapy
X-rays were discovered in 1895 by the German physicist Wilhelm Conrad
Röntgen [25]. He called them x-rays, using the mathematical term commonly used to denominate something unknown. By the turn of the century, x-rays had been tested in basic therapeutic applications in Europe
and America. Notably, the first reports on x-ray treatment of cancer patients came as early as 1896, only seven months after Röntgen´s discovery
[26]. Röntgen also demonstrated how the rays could be employed to make
a radiograph of the bones of the hand on photographic film, thereby laying
the ground to diagnostic radiology. He was awarded the very first Nobel
Prize in Physics in 1901.
Parallel to Röntgen´s breakthrough, Becquerel discovered natural radioactivity in 1898 [27]. He also inadvertently found out about the danger
of radiation, after having left a container with 200 mg of radium in his vest
pocket [28]. Within two weeks, a rash became evident on the skin of the
chest, and an ulceration developed that required several weeks to heal. In
1903, he shared the Nobel Prize in Physics with Pierre and Marie Curie.
Radiotherapy was developed in the early decades of the 20th century.
During this time, the relation between radiosensitivity and mitotic activity of the tissue was investigated. Also, the advantage of fractionation was
discovered. Experiments were performed in which researchers tried to sterilise rams by irradiating their testes with x-rays [28]. If the radiation was
delivered as a single dose, the skin of the scrotum was severely damaged.
However, if the same dose was divided and fractionated over a period of
time, it was possible to sterilise the animals and at the same time spare the
skin from damage. This formed the basis of fractionation in clinical radiotherapy.
Clinical radiotherapy
Today, radiation therapy is one of the most important treatments of cancer.
All patients who have undergone breast conserving surgery, and a majority
of the breast cancer patients who have been mastectomised and are lymph
node positive, are treated with radiotherapy. Even though breast conserving surgery, or the more extensive mastectomy, can remove all disease that
15
I NTRODUCTION
has been detected in the breast or regional lymph nodes, there is a risk
that undetected deposits of tumour cells remain. These tumour cells can, if
left untreated, develop into a local recurrence or set distant metastases and
pose a threat to the life of the patient. Radiation therapy is a local treatment, as opposed to chemotherapy and endocrine therapies, and it reduces
the risk of locoregional recurrence by killing remaining tumour cells and
micrometastases in the irradiated area [29].
About three-quarters of the risk of local recurrence occurs in the first
five years after surgery [29]. Distant recurrence and breast cancer mortality
generally occur later. The risk of local recurrence is higher if the tumour
had spread to the axillary lymph nodes at the time of surgery (as compared
to lymph node-negative disease). The risk is also affected by age, being approximately twice as high in younger as in older women after breast conserving surgery. A meta-analysis of the effect of postoperative radiotherapy showed that the absolute risk reduction was 4% if node-negative breast
cancer patients were treated with radiotherapy after mastectomy (decreasing from a 5-year local recurrence risk of 6% with only surgery, to 2% when
surgery and radiotherapy was combined) [29]. By contrast, the risk of local
recurrence for patients with node-positive disease was 23%, and radiotherapy reduced it to 6%, resulting in an absolute risk reduction of 17%.
Well known side effects of breast cancer radiotherapy are damages to
the skin, radiation fibrosis and changes in the size of the treated breast. The
more serious side effects include damage to the heart and lung tissue, but
the risk of this is reduced as today, radiotherapy dosage is very carefully
planned.
Biological effects of ionising radiation
In the clinic, linear accelerators are used for the treatment of patients. They
produce high energy x-rays at energies of 4-25 MV. Ionising radiation (IR)
can also arise through the natural breakdown of radioactive elements, e.g.
60 Co, that emit γ-rays.
X-rays and γ-rays are uncharged, electromagnetic photon radiations of
very high energy. When they penetrate tissues in the body, they ionise
molecules in their track. It is this ionisation that results in the biological
effects seen after exposure to ionising radiation. During the fraction of a
16
Biological effects of ionising radiation
A dose of 1 Gy causes appr.
1000 base damages
γ-rays
or x-rays
1000 single-strand breaks
40 double-strand breaks
Figure 2: Illustration of the ionisation tracks from radiation with low linear energy transfer (LET). One Gy of absorbed radiation dose produces approximately
1000 tracks. Ionisation tracks caused by high-LET radiation, from e.g. α-particles,
are much more dense.
second that it takes for a photon to pass through a cell, it interacts with electrons, ejecting some of them from atoms (ionisation) and elevating others to
higher energy levels within the atom (excitation) (Fig. 2). These secondary
ejected electrons may excite or ionise other atoms in their vicinity, causing
a chain reaction of ionisations. A dose of 1 Gy produces approximately 105
ionisations/cell [30].
Radiation-induced DNA damage
The most important target of ionising radiation in the cell is the DNA. The
DNA molecule can be damaged by ionising radiation through indirect or
direct ionisation. Indirect ionisation involves the ionisation of some other
molecule in the cell, most often a water molecule. Ionisation of water results in free hydroxyl radicals (OH·), which can then interact with the DNA.
The photon itself can also hit the DNA, causing a cascade of ionisations in
the atoms that build up the DNA molecule.
Ionising radiation induces base damages, single-strand breaks and
double-strand breaks. Similar damages are also produced endogenously
17
I NTRODUCTION
Figure 3: Illustration of a local multiply damaged site in the DNA, created by a
cluster of ionisations. The lesions can be a combination of several different types
of damages.
by reactive oxygen species that are by-products of cellular metabolism, and
double-strand breaks are created under controlled forms during B- and T
cell receptor recombination. However, due to the localised energy deposition, the damage produced by ionising radiation typically results in clustered lesions or multiply damaged sites (MDS) that are different from damages produced by endogenous activities (Fig. 3) [31]. These clustered lesions can consist of a strand break in combination with a base damage, or
several strand breaks in close vicinity to each other so that the base-pairing
bonds in the area between the breaks are insufficient to hold the DNA parts
together.
The termini of DNA ends at a break caused by ionising radiation are
frequently altered as well, as compared to DNA ends generated by endogenous nucleases or bacterial restriction enzymes (often used to induce strand
breaks in experiments designed to study the repair of strand breaks) [31].
These altered DNA ends have to be processed before the break can be repaired. Several enzymes involved in the repair pathways have been found
to possess the ability to process DNA ends; however the excision of damaged bases will frequently result in the loss of bases at the break and the
potential introduction of mutations.
Cellular response to ionising radiation
When a cell is faced with DNA damage, it can attempt to repair the damage,
delay cell division or die (or a combination of these alternatives). The cellular response to radiation-induced damage is influenced by the cell type,
18
Cellular response to ionising radiation
timing (i.e. cell cycle phase in which the damage occurs), the extent of
damage and the integrity of the pathways that govern these responses. As
mentioned before, tumour cells often have deficient apoptotic, cell cycle
regulatory and DNA repair responses due to mutations and other aberrations in these pathways. It has been proposed that the DNA damage response machinery is activated early in tumourigenesis (e.g. by oncogenes),
acting as a barrier to tumour development, and that this checkpoint has to
be inactivated by mutations in critical genes before cancer can arise [32].
Cell cycle arrest
A common cellular response to DNA-damaging agents is cell cycle arrest
due to activation of cell cycle checkpoints. These checkpoints act as barriers
to prevent propagation of cells with a damaged genome. Especially the
G2/M checkpoint is important in the response to ionising radiation, since
cell cycle arrest in the G2 phase allows time for repair of DNA damages in
the replicated genome before cell division.
The ataxia-telangiectasia mutated (ATM) kinase is the most proximal
initiator of signal transduction to the cell cycle machinery after IR-induced
DNA damage. When a cell is exposed to IR, ATM is rapidly activated
and phosphorylates numerous downstream targets, including p53, MDM2,
CHK2, NBS1 and BRCA1 [33, 34, 35]. Several of these targets function to
block progression through the cell cycle (Fig. 4).
A transient cell cycle arrest allows time for the cell to try to repair DNA
damage. Cell cycle arrest can also be prolonged and lead to senescence,
a state in which the cell ceases to divide. Prolonged cell cycle arrest can
ultimately result in cell death, but even though the senescent cell does not
replicate it is still metabolically active and produce secreted proteins that
can have stimulating effects on neighbouring cells [36].
The tumour suppressor p53 has a central role in the cellular response
to DNA-damaging agents, since it coordinates DNA repair with cell cycle
progression and apoptosis [37]. Damage recognition and checkpoint activation leads to p53 phosphorylation, which alters its conformation and
greatly increases its stability. This is a rapid response and p53 levels increase within minutes after DNA damage. p53 has the ability to bind to
DNA and functions as a transcription factor. It induces gene transcription
19
I NTRODUCTION
Cell
cycle
phase
G1
S
G2
M
intra S block
Cyclin B1
Cyclin E
NBS1
Abortive
mitosis
CDK1
GADD45
CDK2
CDK2
T
14-3-3
P
T
p21
Aneuploid
checkpoint
T
CDC25A/C
BRCA1
P
CHK
1/2
p73
P
P
p21
P
c-Abl
T
P
ATM
p53
P
T
P
P
DNA-PK
inhibition
DNA damage
Mdm2
P
stimulation
phosphorylation
T
Mdm2
transactivation
Figure 4: A simplified illustration of the complex networks that are activated
in response to radiation-induced DNA damage. Activation of ATM leads to signalling through several pathways that result in cell cycle arrest. Note that several
proteins that are important for DNA repair also have functions in cell cycle regulation (e.g. BRCA1, DNA-PK and NBS1) [40].
of several cell cycle regulators, including p21, GADD45 and members of
the 14-3-3 family [38]. p53 also induces BAX and other proteins involved in
apoptosis, but suppresses DNA repair through down regulation of RAD51
[38, 39].
Cell death
Ionising radiation induces two different modes of cell death: apoptosis and
mitotic cell death (also called clonogenic cell death or mitotic catastrophe).
Mitotic cell death is thought to be the major mechanism by which solid
tumours are killed by radiotherapy [41]. It occurs during mitosis, or after
20
Cellular response to ionising radiation
several rounds of cell divisions [42]. This mode of cell death is probably
coupled to failure to completely or accurately repair DNA damage. Also,
attempts to enter mitosis before the completion of DNA replication in S
phase can result in mitotic catastrophe [42]. In addition, deficient cell cycle
checkpoints increase the probability of mitotic cell death. Cells that are
unable to halt in G2/M after DNA damage might enter repeated rounds
of failed cell division that result in aneuploid cells and eventually mitotic
catastrophe. It seems that the apoptotic machinery is activated to execute
the mitotic cell death, and that it involves activation of caspase-2, -3 and -9
[42].
Radiation also induces apoptosis, a form of strictly controlled cell death
which is characterised by nuclear condensation, fragmentation of DNA, the
formation of small so called ‘apoptotic bodies´ and subsequent phagocytosis by surrounding cells.
The relevance of apoptosis in the response to clinical radiotherapy has
been widely debated. Radiation readily induces apoptosis in tumours of
hematopoietic and lymphatic origin, whereas it has been stated that DNA
damage-induced apoptosis seems to be the exception rather than the rule
in solid tumours such as breast cancer [43, 44, 45]. On the other hand,
low expression of BCL-2, an anti-apoptotic oncogene that is often overexpressed in breast tumours, was an independent prognostic factor of complete response to preoperative chemotherapy [46]. High levels of apoptosis
in breast tumours treated with preoperative chemotherapy have also been
shown to correlate with clinical response and survival [47]. The relevance
of radiation-induced apoptosis to clinical effect of radiotherapy remains to
be determined.
Repair of radiation-induced damages
Of the different damages induced by ionising radiation, the double-strand
breaks (DSB) are considered to be the most dangerous. Unrepaired DSBs
can lead to loss of chromosomal material, or cell death. If they are repaired
incorrectly, DSBs can lead to mutations and chromosomal rearrangements,
and thereby contribute to tumourigenesis.
Mammalian cells have evolved several different repair mechanisms,
that are specialised in handling different kinds of damages. Base dam-
21
I NTRODUCTION
ages are repaired by base excision repair (BER) or nucleotide excision repair (NER), depending on the number of bases involved. There are several
repair mechanisms that take care of double-strand breaks, of which homologous recombination (HR) and non-homologous end-joining (NHEJ) are
the best characterised. There is also single-strand annealing (SSA), which
is a form of recombination repair.
Non-homologous end-joining
The repair pathways mentioned above differ in their requirement for a
homologous template, and consequently in the fidelity of the repair. HR
uses an intact, homologous DNA sequence as a template, most commonly
the sister chromatide, thereby ensuring accurate restoration of the original
sequence. By contrast, NHEJ does not use a template, and involves the simple re-ligation of the broken DNA ends. NHEJ is mostly precise for simple
breaks with blunt ends, but results in loss of nucleotides when the ends
are not compatible. Before ligation, the ends are often trimmed by nucleases, and any DNA sequence that is lost at the break site will be fixed as a
permanent mutation or deletion after the repair. Although this pathway is
described as ‘non-homologous´, a tiny sequence homology of 1-6 bp (a microhomology) near the DNA break facilitates end-joining [48]. HR requires
longer sequences of homology, generally encompassing 100 bp or more.
Repair of double-strand breaks by NHEJ involves detection of the
strand break by the end-binding heterodimer of the Ku70 and Ku80 proteins (Fig. 5). The Ku heterodimer forms a ring that encircles two turns of
duplex DNA, thereby providing structural support to broken DNA without
obscuring the ends [49]. Ku then recruits DNA-PK catalytic subunit (DNAPKcs) and together they form the DNA-PK complex that holds the broken
ends precisely together to allow ligases to fill in the gap [50, 51]. Upon
binding to Ku70/80, DNA-PKcs is activated by autophosphorylation and
in turn phosphorylates ligase IV that is responsible for the rejoining step.
Damaged ends, or ends with overhang, may have to be processed prior to
ligation. Several nucleases have been implicated for this step, including the
MRE11/RAD50/NBS1 complex, WRN (deficient in the human premature
ageing Werner syndrome) and Artemis (deficient in human severe combined immunodeficiency) [48].
22
Cellular response to ionising radiation
5´
Ku70/80
DNA-PKcs
DNA-PK
Ligase IV/
XRCC4/
XLF
Figure 5: Model of non-homologous end-joining of a double-strand break. First,
DNA ends are bound by the Ku70/80 heterodimer, which then attracts DNA-PKcs
to form the DNA-PK complex. The ends are ligated by the ligase IV complex,
consisting of ligase IV, XRCC4 and XLF.
Interestingly, the Ku heterodimer is also found at the telomeres at the
extreme end of the chromosomes, where it probably functions to prevent
chromosome fusing [52].
Homologous Recombination
There are several subpathways within homologous recombination repair.
This rather complex process is illustrated in (Fig. 6). All homology directed
repair pathways are initiated by resection at the double-strand break to cre23
I NTRODUCTION
ate a 3’-overhang, a process which is facilitated by the MRE11/RAD50/NBS1
complex [53].
One of the most important players in HR is the DNA recombinase
RAD51. The single-stranded 3’ end at the break is coated by several RAD51
monomers to form a nucleoprotein filament. This oligomerisation process is regulated by BRCA2 [54]. RAD51 is part of a multiprotein complex termed BRCC, which contains seven polypeptides including BRCA1,
BRCA2, RAD51 and BRCA1-associated RING domain (BARD1), that possesses ubiquitin E3 ligase activity and enhances cellular survival following
DNA damage [55].
As mentioned previously, this repair mechanism is dependent on sequence homology between the damaged DNA and the template. RAD51
facilitates the search for homology. The next step is strand invasion, in
which the 3´end invades the homologous sister chromatide and base pairs
with the template sequence to form heteroduplex DNA. Strand invasion is
followed by DNA synthesis that copies the template to restore the missing
sequence that was lost at the break point. After synthesis, the invading
strand is released and anneals to the other side of the break. The repair is
completed by ligation of the gaps by a DNA ligase.
It is not clear exactly how the DNA repair pathway choice is made. The
importance of each pathway largely depends on the phase of the cell cycle.
Due to the need of a homologous template for HR, this type of repair is
confided to late S and G2 phases of the cell cycle when a sister chromatide
is available. NHEJ is dominating in the G1 phase. It has been proposed
that the choice depends on whether it is Ku70/80 or RAD52 that first binds
to the DNA ends [56]. In addition, the involvement of each pathway may
be influenced by the complexity of the break, since complex breaks take a
longer time to repair and probably require homologous recombination.
The importance of functional DNA repair is highlighted by the consequences of deficiencies of key proteins. Several rare syndromes have been
described that are caused by mutations in proteins that are critical in the
repair pathways. Some examples are ataxia-telangiectasia (mutated ATM)
[57, 58], ataxia-telangiectasia-like disorder (ATLD, mutated MRE11) [59],
Nijmegen breakage syndrome (mutated NBS1) [60, 61], severe combined
immunodeficiency in mice (SCID, mutated DNA-PKcs) [62] and hereditary
breast cancer (mutations in BRCA1 and BRCA2) [63, 64]. Several of these
24
Cellular response to ionising radiation
1
5
DSB
M
2
R
sliding of
Holliday
junctions
homologous
template
N
5´
6
3´
resection
of ends
strand
release
RAD51
3
7
Holliday
junction
strand
invasion
ligation
4
DNA
synthesis
M
R
N
MRE11/RAD50/NBS1
complex
Figure 6: Homologous recombination is initiated by the creation of singlestranded 3´ends. RAD51 binds to the free end, which then invades the template
strands. New DNA is synthesised. The Holliday junction slides towards the 3´end
until the end is released and can anneal to the complementary strand. Repair is
completed by ligation of the gaps. DSB = double-strand break
syndromes are characterised by hypersensitivity to radiation, chromosomal instability and predisposition to cancer.
DNA repair-associated proteins investigated in this thesis
Cellular responses to DSBs are initiated by recognition of the damage by
sensor proteins. ATM and NBS1 seem to cooperate as primary DSB sensor
proteins [65]. DNA damage recognition by these proteins results in posttranslational modification of downstream mediators, e.g. BRCA1. The mediators then amplify the signal, resulting in a signalling cascade that activates effector proteins such as RAD51, p53 and checkpoint proteins which
execute DNA repair, cell cycle regulation and apoptosis.
25
I NTRODUCTION
Many of these proteins work together in larger protein complexes. NBS1
forms a complex together with the exonuclease MRE11 and RAD50. The
MRE11/RAD50/NBS1 complex has been implicated in both HR and NHEJ
repair of DSBs. BRCA1, BRCA2 and RAD51 function in HR. It has been
suggested that BRCA1 functions as a coordinator of DNA damage responses,
since it is also found in a multiprotein complex called BRCA1-associated
genome surveillance complex (BASC). The BASC contains at least 15 subunits, including ATM, MRE11, RAD50, NBS1, mismatch repair proteins
and BRCA1 itself [66].
Tumour cell resistance to ionising radiation
IR is an extremely efficient DNA-damaging agent with high spatial specificity. Radiotherapy can be used to treat virtually all types of solid tumours,
but there is a relationship between radiocurability and the histological type
of the tumour [30].
Most epithelial tumours, including breast cancer, are more resistant to
ionising radiation as compared to tumours arising from lymphoid cells or
germ cells. Some tumours, e.g. lymphomas and seminomas, are sensitive to radiation and can be cured by low doses, whereas others such as
melanoma and glioblastoma (a brain tumour) are typically radioresistant
and continue to grow even after high doses of radiation [34]. As mentioned before, part of this difference is probably attributable to the tendency of lymphomas to undergo apoptosis in response to genotoxic treatment, whereas solid tumours seem to be relatively refractory to radiationinduced apoptosis [44]. It is however important to remember that tumours
from different patients with the same histological diagnosis can vary in
their responsiveness to radiation.
Other factors, beside tumour origin, that affect radioresistance are tumour size, vascular supply (hypoxia) and cell cycle phase (reviewed in
[34]). Cells are most sensitive to radiation in the G2/M phase, less sensitive
in the G1 phase and the least sensitive during the latter part of S phase. It
has been suggested, however, that the most important element that determines radioresponsiveness is cellular and genetic factors, and that differential expression of critical genes may result in radiation-resistant phenotypes
[34].
26
HER2 and AKT signalling in breast cancer
HER2 and AKT signalling in breast cancer
The HER2 receptor
The human epidermal growth factor receptor-2 (HER2, also called erbB2
since it is homologous to the avian erythroblastosis virus v-erbB) belongs to
a family of transmembrane growth factor receptors. The other family members are epidermal growth factor receptor (EGFR or HER1), HER3 (erbB3)
and HER4 (erbB4). The receptors in the HER family consists of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain, and they function by activating intracellular
signalling pathways in response to extracellular signals.
The extracellular domain of HER receptors can adopt two different conformations: a closed inhibited conformation and an open active one [67].
Ligand binding induces a conformational change to the active state, and
this promotes receptor dimerisation and subsequent transphosphorylation
of the intracellular kinase domains. These phosphorylated tyrosine residues
dock several intracellular signalling molecules, resulting in the activation
of numerous downstream signalling pathways.
Unlike the other family members, HER2 does not alter between the active and inactive conformation. Instead, it is constitutively active and it
lacks the ability to bind a ligand [67]. HER2 also has the strongest catalytic
kinase activity of the HER receptors, and heterodimers containing this receptor have the strongest signalling functions, especially when being in
complex with HER3 [68].
Role in breast cancer
HER2 is overexpressed in approximately 15-30% of breast tumours [69].
Overexpression of the HER2 receptor is due to amplification of a segment
on chromosome 17 at 17q12-q21 that contains many genes, including HER2.
HER2 overexpression is related to a worse prognosis of breast cancer [69].
The notion that HER2 amplification is an early event in human breast
tumourigenesis is supported by the observation that this alteration is seen
in almost half of all in situ ductal carcinomas (a non-invasive growth that
contains abnormal cells) [70, 71].
27
I NTRODUCTION
Besides its function as a growth factor receptor, it has also been proposed that HER2 can act as a nuclear transcription factor [72]. Gene expression profile studies indicate that HER2 amplified breast cancer is a specific
disease subtype, with a unique molecular profile that is maintained during the progression to metastatic disease [73]. In part, this characteristic
gene expression profile of these tumours might be a result of the proposed
transcriptional activities of HER2.
Trastuzumab is a recombinant monoclonal antibody against the extracellular domains of HER2. Treatment with trastuzumab combined with
chemotherapy improves the outcome of women with HER2-positive breast
cancer [74, 75], although resistance is not uncommon and it is estimated
that nearly 15% of the patients receiving trastuzumab based chemotherapy
will progress [76].
The molecular mechanism by which trastuzumab works is uncertain,
but it contributes to apoptosis, down regulates HER2 expression on the cell
surface, alters downstream signalling and suppresses the production of the
angiogenic factor vascular endothelial growth factor (VEGF) [76].
The PI3-K/AKT signalling pathway
The most important function of the HER2/HER3 heterodimer complex
seems to be activation of the phosphatidylinositol 3-kinase (PI3-K). Activation of the PI3-K pathway results in phosphorylation of the serine/threonine kinase AKT mediated by phosphoinositide-dependent kinase (PDK) 1 and 2 [77].
AKT functions in the crossroads of multiple signal transduction pathways that regulate several functions that are critical for normal and cancerous cells, including cell proliferation, cell survival, glucose metabolism,
cell size, epithelial-mesenchymal transition, invasiveness, genome stability
and angiogenesis (Fig. 7) [78, 79]. Activated AKT stabilises cyclin D1 and
enhances degradation of the cyclin-dependent kinase (cdk) inhibitor p27,
resulting in uncontrolled proliferation [77, 80]. It also inhibits BAD and
caspase 9, thereby making the cells more resistant to apoptosis [81, 82].
28
HER2 and AKT signalling in breast cancer
- +
Trastuzumab
E
G
F
R
H
E
R
2
H
E
R
3
H
E
R
2
Increased EGFR
heterodimer signalling
Heregulin
Increased HER3
heterodimer signalling
P
H
E
R
2
H
E
R
2
Increased HER2
homodimer signalling
PI3-K
Ras
PAR6
PI3-K
P
P
PLC
aPKC
AKT
loss of cell polarity
invasion
G1/S
deregulation
proliferation
survival
metabolism
invasion
angiogenesis
Nucleus
HER2-induced
transcription
Figure 7: HER2 overexpression increases HER2-containing dimers of all kinds,
that contribute to tumourigenesis. HER2/EGFR heterodimers stimulate RAS and
PI3-K signalling, thereby driving proliferation and invasion. Activation of PI3K/AKT signalling through HER2/HER3 heterodimers seems to be the critical tumourigenic function of overexpressed HER2. AKT regulates multiple functions
in the cell, including proliferation, evasion of apoptosis, glucose metabolism, cell
size and invasiveness [68].
29
I NTRODUCTION
Connections between AKT signalling and DNA repair
Researchers have found several points of contact between PI3-K/AKT signalling and proteins in the DNA repair pathways (Fig. 8). Overexpression
of NBS1 stimulates tumourigenesis through activation of PI3-K and AKT in
vitro, and this mechanism has been implicated in head and neck squamous
cell carcinoma (HNSCC) [83, 84].
Recently, a connection between the tumour suppressor phosphatase and
tensin homologue (PTEN) and RAD51 was reported [85]. PTEN is a phosphatase that acts in the cytoplasm as a negative regulator of the PI3-K/AKT
signalling pathway, and it is mutated in a wide variety of solid tumours.
Loss of heterozygosity (LOH) at the region containing the PTEN gene is
a common event in breast tumours, but only a small fraction of these tumours have mutations in this gene, indicating that PTEN inactivation through
mutation is not a common mechanism in sporadic breast cancer [86]. Nevertheless, it has been reported that approximately one third of breast tumours have lost or reduced expression of the PTEN protein, and lack of
PTEN expression is associated with aneuploidy in breast cancer [79, 87].
The finding that nuclear PTEN regulates RAD51 reveal a molecular link
between nuclear PTEN and DSB repair.
It has been shown that stimulation of the PI3-K/AKT pathway by
heregulin-β induces phosphorylation of BRCA1 [88]. AKT phosphorylates BRCA1 on the residue Thr-509, which is located adjacent to a nuclear localisation signal (NLS) at residues 503-508. It was recently shown
that this phosphorylation event regulates the subcellular localisation of
BRCA1, leading to nuclear accumulation and increased BRCA1 transcriptional activity [89]. The nuclear functions of BRCA1 include inhibition of
cell growth [90], induction of apoptosis [91], cell cycle regulation [92] and
maintenance of genomic stability [93].
Conversely, BRCA1 has been shown to be a negative regulator of the
AKT pathway [94]. Thus, BRCA1 deficiency increases the kinase activity of
AKT and is associated with nuclear accumulation of this oncogene. These
novel findings might give part of an answer to the puzzling question of
why BRCA1 deficiency leads to tumourigenesis in the breast.
30
Connections between AKT signalling and DNA repair
Heregulin
P
Nucleus
PI3-K
maintenance
of the
genome
T
NBS1
c-MYC
P
PTEN
ubiquitination
Akt
RAD51
P
P
BRCA1
imp.
BRCA1
transcription
inhibition
stimulation
P
T
phosphorylation
transactivation
Figure 8: Connections between PI3-K/AKT signalling and DNA repair. AKT regulates the subcellular localisation and activity of BRCA1. Stimulation of heregulinβ induces AKT-mediated phosphorylation of BRCA1 on Thr-509 next to the nuclear localisation signal. This phosphorylation event increases the nuclear content
of BRCA1, and at least some of its nuclear functions. Conversely, BRCA1 has
been shown to mediate ubiquitination and degradation of AKT. Overexpression
of NBS1 in head and neck tumours stimulates tumourigenesis through activation
of PI3-K/AKT signalling. The nuclear fraction of PTEN regulates RAD51 expression, thereby contributing to maintenance of genomic stability.
It also demonstrates that the signalling networks in a cell, regulating
genomic integrity, cell cycle checkpoints, proliferation, apoptosis and other
functions, are interconnected on many levels.
31
I NTRODUCTION
DNA repair pathways as targets for cancer therapy
Pharmacological inhibition of DNA repair has the potential to sensitise tumour cells to the damaging effects of therapy. Inhibitors have been developed against several of the proteins studied in this thesis, including the
PI3-K/AKT pathway [95], DNA-PK and ATM [96, 97, 98].
Recently, a novel strategy using short DNA molecules that mimic DSBs
and disorganise DNA damage signalling was shown to sensitise tumours
to radiation in vitro and in mouse models [99]. In addition, inhibitors of
poly(ADP)-ribose polymerase (PARP) can be used as sensitisers for cancer
therapies such as temozolomide and IR. Especially BRCA1- and BRCA2mutated hereditary breast cancers are sensitive to PARP inhibitors [100,
101]. PARP is involved in repair of single-strand breaks by BER. Unrepaired single-strand breaks can be converted to double-strand breaks, and
these lesions can not be repaired by HR-deficient cells. The effect of PARP
inhibitors in breast cancer patients with BRCA1/2 mutations is currently
evaluated in phase II clinical trials. This exemplifies that modulation of
DNA repair has potential to become a future strategy to increase tumour
sensitivity to chemo- or radiotherapy.
32
Aims of the thesis
General aim
The general aim of this thesis was to gain deeper insight into the complex
mechanisms that affect tumour cell resistance to ionising radiation.
Specific aims
• To investigate the role of the HER2/PI3-K/AKT signalling pathway
in resistance to radiation-induced apoptosis in breast cancer.
• To study the expression of DNA repair-associated proteins in breast
tumours.
• To examine if the expression of DNA repair proteins is associated
with clinicopathological variables and if it is related to local and distant recurrence-free survival.
• To investigate if the expression of DNA repair proteins is related to
the outcome of radiotherapy.
• To examine if the RAD51 135G/C polymorphism is associated with
RAD51 protein expression, prognosis and effect of radiotherapy or
CMF chemotherapy.
33
Comments on materials
and methods
The following is a discussion on the principles, advantages and disadvantages of methods used in the papers included in this thesis. Specific conditions and reagents used are described in more detail in the papers.
Cell culture
The use of cancer cell lines have allowed considerable advances in the field
of cancer biology and have, for a long time, been the principal model system for breast cancer research [102]. They provide a source of homogenous
material that self replicate without limit. Established breast cancer cell lines
are easy to culture in standard media and can be manipulated in countless ways. Under well defined experimental conditions, cell lines generally
yield reproducible and quantifiable results [102].
Cell lines grown in vitro can not fully capture the complexity of clinical breast tumours. The advantage of having a homogenous population
of one cell type in the culture dish, at the same time excludes the possibility to study the interaction of cancer cells with the microenvironment.
Most of the available breast cancer cell lines are derived from metastases,
and questions have been raised as to how well they represent primary
tumours. However, most investigations have revealed that progression
from primary tumour to metastasis is not accompanied by major changes
in marker expression or histological grade, and the expression of differ35
C OMMENTS ON MATERIALS AND METHODS
ent markers is well correlated when cell lines are compared to their corresponding archival tumour tissue after being grown in vitro for an extended
period of time [103]. The results from experiments performed on cell lines
can not be directly translated to in vivo tumours, but they provide important insights to tumour biology.
The cell lines used in Paper I, MCF-7 and BT-474, are human breast
cancer cell lines that have been widely used in experimental research. We
choose them because they differ in HER2 expression and p53 status.
Patients and tumour material
The breast tumours used in Papers II-IV were collected during two randomised clinical trials conducted in 1976-1990 by the Stockholm Breast
Cancer Study Group [104]. The studies compared postoperative radiation therapy (RT) with adjuvant chemotherapy (CT) in pre- and postmenopausal patients who were at high risk of recurrence. In Papers II and III,
tumours from premenopausal patients were used. In Paper IV, we have
used tumours from both pre- and postmenopausal patients. The postmenopausal trial also included a randomisation between tamoxifen versus no
adjuvant treatment, which means that the patients were randomised to one
of four groups (RT only, RT + tamoxifen, CT only and CT + tamoxifen). Tamoxifen was not considered in the analyses of treatment outcome in Paper
IV.
All patients included in the two trials had histologically verified invasive, unilateral breast cancer. They were treated with modified radical mastectomy. Inclusion criteria were node-positive disease, or a tumour diameter exceeding 30 mm, as measured on the surgical specimen. Exclusion
criteria were inoperable local disease, distant metastases at the time of primary diagnosis, other concurrent cancers, medical contraindications of the
treatment, or surgery that deviated from the protocol.
Radiotherapy was given at a total dose of 46 Gy with 2 Gy per fraction,
5 days per week. Chemotherapy was 12 courses of cyclophosphamide/
methotrexate/5-fluorouracil (CMF). More details on the treatments can be
found in Papers II-IV, and in the report on the original study [104].
This material has several advantages. It is relatively homogenous in
terms of patient characteristics and treatment. Patients have been ran36
Western blot
domised to receive one of two treatments, and randomised materials similar to this are rare today. The patients have not been treated with neoadjuvant chemotherapy prior to surgery, which means that the protein expression in the tumours is unaffected by treatment.
Some difficulties are that the positive effects of radiotherapy on distant recurrence that has been reported complicate the analyses of prediction of treatment effect [105]. Also, these tumours are relatively large and
the frequency of recurrence is high. The patient group is relatively homogenous, which makes it difficult to draw generalised conclusions that apply
to breast cancer patients with other characteristics.
Western blot
Western blot is a method that allows comparison of protein expression between different samples of cell or tissue extracts (Fig. 9). Similar to immunohistochemistry (IHC), this technique relies on antibody detection of
the protein in interest, and the result is dependent on antibody specificity.
In short, samples are lysed and the concentration of proteins in the
lysate is measured. The proteins are denatured and separated on a polyacrylamide gel according to their size. They are then transferred from the
gel to a membrane made of polyvinylidine fluoride (PVDF) or nitrocellulose, to make the proteins accessible to antibody binding. The membrane is
incubated in a blocking solution containing milk or bovine serum albumin
(BSA) to prevent nonspecific binding and subsequently incubated with the
primary antibody. The membrane is washed, incubated with a secondary
antibody directed against the primary antibody, and bound antibodies are
detected and analysed.
Correct handling of the lysates is crucial, since proteins are sensitive to
degradation. Samples should be kept cold and the lysing medium should
contain protease inhibitors. If phosphorylation is to be investigated, phosphatase inhibitors must be used.
Since the sample consists of concentrated cell lysate, the primary antibody can be more diluted than in IHC, typically in the range 1:1000-1:10000.
Antibody dilution, incubation time (30 min - overnight) and incubation
temperature (warmer temperature resulting in more binding, both specific
and nonspecific) have to be optimised for each antibody.
37
C OMMENTS ON MATERIALS AND METHODS
Figure 9: Western blot of NBS1 expression in BT-474 and MCF-7 cells using the
same antibody that was used for IHC staining in Paper II. The bands to the left are
the size marker. The molecular weight of NBS1 is 95 kDa.
The size (molecular weight) of a detected protein is approximated by
comparing the distance that the band has migrated in the gel to the bands in
a size marker (a ‘ladder´, containing a mixture of proteins of known sizes).
Equal loading of the amount of protein in different samples is validated by
repeating the procedure, using an antibody against a structural protein (e.g.
β-actin or tubulin) that should not differ between the samples. The relative
amount of protein in different samples can be approximated by measuring
the intensity of the bands, but it is important to remember that Western blot
is not a quantitative method.
Western blot is a very common and relatively inexpensive method that
can be employed to assess the expression of a wide range of proteins to
which antibodies have been developed. Some drawbacks are that the protein expression is analysed in a lysate and not in single cells, which means
that it gives a mean expression level for all the cells in a sample. Nonspecific background staining is sometimes a problem, but can often be avoided
by using the detergent Tween in washing and blocking buffers, optimising
blocking conditions and keeping antibody concentration and incubation
temperature low.
38
Cell cycle analysis by flow cytometry
Cell cycle analysis by flow cytometry
Cell cycle distribution was analysed by DNA flow cytometry. Exponentially growing cells were stained with propidium iodide (PI) using a detergenttrypsin method according to Vindelöv [106]. PI is a fluorescent dye that
binds to DNA and emits light when the cell passes through a laser beam
in the flow cytometer. Because PI binds to DNA and cells contain different
amounts of DNA depending on where they are in the cell cycle, the proportion of cells in each cell cycle phase can be analysed with this method. This
method can also be used to detect populations of cells that have abnormal
amounts of DNA, e.g. tetraploid cells.
Flow cytometry can be used in a wide range of applications, including
cell sorting and phenotyping. In addition to DNA staining, different antibodies can be used to detect extracellular or intracellular markers, and
it is possible to study several markers at the same time by combining antibodies that are conjugated with different fluorochromes. Cells are also
characterised according to their size and granularity.
Flow cytometry applications that rely on antibodies are afflicted with
the same problems regarding antibody specificity that other immunobased
methods have. Another disadvantage is that this technique requires relatively large amounts of material.
Apoptosis detection
There is a plethora of methods and kits available for apoptosis detection.
Some are based on manual evaluation of the morphological changes associated with apoptosis (e.g. DAPI staining), others measure DNA fragmentation (e.g. TUNEL assay), caspase activation or other alterations in
macromolecules that are unique to apoptotic cells.
In Paper I, apoptosis was detected using an Annexin V assay [107]. During the early stages of apoptosis, phosphatidylserines (PS) are translocated
from the inner side of the cell membrane to the outer layer. Annexin V is a
phosphobinding protein that binds PS with high affinity. Translocation of
PS also occurs during necrosis, so it is necessary to discern apoptotic cells
from necrotic. This is achieved by staining the cells with PI. Since the cell
membrane looses its integrity and becomes leaky in necrotic cells, these
39
C OMMENTS ON MATERIALS AND METHODS
cells are stained by PI, whereas the membrane remains intact during the
initial stages of apoptosis. Thus, cells that are Annexin V+/PI- are apoptotic, whereas cells that are positive for both Annexin V and PI are necrotic
or post-apoptotic necrotic. The cells were analysed in a flow cytometer.
We have also used M30, an antibody that recognises caspase-cleaved
cytokeratin 18 [108]. In Paper I, we used an unconjugated M30 antibody
for detection of apoptosis by flow cytometry, and a fluorescein-conjugated
M30 antibody for immunocytochemistry (Fig. 10).
Most methods that detect apoptosis give a notion of the level of apoptosis at that very moment. However, apoptosis is a process, and it is thus
important to make measurements at several time points. It is also recommended to use at least two different methods. With some methods, it can be
difficult to discern apoptotic cells from necrotic. We chose to use Annexin
V because flow cytometry allows for the rapid analysis of tens of thousands
of cells and the PI staining excludes cells that are necrotic. M30 was chosen as a complement to Annexin V, since this method relies on cleavage of
cytokeratin 18, a process that is separate from PS translocation. The possibility to use M30 in both flow cytometry and immunocytochemistry was
considered an advantage.
Immunohistochemistry
Immunohistochemistry allows detection of proteins in a tissue by the use
of specific antibodies (Fig. 11).
What follows is a brief description of how to perform the method.
Paraffin-embedded (or snap frozen) tumours are sectioned and mounted
on adhesive microscope slides. The sections are deparaffinised in xylene
and rehydrated in a series of ethanol baths of decreasing concentration. For
most antibodies, antigen retrieval is needed prior to staining (see below).
The specimens are incubated with a blocking reagent (see below), washed
and the primary antibody is applied. The dilution of the antibody and incubation time affect the staining results and must be optimised empirically
for each antibody. The slides are washed and incubated with a secondary
antibody, which is enzyme-conjugated (e.g. horseradish peroxidase, HRP)
and directed against the primary antibody.
40
Immunohistochemistry
Figure 10: M30 reactivity (green) is confined to the cytoplasm of apoptotic cells.
Two cells at different stages of the apoptotic process are shown (left). Cells that
have been successfully transfected with constitutively activated AKT are visualised in red (right).
Figure 11: Immunohistochemical staining using an antibody against NBS1. In
normal breast (a), the epithelial cells form ducts in an organised structure with a
clear histology. In tumours (b), the tissue is disorganised. The cells have often lost
attributes that are characteristic for the different cell types. The size of the cells can
also vary and many tumour cells are enlarged as compared to normal epithelial
cells. Original magnification x400.
After washing, the sections are immersed in a substrate reagent solution, e.g. 3,3’-diaminobenzidine tetrahydrochloride (DAB), and the HRP
catalyses a reaction that produces an insoluble, brown end-product in the
cells that express the protein of interest. The specimens can be counterstained; e.g. with heamatoxylin, which stains all nuclei in a blueish colour.
The slides are washed, dehydrated in ethanol/water baths of increasing
ethanol concentration and mounted with cover glasses.
41
C OMMENTS ON MATERIALS AND METHODS
TMA
All IHC stainings in Papers II and III were performed on tissue microrarray
(TMA) slides. The TMA method was introduced in 1998 by Kononen and
colleagues [109]. The arrays are constructed by taking core needle biopsies from paraffin-embedded tumours and re-embedding the biopsies in
a new paraffin block. In this way, it is possible to collect samples from
hundreds of tumours in a few blocks. Tumour heterogeneity could pose a
problem; however, validation studies have shown that if biopsies are taken
in triplicates from each tumour, TMA is comparable to whole-section analysis [110, 111, 112]. This technique has several advantages. In contrast
to whole-section staining, which requires the processing and staining of
hundreds of slides, the microarray technique allows for investigation of a
large set of cases by analysing just a few slides. Since it is possible to stain
all slides at the same time, the problem of interbatch variation is reduced.
The biggest advantage is that this technique reduces the amount of tissue
needed, which is an important aspect when working with archived material.
Antibodies
Antibodies can be either monoclonal or polyclonal. Monoclonal antibodies are typically produced by myeloma cells that are fused with antibodysecreting spleen cells from a mouse that has been immunised with the specific antigen. The resulting cell, a hybridoma, can be cell cultured to produce large quantities of identical monoclonal antibodies. Polyclonal antibodies, on the other hand, are secreted from several different clones of cells
and react with multiple epitopes on the antigen. Due to the greater risk
of unspecific binding of polyclonal antibodies to molecules other than the
target, monoclonal antibodies are often preferred. The greatest advantage
of polyclonal antibodies is that they are not as sensitive to small conformational changes of the target antigen (caused by fixation, glycosylation or
polymorphisms in the target) since several antibodies with different target
epitopes can bind to the same molecule.
Background staining can be a problem with some antibodies. It stems
from hydrophobic or ionic interactions between the antibody and tissue
components other than the target antigen. We used Protein Block Serum42
Immunohistochemistry
Free (DAKO, Carpinteria, CA) to reduce nonspecific staining. This reagent
utilises casein, a hydrophilic protein which has been shown to decrease
nonspecific binding of primary and secondary antibodies in immunohistochemistry. It is also common to add BSA to the buffer in which the antibody
is diluted and to have a detergent, such as Tween, in the washing buffer to
reduce nonspecific staining.
When choosing antibodies, we have reviewed the literature and
searched for antibodies that have been successfully used before and that
are commercially available. We have preferred monoclonal antibodies over
polyclonal, and tried to find antibodies which have been tested for IHC of
formalin-fixed, paraffin-embedded sections.
Fixation and antigen retrieval
One of the most important steps in IHC is the fixation of the tissue to preserve the structures in a lifelike state and prevent degradation. Formalin is
the most commonly used fixative and it forms stable bonds, or ‘crosslinks´,
with macromolecules in the specimen. Fixation can mask the epitopes that
the antibodies are directed against. It is therefore often necessary to process
the sample to restore the reactivity of many antibodies for tissue epitopes, a
procedure referred to as antigen retrieval. Prior to staining, we have heated
the slides in citrate buffer (pH 6.0) in a pressure cooker or microwave oven.
Other antigen retrieval methods include enzymatic digestion and heating
in an alkaline buffer.
Advantages and disadvantages
Immunohistochemistry is widely used and it is one of a few methods that
can be used to study protein expression in archived tissue. The method has
several advantages, including relatively low cost, no need for expensive
and complicated equipment and the possibility to see the morphological
structure of the sample and exactly which cells express a specific protein.
The largest drawback of immunohistochemistry is the risk of subjectivity when grading the staining. To reduce this problem, all stainings were
judged independently by two investigators (K.S. and M.S.A). In cases of
disagreement, the stainings were re-evaluated until consensus was reached.
Grading of the staining was performed blindly.
43
C OMMENTS ON MATERIALS AND METHODS
PCR-RFLP
The polymerase chain reaction (PCR) is one of the most widely used laboratory techniques today. It allows the amplification of a short DNA sequence
into millions of copies, which can then be visualised on an agarose gel or
further analysed using different applications.
The usability of DNA from fixed, paraffin-embedded material depends
on several factors, including (a) the fixative used, (b) how long the specimen was kept in the fixative, (c) the age of the specimen and (d) the length
of the DNA fragment to be amplified. Some fixatives, especially highly
acidic solutions, cause DNA degradation. Fixation with formalin does not
seriously compromise amplification efficiency [113]. The probability of successful amplification of large PCR products decreases with longer fixation
periods. Similarly, DNA from older specimens is more difficult to amplify.
When working with older samples, one way to improve the likelihood of
successful amplification is to choose small target fragments (<200 bp).
In Paper IV, a 157 bp fragment of the RAD51 gene was amplified by
PCR. The PCR products were subsequently digested by the MvaI restriction enzyme, which cleaves DNA at a specific restriction site (Fig. 12). The
RAD51 135G/C polymorphism alters this restriction site. Digested DNA is
separated according to size on an agarose gel containing the DNA-binding
dye ethidium bromide. This restriction fragment length polymorphism
(RFLP) method has been used in many previous studies for the investigation of this polymorphism [114, 115, 116, 117].
T
C C ^A G G -3´
3´- G G T^ C C -5´
A
5´-
^ restriction site
polymorphic site
Figure 12: Restriction site of the MvaI enzyme. Cleavage of the 157 bp amplicon results in two fragments, 86 and 71 bp long. The restriction site is destroyed
by the 135G/C polymorphism, so that the amplicon remains uncleaved in C/C
homozygotes.
44
Statistical analysis
Statistical analysis
In Paper I, we studied the effects of heregulin-β1 and wortmannin on cellular sensitivity to radiation-induced apoptosis. The ratio between treated
and untreated cells were calculated. Student´s t-test was used to test the
difference between the mean of the ratios and 95% confidence intervals (CI)
were calculated.
In Paper II-IV, the significance of the differences between grouped variables (e.g. the association between the expression of protein x and clinicopathological variables such as histological grade) were tested with χ2 test
for contingency tables. In Paper III and IV, χ2 test for trend (Spearman) was
used if a variable contained 3 or more categories.
Survival curves were estimated using the product limit method by
Kaplan-Meier. The role of one (univariate) or a group of variables (multivariate) in survival analysis was estimated using Cox´s proportional hazard regression. Cox analysis was also used to test the interaction between
a variable x and treatment. This multivariate Cox analysis included the
covariates x, treatment and the interaction variable x×treatment. p-values
≤0.05 were considered statistically significant.
45
Results & Discussion
HER2/PI3-K/AKT signalling is important for resistance
to radiation-induced apoptosis (Paper I)
The gene encoding the HER2 (or erbB2) growth factor receptor is amplified in 15-30% of all breast tumours and overexpression of the receptor is
related to resistance to several chemotherapeutic agents [69, 118, 119, 120].
In the first study, we investigated the hypothesis that the HER2 receptor
confers resistance to radiation-induced apoptosis through the PI3-K/AKT
signalling pathway.
We used two different breast cancer cell lines, which differ in HER2 expression and p53 status. The MCF-7 cell line has normal HER2 expression
and functioning p53, whereas BT-474 has constitutively activated AKT due
to HER2 gene amplification (Table 3). In addition, p53 is mutated in this
cell line.
Cell line
MCF-7
BT-474
HER2
normal
overexpressed
AKT activity
upon stimulation
constitutively active
p53 status
wildtype
mutated
Table 3: Characteristics of breast cancer cell lines used in Paper I.
47
R ESULTS & D ISCUSSION
Cell cycle regulation after γ-radiation
Cells were irradiated with 10 Gy γ-radiation and analysed for cell cycle
distribution. Two days after irradiation, the fraction of MCF-7 cells in S
phase was reduced in irradiated cells as compared to the control (Fig. 1e,
Paper I). This decrease of proliferative cells was not seen for the BT-474 cell
line, indicating that MCF-7 was more sensitive to radiation. The reduction
of MCF-7 cells in S phase was accompanied by an accumulation of cells in
G2/M phase, whereas the proportion of cells in G1 phase remained relatively constant over time (Fig. 1d-f, Paper I). This suggests that both the G1
and the G2/M checkpoint were functional in this cell line. In BT-474 cells,
the G1 fraction decreased and the G2/M fraction was increased to the same
extent, indicating that these cells only halted at the G2/M checkpoint (Fig.
1a-c, Paper I).
The ability of cells to halt in the G2 phase allows them to repair any sublethal DNA damage before entering mitosis, and is thus considered to be
an important determinant of radiosensitivity. Whereas a role for p53 in the
G1/S checkpoint has been clearly established, its function in G2/M is less
clear [34]. Our results, showing that only the cell line with functional p53
entered G1 phase arrest, whereas both cell lines had intact G2/M checkpoints, are in line with this.
Radiation-induced apoptosis
Both cell lines were treated with different combinations of heregulin-β1 and
wortmannin, and half of the samples were exposed to 10 Gy γ-radiation.
Radiation-induced apoptosis was higher in MCF-7 cells than in BT-474 cells,
again indicating that MCF-7 cells were more sensitive to the effects of IR
(Fig. 3, Paper I). Heregulin-β1 protected non-irradiated MCF-7 cells from
spontaneous apoptosis. This effect was not seen in BT-474 cells in which
the HER2/PI3-K/AKT signalling pathway was already activated by HER2
overexpression.
When BT-474 cells were pretreated with the PI3-K inhibitor wortmannin prior to irradiation, apoptosis increased as compared to treatment with
wortmannin alone (Fig. 3, Paper I). We propose that the sensitivity of BT474 cells to inhibition of PI3-K signalling is due to the dependency of these
cells on HER2-mediated signalling. Transfection of MCF-7 cells with con48
Paper I
stitutively active m/p-AKT made the cells more resistant to apoptosis (Fig.
5, Paper I).
When assessing radiosensitivity in vitro, it is common to study the ability of cells to form colonies after treatment with radiation (’colony-forming
assay’). Despite several attempts, we did not succeed in growing the cells
in soft agar.
PI3-K is structurally related to DNA-PK and ATM, and wortmannin has
been shown to inhibit all of these PI3-K superfamily members at different
concentrations [121, 122, 123, 124]. Since both DNA-PK and ATM have important roles in DNA damage signalling, inhibition of these proteins might
make cells sensitive to γ-radiation.
For several reasons, we believe that the radiosensitising effect by wortmannin treatment in BT-474 cells is due to inhibition of the PI3-K/AKT
signalling pathway. The IC50 value of PI3-K inhibition by wortmannin is
2-4 nM [121], whereas inhibition of ATM and DNA-PK have higher IC50
values [125]. The dose that we used (100 nM) is considerably lower than in
studies on the other kinases, in which the wortmannin dose was 10-50 µM
[123, 124, 126].
Wortmannin seemed to only have a sensitising effect in BT-474 cells,
which overexpress HER2. Since we have not investigated the expression of
DNA-PK and ATM, we can not rule out the possibility that these kinases
are differently expressed in the cell lines and coincide with the effect of
wortmannin. However, AKT phosphorylation was decreased after treatment with wortmannin, indicating that PI3-K was affected (Fig. 2, Paper I).
In addition, stimulation with heregulin-β1, as well as transfection of MCF-7
cells with activated AKT, made these cells more resistant to apoptosis, thus
supporting the hypothesis that the radiosensitising effect of wortmannin
was mediated by PI3-K/AKT signalling.
In conclusion, the results in Paper I support the hypothesis that the
HER2/PI3-K/AKT signalling pathway is involved in resistance to radiationinduced apoptosis in breast cancer cells. This pathway has been shown to
be dysregulated on several levels in cancer, including HER2 overexpression
and loss of the tumour suppressor PTEN.
49
R ESULTS & D ISCUSSION
DNA repair-associated proteins in breast cancer
(Paper II and III)
In this thesis, we have studied the protein expression patterns of ATM,
MRE11, RAD50 and NBS1 (Paper II), as well as BRCA1, BRCA2 and RAD51
(Paper III). We investigated the role of these proteins in prognosis and prediction of radiotherapy effect in early breast cancer.
Reduced or elevated expression of DNA repair proteins in
breast tumours?
One of the most elementary questions to address in studies that aim to
investigate the prognostic or predictive significance of a protein, is in what
way the expression is altered in tumours as compared to normal tissue. In
the immunohistochemistry studies (Paper II and III), we have used normal
breast epithelium as reference.
NORMAL BREAST
Protein
Intensity
Percentage a
ATM
MRE11
RAD50
NBS1
BRCA1
BRCA2
RAD51
moderate
strong
strong
strong
strong
weak
strong
1-25
51-75
1-25
26-50
51-75
26-50
26-50%
TUMOURS
Reduced
Increased perb
intensity
centage a,b
76%
80%
72%
20%
79%
61%
79%
79%
80%
46%
1%
56%
76%
40%
Table 4: Staining of the different proteins investigated in Paper II and III.
a
b
percentage of positively stained nuclei
as compared to normal breast epithelium
Five of the seven DNA repair-associated proteins that were investigated
in Paper II and III showed strong staining intensity in normal breast epithelium, with the exception of BRCA2 and ATM (Table 4). The staining intensity was reduced in more than 70% of the tumours for all proteins except
BRCA2. The percentage of positive cells in normal breast varied for the
different proteins; yet the percentage of positive cells was increased in 40%
or more of the tumours for all proteins except MRE11. Taken together, this
50
Paper II and III
indicates that the DNA repair proteins were expressed in more cells, but at
a lower level, in breast tumours as compared to normal breast tissue.
There are a couple of reports on the expression of DNA repair proteins
in breast tumours. The results in Paper II, indicating that down regulation
or loss of MRE11, RAD50, NBS1 and ATM is a common event in breast
cancer, are supported by a previous study by Angele et al [127]. Similarly,
BRCA1 expression is often aberrant in sporadic breast cancer due to LOH,
methylation of the promotor region and low mRNA and protein expression
[128, 129, 130, 131].
As for RAD51, its role in tumour development and progression is not
clear. Several studies have reported overexpression of RAD51 in cell lines
and tumours of different origin [132, 133, 134, 135]. However, other lines
of evidence indicate that RAD51 represses tumour progression [136]. In
breast tumours, both high and low RAD51 expression has been reported
[132, 137], but the mechanism behind deregulated RAD51 expression is as
yet unknown.
When the expression of the individual proteins was combined to allow evaluation of the complexes, 44% of the tumours had negative/weak
nuclear staining intensity of the MRN complex and 53% were categorised
as having low expression of the BRCA1/BRCA2/RAD51 complex (Paper
II and III). This implies that the protein expression of these DNA repairassociated proteins is reduced in a considerable proportion of breast tumours.
Significance of the subcellular localisation
In Paper II, a smaller fraction (10%) of the tumours showed cytoplasmic
staining of NBS1. Similarly, MRE11 was found in the cytoplasm in more
than half of the tumours, but not in normal breast tissue (Paper II). This cytoplasmic staining correlated with weak nuclear RAD50 staining intensity.
This correlation between weak nuclear staining of one protein and cytoplasmic staining of another might be explained by a previously reported
observation that alterations in one member of a multiprotein complex can
destabilise some or all of its protein components. NBS1 and MRE11 staining is exclusively nuclear in normal fibroblasts, but diffuse in cells from
patients with ATLD (caused by mutations in the MRE11 gene) [59]. Sim-
51
R ESULTS & D ISCUSSION
ilarly, the nuclear fraction of MRE11 and RAD50 is reduced in NBS cells
(lacking NBS1), with a concomitant increase of protein in the cytoplasm
[60].
Cytoplasmic staining was also observed for BRCA2, both in normal
breast epithelium and in tumours (Paper III). Reduced cytoplasmic staining of BRCA2 in tumours was correlated to ER negativity (p=0.01), but not
to any other clinicopathological factors, nor to the local recurrence-free survival.
RAD51 showed cytoplasmic staining in approximately half of the tumours (53%) (Paper III). A small number of tumours were positive for cytoplasmic staining, but negative for nuclear staining. This expression pattern has been associated with germline mutation of BRCA2 [138]. Though
BRCA2 and RAD51 are primarily nuclear proteins, they have previously
been observed in the cytoplasmic compartment [137, 138, 139, 140].
A large fraction of the tumours (64%) showed cytoplasmic staining
of ATM, whereas normal breast epithelial cells had only nuclear expression (Paper II). Cytoplasmic staining of ATM in breast cancer cells has
been reported previously [141], but the function of ATM in the cytoplasm is not known. Cytoplasmic staining of ATM was correlated to
ER negativity (p=0.03), but not to the rate of local recurrence. Patients
with cytoplasm-positive tumours had no significant benefit of radiotherapy over chemotherapy on local recurrence-free survival (p=0.20), whereas
cytoplasm-negative patients responded to radiotherapy (p=0.03).
Subcellular relocalisation is a means of regulation of protein function,
as can be seen for the oncogenic kinase c-ABL [142], and it is possible that
cytoplasmic accumulation of the MRN proteins (or other enzymes that exert similar activities) impair their function. Since many of the functions of
the DNA repair enzymes involve DNA damage signalling, cell cycle regulation and direct interaction with DNA, we reasoned that nuclear protein
would have the highest impact on DNA repair efficiency. However, we do
not rule out the possibility that cytoplasmic fractions of these proteins may
act as a reserve, which can be shuttled to the nuclear compartment under
some circumstances.
The clinical relevance of cytoplasmic expression of the investigated proteins is not clear. In both Paper II and III, it was the nuclear expression that
was associated with local recurrence-free survival and prediction of radio52
Paper II and III
therapy effect. It is however possible that cytoplasmic expression of ATM
has biological relevance, since the results in Paper II indicated that cytoplasmic ATM staining predicted a poor response to radiotherapy.
Expression patterns of proteins that form a complex together
The nuclear staining intensities for MRE11, RAD50 and NBS1 were highly
correlated, with strong staining of one of the proteins correlating with strong
staining of the other two (all p<0.001) (Paper II). The observation that low
expression of MRE11, RAD50 or NBS1 was rarely found alone is in line with
a previous finding that the components of the complex stabilise each other.
Similarly, low RAD51 expression was correlated to weak BRCA1 staining
(p<0.001) (Paper III). We have also found correlations between the expression of DNA-PKcs and Ku70/80, the components of the DNA-PK complex
that is central in NHEJ repair (unpublished results).
It could be that the expression of these proteins is regulated by related
mechanisms. Another possibility is that the observed correlations are an
artefact, caused by fixation problems or differences in tissue quality between tumours, that affect the staining result. The most likely explanation,
however, is probably that the similarities in expression patterns of proteins
that act in complexes with each other are the result of stabilisation when all
components of the complex are present, as mentioned above.
Reduced expression of complexes is associated with
clinicopathological variables
Negative/weak expression of the MRN complex was correlated with high
histologic grade (p=0.01) and ER negativity (p=0.0001) (Paper II). Similarly, low expression of the BRCA1/BRCA2/RAD51 complex was associated with high histologic grade (p=0.05) (Paper III). This is in line with
several previous studies, in which a correlation between reduced BRCA1
expression and high histologic grade has been a consistent finding [143,
144, 145, 146].
When the proteins in Paper III were analysed individually, RAD51 expression in a low fraction of cells (defined as 0-25% positive nuclei) correlated with high grade and ER negativity (p<0.001 and p=0.006, respectively), as did weak RAD51 intensity.
53
R ESULTS & D ISCUSSION
In contrast to our results, high RAD51 expression (defined as dark staining in >1% of tumour cells) has been shown to correlate with high histologic grade and ER negativity in breast cancer specimens [132]. One problem with IHC is that variations in scoring systems make it difficult to compare the results from different studies. When evaluating the IHC staining,
we chose to categorise the intensity into four groups (-,+,++ and +++), since
the intensity varied between specimens. Variations in staining intensity of
these proteins have been observed by others as well [144, 146].
In conclusion, the results from Paper II and III indicate that there is an
association between reduced expression of these DNA repair complexes
and an aggressive tumour phenotype.
Relation to recurrence-free survival
All patients in Paper II-IV have received either local or systemic treatment.
In analyses of prognosis, we tried to minimise the effect of treatment by
only including the subgroup of patients that received CMF when analysing
local recurrence-free survival, and only the radiotherapy treated patients
in analysis of distant recurrence-free survival. However, we can not completely rule out the possible influence of treatment in the prognosis analyses.
Local recurrence
The percentage of NBS1-expressing tumour cells was an independent prognostic factor, and patients with high expression (>25% positive cells) had
fewer local recurrences (Fig. 2a, Paper II). The MRN complex was not a
prognostic factor of local recurrence-free survival.
Similarly, patients with reduced expression of the BRCA1/BRCA2
/RAD51 complex had significantly more local recurrences as compared to
patients with high expression (Fig. 2a, Paper III). When analysed individually, low RAD51 expression was related to a higher risk of developing local
recurrence (Fig. 2b, Paper III). Both RAD51 and the complex were independent prognostic factors in multivariate analysis.
54
Paper II and III
Distant recurrence
Moderate/strong expression of the MRN complex was related to longer
distant recurrence-free survival as compared with negative/weak expression (Fig. 2b, Paper II). Similarly, patients whose tumours had moderate/strong expression of MRE11 had a better prognosis. MRE11 intensity
and expression of the MRN complex were independent prognostic factors
of distant recurrence. In Paper III, the BRCA1/BRCA2/RAD51 complex
was not related to distant recurrence-free survival.
Deregulated expression of DNA repair-associated proteins seems to result in genomic instability, as evidenced by an increased frequency of chromosomal rearrangements in cells from NBS patients, chromosome aberrations in cells deficient of BRCA1 or BRCA2 and genome instability in cells
after depletion or overexpression of RAD51 [147, 148, 149, 150, 151, 152].
In a recent study, chromosomal instability was shown to predict poor clinical outcome in multiple types of cancer, including breast cancer [153]. Our
findings, that low expression of DNA repair proteins are related to higher
risk of recurrence, are in line with this.
The prognostic significance of some of these proteins have been investigated previously. Studies on the correlation between prognosis and reduced expression of BRCA1 in spontaneous breast tumours have yielded
contradicting results [143, 145, 146]. We found no prognostic value for
BRCA1 expression alone.
In a previous study on RAD51 expression and prognosis in patients
with non-small cell lung carcinoma (NSCLC) of different stages, the cut
off point was chosen to obtain optimal separation between prognostically
different groups [154]. High RAD51 expression (>10%positive cells) was
found to correlate to shorter overall survival in that study.
The prognostic significance of NBS1 has been investigated in other cancer types as well. Overexpression of NBS1 is a marker of poor prognosis
in advanced HNSCC and uveal melanoma [84, 155]. This is in contrast
with our results, that suggested that high expression of NBS1 was related
to good prognosis. As indicated above, moderate/strong expression of
the MRN complex, and the individual proteins as well, were associated
with low histologic grade and ER positivity. It should be noted, however,
that these studies differ in measured endpoint (local recurrence vs overall
55
R ESULTS & D ISCUSSION
survival), tumour type and stage of the disease (early vs advanced). It is
possible that both decreased and increased levels of these proteins lead to
disturbed cellular functions and affect prognosis.
The results in Paper II suggest that the components of the MRN are
prognostic markers in breast cancer. Both RAD51 and the whole BRCA1/
BRCA2/RAD51 complex seem to be related to local, but not distant recurrencefree survival. Overall, results from different studies on the possible prognostic value of these proteins are not conclusive and further studies are
needed.
Can the expression of DNA repair proteins predict the effect of
radiotherapy?
We hypothesised that reduced expression of DNA repair-associated proteins in tumour cells results in impaired repair of radiation-induced damages. This could lead to better eradication of tumour cells by radiotherapy
and be reflected as improved local tumour control. For example, inhibition of NBS1 or RAD51 has been shown to increase radiosensitivity in vitro
[156, 157, 158, 159, 160].
The MRN complex and ATM
Radiotherapy significantly reduces the risk of local recurrence [29]. This
effect was also seen in Paper II and III, in which radiotherapy resulted in
improved local tumour control as compared to chemotherapy (p=0.04). The
greatest benefit of radiotherapy was seen in patients with moderate/strong
MRN expression, whereas patients with negative/weak MRN complex had
no benefit of radiotherapy over CMF on local tumour control (Fig. 3 and
Table 4, Paper II).
The finding that high expression of these proteins was important for the
clinical effect of radiotherapy was unexpected and differed from our original hypothesis. The MRN complex acts as a sensor of DSBs and recruits
ATM to broken DNA molecules [161]. It is possible that down regulation or
loss of the proteins in the MRN complex impairs the ability to detect DNA
strand breaks and results in failure to induce DNA damage signalling, cell
cycle arrest and apoptosis, thereby increasing the risk of relapse. In sup-
56
Paper II and III
port of this, a role for NBS1 in apoptosis induction following DNA damage
was recently reported [162].
A benefit of radiotherapy was also seen in patients with moderate/strong expression of nuclear ATM (Paper II). We performed analyses
on the combined expression of ATM and the MRN complex, and found
that patients that had moderate/strong expression of MRN responded to
radiotherapy, regardless of ATM expression (Fig. 4a-b, Paper II). Among
patients with negative/weak MRN expression there was no significant difference between radiotherapy and chemotherapy on local recurrence, irrespective of ATM expression (Fig. 4c-d, Paper II). One interpretation is that
the MRN complex is more important than ATM in the clinical response to
radiotherapy. Experimental studies indicate that binding of ATM to DNA
is dependent on MRN, whereas MRN can associate with DNA independently of ATM [161]. In addition, MRN seems to serve as an amplifier of
ATM activity [163].
The BRCA1/BRCA2/RAD51 complex
In Paper III, a good response to radiotherapy was seen in patients whose
tumours had low expression of the BRCA1/BRCA2/RAD51 complex (Fig.
3a). When analysed individually, there was a significant benefit of radiotherapy in patients with low expression of RAD51 or weak expression of
BRCA2 (Fig. 3 and Table 4, Paper III). Similarly, there was a trend towards
a difference in local recurrence-free survival between the treatments among
patients with weak BRCA1. In concordance with our results, high expression of RAD51 has been associated with resistance to treatment in different
tumour types [135, 164, 165].
The results from Paper II and III suggest that reduced expression of the
MRN complex predicts a poor effect of radiotherapy in patients with early
breast cancer. Conversely, patients with low expression of the BRCA1/
BRCA2/RAD51 complex in their tumours benefit from radiotherapy. This
indicates that the proteins in different DSB repair pathways contribute differently to the effect of radiotherapy.
57
R ESULTS & D ISCUSSION
Does a polymorphism in RAD51 influence protein
expression, prognosis and the effect of therapy?
(Paper IV)
The 135G/C single-nucleotide polymorphism (SNP) in the 5’ UTR of the
RAD51 gene has been found to elevate breast cancer risk among BRCA2
carriers [114, 116, 166, 167, 168]. We aimed to investigate whether this
polymorphism is related to RAD51 protein expression, prognosis of early
breast cancer and if it contributes to resistance to therapy. Due to the low
frequency of C/C homozygotes, C/C and G/C were grouped and labelled
’C-allele’ and compared with G/G homozygotes in all analyses.
The frequency of C-allele was 15.4% (including two C/C homozygotes).
Presence of C-allele was correlated to ER positivity (p=0.008), but not to any
other clinicopathological variables, nor to the recurrence-free survival.
The functional effect of this SNP in the untranslated region of the gene is
still unclear. The activity of the RAD51 promotor is significantly enhanced
when G is substituted for a C at this site in vitro [169]. On the other hand,
it has been suggested that the SNP affects the alternative splicing within
the 5’ UTR, and that this would result in a lower abundance of the RAD51
protein, but this has not been confirmed on protein level [167]. Since we
observed reduced expression of RAD51 in a majority of breast tumours,
we aimed to investigate whether the deregulated expression was coupled
to the RAD51 135G/C polymorphism. However, no statistically significant
correlation between genotype and RAD51 protein expression was found
(Paper IV).
In Paper III, patients whose tumours had low expression of RAD51
responded well to radiotherapy, in accordance with previous studies on
RAD51 and in vitro radioresistance [158, 170]. The results from Paper IV
suggest that patients who had the C-allele had no benefit of radiotherapy,
whereas patients with the G/G phenotype had significantly less local recurrences when treated with radiotherapy (Fig. 1, Paper IV). The number
of local recurrences in the C-allele group was, however, small and these results need to be confirmed on a larger group of patients before conclusions
can be drawn.
RAD51 has also been implicated in resistance to different chemotherapeutics, e.g. etoposide, doxorubicin, cisplatin and mitomycin C [135, 165,
58
Paper IV
171]. In Paper IV, CMF significantly reduced the risk of distant recurrence the first 20 years in patients who had the C-allele. This benefit from
chemotherapy was not observed in patients who had the G/G genotype,
indicating that CMF effect was related to genotype (Fig. 2, Paper IV). There
was a significant interaction between chemotherapy and C-allele (p=0.02).
The results suggest that patients harbouring the C-allele respond to
CMF, but not to radiotherapy. Most of the chemotherapeutics that have
been investigated in respect to RAD51 overexpression and therapy resistance, including etoposide, cisplatin and mitomycin C, cause strand breaks,
in some ways similar to ionising radiation. Of the drugs in the CMF regimen investigated in Paper IV, only cyclophosphamide is a crosslinking
agent, whereas methotrexate and 5-FU inhibits RNA and DNA synthesis
and do not induce strand breaks. It is possible that the influence of the
RAD51 polymorphism on therapy resistance is different for drugs and radiotherapy, and also for chemotherapeutics with different mechanisms of
action.
Since there was no relation between RAD51 expression and outcome of
CMF treatment in Paper III, it is possible that the connection between the
135G/C polymorphism and response to CMF treatment is not mediated
by changes in protein levels. The uncertainty revolving around the consequences of this SNP at the protein level makes it difficult to speculate about
the molecular mechanism behind the observed connection. It is however
not implausible that the RAD51 135G/C polymorphism could affect cellular phenotype, considering the modifying effect that this SNP seems to
have on BRCA2 mutation penetrance [114, 116, 166, 167, 168].
59
Conclusions
Growing evidence point towards connections between the PI3-K/AKT pathway, DNA repair and genomic stability. A negative regulatory loop involving AKT and BRCA1 indicates that down regulation or loss of DNA repair
proteins might affect cell survival through additional mechanisms other
than impaired DNA repair. Also, NBS1 has been implicated in both apoptosis stimulation and inhibition. Our studies showed that:
• stimulation of the PI3-K/AKT pathway can prevent induction of apoptosis in breast cancer cells in vitro. Inhibition of this pathway rendered
HER2-overexpressing breast cancer cells more sensitive to radiationinduced apoptosis.
• The expression of the MRE11/RAD50/NBS1 complex and the BRCA1/
BRCA2/RAD51 complex on protein level seemed to be reduced in a
considerable portion of breast tumours.
• The expression of proteins that form a complex together was correlated.
• Reduced expression of the complexes was correlated with clinicopathological variables that are associated with poor prognosis.
• NBS1 alone, but not the MRN complex as a whole, was an independent prognostic factor of local recurrence-free survival. Similarly, the
BRCA1/BRCA2/RAD51 complex and RAD51 had prognostic value
in this patient material.
61
C ONCLUSIONS
• The MRN complex, but not the BRCA1/BRCA2/RAD51 complex,
was a prognostic marker of distant recurrence-free survival.
• Moderate/strong expression of the MRN complex, but reduced expression of the BRCA1/BRCA2/RAD51 complex, predicted good response to radiotherapy.
• The RAD51 135G/C polymorphism may be a predictor of therapy
response. Patients with the G/G genotype tended to respond well to
radiotherapy, whereas patients who had the C-allele benefitted from
CMF chemotherapy.
The results could be of clinical importance, since approximately one in
five breast cancer patients have tumours that overexpress HER2 and this
receptor can be targeted by treatment with Trastuzumab. Furthermore, our
results suggest that the different repair complexes contribute differently to
tumour cell sensitivity to radiotherapy. This is probably a reflection of the
complexity of the DNA damage response networks. It would be important
to keep this complexity in mind when developing individualised cancer
treatments that target proteins in the DNA damage signalling and repair
pathways.
Future perspectives
It would be interesting to study the role of the investigated DNA repairassociated proteins in radioresistance in vitro by using stable transfection
of breast cancer cell lines. Previous studies have primarily used short term
inhibition or overexpression of these proteins in cell culture, but it is possible that cells in clinical tumours are adapted to altered expression and
respond differently. Also, the level of expression of key proteins that is
needed for functional repair has as yet not been determined.
Preclinical studies of PARP inhibitors have shown promising results in
breast cancer patients with mutations in BRCA1 or BRCA2. It would be
interesting to study the effect of PARP inhibitors on cells that lack other
key proteins in double-strand break repair pathways, such as RAD51.
62
Acknowledgements
I would like to express my sincere gratitude to everyone who has helped
and supported me in different ways during these years.
Marie Stenmark Askmalm for all you have taught me and for your great
generosity on many levels. Thanks for your never-ending optimism,
support and belief in me.
Olle Stål for allowing me to do my Master´s thesis and welcoming me in
your group without any prior knowledge of me. Thank you for your
wise guidance during these years, your fairness and for sharing your
enthusiasm for science. You connect the dots in the map of signalling
pathways like no one else.
Bibbi, our very own ‘spider in the web´. Without you the lab would probably look like Ground Zero.
Chatarina Malm You always have time to help with all kinds of things and
you cheer me up with your energy.
Bo Nordenskjöld Thank you for your support in general, and the valuable
feedback on my manuscripts in particular.
My co-authors for critical reading of the article manuscripts.
Jan Rzepecki for fact checking the part about radiotherapy in this thesis,
Peter Larsson, Sara Olsson, Lotta Jonson and Erik Angland for your
63
A CKNOWLEDGEMENTS
help with irradiating cells, Najme Wall and Jan Olofsson for classifying the tumours according to the NHG system, Torsten Hägerström
for collecting the archived tumour material, Lilianne Ferraud and
Birgitta Holmlund for constructing the TMA and purifying DNA,
and Lambert Skoog for sharing your great knowledge about histology and evaluation of IHC stainings.
Anna Asklid for enthusiastically working so hard with the genotyping.
Ben Kersley for the thorough proofreading of my thesis and for curing
my hyphenation disease. Plus, now I know 100% more about ball
bearings than I did before.
Dennis Netzell at LiU-Tryck, for excellent help with the printing of this
thesis. I learned many things about cover design and typesetting
choices from working with you.
The Swedish Cancer Foundation, Landstinget i Östergötland and the Lions foundation for economical support of this work.
My friends and fellow PhD students:
Åsa my friend, room mate and valuable book critic. Thanks for teaching
me (with the help of urbandictionary.com) what a ‘** fo sho´ is (a
very dirty expression indeed) and for filling our office with laughter.
Also, thanks for providing a warm shoulder to cry on when I needed
it the most. I hope our endless discussions on molecular mechanisms
in tumour biology, how to hit the notes on ABBA Singstar and, of
course, family life and childhood diseases, will continue for a long
time.
Marie A W my friend and room mate, with whom I have shared the ups
and downs of PhD studies and research. Thanks for giving me the
extra energy needed for thesis writing with your specially designed
kit. You ought to patent that and make a living on it, I think it would
be a hit. You will always be my ‘seniordoktorand´.
Daniella We shared loss and sorrow not long ago - now it is soon time for
us to share joy and pride together when we have reached our goal.
64
Thank you for being such a good and noble friend, both at work and
in our free time. I will miss you deeply. Give me a call whenever you
need anything from your ‘sugar daddy´. Good luck ‘over there´!
Gizeh my very own guru, who welcomed me in your very generous way
when I came to do my Master´s thesis many years ago. Thanks for
all the things you have taught me in the lab, the inspiring and often
philosophic discussions and all the fun things we have done together
with the children. A special thanks for inviting Emma to your heroin
party (or was it ‘Halloween´? Emma was not sure). I know you will
be successful, whatever you choose to do.
Josefine B We have had so much fun together with our four daughters
(and men). When I have finished this thesis, we can celebrate with
lots of sushi.
Piiha-Lotta for being an inspiring friend and a great travel companion on
the trip to Washington D.C.
Agneta, Tove, Andreas, Annica, Tina, Annelie, Xiao-Feng, Birgitta and
Lilianne for creating a good atmosphere. Thanks for all our enjoyable discussions at fredagsfikat and lunches, the many great social
gatherings and for making Onkologen a great place to belong to.
Present and past co-workers at KEF for nice fika discussions and a friendly
atmosphere.
Pia, Håkan and Kerstin for keeping the wheels rolling at KEF.
Present and past members of Syjuntan (Cissi, Daniella, Josefine A, Marie,
Charlotte, Hanna, Johanna and Anna) for the chats about kids, career,
evolution theories, Eurovision Song Contests, ice hockey and how to
make the embroideries pretty on the back side. I wonder when was
the last time we finished a cross stitch project?
Bokcirkeln (Agneta, Daniella, Gizeh, Josefine, Piiha-Lotta, Tove and Åsa)
for fruitful discussions on relationships, research, how to cast movies
and sometimes even the book we have (tried to) read.
65
A CKNOWLEDGEMENTS
Cissi, Dan, Isabelle, Daniella and Johan for all the board game nights during these years. Tasty food, good wine, three kids who get tired
at three different time-points during the evening (synchronisation?
What does that mean?), challenging boardgames and sweet desserts
on top of all that - could there be a better way to spend a Saturday
night? Nope.
Erik and Maria for being good friends, babysitters and perfect neighbours
in the ‘good old days´. I look forward to next New Year´s Eve!
Kicki, Alex, Bettan, Herman, Carro, Ida, Olle, Anne Marie, Gittan and
Arne for welcoming me into your large and warm family. You have
always room for one more around the table.
Björn and Märit You always offer both delicious dinners and fun discussions when we meet. Thank you for encouraging me by showing
interest in both my work and my free time writing projects.
Mum and dad You have always showed that you believe in me. Without
your support, this thesis would probably not exist. I love you both so
very much.
Mikael for being the best little brother and for all those fun times that we
have shared. You left me with many happy memories. Thanks for
showing me the importance of not letting obstacles get in the way of
making dreams come true. I miss you so much. I think of you every
single day, and smile.
Emma my witty and very wise daughter, and Tilda, my ever smiling ‘penntroll´. You have brought so much laughter and joy into my life.
Ola My love and my best friend. ♥
66
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