Download Prostate cancer and bone cell interactions: implications for

Prostate cancer and bone cell
interactions: implications for
metastatic growth and therapy
Annika Nordstrand
Department of Radiation Sciences, Oncology
Department of Medical Biosciences, Pathology
Umeå University 2017
Responsible publisher under swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
New Series No: 1886
ISBN: 978-91-7601-678-7
ISSN: 0346-6612
Cover illustration: Evelina Wendelsten
Figure design: Annika Nordstrand
E-version avaliable at: http://umu.diva-portal.org/
Printed by: Print & Media
Umeå, Sweden 2017
To my family
Table of Contents
Table of Contents
i
Abstract
iii
Populärvetenskaplig sammanfattning på svenska
v
Abbreviations
vii
List of papers
ix
Introduction
1
Prostate cancer
1
General background
1
The androgen receptor
2
Treatment of prostate cancer
2
Normal bone remodeling
3
Osteoclasts
4
Osteoblasts
6
Bone metastasis
6
Osteotropism
6
Mechanisms behind prostate cancer bone metastases
7
Effects of sex hormones on bone
9
Therapeutic strategies for targeting the tumor-bone microenvironment
The insulin-like growth factor system
10
11
IGF-1R signaling
11
Physiological functions of the IGF axis
11
The IGF axis in prostate cancer
12
IGF-1R as a therapeutic target
13
Aims
15
General aims
15
Specific aims
15
Materials and methods
16
In vitro studies
16
Cell lines (Paper I-III)
16
Co-culture model (paper I-III)
16
Cell viability assay (paper II-III)
17
Flow cytometry (paper II)
17
RNA analyses
17
RNA extraction and cDNA synthesis (paper I-III)
17
Quantitative real-time RT-PCR (paper I-III)
18
Gene expression array analysis (paper IV)
18
Protein analyses
19
Protein extraction (paper II)
19
Western blot (paper II)
19
Phospho-receptor tyrosine kinase array (paper II)
19
i
ELISA (paper I-III)
19
Immunohistochemistry (paper (III-IV)
20
Animals and treatments (Paper III)
20
Patient materials (Paper III-IV)
21
Statistics
21
Univariate statistics (Paper I-IV)
21
Multivariate statistics (Paper III-IV)
21
Functional enrichment analysis
21
Results and discussion
22
Paper I
22
Paper II
24
Paper III
27
Paper IV
29
Conclusions
34
General discussion and future perspectives
35
Limitations of experimental models for prostate cancer bone metastasis
35
Bone-derived factors stimulating and/or protecting metastatic tumor cells
38
The need for predictive biomarkers and individualized treatment plans
41
Acknowledgements
43
References
47
ii
Abstract
The skeleton is the most common site of prostate cancer bone metastasis,
and at present, there are no curable treatments for these patients. To further
understand what stimulates tumor cell growth in the bone
microenvironment and to find suitable therapies, reliable model systems are
needed. For this purpose, we have developed an in vitro co-culture system
that can be used to study interactions between tumor cells and murine
calvarial bones. To validate the model, we measured the release of collagen
fragments and monitored changes in expression levels of genes normally
expressed during active bone remodeling.
One of the major reasons why prostate cancer cells colonize bone is the
abundance of tumor-stimulating factors, such as insulin-like growth factors
(IGFs), present in this milieu. We found that the IGF-1 receptor (IGF-1R)
was one of the most highly activated receptor tyrosine kinases in tumor cell
lines stimulated with bone conditioned media. Since IGF-1 is known to be a
strong survival factor for tumor cells, we hypothesized, that concurrent
inhibition of IGF-1R signaling can enhance the effects of apoptosis-inducing
therapies, such as castration. We used our co-culture model to target human
prostate cancer cell lines, PC-3 and 22Rv1, with simvastatin (an inhibitor of
the mevalonate pathway and an inducer of apoptosis), in combination with
anti-IGF-1R therapy. Tumor cell viability declined with either one of the
therapies used alone, and the effect was even more pronounced with the
combined treatment. The hypothesis was also tested in rats that had been
inoculated with rat prostate cancer cells, Dunning R3327-G, into the tibial
bone, and treated with either anti-IGF-1R therapy, castration, or a
combination of both therapies. Immunohistochemistry was used to evaluate
therapeutic effects on tumor cell proliferation and apoptosis, as well as
tumor cell effects on bone remodeling. The tumor cells were found to induce
an osteoblastic response, both in vivo in rats, and in vitro using the coculture model. Interestingly, the therapeutic response differed depending on
whether tumor cells were located within the bone marrow cavity or if they
had leaked out into the knee joint cavity, highlighting the role of the
microenvironment on metastatic growth and therapeutic response.
Therapies targeting the IGF-1R have been tested in clinical trials,
unfortunately with disappointing results. By immunohistochemical
evaluation of bone metastases from patients with castration-resistant
prostate cancer, we found a large variance in IGF-1R staining within this
group of patients. Hence, we postulate that the effects of anti-IGF-1R
therapies could be more beneficial in patients with high tumoral IGF-1Ractivity than in IGF-1R negative cases. We also believe that side effects, such
iii
as hyperglycemia, associated with anti-IGF-1R therapy, could be reduced if
this treatment is administered only to selected patients and for shorter time
periods.
In a separate study, using whole-genome expression data from bone
metastases obtained from prostate cancer patients, we present evidence that
a high activity of osteoblasts is coupled to a high activity of osteoclast.
Moreover, we found that high bone remodeling activity is inversely related to
tumor cell androgen receptor (AR) activity. The results from this study may
be of importance when selecting therapy for patients with bone metastatic
cancer, especially when bone-targeting therapies are considered, and could
aid in the search for novel therapeutic targets.
In summary, we present an in vitro model for studies of the bidirectional
interplay between prostate cancer cells and the bone microenvironment. We
also demonstrate the importance of IGF-1 in prostate cancer bone
metastases and suggest that inhibition of IGF-1R signaling can be used to
treat prostate cancer as well as to enhance effects of other treatments such as
androgen deprivation therapy. Furthermore, we emphasize the possibility of
molecular tumor characterization when designing treatment plans for
individual patients, thereby maximizing the therapeutic effects.
iv
Populärvetenskaplig sammanfattning på
svenska
I Sverige får varje år cirka 10 000 män diagnosen prostatacancer. Det är
därmed den vanligaste elakartade tumörformen bland svenska män. Om
tumören upptäcks i ett tidigt skede, innan den hunnit sprida sig, är
prognosen god och många patienter kan botas helt. Har tumören däremot
spridit sig (metastaserat) till andra organ, finns det ingen botande
behandling. Prostatacancer sprider sig gärna till skelettet. Detta orsakar
stora problem för patienten, som drabbas av skelettsmärta och löper risk för
frakturer.
Dessa
män
behandlas
vanligtvis
med
så
kallad
kastrationsbehandling, dvs man sänker nivåerna av manligt könshormon.
Behandlingen bromsar tumörtillväxten och lindrar symptomen, men efter en
tid blir tumörcellerna kastrationsresistenta och börjar växa igen. För att
möjliggöra utvecklingen av nya behandlingar behövs en ökad förståelse om
varför dessa tumörceller sprider sig till just skelettet och vad som stimulerar
deras tillväxt.
För att vårt skelett ska behålla sin styrka sker hela tiden en nedbrytning av
gammalt ben följt av uppbyggnad av nytt ben. Denna process kallas
benombildning och utförs av två specifika celltyper; osteoklaster som bryter
ner gammalt ben och osteoblaster som bygger upp nytt ben. När metastaser
bildas i skelettet störs balansen mellan bencellerna. Då andra typer av
cancrar sprider sig till skelettet är det vanligast att det leder till
bennedbrytning, men vid prostatacancer är det vanligast att det leder till en
ökad bildning av ben. Det är sedan tidigare känt att tumörceller och
benceller kan kommunicera med varandra och att detta kan stimulera
tumörernas tillväxt i ben. Målet med denna avhandling var att öka
förståelsen kring hur tumörceller och benceller kommunicerar med
varandra.
I denna avhandling presenterar vi en ny metod, ett modellsystem, för
studier av hur tumörceller och benceller kommunicerar med varandra.
Kommunikation mellan celler sker via frisläppning och upptag av olika
molekyler, som bland annat utgörs av så kallade tillväxtfaktorer.
Tillväxtfaktorer är molekyler som tas upp av celler och påverkar hur snabbt
de delar sig. En av dessa faktorer är proteinet IGF-1. Det är känt att benet i
sig är fullpackat med tillväxtfaktorer, såsom IGF-1. Med hjälp av vårt nya
modellsystem fann vi att tumörceller som kommer i kontakt med ben ökar
frisättning av IGF-1 och att detta i sin tur stimulerar tumörtillväxt. Baserat
på denna upptäckt formulerade vi hypotesen att behandlingar som inducerar
tumörcellsdöd i benmetastaser kan bli mer effektiva om man samtidigt
v
blockerar IGF-1-signalering. Först undersökte vi detta i tumörceller
samodlade med skallben från möss och fann att IGF-1-blockering kunde öka
behandlingseffekten av simvastatin (som i sig själv också kunde inducera
tumörcellsdöd). Därefter testade vi hypotesen i en råttmodell för
prostatacancer som behandlades med kastrering. Resultaten från denna
studie visade också att kombinationsbehandlingen gav en ökad effekt,
däremot såg vi att tumörcellerna svarade olika mycket på behandling
beroende på var i djuret de växte.
Det är ännu inte känt varför benmetastaser vid prostatacancer ger en ökad
benbildning medan andra tumörformer oftast ökar nedbrytningen. För att
försöka förstå detta och öka kunskapen kring vilka faktorer som driver på
benombildning i metastaser studerade vi vävnadsprover tagna från
skelettmetastaser hos prostatacancerpatienter. Vi upptäckte att de
metastaser som hade förhöjd osteoblastaktivitet oftast även hade en förhöjd
osteoklastaktivitet. Det vill säga metastaserna hade antingen en hög eller låg
total bencellsaktivitet.
Prostatacancerceller är starkt påverkade av manligt könshormon som
signalerar genom androgenreceptorn. Även benombildning påverkas av
steroider. Eftersom androgenreceptorn spelar en mycket stor roll i just
prostatacancer, men inte i andra cancerformer, undersökte vi den i relation
till
bencellsaktivitet.
Vi
fann
att
patienter
med
hög
androgenreceptorsaktivitet hade en bencellsaktivitet och vice versa. Denna
upptäckt kan vara av betydelse vid val av behandlingsmetod för dessa
patienter. Dessutom undersökte vi vilka signaleringsvägar som verkar driva
på benremodelleringen med förhoppningen att dessa resultat i slutändan
kan leda till nya behandlingsstrategier.
Sammantaget visar denna avhandling att interaktionen mellan
tumörceller och benceller är av yttersta vikt vid skelettmetastaser. Genom att
blockera faktorer från benet som har förmågan att stimulera tillväxten hos
tumörcellerna kan man direkt bromsa metastasers tillväxt samt öka effekten
av andra behandlingar. Vidare stimuleras bencellers aktivitet av tumörcellers
närvaro och det är även centralt att identifiera de signaleringsvägar som
huvudsakligen driver detta. Denna kunskap kan användas för att urskilja
vilka patienter som har störst chans att svara på en viss behandling samt för
att hitta nya möjliga behandlingsmetoder.
vi
Abbreviations
ACP5
Tartrate-resistant acid phosphatase
Actb
Beta actin
ADT
Androgen deprivation therapy
ALP
Alkaline phosphatase
AR
Androgen receptor
BMD
Bone mineral density
BMP
Bone morphogenetic protein
BGLAP
Osteocalcin
BrdU
Bromodeoxyuridine
CATK
Cathepsin K
CTX-I
C-terminal cross-linking telopeptide of type I collagen
DKK-1
Dickkopf-1
ER
Estrogen receptor
ET-1
Endothelin-1
FGF
Fibroblast growth factor
IGF-1
Insulin-like growth factor-1
IGF-1R
Insulin-like growth factor-1 receptor
IGFBP
Insulin-like growth factor binding protein
INSR
Insulin receptor
M-CSF
Macrophage colony-stimulating
MMP9
Matrix metalloproteinase-9
Oc
Osteocalcin
Opg
Osteoprotegerin
OPLS
Orthogonal projections to latent structures
PCA
Principal component analysis
PDGF
Platelet-derived growth factor
PINP
Procollagen type I N-propeptide
PSA
Prostate-specific antigen
PTH
Parathyroid hormone
PTHrP
Parathyroid hormone-related protein
RANK
Receptor activator of nuclear factor-κB
vii
RANKL
Receptor activator of nuclear factor-κB ligand
RUNX2
Runt-related transcription factor 2
SOST
Sclerostin
SPP1
Osteopontin
SRE
Skeletal-related event
TGFβ
Transforming growth factor-beta
TRAP
Tartrate-resistant acid phosphatase
uPA
Urokinase-type plasminogen activator
Vitamin D3
1,25-dihydroxyvitamin D3
Wnt
Wingless-related integration site
viii
List of papers
This thesis is based on the following papers, referred to in the text by their
roman numerals in bold font:
I.
Nordstrand A, Nilsson J, Tieva Å, Wikström P, Lerner UH,
Widmark A (2009). Establishment and validation of an in vitro
co-culture model to study the interactions between bone and
prostate cancer cells. Clinical and Experimental Metastasis,
26(8):945-53
II.
Nordstrand A, Lundholm M, Larsson A, Lerner UH, Widmark
A, Wikström P (2013) Inhibition of the insulin-like growth factor1 receptor enhances effects of simvastatin on prostate cancer cells
in co-culture with bone. Cancer Microenvironment, 6(3):231-40
III.
Nordstrand A, Halin Bergström S, Thysell E, Bovinder-Ylitalo
E, Lerner UH, Widmark A, Bergh A, Wikström P. Inhibition of the
insulin-like growth factor-1 potentiates acute effects of castration
in a rat model for prostate cancer growth in bone. Submitted
manuscript, under revision
IV.
Nordstrand A, Bovinder-Ylitalo E, Thysell, E, Jernberg E, Sead
Crnalic, Widmark A, Bergh A, Lerner UH, Wikström P. Bone
remodeling in relation to androgen receptor activity in prostate
cancer bone metastases. Manuscript
Paper I was reprinted with permission from Springer. For paper II no
permission was needed.
Other papers by the author not appended in the thesis
Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson
L, Breakefield XO, Widmark A (2009) Prostate cancer-derived
urine exosomes: a novel approach to biomarkers for prostate
cancer. British Journal of Cancer, 100(10):1603-7
ix
Introduction
Prostate cancer
General background
Prostate cancer is one of the most common forms of cancer among
men worldwide [1]. In Sweden, approximately 10 000 men are
diagnosed and about 2 500 men die from their disease each year (The
National Board of Health and Welfare, Sweden). The risk of
developing prostate cancer increases with age, though not all suffer
from their disease. It is in fact not uncommon to, at autopsies, find
previously undetected and asymptomatic tumors in the prostates of
elderly men [2]. Although the death rate in prostate cancer has
declined in most western countries, the incidence rate has increased
over the last two decades [1, 3]. There is a large variation in incidence
rate between different parts of the world, with more than a 25-fold
difference between regions with the highest and lowest incidence [1].
Some of this variation might be explained by differences in screening,
but there are also other factors involved, such as diet, environment
and genetic background [1, 4].
One major reason why the reported prostate cancer incidence has
increased over the last decades is the introduction of the prostatespecific antigen (PSA) test [3]. In healthy men, PSA is produced in the
prostate and secreted in ejaculate to aid in liquefying the semen [5].
When tumors are formed in the prostate they can disrupt the normal
architecture within the prostate, resulting in leakage of PSA out of the
prostate gland [6]. Other conditions, such as benign prostatic
hyperplasia and inflammation can, however, also increase PSA
leakage. Nevertheless, serum levels of PSA is used to assess the risk of
prostate cancer. The use of PSA testing means that prostate cancer can
be detected earlier and in more patients. Unfortunately, it also results
in overdiagnosis and even overtreatment of patients that would most
likely not develop clinical symptoms [7]. It is even debated whether
PSA testing provides any survival benefit [8, 9]. Patients with PSA
levels above 10 ng/ml are considered to have a high risk of prostate
cancer and are subjected to ultrasound guided needle biopsy of the
prostate. If the histological examination show that tumor cells are
growing in the prostate, the tumor is graded according to the Gleason
system, which is used to predict prognosis [10]. After detection, digital
rectal exam and bone scintigraphy are some of the methods used to
1
determine whether the disease is local, locally advanced or advanced.
In local prostate cancer the tumor cells have not spread outside the
prostate gland, whereas in locally advanced disease tumor cells have
spread outside the fibrous capsule covering the prostate gland, though
they have not formed distant metastases. If metastases are found at
distant organs, it is classified as advanced prostate cancer.
The majority of patients with advanced prostate cancer will develop
metastases to the bone [11, 12]. Prostate cancer cells generally
metastasize to sites with a high content of red bone marrow, including
the vertebral column, pelvic bones, ribs, skull and the femurs [13].
Patients often experience severe pain, and pathological fractures and
compressions of the spinal cord are also common [14].
The androgen receptor
The prostate is dependent on androgens for its normal development
and function [15]. Androgen signaling is mediated via the androgen
receptor (AR) which is a ligand-dependent transcription factor. The
main circulating androgen, testosterone, is mainly produced by Leydig
cells in the testis. Testosterone can also be converted into a more
potent androgen, dihydrotestosterone (DHT) [16]. AR activation by
androgens initiates a process where the AR dimerizes and translocates
from the cytoplasm into the nucleus. In the nucleus, receptor dimers
bind to promoter regions of specific target genes, such as KLK2, KLK3
(encoding PSA), NKX3-1, STEAP2, and TMPRSS2, and interact with a
number of coregulatory proteins, including HOXB13 and FOXA1 [17].
Treatment of prostate cancer
Local prostate cancer is treated with curative intentions, either by
surgical prostatectomy or radiation therapy. Low risk patients with a
long life expectancy can also be put under active surveillance, where
treatment is initiated upon signs of tumor progression. Low risk
patients with a short life expectancy may instead be subjected to
watchful waiting, meaning that they will receive no treatment until
symptoms of metastasis appear. In advanced prostate cancer where
metastases have formed, the treatment objective shifts from curative
to palliative. Patients are treated with androgen deprivation therapy
(ADT), either by chemical or surgical castration.
ADT lowers androgen levels, resulting in reduced tumor size, relief
of pain associated to bone metastases, and in most patients, a drop in
PSA levels [18]. Unfortunately, within 1-2 years, most tumors become
castration-resistant and relapse [19]. In castration-resistant prostate
2
cancer, tumors have an activated AR, despite low serum levels of
testosterone [20, 21]. Several mechanisms have been suggested for the
reactivation of the AR, including AR amplification and mutation,
aberrant activities of AR co-regulators, and activation of AR through
other signaling pathways [22]. Activation of the AR can also be a result
of intratumoral androgen synthesis. Studies show that even during
ADT, intratumoral levels of testosterone and DHT are similar to levels
within hormone naïve tumors [23]. In castration-resistant patients
with active AR signaling, palliative treatment is continued, usually
with ADT in combination with an AR antagonist. Moreover, relapsing
patients often receive therapies, such as chemotherapy, radiotherapy,
abiraterone (an inhibitor of androgen synthesis) [24], and
immunotherapy [25], to slow down disease progression and reduce
pain. Therapies specifically aimed at targeting the bone
microenvironment are also commonly used and will be described in
more detail in a later chapter.
Normal bone remodeling
To understand why some tumor cells thrive in bone, it is of essence to
understand the natural composition of the bone microenvironment
and the processes that occur at these sites. The bones of our skeleton
are classified as flat bones and long bones. They are both composed of
different proportions of cortical and trabecular bone. Cortical bone is
dense and form the outer layer of bone that surrounds the bone
marrow. Trabecular bone is a light and porous bone located in the
bone marrow compartment [26, 27]. The skeleton remains it
structural integrity via constant remodeling of the bone. This process
is maintained by the tightly controlled balance between two types of
bone cells, osteoclasts and osteoblasts. Old bone is degraded by boneresorbing osteoclasts, followed by the formation of new bone at these
sites, performed by bone-forming osteoblasts.
Bone is composed of an inorganic matrix, consisting mainly of type
I collagen, and an inorganic component, made up of mineral crystals,
which strengthens the bone matrix [28]. The bone matrix is also rich
in osteoblast-produced growth factors, such as insulin-like growth
factors (IGFs), bone morphogenetic proteins (BMPs), transforming
growth factor-betas (TGFβs), fibroblast growth factors (FGFs), and
platelet-derived growth factors (PDGFs) [27, 29]. These proteins are
3
stored in the bone matrix during the formation of new bone and are
released in the bone marrow cavity during resorption.
The balance between osteoclasts and osteoblasts is easily disrupted
in various pathological conditions. For example, in patients with
rheumatoid arthritis or postmenopausal osteoporosis, osteoclast
activity is increased, resulting in excessive bone resorption [26]. In
cancer, when metastases are formed in bone, the balance is also
shifted. Depending on the origin of the primary tumor the
characteristics of the resulting metastases differ widely. Prostate
cancer bone metastases are primarily viewed as sclerotic, with an
increased bone formation, while bone metastases from breast, lung,
and renal cancers are predominantly lytic, with an increased bone
resorption [28, 30].
Osteoclasts
Osteoclasts are derived from cells in the monocyte-macrophage
hematopoietic lineage. The maturation and activation of osteoclasts is
a complex multistep process starting with the differentiation of these
precursor cells into inactive osteoclasts, that later fuse together to
form multinuclear giant osteoclasts. Two crucial factors in
osteoclastogenesis are macrophage colony-stimulating factor (M-CSF)
and receptor activator of nuclear factor-κB ligand (RANKL). M-CSF is
important in the initial phase of osteoclastogenesis [31], while RANKL
is crucial in most steps of osteoclastogenesis and also in the activation
of mature osteoclasts [32, 33]. RANKL is mainly expressed by bone
stromal cells, including osteoblasts, and binds to its receptor (RANK)
on differentiating and mature osteoclasts [28, 34]. Studies that further
support the importance of RANK/RANKL in osteoclastogenesis show
that RANK and RANKL whole-body knockout mice fail to produce
osteoclasts, resulting in severe osteopetrosis, i.e. abnormally dense
bone [35, 36]. RANKL production in osteoblasts and stromal cells is
stimulated by a number of factors, including prostaglandins,
parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 (vitamin
D3) [28]. The formation of osteoclasts can also be directly stimulated
by cytokines, such as interleukin-1 (IL-1) [37].
Under normal conditions, osteoclastogenesis is controlled by the
balance between RANKL and its decoy receptor, osteoprotegerin
(OPG). OPG is a member of the superfamily of tumor necrosis factor
receptors, and is expressed by osteoblasts [34, 38]. Studies have
shown that knockdown of OPG in mice results in lowered bone
4
mineral density (BMD) [39], while overproduction of OPG leads to an
increased density [40].
To resorb bone, activated multinuclear osteoclasts form a
resorption compartment between itself and the bone surface. The
osteoclasts produce acids that lowers the pH within this sealed
microenvironment, resulting in the release of matrix minerals from
the bone [31, 41]. The organic bone matrix is degraded by osteoclastsecreted enzymes, such as tartrate-resistant acid phosphatase (TRAP),
cathepsin K (CATK), and matrix metalloproteinase-9 (MMP9) [41].
For an overview of the most important factors discussed within this
thesis, see figure 1.
Figure 1 Schematic illustration of key players in normal bone remodeling.
Hematopoietic precursor cells can differentiate into multinucleated bone resorbing
osteoclasts. Mesenchymal stem cells can differentiate into bone forming osteoblasts.
During bone formation some osteoblasts differentiate into osteocytes and become
embedded in bone.
5
Osteoblasts
Apart from regulating osteoclastogenesis, the main function of
osteoblasts is to produce new bone. Osteoblasts arise from
mesenchymal stem cells located in the bone marrow stroma. During
the first phase of osteoblastogenesis, when mesenchymal stem cells
commit to becoming osteoprogenitors, BMPs and members of the Wnt
pathways are important regulators [42, 43]. Another essential factor
for osteoblast differentiation is the runt-related transcription factor 2
(RUNX2). Studies show that Runx2 knockout mice have no mature
osteoblast and therefore lack the ability to form bone, resulting in a
skeleton made entirely of cartilage [44, 45].
The differentiation of these osteoblast progenitor cells into mature
osteoblasts starts off with highly proliferating cells, producing alkaline
phosphatase (ALP), that later mature into non-proliferating
osteoblastic cells. These cells then transform from a flat to a cuboidal
appearance and increase their expression of bone matrix proteins,
both noncollagenous proteins, such as osteocalcin (BGLAP) and
osteopontin (SPP1), and collagenous proteins, such as collagen type I
[27, 42]. These bone matrix proteins form the organic matrix, which is
later mineralized by the osteoblasts [42].
After the bone forming phase, most osteoblasts undergo apoptosis,
whereas the remaining cells either differentiate into inactive lining
cells or non-proliferative, terminally differentiated, osteocytes [46].
Osteocytes are embedded into small spaces in the bone matrix, called
lacunae, they can function as mechanosensors and regulate the
function of both osteoblasts and osteoclasts [47]. Osteocytes are
known to express high levels of dickkopf-1 (DKK-1) and sclerostin
(SOST), two inhibitors of Wnt-signaling, and by doing so they block
Wnt-mediated bone formation [42]. They communicate with each
other and other cells, for instance the bone lining cells, via dendritic
processes that run through small tunnels in the bone, termed
canaliculi.
Bone metastasis
Osteotropism
Both prostate and breast cancers are osteotropic, meaning that they
have a tendency to form metastases in bone. It has been estimated
that together they account for over 80 % of patients with metastatic
bone disease [48]. It is still not completely understood why some
6
cancer types almost always disseminate to bone, while others, such as
tumors of the gastrointestinal tract, rarely metastasize to bone [26,
28]. The most popular theory, the ‘seed and soil’ hypothesis, was
proposed already in 1889, by English surgeon Stephen Paget [49]. He
examined autopsy records from 735 women with breast cancer and
found that there was a non-random distribution of metastases to bone
and other organs. From these findings he postulated that certain
tumor cells, the ‘seeds’, will only colonize organs, the ‘soil’, that
provide a favorable environment for the cells. Paget’s theory has been
challenged by many others throughout the years. In 1928 James
Ewing proposed that the anatomical structure of the vascular and
lymphatic channels determines where metastases will form. He
hypothesized that tumor cells from the primary tumor will follow the
blood flow and lodge in the first organ it comes across [50]. Although
Ewing’s theory prevailed for decades it was later shown, by Weiss and
Sugarbaker, that it could mainly account for regional metastasis [51,
52]. For tumor cells to metastasize to distant organs the ‘seed and soil’
theory still persisted [50]. In osteotropic cancers the bone and bone
marrow serve as a congenial soil for tumor cell growth. Red marrow
has a high blood flow and is the preferred site for bone metastases
[53]. Other than the high vascularity, a number of biochemical factors,
such as cell adhesion molecules, cytokines, and chemokines, as well as
physical factors, including acidic pH, hypoxia, and high levels of
extracellular calcium, also support the colonization, survival and
expansion of tumor cells in bone [54].
Mechanisms behind prostate cancer bone metastases
As previously mentioned, bone metastases in prostate cancer are
generally categorized as sclerotic. In other osteotropic cancers, such as
breast, lung, and renal cancers, metastases have a predominantly
osteolytic phenotype [55]. There is however an overlap in bone cell
activities between these two types of metastases. Some osteolytic
metastases have sites with increased osteoblast activities, and some
osteosclerotic metastases show increased osteoclast activities [28, 56–
58]. In fact, bone resorption markers in serum are often higher in
patients with prostate cancer bone metastases compared to patients
without bone metastases, and also compared to breast cancer patients
with bone metastases [59, 60]. Despite prostate cancer bone
metastases having an increased bone formation, the resulting bone is
weaker than healthy bone, and pathological fractures are common in
these patients [61].
7
Why prostate cancer cells have a preference for the bone milieu is
most likely a combination of several factors. The main focus in this
thesis is the abundancy of tumor cell stimulating growth factors, in
particular IGF-1, in the bone microenvironment. It has been
hypothesized that when prostate cancer cells first metastasize to bone
they induce bone resorption, followed by a release of growth factors
from the bone matrix, which in turn stimulates tumor cells to induce
osteoblast activity [53, 58]. Examples of evidence supporting this
theory includes the finding described above, that patients with
prostate cancer bone metastases have increased levels of markers of
both bone resorption and formation, in their blood [59, 60].
Furthermore, it has been demonstrated in mouse models that a
decreased bone turnover prior to the introduction of tumor cells
significantly delays the formation of metastases in bone [62]. If
RANKL is instead activated during the early stages of osteoblastic
bone metastasis, the resulting increase in bone resorption facilitates
metastatic colonization of prostate cancer cells [63].
A number of tumor-derived factors has been suggested as
particularly important in prostate cancer bone metastasis. For
example, prostate cancer patients with osteoblastic metastases have
elevated serum levels of endothelin-1 (ET-1) [64]. ET-1 stimulates
osteoblast proliferation and thereby promote bone formation [65, 66].
One important function of ET-1 is to suppress DKK-1, a negative
regulator of Wnt [67], thereby increasing Wnt signaling. Furthermore,
overexpression of DKK-1 in prostate cancer cells give rise to osteolytic
metastases [68]. Most prostate cancer bone metastases display
increased levels of Wnt-1 [69]. Moreover, members of the BMP family
are also important for the formation of bone metastases in prostate
cancer. They are expressed in both normal and neoplastic prostate
tissue at various levels [70]. Studies have for instance shown that
prostate cancer cells can promote their osteoblast activity via BMP-6
[71]. Some of the tumor-derived factors found to be associated with
formation of bone metastases may also have a connection to the IGF
family of proteins. Urokinase-type plasminogen activator (uPA) has
been suggested to stimulate proliferation of osteoblasts by hydrolyzing
IGF binding proteins (IGFBPs), and in that way increasing free IGF
levels [30]. Similar effects can be seen with tumor-derived PSA, which
can cleave IGFBP3 [72]. PSA can also cleave parathyroid hormonerelated protein (PTHrP), a promoter of osteoclastogenesis, thereby
decreasing bone resorption [73].
8
Effects of sex hormones on bone
Hormones are known to influence skeletal homeostasis during
adulthood and to affect both the size and shape of bones during
growth [74, 75]. The relatively sclerotic nature of bone metastases in
prostate cancer, compared to other cancers, indicates an association
with the AR. The AR is expressed in osteoblasts, and its expression has
been found to increase as the cells mature into osteocytes [76].
Androgens are known be important for maintaining trabecular bone
mass, and it has been indicated that AR signaling can indirectly inhibit
osteoclastogenesis by downregulating RANKL expression in
osteoblasts [77, 78]. Androgens can also be converted into estrogen
and activate the estrogen receptor (ER) in osteoclasts and progenitor
osteoblasts [74]. This results in decreased bone resorption, in part by
inhibition of RANKL synthesis and stimulation of OPG synthesis in
osteoblasts [42] (Summarized in figure 2). Both AR and ERα are
known to interact with other nuclear transcription factors, such as
RUNX2, to modulate transcription. It has been demonstrated that
when RUNX2 binds to either AR, or ERα, this most often results in
inhibited, but in some cases activated, transcription of target genes
[79–81]. This can affect both bone formation and osteoclastogenesis.
Figure 2 Effects of androgens and estrogens on bone remodeling. Androgens can be
converted into estrogens by the enzyme aromatase.
In men, low androgen levels results in bone loss, and the same
effect can be observed in women with menopause associated estrogen
deficiency [74]. In both cases bone loss is associated with increased
9
bone remodeling activities. Germline deletion of AR in male mice
results in low BMD and high bone turnover [82, 83]. In agreement
with these findings, ADT is known to have a negative impact on the
skeleton in prostate cancer patients, causing reduced BMD and an
increased overall risk of fractures [84, 85].
Therapeutic strategies for targeting the tumor-bone
microenvironment
Both ADT and bone metastases themselves, can induce bone pain and
also increase the risk of skeletal-related events (SREs), such as spinal
cord compressions and pathological fractures [13, 14, 84, 85].
Therefore, therapies aimed to specifically target the bone
microenvironment are used in patients with bone metastases.
Bisphosphonates are a class of drugs that binds to bone surfaces, and
are internalized by osteoclasts during bone resorption, resulting in
osteoclast apoptosis and reduced bone resorption [86]. Despite
prostate cancer bone metastases being predominantly osteoblastic, as
previously mentioned, they often have osteolytic components as well,
motivating the use of bisphosphonate therapy in these patients.
Clinical trials have shown that bisphosphonates, such as zoledronic
acid, reduce bone pain and the number of SREs [86, 87], while no
effect in overall survival has been achieved [88]. The RANKL antibody
denosumab is another inhibitor of osteoclastogenesis and bone
resorption, that delays the onset of SREs [89].
Radioisotopes can also be used to specifically target tumor cells in
bone. Radium-223 is an alpha emitter, that due to its chemical
similarity to calcium, can be incorporated into the bone matrix during
bone formation [90, 91]. Since prostate cancer cells induce bone
formation, the uptake is supposedly higher at metastatic sites [92].
The high-energy alpha particles emitted from bone-incorporated
radium-223 delivers an intense and, given its short range, highly
localized radiation dose to surrounding cells [90]. A large phase III
clinical trial by Parker et al demonstrated that radium-223 treatment
increase the overall survival in castration-resistant prostate cancer
patients with bone metastases [92].
In addition to the therapies described here, there are a number of
novel bone targeting agents currently tested in clinical trials. Some of
these will be discussed in later chapters.
10
The insulin-like growth factor system
IGF-1R signaling
The IGF-1R is a transmembrane receptor tyrosine kinase with a high
sequence homology to the insulin receptor (INSR) [93]. Both IGF-1R
and the INSR are composed of two α-chains and two β-chains. The
extracellular α-subunit binds the ligand while the transmembrane βsubunit contains the tyrosine kinase domain and is responsible for
intracellular signaling [94]. Because of the high sequence and
structure similarities between IGF-1R and INSR they can form hybrid
receptors [93]. The IGF-1R can be activated by three ligands, IGF-1,
IGF-2, and insulin. IGF-1 has the highest affinity for the receptor, and
insulin the lowest [94].
IGF binding proteins (IGFBPs) are a group of proteins that binds to
IGFs and modulates their biological activities. For example, IGFBPs
can regulate the half-life of IGFs, and modify their distribution to
different tissues and their bioavailability for receptor binding [95].
There are six different IGFBPs; IGFBP1-5 have similar affinities to
both IGF-1 and IGF-2, whereas IGFBP6 have a higher affinity for IGF2 [96, 97]. Insulin can bind to all IGFBPs, but with a very low affinity
[96, 98].
Upon ligand binding to the IGF-1R α-subunit, the β-subunit
undergoes a conformational change, followed by autophosphorylation of tyrosine kinase residues in the β-subunit [99, 100].
This initiates a cascade of downstream signaling via the PI3K/AKT
pathway the Grb2/RAS/RAF/MAPK pathway, affecting for example
proliferation, differentiation, and transformation [101, 102].
Physiological functions of the IGF axis
The IGF axis is important in both embryonic development and postnatal growth. Even though IGF-1 is expressed only at low levels in
mouse embryos, the majority of IGF-1 knockout mice die before or
soon after birth [103–105]. In addition, those that are born are
markedly underweight compared to wild-type mice, and exhibit a
delayed ossification and infertility [103–106]. In humans, IGF-1
deficiency results in short stature [107].
Disruption of IGF-1R results in similar, but more pronounced,
embryonic defects as seen in IGF-1 knockout mice, and homozygous
null mutants die at birth due to respiratory failure [105, 108].
Knockout studies have also revealed a role of the IGF axis in the
11
development of cardiac and skeletal muscles [105, 109–111], and the
nervous system [106, 112].
Serum levels of IGF-1 increases slowly from birth, peaks during
puberty, and later declines with age [113]. In adults levels of
circulating IGF-1 is determined by both genetic and lifestyle factors
[114]. IGFs are mainly synthesized in the liver, in response to
somatotropin stimulation [115], but they can also be produced in other
tissues such as bone, brain and muscles [116]. Mice with liver-specific
disruption of Igf-1 has a 75 % reduction in levels of circulating IGF-1
[117, 118]. However, they exhibit no significant reduction in overall
postnatal body growth compared to wild-type mice. Indicating that,
despite the liver being the largest producer of serum IGF-1, the local
production in other organs is more important for growth.
The IGF axis in prostate cancer
Numerous studies have implicated the importance of members of the
IGF axis in the development and progression of various cancers.
Elevated levels of IGF-1 in serum has been linked with an enhanced
lifetime risk of developing prostate cancer [119–123]. However, there
has been reports where no association between serum IGF-1 and risk
for prostate cancer was found [124–126].
Furthermore, it has been suggested that the IGF-1R has an essential
role in transformation. For instance, fibroblasts from IGF-1R
knockout mice, cannot transform in response to viral or cellular
oncogenes unless IGF-1R is reintroduced to the cells [127]. Besides
this, IGF-1R is frequently overexpressed by tumor cells in advanced
and metastatic prostate cancer [128–130], although some reports
show that levels of IGF-1R is unchanged or even lowered [123, 131,
132]. Nonetheless, disruption of IGF-1R reduces tumor cell growth
and survival [133–136].
Under normal conditions, prostate stromal cells secrete factors,
such as IGF-1, that stimulates epithelial and vascular cells within the
prostate [137, 138]. It has been shown in vivo, that castration causes a
rapid decline in IGF-1 signals from the stroma [137]. And in patients
there is a correlation between the reduced IGF-1 levels within the
prostate and increased tumor cell apoptosis [139].
Of great interest in prostate cancer is the suggested connection
between the AR and both IGF-1 and IGF-1R. It has been shown that
the AR can upregulate expression of IGF-1R [140–143], the underlying
mechanisms are however not fully understood [144]. The upregulation
12
of IGF-1R in prostate cancer cells results in increased cell proliferation
and invasiveness in response to IGF-1 stimulation [141].
There is also evidence that IGF-1 can influence AR signaling.
According to some reports IGF-1 can activate the AR in a ligandindependent manner [145, 146], which is of particular interest during
ADT. However, Plymate et al, showed that this may only be valid in
primary tumors, and that in metastatic cell lines IGF-1 suppress AR
activity in response to DHT [140]. Studies also show that IGF-1 may
enhance nuclear translocation of AR in the absence of androgens, and
that this effect can be blocked by inhibiting IGF-1R [144, 147]. This
process might be of importance during prostate cancer progression to
an androgen independent disease.
IGF-1R as a therapeutic target
Several clinical trials studying IGF-1R inhibition in cancer has been
performed, however, many of these trials have reported a poor
effectiveness of the drugs as well as problematic side effects (primarily
hyperglycemia) [101, 116]. There are several different approaches to
target the IGF-1R in patients. Currently two methods have been used
on prostate cancer patients in clinical trials; Tyrosine kinase inhibitors
and neutralizing antibodies. Tyrosine kinase inhibitors prevent
autophosphorylation of the tyrosine kinase domain of the receptor,
and thereby the enzymatic activation of downstream proteins [148].
Since the IGF-1R and INSR are highly homologous, tyrosine kinase
inhibitors generally inhibit both receptors, although their affinities for
the two receptors can vary. The compound used within the work of
this thesis, NVP-AEW541, was developed by Novartis and is a small
molecule inhibitor 27-fold more potent against the IGF-1R compared
to INSR [134]. In tumors that depend on both IGF-1R and INSR
signaling the therapeutic effects would most likely be lower with an
IGF-1R-specific drug. And preclinical evidence show that tumor cells
can deploy INSR signaling to adapt to IGF-1R-specific antibodies [149,
150]. NVP-AEW541 has been used in numerous pre-clinical studies of
cancer, where it has been shown to inhibit tumor cell proliferation and
survival both in vitro and in vivo, in a variety of cancer types [151–
153].
A number of monoclonal antibodies targeting the IGF-1R has been
developed. These antibodies are highly specific for IGF-1R and do not
cross-react with the INSR. They bind to the extracellular domain of
the IGF-1R, thereby blocking ligand binding, hence, preventing the
activation of downstream signaling pathways [102]. Other important
13
effects of monoclonal antibodies is that they can downregulate IGF-1R
over time by inducing internalization and degradation of the receptor
[133, 154], and that they might induce antibody-dependent cellular
cytotoxicity [148, 155]. Antibodies have a longer half-life than small
molecule inhibitors and could result in more severe hyperglycemia
[102]. However, Phase I and II clinical trials demonstrate that most of
these antibodies are well tolerated [148]. Cixutumumab (IMC-A12) is
a human monoclonal antibody that inhibits ligand binding to IGF-1R
without blocking insulin binding to the INSR [133]. Several preclinical
studies have demonstrated the antitumoral effects of cixutumumab on
for example prostate, breast, colon, multiple myeloma and pancreatic
cancer cells [133, 135, 136, 144]. In a xenograft model of human
prostate cancer cixutumumab was shown to reduce growth rates of
both androgen-dependent and androgen-independent cancer cells
[156]. Furthermore, cixutumumab, in combination with castration,
has been shown to prolong the time to androgen independent
recurrence, compared with castration alone. [147] At present, this
antibody is tested in several clinical trials, either in combination with
other therapies or as a single agent.
14
Aims
General aims
Skeletal metastases are common in patients with prostate cancer, and
at present, there are no curable treatments for these patients. There
are, most likely, a number of reasons why prostate cancer cells thrive
in bone. It is known that as tumor cells disseminate to bone they alter
the activities of cells responsible for bone remodeling, and by doing so
they create a more favorable environment for themselves.
The general aim of this thesis was to expand our knowledge about
the bidirectional interplay between tumor cells and bone cells, and
thereby to identify possible therapeutic targets and predictive markers
of therapeutic response.
Specific aims
Paper I
To establish an in vitro model system that enables studies of
interactions between tumor cells and bone cells
Paper II
To confirm stimulating effects of bone-derived IGF-1 on prostate
cancer cells and to examine if IGF-1R inhibition can enhance the
inhibitory effects of simvastatin on prostate cancer cell lines cocultured with bone
Paper III
To study acute tumor-inhibiting effects of IGF-1R inhibition in
combination with castration in a rat model of prostate cancer growth
in bone
Paper IV
To characterize bone remodeling activities, in relation to tumor cell
AR activity, in clinical samples of prostate cancer bone metastases,
and thereby identify therapy-predictive markers and possible
therapeutic targets
15
Materials and methods
For detailed descriptions please see the corresponding papers.
In vitro studies
Cell lines (Paper I-III)
Human prostate cancer cell lines PC-3, LNCaP and 22Rv1 were used
for in vitro experiments. PC-3 is an androgen-independent cell line,
originally derived from a clinical bone metastasis and it is known to
induce an osteolytic response in bone [157, 158]. LNCaP is an
androgen-sensitive cell line, derived from a clinical lymph node
metastasis and it induces a mixed, somewhat osteoblastic, response in
bone [158, 159]. 22Rv1 originates from CWR22 cells (derived from a
primary prostate tumor) that were serially passaged in mice [160].
After castration-induced regression the CWR22 cells relapsed, giving
rise to the 22Rv1 cell line. 22Rv1 cells are androgen-sensitive and
induce a mixed response in bone [161].
The rat prostate cancer cell line Dunning R3327-G was used both in
vitro and inoculated into rats. The original Dunning R3327 cells were
derived from a spontaneous prostate tumor in a Copenhagen rat, and
after serial passages these cells gave rise to a number of sublines [162].
The subline used here, Dunning R3327-G, is an androgen-sensitive,
cell line with low metastatic potential [163].
All cell lines were cultured according to the manufacturer’s
instructions.
Co-culture model (paper I-III)
Tumor cells were seeded in their respective culture medium (for more
details, see paper I-III) in 6-well plates. After 24 hours the medium
was replaced with bone culturing medium, and cells were incubated
for another 24 hours. Thereafter, cells received new bone culture
medium and after 3-4 hours calvarial bones on metal grids were
placed in the wells (see paper I). These grids separate the bones from
the tumor cells, but enables signaling through the medium between
the two compartments. The calvarial bones were dissected from 5- to
7-day-old CsA mice, cut into halves, preincubated with indomethacin
for 24 hours. Indomethacin prevents initial bone resorption induced
by an increased release of prostaglandins as a result of dissection
16
trauma [164]. Calvariae were subsequently washed before they were
introduced to the cell cultures. In the different papers various
stimulating factors or inhibitors were added to the co-cultures to study
their effect on the calvariae and/or the tumor cells (vitamin D3 in
paper I, an IGF-1 neutralizing antibody (R&D Systems) or NVPAEW541 (Novartis Pharmaceuticals) and/or simvastatin (SigmaAldrich) in paper II, and DHT in paper III).
Cell viability assay (paper II-III)
Tumor cell viability was determined by the Cell Proliferation kit I
(Roche Diagnostics) according to the manufacturer’s instructions.
Briefly, cells were seeded in 96-well plates and 24 hours later culture
medium was replaced with phenol-free medium with 2.5 % charcoal
stripped FBS. This medium was supplemented with various
concentrations of NVP-AEW541 and/or simvastatin in paper II, and
rat recombinant IGF-1 (R&D Systems), and/or DHT and/or NVPAEW541 in paper III. Cell viability was measured at time points
between 2-7 days.
Flow cytometry (paper II)
Flow cytometry was used to examine the relative levels of apoptotic
tumor cells from co-cultures supplemented with NVP-AEW541 and/or
simvastatin. Tumor cells from co-cultures were harvested and washed,
and apoptosis was assessed using the FITC Annexin-V Apoptosis
Detection Kit (BD Pharmingen) according to manufacturer’s
instructions. Staining was determined by flow cytometry
(FACSCalibur, BD Biosciences FACS) and analyzed using the
CellQuest software (BD).
RNA analyses
RNA extraction and cDNA synthesis (paper I-III)
Total RNA, from cell lines and calvariae, was extracted using the
RNaqueous kit (Ambion) according to the manufacturer’s
instructions. RNA was DNase-treated with TURBO DNase (Ambion)
and RNA concentrations were determined using a Nanodrop ND 1000
(Nanodrop Technologies). RNA was reverse transcribed using the
High Capacity cDNA Reverse Transcription kit (Applied Biosystems;
paper II) or the 1st Strand cDNA Synthesis kit (Roche) with Oligo(dT)15
primers (paper I, III).
17
Quantitative real-time RT-PCR (paper I-III)
To evaluate bone cell activation in calvariae, mRNA levels of osteoclast
activator Rankl and its decoy receptor Opg, together with osteoclast
markers genes Catk, Mmp9 and Trap, and osteoblast marker genes
Alp and Oc, were measured using quantitative real-time RT-PCR.
Quantifications were performed using the ABI PRISM 7900 HT
Sequence Detection System (Applied Biosystems) in combination with
the TaqMan Universal PCR Master Mix (Applied Biosystems) or the
Power SYBR Green Master Mix (Applied Biosystems). Negative
controls were always run in parallel. Resulting quantification data was
analyzed using the ABI SDS software and the standard curve method
(User Bulletin #2, Applied Biosystems). All samples were normalized
to the expression levels of housekeeping genes (Actb in rodent mRNA
and RPL13 in human mRNA).
Gene expression array analysis (paper IV)
Total RNA was extracted from representative areas of fresh frozen
bone metastases sections using the Trizol (Invitrogen) or the AllPrep
DNA/RNA/Protein Mini Kit (QIAGEN) protocols. Nucleic acids were
quantified by absorbance measurements using a spectrophotometer
(ND-1000 spectrophotometer; NanoDrop Technologies). The RNA
quality was analyzed with the 2100 Bioanalyzer (Agilent Technologies,
Santa Clara, CA) and verified to have an RNA integrity number ≥ 6.
Gene expression array analysis was performed using the human HT12
Illumina Beadchip technique (Illumina, San Diego, CA). Data from
two gene expression studies were combined and samples were
included if they contained a minimum of 10 % bone tissue and a tumor
cell content of at least 30 %. Beadchip data was included for all probes
with average signals above two-times the mean background level in at
least one sample per study array. Arrays were individually normalized
using the quantile method (GenomeStudio V2011.1, Illumina) and
each probe was centered by the median of the samples in the
corresponding dataset. Normalized datasets were merged by mapping
Illumina ID and Hugo gene symbol, leaving 13846 probes for further
analysis.
18
Protein analyses
Protein extraction (paper II)
Proteins were extracted in lysis buffer containing 1 % Igepal CA-630
(Sigma-Aldrich, ST. Louis, MO), 20 mM Tris–HCl (pH 8.0), 137 mM
NaCl, 10 % glycerol, 2 mM EDTA and Complete Protease Inhibitor
(Roche Diagnostics). Lysis buffer for cells to be used in the phosphoRTK array also contained 10 μL/mL Halt™ Phosphatase Inhibitor
Cocktail (Thermo Scientific, Rockford, IL). Samples were mixed and
incubated on ice for 30 minutes. Supernatants were isolated following
centrifugation and protein concentration was determined by the BCA
Protein Assay Reagent kit (Pierce Chemical Co.)
Western blot (paper II)
Proteins were separated by 7.5 % SDS-PAGE under reducing
conditions and subsequently transferred to PVDF membranes.
Membranes were blocked in 5 % milk prior to incubation with primary
antibodies targeting the AR (N20, Santa Cruz Biotechnology), to
detect full length AR and AR variants with an intact N-terminal
domain, or the IGF-1R (AF-305-NA, R&D Systems). Following
incubation with the appropriate secondary antibodies (Dako) protein
expression was visualized using ECL Advanced or ECL Plus detection
kits (GE Healthcare). Membranes were stripped and re-analyzed with
primary antibody against actin (Sigma) and the relative AR and IGF1R levels were adjusted for the corresponding actin levels.
Phospho-receptor tyrosine kinase array (paper II)
Starved tumor cells were stimulated with calvaria-conditioned media
for 10 minutes and then lysed on ice as described above. Phosphoreceptor tyrosine kinase arrays (R&D Systems) were performed
according to the manufacturer’s instructions and receptor tyrosine
kinase phosphorylations were detected using the ECL Plus detection
system. Average pixel density was determined using the Quantity-one
software (Bio-Rad Laboratories) and average pixel density of duplicate
spots was normalized to positive control spots.
ELISA (paper I-III)
For measurements of proteins secreted from murine calvariae or
human cell lines during co-culture, conditioned media from the
cultures were collected. Tumor cell-secretion of IGF-1 (R&D Systems)
was determined using a human-specific ELISA. Bone- and bone cell-
19
secreted IGF-1 (R&D Systems), RANKL (R&D Systems), and OCL
(Demeditec Diagnostics GmbH), was measured using mouse-specific
ELISAs. The release of collagen fragments specific for bone resorption
(C-telopeptide fragments of collagen type I, CTX-I, Immunodiagnostic
Systems) and bone formation (Procollagen type I N-propeptide,
PINP, Immunodiagnostic Systems) were also measured in conditioned
media. All assays were carried out according to the manufacturer’s
instructions.
Immunohistochemistry (paper (III-IV)
Sections containing bone tissue were decalcified in formic acid for 48
hours prior to paraffin embedding. Paraffin sections were stained with
hematoxylin-eosin and van Gieson, for morphological analysis.
Immunostainings were performed using primary antibodies against
bromodeoxyuridine (BrdU, 347580, BD Biosciences), cleaved caspase3 (9661L, Cell signaling), IGF-1R (AF-305-NA, R&D Systems), IGF-1
(05-172, Millipore), AR (pG21, 06-680, Millipore), RUNX2 (ab81357,
Abcam), and TRAP (MABF96, Millipore). Negative control sections
were prepared by performing immunostaining procedures without
adding primary antibodies.
For a more detailed description of histochemical and morphological
evaluations, see paper III and IV.
Animals and treatments (Paper III)
Intra-tibial injections of Dunning R3327-G cells were performed in
anesthetized, adult, male Copenhagen rats (Charles River, bred at our
animal facility). Four weeks later half of the rats were treated per oral
gavage with 80 mg/kg/day of NVP-AEW541 dissolved in 25 mM L(+)tartaric acid (Sigma-Aldrich), and the other half with 25 mM L(+)tartaric acid only. On the second day of treatment, half of the treated
and half of the control rats were castrated and the remaining rats
sham operated. At sacrifice, on the fifth day of treatment, rats were
injected i.p. with bromodeoxyuridine (BrdU 50 mg/kg; SigmaAldrich) and one hour later animals were sacrificed. Tumorcontaining tibias, prostate lobes and lungs were dissected and
weighted before fixed in 4 % paraformaldehyde for 48 hours, and
subsequently embedded in paraffin. Six rats were injected with RPMI
only, and sacrificed 5 weeks later. Animal work was carried out in
accordance with protocol approved by the Umeå Ethical Committee
for Animal Studies (permit number A5-15).
20
Patient materials (Paper III-IV)
Bone metastasis samples were obtained from a series of fresh-frozen
and formalin fixed paraffin embedded biopsies collected from patients
with prostate cancer operated for metastatic spinal cord compression
or pathologic fractures at Umeå University Hospital (2003-2015). The
study was approved by the local ethic review board of Umeå University
and participants gave written or verbal consent (Dnr 03-158, Dnr 0426M).
Statistics
Univariate statistics (Paper I-IV)
Correlations between variables were investigated using Spearman
rank test. Groups were compared using the independent samples ttest, paired samples t-test or Mann-Whitney U test. Kaplan-Meier
survival analysis was performed with death due to prostate cancer as
event and follow-up time as time between metastasis surgery and the
latest follow-up examination. A P-value below or equal to 0.05 was
considered statistically significant. Statistical analyses were performed
using the latest version of SPSS software.
Multivariate statistics (Paper III-IV)
Multivariate modelling by means of principal component analysis
(PCA) and orthogonal projections to latent structures (OPLS), was
used to capture variations in whole-genome expression data. Models
were based on IGF-1R immunoreactivity score in paper III and bone
remodeling activity in paper IV. Multivariate statistical analyses were
performed with SIMCA software version 14.0 (Umetrics AB).
Functional enrichment analysis
Enrichment analysis was performed using the MetaCore software
(GeneGo, Thomson Reuters) in order to identify enriched process
networks in the data. Upstream analysis was used to identify
regulators with a probability to be responsible for the observed
enriched process networks, based on the P-value for a calculated
connectivity ratio between actual and expected interactions with
objects in the data.
21
Results and discussion
Paper I
“Establishment and validation of an in vitro co-culture
model to study the interactions between bone and prostate
cancer cells”
In order to understand the underlying mechanisms of prostate cancer
bone metastases and to find new treatments for these patients we need
reliable model systems, both in vitro and in vivo, where interactions
between tumor cells and the bone microenvironment can be studied.
Here we present a two-compartment co-culture system where tumor
cells are cultured together with calvarial explants from mice. The
tumor cells are separated from the calvaria by a metal grid that
enables signaling between the two compartments via the culture
medium. We validated the model using two human prostate cancer
cell lines; PC-3, which is known to be osteolytic, and LNCaP, that have
a mixed/osteoblastic phenotype [158].
To evaluate the effects on bone remodeling induced by the presence
of tumor cells we used a panel of genes expressed by bone cells [42].
Catk, Mmp9 and Trap were used to detect active osteoclasts, and Alp
and Oc were used to detect active osteoblasts. Osteoclast activator
Rankl, and its decoy receptor Opg, were also included in the panel.
Co-culture of calvariae and LNCaP cells initially induced calvarial
transcription of osteoclast marker genes, which was later followed by a
shift towards a more osteoblastic response, with upregulation of
osteoblast marker genes. This resembles the, above described, tumor
cell-induced bone response observed in patients, where increased
osteolytic and osteoblastic activities can be observed simultaneously,
and will be further discussed in paper IV. It also resembles the
sequence of events seen in normal bone remodeling where resorption,
performed by osteoclasts, causes a release of factors that in turn
initiates bone formation.
PC-3 cells induced calvarial transcription of genes associated with
the activation and function of osteoclasts, and a decreased
transcription of osteoblast associated genes. This gene expression
profile was similar to that induced by vitamin D3, which was used as a
positive control for bone resorption. However, the inhibitory effect on
transcription of osteoblast associated genes was more pronounced
22
with PC-3 cells compared to vitamin D3. To examine the actual
biological resorption of bone the levels of collagen fragment CTX-I,
which is released during degradation of bone matrix, was measured in
the co-cultures. Both PC-3 and vitamin D3 stimulated the release of
CTX-I, while LNCaP had no effect on CTX-I levels. This further
supports the observed gene expression profiles. Interestingly, vitamin
D3 was found to have a stronger effect on CTX-I release, compared to
PC-3, and furthermore, gene expression profiles were initially more
lytic in vitamin D3 stimulated calvariae. We speculate that this could
be due to the direct addition of vitamin D3 when initiating the
experiments, compared to the accumulation of resorption stimulatory
factors in the media when PC-3 cells are used.
Gene expression analysis of the prostate cancer cell lines revealed
that PC-3 cells express high levels of genes associated with bone
resorption, such as DKK-1 and PTHRP. These genes were only
expressed at very low levels in LNCaP cells. Moreover, IGF-1R was
highly expressed in both cell lines, even though PC-3 had nondetectable, and LNCaP only very low levels of its ligand, IGF-1. This
indicates that in order to activate this receptor the tumor cells require
IGF-1 produced by other cells. Most likely, PC-3 cells acquire IGF-1 by
inducing bone resorption, which results in degradation of bone
matrix, and thereby a release of IGF-1 and other growth factors from
the matrix [26, 165, 166]. The LNCaP cells might be able to acquire
IGF-1 in the same way, but since they activate bone formation it is
possible that they can be stimulated by IGF-1 produced by osteoblasts
before it is incorporated into the newly formed bone matrix [26, 167,
168]. IGF-1R signaling is further studied in paper II and III.
One of the main concerns when performing research on bone
metastases is the complexity of the bone microenvironment and how
to reproduce this in vitro. Many studies are based on co-cultures of
tumor cells and one, or several, types of bone cells, including bone
marrow endothelial cells or osteoblasts, cultured for instance as
monolayers, in collagen matrices, or in direct contact with each other
[169–172]. These models have advanced our knowledge of for example
what stimulates tumor cells and bone cells, however, they cannot
provide us with information on how tumor cells and bone cells
interact with each other, and the rest of the bone microenvironment,
at the same time. To overcome this limitation tumor cells can instead
be cultured together with bone tissue explants. This provides a more
natural setting, including the extracellular matrix together with all
bone cell types. In our co-culture model presented in this paper the
23
calvarial bone is separated from the tumor cells by a metal grid. Since
not all factors that can stimulate tumor cells or bone cells are secreted,
but require cell-cell or cell-extracellular matrix interactions, it is
possible that by culturing the tumor cells in direct contact with the
bone even more processes can be activated in tumor cells and bone
cells. Furthermore, the extracellular matrix makes bone remodeling
possible which is a very important process in bone metastases,
facilitating the release of different factors important both for tumor
cells and bone cells.
In conclusion, when performing in vitro studies of bone metastases
it is important to keep in mind the benefits and limitations of the
different model systems when selecting a method suitable for the type
of questions that are asked. In this work we present a co-culture model
that can be used to study tumor cell-mediated activation of bone cells
and bone remodeling, and the effects of the bone microenvironment
on tumor cells. The model is simple to use and the experimental
settings can easily be manipulated. The use of this model in
comparison to other available models will be discussed further in the
general discussion.
Paper II
“Inhibition of the insulin-like growth factor-1 receptor
enhances effects of simvastatin on prostate cancer cells in
co-culture with bone”
Within this paper we found that tumor cells were stimulated by coculture with calvarial explants from mice. To understand which bonederived factors could be responsible for this, we stimulated prostate
cancer cell lines (PC-3 and 22Rv1) with calvaria-conditioned medium
and examined the phosphorylation status of 42 receptor tyrosine
kinases. The IGF-1R was found to be one of the most highly activated
tyrosine kinase receptor in both cell lines. In addition to this, we found
that, during co-culture, both cell lines induced calvarial release of IGF1 into the media. Only very low levels of human IGF-1 from the
prostate cancer cells were found in the co-culture media, and neither
the absence, nor the presence, of calvariae had any effects on these
levels. We also found that by adding a neutralizing antibody targeting
murine IGF-1 to the co-cultures the calvariae-stimulated increase in
PC-3 proliferation could no longer be observed. No significant change
was seen in number of 22Rv1 cells, indicating that there are additional
24
calvaria-derived factors that can affect their growth. Possible
candidates will be further discussed in the general discussion. Taken
together, these results show that tumor cells can induce the release of
bone-derived IGF-1, resulting in IGF-1R activation, and stimulation of
tumor cell growth. Tumor cell-response to ADT is generally less
pronounced in bone metastases compared to primary tumors. One
reason for this might be that the bone milieu is rich in tumor
stimulating growth factors, such as IGF-1. We hypothesize, that by
blocking IGF-1 tumor cell-stimulation the tumors would have a
stronger response to apoptosis-inducing therapies, in this case
simvastatin.
A previous study from our group showed that prostate cancer bone
metastases contain high levels of cholesterol [173]. And it has been
proposed that cholesterol targeting drugs, namely statins, can induce
apoptosis in prostate cancer cells in vitro [174–176]. Here we used
simvastatin in combination with a small molecule IGF-1R kinase
inhibitor, NVP-AEW541. The drugs were tested on tumor cells only,
but also added to the co-cultures. We found that tumor cell numbers
decreased when cells were treated with either drug alone, and that
when the drugs were combined the inhibitory effect was enhanced.
The effects on apoptosis were also more pronounced when a
combination of the drugs was used. The fact that 22Rv1 cells were
unaffected by neutralization of calvaria-derived IGF-1 in the cocultures, but inhibited by NVP-AEW541, could indicate a possible
autocrine stimulation of IGF-1R, or that IGF-1R is transactivated by
other ligands.
Statins are one of the most frequently prescribed drugs worldwide,
mainly used to prevent cardiovascular diseases [177]. Due to the
widespread use of statins, it has been possible to perform large
epidemiological studies, revealing that statins might be associated
with a reduced risk of developing prostate cancer, in particular a more
aggressive prostate cancer [178–180]. The results from these reports
are however controversial, since other studies have shown that there is
no direct association between statin use and risk of developing
prostate cancer [181, 182]. To further elucidate this potential
relationship more studies has to be performed, both to understand the
mechanisms behind the potential anti-cancer properties of statins,
and to learn the optimal timing for administration of the drug, and
what type of statins to use [183]. For the work presented in this paper
it is also interesting to note that statins and IGF-1R inhibitors might,
at least in part, affect the same intracellular pathway, the PI3K-Akt
25
pathway [175]. Statins have also been shown to downregulate
expression of IGF-1R in tumor cells [184, 185]. We found that, in PC-3
cells, both IGF-1R mRNA and protein levels were reduced in response
to simvastatin alone, and in combination with NVP-AEW541. This
effect could not be observed in 22Rv1 cells, where the only significant
change found, was increased IGF-1R mRNA levels after NVP-AEW541
treatment.
It has been indicated that statins can affect expression and
degradation of the AR in some prostate cancer cell lines [186]. We
investigated this in 22Rv1 cells co-cultured with calvariae and treated
with simvastatin. Protein levels of full-length AR were unaffected,
while mRNA and protein levels of the constitutively active AR-V7
splice variant were reduced in response to simvastatin. It has been
reported that expression of ligand-independent AR variants, such as
AR-V7, promotes the development and growth of castration-resistant
prostate cancer [187–190], supporting the idea that statins can reduce
the development of aggressive prostate cancer and suggesting that
statins might be of benefit to patients expressing AR variants. Another
interesting aspect of statin use in prostate cancer patients is the
connection to steroidogenesis. One mechanism by which the AR can
be reactivated, as prostate cancer becomes castration-resistant after
ADT, is de novo intratumoral androgen synthesis [23, 191].
Cholesterol is a precursor in the multi-step pathway of androgen
synthesis [192], and it has been shown that cholesterol levels are
higher in bone with prostate cancer metastases compared to bone
without tumors [173]. Therefore it has been suggested that
administration of cholesterol-lowering drugs, such as statins, may be
able to reduce intratumoral steroidogenesis, and thereby delay the
development of a castration-resistant cancer [193].
In summary, we show that prostate cancer cells induce the release
of growth factors such as IGF-1 from bone, which can in turn stimulate
tumor cell growth. Simvastatin induced apoptosis and reduced
proliferation of prostate cancer cells co-cultured with bone, and by
combining simvastatin with IGF-1R inhibition the therapeutic effects
were further enhanced. Thus, inhibition of the IGF-1R might be a way
to improve effects of therapies that induces apoptosis in prostate
cancer cells in a bone milieu. Simvastatin effects in patients with ARV7 positive metastases deserve further evaluation.
26
Paper III
“Inhibition of the insulin-like growth factor-1 potentiates
acute effects of castration in a rat model for prostate
cancer growth in bone”
The theories proposed in paper II were further tested in paper III,
advancing from an in vitro model to an in vivo rat model. Within this
study the acute effects of NVP-AEW541 in combination with
castration, the conventional treatment for advanced prostate cancer,
were investigated. Treatment response (proliferating and apoptotic
tumor cells) was evaluated 4 days after castration, when the maximum
effects on apoptosis are normally found. The rat prostate cancer cell
line, Dunning R3327-G, was found to be a suitable model since we
demonstrated that these cells were stimulated by IGF-1 and DHT,
responded to NVP-AEW541 therapy, and were found to induce an
osteoblastic response in bone in vitro and an overall increase in bone
remodeling in vivo.
To evaluate the response of these treatments, Dunning R3327-G
cells were injected into the tibia of male rats. Five weeks after
injection, established tumors were found in the bone marrow cavity
and, occasionally, also outside the bone in the knee joint cavity and in
muscles. Interestingly, we found that the effects of castration differed
depending on where the tumor cells were located. Outside the bone,
castration resulted in decreased proliferation and increased apoptosis
in tumor cells. In contrast, proliferation and apoptosis in tumor cells
within the bone marrow cavity was unaffected by castration.
Importantly, NVP-AEW541 was able to reduce proliferation both
alone, and even further, in combination with castration, independent
of tumor site. Apoptosis was however only induced outside tibia in
response to NVP-AEW541. Comparing the basal apoptotic rates, we
found that tumor cells located in the bone marrow had a significantly
higher apoptotic rate compared to tumor cells outside the bone. We
speculate that this might affect the response to treatment, and that it
could be a result of the hypoxic environment in the bone marrow and
its low androgen levels [194, 195]. Recently, Halin et al [194], reported
similar findings using Dunning R3327-G in rats. Castration had no
effect on tumor cells growing in the bone marrow cavity, while tumor
cells growing orthotopically, in the rat ventral prostate, displayed a
decreased proliferation and increased apoptosis in response to
castration. Castration-induced apoptosis has also been observed in an
AR-negative subline of Dunning R3327, AT-1, growing in rat ventral
27
prostate [196]. Thus, it is evident that the tumor microenvironment
plays a large role in response to castration. In the prostate, ADT
affects epithelial-derived tumor cells both directly, via reduced
stimulation of the AR, and indirectly by altering stromal cells,
resulting in for example reduced IGF-1 levels [137, 197]. The reasons
why we observe castration-induced effects in Dunning R3327-G cells
outside the tibial bone, but not within the bone, is not known. It is
possible that the microenvironment of the knee joint cavity and the
skeletal muscles is androgen-sensitive and thereby influence the
tumor cells in a similar way as described in localized prostate cancer.
It could also be caused solely by the direct effect on AR signaling in the
tumor cells. The lack of therapy-induced effects within the bone might
in addition to the reason discussed above be explained by the presence
of tumor-stimulating factors, other than IGF-1. This needs to be
further examined in the Dunning model, and whether or not this is
also true in patients.
The hypothesis investigated in this paper was recently tested in a
phase II clinical study where prostate cancer patients were treated
with a combination of ADT and the IGF-1R targeting cixutumumab
[198]. The treatment was well tolerated, however, the primary
endpoint of the study, a difference in the rate off undetectable PSA
after 28 weeks, was not reached, and therefore the trial was
terminated. In our study we show that tumor cell IGF-1R
immunoreactivity varies between patients, indicating that not all
patients can be expected to respond to this therapy. Therefore, we
believe that there is a need for predictive markers that can identify the
subgroup of patients that are most likely to benefit from inhibition of
IGF-1R signaling. This subject will be further debated in the general
discussion.
Gene expression profiles and measurements of calvaria-released
collagen fragments revealed that Dunning R3327-G cells were able to
induce a sclerotic bone response in vitro. However, in rats, bones with
Dunning R3327-G cells were found to have increased activities of both
osteoclasts and osteoblasts. This resembles the findings we make in
prostate cancer metastases from patients described in paper IV. It is
also in line with other studies, where elevated serum levels of bone
resorption markers can be found in patients with sclerotic bone
metastases [28]. We currently don´t know the reasons to why
resorption is induced by Dunning R3327-G in vivo, but not in vitro.
As discussed above, the use of a rat cell line with a murine calvaria in
vitro may hinder some of the communication between the tumor cells
28
and the bone, but several other explanations might exist for this
finding. There was no increase in bone mass in rats even though
osteoblast activity was highly stimulated. The mechanisms behind this
might be an increased number of osteocytes differentiating from
osteoblasts. Osteocytes produce high levels of RANKL [199], which in
turn activates osteoclasts and consequently increases bone resorption.
These results support the ‘seed-and-soil’ theory previously described,
and results in a release of IGF-1 and other growth promoting factors
yet to be identified from the bone matrix.
In summary, using the Dunning R3327-G model we show that
inhibition of pro-survival IGF-1R signaling has the potential to
enhance the acute therapeutic effects of castration in prostate cancer.
We also reveal that tumor cell response to both castration and antiIGF-1R therapy is greatly dependent on the tumor cell
microenvironment, and tumor-protecting factors present in the bone
needs to be identified. Moreover, we found that Dunning R3327-G
cells induce bone remodeling by activation of both osteoclasts and
osteoblasts in rat tibia, demonstrating the relevance of using this
model for studies of tumor cell and bone cell interactions and also to
investigate the effects of treatments aimed at prostate cancer bone
metastases.
Paper IV
“Bone remodeling in relation to androgen receptor activity
in prostate cancer bone metastases”
The osteoblastic nature of prostate cancer bone metastases in
comparison to other cancers, such as breast, lung and renal cancers,
where bone metastases are generally classified as osteolytic, indicates
an association to the AR. In this study we aimed to characterize the
ongoing bone remodeling activities in prostate cancer bone
metastases, and to investigate how bone remodeling relates to AR
activity.
Using whole-genome expression analysis, together with histological
evaluations, we studied bone remodeling activities in bone metastases
from prostate cancer patients. We defined a set of genes, similar to the
gene profiles used to follow bone remodeling in calvariae in co-culture
(Paper I, II), to represent osteoclasts (ACP5; encoding the TRAP
protein, CTSK, and MMP9), osteoblasts (ALPL, BGLAP, and RUNX2),
and osteocytes (SOST) [42]. The transcript levels of these genes
revealed high osteoblast activity to be coupled to high osteoclast
29
activity. In other words, patients either had high or low over all
activity of bone remodeling. This was further verified using
immunohistochemistry. The fraction of TRAP-positive osteoclasts and
RUNX2-positive osteoblasts lining the bone surface was positively
correlated to the expression of their respective genes. The correlation
was, however, relatively weak, which could be a result of heterogeneity
between samples. Throughout the histological examinations
performed within this work we found a large heterogeneity, both
between different tissue specimens taken from the same metastasis,
but also within samples taken from the same piece of tissue. For
example, areas of high bone remodeling were often found in the same
tissue section as areas with old bone and no apparent bone cell
activity. This is important to keep in mind when comparing the results
from the various analyses performed here. One should also note that
the metastases used in this study are taken from one site, after spinal
cord compression, and may not represent the whole tumor burden.
Measuring markers of bone remodeling in serum may provide a better
picture of bone remodeling activities in patients.
To investigate what distinguishes patients with high bone
remodeling activity from patients with low activity, we compared
RUNX2 and TRAP immunoreactivity to a number of clinical variables.
Results showed that RUNX2 and TRAP immunoreactivity was
associated to patient age at diagnosis, implying that older patients
have more active bone remodeling. It has been shown that androgen
levels decrease with age [200], however, it is not known if this
influences our results since most of the included patients have
received ADT. Out of 35 patients, four had undergone chemotherapy,
and interestingly, we found that these patients had significantly lower
RUNX2 immunoreactivity, while TRAP immunoreactivity was
unaffected. This indicates that chemotherapy might select for
development of osteolytic metastases, and needs to be further
explored, both functionally in animal models, and in a larger cohort of
patients.
Moreover, serum PSA levels at metastatic surgery was found to be
inversely correlated to RUNX2 and TRAP immunoreactivity. To
further investigate this finding, we analyzed the whole-genome
expression data and found that bone remodeling activity was inversely
correlated to genes indicating tumor cell AR activity [201], specifically,
the AR itself, AR co-regulators FOXA1 and HOXB13, and AR regulated
genes KLK2, KLK3, NKX3-1, STEAP2, and TMPRSS2. As previously
described, osteoblasts have both AR and ERα, and activation of these
30
receptors results in increased bone formation, and an indirect
inhibition of osteoclast formation. Estrogens can also inhibit
osteoclastogenesis directly, by binding to the ERα found on
osteoclasts. In addition, androgens can be converted to estrogens and
thereby stimulate the ERα. When levels of these sex hormones drop,
bone remodeling increase, resulting in bone loss. It is known that
increased levels of bone remodeling markers can be found in serum of
patients undergoing ADT, and that a side effect of this treatment is
reduced bone density [85]. Most of the metastases included in our
study were taken from castration-resistant patients, thus they were
likely to have increased bone remodeling activity as a consequence of
ADT. From this, we further hypothesized that some metastases may
have regained AR activity via intratumoral steroidogenesis and then
also have regained the protective effects exerted by androgens on
bone. The net effect of bone remodeling in some metastases included
in the study might then be predominantly osteoblastic metastases, as
expected in prostate cancer patients, and it would be interesting to
compare these findings in relation to scintigraphy examinations as
well as to intratumoral androgen and estrogen levels. Based on our
results, however, this does not seem to the result of an overall high
tumor AR activity, but might as discussed above, still be depending on
direct stimulation of androgens or estrogens on bone cells, something
that could be verified by immunohistochemical evaluation of AR and
ERα in bone cells. Aromatase, the enzyme converting androgens to
estrogens [202], could also be evaluated by immunohistochemical
staining, although expression of CYP19A1, the gene encoding
aromatase, was not detected in our data. Furthermore, we speculate
that other tumor-secreted factors may be responsible for shifting the
balance between bone formation and bone remodeling towards a more
osteoblastic phenotype.
By secreting factors that stimulate osteoblasts or osteoclasts, tumor
cells can initiate a vicious cycle, where tumor cells and bone cells
stimulate each other. Since tumor cell AR activity was not found to be
the driving force behind the increased bone remodeling seen in a
subgroup of metastases, we focused on pathways that were enriched in
these metastases. Ontology analysis revealed that the most enriched
process in metastases with high bone remodeling was associated to
ossification and bone remodeling, which can be expected since the
model is based on a number of bone cell markers. Other highly
enriched processes involved epithelial-to-mesenchymal transition, cell
adhesion, proliferation, and BMP and GDF signaling. Interestingly,
31
inflammation, was also found among the most enriched processes.
Bone traumas, such as fractures, are known to induce inflammation
followed by bone repair [203].
Next, we identified probable upstream regulators that could be
responsible for inducing the increased bone remodeling activities, and
found that BMP signaling is a possible driver. Expression of BMP2,
BMP4, and other factors involved in BMP signaling was highly
enriched in bone metastases with high bone remodeling activity. BMP
signaling involves specific SMADs that translocate to the nucleus to
activate transcription. SMADs can form complexes with RUNX2, and
thereby stimulate the transcription of genes involved in
osteoblastogenesis and bone formation [204]. Other than being
involved in bone formation, BMPs have also been found to be involved
in several developmental processes that in the end have the potential
to stimulate tumor cells. SPP1 was another interesting gene whose
expression was found to be correlated to increased bone remodeling.
BMP2 signaling, via the RUNX2-SMAD complex, can activate
transcription of SPP1. It has been reported that osteopontin, the
protein encoded by SPP1, can stimulate tumor cells proliferation and
invasiveness, and that it can instigate the growth of otherwise
quiescent metastases [205–207]. Whether BMP2, BMP4, and SPP1
are expressed by the tumor cells cannot be determined from gene
expression analyses performed within this work, but will need
immunohistochemical evaluation. It would also be interesting to
evaluate the expression of these factors in relation to the total tumor
burden of the patients.
Revealing the ongoing bone remodeling processes within bone
metastases can be useful when selecting therapy for patients with bone
metastatic cancer. As previously described, therapies targeting the
bone microenvironment have been proven beneficial for patients with
prostate cancer bone metastases. Bisphosphonates inhibits osteoclasts
and successfully reduce the number of SREs. The same effects can be
obtained with the RANKL inhibitor denosumab. In this study,
expression of the gene encoding RANKL was undetectable in the array
data in most metastases and it was therefore excluded from analysis.
RT-PCR and/or immunohistochemical evaluation of RANKL in the
studied metastasis cohort might still be done to evaluate RANKL
expression in relation to the observed bone remodeling activity and if
it could aid in determining a subgroup of patients likely to respond to
denosumab treatment. Another interesting therapy for bone
metastatic cancer is radium-223 which is incorporated into bone in
32
areas with a high bone turnover. It seems plausible that the bone
remodeling activity within metastases may be of importance for
patient response to radium-223 [208].
As indicated in this work, using therapies directed at BMP signaling
may also be a way of targeting bone metastases. Studies that further
signify the use of BMP inhibition show that the natural BMP inhibitor
noggin can inhibit the expansion of PC-3 cells in vitro, and that
inhibition of BMP receptors can inhibit ovarian cancer cell growth in
vivo [209, 210].
In conclusion, we demonstrate that high osteoblast activity is
coupled to high osteoclast activity in prostate cancer bone metastases,
and that high bone remodeling activity is inversely related to tumor
cell AR activity. All of these variables might be of use when selecting
therapies for patients, especially when bone-targeting targeting
therapies are considered. Our results also provide some insight when
searching for possible therapeutic targets in bone metastases. We
found that BMP levels were high in metastases with high bone
remodeling activity, and suggest that its importance in osteoblastic
metastases and its suitability as a therapeutic target should be further
examined.
33
Conclusions

The co-culture system described in this thesis provides an easily
manipulated, simple to use, in vitro model to study interactions
between tumor cells and bone cells

Prostate cancer cells induce the release of IGF-1 from bone,
which can in turn stimulate tumor cell growth

Inhibition of the IGF-1R has the potential to enhance the
therapeutic effects of both castration and simvastatin

The microenvironment is important for tumor cell-response to
both castration and IGF-1R inhibition

Dunning R3327-G cells induce an osteoblastic response mouse
calvariae in vitro and increased bone remodeling in rat tibial
bone

A high osteoblast activity is coupled to a high osteoclast activity
in prostate cancer bone metastases, in the experimental
Dunning R3327-G model and in clinical samples

There is an inverse relation between bone remodeling activity
and tumor cell AR activity in clinical bone metastases that may
be of importance for patient response to conventional therapies
34
General discussion and future
perspectives
At present, there is no cure for patients with prostate cancer bone
metastases. This highlights the importance of expanding our
knowledge of the molecular processes occurring within these
metastases, and thereby finding therapy-predictive markers and novel
potential therapeutic targets. The work presented within this thesis
was in part aimed at finding model systems where tumor cell
interactions with bone cells and the bone itself can be studied. We also
utilized the models presented here, to further investigate the potential
of IGF-1R inhibition in bone metastases as a therapy to be used in
combination with other treatments. Moreover, our characterization of
bone remodeling activities in clinical samples of prostate cancer bone
metastases might be useful when searching for novel therapies and
markers predicting therapy-response to already available treatments.
Limitations of experimental models for prostate
cancer bone metastasis
The metastatic cascade is a complex, multistep process and it would be
ideal to have a pre-clinical model of prostate cancer bone metastasis
where all of these steps occurred spontaneously. In such model
primary tumors would form in the prostate, and after some time
intravasate into the blood and/or lymph vessels, followed by
extravasation from the circulation and formation of distant metastases
with growth starting in close contact with bone marrow.
Unfortunately, there are no such animal models and instead
researchers have to use models that replicate a chosen stage of the
metastatic cascade [211–214]. Dogs develop prostate cancer at a
relatively high frequency and they occasionally develop metastases to
the bone [212]. Their disease is most often hormone-insensitive and
tumors frequently arise in castrated dogs, therefore they might be
more similar to castration-resistant prostate cancer in humans [215].
Performing large studies on dogs would be difficult and very expensive
since it is a disease that affects elderly dogs and only a few percent of
dogs develop spontaneous cancers [212, 216]. Spontaneous bone
metastases can also arise in rats. The Dunning cell line used in our
35
study was originally isolated from a spontaneous tumor in the prostate
of a Copenhagen rat. Multiple sublines, with various phenotypes, that
can form metastases at different sites, have later been developed
[162]. Unfortunately, none of the Dunning sublines are able to
spontaneously metastasize to bone.
A number of animal models can mimic the pathology of human
primary prostate cancer, but there are no animal models that
adequately mimics the formation of bone metastases of human
prostate cancer [213]. Most studies are performed in mice and rats
since they can be genetically modified, are relatively inexpensive, and
share a high organ homology with humans [211–214].
In the work presented within this thesis we used a syngeneic rat
model, meaning that tumor cells are implanted into the same strain of
rat as the tumor cells were originally derived from. With this type of
model, the cancer cells and the host animal are immunologically
compatible and therefore the rats have an intact immune system. This
is a major advantage, since it is known that the immune system plays
an important role in cancer progression [217]. One drawback with
using a model where the tumor cells are not of human origin is that
drugs designed to be directed at a specific target in human tumor cells
may not be as efficient in rodent tumor cells. Using xenograft models,
human cancer cells can be inoculated into rodents, most commonly,
mice [211, 212]. These animals have to be immunocompromised,
otherwise the host’s immune system would reject the tumor cells,
meaning that the effects of immune cells will be lost in these animals.
With this kind of model there is also the uncertainty of whether some
of the signaling between tumor cells and bone cells is lost because of
interspecies signaling incompatibilities [218]. To overcome this
potential problem, xenograft models where either human bone
fragments, or tissue-engineered bone constructs consisting of human
cells and matrix proteins, are implanted subcutaneously into
immunodeficient mice [219–222].
Since spontaneous metastases are uncommon in rodents, tumor
cells have to be injected, either via tail vein, intracardiac, orthotopic,
or, as in our study, intraosseous injections [214]. Direct injections into
bone, in this case tibia, is a reproducible model system. Since the
tumor cells are forced into the bone marrow cavity, this model cannot
be used to study early metastatic events. Instead it represents already
established metastases, and it allows for studies of interactions
between cancer and bone cells and also the bone microenvironment.
The fact that rats have larger bones than mice makes them easier to
36
work with, both regarding the precision work of injecting the cells, and
also the subsequent analyses of the bones. A drawback with rats is
however that the number of transgenic and knockout rat strains is
much lower than with mice. And there are far less analytic reagents for
cancer research available for rats.
Taken together, there is no ideal animal model for studies of
prostate cancer bone metastases. There are, however, a variety of
animal models available that can be used for studies of different stages
of the metastatic cascade. Hence, it is important that researchers are
familiar with the strengths and weaknesses of the specific models to
design appropriate strategies to address their specific questions.
In the co-culture model presented in this paper we used human
prostate cancer cell lines and murine calvariae. The use of different
species within the same model system can be viewed both as an
advantage and a disadvantage. By using different species, the
subsequent analyses can be performed using species-specific primers
for gene expression analysis and species-specific ELISAs to study
factors secreted into the media from the different compartments. In
paper III we used a rat prostate cancer cell line, Dunning R3327-G, for
co-cultures with calvariae. Because of the species similarities between
rat and mouse we could not measure the release of IGF-1 from the
calvaria without detecting rat IGF-1. Thus, in that specific case, using
species that are highly related was unfavorable. A more critical
drawback with the use of two different species is that there might be
lack of species-specific osteotropism. Since not all proteins are
identical amongst all species, it is possible that this could impair some
interspecies communication. Using a mouse model of breast cancer,
Kuperwasser et al [222] demonstrated that orthotopic inoculation of
human breast cancer cell lines into the mammary fat pads of mice can
result in spontaneous metastasis to ectopically implanted human bone
plugs. Metastases were not formed in the skeleton of the mice,
highlighting the species-specific osteotropism. In another, recently
published, study, human breast- and prostate cancer cells were cocultured with human bone tissue collected from patients undergoing
total hip replacement [223]. This is a very promising model, not only
because one single species is used, but also since bones from patients
with, for example, an appropriate age for prostate cancer studies can
be used. Even though a lot can be learned by using calvarial explants
from mice that are only up to one week of age, the use of bones from
elderly men might provide researchers with even more knowledge
37
about the disease. Another interesting feature of this model is the
presence of bone marrow cavities and viable bone marrow since this is
where tumor cells normally form metastases. There is, however, the
difficulty of finding suitable patients and the fact that every patient is
different. Factors such as age, lifestyle, and diseases might therefore
make it difficult to reproduce studies. Using calvarial bones from
inbred mouse strains yields less variation and might be more
appropriate for certain studies.
Another factor that should be considered when studying bone
metastases in vitro is hypoxia. The bone marrow is normally highly
hypoxic [195], and furthermore, hypoxia is known to affect tumor cell
progression [224]. Despite this, most studies using, the above
described, in vitro models are carried out under normoxic conditions
[169–172], with only a few exceptions [223, 225]. It would be fairly
easy and very interesting to test how hypoxia affects both tumor cells
and bone cells in our model system.
In conclusion, there are a number of in vivo and in vitro models
available for studies of tumor cell and bone cell interactions. No model
is ideal and the strengths and weaknesses of the models needs to be
considered when designing specific experiments.
Bone-derived factors stimulating and/or protecting
metastatic tumor cells
The relatively low response to therapies targeting bone metastases
suggests that there are factors present in the bone milieu that
stimulates the tumor cells. In paper II, we found that one of the most
highly activated tyrosine kinase receptor after stimulation with boneconditioned medium, was IGF-1R. We were able to show that prostate
cancer cell line PC-3 was stimulated by IGF-1 from the bone and that
this effect could be diminished by neutralization of IGF-1. Prostate
cancer cell line 22Rv1 was also found to be stimulated by boneconditioned medium. However, neutralization of IGF-1 was not
sufficient to reduce this effect, suggesting that other bone-derived
factors are stimulating the tumor cells.
To discover factors that in addition to IGF-1 may originate from the
bone microenvironment and play a role in protecting and/or
stimulating metastatic tumor cells we will make use of the phosphoarray data presented in paper II, and which we also have available for
prostate cancer cell line LNCaP (unpublished data). In agreement with
38
PC-3 and 22Rv1 cells, LNCaP cells also showed phosphorylation of the
IGF-1R by stimulation of calvaria-conditioned medium (data not
shown), but as can be seen in figure 3 other tyrosine kinase receptors
were highly activated as well.
Control
Stimulated
PC-3
LNCaP
22Rv1
EphA1 1.21
2.74
0.55
EphA2 1.06
3.38
0.54
EphB2 0.77
1.71
2.82
ErbB2 1.56
1.54
2.64
ErbB4 0.30
2.19
2.76
MCSF-R 2.30
0.87
2.06
MuSK 3.75
0.96
1.56
ROR1 1.96
3.08
1.14
Tie-2 0.96
0.92
5.52
50
50
50
Phosphorylation status (% of control)
Figure 3 Relative phosphorylation levels of tyrosine kinase receptors in prostate cancer
cell lines PC-3, LNCaP and 22Rv1, before and after stimulation with calvariaeconditioned medium for 10 minutes. Numbers indicate the ratio between control cells
and stimulated cells.
ErbB2, also known as HER2, was found to be activated by boneconditioned medium in all three cell lines. ErbB2 has no known ligand
and is instead activated via heterodimerization with other receptors of
the ErbB family, for instance EGFR and ErbB4. The EGFR was the
most highly phosphorylated receptor in all three cell lines before
stimulation, and only a small increase was seen after stimulation.
ErbB4 phosphorylation increased in both 22Rv1 and LNCaP cells
upon stimulation. In PC-3 cells, however, phosphorylation was low
prior to stimulation, and it even decreased after. This indicates that, at
least in PC-3 cells, ErbB2 must be activated by other means. One
possibility is crosstalk between ErbB2 and other receptors, such as
EphA2 [226].
39
Interestingly EphA1 and EphA2 were also among the most highly
activated receptors after stimulation with calvaria-conditioned
medium. EphA receptors are activated by membrane-bound ephrinA
ligands, and play an important role in the interplay between
osteoclasts and osteoblasts. However, the tyrosine kinase receptors of
tumor cells in this experiment were not activated by direct cell-cell
contact with bone cells, but by something found in the medium. It is
possible that calvarial bone cells release exosomes with ephrin ligands
into the medium, however, so far only ephrinBs have been found in
exosomes [227]. There is also evidence that ephrinAs can be released
from the cell surface and thereby activate its receptors [228, 229].
MCSF-R was highly activated in PC-3 and 22Rv1 cells. This receptor
is activated by CSF-1 that has been found to be expressed by several
cells in the bone, including osteoblasts. M-CSF is, together with
RANKL, the two major regulators of osteoclastogenesis [41]. In
prostate cancer, increased expression of both MCSF-R and its ligand
in primary prostate cancer is associated with metastatic potential
[230]. Our transcriptomic studies on bone metastases revealed that
expression of CSF1R, the gene encoding this receptor, was highly
associated to bone remodeling activity (supplementary tables, Paper
IV).
MuSK and ROR1 are closely related receptors [231]. MuSK is
activated by LRP4 and ROR1 by WNT5A [232, 233]. ROR1 is
expressed in prostate cancer tissue but not in normal prostate [234],
and its involvement in Wnt signaling makes it an interesting target in
cancer therapy [235].
Tie-2, which was found to be activated in 22Rv1 cells, is normally
expressed by osteoblasts, endothelial cells, and hematopoietic stem
cells. The receptor is activated by Angoipontin-1 (Ang-1), which is
secreted large amounts by osteoblasts. It has been demonstrated that
osteoblasts can regulate the stemness of hematopoietic stem cells by
secreting Ang-1 and thereby activating Tie-2 [236]. Interestingly, Tie-2
overexpression in prostate cancer cells has been found to enhance
their chemoresistance and increase their ability to adhere to stromal
cells such as osteoblasts [237].
One approach to further investigate the potential importance of the
receptors presented in figure 3, could be to neutralize the ligands or to
use inhibitors targeting the specific receptor, as done in paper II with
IGF-1R and its ligand. Using an animal model, for instance the
Dunning rat model presented in paper III, the effects on tumor cells
could be further evaluated.
40
In summary, understanding what bone-derived factors that are able
to stimulate tumor cells might provide more insight into the biology
behind prostate cancer bone metastasis, and possibly provide us with
potential therapeutic targets. However, it need to be kept in mind that
in some signaling pathways, activation is dependent on a direct
contact between tumor cells and bone cells, or tumor cells and the
bone itself.
The need for predictive biomarkers and individualized
treatment plans
The concept of cancer treatment is gradually changing from the broad
use of agents that are systemically toxic, to individualized, targeted
and hopefully more effective therapies. To better guide the therapeutic
decision making, we need suitable predictive markers to find the
subgroups of patients that are most likely to respond to a specific
therapy.
A previous study from our group, where the same metastatic
samples as here (paper III & IV) were analyzed, identified two
subgroups among castration-resistant bone metastases. One subgroup
with high AR and metabolic activities, but low cellular immune
responses, and another subgroup with low AR and metabolic
activities, but high cellular immune responses [201]. In paper III,
when analyzing gene expression in relation to tumor cell IGF-1R
immunoreactivity, we found similar subgroups. Metastases with high
IGF-1R levels showed a high similarity to the previously identified
group with high AR activity. We speculate that the AR-driven
metastases are more likely to respond to the wide range of ARtargeting agents available, and furthermore, that they might benefit
from adding IGF-1R targeting therapies.
The group of non-AR-driven metastases are, unfortunately, not
likely to respond to AR-targeting therapies, and therefore have less
treatment options. It is, however, possible that since they were found
to have a high cellular immune response; including tumor cell
expression of MHC class I expression and metastasis infiltration of
cytotoxic T cells, but also of immunosuppressive macrophages and
myeloderived suppressor cells, they may be susceptible to immunestrengthening therapies. Furthermore, in paper IV, we found that the
non-AR-driven subgroup of patients had increased bone remodeling
activities, signifying the use of therapies targeting the bone
41
microenvironment in these patients. There are assays available for
measuring levels of bone turnover markers, such as ALP, PINP and
OC indicating osteoblastic activity, and CTX-I, TRAP and osteopontin,
indicating osteolytic activity, in patient serum or urine. It is possible
that these could function to mirror the overall ongoing bone
remodeling in a patient and, thus, as predictive markers for response
to bone targeting therapies.
Reviewing the literature on IGF-1R-targeted therapies in prostate
cancer bone metastasis, it becomes very clear that a marker to predict
response to IGF-1R inhibition is urgently needed. The number of preclinical studies presenting evidence of the importance of the IGF axis
in tumor development and progression, led to the initiation of several
phase II and III trials targeting IGF-1R signaling. Most clinical trials
have sadly reported disappointing results [101], and it is of course
possible that the findings from in vivo and in vitro experiments does
not translate to human disease. However, the fact that a subset of
patients show beneficial response to anti-IGF-1R treatments indicates
that we should not discard IGF-1R inhibition as an interesting option
for prostate cancer patients. Instead, we need to find predictive
biomarkers that can identify the subgroup of responding patients. To
the best of our knowledge, no such markers have been used for
selecting therapy or even for retrospectively evaluating response in
prostate cancer patients. However, higher levels of IGF-1 in plasma
prior to treatment has been indicated to predict therapy response in
non-small cell lung cancer and pancreatic adenocarcinoma [238, 239].
Expression of the IGF-1R has also been suggested as a predictive
marker in, for example, non-small cell lung cancer and osteosarcoma
[240, 241].
The shift towards individualized treatment plans has made it more
important than ever to find methods for up-front, prospective
monitoring of biomarkers, and liquid biopsies could be a realistic
substitute to more invasive tissue biopsies. As discussed above, serum
and plasma samples could be useful for monitoring markers of bone
remodeling. Other markers as the membrane-bound IGF-1R will be
better assessed at the RNA level in [100]. Furthermore, exosomes,
thrombocytes, and even free circulating RNA and/or DNA may also
carry tumor-derived markers and important information from the
tumors [242–246].
42
Acknowledgements
Här kommer det äntligen! Mitt tacktal till alla som stöttat och peppat
under åren. Eftersom jag varit sängliggande i feber i flera dagar nu har jag
kommit på en väldigt lång lista på människor att tacka.
Först och främst vill jag tacka min fenomenala huvudhandledare
Pernilla Wikström som tog sig an mig när Jonas bestämde sig för att
fly landet för en herrans massa år sen. Du är inte bara en fantastisk
forskare, mentor och förebild, utan även en väldigt omtänksam person
som jag lärt mej så otroligt mycket av under denna tid. Att vi dessutom
numera jobbar i skift gör oss till ett grymt effektivt lag!
Min biträdande handledare Anders Widmark, ”if you’ve got the time,
I’ve got the money, honey!” och precis så blev det, en väldans massa tid
tog det och jag har säkerligen kostat en del också! Tack att du alltid är så
positiv!
Min andra biträdande handledare Jonas Nilsson, som kanske gjorde sin
största insats inom det här projektet när jag började på labbet för 10 år(!)
sedan. Tack för allt du lärt mig! Du är en idéspruta utan dess like. Tack
också för allt lunchsällskap (även om jag ibland surnat till när du ”ska
bara”….Alfons!).
Anders Bergh och Ulf Lerner, er tankar kring mina projekt är alltid
lika värdefulla och man skulle kunna säga att jag ser er som mina två
inofficiella biträdande handledare! Anders din fantastiska förmåga att ta
ett, eller flera, steg bakåt och se helheten är något som vi alla borde bli
bättre på! Ulf, jag vet ingen annan som med sådan inlevelse kan berätta
om vad som helst som är benrelaterat (eller fågelrelaterat för den delen)
som du. Du är en sann inspiration!
Extra stort tack till mitt mest frekventa fikasällskap, Terese, Ulrika,
Kristina, Emma, Gunnar, Linda, Anna D, Sophia, Micke K,
Yvonne och Annika. Det är alltid skönt att sitta ner och prata om allt
från hönsfarmar och norrsken till hockeysöner och utförsåkning. Extra
tack till Terese som alltid varit min tepaus-kompanjon…förlåt för att jag
övergick to the dark side. Ulrika, tack för alla hejarop! Yvonne, för att
du fightas för mina rättigheter.
Håkan, för att du hela tiden uppmuntrar oss att bredda vår kunskap
inom allt som har med cancer att göra! Camilla, tack för all pepp och alla
samtal om allt möjligt här i livet! Samuel, en större nörd får man leta
efter. Tack för allt onödigt vetande du lär oss! Mikael J, min partner in
crime när det kommer till att sätta ihop fula bildspel till disputationsspex.
Lotta, du ska bara veta hur många av dina middagstips jag faktiskt
43
använt! Daniel tack för att du fyllde mitt skrivrum med så mycket lådor
att ingen annan fick plats att sitta där! Lee-Ann I’m sorry for abandoning
you at the office like that, several years together, and then I suddenly leave
without a warning! Thanks for all the nice long chats. Tack också till
övriga på labbet eller med anknytning till labbet som hjälper till att göra
det till den fina arbetsplats det är: Mahmood, David, Britta, Henrik,
Martin, Markus, Simona, Carl, Charlotte, Anna C, Agneta,
Parviz, Kjell, Roger och Beatrice och alla andra som kommit och gått.
Maria, Pia, Karin, Clas, Terry, Carina, Åsa, Monica. Ni är många
som på olika sätt hjälp till med det administrativa. Stort tack till er alla!
Mina doktorandkollegor i Pernillas grupp: Erik, Erik och Helena Ni är
så sköna människor alla tre och lycka till med resten av er tid som
doktorander! Emma tack för att du skrev en så bra bok som jag har
kunnat ta lärdom av nu när jag själv skrivit.
Tack också till övriga medlemmar i prostatagruppen. Sofia, att få hänga
med dig och gnagarna på djurhuset är alltid ett sant nöje! Eller…kanske
inte den där gången vi skulle ta hand om galna kamikaze-hopp-möss.. det
var lite obehagligt.. Men alla andra gånger! Tack för allt du lärt mig! Elin,
vad skulle vi göra utan dig?! Tack för allt arbete du bidragit med och för
att du är en så härlig tjej! Pernilla A, du är grym! Du fick slita hårt där
ett tag med alla mina färgningar och jag hoppas att du förstår hur mycket
vi uppskattar ditt arbete! Marie tack för det labbarbete du bidragit med
och för att det alltid är lika trevligt att umgås med dig! Siggan, den
skickligaste jag träffat när det kommer till arbete med djur! Tur du lärt
Sofia så mycket att hon kan ta över efter dig. Du är alltid så härligt positiv!
Maria B, när det gäller allt som har med laborativt arbete att göra kan du
vara den mest kunniga person jag någonsin träffat på! Stina, tack för att
du alltid är så hjälpsam! Tack också för att du alltid varit ett så trevligt
resesällskap på diverse konferensresor. Tack också till Mona, Hani,
Christina, Kerstin, Maria N, Birgitta, Elisabeth, Susanne, Peter
samt Maréne och hela din grupp.
Personalen på Ulfs lab, framförallt Anita, Inger och Ingrid för allt ni är
ett så trevligt gäng och för att ni alltid tagit er tid att svara på mina frågor.
Tack också till personalen på djurhuset som gjort sitt bästa för att försöka
könsbestämma en vecka gamla möss. Jag vet att det inte var enkelt!
Alla ni som under dessa år på olika sätt förgyllt livet utanför jobbet!
Tack till David & Britta, Linda, Mattias & Sofia, Henke & Vickan,
Åsa & Frasse, Kräftskivegänget, Knatti, Benny & Carina, som på
olika sätt underhållit med middagar, festligheter, lunchsällskap,
golfrundor, hemmagjorda musikquiz, allsång långt in på småtimmarna
44
och ett stort antal afterworks på Rött. Johan & Leonora, it is always fun
to see you and your wonderful girls! Please come here more often now
that you have the house.
Ett extra stort tack till:
Den så kallade ”Umeåfamiljen”: Annasara, Andreas, John, Marie,
Reine¸ Sofia. Ni är anledningen till att jag gick från att känna att flytten
till Umeå var ett av de dummaste beslut jag tagit till att det var ett riktigt
bra beslut. Vi har hunnit med så många roliga upptåg genom åren och
våra mysiga pysselkvällar är bland det bästa jag vet!
Nitte & Maria, sättet att umgås må ha gått från sena kvällar och
Stoneloadspelningar, till skidresor och grillutflykter, men vad vi än hittar
på är det alltid grymt! Och ett extra jättetack Nitte för att du förstår
vikten i att ta en paus och bara prata strunt! Och för alla gånger du råkat
vara duktig och tagit med en matlåda, men valt att dissa den för att det är
tråkigt att äta i en låda. Ante, tack för alla luxuösa middagar,
kemilektioner på Bishops, och inte minst den fantastiska ”one time”drinken! Det känns lite tomt i vår soffa sen du slutade sitta där var och
varannan helg! Soma, tack för alla år som roomiesar, alla mysiga
fikadejter och för att du underhåller mej med roliga historier även när vi
inte ses. Erik & Hanna, vare sig det är middagshäng eller matchhäng är
det alltid lika trevligt! Utom när stämningen blir lite bitter vid förluster
då.. Snart kanske vi kan återuppta golfen, hoppas på färre smileysar i
scorekorten bara. Cissi & Björn, fortsätt alltid vara de sköna och roliga
människor ni är! Och sluta aldrig lyssna på bra musik  Lina, varför var
du tvungen att flytta till världens ände!? Jag är så glad att du hamnade på
labbet, för alla struntpratsträffar i SELDI-rummet, festivaliga resor, tvspelshäng och allt annat kul vi hunnit med! Johanna & Tommy, ni är de
bästa grannar (med ett antal hus mellan) man kan tänka sig!! Tack för allt
häng, vare sig det är några timmars kaffepaus med gofika eller getingsafari
i Lycksele! Det känns himla bra att ha er som stöd när tjejerna blir stora
och flyttar till ”Bryssele”.
Jag är så glad att Calle kom med en så fin släkt på köpet! Tack för all tid vi
får spendera med er, alla middagar, semesterresor, och alla högtider och
kalas vi får fira med er! Mina fantastiska svärföräldrar Marre och
Göran! Vad skulle vi göra utan er?! Tack för att ni alltid finns där för oss
och för att ni drar ihop familjen så att vi alla får träffas så pass ofta som vi
gör! Carin, älskade Carin! Tack för alla kvällar du och jag planlöst
vandrat runt på Berghem, det är som balsam för själen! Team win for life
 Och tur att du skaffat dig en så toppenbra familj!!! Erik, nu när jag har
slutat upp med att vara gravid/ha en pytteliten bebis/skriva avhandling så
är jag äntligen redo för den där Liverpool resan! Wille och Isac,
bröderna som lärt mej mer om pokemons än jag någonsin trodde att jag
45
skulle kunna. Tack för att ni är sånna fina grabbar och att ni tar hand om
era småkusiner på ett sånt fint sätt!
Emelie, väääärldens bästa syster, och hon är min! Trots att du är några
timmar bort så muntrar du upp min vardag nästan varje dag. Och Emil
du är grym du med! Tack för allt du lärt oss om viktiga saker som ellära
och hur man ska fota småfiskar för att få dem att se större ut  Finaste
Isabelle, moster Annos lilla älskling som snart ska bli stortjej, hur galet
är inte det?! Önskar jag fick krama på dig varje dag! Mormor Starka,
fina, underbara mormor! Det är synd att avståndet är så långt, annars
skulle vi komma och fika hos gammelmormor ”gung-gung” mest hela
tiden! Mamma och Pappa, för att ni alltid låtit mig gå min egen väg och
alltid stöttat vad jag än gjort! Ni är helt enkelt BÄST!
Mina älskade flickor, Olivia och Thilda! Underbara ungar! Ni är
anledningen till att jag gick in i ”maximum efficiency”-läge under dagarna
så att jag kunde åka hem och krama på er på kvällarna! Men det allra
största tacket måste ändå gå till dig Calle! Du har som vanligt varit mitt
allra största stöd! Du har peppat mig, påmint mej om ”tänk Niva!”, gått på
rekordsnabba lunchdejter, tagit hand om allt som har med barnens
överlevnad att göra, läst igenom en stor del av det jag skrivit och kommit
med bästa kritiken. Jag vet att det varit en tuff period, men du kämpade
dig igenom den! Jag älskar dig!
46
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