angiogenesis regulation in hormone dependent breast- and ...382707/fulltext01.pdf · abstract a...
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Linköping University Medical Dissertations
No. 1216
Angiogenesis regulation in hormone dependent
breast- and ovarian cancer
Christina Bendrik
Division of Oncology Department of Clinical and Experimental Medicine
Faculty of Health Sciences, SE-581 85, Linköping, Sweden
Linköping 2011
© Christina Bendrik, 2011
ISBN 978-91-7393-280-6
ISSN 0345-0082
Cover: H&E immunohistochemical staining of MCF-7 xenograft section.
Published articles and figures have been reprinted with the permission of the respective
copyright holder:
Paper I: Elsevier Ltd.
Paper II: The American Association of Cancer Research (AACR) Inc.
Paper IV: The American Association of Immunologists, Inc., Copyright 2009.
Printed in Sweden by LiU-tryck, Linköping 2011
Till Tor och Aris
SUPERVISOR Charlotta Dabrosin, MD, PhD, Professor
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University, Linköping
OPPONENT
Malin Sund, MD, PhD, Associate Professor
Department of Surgical and Perioperative Sciences
Faculty of Medicine
Umeå University, Umeå
COMMITTEE BOARD
Mårten Fernö, PhD, Professor
Department of Clinical Sciences
Faculty of Medicine
Lund University, Lund
Agneta Jansson, PhD, Associate Professor
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University, Linköping
Ingemar Rundquist, PhD, Professor
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University, Linköping
Abstract
Angiogenesis is a key event in tumor progression and a rate-limiting step in the establishment
of a clinical cancer disease. The net balance of pro- and anti-angiogenesis mediators in the
tissue dictates the angiogenic phenotype of a tumor. Matrix metalloproteinases (MMPs) are
major regulators of extracellular matrix turnover and have for long been associated with pro-
tumorigenic activities due to their tissue degradation capacities. However, broad-spectra
MMP inhibitors as anti-tumor therapy in clinical trials have failed, and it has now become
evident that several MMPs may induce biological activities beneficial to the host, such as
suppressed angiogenesis. In this thesis the protective role of specific MMPs in breast and
ovarian tumor tissues was further demonstrated.
The process of angiogenesis is essential for physiological functions in the female reproductive
tract, where sex steroids regulate new blood vessel formation and regression in each cycle.
Despite progress made during the past years, our knowledge in sex steroid regulation of
angiogenesis in hormone-dependent tumor tissues remains limited. Tamoxifen is a
cornerstone in the treatment of estrogen receptor (ER)-positive breast cancer. The therapeutic
value of tamoxifen in the treatment of ER-positive ovarian cancer is to date less investigated.
The results presented in this thesis suggest that tamoxifen may induce anti-tumorigenic
responses in ER-positive ovarian cancer by means of both anti-proliferative and anti-
angiogenic mechanisms. In experimental models of human ovarian cancer in vitro and in vivo,
tamoxifen treatment increased extracellular levels of MMP-9 and enhanced generation of the
angiogenesis inhibitor endostatin which resulted in significantly decreased angiogenesis and
tumor growth. Low levels of MMP-9 and endostatin in ascites collected from ovarian cancer
patients suggest a possibility to therapeutically enhance MMP-9 by administration of
tamoxifen, and thereby counteract angiogenesis in ovarian tumors by increased generation of
anti-angiogenesis fragments, such as endostatin.
The significance of enhanced MMP activities in tumor tissues was further investigated by
experimental models of intratumoral MMP gene transfer to human breast tumor xenografts,
which were assessed by using microdialysis. Treatment of tumors with MMP-9 or MMP-3
resulted in dose-dependent inhibition of tumor growth. Low dose of either MMP induced
tumor stasis whereas a higher dose induced significant tumor regression. MMP-9 and
tamoxifen exerted synergistic therapeutic effects on breast tumor angiogenesis and growth
whereas gene transfer of the MMP-inhibitor TIMP-1 counteracted the beneficial effects
induced by tamoxifen.
Further on, we confirm the pro-angiogenic potential of estradiol by demonstrating a
significant correlation between local levels of estradiol and the pro-angiogenic cytokine IL-8
in normal human breast tissues and in ER/PgR-positive breast cancers of women. Estradiol-
induced IL-8 secretion was additionally confirmed in normal human whole breast biopsies in
culture and in experimental human breast cancer in vitro and in vivo.
In conclusion, the results of this thesis may hopefully increase the overall understanding of
several mechanisms involved in angiogenesis regulation and may additionally be useful in the
development of novel approaches for targeted therapy in the treatment of hormone-sensitive
breast- and ovarian cancer.
“εις υγείαν”
Table of Contents
POPULÄRVETENSKAPLIG SAMMANFATTNING...................................................................... 9
LIST OF PAPERS ............................................................................................................................... 11
ABBREVIATIONS ............................................................................................................................. 13
BACKGROUND .................................................................................................................................. 15
CARCINOGENESIS AND TUMOR PROGRESSION .................................................................................. 15
HORMONES AND CANCER .................................................................................................................. 15
BREAST AND OVARIAN CANCER ........................................................................................................ 16
Epidemiology ................................................................................................................................ 16
Breast cancer ........................................................................................................................................... 16
Ovarian cancer ........................................................................................................................................ 17
Breast and ovarian anatomy and sites of cancer origin ............................................................... 18
ESTROGENS ....................................................................................................................................... 20
Synthesis ....................................................................................................................................... 20
Estrogen receptors ........................................................................................................................ 21
Tamoxifen and its mechanisms of action ...................................................................................... 22
ANGIOGENESIS AND THE TUMOR MICROENVIRONMENT ................................................................... 24
Tumor microenvironment ............................................................................................................. 24
Tumor angiogenesis ...................................................................................................................... 24
Regulators of angiogenesis ........................................................................................................... 25
Activators of angiogenesis ...................................................................................................................... 26
Interleukin-8 ....................................................................................................................................... 27
Endogenous inhibitors of angiogenesis................................................................................................... 28
Endostatin .......................................................................................................................................... 28
Matrix metalloproteinases ............................................................................................................ 30
MMP inhibitors in clinical trials ............................................................................................................. 32
Individual MMPs in cancer ..................................................................................................................... 32
Tissue inhibitors of metalloproteinases ................................................................................................... 34
AIMS OF THE THESIS ..................................................................................................................... 35
COMMENTS ON MATERIALS AND METHODS ........................................................................ 37
EXPERIMENTAL MODELS ................................................................................................................... 37
Cancer cell lines (I – IV) .............................................................................................................. 37
HUVECs (IV) ................................................................................................................................ 38
Normal breast tissue in culture (IV) ............................................................................................. 39
Hormonal treatments of cells/tissues in culture (I, IV) ................................................................. 39
Study populations (I, IV) ............................................................................................................... 41
Paper I ..................................................................................................................................................... 41
Paper IV .................................................................................................................................................. 42
Animal models (I – IV) .................................................................................................................. 44
Assessment of tumor growth (I – IV) ............................................................................................ 45
The microdialysis technique (II, III, IV) ....................................................................................... 45
Direct quantification of tumor MMP-9 activity in vivo (II) .......................................................... 48
Gene transfer studies (II, III) ........................................................................................................ 49
Cell viability assay (I, IV) ............................................................................................................. 51
Quantification of proteins and sex steroids (I – IV) ..................................................................... 52
Immunohistochemistry (I – IV) ..................................................................................................... 53
Assessment of angiogenesis .......................................................................................................... 54
REVIEW OF THE STUDY ................................................................................................................ 55
PAPER I .............................................................................................................................................. 55
Tam enhanced MMP-9 activity and generation of endostatin in ER-positive ovarian cancer ........... 55
PAPER II-III ....................................................................................................................................... 57
AdMMP-9 affected tumor levels of both pro- and anti-angiogenesis mediators with decreased tumor
angiogenesis as a net result ................................................................................................................ 58
Gene transfer of TIMP-1 counteracted Tam-induced tumor regression ............................................. 59
AdMMP-9 and AdMMP-3 induce breast cancer regression in a dose-dependent manner................. 60
PAPER IV ........................................................................................................................................... 61
E2 regulates extracellular IL-8 levels in normal and malignant breast tissue...................................... 62
E2-induced IL-8 secretion increases HUVEC proliferation and tumor angiogenesis ......................... 62
CONCLUSIONS .................................................................................................................................. 65
CONCLUDING REMARKS .............................................................................................................. 67
ACKNOWLEDGEMENTS ................................................................................................................ 71
REFERENCES .................................................................................................................................... 73
9
Populärvetenskaplig sammanfattning
Bröst- och äggstockscancer hör till de s.k. hormonberoende tumörtyperna där kroppsegna
(endogena) eller utifrån administrerade (exogena) hormoner anses vara en bidragande orsak
till uppkomst av sjukdom och fortsatt tumörtillväxt. En av tio kvinnor i Sverige löper risk att
drabbas av bröstcancer under sin livstid. Äggstockscancer är den gynekologiska cancerformen
med högst dödlighet beroende på att sjukdomen oftast upptäcks i sent skede. Den anti-
östrogena behandlingen tamoxifen är en av de mest använda medicinska behandlingarna mot
bröstcancer. Trots likheter i uttryck av hormonreceptorer vid bröst- och äggstockcancer är
anti-östrogen terapi inte utvärderat som ett primärt behandlingsalternativ vid äggstockscancer.
Nybildning av blodkärl till tumören, angiogenes, är en förutsättning för tumörtillväxt och för
spridning av cancerceller till andra organ. Angiogenesprocessen styrs av balansen mellan
stimulerare och hämmare i vävnaden men hur denna balans är reglerad i bröst och
äggstockstumörer är till stora delar okänt. Enzymaktivitet till exempel i form av matrix
metalloproteinaser (MMP) i tumören är en viktig faktor som kan påverka tumörtillväxt både i
positiv och negativ riktning genom att frisätta angiogenes-stimulerare och/eller inhibitorer
från vävnaden som omger cancercellerna, mikromiljön. Mikrodialys, en av flera tekniker som
använts i denna avhandling, är en minimalt invasiv metod som ger möjlighet att studera
mikromiljön i levande vävnad. Flera av de proteiner som är viktiga i regleringen av
angiogenes kan med hjälp av denna teknik hämtas ut från mikromiljön och på ett unikt sätt
studeras.
Syftet med denna avhandling var att få djupare insikt i angiogenesreglering i hormonberoende
bröst- och äggstockscancer.
I avhandlingens första delarbete undersöktes effekterna av östrogen och tamoxifen på MMP-
aktivitet i experimentell äggstockscancer. Vi visar att östrogen sänkte och tamoxifen höjde
enzymaktiviteten av MMP-9 i hormonberoende äggstockscancer. Tamoxifen-behandlingen
resulterade även i ökad frisättning av endostatin (en potent hämmare av angiogenes), minskad
angiogenes och hämmad tumörtillväxt. Analyser av bukvätska från äggstockscancerpatienter
visade låga nivåer av MMP-9 och endostatin. En ökning av dessa proteiner, och därmed en
minskad tumörtillväxt, med tamoxifenbehandling till dessa patienter skulle därför kunna vara
möjlig.
I delarbete två och tre undersöktes effekterna av ökad MMP-aktivitet i tumörvävnaden med
avseende på tumörtillväxt och angiogenes genom att utföra genterapi på experimentell
bröstcancer. Ökade nivåer av MMP-3 och MMP-9 med hjälp av genterapi resulterade i
hämning av angiogenes och tumörtillväxt. Tamoxifen och MMP-9 i kombination gav en
10
synergistisk behandlingseffekt medan genterapi med en hämmare av MMP, TIMP-1,
motverkade tamoxifenets effekter på angiogenes och tumörtillväxt.
I avhandlingens fjärde delarbete demonstreras en stark koppling mellan östrogen och det
angiogenesstimulerande proteinet IL-8 i normal bröstvävnad hos friska forskningspersoner, i
brösttumörer på kvinnor med hormonberoende bröstcancer och i experimentella modeller av
östrogenberoende human bröstcancer.
Sammanfattningsvis kan resultaten från de här studierna ge ökad förståelse för
angiogenesreglering i bröst- och äggstockscancer och på sikt ge förbättrade
behandlingsmöjligheter för de kvinnor som drabbas av dessa sjukdomar.
11
List of Papers
This thesis is based on the following original papers, which will be referred to in the text by
their Roman numerals (I-IV):
I Christina Bendrik, Lisa Karlsson, and Charlotta Dabrosin
Increased endostatin generation and decreased angiogenesis
via MMP-9 by tamoxifen in hormone dependent ovarian
cancer
Cancer Letters (2010) 292: 32-40
II Christina Bendrik, Jennifer Robertson, Jack Gauldie, and
Charlotta Dabrosin
Gene transfer of MMP-9 induces tumor regression of breast
cancer in vivo
Cancer Research (2008) 68: 3405-12
III Christina Bendrik and Charlotta Dabrosin
MMP-3 and MMP-9 gene transfer decrease growth and
angiogenesis in breast cancer xenografts in vivo
Manuscript
IV Christina Bendrik and Charlotta Dabrosin
Estradiol increases IL-8 secretion of normal human breast
tissue and breast cancer in vivo
The Journal of Immunology (2009) 182: 371-78
12
13
Abbreviations
Ad Adenovirus
ADAM A Disintegrin And Metalloproteinase
AI Aromatase inhibitors
ANOVA Analysis of variance
CYP Cytochrome P450
DNP Dinitrophenol
E1 Estrone
E2 Estradiol
E3 Estriol
E1S Estrone sulphate
ECM Extracellular matrix
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
ELISA Enzyme-linked immunosorbent assay
ER Estrogen receptor
ERT Estrogen replacement therapy
FGF Fibroblast growth factor
FIGO International Federation of Gynecology and Obstetrics
GFP Green fluorescent protein
HRT Hormone replacement therapy
HUVEC Human umbilical vein endothelial cells
IARC International agency for research on cancer
IHC Immunohistochemistry
IL Interleukin
IP Infectious particles
IU Infectious units
MMP Matrix metalloproteinase
MT-MMP Membrane-type MMP
MVD Microvessel density
MWCO Molecular weight cut-off
14
OC Oral contraceptives
P4 Progesterone
PDGF Platelet-derived growth factor
Pfu Plaque-forming units
PgR Progesteron receptor
PlGF Placental growth factor
SEM Standard error of the mean
SERM Selective estrogen receptor modulator
SSRI Selective serotonin reuptake inhibitors
Tam Tamoxifen
TIMP Tissue inhibitor of metalloproteinase
TNF Tumor necrosis factor
TNM Tumor size, node involvement, metastasis status
TU Transducing units
VEGF Vascular endothelial growth factor
WHI Women´s Health Initiative
15
Background
Carcinogenesis and tumor progression
The development of normal human cells into malignant cells and further progression to a
solid tumor and a clinical cancer disease is a complex and multi-step process. The process is
initiated due to accumulation of genetic and/or epigenetic alterations that allow for evading
normal regulation of cell proliferation and homeostasis. Genetic alterations leading to cancer
may be inherited (germ-line mutations), induced by viral agents, ionizing irradiation,
genotoxic compounds, or sporadically induced by other means. Genetic lesions may randomly
occur at any location of the genome but will most often damage parts that are of minor
importance of cell faith. Genetic alterations leading to the activation of oncogenes or loss of
tumor-supressor genes however, may initiate the tumorigenic pathway that progressively
drives the transformation of normal cells into malignant tissue (Hanahan et al. 2000). Tumor
development requires that the cells obtain certain functional capabilities such as a limitless
replicative potential, evasion of programmed cell death (apoptosis), and self-sufficiency in
growth signals (Hanahan et al. 2000). In addition, tumor growth is angiogenesis dependent
(Folkman 1971). A small lump of malignant cells need to overcome normal vessel quiescence
of the tissue and initiate the recruitment of a capillary network. A tumor that fail to induce
angiogenesis will not reach a size beyond 1-2 mm (Folkman 1972).
Hormones and cancer
Endogenous and exogenous hormones may trigger the carcinogenic pathway in a number of
human tissues. Sporadic tumors of the breasts and ovaries are considered to be hormone
related diseases, and so are also cancers of the endometrium, prostate, testis, thyroid, and the
bone (osteosarcoma) (Henderson et al. 2000; Persson 2000). This group of cancers are
proposed to share a common mechanism of carcinogenesis; endogenous and/or exogenous
hormones drive cell proliferation, thus increasing the number of cell divisions and the risk for
random genetic errors (Henderson et al. 1982; Henderson et al. 2000). Estrogens, the
hormones of interest in this thesis, have been shown to influence cell proliferation rates but
may also be metabolized into genotoxic compounds with direct or indirect abilities to induce
DNA damage (Cavalieri et al. 2000; Yager 2000; Liehr 2001; Yue et al. 2003). According to
IARC (International Agency for Research on Cancer), the evidence are sufficient to classify
estrogens as carcinogens to humans (IARC 1998). Angiogenesis is crucial for a functional
ovulation and menstrual cycle, a process strictly regulated by sex steroids. The influence of
sex steroids on the angiogenesis process in ovarian tumors is unclear. We do know however,
16
that estrogens affect tissue levels of several mediators involved in the angiogenesis process in
both normal breast tissue and in breast tumors (Dabrosin 2003; Dabrosin 2005; Garvin et al.
2005; Garvin et al. 2006; Nilsson et al. 2006; Garvin et al. 2008; Nilsson et al. 2010). The
fact that breast cancer may be a causal effect of increased endogenous levels of sex steroids
has been known for more than a hundred years. The first known case where oophorectomy
was used as endocrine therapy in breast cancer was reported in the Lancet 1896 (Beatson
1896).
Breast and ovarian cancer
Epidemiology
Breast cancer
Breast cancer is by far the most common form of cancer in women worldwide, with high
incidence rates in the Western World (Garcia et al. 2007). In Sweden, approximately 7000
women are diagnosed each year, meaning that breast cancer affects one in ten women during
lifetime (Socialstyrelsen 2009). The incidence rates have been rising over the past decades, a
rise which is hypothesized to be due to changes in reproductive patterns but also because of
improved screening and higher detection rates. The last years however, a decrease in breast
cancer incidence was reported from several Western countries, Sweden included (Keane et al.
1997; Ravdin et al. 2007; Canfell et al. 2008; Parkin 2009; Seradour et al. 2009; Lambe et al.
2010). This decrease is believed to be associated with reduced intake of hormone replacement
therapies (HRT), as a result of the 2002 release of the report from the Women´s Health
Initiative (WHI) hormone trial (Rossouw et al. 2002). Although the prognosis of breast cancer
in developed countries is rather good due to early detection and improved treatment, the
mortality rate in low resource countries continues to increase (Garcia et al. 2007).
The relationship between breast cancer and sex steroids is widely recognized although a
complete understanding of the precise mechanisms is lacking. Epidemiological data have
revealed that long-term exposure of endogenous or exogenous hormones, as in a long
menstrual history (early menarche and late menopause), nulliparity, late full term pregnancy,
and intake of oral contraceptives (OC) or HRT increase the risk of disease, whereas increasing
parity and long-term breastfeeding are considered to be protective (McPherson et al. 2000;
Akbari et al. 2010; Ma et al. 2010). Lifestyle factors associated with higher breast cancer risk
include high socioeconomic status, low physical activity and over-weight (Robert et al. 2004;
Lahmann et al. 2007; Rose et al. 2009; Vona-Davis et al. 2009). A family history of breast
cancer significantly increases the susceptibility of disease, and carriers of the dominant high-
penetrance genes BRCA1 or BRCA2 confers substantially increased risk of developing tumors
of both breasts and ovaries (Antoniou et al. 2003). Inherited genetic predisposition is however
estimated to count for less than 10% of breast cancer cases (McPherson et al. 2000; Foulkes
2008).
17
Ovarian cancer
Ovarian cancer is the sixth most common cancer among women worldwide and the
gynaecologic malignancy with poorest prognosis (Garcia et al. 2007). The annual incidence
of ovarian cancer is high in Scandinavia (15/100 000) and in Sweden approximately 800
women are diagnosed each year (Socialstyrelsen 2009). Ovarian tumours demonstrate a silent
progression and women are often asymptomatic until tumors have reached a size that affect
nearby organs. Patients typically present in the advanced stages III or IV and have poor
prognosis, with a 5-year survival of approximately 30% (Hanna et al. 2006). Extensive
surgery and recent advances with multiple-agent chemotherapy have only moderately
improved survival rates (Johnston 2004).
The pathogenesis of ovarian cancer is poorly understood but reproductive factors have been
suggested to be crucial in the aetiology of disease. Fathalla proposed the “incessant ovulation”
theory in 1971, suggesting that increasing numbers of ovulations enhances the risk of ovarian
cancer, as the traumatized epithelium of ruptured follicles is recurrently repaired and exposed
to high estrogen concentrations of the follicular fluid (Fathalla 1971). The “gonadotrophin”
hypothesis published by Stadel in 1975 suggests that high levels of pituitary gonadotrophins
induce malignant transformation by stimulating the ovarian surface epithelium (Stadel 1975).
High levels of gonadotrophins are detected during early postmenopausal years and coincide
with high age-specific incidence of ovarian cancer. A third theory proposes that androgens,
which are increased in obesity and in the postmenopausal state, stimulate carcinogenesis
while progesterone is protective (Risch 1998). These theories are supported by
epidemiological research and experimental data where the importance of sex steroids in
ovarian cancer aetiology has been confirmed. However, up to date there is no consensus
regarding the mechanisms behind sex steroid-induced carcinogenesis of the ovary, and the
complex issue of hormonal control of initiation and progression of ovarian tumors is far from
being solved.
Epidemiological studies show that the risk of ovarian cancer is related to total number of
lifetime ovulations (Purdie et al. 2003). Pregnancy and lactation significantly protects women
against ovarian cancer and the benefit is enhanced with increasing number of pregnancies and
period of lactation (Chiaffarino et al. 2001; Titus-Ernstoff et al. 2001; Riman et al. 2002).
There is in addition strong evidence that the use of combined OC confers protection, possibly
by inhibiting ovulation but also by affecting endogenous levels of hormones acting upon the
ovary (Bosetti et al. 2002; McGuire et al. 2004). Estrogen replacement therapy (ERT) for
relieve of climacteric symptoms has been shown to increase the risk whereas intake of HRT
containing both estrogen and progestins not was associated with increased ovarian cancer risk
as compared to non HRT users (Riman et al. 2002). The evidence for age at menarche and
menopause is conflicting; some studies show that late menarche and early menopause
decreased ovarian cancer risk whereas other studies have shown no association of these
factors (Franceschi et al. 1991; Chiaffarino et al. 2001; Schildkraut et al. 2001; Titus-Ernstoff
et al. 2001; Riman et al. 2002). Elevated plasma levels of sex steroids in women with ovarian
tumors, levels that additionally have been shown to correlate with tumor size and to increase
prior to tumor recurrence, further indicate the fact that ovarian tumors are endocrine related
18
and hormone dependent (Heinonen et al. 1982; Mango et al. 1986; Mahlck et al. 1988). The
risk of ovarian cancer is increased in women with a family history of the disease but also in
women with a family history of other cancers, such as cancer of the breast, but also cancer of
stomach, intestine, lung, and lymphoma (Hanna et al. 2006).
Breast and ovarian anatomy and sites of cancer origin
The breasts and ovaries are highly hormone-sensitive organs. Many women experience
tenderness of her breasts and ovaries at certain phases of the menstrual cycle due to hormonal
influences of these tissues. Sex steroids are crucial for normal mammary gland development,
proliferation, and differentiation (Anderson et al. 1998). Estrogens induce growth of the
extensive ductal system of the breasts and are in addition responsible for the characteristic
external appearance of the female breast by causing development of stromal tissues as well as
the deposition of fat. The development of lobule and alveoli are dependent on the influence
of estrogens but the determinate growth and function of these structures are caused by
progesterone and prolactin. The post-pubertal female breast contains thousands of hormone-
sensitive lobule (Figure 1).
Figure 1. Breast anatomy.
A. Ducts
B. Lobules
C. Dilated section of duct to hold milk
D. Nipple
E. Fat
F. Pectoralis major muscle
G. Chest wall/rib cage
Enlargment of duct.
A. Normal duct cells
B. Basement membrane
C. Duct lumen
(Adapted from www.breastcancer.org).
19
Each of these potentially milk-producing micro-glands is drained into a terminal duct which
in turn is attached to the main duct system. This unit, which is called the terminal ductal-
lobular unit, is the most common site of breast tumor origin (Juncker-Jensen et al. 2009). The
lobules have been identified in four different structure types, type 1-4, depending on the
developmental stage they are representing (Russo et al. 2005). Type 1 with only a few
ductules per lobule represents the most undifferentiated lobule and is present in the immature
breast before menarche. Type 2 has more ductules per lobule and exhibits a more complex
morphology. Type 1 and 2 is transformed into type 3 lobule during first and second trimesters
of pregnancy and exhibit more numerous ductules per lobule. Type 4 represents the fully
differentiated condition and this transformation occurs only during lactation. Epithelial cells
of lobule type 1 and type 2 have high proliferative capacity and are susceptible to malignant
transformation, whereas cells of differentiated lobule type 3 and type 4 are more refractory to
carcinogenesis (Russo et al. 2005).
Figure 2. Ovarian anatomy.
(Adapted from www.colorado.edu)
20
Synthesis and secretion of estradiol from the ovaries increases approximately 20-fold at
puberty under the influence of the pituitary gonadotropic hormones and during this period
estradiol promotes growth of the female reproductive organs including ovaries and regulates
adaptation to a functional adult reproductive system. From puberty to menopause regulation
and physiological functions of the ovaries are highly associated with sex steroids in autocrine,
paracrine, and endocrine manners (Guyton 2000).
There are different histological types of ovarian tumors depending on the site of cancer origin.
The most common site of tumor origin is the ovarian surface epithelium (more than 90% of
all malignant ovarian neoplasms) but tumors may also origin from germ cells or stromal
components of the ovary (Figure 2). The epithelial ovarian tumors are further divided into
several histopathological subgroups where among serous adenocarcinoma is the most usual
subtype (Booth et al. 1989; Riman et al. 1998). The term “ovarian cancer” in this thesis is
used for epithelial tumors of the ovary.
Estrogens
Synthesis
In healthy, premenopausal non-pregnant women, the major source of estrogen is the ovaries,
although small amounts also are secreted by the adrenal cortices. Sex steroids are synthesized
by the ovaries from cholesterol derived from the blood (Figure 3). Aromatization is the last
step in estrogen formation, a reaction which is catalyzed by the P450 aromatase
monooxygenase enzyme complex that converts androgens into estrogens through steps of
hydroxylation (Guyton 2000; Gruber et al. 2002). The aromatase enzyme is found at high
levels in the granulose cells of the ovarian follicle and at lower levels in peripheral tissues of
the body such as adipose tissue, brain, and muscle (Simpson et al. 1993). Estrogens exist in
three forms; estrone (E1), 17β-estradiol (E2), and estriol (E3) where E2 is the most potent form
and the principal estrogen secreted by the ovaries. At menopause the ovarian production of
sex steroids decreases, and the major estrogen found in the blood of postmenopausal women
is estrone sulphate (E1S) which derives from peripherally aromatized androstenedione into E1
which is rapidly sulphated into E1S by a number of sulfotransferases distributed throughout
the body (Hobkirk 1993). The circulating levels of E1S is approximately eight times higher
than E1 levels (1500 pmol/L as compared to 200 pmol/L) and 40 times higher than those of E2
(<40 pmol/L) (Hobkirk 1993; Stanway et al. 2007). However, breast tissue E2 levels of
postmenopausal women have been shown to be 10-20 times higher than corresponding
plasma levels and comparable with those of premenopausal women (van Landeghem et al.
1985; Vermeulen et al. 1986). In breast tumor tissues the concentrations of E2 have been
shown to be increased as compared to normal breast, suggesting a specific tumor biosynthesis
and E2 accumulation in this area (van Landeghem et al. 1985; Pasqualini et al. 1997).
Enhanced activities or disregulated function of enzymes involved in the different steps of E2
21
bioformation may be one important factor of increased local breast tumor production of
estrogens (Chetrite et al. 2000; Pasqualini et al. 2005; Gunnarsson et al. 2008; Sasano et al.
2008; Subramanian et al. 2008).
Estrogen receptors
The diverse biological effects of estrogens in the human body are mediated through
interaction with estrogen receptors (ERs). ERs exist in two isoforms (ERα and ERβ) which
are distributed with distinct cell-specific expression pattern in a broad spectrum of tissues
throughout the body. Studies in mice lacking ERα or ERβ have revealed overlapping but also
unique roles by the two ERs in vivo (Matthews et al. 2003). After the discovery of ERβ it was
found that several tissues that before were considered as estrogen-insensitive were ERβ
positive and estrogen-responsive (Kuiper et al. 1996; Kuiper et al. 1997; Morani et al. 2008).
In cells and tissues expressing both the receptors, ERβ has been suggested to counteract ERα-
induced effects (Strom et al. 2004; Morani et al. 2008; Hartman et al. 2009). In the classical
mechanism of action, estrogen molecules diffuse into the cell and bind to ER located in the
nucleus, resulting in a conformational change of the ER which allows for interaction with co-
regulator complexes that either activates or represses transcription of target genes (Klinge
2000; Dahlman-Wright et al. 2006). The resulting physiological response in form of mRNA
and protein fluctuations following estrogen exposure takes place within hours when acting
Cholesterol
Pregnenolone
Progesterone
P450 side chaincleavage enzyme
3ßHSD
17a-OHPregnenoloneP45017ahydroxylase
17a-OHProgesterone
P45017a
hydroxylase
3ßHSD
DHEA
Androstenedione Estrone
Testosteron 17ß-Estradiol
P45017a
hydroxylase
P45017a
hydroxylase 17ßHSD
P450aromatase
P450
aromatase
Figure 3. Gonadal synthesis of sex steroids.
22
through this classical, genomic pathway (Dahlman-Wright et al. 2006; Deroo et al. 2006).
Estrogen exposure may additionally induce rapid (non-genomic) responses within seconds or
minutes in cells and tissues via membrane-bound ERs. Rapid effects of estrogens include the
formation of cAMP, activation of the MAP kinase signaling pathway, Ca2+
influx or release
from intracellular stores, and immediate increase in NO production amongst others
(Falkenstein et al. 2000; Kim et al. 2008).
In normal breast epithelium ER expression is very low and enhanced levels have been
correlated with increased risk of breast cancer (Khan et al. 1994). In breast cancer, ER status
is a well established predictor of response to endocrine therapy. Approximately 70% of breast
tumors are considered to be ER+/PgR+, the phenotype with the most favourable response to
endocrine therapy and cancer-specific survival (Anderson et al. 2001; Cordera et al. 2006).
When discussing ER status in breast cancer it refers to the ERα subtype as routine clinical
screening of breast tumor specimen detects ERα only. In Sweden, approximately 78% of
breast tumors are ER-positive, as reported during 2008-2010 (INCA).
Ovarian primary tumors are not routinely screened for ER status. However, several studies
show that approximately 40-50% of ovarian tumors are ER+/PgR+ (Ford et al. 1983;
Kauppila et al. 1983; Sutton et al. 1986; Enmark et al. 1997; Pujol et al. 1998; Lindgren et al.
2004). In normal ovaries, ERβ seem to be the predominant ER whereas the ratio ERα:ERβ is
increased in ovarian tumors, suggesting an anti-tumorigenic role of ERβ in this tissue which
may be lost during tumor progression (Brandenberger et al. 1998; Pujol et al. 1998; Li et al.
2003).
Tamoxifen and its mechanisms of action
Tamoxifen is a non-steroidal anti-estrogen, well established in the medical treatment of ER
positive breast cancers. Long term (5 years) adjuvant tamoxifen therapy significantly
improves recurrence rates and survival, irrespectively of age and menopausal status
(EBCTCG 1998). Despite the beneficial therapeutic effects demonstrated in hormone-
dependent breast cancers, tamoxifen is rarely used as a treatment option of hormone-sensitive
ovarian cancers. Clinical trials evaluating tamoxifen in ovarian cancer patients are few and
performed on heavily pre-treated patients with relapsed disease, some of them refractory to
chemotherapy, and in most cases with unknown ER-status (Hatch et al. 1991; Markman et al.
1996; Benedetti Panici et al. 2001; Perez-Gracia et al. 2002). The relevance of tamoxifen in
the adjuvant settings of ovarian cancer has never been assessed.
Tamoxifen is metabolically transformed by the cytochrome P450 enzyme system in the body
and give rise to a number of metabolites with varying circulating concentrations and
therapeutic efficacies (Figure 4). Amongst the most important are 4-hydroxy-tamoxifen and
endoxifen in terms of their ability to inhibit estrogen-induced proliferation (Stearns et al.
2003; Desta et al. 2004).
23
Tamoxifen belongs to the category of selective estrogen receptor modulators (SERMs) acting
as an agonist or antagonist depending on the target tissue. Agonistic (estrogenic) effects are
exerted in bone, endometrium and on the blood lipid profile, whereas antagonistic (anti-
estrogenic) effects are seen in breast tissue (Osborne et al. 2000; Clarke et al. 2001). The
differential tissue effects induced by tamoxifen may depend on expression levels and ligand-
receptor interactions of the two ER subtypes, ERα and ERβ (Katzenellenbogen et al. 2000).
ERα positivity is a well established predictor of favourable response to tamoxifen, whereas
the role of ERβ in tamoxifen therapy is contradicting. It has been suggested that low ERβ
expression is associated with tamoxifen resistance (Esslimani-Sahla et al. 2004), and
coexpression of ERα and ERβ indicative of poorer prognosis (Speirs et al. 1999). In a recent
study however, it was demonstrated that high expression of ERβ in ERα negative tumors was
predictive for favourable response to tamoxifen (Gruvberger-Saal et al. 2007). This could
explain why a subgroup of patients with ERα negative tumors demonstrate responsiveness to
tamoxifen (EBCTCG 1998).
Tamoxifen has additionally been shown to induce non-ER mediated cellular events with
potential anti-tumorigenic implications. These events include apoptotic and antioxidative
cellular processes which may enhance the anti-tumorigenic effects exerted by tamoxifen
(Clarke et al. 2001; Mandlekar et al. 2001).
Figure 4. Metabolic pathways of tamoxifen.
Modified from Stearns et al.; JNCI, 95:1758-1764, 2003
24
Angiogenesis and the tumor microenvironment
Tumor microenvironment
The tumor microenvironment is a heterogeneous compartment consisting of different cell
types and stromal components, all with capacities to affect tumor progression in both negative
and positive ways. In addition to the cancer cells themselves, the tumor tissue is composed of
resident cells such as fibroblasts and endothelial cells, and of infiltrating immune cells such as
macrophages and lymphocytes. Large amounts of cell-secreted bio-active products including
components of the extracellular matrix (ECM), cytokines, chemokines, growth factors and
proteolytic enzymes regulate the complex cross-talk between tumor cells and other cells. Cell-
cell and cell-microenvironment interactions are suggested to modify different steps of tumor
progression including proliferation, differentiation and invasiveness. Matrix
metalloproteinases (MMPs) are largely involved in the cross-talk of interacting components
of a tumor due to their tissue remodelling capacities and by enzymatic cleavage and release of
stromal and cell-bound molecules with abilities to affect both pro- and anti-tumorigenic
activities.
The impact of the tissue microenvironment on tumor growth has been recognized for more
than a 100 years, since Paget hypothesized the “seed and soil” theory in 1889 (Paget 1889).
During recent years the important role of stromal components on tumor behaviour has
become more evident. Subtypes of tumor stroma corresponding to good or poor outcome
breast cancers have been identified by studying changes in gene expression patterns in breast
tumor stroma (Finak et al. 2008). The prognostic information of the stroma may lead to
identification of useful biomarkers in cancer and in designing appropriate treatments, as
reviewed by Sund & Kalluri (Sund et al. 2009). The fact that malignant transformation of
cells per se is not enough to induce cancer and that the process also requires a certain stromal
phenotype is termed “cancer without disease” (Folkman et al. 2004). The microenvironment
has been shown to influence tumor-induced angiogenesis, which may be illustrated by a study
where breast tumors of the same origin but implanted into different tissues showed diverse
angiogenic responses (Monsky et al. 2002).
Tumor angiogenesis
All cells and tissues are dependent on a regular supply of oxygen and nutrients and so is the
development of a solid tumor dependent upon its ability to induce vessel growth into the mass
of dividing tumor cells. Angiogenesis is a fundamental process by which new blood vessels
are formed. During embryogenesis the primordial formation of a vascular network is
established through vasculogenesis; where progenitor cells (angioblasts) differentiate into
endothelial cells which organize into luminal structures. This vasculature is extended by
angiogenesis; i.e., the sprouting of new capillaries from the preexisting network (Risau 1995).
Endothelial cell proliferation is very high during embryogenesis and during postnatal
development, whereas the vasculature in the normal adult body is quiescent. In the adult, new
25
vessels are formed only through angiogenesis (Risau 1995). Angiogenesis occurs at tissue
trauma and wound repair and in the female reproductive tract where it is crucial for a
functional ovulation, menstruation and implantation. During these conditions angiogenesis is
a strictly regulated process, turned on for a brief period of time and then inhibited (Folkman et
al. 1992). New blood vessel formation and regression in each female reproductive cycle is
strictly regulated by sex steroids (Hyder et al. 1999; Losordo et al. 2001; Ramakrishnan et al.
2005). Less is known about how sex steroids influence and regulate the process of
angiogenesis in hormone-dependent malignant tissues.
The process, in which new blood vessels grow out of existing vessels of the tissue to sustain
tumor expansion, is known as tumor angiogenesis. Microscopic tumors that fail to induce
angiogenesis result in dormant tumors without the ability to progress in size. Clinical and
experimental evidence suggest that human tumors may persist for long periods of time as
microscopic lesions that are in a state of dormancy (Black et al. 1993; Demicheli et al. 1994;
Udagawa et al. 2002). Pathologists performing autopsies on individuals who died in car
accidents or other trauma documented the presence of microscopic carcinomas in situ in the
breast of 39% of women age 40 to 50 years, whereas only 1% of women are ever diagnosed
with breast cancer during life in the same age range (Black et al. 1993). Even more strikingly,
microscopic carcinomas were found in the thyroid of more than 98% of individuals in the age
of 50 to 70 years who died of trauma, whereas only 0.1% are diagnosed with this type of
cancer during life (Black et al. 1993).
The link between the vascular system and malignant growth in man was suggested to be
important already for more than a 100 years ago, although without providing underlying
mechanisms or experimental proof (Goldmann 1908). The importance of angiogenesis for the
growth of solid tumors is now well recognized. The fact that tumor growth is angiogenesis-
dependent and the idea that anti-angiogenic therapy could be used in the treatment of cancer
was first proposed in the beginning of 1970 (Folkman 1971; Folkman 1972; Gimbrone et al.
1972).
Regulators of angiogenesis
Prevailing evidence suggest that angiogenesis in physiological as well as in pathological
states is regulated by the net balance of positive and negative effectors of endothelial cell
proliferation and migration in the tissue (Folkman 2003). These regulators of angiogenesis
may be secreted by the tumor cells, by stromal cells or by immune cells. In healthy tissues
inhibitors of angiogenesis are in excess and keep the tissue vascularisation in a quiescent state
(Figure 5). Normal endothelial cells have the longest life among all cells except for those of
the nervous system, with only one in 10 000 in a proliferating state (Engerman et al. 1967).
Tumor-induced angiogenesis is thought to be a result of increased production and secretion of
activators of angiogenesis by the tumor or by down-regulation of normal expression level of
26
inhibitors (Hanahan et al. 1996; Sund et al. 2005). An increase of pro-angiogenesis molecules
or a decrease of anti-angiogenesis molecules would tip the balance to favour an angiogenic
state in the tissue. It has been shown that several angiogenesis regulatory molecules are stored
in the extracellular environment, bound to different molecules of the ECM, or are fragments
of larger ECM components and releasable by specific enzymes (Folkman 2003). Therefore,
proteolytic activities and ECM remodelling events within the tumor microenvironment are
important aspects of the angiogenesis regulatory process (Mott et al. 2004; Sottile 2004).
Activators of angiogenesis
There are several pro-angiogenic proteins identified which may be produced by a tumor, such
as vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factor
(aFGF and bFGF), platelet-derived growth factor (PDGF), interleukin-8 (IL-8), epidermal
growth factor (EGF), tumor necrosis factor-alpha (TNF-α), and placental growth factor
(PlGF) (Folkman 2003) (see Table I). One or more of these pro-angiogenesis proteins may be
triggered by oncogenes and over-expressed during the switch to the angiogenic phenotype of
a tumor (Hanahan et al. 1996). VEGF is a potent pro-angiogenic protein expressed by many
breast tumors at the time of diagnosis. There are indirect angiogenesis inhibitors targeting
VEGF, such as Iressa (tyrosine kinase-inhibitor), Avastin (anti-VEGF antibody) and SU
11248 (VEGF receptor blocking agent). An indirect angiogenesis inhibitor targets an
oncogene or its product, or the receptor for that product, whereas direct angiogenesis
inhibitors, such as endostatin, target the microvascular endothelial cells of the tumor and
blocks the endothelial cell response to pro-angiogenesis proteins (Kerbel et al. 2002; Folkman
2003). Direct angiogenesis inhibitors are suggested to be less likely to induce acquired drug
resistance, as they target genetically stable endothelial cells rather than mutating cancer cells
with genomic instability (Kerbel et al. 2002).
Figure 5. The angiogenesis balance. The
net tissue level of pro- and anti-angiogenesis
mediators dictate whether the endothelial
cells will be in a quiescent or an angiogenic
state. It is believed that increased tumor
production of activators or a decrease of
inhibitors in the tumor microenvironment
mediate the angiogenic switch, a key event
in tumor progression.
27
Interleukin-8
IL-8 (or CXCL8) is a pro-inflammatory cytokine originally identified as an activator and
chemoattractant for neutrophils but also recognized to possess pro-tumorigenic and pro-
angiogenic properties as well (Koch et al. 1992; Strieter et al. 1992). Over-expression of IL-8
has been demonstrated in several tumor types, including breast- and ovarian cancer, and IL-8
has been suggested to have a significant impact on tumor progression due to its ability to
affect multiple cell types of the tumor microenvironment (Xie 2001; Waugh et al. 2008). IL-8
activates several intracellular signalling pathways of immune- and non-immune cells
(including cancer cells and endothelial cells) through interactions with two cell-surface
receptors, CXCR1 and CXCR2. Downstream events of IL-8 signalling include activation of
several pathways, such as PI3K/Akt, Src-kinases and FAK, and MAPK signalling cascades,
which results in enhanced transcriptional activity of proteins involved in proliferation,
migration and invasion (Waugh et al. 2008). In breast cancer patients, high expression of IL-8
and its receptors have been associated with poor prognosis, and experimental studies have
demonstrated a strong correlation between the metastatic potential of breast cancer cells and
IL-8 expression level (Miller et al. 1998; De Larco et al. 2001; Benoy et al. 2004). Several
stress factors of the tumor microenvironment have been shown to trigger the production of IL-
8, including hypoxia (via activation and cooperation of NFκB and AP-1), acidosis, NO, and
cell density (Xie 2001). Sex steroids are suggested to have a role in IL-8 regulation; however
existing data are contradicting and seem to vary in different tissues (Kanda et al. 2001;
Bengtsson et al. 2004; Suzuki et al. 2005). In vitro studies of breast cancer cell lines may
indicate an over-expression of IL-8 predominantly in ER negative cells as compared to ER
positive cells (Freund et al. 2003; Lin et al. 2004). However, this does not rule out an
estradiol regulation of this protein.
Activators Inhibitors
VEGF Thrombospondin
bFGF
aFGF
IL-8
Angiogenin
PDGF
TNF-α
Angiostatin
Tumstatin
Endostatin
Canstatin
Arresten
Interferon-α
Table I.
Examples of activators and inhibitors of angiogenesis.
28
Endogenous inhibitors of angiogenesis
Endogenous inhibitors of angiogenesis are proteins or fragments of proteins formed in the
body with capacities to inhibit blood vessel formation (Nyberg et al. 2005). They act by
blocking endothelial cell cycle progression and/or activating apoptotic pathways of the
endothelial cells. Several of the known anti-angiogenesis mediators are fragments derived
from different ECM components, such as collagens of the vascular basement membranes
(Kalluri 2003; Sund et al. 2004). Collagen type IV, the main component of all basement
membranes, is precursor protein of the angiogenesis inhibiting fragments arresten, canstatin,
and tumstatin, although they are localized at different α-chains of the collagen (Colorado et al.
2000; Kamphaus et al. 2000; Maeshima et al. 2000). Among the type IV collagen-derived
anti-angiogenic fragments, tumstatin (28 kDa fragment of the α3-chain) may be the most
extensively studied. Tumstatin induces apoptosis in proliferating endothelial cells through the
binding to αvβ3 integrin on the cell surface leading to inhibition of mTOR (mammalian target
of rapamycin) (Maeshima et al. 2000). Tumstatin exert similar effect as rapamycin (a small-
molecule inhibitor of mTOR) with the exception that tumstatin only affects proliferating
endothelial cells (Maeshima et al. 2002). Mice with genetic deletion of Col IVα3 show
accelerated tumor growth which is reversed by the supplementation of recombinant tumstatin
(Hamano et al. 2003). Additionally, mice deficient in MMP-9, which efficiently generates
tumstatin from collagen type IV, show decreased levels of circulating tumstatin and
accelerated tumor growth (Hamano et al. 2003). Arresten was more recently characterized
and was shown to inhibit angiogenesis by downregulating the anti-apoptotic molecules Bcl-2
and Bcl-xL in endothelial cells through interactions with cell surface α1β1 integrins (Nyberg
et al. 2008).
Type XVIII collagen is the precursor protein of endostatin, which is discussed in a separate
section. Other examples of endogenous inhibitors of angiogenesis derived from ECM
precursor proteins are anastellin generated from fibronectin and endorepellin derived from the
proteoglycan perlecan, amongst others (Yi et al. 2001; Mongiat et al. 2003; Bix et al. 2006).
There are additionally several cell-secreted growth factors with anti-angiogenic properties,
including the interferons and some of the interleukins (IL-4, IL-12 and IL-18) (Brouty-Boye
et al. 1980; Nyberg et al. 2005). Angiogenesis inhibitors may also be derived from blood
coagulation factors, such as angiostatin (a fragment of plasminogen), cleaved and intact forms
of antithrombin, platelet factor-4 and thrombospondins (Good et al. 1990; O'Reilly et al.
1994; O'Reilly et al. 1999; Larsson et al. 2000).
Endostatin
Endostatin is a 20 kDa fragment cleaved from the C-terminus of type XVIII collagen and a
potent inhibitor of angiogenesis (O'Reilly et al. 1997). Type XVIII collagen, a heparin
sulphate proteoglycan, is one of several proteins composing the basement membranes (Kalluri
2003). Endostatin may be generated by enzymatic activity of several proteases in the tumor
microenvironment, including specific MMPs and elastase (Wen et al. 1999; Ferreras et al.
2000; Heljasvaara et al. 2005). MMP-3, -7, -9, -13 and -20 efficiently cleaves endostatin from
29
collagen XVIII in vitro (Heljasvaara et al. 2005). Physiological levels of circulating
endostatin of healthy individuals range from 20-50 ng/mL (Hefler et al. 1999; Zorick et al.
2001). The concentration in serum may be higher than in plasma due to release of endostatin
from platelets, as platelets are found to sequester endostatin for later release at aggregation
(Ma et al. 2002). Endostatin inhibits angiogenesis by affecting a wide range of endothelial
cell functions through interactions with several cell surface receptors, including α5β1, ανβ3,
and ανβ5 integrins (Rehn et al. 2001; Sudhakar et al. 2003). Downstream intracellular events
affect a wide range of transcription factors leading to cell cycle arrest, inhibition of
migration/invasion and tube formation, and induction of apoptosis (O'Reilly et al. 1997;
Dhanabal et al. 1999). Pan-genomic array analyses of endostatin influence on endothelium
have shown that endostatin has a major function in the regulation of genes coding for proteins
involved in angiogenesis (Abdollahi et al. 2004). For example, endostatin upregulates levels
of thrombospondin, another major inhibitor of angiogenesis which has been shown to be
suppressed during the angiogenic switch and downregulates levels of cell survival factors
such as HIF-1α, NF-κB, AP-1 and STATs (Abdollahi et al. 2004).
The ability of endostatin to inhibit angiogenesis and tumor growth in vivo has been
extensively studied and demonstrated in animal models (Boehm et al. 1997). Endostatin-
deficient mice show enhanced level of angiogenesis and 2- to 3-fold accelerated tumor growth
as compared to wild-type mice (Sund et al. 2005). Correlative clinical evidence also suggests
a tumor suppressive role of endostatin; individuals with Down syndrome have approximately
1.6-fold higher circulating levels of endostatin due to presence of three copies of the gene
coding for collagen XVIII, which is located on chromosome 21, and very low incidence of
solid tumors (Zorick et al. 2001; Xavier et al. 2009). Experimental studies, using transgenic
mice expressing the same moderate increase in circulating endostatin showed suppressed
angiogenesis and a 3-fold reduction in tumor growth (Sund et al. 2005).
Endostatin was the first angiogenesis inhibitor to reach clinical trials (Quesada et al. 2006).
Initial phase I trials, which included patients with various tumor types, such as breast, lung,
liver, colorectal, ovarian, pancreatic and kidney cancers, indicated that recombinant
endostatin was a well tolerable drug without any significant toxicity (Eder et al. 2002; Herbst
et al. 2002; Thomas et al. 2003). In these trials however, minor or no tumor response were
observed. Phase II studies evaluating recombinant human endostatin were initiated in 2002
but were later on terminated due to poor clinical responses (Kulke et al. 2006). However,
parallel trials evaluating recombinant endostatin conjugated to a zinc-binding peptide (ZBP-
endostatin) were performed in China during these years, and were in 2005 approved by the
State Food and Drug Administration of China as a cancer drug (Endostar) for the treatment of
non-small cell lung cancer. Endostar was suggested to have a more stable structure and
therefore more potent in anti-cancer therapy (Fu et al. 2009). The addition of Endostar to
standard chemotherapy resulted in significant improvement in response rate and survival
benefit in non-small cell lung cancer patients (Wang et al. 2005). Results from Chinese trials
reporting beneficial anti-tumorigenic effects of Endostar alone in peer-reviewed journals are
yet to be published.
30
There are studies demonstrating a biphasic effect of recombinant endostatin-induced
inhibition of tumor growth, with a U-shaped dose-response curve, and with optimal efficacy
between very low and very high dose depending on the tumor type (Celik et al. 2005). This
may suggest that further investigations evaluating effective dosing of recombinant endostatin
may improve therapeutic efficacy. Additionally, there are several reports suggesting that
endogenous inhibitors of angiogenesis, including endostatin, may be increased
pharmacologically by the intake of orally available molecules (Folkman 2006). Increased
generation of physiologically occurring anti-angiogenic fragments may be a more favourable
approach to target tumor growth as compared to administration of recombinant proteins.
Matrix metalloproteinases
Matrix metalloproteinases (MMPs) are a family of multifunctional zinc-dependent
endopeptidases important in ECM remodelling and cell-matrix modifications, and therefore of
major interest as mediators of angiogenesis and tumor progression. The discovery of an
amphibian interstitial collagenase, for almost 50 years ago, led to identification of
approximately 25 structurally related enzymes with clinical relevance in man, later called
MMPs (Gross et al. 1962). The expression of MMPs is crucial in many fundamental
physiological events; such as organ development during embryogenesis, wound healing,
angiogenesis, uterine and mammary involution, menstruation and ovulation, and in
inflammatory processes (Hulboy et al. 1997; Vu et al. 2000; Page-McCaw et al. 2007). In the
normal female reproductive tract, temporal and spatial expression of several MMPs, including
MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-10 govern tissue remodelling in the
different phases of the menstrual cycle and pregnancy (Hulboy et al. 1997). As these
processes are under strict control of sex steroids, it is likely – although not fully understood –
that sex steroids are involved in the regulation of MMP activities in these tissues. In the rat,
removal of all endogenous steroid hormones by oophorectomy and adrenalectomy resulted in
complete loss of uterine collagenase synthesis and activity, a synthesis which was restored
after implantation of estradiol-releasing pellets (Anuradha et al. 1993). However, the
knowledge of sex steroid regulation of MMP expression in hormone dependent malignant
tissues is limited.
MMP members are products of different genes but share a common catalytic domain
containing the binding site for Zn2+
, the binding of which is essential for proteolytic activity
(Visse et al. 2003; Nagase et al. 2006). All MMPs are synthesized with a pre-domain which
targets the MMP for extracellular secretion or membrane localization, and a pro-domain
responsible for maintaining the enzyme inactive until proteolytic activity is required (Figure
6). The active form of MMP-7 contains the minimum catalytic domain only, whereas all other
MMP members have additional domains that contribute to their individual characteristics.
Most MMPs contain a hemopexin domain attached at their C-termini, which encodes a
specific four-bladed β-propeller structure mediating protein-protein interactions (Page-
31
McCaw et al. 2007). MMP-2 and MMP-9 additionally contain fibronectin type II repeats inserted into the catalytic domain, which mediate binding to collagens. Although most MMPs identified are secreted enzymes, some of the members are membrane-type MMPs (MT-MMPs) and localized to the cell-surface due to transmembrane domains and cytoplasmic tails (MMP-14, MMP-15, MMP-16, and MMP-24). MMP-17 and MMP-25 are cell surface bound through glycosylphosphatidylinositol linkages (Nagase et al. 2006).
The activity of MMPs is controlled at several levels; at transcriptional and protein synthesis level, at levels of secretion, at post-translational level in the extracellular space where enzymatic removal of the pro-domain is required for enzymatic activity, by the presence of natural inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs) and α2-macroglobulin, and by protease degradation (Chakraborti et al. 2003; Page-McCaw et al. 2007). Additionally, the availability of substrate determines the degree of MMP action. The fact that MMP action is to a large extent regulated in the extracellular space implies the difficulties to interpret MMP activities from RNA expression patterns, and the complex issue of MMP regulation remains to a large extent unknown. The MMP substrate specificity determines the bio-specific response of MMP activities in the tissue. MMP function is mainly associated with degradation of structural components of ECM. Recent studies reveal however, that the MMP degradome (MMP proteolytic substrates) is much more comprehensive than earlier believed and include a large number of matrix and non-matrix proteins with known and unknown biological functions (Cauwe et al. 2007;
Figure 6. Domain structures of MMPs. All MMP are synthesized with a signal peptid (pre-domain) which is cleaved during transport through the secretory pathway and a pro-domain which
keeps the MMP in a latent form until enzymatic activity is required. MMP-2 and MMP-9 have additionally fibronectin type II repeats inserted into the catalytic domain.
Zn
Signal peptide
Pro-domaine Catalytic domain
Hemopexin domain
Fibronectin type II repeats
domain
Hemopexin
Membrane linkage
32
Morrison et al. 2009; Rodriguez et al. 2010). It has become evident that MMPs have the
ability to regulate cell behaviour through finely tuned and tightly controlled proteolysis of a
wide variety of molecules, including growth factors, other proteases, cell-adhesion molecules,
cytokines and chemokines, tyrosine kinase receptors (such as epidermal growth factor
receptor (EGFR)), and pro-and anti-angiogenic fragments to mention a few (Mott et al. 2004;
Morrison et al. 2009; Kessenbrock et al. 2010; Rodriguez et al. 2010).
MMP inhibitors in clinical trials
The role of MMPs in cancer is traditionally associated with their degrading capacities of ECM
components and they have therefore been viewed as facilitators of tumor invasion (Liotta et
al. 1990; Kleiner et al. 1999; Stamenkovic 2000). High expression of several MMPs has been
reported in almost all types of tumors, which led to the development of MMP inhibitors and
clinical trials where MMP activities were evaluated as anti-tumor targets. Broad-spectrum
inhibitors such as marimastat and batimastat were promising in animal setups, but when given
to cancer patients no efficacy could be observed, and side-effects in terms of musculoskeletal
pain and inflammation were reported (Coussens et al. 2002). More specific attempts, such as
in the use of prinomastat and tanomastat (inhibitors of MMP-2 and MMP-9) in clinical trials
including patients with non-small cell lung cancer, prostate cancer and pancreatic cancer,
resulted in early termination due to lack of efficacy or poorer survival than placebo-treated
patients (Coussens et al. 2002). The failure of these trials in combination with large amounts
of research data have revealed complex roles of MMP actions in tumor biology, and evidence
of both pro- and anti-tumorigenic activities (Coussens et al. 2002; Overall et al. 2006; Lopez-
Otin et al. 2007; Martin et al. 2007). Much more knowledge is warranted on specific MMP
actions in normal and malignant tissues prior new attempts of targeting these enzymes in anti-
cancer trials.
Individual MMPs in cancer
MMP-2 and MMP-9 have for long been associated with basement membrane degradation due
to their early classification as type IV collagenases, and therefore to a large extent considered
responsible for pathological tissue-invasive events. Although a strong correlation between
MMP-2 or MMP-9 expression and basement membrane remodelling events is established, the
landmark studies assigning the type IV collagenolytic activity to MMP-2 and MMP-9, have
recently been called into question, as the results from those early in vitro experimental setups
may not hold true in vivo (Mackay et al. 1990; Mackay et al. 1990; Wheatcroft et al. 1999;
Rowe et al. 2008). Moreover, it has been demonstrated that MMPs are not exclusively
required for tumor cells to invade a tissue, as transmigration of cancer cells may occur by
their adoption of amoeba-like movements, similar as in immune surveillance where basement
membranes are traversed by billions of cells daily in healthy tissues (Wolf et al. 2003; Jodele
et al. 2006; Rowe et al. 2008).
33
MMP-9 has been reported to affect tumor angiogenesis in both directions. MMP-9 was
suggested to initiate the angiogenic switch in experimental studies of pancreatic cancer by
increasing VEGF bioavailability (Bergers et al. 2000). In this study it was demonstrated by
IHC that MMP-9 not was expressed by the tumor cells but by a small number of cells close to
the existing vasculature, suggested as infiltrating inflammatory cells, and in stromal cells of
the tumor. Interestingly, in breast cancer there are studies indicating that stromal expression
of MMP-9 would induce pro-tumorigenic activities, whereas tumor cell-derived MMP-9
would be protective (Pellikainen et al. 2004).
MMP-9 has been shown to decrease tumor growth in several studies by the release of anti-
angiogenesis fragments. Hamano et al. showed that MMP-9-knockout mice have decreased
levels of the anti-angiogenic fragment tumstatin and accelerated growth of tumors (Hamano et
al. 2003). Pozzi et al. demonstrated that reduced plasma levels of MMP-9 leads to decreased
generation of anti-angiogenic angiostatin from plasminogen and a consequent increase of
tumor growth and vascularization, and also that enhanced MMP-9 expression caused reduced
tumor growth in vivo (Pozzi et al. 2000; Pozzi et al. 2002). MMP-9 together with MMP-2 and
MMP-12 has been demonstrated to generate significant amounts of angiostatin by degradation
of plasminogen (Cornelius et al. 1998). Further on, our group reported that estradiol
decreased and tamoxifen increased the levels and activities of MMP-9 in experimental breast
cancer in vitro and in vivo, and tamoxifen-treated mice exhibited enhanced levels of
endostatin and decreased tumor growth as compared to estradiol-treated animals (Nilsson et
al. 2006; Nilsson et al. 2007).
MMP-3 is another MMP member with contradicting roles in cancer, as reported from several
studies. MMP-3 has been suggested to be protective in squamous cell carcinoma, as absence
of MMP-3 resulted in faster initial tumor growth rate and more aggressive phenotype of
tumors when MMP-3 null mice were exposed to chemical carcinogens, as compared to wild-
type mice (McCawley et al. 2004). In breast cancer MMP-3 levels were reported to be
upregulated, and MMP-3 was suggested to promote the epithelial-mesenchymal transition and
malignant transformation of breast epithelial cells and to induce genomic instability by
increased production of reactive oxygen species (Lochter et al. 1997; Sternlicht et al. 1999;
Radisky et al. 2005). In contrary, MMP-3 expression has been shown to be associated with
benign and low stage breast cancers, and that its expression frequently is lost in advanced
stages of disease (Brummer et al. 1999; Nakopoulou et al. 1999). Transgenic mice over-
expressing MMP-3 in their mammary glands developed fewer chemical-induced mammary
tumors than control mice, a fact that may be explained by the observed accelerated rate of
apoptosis in MMP-3 over-expressing cells (Witty et al. 1995).
Several MMP members have been recognized to influence inflammation in terms of both pro-
inflammatory and anti-inflammatory actions and the same MMP can have different roles in
different conditions (Page-McCaw et al. 2007). MMPs facilitate recruitment as well as
clearance of inflammatory cells in the tissue by cleaving inflammatory mediators from ECM,
resulting in a tightly regulated inflammatory response (Overall et al. 2002). As tumor
progression to some extent also is associated with inflammation, it has been hypothesized that
34
the clinical observations of upregulated levels of certain MMPs in tumor tissues might reflect
their roles of triggering and controlling inflammation (Page-McCaw et al. 2007).
Tissue inhibitors of metalloproteinases
The multifunctional role of MMPs may also be extended to their main endogenous inhibitors,
the TIMPs. The mammalian TIMP family has four members (TIMP-1, TIMP-2, TIMP-3, and
TIMP-4), which share structural homology at the protein level and reversibly block MMP
activity in a 1:1 stoichiometric ratio (Nagase et al. 2006; Stetler-Stevenson 2008). MMP
activity inhibition is accomplished through co-ordination of the Zn2+
of the MMP catalytic
domain by amino and carbonyl groups of the TIMP N-terminal cysteine residue (Stetler-
Stevenson 2008). TIMP-1 is the prototypic inhibitor for most MMP family members although
a poor inhibitor of MT-MMPs (Baker et al. 2002). TIMP-2 may, in addition to its inhibiting
properties, also interact with MT1-MMP to facilitate the activation of MMP-2 (Lambert et al.
2004). TIMP-3 predominantly inhibits the family of A Disintegrin And Metalloproteinase
(ADAM), a subgroup of MMPs, whereas less is known about TIMP-4 (Brew et al. 2000;
Stetler-Stevenson 2008).
Increasing amounts of data demonstrate that TIMPs exert biological activities not only
associated with MMP inhibition and actually may induce tumor promoting activities such as
cell growth stimulation and suppression of apoptosis (Guedez et al. 1998; Luparello et al.
1999; Lambert et al. 2004; Liu et al. 2005). Elevated levels of TIMP-1 is associated with
poorer prognosis in several types of cancer, breast and ovarian cancer included (Manenti et al.
2003; Rauvala et al. 2005; Kopitz et al. 2007; Kuvaja et al. 2007; Kuvaja et al. 2008; Oh et
al. 2010). TIMP-1 has additionally been suggested to have predictive value in metastatic
breast cancer as high levels have been shown to associate with poor response to chemotherapy
as well as to endocrine therapy (Schrohl et al. 2006; Lipton et al. 2007; Lipton et al. 2008;
Klintman et al. 2010).
35
Aims of the Thesis
The general goal of the present study was to explore angiogenesis regulation in hormone-
dependent breast- and ovarian cancer.
The specific aims of individual papers included in this thesis were the following:
Paper I: To investigate if estradiol and tamoxifen affect MMPs, TIMPs and endostatin
levels in hormone-dependent ovarian cancer and to explore the levels of these
proteins in ascites from ovarian cancer patients.
Paper II: To investigate if MMP-9 and TIMP-1 gene transfer with or without tamoxifen to
hormone-dependent breast tumor xenografts affect long term tumor growth and
angiogenesis.
Paper III: To investigate if MMP-3 and MMP-9 affect long term growth of breast cancer
xenografts in a dose-dependent manner.
Paper IV: To investigate if estradiol regulates IL-8 in normal and malignant breast tissue.
36
37
Comments on Materials and Methods
This chapter features comments on experimental methods used in paper I-IV. The main
purpose is to provide a general understanding of each method as well as to discuss possible
methodological draw-backs. For more detailed information of a specific experimental set-up,
please refer to the materials and methods section of each paper.
Experimental models
Tumor angiogenesis is governed by pro- and anti-angiogenesis mediators in the tissue. These
mediators are expressed and secreted either by tumor cells or other cell types in the tissue or
released by proteolytic cleavage from extracellular matrix components of the surrounding
stroma, in response to cell-cell or cell-stromal interactions. When investigating tissue levels of
factors involved in angiogenesis it is imperative to use experimental models that reflect the in
vivo complexity of the tumor microenvironment.
Cancer cell lines (I – IV)
Cell lines are used in many areas of biomedical research and particularly in the field of
cancer. In fact, a considerable part of the total knowledge of cancer is based on experimental
in vivo and in vitro studies of human cancer cell lines (Masters 2000; Lacroix et al. 2004; van
Staveren et al. 2009). An established human cancer cell line provides a pure system with
reproducible results when used with the same protocol and stage of cells, and avoids
interference of variations between individuals. The available commercial cell lines are most
often easy cultivated and represent an unlimited source of material. Their use in vitro
additionally bypasses ethical issues that may be associated with animal and human
experiments. Cancer cell lines are often issued from metastatic tumors and are frequently
debated as being unrepresentative of the tumor of origin, even though comparative studies of
cell lines and their corresponding tumors show that the cultures retain many of the original
tumor properties – such as ER expression – for a long time (Wistuba et al. 1998; Wistuba et
al. 1999; Masters 2000). There are however some important aspects that should be considered
when using cell lines; the genomic instability of cancer cells may lead to cell line variants, by
limiting the number of passages and always come back to frozen stock of early passage cells
this risk could be prevented (Hughes et al. 2007); cross-contamination between cancer cell
lines has been reported but can be verified by genotyping (Nelson-Rees et al. 1981; Rae et al.
2007; Schweppe et al. 2008); and finally to screen your cell line for mycoplasma as this
infection may change the properties of the culture severely (Young et al. 2010). In this thesis
38
the MCF-7 cell line was chosen for experimental set-ups on breast cancer, and BG-1 and
OVCAR-3 cell lines were used for ovarian cancer investigations.
The MCF-7 breast adenocarcinoma cell line was established in 1973 and issues from a pleural
effusion removed from a woman with metastatic breast cancer (Soule et al. 1973). Similar to
the majority of human breast tumors, MCF-7 are ER/PgR-positive making them suitable for
hormone-dependent investigations (Lacroix et al. 2004). In addition, their responsiveness to
estrogen has been shown to be retained over a sustained period of cell culture (Lippman et al.
1976).
BG-1 cells were derived from a patient with stage III, poorly differentiated ovarian
adenocarcinoma, and express high levels of ER and PgR (twice as much as MCF-7 cells),
making this cell line an excellent model for investigating hormone responsiveness of ovarian
cancer tissue (Baldwin et al. 1998). In addition, BG-1 produces and secretes the ovarian
tumor marker CA-125 into the culture medium in quantities sufficient to be clinically
significant (Geisinger et al. 1989). CA-125 is a high molecular weight glycoprotein which is
found in elevated levels in serum of the majority of women with ovarian cancer, whereas the
circulating level of CA-125 in serum of healthy women is absent or low (Ganrot 1997). CA-
125 is however a non-specific ovarian tumor marker and is not suitable for screening, as the
levels may be increased in other conditions such as pregnancy, endometriosis, pelvic
inflammatory disease, and in normal menstruation. The primarily use of the CA-125 marker
is to monitor therapy during ovarian cancer treatment.
OVCAR-3 origins from malignant ascites of a patient with progressive ovarian
adenocarcinoma and were originally described as ER-positive (Hamilton et al. 1983). There is
however contradicting data regarding ER-status and estrogen-responsiveness of OVCAR-3,
and some studies suggest that these cells do not express a functional ER (Lindner et al. 1997;
O'Donnell et al. 2005). Experiments performed in our laboratory on cell proliferation did not
observe any estrogen-induced growth response in OVCAR-3 and ERα was not detected by
immunocytochemistry.
HUVECs (IV)
Human umbilical vein endothelial cells (HUVECs) were used in paper IV to investigate the
proliferative potential of IL-8 on endothelial cells. Primary endothelial cells have a limited
lifespan. In angiogenesis research, immortalized endothelial cell lines or cell lines with
prolonged lifespan are commonly used as in vitro model systems (Bouis et al. 2001).
HUVECs were preferred in this study, as they are more likely to retain traits and
characteristics of normal endothelial cells in vivo as compared to permanent endothelial cell
lines. Endothelial cells express ER and are targets for estrogens (Kim-Schulze et al. 1996;
Venkov et al. 1996). Estrogens induce multiple effects on the endothelium, including gene
transcription, protein synthesis, and oxidative metabolism, resulting in physiological
consequences such as vasodilation and angiogenesis among others (Losordo et al. 2001;
Garvin et al. 2005; Kim et al. 2008; Miller et al. 2008).
39
In this study, fresh umbilical cords were obtained at the delivery ward at Linköping
University Hospital and HUVECs were isolated by enzymatic digestion according to a
modified version of the method described by Jaffe et al. (Jaffe et al. 1973). Female donors
were used since the majority of breast cancer patients are women. The experiment was
conducted on cells from a single donor and repeated on cells isolated from another donor.
Due to the limited lifespan of HUVECs, all experiments were conducted on cells from
passages 2-3.
Normal breast tissue in culture (IV)
Sex steroids are involved in hormone-dependent tumorigenesis, but have also a crucial role in
normal physiology of the same tissues. When investigating the effects of sex steroid exposure
regarding the expression of a certain protein in a tumor it is also of major interest to evaluate
the physiologic profile in correlating normal tissue exposed to hormones. Lack of convenient
models has resulted in limited knowledge in this area. For this purpose a method of culturing
whole normal breast tissue ex vivo has previously been set up and used in this laboratory
(Garvin et al. 2006). Briefly, breast tissue biopsies, containing intact epithelium, stroma and
adipose tissue, are produced from human breast tissue obtained from healthy pre-menopausal
women undergoing routine reduction mammoplasty. None of the women included in this
thesis were taking contraceptives or had any other kind of ongoing hormonal treatment. This
method allows for culture of whole breast tissue in vitro for up to one week with verified
preserved morphology and structural integrity, and with immunohistochemical analyses
confirming sustained expression of ER, PgR, and the proliferative marker Ki-67 (Garvin et al.
2006). There were not any detectable differences in breast tissue morphology after one week
of treatment with or without the addition of serum to the culturing media. This is of
importance as investigations of hormone-induced effects on cells and tissues require a serum-
free milieu to avoid that serum-containing confounding factors affect the results. The
occurrence of inter-individual differences in biological response must be considered and is a
limitation but also strength of the method, as all treatment groups origin from one donor and
is repeated on another.
Hormonal treatments of cells/tissues in culture (I, IV)
In this thesis, ovarian cancer cells, breast cancer cells and normal breast tissue biopsies were
exposed to hormones and treated for one week. During treatments the hormone medium was
changed and fresh hormones administered to the cultures every 24 hours. Serum-free
culturing media was used for all experiments because physiological levels of steroid
hormones are present in serum (Esber et al. 1973), and estradiol concentrations as high as 10
nM have been found in some fetal calf sera (Bodgen et al. 1974). Decreased hormone levels
of serum may be achieved by charcoal-filtration, which is commonly used for this kind of
experiments. Analyses performed on charcoal-filtrated serum in our laboratory revealed
40
however that low estradiol levels still were detectable, therefore serum-free medium was
preferred. We additionally excluded cell culture media containing phenol red, as this
compound has been shown to exert estrogenic effects (Berthois et al. 1986). The naturally
occurring 17β-estradiol was used in all experiments. Given the biphasic dose-response of
estradiol we aimed to use physiological concentrations at all times. The term physiological
may not be suited when discussing in vitro studies, our aim was however to use hormone
concentrations that reflect the in vivo situation. Plasma levels of sex steroids in
premenopausal, non-pregnant women fluctuate throughout the different phases of the
menstrual cycle. Plasma estradiol in these women range between 100-1500 pM, with the
lower concentrations in the follicular phase and increased levels in the luteal phase, and with
the highest peak measured just prior to ovulation (Ganrot 1997). The concentration of
estradiol in plasma of postmenopausal women is decreased and range between 40-150 pM
(Ganrot 1997). Breast tissue concentrations of estradiol have been shown to be similar in pre-
and post-menopausal women despite the large differences in peripheral plasma levels (van
Landeghem et al. 1985). In malignant human breast tissue the concentration of estradiol may
be significantly increased as compared to non-malignant breast tissue (Pasqualini et al. 1997;
Chetrite et al. 2000; Clarke et al. 2001). We used the physiological concentration of 1000 pM
estradiol for the treatment of normal breast tissue biopsies as well as for the treatment of
breast- and ovarian cancer cells.
For progesterone (P4) treatments of normal breast tissues we used a concentration of 10 nM as
plasma levels in premenopausal, non-pregnant women range between 0.5-40 nM, and the
chosen concentration is in line with circulating levels in the luteal phase of the menstrual
cycle (Ganrot 1997).
Tamoxifen was used in a concentration of 1 μM for the treatment of MCF-7 breast cancer
cells, which is equivalent to therapeutic serum concentrations in breast cancer patients (FASS
2010). For the treatment of ovarian cancer BG-1 and OVCAR-3 cells, tamoxifen was used in
the concentration of 2 μM. Tamoxifen is rarely used as a treatment strategy in ovarian cancer
patients. However, it may be used as a second line choice of pharmaceutics in metastatic
ovarian cancer disease, and these women are usually administered with double dose of
tamoxifen, as compared to women that are taking tamoxifen as a treatment against hormone-
dependent breast cancer (Perez-Gracia et al. 2002). In addition, the 2 μM concentration of
tamoxifen used in our study is also in line with in vitro studies evaluating the anti-
proliferative effects induced by tamoxifen exposure to ovarian carcinomas reporting a better
response by 2 μM than by 1 μM (Lazo et al. 1984; Nash et al. 1989).
As discussed in the background, tamoxifen is extensively metabolized by the cytochrome
P450 (CYP) enzyme system into several active metabolites, such as 4-hydroxy-tamoxifen and
endoxifen (Lim et al. 2005) (Figure 4). These two metabolites are approximately 100-fold
more potent than the parent drug to antagonize ER (Stearns et al. 2003). 4-hydroxy-tamoxifen
continues to be used by researchers for in vitro studies of tamoxifen, even though human
tamoxifen pharmacokinetic data not support a major role for 4-hydroxy-tamoxifen in vivo
given the fact that steady-state plasma concentrations of this compound are very low (< 10
nM) (Borges et al. 2006). The plasma concentrations of endoxifen in breast cancer patients
41
that are taking tamoxifen chronically are approximately 10-fold higher than those of 4-
hydoxy-tamoxifen, suggesting that endoxifen is likely to be more important clinically (Jin et
al. 2005; Lim et al. 2005; Lim et al. 2006). Recent data indicate that endoxifen but not 4-
hydoxy-tamoxifen induces degradation of ER-α in addition to its antagonistic effects on the
transcriptional level (Wu et al. 2009). However, endoxifen is formed almost exclusively by
the polymorphic CYP2D6 (Desta et al. 2004), and genetic variations of the gene coding for
CYP2D6 may result in significantly decreased plasma concentrations of endoxifen (Hoskins
et al. 2009). A recent clinical study, involving over 1 300 women with breast cancer and
tamoxifen treatment, reported an association between CYP2D6 polymorphisms and clinical
outcome (Schroth et al. 2009). On the contrary, other studies report no such association
(Wegman et al. 2005; Wegman et al. 2007; Okishiro et al. 2009). Several drugs are
metabolized through the CYP2D6 metabolic pathway and have been suggested to interfere
with the efficacy of CYP2D6 activity and generation of tamoxifen metabolites (Stearns et al.
2003; Jin et al. 2005; Borges et al. 2006; Sideras et al. 2010). Biotransformation of tamoxifen
occurs mainly in the liver, but the responsible CYP enzymes are expressed in normal and
cancerous tissues throughout the body (Romkes-Sparks et al. 1994; Guidice et al. 1997;
Nagai et al. 2002). Expression of genetic variations of CYP2D6 in normal as well as
cancerous breast and ovarian tissues has been reported (Huang et al. 1996; Huang et al. 1997;
Downie et al. 2005). As the physiological role of 4-hydroxy-tamoxifen may be questioned
and endoxifen is not yet commercially available, the mother-substance tamoxifen has been
used in all experiments.
Study populations (I, IV)
Paper I
In paper I, ascites was collected by abdominal paracentesis from 17 women diagnosed with
advanced ovarian carcinoma (Table II). The local ethical committee approved the study and
all women gave their informed consent. None of the women had any ongoing treatment at the
time of collection. The ovarian tumor stage in these women ranged between III and IV (FIGO
classification; staging system developed by the International Federation of Gynecology and
Obstetrics) and patients were radically operated with various degree of tumor burden, ranging
from minor to massive tumor load. All tumors demonstrated serous papillary adenocarcinoma
histology, as determined at the Department of Pathology and Cytology, University Hospital of
Linköping.
Ascites is the accumulation of serous fluid in the peritoneal cavity and is a characteristic event
in ovarian cancer patients (Berek 2003). The accumulation of ascites is due to increased
capillary permeability and the following increased capillary filtration from tumors located
inside entire or restricted parts of the peritoneal cavity. In addition, tumor obstruction of the
mesenteric lymphatic system results in decreased capacity of the normal peritoneal fluid
outflow. The presence of ascites may facilitate the spread of disseminated cancer cells from
42
the site of origin to other sites in the peritoneal cavity. The location of ovarian tumors implies
a close contact between ascites and ovarian tumor tissue. Hence, the ascites contains secreted
products from the tumoral tissue, making this fluid an interesting target for investigations of
ovarian cancer characteristics.
Table II.
Ovarian cancer patient and ascites characteristics.
Patient
No.
Age Stage
(FIGO)
Estradiol
(pg/mL)
Progesteron
(ng/mL)
MMP-2
(ng/mL)
MMP-9
(ng/mL)
TIMP-1
(ng/mL)
TIMP-2
(ng/mL)
Endostatin
(ng/mL)
1
77
III
37
0.6
54.6
0.0
5169
517
14.7
2 65 IV 0 0.7 61.8 0.4 4486 319 24.9
3 68 IV 0 0.8 52.7 0.0 3463 520 14.7
4 61 IV 0 0.1 66.6 1.3 3793 497 20.9
5 72 III 0 0.2 62.8 0.2 3268 345 22.9
6 51 IIIB 39 0.2 61.3 6.1 1875 239 24.3
7 82 IV 0 0.3 70.6 0.3 2848 319 26.4
8 67 IV 0 0.3 62.0 2.7 4961 410 19.3
9 56 IIIC 0 0.4 56.2 0.0 3060 391 20.3
10 65 III 0 0.2 55.0 1.8 2151 395 19.5
11 56 IIIC 0 0.3 58.6 0.1 5771 809 23.5
12 71 IIIC 0 0.2 73.4 3.0 6110 626 34.6
13 79 IV 0 0.2 57.4 1.2 2386 547 15.5
14 69 III 37 0.5 55.9 0.4 3210 444 24.2
15 66 IIIC 0 0.5 63.7 0.1 3884 562 24.2
16 59 IIIC 0 0.2 56.1 1.0 3590 361 28.5
17 34
IIIC 106
0.2
60.3
0.2
3264
270
25.4
Modified from Bendrik et al., Cancer Letters 2010, 292; 32
FIGO III: Microscopic peritoneal metastasis beyond pelvis and/or regional lymph node metastasis;
IIIB: Macroscopic peritoneal metastasis ≤ 2 cm; IIIC: Macroscopic peritoneal metastasis > 2 cm; IV:
Distant metastasis.
Paper IV
In paper IV, microdialysis was used to sample IL-8 from normal human breast tissue in pre-
and postmenopausal women, and preoperatively in breast cancer patients. The local ethical
43
committee approved the study, and all women gave their informed consent. The healthy
volunteers consisted of 12 premenopausal women at the age of 22-30, with a history of
regular menstrual cycles (cycle length 27-34 days). Four women were investigated in the
follicular phase and eight in the luteal phase. There were additionally six postmenopausal
women in the healthy volunteer group, with an age of 52-55 years. None of the subjects had
any ongoing sex steroid containing medication, such as contraceptives or hormone
replacement therapies, or had any such treatment for the past 3 months. Postmenopausal
status was defined by having no spontaneous menstrual bleeding for at least 1 year in women
older than 50.
Table III.
Breast cancer patient and tumor characteristics.
1 2 3 4 5 6 7 8 9 10
Age
67
59
51
86
83
66
56
68
65
57
Histology lobular ductal ductal ductal ductal ductal ductal lobular lobular ductal
Size (mm) 18 9 20 60 20 20 17 15 50 21
Stage (T) 1 1 1 3 1 1 1 1 2 2
ER (%) 50 80 100 80 100 80 100 75 75 75
PgR (%) 100 10 0 80 100 0 0 0 50 0
Modified from Garvin & Dabrosin, BMC Cancer 2008, 8; 73
T1: Tumor 2 cm or less in greatest dimension without axillary lymph node metastasis; T2: Tumor less
than 2 cm and presence of axillary node metastasis, or tumor 2-5 cm with or without spread to local
lymph nodes; T3: Tumor more than 5 cm in greatest dimension with or without lymph node
metastasis. All tumors extending to chest wall or engagement of skin. Inflammatory carcinoma,
regardless of lymph nodes.
The 10 breast cancer patients who kindly volunteered to participate in this study were all
postmenopausal women with an age of 51-86 years. Seven of these ten women were
diagnosed with invasive ductal carcinoma, and three with invasive lobular carcinoma. All of
the tumors were ER-positive and 5 out of 10 tumors were also PgR-positive. Tumor histology,
size, and stage (TNM classification; tumor size, node involvement, metastasis status) were
determined at the Department of Pathology and Cytology, University Hospital of Linköping.
Patient characteristics are provided in Table III.
44
Animal models (I – IV)
In vivo investigations of human cancers are largely facilitated by the availability of various
immunodeficient mice strains that allow for the establishment and growth of human tumor
xenografts. The nude mouse has a mutation in the Foxn1 gene, resulting in deteriorated or
absent thymus and greatly reduced number of T-cells. Due to the non-functional immune
system, the nude mouse can receive foreign cells and tissues without the rejection response
that normally occurs. The phenotype of this mouse is lack of body-hair, giving it its “nude”
nickname. The absent immunological response is a prerequisite for tumor implantation and
growth but may also be considered as a draw-back in cancer investigations as the model fails
to reflect the real in vivo situation with a competent immune system. However, although
athymic nude mice lack a complete functional immune system, they have been shown to
demonstrate higher cytotoxic activity of NK cells and macrophages than euthymic mice
(Budzynski et al. 1994). This fact indicates that certain immunological responses on tumor
induction and progression indeed may be investigated using the athymic mouse model.
Interspecies differencies between human and murine tumors suggests that the human tumor
xenograft model is superior to spontaneous murine cancer models (Anisimov et al. 2005). In
addition, human xenografts in nude mice confer an important advantage over the use of
murine mammary carcinoma models in terms of hormone responsiveness. Mouse mammary
tumors express low levels of estrogen and progesterone receptors and are poorly responsive to
hormones, in contrast to human breast tumors of which approximately 70% are hormone
receptor positive (Clarkson et al. 2004). Xenografts of human tumors, or tumor cell lines in
nude mice has been shown to reproduce the histology and metastatic pattern of most human
tumors at advanced stage (Cespedes et al. 2006).
In this thesis, we used the Balb/cA model of nude mice. Female mice at the age of 6-8 weeks
at the time of purchase were oophorectomized and supplemented with 17β-estradiol in the
form of subcutaneous 3-mm pellets (0.18 mg/60-day release) implanted in the neck. The
pellets ensure a continuous and stable release of estradiol, with serum concentrations of 150-
250 pM as confirmed by serum analysis in our laboratory (Dabrosin 2003). These levels
represent physiological concentrations of estradiol as observed in both human and mice
during the estrous cycle. Tamoxifen was administered by subcutaneous injections, 1 mg every
2 days in MCF-7 tumor-bearing mice, yielding serum concentrations comparable to
therapeutic levels in breast cancer patients (FASS 2010). For ovarian cancer studies,
tamoxifen was administered to BG-1 tumor-bearing mice in a dose of 2 mg every 2 days.
These concentrations are equivalent to the levels of tamoxifen used for treatment of MCF-7
and BG-1 cancer cells in vitro. As both MCF-7 and BG-1 cells are highly estrogen-dependent
cancer cells, tumor growth in an untreated control group is unfortunately not achievable. The
continuous treatment of estradiol in physiological levels upon the addition of tamoxifen
reflects the tumor microenvironment in both pre- and post-menopausal breast cancer patients,
and would most probably also reflect the hormonal exposure of ovarian tumors if tamoxifen
was used as a treatment strategy in ovarian cancer patients (van Landeghem et al. 1985).
45
Assessment of tumor growth (I – IV)
Human breast and ovarian cancer cells injected subcutaneously on the flank or in the
mammary gland of Balb/cA resulted in palpable tumors in approximately 10 days. Tumor
growth was monitored by measuring length, width, and depth of the tumor with a calliper
every 2 or 4 days throughout the experiments. Tumors were size-matched and exposed to
treatment with tamoxifen or vectors at approximately identical tumor volume. Monitoring
tumor growth by using a calliper is a standard approach in xenograft animal models when the
tumors are subcutaneous and visible. It is a rapid, non-invasive method to follow the growth
of the tumor throughout the experiment. At the end of experiments, tumor sections from all
explanted tumors were in addition exposed to H&E staining to verify absence of necrosis and
to assure viable tumor cells. In studies of internal xenograft tumors the standard assessment is
a single experimental end-point measurement of tumor volume and/or weight. MCF-7 cells
used in our human breast tumor xenograft studies are considered to have a low invasive
potential and rarely metastasize. In xenograft studies using tumor cells with more invasive
properties and where the aims involve localisation and quantification of metastases, the
assessment of bioluminescence may be a more convenient, but also technically more
advanced and complex method. The tumor cells are then transfected with the gene for
luciferase or green fluorescent protein (GFP) prior to injection, whereafter the tumor cells and
the tumor load may be followed over time in the same animals by specific detection systems
(Jenkins et al. 2003; Jenkins et al. 2005; Klerk et al. 2007).
The microdialysis technique (II, III, IV)
Microdialysis is a technique that allows for investigations of the extracellular chemistry of an
individual organ or tissue (Ungerstedt 1991). The principle of the technique is to mimic the
function of a capillary blood vessel (Figure 7). The microdialysis system consists of a
microdialysis pump and a microdialysis catheter (which is also called a probe). The probe is a
hollow fibre with a semipermeable membrane at the tip of the probe, which is inserted into
the tissue of interest, and perfused with a continuous flow of perfusate that after a certain
equilibrium time allows for passive diffusion of interstitial molecules over the membrane. The
perfusion fluid enters the probe through the inlet tubing at a constant flow rate (generally 0.5-
5 μL/min), passes the membrane and is transported through the outlet tubing and collected in
microvials (Ungerstedt 1991; Plock et al. 2005) (Figure 7). The fluid balance is crucial and
the recovery of perfusion fluid should be 100%; meaning, the same volume that enters the
microdialysis probe should also exit to ensure a diffusion-driven sampling of molecules.
Depending on the pore size of the membrane (the molecular weight cut-off (MWCO)),
various concentrations of dextrans (neutral polysaccharides) is added to the perfusion fluid to
maintain the osmotic pressure and to avoid ultrafiltration and loss of perfusion fluid into the
tissue (Rosdahl et al. 2000; Dahlin et al. 2010). In present studies, a colloid (40 g/L dextran-
70 and 154 mM NaCl) was added to the perfusion fluid.
46
The most important parameter in microdialysis is the extraction efficiency, which also can be
termed the relative recovery. The recovery of a given substance is dependent on several
factors, such as the MWCO of the membrane, the flow rate of the perfusate, the length of the
membrane, and the ability of the molecule to cross the membrane due to its characteristics,
electrical charging of side chains etc. (Figure 8). The recovery of a certain molecule can be
evaluated by in vitro experiments, and is defined as the concentration of the analyte in a
perfusate divided by the concentration of a standard solution on which the microdialysis is
performed. The mean in vitro recovery value was 6 ± 0.06% for VEGF at a flow rate of 0.6
μL/min, 33 ± 13% for endostatin at 1 μL/min and 15.3 ± 0.3% for IL-8 at a flow rate of 1
μL/min. In vitro recovery can only be an estimate of in vivo recovery since additional factors
such as interstitial pressure, blood flow, and body temperature may affect the diffusion of
substances (Ungerstedt 1991; Plock et al. 2005). It should be pointed out, that microdialysis
in this thesis was used to compare interstitial levels of specific analytes between groups, not
to quantify the absolute levels of the analytes in tumors or other tissues. Therefore, all
microdialyses values are presented as original data.
Figure 7.
The microdialysis probe mimics a blood capillary inserted into the tissue. Chemical substances from
the extracellular fluid diffuse across the dialysis membrane of the probe and are carried out by a flow
of perfusion fluid. The collected dialysates may thereafter be analyzed for substances of interest.
Adapted from CMA Microdialysis.
47
Microdialysis was originally applied in neurosciences for the measurement of
neurotransmittors (Ungerstedt 1991). An important application of the microdialysis technique
is still in the field of brain tissue studies, and microdialysis is widely used clinically for
monitoring neurochemistry changes after brain injury, but its use has also spread to diverse
areas in biomedical sciences. Microdialysis is today applied for investigations of extracellular
chemistry in a variety of peripheral tissues, such as muscle, skin, heart, lung, and kidney
amongst others (de la Pena et al. 2000; Rosdahl et al. 2000; Herkner et al. 2002; Mantovani et
al. 2002; Kennergren et al. 2003; Plock et al. 2005; Samuelsson et al. 2006; Farnebo et al.
2010). C. Dabrosin introduced and developed microdialysis as an application for in vivo
investigations of normal human breast tissue and of human breast cancerous tissue (Dabrosin
et al. 1997; Dabrosin 2003; Garvin et al. 2003; Dabrosin 2005; Garvin et al. 2008).
In this thesis, microdialysis was used to quantify MMP activity in human breast tumor
xenografts in nude mice after intratumoral gene transfer of MMP-9 and TIMP-1. Sampling of
extracellular fluids from these tumors by microdialysis allowed for investigations on how
VEGF levels as well as endostatin generation are affected by tumor over-expression of
human genes of MMP-9 or TIMP-1. Endostatin levels were in addition quantified in
microdialysates collected from human breast tumor xenografts in mice treated with low or
high dose of intratumoral MMP-3 and MMP-9, to investigate if the generation of endostatin
by MMP activities was dose-dependent. Microdialysis was also applied for the sampling of
IL-8 and estradiol in normal human breast tissue and in subcutaneous abdominal fat in pre-
and postmenopausal healthy female volunteers, and pre-operatively in breast cancers of
women. Microdialysates collected from experimental breast cancers in mice were also
investigated regarding levels of IL-8 and correlated to estradiol and tamoxifen treatment and
microvessel area of the tumors.
Figure 8.
The recovery of a given substance is
dependent on several factors, such as
the MWCO of the membrane, the flow
rate of the perfusate, the length of the
membrane, and the ability of the
molecule to cross the membrane.
Adapted from CMA Microdialysis.
48
Changes in expression levels of a large amount of bio-active molecules in the tumor
microenvironment are necessary steps in cancer development and progression. Hence, in vivo
methods investigating these changes in the extracellular compartment of the tumor are crucial.
Microdialysates collected from tumoral tissues reflect the chemical composition of the
tumoral extracellular milieu, which encounters release/uptake of molecules from – not only
the cancer cells themselves – but from all cell types present in the tumor, as well as bio-active
molecules enzymatically released from stromal components, such as collagens.
Unfortunately, the localization of ovarian tumors implies the difficulties of using
microdialysis as a tool for in vivo investigations of angiogenesis preoperatively in human
ovarian cancer studies. However, regulatory mediators of angiogenesis may be investigated
with this technique in human ovarian tumor xenografts implanted subcutaneously in nude
mice.
The main application for the microdialysis method has been sampling of small molecules
(e.g., glucose, urea, pyruvate, and lactate) from tissues, and microdialysis probes with
membrane pores exceeding 100 000 Da MWCO are not yet commercially available. Increased
pore size of the membrane is associated with increased ultra-filtration and loss of fluid into
the tissue. This implies a limitation for investigations of extracellular levels of larger
molecules, such as enzymes, cytokines, and other proteins. Tissue levels of MMP-9 for
example, can not be investigated by microdialysis as the properties of the molecule not allow
for diffusion over the membrane. However, the microdialysis technique may be used for
investigating levels of enzymatic activity in the tissue, a method that in this study was used
for determination of tumor MMP-9 activity and which is further discussed in the next section.
A potential drawback of microdialysis that has to be taken into consideration is the fact that
tissue trauma may be caused by the insertion of the probe. Tissue trauma will lead to an
inflammatory response which may affect tissue levels of the molecule of interest. This aspect
is particularly important when investigating mediators that are involved in/affected by the
inflammatory response.
Direct quantification of tumor MMP-9 activity in vivo (II)
Increased levels of MMPs in a tissue as detected by specific antibodies utilizing methods such
as IHC or ELISA, does not necessarily correlate with the enzymatic activity of the same
MMP in the tissue. MMPs are secreted from the cell as pro-enzymes and require proteolytic
removal of the pro-domain in order to exert their activities. In addition, activated MMP
enzymes may be bound to TIMPs and unable to have any function even though present in the
tissue. We have previously employed an approach for direct investigations of MMP activity in
breast tumor tissue of nude mice, using the microdialysis technique (Nilsson et al. 2006). In
paper II, this method was used to investigate the intratumoral activity of MMP-9 in vivo after
gene transfer of MMP-9 and TIMP-1. One week after adenoviral injections, mice were
anesthesized and microdialysis probes with 20-kDa molecular mass cut-off membrane were
49
inserted intratumorally. As perfusion fluid a quenched, fluorescent MMP-2/MMP-9-specific
substrate dissolved in physiological solution was used. The intact substrate consists of a
fluorescent Trp residue and a dinitrophenol (DNP) quenching group on the N-terminus.
During perfusion, the substrate will passively diffuse from the inner side of the microdialysis
membrane into the tissue. When active MMP-9 is present in the tissue, the quenching part of
the substrate will be enzymatically cleaved off which allows for continuous recording of
fluorescence. The 20-kDa cut-off allows for diffusion of the substrate into the tumor tissue
but excludes the direct traversing of MMP-2 (72 kDa) and MMP-9 (92 kDa) from the
interstitial space over the membrane. The fluorescence in the collected microdialysates will
thereby directly correlate with the MMP activity in vivo. The entire microdialysis system was
protected from ambient light to prevent fading of the substrate and after an equilibrium period
of 30 minutes, the dialysates were collected in 30-minutes intervals into chilled tubes and
immediately subjected to fluorometry measurements.
This fluorogenic MMP-2/-9-specific substrate was additionally used in paper I for in vitro
studies, investigating the activity of MMP-9 in conditioned media from hormone-treated
ovarian cancer BG-1 cells. Briefly, conditioned media collected after one week of BG-1
exposure to estradiol or tamoxifen, was mixed 1:1 with the fluorogenic substrate in a dark 96-
well plate. After 20 minutes of gentle agitation and protected from light, fluorescence was
determined and correlated to total protein of the cell lysates of each sample.
Gene transfer studies (II, III)
Overexpression of MMPs and TIMPs of human breast tumor xenografts was achieved by
intratumoral delivery of adenoviral vectors containing the human gene of MMP-3, MMP-9, or
TIMP-1. Adenoviruses (Ads) are widely used as mammalian gene transfer vectors due to their
high efficiency in gene transduction to a wide range of cells in both quiescent and
proliferating states from various species, their high production titer, and their inability to
integrate the host genome (Yeh et al. 1997; Dormond et al. 2009). The Ad is a non-enveloped
icosahedral capsid comprising approximately 30-40 kb of linear double-stranded DNA. The
viral DNA molecule is left free in the nucleus of the host cell and not incorporated in the
genome of the host. This extra DNA is transcribed and expressed as other genes, but will not
undergo replication at host cell division and therefore the viral DNA expression is lost in
descendant cells. Ads have been extensively used as vectors for high level protein production
in cultured cells, for recombinant vaccines, and for gene transfer where a transient transgene
expression is desired (Hitt et al. 1997; Palmer et al. 2008). Adenoviruses are dependent on the
early region 1 (E1) of the viral genome for ability to replicate. The E1 region is therefore
deleted in recombinant adenoviral vectors and the virus is replication-deficient, an important
aspect regarding the safety for human gene therapies.
50
The vectors used in the present study were constructed and transgene expression verified by
co-authors of paper II. Briefly, cDNAs from MMPs and TIMPs were cloned into a shuttle
vector (pDC316). The recombinant pDC316 vector and a viral E1-deficient vector were co-
transfected into 293 cells, a human kidney cell line which has been stably transfected with the
E1 region of the viral genome. Following co-transfection, the two DNA species undergoes
homologous recombination within these cells resulting in a recombinant Ad vector. The Ad
vector replicates and is packaged into infectious virions in the 293 cells, which cause plaques
to form on the monolayer of the cells (Palmer et al. 2008). The infectious viral concentration
is often reported as plaque-forming units (pfu), as in the present study, but may also be
referred to as infectious units (IU), infectious particles (IP), or transducing units (TU) among
others (Dormond et al. 2009). In the present study a control Ad vector (Addl70) was used in
all set-ups. The Addl70 is of identical construct as AdMMP and AdTIMP, with the only
exception that it does not contain any human gene sequence. Addl70 was injected
intratumorally in the same concentrations as AdMMPs and AdTIMPs.
Transgene activity of MMP-9 and TIMP-1 was evaluated by zymography and reversed
zymography respectively. Bronchoalveolar lavage from AdMMP9 or AdTIMP1-infected rats
was prepared and loaded onto SDS-PAGE gels containing gelatin, with the addition of
gelatinases in the gel used for reversed zymography and evaluation of AdTIMP1 activity.
TIMP-1 activity was visualized as a dark band against clear background, and MMP-9 as a
clear band against a dark background (Figure 9A and B).
Naturally occurring Ads are common human pathogens and exposure is generally associated
with low risk of causing serious disease. Ad infection is usually associated with a mild
respiratory illness with symptoms such as sore throat, cough, fever, and runny nose (common
cold). Most people are largely exposed to Ads in childhood and adults have therefore in
general acquired a solid immunity. The risk factor may be increased however, when exposed
to genetically altered viruses, depending on which gene is expressed. When performing Ad
gene transfer on animals, virus may be shed in animal feces for up to 10 days after
administration. Precaution should be taken with contagious waste as contact with mucous
membranes or cuts in the skin may cause exposure. Although the vectors are replicative-
defective there is a minor risk that the inoculated virus combines with naturally occurring
viruses in animals or people and regain their ability to replicate. In addition, there is also a
possibility that some of the Ad vectors could be contaminated with replication-competent
virus.
51
A
B
Cell viability assay (I, IV)
In paper I, the responsiveness of ovarian cancer BG-1 and OVCAR-3 cells to estradiol and
tamoxifen regarding cell proliferation was evaluated by using the Cell Titer 96® Aqueous One
Solution Cell Proliferation Assay (MTS assay). There are a number of methods that can be
employed for investigating the rate and amount of cell proliferation. Traditionally, cell
proliferation in vitro may be determined by direct counting of cells and determination of the
mitotic index. This method is however labor-intense and not practical for evaluating large
numbers of samples. Since cellular proliferation requires the replication of cellular DNA,
other methods is based on monitoring the synthesis of DNA. Most commonly used is [3H]-
thymidine for incorporation and labelling the DNA of mitotically active cells. The use of
radio-isotopes and the handling of radio-active disposals are disadvantages of these kinds of
assays as is the need for specialized and expensive equipment. The MTS assay is indirectly
measuring cell proliferation as it determines the number of viable cells in culture. It is a
convenient, non-toxic method performed in 96-well microplates and the results are easily
Contr Addl-70 AdTIMP-1 AdTIMP-1
5 x 108
pfu 1 x 109
pfu
Contr AdMMP-9
1 x 109
pfu
Figure 9.
Adenoviral vector efficacy and transgene
activity of MMP-9 and TIMP-1 was
evaluated by zymography and reversed
zymography respectively. Bronchoalveolar
lavage from AdMMP-9 and AdTIMP-1-
infected rats was loaded onto SDS-PAGE
gels. A) MMP-9 activity was visualized as
a clear band against a dark blue background
and B) TIMP-1 activity as a dark band
against a clear background.
52
quantified with a standard ELISA reader. The assay contains the MTS tetrazolium compound
(also called Owen´s reagent) which is bioreduced by cells into a coloured formazan product
that is soluble in cell culture media. The conversion is accomplished by NADPH or NADH
produced by dehydrogenase enzymes in metabolically active cells. The quantity of formazan
as measured by the amount of 490 nm absorbance is directly proportional to the number of
living cells in culture.
The MTS assay was additionally employed in paper IV to investigate the effects of estradiol-
induced IL-8 on HUVEC growth in vitro. HUVEC viability was determined after three days
of culture in conditioned media collected from breast cancer MCF-7 cells treated with
estradiol alone or in combination with the human anti-IL8-antibody, ABX-IL8. HUVEC
viability after exposure of recombinant IL-8 was also investigated.
The choice of method to measure proliferation is depending on the specific aim of the
investigation. If the aim is to investigate the direct mitotic effect of a compound of interest
another method might be superior. In these studies the general aim was to evaluate and
compare the amount of living cells after a certain time of exposure with different compounds.
Quantification of proteins and sex steroids (I – IV)
In this study, quantitative determinations of proteins in tumor tissue homogenates,
microdialysates, cell culture supernatants, and ascites were performed using commercial kits
employing the ELISA (enzyme-linked immunosorbent assay) technique. Briefly, these assays
use the sandwich type of technique, were 96 microplate wells are pre-coated with a
monoclonal antibody specific for the protein of interest. Samples and standards of
recombinant protein are added to the wells and the protein of interest, if present in the
samples, will bind to the immobilized antibodies in the wells. To sandwich the protein-
primary antibody complex a secondary enzyme-linked polyclonal antibody also specific for
the given protein is added. Repeated washing-steps ensure the removal of unbound sample
and excess enzyme. The addition of a substrate specific for the enzyme results in color
development or emission of light which is proportional to the amount of protein bound to the
primary antibody of the initial step. A luminometer or a spectrophotometer is used for
measuring the light/color intensity dependent on which substrate is used.
Due to the fact that microdialysis yields a limited dialysate volume, the Fluorokine MAP
cytokine assay (luminex system) was used for determination of IL-8 levels in microdialysates
from normal breast tissue and abdominal subcutaneous fat, from breast cancers preoperatively
in women, and from breast tumors in nude mice (paper IV). This method is more sensitive as
compared to the Quantikine IL-8 ELISA (0.39 pg/ml as compared to 3.5 pg/ml) and allows
for measurements of multiple proteins simultaneously in a limited sample of volume. Color-
coded beads, pre-coated with analyte-specific capture-antibodies for proteins of interest (in
this case anti-IL-8) are pipetted into wells together with samples and standards. In the second
step biotinylated detection antibodies specific for IL-8 are added and form an antibody-
antigen sandwich on the beads. Streptavidin-conjugated phycoerythrin is added to the wells
53
and the measurement is performed on a Luminex® 100TM
analyzer. The detection system is
accomplished by two lasers, one that is classifying the bead and determines the analyte that is
being detected. The second laser determines the magnitude of the signal from the streptavidin-
conjugated phycoerythrin, which is proportional to the amount of bound analyte.
For the quantification of estradiol and progesterone in plasma and microdialysates from
healthy volunteers and breast cancer patients (paper IV), we used commercial ELISA kits
employing a detection technique based on the principle of competitive binding. Instead of the
addition of an enzyme-linked polyclonal antibody to sandwich the hormone of interest
(estradiol or progesterone), a horseradish peroxidase (HRP) conjugated to the hormone is
added to the sample. This HRP-hormone-conjugate competes with estradiol or progesterone
present in the sample for binding to the immobilized primary antibodies in the wells. The
amount of bound peroxidase-conjugate is inversely proportional to the concentration of the
given hormone in the sample.
Immunohistochemistry (I – IV)
Immunohistochemistry (IHC) is a common method that allows for detection of proteins in a
tissue by the use of specific antibodies. In this thesis, IHC was used to confirm the presence
of MMP-9 in BG-1 tumors, von Willebrand´s factor (Factor VIII) in BG-1 tumors, MCF-7
tumors, and normal breast tissue biopsies, and IL-8 in normal breast tissue biopsies and MCF-
7 tumors in nude mice.
Before the procedure of IHC staining can take place, tissue preparation by fixation is an
essential event to preserve tissue architecture and cell morphology. The chemical fixation of
4% paraformaldehyde may be the most widely-used fixation of whole tissue specimen, and
was used in all our experiments. Paraformaldehyde rapidly penetrates the tissue and arrests
enzymatic degradation. It also denatures and cross-links proteins, leading to tissue hardening.
MCF-7/BG-1 tumors were rapidly removed from euthanized mice and immersed into
paraformaldehyde. After approximately 48 hours of fixation (the time of fixation depends on
the thickness of the tissue), tissue specimen were dehydrated by incubation steps in increasing
percentage of ethanol and a final change in xylene before embedded into paraffin blocks (
dehydration and paraffin-embeddening were performed by staff at the Division of Pathology).
Paraffin-embedded tissues were subsequently cut into 3-5 μm sections and subjected to IHC.
Primary monoclonal antibodies were used in the present study for detection of proteins of
interest, with the only exception of using a polyclonal rabbit antibody against human von
Willebrand´s factor. Monoclonal antibodies derive from a single clone of B-cells and bind to
one single, specific epitope of the protein, whereas polyclonal antibodies derive from multiple
B-cells and recognize different epitopes on the protein of interest, a fact that may enhance the
risk of cross-reactivity and background staining. For detection of primary antibodies bound to
the protein of interest we used the HRP-DAB detection system. The enzyme-substrate
reaction yields a brown color at the primary antibody binding site. To visualize the tissue,
sections were counterstained with Mayer´s haematoxylin, mounted and investigated under a
54
light microscope. For all experiments, negative controls incubated without the primary
antibody did not show any staining. All scoring was conducted blinded to treatment group and
by two observers.
Assessment of angiogenesis
In the present study, scoring of tumor angiogenesis in human breast and ovarian cancer
xenografts was assessed by measuring intratumoral microvessel density (MVD). This method
was originally described by Weidner et al. and has been widely used as a standard amongst
techniques to quantify vascularity in clinical specimen (Weidner et al. 1991). Tumor sections
are scanned at low magnification to identify areas with the highest degree of vascularity (the
so-called “hot spots”), whereafter the number of vessels in each hot spot area is counted at
high power (x200 or more) magnification. Microvessel density as measured by the hot spot
method of Weidner has been shown to be a valuable prognostic indicator for a wide range of
tumor types (Weidner et al. 1991; Heimann et al. 1998; Gasparini 2001; Hlatky et al. 2002;
Uzzan et al. 2004). Tumor vasculature in our experiments was evaluated by IHC staining of
the endothelial cell-specific marker von Willebrand´s factor (Factor VIII antigen). Other
commonly used endothelial cell markers are CD31, CD34 and CD105 (Horak et al. 1992),
with contradicting opinions regarding which marker is the most reliable. Lymphatic vessels
may also be identified when staining against von Willebrand´s factor, whereas the anti-CD31
antibody may cross-react with infiltrating plasma cells, and the antibody detecting CD34 also
stains a variety of stromal cells (Heimann et al. 1998; Fox et al. 2004). The polyclonal rabbit
anti-human von Willebrand´s factor antibody used in our model has been validated for
paraffin-embedded tissues, and identifies the protein in murine endothelial cells, as the
infiltrating endothelial cells that form vessels in the human xenografts are from the host.
Specific softwares (ImageJ, NIH; and Easy Image Measurement Software, Bergstrom
Instruments) were used to calculate the percentage of positive vessel staining, aiming to
minimize subjectivity in vessel quantification, in at least three hot spots/tumor section. The
mean percentage of microvessel area was calculated for each tumor section, and the
quantification was performed blinded to treatment group on sections from three or five tumors
of each group. In this study, vessel quantification of human tumor xenograft sections was
used as a tool to compare the angiogenic response of different treatments. Human xenografts
are not comparable to human primary tumors regarding vascularization. Xenografts are much
more homogenous due to origin from one clone of cancer cells, and are less infiltrated by
other cell types than human tumors which may exhibit a large variation in vascularization
(Marson et al. 1999; Lacroix et al. 2004). Another way to investigate the angiogenic response
after different treatments is comparing levels of angiogenesis mediators in the tissue, which in
our studies was assessed by investigating microdialysates collected from the tumors.
55
Review of the study
Paper I
The project resulting in Paper I aimed to investigate if E2 and Tam exposure affects
extracellular levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and the generation of endostatin in
experimental models of hormone-dependent ovarian cancer. Our group has previously
reported an E2 and Tam-induced regulation of these proteins in experimental ER/PgR-positive
breast cancer in vitro and in vivo, and where the Tam-induced increase of extracellular MMP
protein levels and activities resulted in enhanced generation of endostatin (Nilsson et al. 2006;
Nilsson et al. 2007). Breast and ovarian tumors share several common characteristics, such as
hormone-sensitivity and expression of endocrine receptors. However, Tam is rarely used as a
treatment strategy in ovarian cancer. The project included in vitro studies of the established
ovarian cancer BG-1 and OVCAR-3 cell lines, analyses of ascites collected from ovarian
cancer patients, and in vivo studies of ovarian cancer xenografts established in nude mice.
Tam enhanced MMP-9 activity and generation of endostatin in ER-positive ovarian cancer
In Paper I, we report a regulatory effect by E2 and Tam on human ER-positive ovarian cancer
BG-1 cells in culture regarding extracellular MMP-9 protein level and activity. Seven days of
Tam exposure to BG-1 cells resulted in significantly increased levels of secreted MMP-9
protein and activity, whereas exposure of BG-1 to physiological levels of E2 decreased MMP-
9 levels and activity in conditioned media (as compared to control-treated cells). MMP-2
protein could not be detected in BG-1 supernatants in any of the treatment groups. BG-1 cell
cultures treated with Tam showed significantly increased levels of endostatin, as compared to
E2 exposed and control-treated BG-1 cells. The addition of a neutralizing, specific anti-MMP-
9 antibody to the last 24 h of Tam treatment decreased endostatin levels as compared to Tam
treatment alone, indicating that the generation of endostatin is MMP-9 dependent (Figure 10).
Extracellular levels of TIMP-1 and TIMP-2 was not significantly altered by either E2 or Tam
exposure as compared to controls. BG-1 xenografts in Balb-C nude mice treated with Tam
showed significantly increased levels of MMP-9 and endostatin, and reduced tumor
vascularity as compared to E2-treated xenografts. In addition, Tam treatment decreased BG-1
cell proliferation in vitro, as well as ovarian cancer xenograft growth in vivo.
Analyses of ascites, collected from non-treated ovarian cancer patients, showed that
extracellular levels of MMP-9 in this compartment were absent or very low. Protein levels of
MMP-2 in ascites however were higher, and were significantly correlating with levels of
endostatin, further demonstrating the physiological relevance of MMP activities and
endostatin generation. As MMP-2 is much less efficient in the cleavage/release of endostatin
from collagen XVIII as compared to MMP-9 (Ferreras et al. 2000), our results suggest a
56
therapeutic possibility of increasing MMP-9 by the use of Tam and thereby enhance the
generation of anti-angiogenesis fragments.
These results from in vitro and in vivo experimental set-ups on hormone-dependent ovarian
cancer are in line with previously published data from our group, where a regulatory function
was shown by E2 and Tam on MMP activities and endostatin generation in experimental ER-
positive breast cancer (Nilsson et al. 2006; Nilsson et al. 2007).
Women with ovarian cancer have the highest mortality rate amongst patients diagnosed with a
gynaecological cancer. In Europe, approximately one third of women that receive ovarian
Figure 10.
Increased endostatin generation of cultured BG-1 is mediated via tamoxifen-induced MMP-9 activity.
A. Extratracellular levels of endostatin in conditioned media from hormone-treated BG-1 cells in culture.
B. Addition of a specific anti-human MMP-9 neutralizing antibody decreased tamoxifen-induced
increase of endostatin.
57
cancer diagnosis are alive after five years (Sant et al. 2003; Bray et al. 2005). The poor
prognosis is mainly due to advanced stage of disease at the time of diagnosis. The initial
response-rate of the cytotoxic drugs used in the treatment of ovarian cancer, usually platinum
and taxanes, is generally high but most women relapse and die from their disease. Although
epidemiological data indicate the importance of hormones in the aetiology of ovarian cancer,
endocrine therapy is rarely used as a treatment option in ovarian cancer patients (Rao et al.
1991; Henderson et al. 2000; Lindgren et al. 2004). Clinical trials evaluating Tam as a
therapeutic in ovarian cancer are mainly performed on heavily pre-treated women with
relapse, in most cases refractory to cytotoxic drug therapy, and with unknown endocrine
receptor status (Hurteau et al. 2010; Williams et al. 2010). Considering the therapeutic value
of Tam in the treatment of ER-positive breast cancers, our results may indicate that ER-
screening of ovarian tumors could have a significant clinical relevance, and that Tam might be
proven useful as an adjuvant treatment in women with endocrine-sensitive primary ovarian
tumors.
Paper II-III
MMPs are involved in a diverse range of cellular and stromal activities to maintain normal
tissue homeostasis, and the complex network of functions by these enzymes are far from
being fully understood. The effects of MMP activities in malignant tissues are reported
contradicting. MMPs are however due to their ECM degrading capacities generally discussed
in terms of pro-tumorigenic activities and as facilitators of tumor invasion and metastases.
Large efforts have been made on the development of synthetic MMP inhibitors as a treatment
strategy for several cancer types but so far clinical trials evaluating these drugs in cancer
patients have been reported un-successful (Coussens et al. 2002; Overall et al. 2006).
Increasing amounts of evidence actually indicate a protective role of several MMPs in tumor
progression through increased generation of anti-angiogenesis mediators in the tumor
microenvironment (Cornelius et al. 1998; Pozzi et al. 2000; Pozzi et al. 2002; Hamano et al.
2003; Lopez-Otin et al. 2007; Martin et al. 2007). The beneficial effects of Tam in the
treatment of ER-positive breast cancers are well established. In experimental breast cancer
setups we have previously shown that Tam exerts growth inhibiting effects but also
enhancement of MMP-9 expression and activities.
In Paper II-III, we aimed to investigate the effects of specific MMPs/TIMPs over-expression
regarding growth and angiogenesis in human hormone-dependent breast tumor xenografts
established in nude mice with or without Tam treatment. Adenoviral vectors expressing the
human genes of MMP-9, MMP-3 or TIMP-1 were administered as intratumoral injections to
tumor size-matched, oophorectomized and E2-supplemented nude mice. Microdialysis was
performed one week after vector administration to sample extracellular tumor endostatin and
VEGF, and tumor growth was followed for 24 and 36 days respectively. In Paper III, a
possible dose-dependent relationship of AdMMP-3/AdMMP-9-induced effects in MCF-7
58
xenografts was evaluated by using two concentrations of Ad vectors, which are referred to as
high and low dose.
AdMMP-9 affected tumor levels of both pro- and anti-angiogenesis mediators with
decreased tumor angiogenesis as a net result
Efficient gene transfer of AdMMP-9 and AdTIMP-1 was confirmed by direct quantification
of tumor MMP-9 activity in vivo by microdialysis, and by quantifying MMP-9 and TIMP-1
protein in tumor homogenates. MMP-9 has been proposed as a component of the angiogenic
switch by increasing VEGF levels in experimental pancreatic tumor tissue in early stage of
tumor progression in (Bergers et al. 2000). We have previously shown that microdialysis is a
valid technique for the sampling of soluble VEGF from the tissue (Dabrosin 2003; Dabrosin
et al. 2003; Garvin et al. 2003; Dabrosin 2005). Here, we sampled VEGF directly in vivo one
week after gene transfer of Addl70, AdMMP-9 or AdTIMP-1. We show that Tam
significantly decreased extracellular soluble levels of VEGF in situ in all treatment groups.
We also report that injections with AdMMP-9 induced a slight but non-significant increase in
VEGF levels in animals without Tam treatment. The MMP-9-induced increase of soluble
VEGF in our study is in line with previous mentioned results from Bergers et al. (Bergers et
al. 2000). However, as all of the AdMMP-9-treated tumors in our study exhibited decreased
angiogenesis, the increase in extracellular soluble VEGF by AdMMP-9 did not seem to have
enough biological importance to affect the angiogenic switch (Figure 11).
The decrease in angiogenesis by AdMMP-9 may, at least in part, be due to significantly
increased levels of tumor endostatin. As endostatin is a cleavage fragment derived from the
precursor molecule collagen XVIII by enzymatic cleavage, and its presence exclusively is
located to the extracellular space, it is imperative that endostatin is quantified in this
Figure 11.
Microvessel area in MCF-7
xenografts 24 days after gene
transfer of Addl70, AdMMP9,
or AdTIMP1.
59
compartment. By using microdialysis as a tool for investigating tumor levels of soluble
endostatin the risk of quantifying collagen XVIII is excluded, as the precursor protein is too
large to pass over the membrane of the probe. Previous results from our group were
confirmed as Tam significantly increased tumor endostatin levels as compared to E2 treatment
alone (control-vector group). The Tam-induced increase of endostatin was seen in all
treatment groups. AdMMP-9 treatment alone induced increased levels of endostatin similar to
those of Tam-treated mice. The combined treatment of AdMMP-9 and Tam synergistically
increased tumor endostatin levels with approximately 100% as compared to control treatment.
Gene transfer of TIMP-1 counteracted Tam-induced tumor regression
TIMPs have been demonstrated to have multiple biological functions extended from MMP
inhibition. Increasing amounts of data show that TIMPs have a dual role in cancer and may
exert tumor promoting activities such as cell growth stimulation and suppression of apoptosis
(Hayakawa et al. 1992; Guedez et al. 1998; Luparello et al. 1999; Liu et al. 2005). Elevated
TIMP-1 levels have been associated with poorer prognosis in several cancer types including
breast and ovarian cancer (Manenti et al. 2003; Rauvala et al. 2005; Kopitz et al. 2007;
Kuvaja et al. 2007; Lipton et al. 2007; Kuvaja et al. 2008; Oh et al. 2010). In our study we
show that MCF-7 xenografts exposed to TIMP-1 gene transfer had similar tumor progression
as tumors treated with the empty control vector (Figure 12).
Figure 12.
Tumor volume at the end of
experiment, 24 days after
intratumoral delivery of
AdMMP9, AdTIMP1, or empty
control vector (Addl70-3).
60
Moreover, we show that tumors exposed to the combined treatment of Tam and TIMP-1 had
significantly larger tumor volume as compared to tumors treated with Tam and control-vector,
indicating that TIMP-1 to some extent counteracted the therapeutic effects of Tam (Figure
12).
These results are in line with previous studies emphasizing Timp-1 as a tumor-enhancing
agent, and with recently performed clinical studies where metastatic breast cancer patient with
elevated TIMP-1 levels had significantly reduced responsive rate to endocrine therapy
(letrozole and Tam), as compared to patients with normal TIMP-1 levels (Lipton et al. 2008).
AdMMP-9 and AdMMP-3 induce breast cancer regression in a dose-dependent manner
The specific type of MMP and the level of its expression may be crucial determinants for
tumor behaviour. Over-expression of MMP-9 resulted in significant tumor regression in our
model of breast cancer in Paper II when tumor growth was followed for 24 days (Figure 12).
In Paper III, the same model was used with the exception that half and double dose of MMP-9
gene transfer was evaluated (0.5 x 109 pfu and 2.0 x 10
9 pfu) as compared to Paper II. In
addition, we performed intratumoral gene transfer of MMP-3 in high and low dose in this
experimental set-up, as MMP-3 is another member of the MMP family where both tumor-
promoting as well as protective roles have been reported. Anti-tumorigenic effects induced by
MMP-3 involve increased rates of apoptosis and enzymatic release of anti-angiogenic
mediators amongst others (Witty et al. 1995; Farina et al. 2002; Lopez-Otin et al. 2007).
MMP-3 has been shown to generate endostatin from collagen XVIII with a similar efficacy as
MMP-9 (Ferreras et al. 2000; Heljasvaara et al. 2005).
In Paper III, tumor growth was followed for 36 days after high and low dose of AdMMP-3
and AdMMP-9 intratumoral gene transfer. Treatment with low dose of either AdMMP-3 or
AdMMP-9 resulted in tumor stasis whereas high dose induced a potent tumor regression.
High dose AdMMP-9-treated tumors were significantly smaller than tumors treated with low
dose MMP-9. No statistical difference was seen when comparing tumor size of high and low
dose of AdMMP-3 (see Figure 1 in Paper III). Endostatin levels were significantly increased
in groups from both high and low MMP-3 or MMP-9 gene transfer, as compared to tumors
treated with the empty control vector. Higher levels of endostatin were detected after MMP-9
gene transfer compared to MMP-3 gene transfer, indicating a more efficient in vivo cleavage
of collagen XVIII by MMP-9 than by MMP-3. However, recombinant endostatin has been
shown to exhibit biphasic effects on endothelial cell migration in vitro and on tumor growth
inhibition in vivo, and therefore higher levels of endostatin does not necessarily assure a more
potent anti-angiogenic response of the tumor than with lower levels of endostatin (Celik et al.
2005). Here, endostatin levels were analyzed in both microdialysates and tumor homogenates.
Higher level of endostatin found in homogenates as compared to levels quantified in
microdialysates depends on the relatively lower tissue recovery of endostatin in vivo by
61
microdialysis but may also be due to cross-reactivity of the antibody to the precursor
molecule collagen XVIII present in the tumor tissue samples. Increased endostatin generation
in homogenates may additionally be a result of enhanced enzymatic activity during the
homogenization process of tumor tissues. The relatively minor difference in endostatin
generation in tumors and dialysates one week after adenoviral vector delivery comparing high
dose to low dose MMP-3/MMP-9 gene transfer, but with a major difference in tumor
regression by the end of experiment, suggest that additional anti-tumorigenic mechanisms,
including generation of other anti-angiogenesis fragments, are involved in the MMP-
dependent tumor regression. Quantification of microvessel area in tumor sections revealed
significantly decreased tumor vascularity after both high and low MMP-3 and MMP-9
treatment as compared to controls. There was a clear dose-related effect on microvessel area
from both MMP-3 and MMP-9 treatment as tumors treated with the higher dose were
significantly less vascularized compared to tumors treated with low dose (see Figure 3 in
Paper III).
Paper IV
Altered expression of growth factors and their receptors is a common phenomenon in
carcinogenesis and tumor progression. The pro-inflammatory chemokine IL-8, primarily
associated with promotion of neutrophil chemotaxis and degranulation, has additionally been
shown to exert pro-tumorigenic activities by enhancing endothelial and cancer cell
proliferation and migration (Xie 2001). Increased IL-8 expression has been observed in a
variety of cancer types including tumors of the breast and ovaries, as compared to the
correlating normal tissues (Green et al. 1997; Merogi et al. 1997; Ivarsson et al. 1998). In
breast cancer, high serum levels of IL-8 have been associated with poor prognosis (Benoy et
al. 2004). Besides the direct angiogenic effects by IL-8 itself, high tumor expression may lead
to elevated migration and infiltration of leukocytes into the tumor microenvironment, cells
which in turn may produce large quantities of angiogenic factors. Tissue levels of IL-8 are
dependent on the release by various cell types present in the tissue and by a number of
microenvironmental factors including inflammatory signals, hypoxia, nitric oxide, and
exposure to chemical stress induced by cytotoxic therapies (Lee et al. 1996; Watson et al.
1998; Duan et al. 1999). Contradicting data have been reported regarding the role of sex
steroids in the regulation of IL-8 in various cells and tissues (Kanda et al. 2001; Bengtsson et
al. 2004; Lin et al. 2004; Azenshtein et al. 2005; Seeger et al. 2006). In experimental breast
cancer, over-expression of IL-8 has been suggested to be more frequent in ER-negative than
in ER-positive breast cancer cells (Freund et al. 2003). This however does not rule out a
hormone-induced regulation of IL-8.
In Paper IV, a profound investigation was performed on how E2 affects extracellular levels of
IL-8 in normal and in malignant human breast tissues. Additionally, the physiologic response
of IL-8 on endothelial cell proliferation in vitro and tumor angiogenesis in vivo was
62
investigated. Microdialysis was used to sample IL-8 from normal human breast tissue in pre-
and postmenopausal women, preoperatively in tumor tissue of breast cancer patients, and in
breast cancer xenografts in nude mice. Whole normal breast tissue and breast cancer cells
were exposed to hormonal treatments in vitro and HUVECs were treated with hormones,
recombinant IL-8, conditioned media collected from hormone-treated breast cancer cells, and
the neutralizing ABX-IL-8 antibody.
E2 regulates extracellular IL-8 levels in normal and malignant breast tissue
We report a significant correlation between E2 and IL-8 levels in normal breast tissue in vivo,
as quantified in microdialysates, whereas no correlation between E2 and IL-8 was found in
subcutaneous abdominal fat tissue of the same women. These results were further confirmed
ex vivo by using a model of normal human whole breast biopsies in culture. IL-8 secretion
was significantly increased from biopsies exposed to E2 for seven days, as compared to
biopsies cultured without hormones. The co-treatment of E2 and P4 did not significantly alter
the breast biopsy IL-8 secretion as compared to E2 treatment alone.
Microdialysis was additionally performed intratumorally, pre-operatively, in women with
breast cancer. Here, we could see a significant correlation between local E2 and extracellular
IL-8 in tumors that were positive for both ER and PgR. In vitro studies of ER+/PgR
+ MCF-7
breast cancer cells revealed that IL-8 levels in supernatants of E2 treated cells were
significantly increased, as compared to supernatants from control-treated cells, an effect that
was further enhanced by increased concentrations of E2. The addition of Tam to the E2
treatment counteracted the E2-induced increase of secreted IL-8. To further explore the E2
induced increase of IL-8 secretion of human breast cancer cells, and to verify whether the
inhibition of E2 released IL-8 by Tam in vitro also was valid in whole tumor tissue, MCF-7
xenografts were established in nude mice, subcutaneously on the hind flank and in the
mammary fat pad. Microdialysis was performed to sample extracellular tumor IL-8 from mice
treated with E2 alone or with the additional treatment of Tam. Tam-treated mice had
significantly lower tumor IL-8 levels as compared to mice treated with E2 only. The level of
decrease of IL-8 by Tam was similar in subcutaneous xenografts as in xenografts of the
mammary fat pads. However, the absolute levels of tumor IL-8 were lower in the mammary
fat pad tumors, due to the use of shorter microdialysis membranes. Anti-IL-8 IHC staining of
tumor sections confirmed a significantly weaker staining of IL-8 in Tam-treated mice as
compared to E2 treatment alone (figure 13A).
E2-induced IL-8 secretion increases HUVEC proliferation and tumor angiogenesis
To investigate the effect of IL-8 on endothelial cell proliferation, HUVECs were isolated and
cultured with recombinant IL-8 with or without the addition of a neutralizing human anti-IL-8
antibody (ABX-IL-8). The significantly increased proliferation of HUVECs induced by
recombinant IL-8 treatment was almost completely abolished by the use of ABX-IL-8. These
results confirm previous studies demonstrating IL-8 as an endothelial cell mitogen (Xie
63
2001). To further investigate if the increased IL-8 secretion of human breast cancer cells
exposed to E2 had any physiological significance on endothelial cell proliferation, HUVECs
were cultured in conditioned media collected from MCF-7 cells after 7 days of treatment in
the presence or absence of E2. A significant increase in HUVEC proliferation was seen after
three days of culture in conditioned media from E2 treated breast cancer cells, as compared to
HUVECs treated in media from breast cancer cells without E2 exposure. We also show that
the addition of ABX-IL-8, either as combined treatment with E2 to MCF-7 cultures, or freshly
added on HUVECs, significantly decreased proliferation. The addition of ABX-IL-8 directly
to HUVEC cultures was more efficient than ABX-IL-8 added during MCF-7 cultures. In vivo,
the significant increase of anti-IL-8 staining in MCF-7 xenografts treated with E2 alone was
additionally followed by increased vessel density (Figure 13A and B).
Taken together, these results suggest that E2 has a significant effect on IL-8 secretion both in
normal breast tissue and in breast cancer in vitro and in vivo.
A B
Figure 13.
A. Anti-IL-8 immunohistochemistry of MCF-7 xenograft sections. Representative sections from
estradiol (E2) and estradiol+tamoxifen (E2+T) treated animals. Seven out of nine tumors were scored as
intensely stained in the E2 group whereas one out of six tumors showed intense staining in the E2-T
group, p = 0.04.
B. Microvessel area in solid MCF-7 xenografts, *, p = 0.01.
64
65
Conclusions
Estradiol and tamoxifen regulate MMP-9 activity and endostatin generation in ER-
positive ovarian cancer. Tamoxifen increased MMP-9 activity and enhanced endostatin
generation in estrogen-responsive ovarian cancer in vitro and in vivo. Ovarian tumor
xenografts of nude mice treated with tamoxifen exhibited reduced vascularization and
decreased tumor area. Low levels of MMP-9 in ascites from ovarian cancer patients suggest
that tamoxifen may have therapeutic implications in ovarian cancer by enhancing the
generation of anti-angiogenesis fragments via increased MMP-9 activity.
MMP-9 gene transfer significantly reduced tumor angiogenesis and growth of human
ER-positive breast cancer xenografts in nude mice. A synergistic effect was seen by
tamoxifen and MMP-9 treatment regarding generation of endostatin and long term tumor
growth inhibition. TIMP-1 gene transfer had no therapeutic effect on tumor growth by itself
and counteracted tamoxifen-induced tumor regression.
Anti-tumorigenic effects induced by MMP-3 and MMP-9 are dose-dependent.
Intratumoral gene transfer with low and high dose of MMP-3 or MMP-9 revealed that low
dose of either MMP induced tumor stasis of solid MCF-7 breast tumors in vivo whereas high
dose resulted in significant tumor regression. Tumor vascularization was significantly reduced
in all tumors exposed to either low or high dose of MMP-3 or MMP-9 as compared to control
treatment but more pronounced anti-angiogenic effects were seen with high dose. The
relatively modest variations in endostatin generation comparing high and low dose MMP-
3/MMP-9 suggest that additional anti-tumorigenic mechanisms are involved in the significant
tumor regression induced by high dose MMP gene transfer.
Estradiol regulates extracellular levels of IL-8 in normal and malignant breast tissue.
A significant positive correlation of extracellular IL-8 and estradiol was found in normal
breast tissue of pre-and postmenopausal women and preoperatively in ER+/PgR+ tumors of
breast cancer patients in vivo. Estradiol increased IL-8 secretion of normal human breast
biopsies and in MCF-7 breast cancer cells in culture. HUVEC cell proliferation induced by
estradiol-treated MCF-7 cell media was significantly inhibited by an anti-IL-8 Ab, ABX-IL-8.
Tamoxifen inhibited the estradiol-induced IL-8 secretion in MCF-7 cultures in vitro and in
solid MCF-7 tumors in vivo. Tamoxifen-treated tumors exhibited decreased anti-IL-8 staining
and microvessel area as compared to tumors treated with estradiol.
66
67
Concluding remarks
Angiogenesis is a crucial and rate-limiting step in tumor progression and a necessary event for
the establishment of a clinical cancer disease. The net balance of pro- and anti-angiogenesis
mediators in the tumor microenvironment is considered to dictate the angiogenic phenotype of
a tumor. Normal quiescent vessel homeostasis may be evaded by increased levels of
activators of angiogenesis or decreased levels of angiogenesis inhibitors. Stimulators and
inhibitors of endothelial cell proliferation, migration and tube formation may be secreted by
several cell types and may additionally be sequestered in the tumoral stroma and released by
ECM turnover. By using microdialysis it is possible to investigate mediators involved in the
crucial intercellular crosstalk in this environment. MMPs are major regulators of ECM
remodelling and may affect tumor progression in both positive and negative ways by
enzymatic cleavage and release of bioactive molecules. MMPs are generally viewed as
bulldozers, facilitating tumor growth and metastases. However, broad-spectra MMP inhibitors
as anti-tumor therapy have failed, and it has now become evident that several MMPs may
induce biological activities beneficial to the host, such as suppressed angiogenesis. This is
clearly demonstrated in this thesis, were it was shown that gene transfer of MMP-9 and
MMP-3 induced decreased angiogenesis and significant tumor regression. More studies
elucidating the roles of individual MMPs in tumor tissues are of outmost importance before
MMPs can be used as anti-tumor targets.
Breast- and ovarian tumors belong to the group of hormone-dependent cancers where sex
steroids are considered to have a major impact on the malignant transformation and further
tumor growth. Despite progress made during the past years, our knowledge in sex steroid
regulation of angiogenesis in hormone-dependent tumor tissues remains limited. Tamoxifen
has been used as endocrine therapy in ER positive breast cancer for several decades with well
established beneficial therapeutic results. The therapeutic value of tamoxifen in the treatment
of hormone-dependent ovarian cancer is to date less investigated. The results presented in this
thesis suggest that tamoxifen may induce anti-tumorigenic responses in ER positive ovarian
cancer by influencing MMP-9 activities and generation of endostatin. These anti-angiogenic
effects by tamoxifen have previously been demonstrated in hormone sensitive breast cancer
and may be important mechanisms behind the anti-tumorigenic properties of this medicine
(Nilsson et al. 2006; Nilsson et al. 2007). Ascites collected from ovarian cancer patients
contained low concentrations of MMP-9, suggesting a possibility to therapeutically increase
MMP-9 by the administration of tamoxifen, and thereby enhance the generation of anti-
angiogenesis fragments, such as endostatin.
The significance of the tamoxifen-induced increase of MMP-9 was further investigated by
performing intratumoral gene transfer of MMP-9 and TIMP-1 to human ER-positive breast
tumors established in nude mice. We show that MMP-9 treatment induced a significant tumor
68
regression as compared to controls. Although MMP-9 over-expression slightly enhanced
tumor levels of soluble VEGF, the increase in endostatin generation was more pronounced
and the net result was significantly decreased angiogenesis. Low or absent levels of MMP-9
has previously been demonstrated to increase tumor growth as the generation of molecules
involved in anti-angiogenic actions is decreasing (Pozzi et al. 2002; Hamano et al. 2003). To
our knowledge, this is however the first time where MMP-9 has been used as anti-tumor
therapy. Tumors treated with TIMP-1 exhibited tumor progression in a similar manner as
control treatment indicating that no anti-tumorigenic effects were induced by this protein. In
contrary, the combined treatment of TIMP-1 and tamoxifen revealed that TIMP-1
counteracted the anti-tumorigenic effects seen by tamoxifen alone. Our results are in line with
previous studies demonstrating reduced responsive rate to endocrine therapy in patients with
high TIMP-1 levels (Lipton et al. 2008). The results from TIMP-1 gene transfer further
confirm the protective role of MMP-9 in tumor biology. The combined treatment of MMP-9
and tamoxifen induced a synergistic effect on tumor regression.
In this thesis we additionally present data on dose-dependent effects induced by MMP-9 and
MMP-3 gene transfer. In our experimental set-up high dose of either MMP induced a more
pronounced anti-tumorigenic response as compared to low dose. The mechanisms behind the
MMP-3/MMP-9-induced tumor regression may be several. Here, we show that tumor levels
of endostatin were increased but probably multiple other proteins involved in both pro- and
anti-angiogenic activities are affected by enhanced MMP activities, and the anti-tumorigenic
mechanisms of MMP-3 and MMP-9 seen in this thesis is yet to be completely elucidated.
Increasing the levels of angiogenesis inhibitors, such as endostatin, is a challenging approach
to prevent non-angiogenic in situ carcinomas from progressing to a clinical cancer disease,
and would probably imply a safe form of long-term anti-cancer therapy. Unfortunately,
although recombinant endostatin efficiently inhibits angiogenesis and tumor growth in
experimental set-ups, the results from clinical trials show poor clinical effect on the
progression of cancer. The use of purified recombinant endostatin as anti-tumor agent has
faced several problems including short half-life, difficulties with production and reproduction,
and routes of administration. To modify the endogenous levels of angiogenesis inhibitors, for
instance by MMP activities, rather than administrating recombinant proteins, may therefore
be a more favourable approach in targeting tumor growth.
Another interesting research approach is the link between angiogenesis and inflammation in
cancer. The grade of inflammation in a tumor may have a major impact on prognosis and
response to treatment, and some data suggest that an intense inflammatory response may be
beneficial as compared to low-grade tumor inflammation (Hagemann et al. 2007; Talmadge et
al. 2007; Chew et al. 2010). MMP-9 has been suggested to have pro-inflammatory potential,
and is in some contexts, such as atherosclerosis and coronary heart diseases, considered as an
inflammatory marker. The links between specific MMP expression and its level of activity,
and inflammatory responses in tumors and how this may affect clinical outcome remain to be
investigated.
69
Estradiol has previously been shown to tip the scale in favour to a pro-angiogenic milieu in
the breast by influencing the bioactivity of VEGF (Garvin et al. 2005; Garvin et al. 2006;
Garvin et al. 2008). In this thesis, the angiogenic potential of estradiol was further confirmed
by demonstrating a significant correlation between local levels of estradiol and the pro-
angiogenic cytokine IL-8 in normal human breast tissues in pre- and postmenopausal women
and in ER/PgR-positive breast cancers of women. The impact of estradiol on IL-8 secretion
was additionally demonstrated in normal human whole breast biopsies in culture and in
experimental human breast cancer in vitro and in vivo. Tamoxifen inhibited the estradiol-
induced increase of IL-8 in experimental breast cancer. Tamoxifen treatment reduces breast
cancer incidence with 40%, but it may also induce severe side effects such as endometrial
cancer and thromboembolism (EBCTCG 1998; Fisher et al. 2005). Therefore, there is need
for more selective compounds in the prevention and treatment of breast cancer. The
significant impact of estradiol on extracellular levels of IL-8 shown in this study suggests that
IL-8 may be a novel approach for targeted therapy in the treatment of hormone-sensitive
breast cancers.
70
71
Acknowledgements
Jag vill härmed framföra ett varmt tack till alla de personer som på ett eller annat sätt varit
delaktiga i den process som lett fram till färdigställandet av denna avhandling. Särskilt vill jag
tacka:
Min handledare, Charlotta Dabrosin, för att du gav mig möjligheten att utföra dessa
intressanta projekt och att skriva den här boken. Jag beundrar din aldrig sinande forsknings-
dedikation och dina djupa kunskaper i de viktiga ämnesområden som handlar om bröst,
hormoner och angiogenes. Tack för den här tiden!
Till de kvinnor, med eller utan cancerdiagnos, som deltog i studierna och därmed bidrog till
att viktiga delar av denna forskning kunde utföras.
Ulrika W Nilsson & Stina Garvin, mina fina f.d. doktorandkollegor för gott samarbete och
för stöd från när & fjärran! Speciellt tack till Ullis, min “storasyster” i MMP-projekten för att
du banade vägen med dina arbeten!
Annelie Abrahamsson, för ditt goda labtekniska kunnande och din positiva attityd!
MedBi-student och medförfattare av papper I, Lisa Karlsson, för idogt odlande av
ovarialcancerceller och för gott samarbete.
Co-authors of paper II, Jack Gauldie & Jennifer Robertson, for excellent collaboration and
for providing us with the adenoviral vectors.
Birgitta Frånlund, för otaliga faktor VIII-färgningar och för generös och skicklig rådgivning
vid immunhistokemiska dilemman.
Siv Andersson & Kerstin Andersson på onkologiska kliniken, för insamlande av ascites.
Danne Linghammar, för dina excellenta insatser och ditt trevliga sällskap på
djuravdelningen.
Kerstin Hagersten, din närvaro på Klinisk Experimentell Forskning har betytt otroligt
mycket för mig!
Anette Wiklund, Håkan Petri & Lena Arvidsson, utan er hade det här aldrig gått vägen!
Håkan Wiktander & Pia Karlsson, för praktisk hjälp och trevligt sällskap!
72
Alla fina kollegor på Avd för Onkologi, ingen nämnd men heller ingen glömd, för goda
fikastunder, inbjudningar till journal clubs och seminarier, och för trevligt sällskap i San
Antonio!
Alla kollegor från olika forskargrupper samlade på Klinisk Experimentell Forskning, för
många goda skratt och emellanåt även givande diskussioner under de gemensamma stunder vi
spenderat i fikarummet! En stor guldstjärna för alla underbara torsdags-frukostar! Ett speciellt
tack till Lena Berglert för leverans av ekologiska ägg!
Lisa Karlsson, Camilla Fredriksson & Sofia Pettersson, jag saknar Er! Önskar er all lycka
i den riktiga världen!
Kristina Briheim & Kristina Warstedt, för alla genuint trevliga stunder på och utanför
jobbet!
Saàd Salim, for all the good laughs and funny stories! Hope You´re doing alright and don´t
forget our trip to India!
Johan Junker & Anders Carlsson, för otroliga grillkvällar på KEF, god whisky,
oförglömliga musikstunder (om än i tveksam genre) men framförallt er förmåga att lätta upp
tillvaron på jobbet!
Martha Sjöberg, hypnosterapeut Ersta Sjukhus för givande möten! Stranden är fantastisk!
Cristina Blendea, för din otroligt vackra röst!
Nikos, ευχαρηζηό για όλα! Χωρίϛ εζέυα δεη είχα θηάζι μέχρι εδώ…
Min älskade mamma, mina kära systar Maria & Anna, min fina svåger Peter och alla
underbara syskonbarn, tack för ert stöd under de här åren – i stort som i smått! Pappa, Stig
Bendrik, jag saknar dig otroligt mycket men tror mig höra din närvaro i form av en
trumvirvel emellanåt …
Sist men inte minst, Tor & Aris mina älskade söner, för ert tålamod och för alla spännande
fotbollsmatcher och underbara musikframträdanden som gett/ger mig otrolig gädje! Ni har
verkligen förmåga att sätta guldkant på min tillvaro! Vad kan jag säga? Äntligen! Nu är det er
tur…
This study was kindly supported by grants from the Swedish Cancer Society; the
Swedish Research Council; Linköping University Hospital Research Funds; and
Familjerna Carl och Axel Molin i Motala Foundation
73
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