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Ovarian Cancer Cells Exhibit Aberrations in the Wnt Signaling Pathway, which Affect Cell Proliferation and Cadherin Expression By Emilia Liana Falcone A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science Department of Experimental Medicine McGill University, Montreal August, 2006 © Copyright by Emilia Liana Faleone (2006)
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~ Ovarian Cancer Cells Exhibit Aberrations in
the Wnt Signaling Pathway, which Affect Cell
Proliferation and Cadherin Expression
By
Emilia Liana Falcone
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science
Department of Experimental Medicine
McGill University, Montreal
August, 2006
© Copyright by Emilia Liana Faleone (2006)
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Table of Contents
1. Abstract ....................................................................................... 4
1.1 English Version ......................................................................... 4
1.2 French Version (Résumé) ........... ................................................... 5
2. Introduction ................................................................................. 6
2.1 Introduction .............................................................................. 6
2.2 Objectives .............................................................................. 10
3. Literature Review ......................................................................... 11
3.1 Epidemiology and histopathology of ovarian cancer ............................. 11
3.2 The Wnt signaling molecule ......................................................... 12
3.3 The Wnt signaling pathways ......................................................... 13
3.3.1 The Wntl~-catenin pathway ...................................................... 14
3.3.2 The WntlCa2+ pathway ............................................................ 14
3.3.3 The Pl anar cell polarity pathway ................................................. 15
3.4 W nt/~-catenin signaling and tumorigenesis ....................................... 15
3.5 Cadherins and ovarian tumorigenesis .............................................. 17
4. Materials and Methods .................................................................. 20
4.1 Celllines and culture .................................................................. 20
4.2 RNA extraction ........................................................................ 21
4.3 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) ................. 22
4.4 Isolation of cytosolic fractions ...................................................... 24
4.5 Immunoblotting ........................................................................ 24
2
.,....--.. 4.6 Transfections and luciferase assays ................................................ 25
4.7 MTT assays ............................................................................ 26
4.8 Statistical analyses ..................................................................... 27
5. Results ... .................................................................................... 28
6. Figures ................................................................................... .... 31
Table 2 ....................................................................................... 32
Figure 2 ....................................................................................... 34
Figure 3 ....................................................................................... 35
Figure 4 ....................................................................................... 37
Figure 5 ............................................................ '" ..... , .................. 39
Figure 6 ....................................................................................... 41
Figure 7 ....................................................................................... 43
Figure 8 ....................................................................................... 44
Figure 9 ........................................ '" ............................................ 46
Figure 10 ...................................................................................... 48
Figure Il ..................................................................................... 49
7. Discussion ... ................................................................................ 52
8. Conclusion ...... ............................................................................ 62
9. Acknowledgments ........................................................................ . 63
10. References ................................................................................. . 64
3
1. Abstract
1.1 Abstract
Ovarian cancer is the leading cause of death from a gynecological malignancy in
North America. It is an incomplete understanding of the early molecular events in ovarian
carcinogenesis which limit our ability to diagnose and effectively treat this disease at a
stage when it is still curable. The Wntl~-catenin canonical signaling pathway is involved
in development, wound repair, and tumorigenesis. Studies examining the involvement of
Wntlcanonical signaling in ovarian carcinogenesis, however, have only recently begun to
emerge. In this study, we hypothesize that ovarian cancer cells exhibit aberrations in
Wntlcanonical signaling, which may cause and/or effect ovarian tumorigenesis. Our
objectives were therefore to 1) determine whether ovarian cancer cells exhibit alterations
in the expression patterns of Wnt signaling pathway components, 2) determine whether
ovarian cancer cells exhibit functional aberrations in response to Wntlcanonical
stimulation and 3) determine whether these aberrations affect cell proliferation and
cadherin expression. Our study shows that ovarian cancer cells exhibit alterations in the
expression profiles of Wnt signaling pathway components and that these cells display
aberrations at different levels of the Wntlcanonical signaling pathway, which in turn,
modulate cell proliferation, and cadherin expression. These results suggest that
Wntlcanonical signaling may be involved in ovarian tumorigenesis and that further study
of this involvement may contribute ta a better understanding of early molecular events in
ovarian cancer.
4
---~ 1.2 Résumé
Le cancer ovarien est la principale cause de mort d'une malignité gynécologique en
Amérique du Nord. Ceci est du à un manque de connaissance des événements
moléculaires qui précipitent la carcinogenèse ovarienne. La VOle de signalisation
canonique de Wnt/~-catenine est impliquée dans le développement embryologique, la
guérison des blessures et la carcinogenèse. Parcontre, des études examinant la voie de
signalisation de Wnt/~-catenine dans le cancer ovarien viennent tout juste d'émerger.
Tenant compte de ces études, nous avons proposé l'hypothèse qu'une aberration au
niveau de la voie de signalization Wnt/~-catenine pourrait être une cause et / ou un effet
de la carcinogenèse ovarienne. Nos objectifs étaient donc de 1) déterminer si des cellules
ovariennes cancéreuses manifestent des changements d'expression de divers composantes
de la voie de signalization Wnt/~-catenine, de 2) déterminer si ces mêmes cellules
cancéreuses manifestent des aberrations au niveau de la fonction de la voie de
signalization Wnt/~-catenine et de 3) determiner si ces aberrations influencent la
prolifération de cellules et l'expression des molécules cadhérines. Notre étude montre que
les cellules ovariennes cancéreuses subissent des changements au niveau de l'expression
de divers composantes de la voie de signalization Wnt/~-catenine et que ces cellules ont
des aberrations à divers niveaux de la voie de signalization Wnt/~-catenine. De plus, ces
aberrations exercent des effets sur la prolifération de cellules cancéreuses et l'expression
des molécules cadhérines. Nos résultats suggèrent que la voie de signalization Wnt/~
catenine pourrait être impliquée dans la carcinogenèse ovarienne et qu'une meilleure
comprehension de cette implication aiderait au diagnostique et/ou au traitement du cancer
ovarien.
5
2. Introduction
2.1 Introduction
Ovarian cancer is the leading cause of death from a gynecological malignancy in
North America (Jemal et al., 2004). This is the result of a failure to detect ovarian
malignancies before they have reached an advanced stage (Kumar et al., 2003). As is the
case with other malignancies, it is an incomplete understanding of the initiating events
and early stages of ovarian carcinogenesis which limit our ability to diagnose or
effectively treat this disease at a stage when it is still curable.
Eighty to eighty-five percent of all ovarian neoplasms are thought to be derived
from the ovarian surface epithelium (OSE) (Berchuck et al., 1997; Kumar et al, 2003).
The currently dominant hypothesis is that the repeated wounding and repair induced by
ovulation (Fathalla, 1971) leads to irreversible oxidative DNA damage, which induces or
promotes tumorigenesis of the OSE (Purdie et al., 2003; Murdoch and Martinchick, 2004).
Ovarian neoplasms are divided into serous, mucinous, endometrioid and c1ear cell
histological subtypes (Kumar et al., 2003; Scott and McCluggage, 2006). The OSE is
embryologically derived from the coelomic mesothelium, which also gives rise to the
Müllarian ducts (Moore and Persaud, 2003). In view of the phenotypic similarities
between Müllerian duct derivatives and the various ovarian cancer subtypes, it is believed
that the histological variability of ovarian epithelial neoplasms is secondary to the
embryologie al derivation of the OSE from the eoelomie mesothelium (Scott and
McCluggage, 2006).
The WntJ~-catenin canonical signaling pathway is involved in development
(Reviewed in Wodarz and Nusse, 1998), wound repair (Labus et al., 1998; Okuse et al.,
6
2005; Fathke et al., 2006) and tumorigenesis (reviewed in Polakis, 2000). Interestingly,
aIl of the se processes appear to be related either directly or indirectly to ovarian
carcinogenesis.
It has been reported that various compartments of rodent ovaries (namely the OSE)
display distinct expression patterns of Wnt signaling molecules (Hsieh et al., 2002;
Ricken et al., 2002), and that certain Wnts are expressed in human ovarian cancer cell
lines (Ricken et al., 2002; Rask et al., 2003). Moreover, differences in the expression and
function of Wntlcanonical signaling pathway components have been reported in normal
versus cancerous OSE cells (Rask et al., 2003) and studies by Shedden et al. (Shedden et
al., 2005) suggest that ovarian cancer subtypes display distinct genetic aberrations of
Wnt/canonical signaling pathway components.
In view of these findings, and the well-established involvement of the
Wntlcanonical signaling cascade in tumorigenesis, we hypothesize that ovarian cancer
ceIls exhibit aberrations in Wntlcanonical signaling, which may cause and/or effect
ovarian tumorigenesis.
Wnts are secreted glycoproteins that bind to Frizzled (Fz) seven-transmembrane
spanning receptors, allowing for either cytosolic accumulation of ~-catenin and
subsequent transcriptional co-activation of target genes, mobilization of intracellular
Ca 2++, or activation of protein kinases (Nusse and Varmus, 1992). In the canonical
pathway, Wnts bind Fz receptors in association with the co-receptors LDL-receptor
related protein (LRP)-5/6 (He et al., 2004), leading to the inhibition of ~-catenin
phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3~ (GSK3~),
within a large cytoplasmic complex inc1uding Disheveled (Dvl), a cytoplasmic
7
phosphoprotein, Adenomatous Polyposis Coli (APC) and Axin, both of which are tumor
suppressor proteins (reviewed in Hulsken and Behrens, 2002). Inhibition of ~-catenin
phosphorylation impairs its degradation by the ubiquitin/proteosome pathway, resulting in
the accumulation of the uncomplexed cytosolic molecule. Uncomplexed ~-catenin then
translocates to the nucleus, where it interacts with T -cell factor/ Lymphoid enhancer
factor (TCF/LEF) and activates target genes (e.g. c-myc, l-myc, cyclin D, vascular
endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), E-cadherin,
N-cadherin) (Nusse and Varmus, 1992; Giles et al., 2003; Hallikas et al., 2006).
Aberrations in the Wntlcanonical pathway le ad to inappropriate ~-catenin-mediated
activation or silencing of these target genes, thereby allowing for uncontrolled cell
proliferation and/or alterations in cell differentiation (reviewed in Polakis, 2000).
LRP S/6
}
The B-<:atenin . Den ructlon
Complex
Figure 1. The Wntlcanonical signaling pathway (adapted from Veeman et al. 2003)
8
In addition to being implicated in the Wntlcanonical pathway, there are ~-catenin
molecules, which are sequestered to the plasma membrane through their interaction with
cadherin ceIl adhesion molecules and a-catenin. Although the mechanism underlying the
selection of ~-catenin molecules to the plasma membrane versus the ceIl nucleus remains
to be elucidated, it has been suggested that the ~-catenin molecules targeted to the plasma
membrane possess specific cadherin and a-catenin binding sites (Gottardi and Gumbiner,
2004).
Previous studies, including those from our laboratory, have confirmed the
expression of Wnt ligands and Fz receptors in rodent and human OSE (Ricken, 2002,
Richards, 2003). In addition, Rask et al. (Rask et al., 2003) have reported the expression
ofWntlcanonical signaling pathway components (i.e. p-catenin, LEF-l, GSK3~ and APC)
in human OSE. They also demonstrated that ~-catenin and GSK3~ expression is
increased in ovarian cancer. FinaIly, the y showed that p-catenin and LEF-l could be co
immunoprecipitated in ovarian tumours, but not in normal OSE. These findings suggest
that components of the Wntlcanonical signaling pathway are expressed in both normal
and cancerous OSE, but that there may be increased activation of the canonical signaling
pathway in ovarian cancer ceIls.
Analyses of genetic mutations according to ovarian cancer histological subtypes
have revealed that ovarian serous carcinomas show frequent mutations of the tumor
suppressor p53 (Katabuchi et al., 1998), while 85% of mucinous carcinomas exhibit
mutations in the K-RAS proto-oncogene (Hough et al., 2000). With respect to Wnt
signaling, 40% of ovarian endometrioid adenocarcinomas (OBAs) exhibit mutations in
the CTNNBl gene, which encodes p-catenin (Wu et al., 2001; Shedden et al., 2005).
9
Although mutations in the CTNNB 1 gene are the most common mechanism of p-catenin
deregulation in OEAs, p-catenin deregulation may also result from mutations in the APC,
AXIN1, and AXIN2 genes (Wu et al., 2001; Shedden et al., 2005). These data suggest
that specific histological subtypes of ovarian carcinoma are afflicted by distinct genetic
mutations. In particular, OEAs exhibit genetic mutations contributing to p-catenin
deregulation, thereby further supporting the contention that deregulation of the
Wntlcanonical signaling cascade may contribute to ovarian carcinogenesis.
2.2 Objectives
Based on the reported expression of Wntlcanonical signaling pathway components
in normal and cancerous OSE, and the aforementioned studies suggesting that canonical
signaling may be deregulated in ovarian carcinogenesis, we further examined whether
cancerous OSE celI lines (in comparison to normal OSE) exhibit alterations in the
expression patterns of Wnt signaling pathway components. We further set out to
determine whether ovarian cancer celIs exhibit functional aberrations in response to
Wntlcanonical stimulation, and whether these aberrations affect celI
proliferationldetachment and cadherin expression.
Our study shows that ovarian cancer celIlines exhibit alterations in the expression
profiles of Wnt signaling pathway components. We also found that ovarian cancer ceIl
lines display aberrations at different levels of the Wntlcanonical signaling pathway, which
in turn, modulate celI proliferationldetachment, and cadherin expression.
10
3. Literature Review
The following chapter represents an overview of the relevant ovarian cancer, Wnt
signaling and cadherin literature. Although it is by no means exhaustive, this review has
been designed with the intent of complementing the reader' s understanding of the present
research study.
3.1 Epidemiology and Histopathology of Ovarian Cancer
Despite representing only 4% of cancers in women, ovarian cancer is the leading
cause of death from a gynecological malignancy in North America (Jemal et al., 2004).
This is the result of failure to detect ovarian malignancies before they have reached
advanced stages. Ovarian cancer, which is staged according to the International
Federation of Gynecology and Obstetrics (F1GO) staging system, is most often detected
at F1GO stage 3, which is associated with a 23-41 % five-year survival rate (Kumar et al.,
2003).
The majority of ovarian neoplasms (80-85%) are thought to be derived from the
OSE and can be divided into the following most common histological subtypes: serou s,
mucinous, and endometrioid (Kumar et al., 2003). These histological subtypes represent
approximately 75%, 20% and 2% of epithelial ovarian tu mors respectively. Less
commonly, clear cell, transitional (Brenner tumor), undifferentiated, and mixed epithelial
ovarian turnors have been reported (Kurnar et al., 2003). Each histological type of
ovarian epithelial neoplasia encompasses a spectrum of tu mors ranging from benign to
borderline to malignant. Seventy and eighty-five percent of serous and mucinous tumors,
11
respectively, tend to be benign while endometrioid and clear cell tu mors tend to have a
high malignant potential (Kumar et al., 2003).
The OSE is derived from the coelomic mesothelium, which also gives rise to the
MülIerian duct and its derivatives. MülIerian duct derivatives include the fallopian tubes,
the uterus, the cervix, and the upper two thirds of the vagina (Moore and Persaud, 2003).
It has been hypothesized that the high morphological variability of ovarian epithelial
neoplasms is secondary to the embryological derivation of the OSE from the coelomic
mesothelium, which may lend to the OSE the intrinsÏc capacity for divergent
differentiation along the MülIerian pathways (Auersperg et al., 1997; Scott and
McCluggage, 2006). This capacity for divergent differentiation has been reflected in
reported findings of foci of ciliated or mucinous epithelium (similar to the fallopian tube
and endometrium, respectively) within the surface lining of the ovary (Auersperg et al.,
1994, 1997). Interestingly, histological examination of ovarian caricinomas has
uncovered marked similarities between reported ovarian epithelial neoplasm subtypes and
MülIerian du ct derivatives. More specifically, serous tu mors resemble fallopian tube
epithelium, while mucinous tumors reportedly resemble endocervical epithelium.
Endometrioid tumors, as the name implies, are likened to the uterine endometrium, and
clear ceIl tu mors are reported to resemble mesonephric cells (Scott and McCluggage,
2006).
3.2 The Wnt Signaling Molecule
In humans, 19 Wnt genes have been identified and chromosomally located. AlI
Wnt proteins are similar in size, ranging in molecular weight from 39 to 46 kDa (Miller,
12
2001). Wnts are secreted lipid-modified glycoproteins that are palmitoylated on a
conserved cysteine, which facilitates their targeting to the cell membrane. Once secreted,
Wnt proteins associate with glycosaminoglycans in the extracellular matrix, and thus
remain close to the cell surface (Miller, 2001). Nevertheless, it is possible to collect
active Wnt from the medium of cultured cells. In addition, data have indicated that Wnts
can function as concentration-dependent, long-range morphogenic signaIs that can act on
distant neighbors (Reviewed in Logan and Nusse, 2004). The exact mechanism
underlying this process, however, remains to be elucidated.
3.3 The Wnt Signaling Pathways
When binding to Frizzled (Fz) receptors, Wnts can activate at least three distinct
intracellular signaling pathways: 1) the Wnt / ~-catenin canonical signaling pathway, 2)
the Wnt / Ca 2+ pathway and 3) the planar cell polarity (PCP) pathway (Boutros et al.,
2000; Miller, 2001).
Distinct sets of Wnt molecules can activate each of these pathways and lead to
unique cellular responses (Miller, 2001). This finding was first observed in studies where
either Wntl, Wnt3a, Wnt8 or Wnt8b was overexpressed in Xenopus, which in tum led to
the specific stimulation of the Wnt/~-catenin pathway. In contrast, overexpression of
Wnt4, Wnt5a or Wnt11 was found to stimulate the Wnt / Ca 2+ pathway (Kuhl et al., 2000;
Miller, 2001). Fz receptors can be separated into similar functional groups based on their
ability to activate one pathway versus another in overexpression assays (Kuhl et al.,
2000). Although these classifications may provide a usefui tool for predicting the
function of Wnts and Fzs, the relationship between specific Wnts and the intracellular
13
pathway expected to be stimulated is not fixed. For ex ample , overexpression of Wnt5a in
combination with Fz5 in Xenopus embryos was found to activate the Wntl~-catenin
pathway as opposed to the Wnt / Ca 2+ pathway (He et al., 1997). This finding suggests
that the activity of Wnts in vivo may be determined by the repertoire of Fz receptors
present at the cell surface.
3.3.1 The Wnt/p-catenin pathway
The canonical Wntl~-catenin pathway is intensely studied and is described in the
previous chapter. Briefly, signaling through this pathway depends upon the cytosolic
accumulation of ~-catenin. In the absence of Wnt stimulation, ~-catenin is targeted for
degradation by a multi-protein destruction complex. Wnt signaling therefore antagonizes
the destruction complex, leading to the cytosolic accumulation of p-catenin and
subsequent activation of target genes (reviewed in Hulsken and Behrens, 2002).
3.3.2 The Wnt/Ca2+ pathway
Xenopus studies have suggested that the WntlCa2+ pathway can be activated by a
distinct group of Wnt ligands and Fz receptors, which include Wnt4, Wnt5a, Wnt11 and
Fz2 (Kuhl et al., 2000). This pathway involves activation of a heterotrimeric G protein,
an increase in intracellular Ca2+, and activation of calciumlcalmodulin-regulated kinase II
(CamKII) and protein kinase C (PKC). The downstream targets of CamKII and PKC are
currently unknown, but it has been shown that activation of the Wnt/Ca2+ pathway can
antagonize the Wnt/p-catenin pathway in Xenopus (Topol et al., 2003). It is, however,
unclear at what Ievel this interaction oecurs and whether it aIso oecurs in mammals
(Veeman et al., 2003).
14
3.3.3 The Planar Cell Polarity Pathway
The WntIPCP signaling pathway controls cell polarity (which can be inferred as a
function of cell shape and cell movement) through the regulation of cytoskeletal
organization. The PCP pathway overlaps with the canonical signaling pathway insofar as
it requires Fz receptors and Dvl molecules. It diverges downstream, however, in that it
does not involve Axin, GSK3~, or ~-catenin (Axelrod et al., 1998). The PCP pathway
was originally described in Drosophila, where it was found to control the polarity or in
other terms, the orientation of hairs, bristles and ommatidia. In vertebrates, PCP
signaling was found to control polarized cell movements during gastrulation and
neurulation (Veeman et al., 2003). The regulation of gastrulation movements in
vertebrates requires the activity of Wntl1, which may signal through Fzd7 to regulate
protrusive activity during convergent extension (Miller, 2001).
3.4 Wnt/p-Catenin Signaling and Tumorigenesis
~-catenin is the central and essential component in the W nt canonical signaling
cascade. It is also an integral component of the cadherin cell adhesion complex since it
links cadherins at the plasma membrane to the actin cytoskeleton through a-catenin and
a-actinin (Kemler, 1993). In the Wnt canonical signaling pathway, ~-catenin functions as
a transcriptional activator in conjunction with LEFffCF DNA binding proteins (Giles et
al., 2003). The need to balance the adhesive and transcriptional functions of ~-catenin is
becoming increasingly apparent from the study of Wntl~-catenin signaling in
tumorigenesis.
15
Tumor genetics revealed that mutations in members of the Wnt/~-catenin pathway
occur in approximately 90% of colorectal cancers as weIl as in other cancer types, su ch as
hepatocellular carcinomas and gastric cancers (Morin et al., 1997). Mutations that
activate the Wnt-~-catenin pathway promote stabilization of ~-catenin and indu ce its
nuclear accumulation (Giles, 2003; Polakis, 2000). This results in activated gene
transcription, altered cell migration, and cell polarity. Inactivating mutations in the APC
gene product, a protein that promotes the degradation of cytosolic ~-catenin, have been
found in up to 85% of sporadic colorectal cancers (Morin et al., 1997). Other mutations
that activate this pathway affect the function ofAxin, a protein that promotes the
formation of the ~-catenin degradation complex (Liu et al., 2000). Furthermore,
activating mutations in the CTNNB 1 gene itself have been reported in approximately
10% of colorectal cancers and in up to 40% of hepatocellular carcinomas (Morin et al.,
1997).
In the adenoma-carcinoma sequence of colon carcinogenesis, loss of APC
function is one of the earliest events in dysplastic transformation and leads to the
formation of adenomas (Bienz and Clevers, 2000). Loss of APC alone, however, is not
sufficient for progression to carcinomas and metastasis. Further mutations in different
pathways, su ch as activating mutations in Ras, and loss-of-function mutations in p53, are
also required (Vogelstein and Kinzler, 2004). Moreover, ~-catenin is found in the nucleus
only at the invasive front of late dedifferentiated, mesenchyme-like tumor ceIls, but not in
the central area of the primary tumors, where it localizes to the plasma membrane
(Brabletz et al., 2001). Therefore, other signaling components might be required for the
nuclear accumulation of ~-catenin.
16
During the development of carcinomas, cells undergo an epithelial-mesenchymal
transition (EMT), which is characterized by loss of cell-cell adhesion and increased cell
motility (Hu ber et al., 2005). The hallmark of EMT is the loss of function of E-cadherin
and the subsequent dissociation of the E-cadherin-~-catenin-a-catenin complex from the
membrane (Brabletz et al., 2005). Dissociation of adherens junctionscan also be induced
by phosphorylation of crucial tyrosine residues in components of these complexes (Roura
et al., 1999). Interestingly, overexpression of tyrosine kinases, and mutations in tyrosine
phosphatase genes that might catalyze these phosphorylation events have been reported in
tu mors (Wang et al., 2004). Loss of E-cadherin-mediated cell adhesion correlates with
increased ~-catenin-dependent transcription (Gottardi et al., 2001). In addition, these
changes correlate with po or prognosis in tumors (Thiery, 2002).
3.5 Cadherins and Ovarian Tumorigenesis
Cadherins are a family of integral membrane glycoproteins that mediate calcium
dependent ceIl-cell adhesion in a homotypic manner (reviewed in Braga, 2000). The
members of this family of cell adhesion molecules (CAMs) include the classical (Type 1)
cadherins, namely, E-, N- and P-cadherin. These cadherins have been shown to play an
important role in the establishment of intercellular junctions and cell polarity, as well as
in the maintenance of tissue integrity (Takeichi, 1994; Braga, 2000). Since cell-cell
associations are often disorganized in tumors, and must be compromised in invasive and
metastatic tumors, changes in cadherin expression or function have been implicated as
important components in tumorigenesis (Takeichi, 1993). Furthermore, as cadherins are
involved in cell sorting and tissue morphogenesis (Takeichi, 1995), inappropriate or
17
altered levels of cadherin expression within tissues may provide a mechanism for the
emergence of metaplastic or dysplastic derivatives which could be responsible for the
establishment of the preneoplastic state. An association between the loss or decreased
expression of E-cadherin with the emergence of poorly-differentiated invasive
carcinomas has been noted and has led to the characterization of the gene encoding E
cadherin as a tumor suppressor gene (Inoue et al., 1992; Takeichi, 1993). Peralta-Soler et
al. (Peralta-Soler et al., 1997) have shown that differential expression of E- and N
cadherin may be used to distinguish between different ovarian cancer histological
subtypes. That is, mucinous ovarian cancers express E-cadherin but not N-cadherin,
whereas serous and endometrioid ovarian cancers express both cadherin subtypes.
Interestingly, endometrioid ovarian cancers have the most malignant potential, followed
by serous and mucinous subtypes (Kumar et al., 2003).
As previously mentioned, studies have shown that EMTs occur during the
progression of epithelial cancers, thereby endowing these tu mors with increased motility
and invasiveness. Multiple oncogenic pathways including the Wntlcanonical signaling
pathway induce EMT, a hall mark feature of which is the down-regulation of epithelial
(E)-cadherin (Huber et al., 2005). Interestingly, studies suggest that the opposite may be
true in ovarian cancer. It is hypothesized that the repeated rupture and healing of the OSE,
as a consequence of ovulation, leads to tumorigenesis, which begins with the formation of
inclusion cysts situated in the ovarian stroma (Maines-Bandera and Auersperg, 1997;
Auesrperg et al., 1999). These, as weIl as ovarian tumors have been shown to express E
cadherin, while normal OSE has been shown to express only N-cadherin (Auersperg et al.,
1997, 1999 and 2001). Moreover, studies examining the transfection of normal OSE cells
with E-cadherin have shown that these cells develop inclusions cysts and begin to assume
18
a more invasive phenotype (Auersperg et al., 1999). These findings suggest that the OSE
may undergo a mesenchymal to epithelial phenotype transformation, which contrasts with
the well-known phenomenon of EMT that occurs during tumorigenesis in other organs.
To summarize, the present chapter has reviewed the most relevant histo
pathological aspects of ovarian cancer while introducing important aspects of Wnt
signaling, and describing the ways in which the Wntlcanonical signaling pathway is
related to cadherin molecules. Moreover, this chapter has described the concept of EMT
and alluded to the fact that ovarian cancer may not behave like most malignancies, insofar
as ovarian tumorigenesis may follow a mesenchymal to epithelial transition. This chapter
has been intended to compliment the present study and provide a framework for its
rationale, which is described in the following "Materials and Methods" chapter.
19
4. Materials and Methods
4.1 Cell Lines and Culture
Five human ovarian cancer celllines are maintained in our laboratory. Four of the
cell lines (SKOV3, CAOV3, OVCAR3, and SW626) were obtained from ATCC
(Manassas, VA, U.S.A.) and were provided by Dr. Derek Lobb (McMaster University,
Hamilton, ON, Canada). The fifth cellline, termed HEY, was a gift from the late Dr. J.
Dorrington (Banting and Best Institute, University of Toronto, Toronto, ON, Canada).
The provenance and characteristics of HEY cells have been reported previously (Buick,
1985). The origin of SW626 cells is unclear, but recent studies indicate that these cells
are probably of colonic rather than ovarian origin (Furlong et al., 1999). We have,
however, continued to use SW626 cells since they provide an interesting comparison with
the bana fide ovarian cancer cells.
AlI of the ovarian cancer lines, with the exception of OVCAR3, were cultured in
MEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 0.1 mM
non-essential amino acids (Life Technologies Inc., Burlington, ON). OVCAR3 cells were
maintained in RPMI 1640 containing 20% FBS, 2 mM L-glutamine and 0.1 mM non
essential amino acids (Life Technologies Inc.). An immortalized human OSE cell line
(IOSE398; (Auersperg et al., 1994) was generously provided by Dr. Nelly Auersperg
(University of British Columbia, Vancouver, BC, Canada) and was cultured in an 1:1
mixture of M-199 and MCDB (Sigma-Aldrich, Oakville, ON, Canada), 5% FBS, and
50J..lg/ml gentamicin (Life Technologies Inc.).
Wnt3a-expressing L cells were provided by Dr. Daniel Dufort (McGill University,
Montreal, QC, Canada) and were cultured in DMEM containing 10% FBS and antibiotics.
20
Conditioned media (CM) were derived from these cens (Wnt3aCM) and from control L
cens (controICM) by seeding the cens at approximately 30% confluence and collecting
the media after three days of culture. The conditioned media were cleared of cellular
debris by centrifugation (300xg for 10 min) and stored at 4°C after filtration through a 0.2
~m filter (Millipore, Mississauga, ON, Canada). Bioactivity of the Wnt3aCM was
assessed by its ability to transform C57MG cells (Shibamoto et al., 1998).
4.2 RNA Extraction
Total RNA was recovered from each cell line using Trizol (Life Technologies,
Inc.) as per the manufacturer's instructions. Cells were washed with sterile phosphate
buffered saline (PBS) and lysed directly in a culture dish by adding 1ml Trizol per 10 cm2
of culture dish surface area. Subsequently, 0.2 ml chloroform per 1 ml Trizol was added
to each sample. Samples were then centrifuged at 12000 x g for 15 min at 4°C to allow
for separation of the mixture into a lower phenol-chloroform phase, an interphase and an
upper aqueous phase containing RNA. Following alcohol precipitation, RNA from each
cell line was reconstituted in 10 ~l water. RNA was quantified by measurement of the
absorbance at 260 nm, and purity of the preparation assessed by the ratio of the
absorbance at 260 and 280 nm. RNA integrity was assessed by 1% agarose gel
electrophoresis in TAE buffer (0.04M Tris, O.OOIM EDTA and 0.02M acetic acid)
(Sambrook and Russel, 2000). Gels were visualized by ethidium bromide staining and
UV trans-illumination. The presence of sharp 28S and 18S rRNA bands with the
intensity of the 28S band being approximately twice that of the 18S band confirmed RNA
integrity (Sambrook and Russel, 2000).
21
4.3 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Each reverse transcription reaction was perforrned in a final volume of 20 III and
utilized 1 Ilg total cellular RNA, 250 ng random hexamers, 4 III 5X-first strand buffer, 2
III 0.1 M dithiothreitol (DTT), 1 III 10 mM dNTP mix, and 200 U Moloney murine
leukemia virus (MMLV-RT) (lnvitrogen, Burlington, ON, Canada). Separate reactions,
lacking reverse transcriptase, were perforrned for each RNA preparation in order to
control for potential genomic DNA contamination.
Polymerase chain reactions (PCR) were conducted in a final volume of 25 Ill,
which consisted of 2.51l1 lOX PCR buffer, 2 III cDNA, 1/lM forward and reverse primers,
2oo/lM dNTPs, 1.5mM MgCh, and 2U Taq DNA polymerase (Amersham Pharmacia,
Baie D'Urfe, QC, Canada). An initial denaturation at 95°C for 3 min was followed by 35
cycles of 95°C denaturation for 30 sec, annealing at 55°C for 30 sec, and extension at
72°C for 30 sec. A final extension at 72°C for 5 min and cooling to 4°C terminated the
reaction. PCR products were resolved in 1 % agarOse gels by electrophoresis in T AE
buffer (O.04M Tris, O.OOlM EDTA and O.02M acetic acid) (Sambrook and Rus.sel, 2000)
and visualized by ethidium bromide staining and UV trans-illumination.
In order to assess for differences in the expression patterns of Wntlcanonical
signaling components between non-cancerous and cancerous OSE cell lines, we sought to
examine the gene expression of selected subsets of Wntlcanonical pathway components,
namely Wnt2b, -3a, -5a, -7a, -11, Fz 1, -3, -7, -9, LRP5, -6 and Dvll, -2, -3. Wnt2b, -5a,
and -11 were selected based on the intention to include in the study both canonical and
non-canonical Wnts Our selection was further defined based on data from our previous
study (Ricken, 2002), which confirrned the expression of Wnt2b, -5a and -11 in rat and
22
human OSE. Wnt3a, -7a and Fzl, -3, -7 were selected based on a study by Hsieh et al.
which confirmed their expression in mouse ovaries (Hsieh et al., 2002). We included Fz9
based on its reported interaction with Wnt7a (Winn et al., 2005), and further examined
LRP5, -6 and Dvll, -2, -3 due to their reported functional importance in the
Wntfcanonical signaling pathway (He et al., 2004; Boutros and Mlodzik, 1999), and due
to the absence of studies having previously addressed the se signaling components in the
ovary.
The primer sequences utilized for the different gene products that were examined
are summarized in Table 1. Primers were designed, where su ch information was available,
so that the PCR products would span exon-intron borders, permitting detection of
potential genomic DNA contamination.
Gene 1 Primer Sequence Size (bp)
Wnt2b/13 5' GAT TCC TGA AGC TGG AGT GC 349 3' CGG CTC CCA TAC TGT GTT GA
Wnt3a 5' CTT TGC AGT GAC ACG CTC AT 375
3' GTG CTT CTC CAC CAC CAT CT
Wnt5a 5' GGA CCA CAT GCA GTA CAT CG 312 3' GCG GTA GCC ATA GTC GAT GT
Wnt7a 5' CAA GGC CAG TAC CAC TGG GA 307 3' GGC TCC ACG TGG ACG GCC TC
Wnt11 5' CGT GTG CTA TGG CAT CAA GT 226 3' GCT CAA TGG AGG AGC AGT TC
Fz1 5 CAT TTG GTC AGT GCT GTG CT 380 3' CGG CCA GGT GAA AAT ACT GT
Fz3 5' ATG GAA TAT GGA CGT GTC ACA C 432 3' GAT AAC GGA ATC TTG TGA CAT C
Fz7 5' CGA CGC TCT TT A CCG TTC TC 499 3' GAG GTA GAC GAA CAG AGG CG
Fz9 5' CT A TTT CCA CAT GGC TGC CT 357 3' GAA GTC CAT GTT GAG GCG TT
LRP5 5' CCG GAA GAT CAT TGT GGA CT 311 3' TGG ATG TCC ATG GGT GAG TA
LRP6 5' ACT GT A TCC CTG TGG CTT GG 439 3' TCC CTT CAT ACG TGG ACA CA
Dvl1 5' ACC CTG AAC CTC AAC AGT GG 200 3' CCC TTC ACT CTG CTG ACT CC
Dvl2 5' CTC ATG ACC AGe GAG CTG GA 230 3' CTT CTC CAT GTT TAG CGT GA
Dvl3 5' GCT AAA TGG AAC TGC GAA GG 187 3' CCG CTT GTG TCT TCT CAT CA
Table 1. PCR primer sequences for selected Wnt signaling pathway components.
23
4.4 Isolation of Cytosolic Fractions
Cytosolic fractions of cells were isolated in order to assess by Western blot
analysis levels of cytosolic ~-catenin. Cells were seeded in 100 mm dishes and incubated
overnight to allow for cell attachment. The following day, culture media were replaced
and cells were treated with final concentrations of 20mM NaCl, 20mM LiCI, 50% CM, or
50% Wnt3aCM. Twenty-four hr later, cells were scraped into IX PBS, centrifuged at
1000 rpm for 10 min, resuspended in homogenization buffer (20 mM HEPES pH 7.5, 10
mM KCL, 1.5 mM EDT A) supplemented with aprotinin (2 !lg/ml), leupeptin (2 !lg/ml),
Na3 V04 (1 !lM), PMSF (100 !lglml) (Sigma-Aldrich), and kept on ice for 25 min. Cells
were passed through a 22 gauge needle 3 times, and sonicated in order to ensure complete
cell lysis. They were then centrifuged at 100,000xg for 30 min at 4 oc. The resulting
supernatants were collected as cytosolic fractions and their prote in content was evaluated
using a modified Lowry assay (Hartree, 1972).
4.5lmmunoblotting
In order to assess the functionality of the Wntlcanonical signaling pathway, we
examined the effects of canonical (Wnt3a and LiCl) stimulation on cytosolic ~-catenin
levels by Western blot analysis. Given that cadherins are functionally related to the
Wntlcanonical pathway via structural association with ~-catenin and that both E- and N
cadherin are target genes of the Wntlcanonical signaling pathway, we further sought to
determine via' Western blot analysis wh ether Wntlcanonical pathway stimulation
correspondingly alters N- or E-cadherin expression.
24
Western blot analyses were performed as deseribed in Harlow and Lane (Harlow
and Lane, 1988). Briefly, protein samples (10-40 f..lg eytosolie proteins when assessing ~
eatenin levels versus total eell lysates when assessing eadherin levels) were resolved on
10% SDS-PAGE gels and transferred eleetrophoretieally onto polyvinylidene fluoride
(PVDF) membranes (Bio/Can Scientifie Ine., Mississauga, ON, Canada). Protein transfer
was confirmed by staining with Poneeau-S (Sigma-Aldrich). Membranes were ineubated
in bloc king buffer (I-Block, Bio/Can Scientific) and incubated overnight at 4°C with
either anti-~-catenin, anti-N-cadherin, anti-E-cadherin (Transduction Laboratories,
Lexington, KY, U.S.A.) or anti-actin (Santa Cruz, Santa Cruz, CA, U.S.A.) antibodies,
which were used to normalize for protein loading. Following incubation with the primary
antibodies, the blots were incubated with secondary antibody (alkaline phosphatase
conjugated donkey anti-mouse IgG or anti-goat IgG (when using anti-actin» (Jackson
Laboratories, West Grove, PA, U.S.A.). Finally, protein bands on the blots were
visualized by incubating the blots with ECF as per the manufacturer's instructions
(Amersham Biosciences) and usmg a STORM 9600 Phosphorimager® (Amersham
Biosciences). Densitometric analysis with ImageQuant TL software (Amersham
Biosciences) was used to quantify the protein bands.
4.6 Transfections and Luciferase Assays
In order to further assess the functionality of the canonical signaling pathway in
cancerous OSE cells, we set out to examine TCFILEF-mediated reporter gene activity in
response to canonical stimulation. Cells were seeded in 35 mm diameter dishes and
transfected the following day, at an estimated 50 % confluence, with either 0.3 Jlg
25
TOPFLASH or FOPFLASH plasmids (Van de Wetering et al., 1991, 1996), and 0.1 flg of
an expression vector coding for the ~-galactosidase gene (used as an internaI control for
transfection efficiency) (Upstate Biotechnology Inc., Lake Placid, NY, U.S.A.). Cells
were transfected using Lipofectamine reagent as per the manufacturer' s protocol
(Invitrogen). The following day, culture media were replaced and cells were treated with
final concentrations of 20mM NaCI, 20mM LiCI, 50% CM, or 50% Wnt3aCM. Twenty
four hr later, cells were washed once with PBS and lysed in 250 III lysis buffer (l %
Triton X-100, 15 mM magnesium sulphate, 4 mM EGTA, 1 mM dithiothreitol and 25
mM glycylglycine) (Fisher) on ice. The luciferase activity of each sample was measured
in an EG&G Berthold Luminometer (EG&G Optoelectronics, Vaudreuil, QC, Canada)
using 45 III ceIllysate and normalized to ~-galactosidase activity (Valderrama-Carvajal et
al., 2002).
4.7 MTT Assays
In view of our observed ceIlline-specific responses to Wnt3a and LiCI stimulation
on cytosolic ~-catenin levels, and TCF/LEF reporter gene activation, we next examined
whether there were changes in cell growth and proliferation. The effects of LiCI or
Wnt3aCM treatment on growth and proliferation of the cell lines were examined using
MTT assays (Kost et al., 1995). Briefly, 5-6000 cells were seeded in 100 ~l medium in
individu al wells of a 96-well culture dish (Corning Inc., Corning, NY, U.S.A.). After an
overnight incubation to allow for cell attachment, culture media were supplemented to
yield a final concentration of either 20mM LiCI (Hedgepeth, 1997) or 50% Wnt3aCM
(Shibamoto et al., 1998). Control wells received the equivalent concentrations of NaCl or
26
CM, respectively. At 0, 24, 48, or 72 hr post-treatment, 10 III MTT reagent
(methylthiazoletetrazolium, 5 mg/ml in sterile PBS) (Sigma-Aldrich) were added to each
weIl and the plate incubated for an additional 4hr at 3ic. The resulting formazan
product was dissolved by the addition of lOO1l1 solubilization solution (20% SDS, 50%
dimethylformamide) to the wells and incubated ovemight at 3ic. Absorbance at 570 nm
was determined using a plate reader (Thermo Max, Molecular Devices, Sunnyvale, CA,
U.S.A.). Absorbances were corrected for background by subtracting the mean absorbance
values derived from wells containing the equivalent volume of cell-free media.
Preliminary experiments were conducted to establish the range of cell concentrations
providing linear absorbance responses for each of the celllines.
4.8 Statistical Analyses
MTT assays were repeated three times, while transfections were performed twice.
The data are shown as averages with corresponding standard error means (SEMs).
Results for the MTT assays were analyzed by ANOV A and multiple comparisons were
made after the application of the Bonferroni correction. Transfection data were analysed
using the student's t-test. Differences were considered significant for p < 0.05.
AIl statistical analyses were performed using Systat software (Systat Software Inc.,
Point Richmond, CA, U.S.A.).
27
5. Results
5.1 OSE Cell Lines Display Distinct Expression Patterns of Wnt Signaling Components
Table 2. (please see Chapter 6. for aIl Figures and Tables referenced in this
chapter) summarizes the RT-PCR results for the selected Wnt signaling components in
OSE ceIls, whereas photographs of representative agarose gels containing PCR products
stained with ethidium bromide are shown in Figure 2. Wnt2b, Wnt7a, Fzl and Fz9 were
expressed in aIl ceIl lines examined. Wnt2b transcripts were detected in aIl ceIl lines,
including IOSE398 ceIls, while Wnt3a mRNA was not detected in any of the OSE lines.
Wnt5a, and -11 transcripts were not detected in IOSE398, but were present in the other
lines. Interestingly, Wnt7a mRNA was not detected in either IOSE398 or SW626 cells.
Fzl,-3 and -7 were expressed in all celllines, while Fz9 transcripts were not detected in
CAOV3, OVCAR3, and SW626 ceIls. LRP5 mRNA was detected in aIl ceIllines, while
LRP6 mRNA was detected only in the cancer celllines. Dvll and Dvl2 were detected in
aIl ceIllines. Dv13 mRNA was expressed in aIl of the ceIllines except SW626 ceIls.
5.2 Wnt3a and LiCl1ncrease CytosoIic p-Catenin and TCF/LEF-Mediated Reporter Gene Activation in a Cell Line-Specific Manner
Figure 3. shows Western blot analyses for cytosolic ~-catenin levels in Wnt3a-
and LiCI-treated ceIls. Wnt3a stimulation caused an increase in cytosolic ~-catenin levels
in IOSE398, HEY and SW626 cells, while Liel stimulated increases in cytosolic ~-
catenin levels in IOSE398, SKOV3, HEY and CAOV3 ceIls.
Figure 4. illustrates the effects of Wnt3a1LiCI-stimulation on ~-catenin TCFILEF-
mediated reporter gene activation. In agreement with the increases in cytosolic ~-catenin
28
levels noted in REY ceIls, both Wnt3a and LiCI-treated REY cells displayed significantly
increased reporter gene activity. In contrast, canonical stimulation of all other cell lines
examined, including SKOV3 cells (data not shown) showed no change in reporter gene
activation compared to controls.
5.3 Treatment with Wnt3a or LiCI Causes Differentiai Effects on Cell Proliferation and Cell Detachment in OSE Cell Lines
Changes in cell proliferation were assessed by MTT assays of cells following
72 hr treatment with Wnt3a (Figure 5.) or LiCI (Figure 6.). Wnt3a treatment of IOSE398
cells resulted in decreased proliferation relative to controls, while having no effect on
cancerous OSE celllines (Figure 5.).
After the first 24 hr of treatment, LiCI-stimulated IOSE398 cells showed a
decrease in proliferation. Similarly, LiCI-treated SKOV3, REY and CAOV3 cells
showed a marked decrease in proliferation relative to control s, while OVCAR3 and
SW626 cells were not affected by LiCI stimulation (Figure 6.).
Figure 7. depicts the distinct morphologie al and growth characteristics of the OSE
cell lines examined in this study. In culture, N-cadherin-expressing SKOV3 and REY
cells exhibited fibroblastic-like morphologie al characteristics. In particular, REY cells
assumed a more stellate appearance, while growing in a disorganized and overlapping
manner. In contrast, the exclusively E-cadherin-expressing OVCAR3 cells displayed a
classical epithelial morphology characterized by a highly organized cellular monolayer of
tightly apposed polygonal cells assuming a cobble-stone appearance. SW626 cells grew
as an epithelial-like monolayer of polygonal cells (Figure 7.).
29
Figures 9A. and 9B. show that 24 hr treatment with Wnt3a resulted in increased
ceIl detachment of IOSE398 ceIls from the culture dish. Figures 1OA. and lOB. show that
24 hr LiCl treatment resulted in increased ceIl detachment from the culture dish of both
IOSE398 and HEY ceIls in comparison to NaCl-treated control cells. None of the
treatments appeared to affect the ceIl morphology of attached ceIls.
5.4 LiCI and Wnt3a Increase N-Cadherin and E-Cadherin Levels in a Cell Line-Specific Manner
The expression pattern of N- and E-cadherin in OSE ceIls is shown in Figure 11.
IOSE398, SKOV3, and HEY ceIls expressed N-cadherin, whereas CAOV3 cells
expressed both N-and E-cadherin. OVCAR3 and SW626 ceIls expressed exclusively E-
cadherin.
Western blot analyses of E- and N-cadherin levels in response Wnt3a1LiCI-
stimulation are shown in Figures 12A.-12D. Wnt3a treatment increased N-cadherin
expression in SKOV3 ceIls and decreased N-cadherin expression in HEY cells.
Moreover, Wnt3a treatment decreased E-cadherin expression in OVCAR3 ceIls and
increased its expression in SW626 ceIls. LiCI increased N-cadherin expression in aIl N-
cadherin-expressing ceIl lines. It also increased E-cadherin expression in CAOV3 cells
and decreased E-cadherin expression in SW626 ceIls.
30
6. Figures
31
IŒE98 SKOV3!lEY iCaOV3:0YCAR31SW)26 ""'" "'" """"'" '" ""'T"""""" ""'," T" """""""'1' """""""""""T"""""" "'J"
rw."-'--'-·'" ---'-'-+-----_", ,.-. --- I-,'------·-t---'--"--i._ 'J '1-Wt2b'·,-----'-"l--3'" j"~, -+'-'-'----1---,-'-+-'-.,."+---~- "'0 ----+--'-.,,-t--'-"'-"+-.---"- +'~-+''''---
r~'---,--""'- _ l "-~--'-=--" _ 1'-"-'-~'-1 i 4':"'-~-"';,1'----'----1-'" "---"~"~-- .----.... --.--11
.-----.,--'----- - j 'Wt5a -,--- i .---- '. .
l''----''------t ,·,'-·,,:'----+1' '-'--':++-"'---T-'±---:"-- + + + 1
,W1:7a . + + + 1W111 ! --,.-+ 1 + i + +
Fzl + Fz3 + .Fz7 + IF19 +
lRP5 i + lRP6
i LMl ! + 1
1 + 1 +
Table 2.
i :
+1 + 1
+ + 1
+ +
+ 1 + , 1
i
i + + ,
1
+ +
i
! i
+ + i
+ + ,
+ +
+ + +
+
+
ND ND ND
, 1
i
+ + +
+
+
+
+
+
+
+ + +
+ + J
i 1
! i + 1 +
32
Table 2. OSE celllines display distinct expression patterns of Wnt signaling components.
This table summarizes the RT-PCR analyses of Wnt signaling components expressed in
one normal OSE line (IOSE398), four cancerous OSE lines (SKOV3, REY, CAOV3,
OVCAR3) and one cell line of probable colon cancer origin (SW626). Results of PCR
products derived from amplification with specifie primers for Wnt2b, -3a, -5a, -7a, -11,
Fzl, -3, -7, -9, LRP5 and 6, and Dvll, -2, -3 are shown. Amplified products were
resolved on 1 % agarose gels and visualized by ethidium bromide staining. In this table (+)
denotes the detection of the PCR product for a given primer, whereas (-) indieates that the
PCR product was not detected.
33
Wnt2b
Wnt7a
Fz 1
Fz9
Figure 2. Representative RT -PCR analyses of Wnt signaling pathway components expressed in IOSE398, SKOV3, HEY, CAOV3, OVCAR3 and SW626 cells.
PCR products derived from amplification with specific primers for Wnt2b, Wnt7a, Fzl
and Fz9 are shown. Wnt2b and Fzl are expressed in all celllines. Neither IOSE398 nor
SW626 express Wnt7a, and Fz9 is not expressed in CAOV3, OVCAR3 and SW626 cells.
Amplified products were resolved on 1 % agarose gels and visualized by ethidium
bromide staining. Similar analyses with at least two different RNA preparations from the
same celllines provided the same results.
34
__ ---------C-~-V-3--------__ __----------I~-E----------_ ~ .f d' /";; ~
Cytosolic B-catenin --
15+----------
10+----==----
r .j' Cytosolic ~
B-catenin
Actin ~.
HEY ...A-
~
~~
SKOV3
Cytosolic B-catenin
.f
Actin ___ _ ..... , ... ,'~c
50 ,..------;;::
40+--------
30 t--t:!:::> 1---
10 t--L":;":'I---
Figure 3.
/'.......
d'
-....
~:;
• a ~
/if'
li •
-92KD
'~' ~.i! !. -45KD
OVCAR3
r~ ~
.A...
~ ~} • a - - • -92KD
i --- ~ -45KD
SW626
r~ ~D ..A...
d' /3r'-
• -92KD
• -.- -' -' -45KD 'IJIt
35
Figure 3. Wnt3a and Lie) increase cytosolic p-catenin in a cellline-specific manner.
Figure 3. depicts representative Western blots for cytosolic fractions of CAOV3,
IOSE398, HEY, OVCAR3, SKOV3 and SW626 cells probed with anti-p-catenin
antibody and anti-actin antibody (in order to normalize for protein loading). Given the
significant difference in molecular weight between p-catenin and actin, blots were divided
and subsequently probed with the appropriate antibody. The bar graphs below each
Western blot represent the numerical values (obtained by densitometric analysis using
ImageQuant TL software) attributed to the bands probed with p-catenin after they were
normalized for protein loading using the numerical value attributed their respective actin
band. The molecular weights for p-catenin and actin are indicated in the right-sided panel
of each Western blot. Prior to performing the Western blots analyses, cells were treated
for 24 hours with either NaCI, LiCI, CM or Wnt3a.
36
HEY 7.5
5.0
5 CI::
2.5
0.0 Na Li CM Wnt-3a
Treatm ents
CAOV3
1.5
1.0
0.5
0.0 Na Li CM wnt3a
Treatm ent
OVCAR3 1.5
1.0
0.5
0.0 Na Li CM Wnt-3a
Treatments
Figure 4.
37
Figure 4. Wnt3a and LiC) increase TCFILEF -mediated reporter gene activation in a cellline-speciflc manner.
HEY, SKOV3 (data not shown), CAOV3 and OVCAR3 cens were transfected with
TOPFLASH plasmids, and treated for 24 hr with NaCI, LiCI, CM or Wnt3a after which,
luciferase activity for each sample was measured (expressed in Relative Light Units
(RLU)). Wnt3a and LiCl treatment le ad to increased luciferase activity (by increased
TCFILEF-mediated reporter gene activation) in HEY cens. TCFILEF-mediated reporter
gene activation was significantly altered in response to Wnt3a or LiCI treatment in
SKOV3 (data not shown), CAOV3 or OVCAR3 cens. Transfections were performed
twice and data are shown as averages with corresponding standard error means (SEMs).
Transfection data were analysed using the student's t-test. Differences were considered
significant for p < 0.05.
38
IOSE398 3
--0- control media
---Wnt3a
o 24 48 72
lime (br)
HEY 6
--a- control media ... Q,l
---Wnt3a ~
E /J/'-=,"",4 = -~ /"'" _0 -- ...... Q,l ..
---\,1 ... /",
=-= 2 ,,---.- .! Q,l .. t)I)!:,
= = ..c: 0 U
i i i i
0 24 48 72
lime (br)
OVCAR3 2.5
--a- control Iredia ... ~ 2.0 __ Wnt3a E ;:= 1.5 _0 -- ~ ~ ~ 1.0 ------------0----------... ---·-----·-=; .- .:! ~-Ë 0.5 .:;
= ..c: U 0.0
Figure 5.
1
o i i
24 48
lime (br)
i 72
4
1.25 ... Q,l 1.00 ~
E---= <:> = ~ 0.75 = .... Q,l Q,l \,1 ~ 0.50 =:0: .- = Q,l-
:..Ë 0.25 = ..c: U 0.00
4
SKOV3 --0- control media
---Wnt3a
i o
i 24
CAOV3
lime (br)
i 48
~ ' ...... '" ...... -. -- ------!----'t----
--a- control Iredia
--Wnt3a
i i i
0 24 48
lime (br)
SW626
--a- control media
---Wnt3a
i o
1 i 24 48
lime (br)
i 72
=11
1
72
72
39
Figure 5. Treatment with Wnt3a causes difTerential efTects on cell proliferationlgrowth.
MTT cell viability assays were performed on IOSE398, SKOV3, REY, CAOV3,
OVCAR3, and SW626 cells after 0, 24, 48 and 72 hr of treatment with Wnt3a
conditioned media versus control media. MTT assays were repeated three times and
relative absorbance values are shown as averages with corresponding standard error
means (SEMs). Absorbance values obtained for MTT assays at different time points are
expressed relative to the values obtained at 0 hr for each ceIlline. Results for the MTT
assays were analyzed by ANOV A and multiple comparisons were made after the
application of the Bonferroni correction.
40
./ IOSE398 2 --c- MedialNaCI
-LiCI
6
2
1
o
HEY
1
24
Time (br)
1
48
-0(1- Media/NaC)
-LiC)
i o
i i 24 48
Time (br)
CAOV3
-0- Media/NaCI
--LiC)
1 o
i i
24 48
Time (hr)
Figure 6.
1
72
i 72
1
72
4
.. ~ ..c e =--. =-_co - -~ ... ... ~
== oz .... .! ~ ... ~,!;:,
= = ..c: U
SKOV3 -0(1- MedialNaCI
--LiCI A ..-"' ............ ..---
_~_._--a'
.. __ .. - ----1
--~ ~
1 o
i 24
Time (br)
i 48
i 72
OVCAR3 2.5
2.0
1.5
1.0
0.5
0.0
4
-0(1- Media/NaCI
i o
i i 24 48
Time (br)
SW626
-0(1- Media/NaCI
--LiCI
1 i i
o 24 48
Time (br)
i 72
1
72
41
Figure 6. Treatment with Liel causes ditTerential etTects on cell proliferation/growth.
MTT cell viability assays were performed on IOSE398, SKOV3, REY, CAOV3,
OVCAR3, and SW626 cells after 0, 24, 48 and 72 hr of treatment with LiCI versus NaCI
(control). MTT assays were repeated three times and relative absorbance values are
shown as averages with corresponding standard error means (SEMs). Absorbance values
obtained for MTT assays at different time points are expressed relative to the values
obtained at 0 hr for each cellline. Results for the MTT assays were analyzed by ANOV A
and multiple comparisons were made after the application of the Bonferroni correction.
42
IOSE398 SKOV3 HEY
CAOV3 OVCAR3 SW626
Figure 7. Phase contrast microscopie images of IOSE398, SKOV3, HEY, CAOV3, OVCAR3 and SW626 cells.
SKOV3 cells exhibit fibroblastic-like morphological characteristics (arrows). HEY cells
assume a more stellate appearance, while growing in a disorganized and overlapping
manner (arrows). E-cadherin-expressing OVCAR3 cells display a classical epithelial
morphology characterized by a highly organized cellular monolayer of tightly apposed
polygonal cells assuming a cobble-stone appearance (arrows). SW626 cells grow as an
epithelial-like monolayer of polygonal cells (arrow).
43
IOSE398 SKOV3 HEY
CM CM CM
Wnt3a Wnt3a Wnt3a
A
CAOV3 OVCAR3 SW626
CM CM CM
Wnt3a Wnt3a Wnt3a
B
Figure 8.
44
Figure 8. Pbase contra st microscopie images of IOSE398, SKOV3, DEY, CAOV3, OVCAR3 and SW626 cells in response to Wnt3a treatment.
IOSE398, SKOV3, HEY, CAOV3, OVCAR3 and SW626 cells were treated with Wnt3a
versus control media (CM) for 24 hr. Wnt3a promotes cell detachment exclusively in
IOSE398 cells (arrows).
45
IOSE398 SKOV3 HEY
NaCI NaCI NaCI
LiCI LiCI LiCI
A
CAOV3 OVCAR3 SW626
NaCI NaCI NaCI
LiCI LiCI LiCI
B
Figure 9.
46
r- Figure 9. Phase contra st microscopie images of IOSE398, SKOV3, HEY, CAOV3, OVCAR3 and SW626 cells in response to LiC) treatment.
IOSE398, SKOV3, HEY, CAOV3, OVCAR3 and SW626 cells were treated with LiCI
versus NaCI (control) for 24 hr. LiCI promotes cell detachment in IOSE398 and HEY
cells (arrows).
47
N-CAD -140 KD
-120 KD E-CAD
Figure 10. OSE celllines exhibit distinct cadherin expression patterns.
Total celllysates of IOSE398, SKOV3, HEY, CAOV3, OVCAR3 and SW626 cells were
analyzed by Western blot techniques. The blots were probed with anti-N-cadherin
antibodies (top panel) and anti-E-cadherin antibodies (bottom panel). Estimated
molecular weights of N-cadherin and E-cadherin are indicated in the right-sided panel.
SW626 cells N-cadherin is expressed in IOSE398, SKOV3, HEY and CAOV3 cells,
while E-cadherin is expressed in CAOV3, OVCAR3 and SW626 cells. The second band
appearing below the 120 KD band reflecting E-cadherin expression in SW626 cells
represents an unphosphorylated variant of E-cadherin.
,--
48
N-CAD -140 KD
Actin -45 KD
6,--------------------------5+-----
lia
N-CAD -. 111. ______ ,.=_-- - • • • -140 KD
Actin . .a; :~~
~.------------------------ 100,----------------------------
3)+-----
ro~------------~
25 +---------
Na(]
Figure 11.
49
OVCAR3
--------~~--------~ r ~'\ ô Ô ,,~ ~~V &' ~~
E-CAD - a ____ •• __ _ -120 KD
Actin -45 KD
3r---------------------------
2_!__----
liO
SW626
--------~~---------r ~ Ô ,.., _, ,,~
~~ ..y.\o.i G~' ~~
E-CAD -120 KD al_ ==:: •
Actin
100,---------------------------,
120;---------------
oo~-------------
~-!----------------
Na(] ua CM
-- Figure 11. (Continued)
50
Figure 11. Liel and Wnt3a increase N-cadherin and E-cadherin levels in a celllinespecifie manner.
Figure 11. depicts representative Western blots probed with either anti-N-cadherinand
anti-actin antibodies (CAOV3, IOSE398, SKOV3 and HEY) or anti-E-cadherin and anti-
actin antibodies (CAOV3, OVCAR3 and SW626). Total cell fractions were used for each
blot. Given the significant difference in molecular weight between both E- and N-
cadherin and actin, blots were divided and subsequently probed simultaneously with the
appropriate antibody. The bar graphs below each Western blot represent the numerical
values (obtained by densitometric analysis using ImageQuant TL software) attributed to
the bands probed with either N- or E-cadherin after they were normalized for protein
loading using the numerical value attributed their respective actin band. Estimated
molecular weights for N-/E-cadherin and actin are indicated in the right-sided panel of
each Western blot. Prior to performing the Western blots analyses, cells were treated for
24 hr with NaCl, LiCI, CM or Wnt3a. The second band appearing below the 120 KD
band depicting E-cadherin expression in SW626 cells represents an unphosphorylated
variant of E-cadherin.
51
7. Discussion
Although the molecular basis of ovarian tumorigenesis is increasingly examined,
our recognition of precursor lesions remains limited (Bell, 2005; Scott and McCluggage,
2006). As a result, ovarian cancer is often diagnosed at a late stage, and the prognosis is
invariably poor (Scott and McCluggage, 2006). The involvement of Wnt signaling in
tumorigenesis is weIl established. Studies examining Wnt signaling in ovarian cancer,
however, have only recently begun to emerge. Underlying the majority of these studies
are correlations between histological subtypes of ovarian tumors and patterns of genetic
aberrations and/or target gene activation (Schwartz et al., 2003; Shedden et al., 2005).
In this study, we examined the expression profiles of Wnt signaling pathway
components, as weIl as the functionality of the canonical pathway in both normal and
cancerous OSE cell lines. Our study showed that ovarian cancer cell lines exhibit
distinct aberrations in the Wnt signaling pathway, which affect cell proliferation
and/or cell adhesion molecule expression. These aberrations may either give rise to
or are a consequence of ovarian tumorigenesis.
Our examination of gene expression profiles based on selected Wnt signaling
pathway components revealed that ovarian cancer cell lines exhibit distinct gene
expression patterns. Wnt2b, however, was found to be expressed in all celllines studied.
Nevertheless, it has been reported that there are two Wnt2b splice variants (Wnt2b
isoforms 1 and 2) (Katoh, 2005). The Wnt2b isoform 2 has been found to activate the
canonical signaling pathway and is upregulated in stem cells and esophageal/gastric
52
cancers (Katoh, 2005). In view of these findings, it would be pertinent to deterrnine
whether ovarian cancer cens express greater levels of Wnt2b isoforrn 2.
Expression of Wnt7a, also a canonical Wnt, was found to be acquired in an
cancerous OSE cell lines exarnined. Consistent with this finding are studies reporting de
nova or increased Wnt7a expression in colorectal, pancreatic and gastric cancer ceIllines
(Kirikoshi and Katoh, 2002). These data suggest that Wnt7a may help promote
tumorigenesis in OSE through aberrant activation of the Wntlcanonical signaling pathway.
Interestingly, our data show that cancerous OSE cell lines also acquire the
expression of non-canonical Wnts (i.e. Wnt5a and Wntll). It has been suggested that
non-canonical Wnt signaling antagonizes canonical signaling and that this antagonism
may inhibit tumor progression (Veeman et al., 2003). Wnt5a expression levels, however,
have been found to be up-regulated in gastric cancer, lung cancer, and melanomas (Katoh,
2005). Moreover, Wnt5a-mediated signaling can enhance the motility and invasiveness
of melanoma cells (Weeraratna et al., 2002). Through their association with either Fz3
or -6, it has been reported that Wnt5a and -11 can activate the WntIPCP signaling
pathway (Katoh, 2005). It has further been suggested that aberrant activation of the PCP
signaling pathway in human cancer promotes invasion and metastasis (Katoh, 2005).
Although the classification of Wnts according to their capacity to stimulate the canonical
signaling pathway versus non-canonical pathways is not absolute (Miller, 2001), the
acquisition of non-canonical Wnt expression in OSE tu mors may help promote invasion
and metastasis.
In our examination of Fz expression patterns, we noted that Fz9 was not expressed
in CAOV3, OVCAR3 and SW626, aIl of which are E-cadherin-expressing ceIllines. Fz9
has been reported to require Wnt2-ligand binding in order to activate the canonical
53
signaling pathway (Karasawa et al., 2002). Interestingly, increases in Wnt2 expression
have been correlated with decreases in cell surface E-cadherin expression and increased
metastatic potential in gastric cancers (Cheng et al., 2005). The absence of Fz9
expression in E-cadherin-expressing cancerous OSE cell lines suggests that these ovarian
cancer cells may be resistant to Wnt2-stimulation and thereby exhibit reduced metastatic
potential.
LRP5/6 co-receptors are necessary components in Wntlcanonical signal
transduction (He et al., 2004). Our data shows that ovarian cancer cells gain LRP6
expression. Although LRP5 and -6 display a certain level of functional redundancy,
LRP6 is reportedly more influential than LRP5 in embryogenesis (He et al., 2004).
Moreover, Bafico et al. (Bafico et al., 2004) report that siRNA-mediated inhibition of
LRP6, but not LRP5, results in decreased levels of uncomplexed ~-catenin in Wnt3a
stimulated ovarian cancer cells. In agreement with this finding He et al. (He et al., 2004)
have reported that Wntl, -3a, and -7a rely more heavily upon LRP6 for canonical signal
transduction. These findings suggest that LRP6 may be a more powerful transducer of
canonical signaling, and its acquisition in ovarian cancer ce Ils may promote
tumorigenesis.
Our study reports Dvll, -2 and -3 expression in all OSE cell lines. Dvls are
versatile scaffold molecules known to have the ability to channel Wnt signaIs towards
canonical, WntlPCP and WntlCa2+ pathways (Wallingford and Habas, 2005). The
mechanisms through which Dvl molecules exhibit pathway specificity remain to be
elucidated. Nevertheless, it appears that pathway specificity may be influenced by cell
type and cellular environment (Wallingford and Habas, 2005). Therefore, despite the
ubiquitous expression of aIl three Dvl molecules in the OSE cell lines, the Wnt signal
54
transduced may differ between celllines given that tumorigenesis can alter cell phenotype
and by extension, cellular environment.
Our next objective was to assess the functionality of the Wntlcanonical signaling
pathway by evaluating cytosolic p-catenin levels and TCFILEF reporter gene activation in
response to Wnt3a (a biological activator of the canonical signaling pathway) and LiCI (a
synthetic activator of the canonical pathway, which promotes the accumulation of
cytosolic p-catenin via the inhibition of GSK-3p (Redgepeth et al., 1997)). Our data
suggest that ovarian cancer cell lines exhibit aberrations at different levels of the
Wntlcanonical signaling pathway.
Similar to non-cancerous IOSE398 cells, our data show that N-cadherin
expressing SKOV3 and CAOV3 cells display decreased cytosolic p-catenin levels in
response to Wnt3a-treatment, whereas aIl N-cadherin-expressing ovarian cancer ceIllines
display increased cytosolic p-catenin levels in response to LiCI treatment. Excluding
REY cells, however, increased cytosolic p-catenin levels did not correlate with increased
TCFILEF-mediated reporter gene expression, which represents the endpoint of the
canonical signaling pathway (Tolwinski et al., 2004). OVCAR3 cells (which only
express E-cadherin), showed no change in cytosolic p-catenin levels or TCFILEF
mediated reporter gene expression in response to either treatment. Interestingly, REY is
the only OSE cell line which displayed increased cytosolic p-catenin levels (in response
to both Wnt3a and LiCI) that subsequently correlated with increased TCFILEF-mediated
reporter gene expression. These findings suggest 1) that the Wntlcanonical signaling
pathway is functionally intact in REY ceIls, but not in SKOV3, CAOV3 or OVCAR3
cells, and 2) that REY cells (but not SKOV3 and CAOV3 cells) have acquired an
alteration in the canonical signaling pathway upstream of GSK3p that facilitates
55
Wntlcanonical signaling in this cell line. OVCAR3 ceIls, on the other hand, have
acquired an aberration downstream of GSK-3~, which renders this ceIlline more resistant
to canonical stimulation.
~-catenin binds to the cytoplasmic domain of type 1 cadherins and the genes
encoding E- and N-cadherins are target genes of ~-catenin-mediated canonical signaling
(Nelson and Nusse, 2004). In view of the convergence of the ~-catenin and cadherin
pathways, as weIl as the association of ~-catenin-mediated canonical signaling with
increased cell proliferation, we examined the effects of canonical stimulation on cell
proliferarion, and cadherin expression. Our data showed that canonical stimulation
correlated with distinct effects on cell proliferation and cadherin expression in ovarian
cancer cells.
In all N-cadherin expressing cell lines, including IOSE398 ceIls, LiCI-treatment
induced increased cytosolic ~-catenin levels, which correlated with decreased cell
proliferation and increased N-cadherin expression. Given that LiCI-mediated increased
cytosolic ~-catenin levels did not correlate with increased TCFILEF-mediated reporter
gene transcription in SKOV3 and CAOV3 cells, it seems unlikely that increased N
cadherin expression in the se cell lines is secondary to TCFILEF-mediated target gene
transcription. Recent studies have shown that GSK3~ inhibition promotes transcription
of Snail, a zinc finger transcription factor (Rosano et al., 2005) which can modulate
cadherin expression by acting alone or in concert with the Wntl~-cateninfTCFILEFaxis
(Yook et al., 2005; Bachelder et al., 2005). Thus, the increase in N-cadherin expression
in response to LiCI-treatment may be secondary to an alternate transcription factor which
is also upregulated by GSK3~ inhibition, su ch as Snail.
56
Interestingly, in OVCAR3 and SW626 ceIls, which only express E-cadherin,
neither cell detachment nor proliferation were affected by Wnt3a/LiCl-treatment. E
cadherin levels, however, were significantly increased in SW626 cells in response to
Wnt3a-treatment. Being of colonic origin, SW626 cells exhibit constitutively activated P
catenin secondary to an APC mutation (Furlong et al., 1999). Thus, Wnt3a-treatment
induced only a slight increase in cytosolic p-catenin levels in SW626 cells. Nevertheless,
this increase in cytosolic p-catenin correlated with increased E-cadherin target gene
transcription. This finding suggests Wnt3a-treatment in colonic tumors may stimulate an
altemate signaling pathway resulting in increased E-cadherin expression.
Overall, cell detachment was only visibly increased in IOSE398 and REY cells.
Moreover, Wnt3a-treatment promoted cell detachment and decreased cell proliferation in
IOSE398 cells, but did not alter cell detachment or proliferation in any cancerous OSE
cell Hnes. Although activation of the canonical signaling pathway typically results in
increased cell proliferation (especially in tumorigenesis) (reviewed in Polakis, 2000),
studies have shown that p-catenin may promote apoptosis in several cell systems (Kim et
al., 2000). In addition, a more recent study reports that p-catenin may control cell cycle
and apoptosis in normal, and transformed epidermal keratinocytes (Olmeda et al., 2003).
Thus, in normal OSE, canonical stimulation may result in decreased cell proliferation
secondary to canonical-mediated cell cycle regulation and induction of apoptosis, while
ovarian cancer cells may acquire a resistance to this effect, which compromises tumor
progression.
Through the examination of gene expression profiles and canonical pathway
functionality, this study has contributed to a deeper understanding of the molecular
changes in OSE tumor progression. Nevertheless, certain limitations must be considered
57
in the interpretation of these data. Firstly, in our examination of canonical signaling
pathway functionality through TCFILEF-mediated reporter gene activation, we
acknowledge that TCFILEF reporter gene assays remain to be completed for both the
IOSE398 and SW626 cells. Moreover, all TCFILEF reporter gene assays should be
repeated at least two more times in order to determine the significance of our findings, as
these experiments were only performed once. Overall, these data wou Id greatly improve
our assessment of canonical pathway functionality in all celllines.
The second limitation of this study pertains to the Western blots examining ~
catenin and cadherin levels in response to Wnt3a and LiCI treatment. Again, these
experiments were only performed once, and should be repeated at least two more times in
order to more accurately determine the significance of the observed differences.
The third limitation of this study is one which is universal in the study of ovarian
cancer, given that common laboratory animaIs do not develop cancer of the OSE
(Damjanov, 1989). In fact, only hens and humans develop tumors of the OSE
(Fredrickson, 1978; Murdoch et al., 2005). Therefore, human OSE cancer must by
necessity be studied in culture (Auesrperg, 2003). While the use of cell cultures has its
advantages, the fact that cultured OSE is no longer influenced by the paracrine secretion
of hormonal factors from neighboring cell types, coupled with the difficulty in obtaining
adequate cell numbers in culture without introducing major changes from the in vivo
phenotype, remain major caveats (Auersperg, 2003). Therefore, the possibility that su ch
changes render the cells non-representative of their source in vivo must be taken into
consideration in the interpretation of data.
As previously established, most ovarian malignancies are epithelial in type
(Auesrperg et al., 2001; Kumar et al., 2003). The most widely accepted hypothesis at
58
present is that OSE tu mors are the result of incessant wounding and repair of the OSE
secondary to ovulation (FathaIla, 1971). Studies have shown that Wnt molecules are
important mediators of wound repair (Fathke et al., 2006). For instance, ~-catenin
mediated canonical signaling has been shown to play an important role in promoting cell
proliferation during cutaneous wound healing and its deregulation is associated with
hyperplastic cutaneous scaring (Cheon et al., 2005). Other studies have shown that
canonical pathway stimulation is capable of inducing adult mammalian skin regeneration
(Fathke et al., 2006). Regeneration implies that the cells repopulating a wound are
plu ri pote nt and can therefore respond to factors promoting their differentiation into skin
appendages, thereby ensuring architectural integrity. Although regeneration is not seen in
most mammalian adult organ systems, there is evidence that stem cells or cells capable of
restoring the original tissue are present within multiple adult tissues (Tumbar et al., 2004;
Wagers and Weissman, 2004; Korbling and Estrov, 2003; Alonso and Fuchs, 2003).
Interestingly, the canonical Wnt signaling cascade has also emerged as a critical
regulator of stem cells. The association between Wnt signaling and cancer has further
raised the possibility that tightly regulated self-renewal mediated by Wnt signaling in
stem and progenitor cells may be subverted in cancer cells in order to allow for malignant
proliferation. Studies examining stem cell deregulation in colon cancer and leukemia
support this possibility (reviewed in Reya and Clevers, 2005).
When interpreting our findings within the context of the involvement of Wnt
signaling in wound repair, stem cell renewal and tumorigenesis, we believe that the OSE
is composed of stem cells which, during tumorigenesis, may assume a more differentiated
morphology such as that of a Müllerian du ct derivative. Whether or not tumorigenic cells
59
assume a more differentiated morphology depends on many factors, inc1uding cellular
environment, and intrinsic cell prograrnrning.
The OSE is derived from the coelomic epithelium and thereby has the intrinsic
capacity for divergent differentiation along the Müllerian pathways (Auersperg et al.,
1997). Interestingly, histological examination of ovarian caricinomas has uncovered
marked sirnilarities between reported ovarian epithelial neoplasm subtypes and Müllerian
duct derivatives. More specifically, serous tumors resemble fallopian tube epithelium,
mucinous tu mors resemble endocervical epithelium and endometrioid tumors are likened
to the uterine endometrium (Scott and McCluggage, 2006). Unfortunately, the
histological subtype of the tu mors from which our ovarian cancer celllines were derived
is unknown.
The differentiation of Müllerian duct derivatives depends on the spatio-temporal
expression patterns ofWnt4, Wnt5a and Wnt7a (Heikkila et al., 2001; Carta and Sassoon,
2004). Interestingly, the OSE cancer celllines studied express both Wnt5a and Wnt7a,
while the non-cancerous IOSE398 cells do not. The expression of E-cadherin in certain
ovarian tumors is also consistent with a more differentiated phenotype. This is due to the
fact that E-cadherin is correlated with an epithelial morphology, and less invasive
behavior (Takeichi, 1993) in contrast to N-cadherin, which has been shown to promote
cell mobility (Gumbiner, 2005). E-cadherin may also decrease canonical signaling by
sequestering active ~-catenin to the cell membrane (Gottardi et al., 2001). Moreover, the
lack of Fz9 expression (with its associated refractoriness to Wnt2 stimulation) in E
cadherin- expressing celllines may reflect a less pluripotential state secondary to the loss
of responsiveness to a canonical Wnt, which interestingly, is expressed by neighboring
granulosa cells (Ricken et al., 2002).
60
Our examination of Wntlcanonical signaling functionality and consequential
effects on cell proliferation and cadherin expression further support the possibility that
ovarian cancer cells may lose pluripotentiality through tumorigenesis. Our findings that
E-cadherin expressing cells show no change in TCFILEF-mediated reporter gene
expression, cell proliferation, cell detachment or cadherin expression in response to
canonical stimulation support the possibility that these cells have became more resistant
to canonical signaling, and its effects on stem cell renewal and wound repair.
61
8. Conclusion
Ovarian cancer is the most common cause of death from a gynecological
malignancy. Our incapacity to diagnose and treat this disease before it has reached an
advanced stage is due to a suboptimal knowledge of the molecular events, which indu ce
and/or promote ovarian cancer. Based on their involvement in development, wound
healing, stem cell regulation, and tumorigenesis, Wnt molecules represent likely
candidates to be involved in ovarian tumorigenesis. Taking into account the recent
emergence of studies examining Wnt signaling in ovarian cancer, we hypothesized that
ovarian cancer cells exhibit aberrations in Wntlcanonical signaling, which may be a cause
and/or effect of ovarian tumorigenesis. Therefore, we examined the expression profiles
of Wnt signaling pathway components, as well as the functionality of the canonical
pathway in both normal and cancerous OSE cell lines. Our study showed that ovarian
cancer cell lines exhibit distinct aberrations in the Wnt signaling pathway, which affect
cell proliferation and/or cell adhesion molecule expression. Based on the analysis of our
results, we proposed that the OSE may be populated by stem cells, which under the
influence of Wnt may or may not assume a more differentiated morphology (i.e. that of a
Müllerian duct derivative). While this is a preliminary study, we believe that it has
contributed to a better understanding of the molecular events underlying ovarian
tumorigenesis.
62
9. Acknowledgements
1 would like to begin by thanking Elegua, Oshun, Olokun, Shango and the rest of
my Orisha army that accompanies me through thick and thin, and gives me the tools that 1
need to keep on fighting ...
1 would like to thank my supervisor, Dr. Riaz Farookhi, for teaching me how to
think more critically and for his support in the production of this thesis. 1 would also like
to thank Maria Kontogiannea for her incredible moral and technical support, Ken
"KenneU" Finson for his advice and unique sense of humor, as well as the "gang" in the
OB-GYN research division at the RVH, including: Patrick, Cassandre, Patricia, Olivia,
Maude and Kevin.
Naturally, support cornes in different forms, thus 1 would also like to express my
appreciation to Dr. Derek Lobb for consistently providing us with cells, Dr. Nelly
Auersperg for her IOSE cells and for teaching me an important lesson about research, Dr.
J.J. Lebrun and his te am for their assistance with the luciferase assays, Dr. Blaschuck and
Normand for their materials and technical assistance, Dr. Clark and Dr. Dufort for their
ongoing support, and finally, the MUHC, the FRSQ, the CIHR and the Faculty of
Medicine at McGill for their financial support.
On a personal note, 1 would like to thank my Godfather Ricardo and Madrina
Susana for their guidance and support, and for propelling me to complete everything that
1 start, my mother for her unconditional love and support, my father for his reassurance,
crazy computer skills and help at all odd moments, Dr. MacNamara for her solid advice,
the ortho sport team for letting me escape the OR to go work on my thesis, and last but
not least, Lil' T, who up to now has got my back.
63
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