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Cdh11 Acts as a Tumor Suppressor in a Murine Retinoblastoma Model by Facilitating Tumor Cell Death
by
Christine Laura Yurkowski
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Molecular Genetics University of Toronto
© Copyright by Christine Laura Yurkowski 2010
ii
Cdh11 Acts as a Tumor Suppressor in Murine Retinoblastoma
Model by Facilitating Tumor Cell Death
Christine Yurkowski
Master of Science
Molecular Genetics
University of Toronto
2010
Abstract
Retinoblastoma, a rare childhood cancer of the retina, is characterized by loss of both alleles of
the RB1 gene. However, additional mutational events are required for malignancy. CGH studies
described common chromosomal changes indicating potential oncogenes and tumor suppressor
genes. Previous work in the lab implicated Cadherin-11 (Cdh11) as a tumor suppressor after
analysis in human retinoblastomas and in the simian virus 40 large T-antigen induced murine
retinoblastoma model (TAg-RB) showed loss of Cdh11 expression. TAg-RB mice crossed with
Cdh11-/-
mice, revealed faster growing tumors in mice null for Cdh11. This thesis focused on
defining the tumor suppressor role of Cdh11 in retinoblastoma progression. The results showed
in vitro and in vivo evidence that Cdh11 was promoting apoptosis and not suppressing
proliferation. We also observed an increase in invasion markers upon the loss of Cdh11. We
conclude that Cdh11 acts as a tumor suppressor in retinoblastoma, through promotion of
apoptosis.
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Acknowledgments
I would like to thank my supervisor, Dr. Brenda Gallie, for giving me the opportunity to
join her lab and be part of her amazing research team, which has vastly contributed to the study
of retinoblastoma. Her passion for science, medicine, life and helping others is truly
inspirational and she has served as such an incredible mentor. I admire her for her dedication
towards everything she does, her many accomplishments and her constant support and desire to
help everyone around her succeed. She is a big reason why I was able to take so much away
from my time in the lab and why I will always remember my experiences so fondly.
I would also like to thank my committee members for providing me with necessary
guidance and support along the way. It was greatly appreciated and will always be remembered.
Being around positive working environments is wondrous for your motivation and your
desire to want to come to work every day. I can honestly say the environment in the lab was one
of the most positive and supportive atmospheres I‟ve ever known. Everyone in the lab is kind,
generous, and knowledgeable. I would love to dearly thank all past and present members of the
lab (Ying Guo, Tim To, Helen Dimaras, Mellone Marchong, Ghada Kurban, Brigitte Theriault,
Stephanie Yee, Clarellen Spencer, Dr. Sanja Pajovic and Lucy Fuccillo). I wish you all the
absolute best in life. On a more personal note, I would like to thank Dr. Sanja Pajovic for her
constant guidance and support. You are an integral part of the lab, and it would absolutely not be
the same without you around, as you are always the greatest help to everyone and always have
something to say to put a smile on our faces. Clarellen Spencer also played an integral part in
my time at the lab, helping me with various protocols and especially in handling the mice. Your
patience is truly a virtue and your amazing stories of your travels and your generous treats are
always a delight. Brigitte Theriault and Ghada Kurban are such knowledgeable people and were
always of tremendous help if I ever needed anything. I will always treasure our morning
conversations in the office to get our days started on the right foot, your laughter and our many
many lunches to our favourite destinations. Lucy Fuccillo constantly manages to keep everyone
on task and organized on a daily basis. Not only does it feel you are a constant life saver, but in
addition to that, are one of the most positive people I know. Your pure zest for life and
involvement in so many wonderful activities is truly admirable. Lastly, to Stephanie Yee, who
was not only alongside for the ride as my peer, but became an amazing and dear friend. I feel
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my experience was that much more special because we got to share everything from the process
of studying for a Master‟s Degree, to personal highs and lows, daily activities, interests, dreams,
concerns, laughs and life. I will never forget our endless conversations and will always deeply
value your friendship.
Finally I‟d like to thank my family and friends who are always there for me and are
constantly sources of love, support and joy. My friends are extended members of my family, and
since my parents are in Winnipeg, I treasure their loyalty, endless love and generosity that much
more. To my parents, Katherine and Malcolm Yurkowski, who have always supported me in
anything I did in life, I have learnt so much from you and continue to do so every day. Your
generosity is the reason I am in Toronto today and the reason I am able to continue following my
dreams. Although you are not in the same city as me, I am lucky enough to be able to be a short
plane ride away and visit you as much as I need to. I am blessed to have such caring parents who
always want the best for me, and you are and always will be the biggest part of my success.
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Table of Contents
Acknowledgments ........................................................................................................................ iii
Table of Contents .......................................................................................................................... v
List of Tables .............................................................................................................................. viii
List of Figures ............................................................................................................................... ix
List of Abbreviations .................................................................................................................... x
List of Appendices ...................................................................................................................... xiii
Chapter 1 ....................................................................................................................................... 1
1.1 Retinoblastoma – The Disease ......................................................................................... 1
1.2 Current Treatment of Retinoblastoma ........................................................................... 2
1.3 Retinoblastoma Gene and Protein (pRB) ....................................................................... 3
1.4 Mouse Studies .................................................................................................................... 4
1.4.1 RB1-/- Mouse Models ............................................................................................ 4
1.4.2 TAg-RB Model Of Retinoblastoma ....................................................................... 5
1.5 Retinal Development ......................................................................................................... 6
1.6 Retinoma – The Benign Precursor .................................................................................. 7
1.7 Genomic Changes Driving Progression to Retinoblastoma .......................................... 8
1.7.1 Gain at 1q ............................................................................................................... 9
1.7.2 Gain at 6p ............................................................................................................. 10
1.7.3 Gain at 2p ............................................................................................................. 11
1.7.4 Loss at 16q ............................................................................................................ 11
1.8 Cadherin Biology ............................................................................................................ 13
1.8.1 Cadherin Structure .............................................................................................. 13
1.8.2 Cadherin Regulation and Expression ................................................................. 15
1.8.3 Cadherins in the Mammalian Retina .................................................................. 18
vi
1.8.4 Cadherin-11 and its Isoforms .............................................................................. 20
1.8.5 Cadherin-11: Implicated Roles ............................................................................ 21
1.8.6 Cadherins in Cancer Progression ....................................................................... 23
1.9 Cadherin-11 as a Tumor Suppressor in Retinoblastoma Progression ....................... 26
1.10 Apoptosis .......................................................................................................................... 27
1.10.1 Extrinsic Pathway ................................................................................................ 28
1.10.2 Intrinsic Pathway ................................................................................................. 29
1.11 Project Aims and Hypothesis ......................................................................................... 30
1.11.1 To examine if Cadherin-11 is promoting apoptosis in TAg-RB tumors and
define the acting apoptotic pathway ..................................................................... 30
1.11.2 To determine if the loss of Cadherin-11 expression leads to invasive and
aggressive tumors through expression of invasion markers ................................. 30
Chapter 2 ..................................................................................................................................... 31
2.1 Mouse Models .................................................................................................................. 31
2.2 Genotyping ....................................................................................................................... 31
2.3 Histology and Slide Selection .......................................................................................... 32
2.4 Immunohistochemistry .................................................................................................... 33
2.5 BrdU Incorporation ......................................................................................................... 36
2.6 Image Analysis .................................................................................................................. 36
2.7 Statistical Analysis ........................................................................................................... 37
2.8 Tissue Culture and Cell Lines ......................................................................................... 37
2.9 RNA Isolation ................................................................................................................... 38
2.10 Protein Isolation ............................................................................................................. 39
2.11 Stealth RNAi ................................................................................................................... 40
Chapter 3 ..................................................................................................................................... 41
3.1 Cadherin-11 acts a tumor suppressor in TAg-RB tumors through promotion of
apoptosis ........................................................................................................................... 41
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3.1.1 Active Apoptotic Pathway in TAg-RB Mice ........................................................ 41
3.1.2 Quantification of Apoptotic Protein Expression in TAg-RB Tumors................ 43
3.1.3 Analysis of Cadherin-11 in TAg-RB Derived Cell Lines.................................... 45
3.1.4 Apoptotic Protein Expression in a TAg-RB Derived Cell Line .......................... 46
3.1.5 Cdh11 and Proliferation in TAg-RB Tumors .................................................... 48
3.2 Loss of Cdh11 in TAg-RB Tumors Increases Invasion Potential ................................ 51
3.2.1 Knockdown of Cdh11 in a TAg-RB Cell Line Leads to an Increase of EMT
Markers and Metalloproteases ............................................................................ 51
3.2.2 β-catenin mRNA and Protein Levels Increase Upon Knockdown of Cdh11 .... 53
Chapter 4 ..................................................................................................................................... 55
4.1 Cdh11 as a Tumor Suppressor in Retinoblastoma Progression .................................. 55
4.2 Cdh11 Acts as a Tumor Suppressor by Promoting Apoptosis ..................................... 56
4.3 Loss of Cdh11 and Invasion ............................................................................................. 59
4.4 Cdh11 in Cancer Progression .......................................................................................... 61
4.5 Future Directions ............................................................................................................. 63
4.5.1 The Mechanism of Influence of Cdh11 on Apoptosis and Invasion Potential . 63
4.5.2 Mechanism of Cdh11 Downregulation in Retinoblastoma ................................ 65
4.6 Summary and Significance .............................................................................................. 66
References .................................................................................................................................... 68
viii
List of Tables
Table 1 Antibodies. ....................................................................................................................... 35
Table 2 RT-PCR Primers. ............................................................................................................. 39
ix
List of Figures
Figure 1.1 Retinoblastoma Genetic: Multistep Model. ................................................................... 8
Figure 1.2 Chromosome 16: Positioning of CDH11 and Cadherin Cluster. ................................ 13
Figure 1.3 Classical Cadherin Structure and Regulation. ............................................................. 18
Figure 1.4 Cadherin-11 and its Isoforms. ..................................................................................... 21
Figure 1.5 Cadherin-11 as a Tumor Suppressor in Retinoblastoma Progression. ........................ 27
Figure 1.6 Apoptotic Pathways. .................................................................................................... 30
Figure 3..1 Expression of Apoptotic Proteins within TAg-RB Tumors. ...................................... 42
Figure 3.2 Acting Apoptotic Pathway in Retinal Tumors of TAg-RB Mice. ............................... 44
Figure 3.3 Differences in Apoptotic Protein Expression in TAg-RB Tumors at PND84. ............ 45
Figure 3.4 Cdh11 Protein Expression in TAg-RB Derived Cell Lines. ........................................ 46
Figure 3.5 Cdh11 Knockdown Leads to Decreased Caspase-3 Expression. ................................ 47
Figure 3.6 Quantifying the Percentage of BrdU Proliferating Cells as a Function of Tumor Area.
....................................................................................................................................................... 49
Figure 3.7 Average Area of Proliferation Cells Per Tumor Area at PND84. ............................... 50
Figure 3.8 Knockdown of Cdh11 Leads to Increased mRNA Expression of EMT and Invasion
Markers. ........................................................................................................................................ 52
Figure 3.9 Cdh11 Knockdown Leads to Upregulation of ϐ-catenin mRNA and Protein
Expression Levels. ........................................................................................................................ 54
x
List of Abbreviations
ADAM A Disintegrin and Metallopeptidase
AJ Adherens Junction
AP Alkaline Phosphatase
APAF1 Apoptotic Peptidase Activating Factor 1
APC Adenomatosis Polyposis Coli
BAX Bcl-2-associated X Protein
BH3 Bcl-2 Homology Domain 3
BrdU Bromodeoxyuridine
BSA Bovine Serum Albumin
Cadherin-11 (i) Cadherin-11 intact isoform
Cadherin-11 (s) Cadherin-11 secreted isoform
Cadherin-11 (v) Cadherin-11 variant isoform
CAM Cadherin Cell Adhesion Complex
CDH Cadherin
CDK Cyclin Dependent Kinase
CGH Comparative Genomic Hybridization
CNS Central Nervous System
CP Cytoplasmic Domain
DAB Diaminobenzidine
DAPI 4‟,6-diamidino-2-phenylindole
DEK DEK oncogene (DNA binding)
dNTP Deoxynucleoside Triphosphate
E2F E2F Transcription Factor
EBR External Beam Therapy
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EC Extracellular Domain
ECL Enhanced Chemiluminescence
ED Embryonic Day
EMT Epithelial to Mesenchymal Transition
F-W Flexner Wintersteiner Rosette
FADD Fas-associated Death Domain
FGFR Fibroblast Growth Factor Receptor
FLIP Caspase-8 (FLICE)-like Inhibiting Protein
GCL Ganglion Cell Layer
GSK-3ϐ Glycogen Synthase Kinase-3ϐ
H-W Homer Wright Rosette
H&E Hematoxylin and Eosin
HAV Histidine Alanine Valine
HRP Horseradish Peroxidase
INL Inner Nuclear Layer
IP Intra-Peritoneal
KIF Kinesin Family Member
LEF Lymphocyte Enhancer Binding Factor
L-Hϐ Luteinizing Hormone Beta
LOH Loss of Heterozygosity
M1, 2, 3…, n Mutational Event 1, 2, 3…, n
MAPK Mitogen-activated Protein Kinases
MMP Matrix Metalloproteinase
MYCN v-myc Myelocytomatosis Viral Related Oncogene, Neuroblastoma
Derived
ONL Outer Nuclear Layer
xii
PCNA Proliferating Cell Nuclear Antigen
PFA Paraformaldehyde
PI3K Phosphoinositide 3-kinases
PND Post Natal Day
QAV Glutamine Alanine Valine
QM-PCR Quantitative Multiplex PCR
RB Retinoblastoma
RTK Receptor Tyrosine Kinase
RT-PCR Reverse Transcriptase PCR
SCID Severe Combined Immunodeficiency
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
TAg SV40 Large T-antigen
TAg-RB SV40 Large T-antigen Induced Murine Retinoblastoma Model
TBP TATA Box Binding Protein
TBS Tris Buffered Saline pH 8.0
TBST Tris Buffered Saline pH 8.0/ Tween-20
TCF T-cell Factor
TGFϐ Transforming Growth Factor Beta
TM Transmembrane Domain
TNE Tris-Sodium-EDTA Buffer
TNF Tumor Necrosis Factor
TRAIL TNF-related Apoptosis-inducing Ligand Receptor
Wnt Wingless Type
xiii
List of Appendices
Parts of this thesis were submitted for publication and are currently in press in PLOs Genetics as
follows: Cdh11 acts as a tumor suppressor in a murine retinoblastoma model by facilitating
tumor cell death. Mellone N. Marchong, Christine Yurkowski, Clement Ma, Clarellen Spencer,
and Brenda L. Gallie.
1
Chapter 1
Introduction
1.1 Retinoblastoma – The Disease
Retinoblastoma is the most common childhood intraocular malignancy that occurs in
approximately 1 in 20,000 births (Balmer et al., 2006). The disease is initiated by two
mutational events in the developing retina, resulting in the loss of both alleles of the RB1 gene.
The disease exists in two forms: hereditary and non-hereditary. Hereditary cases are
characterized by an inherited mutation in the RB1 gene that is present in all cells in the body. A
second spontaneous mutation in the retina results in the loss of the remaining normal allele.
These patients often develop multiple tumors in both eyes (bilateral retinoblastoma), and
furthermore leave the patient more susceptible to secondary malignancies later in life
(Abramson, 1999; Marees et al., 2008). The non-heritable form arises by two sporadic
mutational events occurring within the same cell, and results in unilateral retinoblastoma,
affecting one eye. The observation that children with bilateral retinoblastoma had an earlier age
of diagnosis compared to children with unilateral retinoblastoma was the basis of Knudson‟s
“two-hit” hypothesis (Knudson, 1971). This concept was expanded to suggest the two „hits‟
consisted of inactivation of the predisposing gene that would normally suppress retinoblastoma
2
(Comings, 1973), now a general idea of the genetic basis of cancer, in which at least two
mutations are necessary for cancer initiation.
1.2 Current Treatment of Retinoblastoma
Retinoblastoma is readily curable today in the developed world, including good prognosis of
visual function. Treatment strategies usually involve a multidisciplinary approach and depend
on constant re-evaluation with survival and salvage of visual function as the primary aim
(Balmer et al., 2006). Most patients presenting with unilateral retinoblastoma are advanced
cases and are usually cured (>96%) by enucleation (Chintagumpala et al., 2007). In children
with bilateral retinoblastoma, treatment often entails multimodality therapy (Chintagumpala et
al., 2007). Often, the eye and sometimes vision can be saved. Therapies include brachytherapy,
thermotherapy, laser photocoagulation, cryotherapy, chemotherapy and external beam therapy
(EBR) (Balmer et al., 2006). The choice between these therapies lies within the size and severity
of the tumor. Most often, chemotherapy will be used in order to shrink the tumor so local
therapies can be used. Primary chemotherapy can shrink the tumor volume by over 50% (Chan
et al., 2005; Shields et al., 1997). This then allows local therapies such as cryotherapy and laser
therapy to eradicate the remaining disease (Chan et al., 2005; Chintagumpala et al., 2007).
EBR is often used as a last resort. It can be effective in curing retinoblastoma, but is proven to
have major long-term side effects, with a reported increase in second cancers in patients
compared to those who do not receive EBR (Abramson and Frank, 1998). In cases of
extraocular disease, patients often have a very poor prognosis, however few patients may benefit
from a combination of EBR and chemotherapy (Chantada et al., 2005). Current therapies
contribute significantly to vision salvage and overall cure rates, however they are not without
significant morbidities. Therefore, emphasis has recently been placed on developing therapies
with fewer long term morbidities, which requires a better understanding of the pathogenesis of
3
retinoblastoma. In an attempt to do so, the use of preclinical models has been important
(Chintagumpala et al., 2007; Hurwitz et al., 1999).
1.3 Retinoblastoma Gene and Protein (pRB)
RB1 was the first tumor suppressor gene to be discovered (Cavenee et al., 1983; Godbout et al.,
1983). It was mapped to chromosomal region 13q14 (Sparkes et al., 1980), and was first cloned
a few years after (Dryja et al., 1986; Friend et al., 1986; Fung et al., 1987; Lee et al., 1987).
RB1 encodes the retinoblastoma protein pRB, belonging to the „pocket protein‟ family. This
family of proteins, including p107 and p130, all share similar and redundant functions in
regulation of the cell cycle, modulating members of the E2F family of transcription factors,
which regulate the expression of genes required for cell proliferation (Cao et al., 1992;
Chellappan et al., 1991; Cobrinik, 2005). The pRB protein has three distinct domains; the N-
terminus, R motif and A/B „pocket‟. The „pocket‟ is necessary for biological function, including
transcriptional regulation and interaction with viral and cellular proteins (DiCiommo et al., 2000;
Kouzarides, 1995). Specifically, pRB interacts with E2F transcription factors to inhibit their
activity. This negatively regulates the cell cycle, halting it in G1 phase. This occurs with pRB in
a hypophosphorylated state. Upon phosphorylation by cyclin dependent kinases (CDK) and
cyclin complexes, pRB releases the E2Fs, allowing transcription of their targets to commence,
subsequently allowing the re-entry to the cell cycle and promoting cell division (Mittnacht,
1998).
Although all three family members show similar functions, only pRB has proven to have tumor
suppressor abilities in human tumors (Lipinski and Jacks, 1999). Mutations in both RB alleles
initiate disease in a variety of cancers, including retinoblastoma, an increase of incidence of
osteosarcoma developing in children and teenagers, small lung carcinomas, and in cancers of the
4
breast, prostate and bladder (Bookstein and Lee, 1991; Deshpande and Hinds, 2006; Harbour et
al., 1988). Additionally, pRB is commonly inactivated in many other types of tumors, through
mutation, viral oncoprotein or by alteration of other proteins involved in the same pathway.
Certain viruses, such as simian virus 40 large T antigen (TAg), human papilloma virus , E7
(HPV), and adenovirus (E1a), consist of an LxCxE motif, required for viral transformation,
allowing them to bind the pRB and „pocket‟ family members at the A/B pocket domain,
specifically the repressor motif (Dyson et al., 1989; Felsani et al., 2006). Inactivation of pRB,
therefore, leads to abnormal division of cells and continual proliferation due to the inability to
exit the cell cycle.
1.4 Mouse Studies
1.4.1 RB1-/-
Mouse Models
Generating mouse models to study retinoblastoma development has been extremely difficult.
Firstly, spontaneous retinoblastoma seems to be almost exclusively a human disease, seen very
rarely in other species. Mice with a RB+/-
genotype show no retinal phenotype, however develop
both pituary tumors and thyroid carcinomas (Jacks et al., 1992). Secondly, genetic RB-/-
knockout proved to be lethal in early embryonic stages, around ED14.5, initially thought to be
due to defects in neural and erythroid systems and massive apoptosis (Clarke et al., 1992; Jacks
et al., 1992; Lee et al., 1992). Instead, this was later found to be due to hypoxia because of
placental defects, as when RB-/-
embryos were studied with wildtype placental, they survived till
birth, but later died due to respiratory problems (de Bruin et al., 2003; Wu et al., 2003). Studies
then focused on producing chimeric mice, but these mice only showed pituitary tumor
development, and no retinoblastoma phenotype (Maandag et al., 1994). A breakthrough came
when these chimeric mice were generated by knocking out both RB1 and one of its family
members, p107 or p130 ((Dannenberg et al., 2004; Robanus-Maandag et al., 1998). The need
5
for RB and at least one of its family members to be lost for retinoblastoma development in mice
was confirmed by the use of conditional knockout models (CKO), generating heritable mouse
models of retinoblastoma ((Chen et al., 2004; MacPherson et al., 2004; Zhang et al., 2004).
1.4.2 TAg-RB Model Of Retinoblastoma
Transgenic mouse models provided the earliest heritable models of retinoblastoma, involving the
expression of viral oncoproteins. Extensively used for study of retinoblastoma is the simian
virus 40 (SV40) large T-antigen induced model of retinoblastoma (TAg-RB). This model arose
fortuitously, as it was originally intended for the study of pituitary adenoma. One founder
presented with bilateral retinoblastoma tumors, from high expression levels of TAg within the
retina (Windle et al., 1990). The transgene is under control of the luteinizing hormone-beta (LH-
beta) promoter, and integration into an unknown genomic control element at chromosomal
location 4p directing transgene expression to cells in the retina, where LH-beta is not normally
expressed. This model gave rise to hereditary, multifocal retinoblastoma presenting from 1
month of age. These tumors resemble human retinoblastomas (Windle et al., 1990), and
originate from the inner nuclear layer (Pajovic et al, unpublished data). Ultrastructurally, these
tumor cells present as microtubules in the characteristic “9+0” arrangement, also seen in normal
photoreceptor cells, a distinguishing characteristic of human retinoblastomas. Histologically,
they contain two multicellular structures common to human retinoblastomas. Flexner-
Wintersteiner rosettes (F-W) consist of a single layer of cuboidal cells surrounding a central
lumen. They resemble primitive photoreceptors and are unique to retinoblastoma. Homer-
Wright rosettes (H-W) are single-layer rows of tumor cells that surround a central area filled
with neurofibrils (Mills et al., 1999; Ts'o et al., 1969; Windle et al., 1990; Wippold and Perry,
2006). These are also highly characteristic of neuroblastoma and medulloblastoma. The TAg-
RB model not only presents histologically similar tumors to human retinoblastoma, but also
6
proves to have similar expression changes associated with tumor development, presented in
human retinoblastoma as gain and overexpression of KIF14, E2F3 and DEK (Corson et al.,
2005; Orlic et al., 2006), as well as loss of p75NTR
expression (Dimaras et al., 2006) and genomic
and expression loss of CDH11 (Marchong et al., 2004). The TAg-RB model is the most used
for pre-clinical and therapeutic studies relevant to human retinoblastoma. Transgenic models are
important as they allow therapeutic manipulation not otherwise possible. Studies have included
use of a variety of chemotherapeutic agents, radiation and use of attenuated viruses to treat
tumors (Kang et al., 2009; Mills et al., 1999; Sobrin et al., 2004; Suarez et al., 2007; Tsui et al.,
2008). Although it is similar to human disease, the TAg-RB model presents some limitations in
the study of human retinoblastomas. As the virus binds to pRB family member, p53 and other
proteins, it is possible other pathways are affected in this model not affected in the human form
of the disease. However, interspecies differences in the mechanisms of tumorigenesis, provides
general limits to uses of all mouse models (Dyer and Bremner, 2005; Mills et al., 1999).
1.5 Retinal Development
The retina is a sheet of neural tissue that takes in input signals from light and sends this
information to the brain to determine what we are seeing. It is first formed by an invagination of
the neural tube, marked by an appearance of an optic pit. This leads to the formation of the optic
vesicles. Next, a lens placode is formed, a thickening of the surface ectoderm, which is in
contact with the optic vesicle. Invagination of these two structures together leads to the lens and
a two layered optic cup, the inner layer forming the neural retina [reviewed in(Chow and Lang,
2001; Young, 1985a; Young, 1985b). This close contact is necessary for neural retinal
development and studies have shown fibroblast growth factor (FGF) plays a role in signaling
neural retinal development during this time period (Guillemot and Cepko, 1992).
7
The retina contains seven main different types of cells. The earliest step in development is
production of retinal cells by the „progenitors‟ of the neurepithelial layer of the optic cup. These
cells migrate to form one of three layers after they have undergone the last mitosis. These cells
start to form synapses with eachother, upon which proper processing is crucial. Patterning and
proper ratios of each cell type are dependent on these early processes and is necessary for proper
retinal and visual development (Cepko et al., 1996; Young, 1985a). The three major layers of
the developed retina are the outer nuclear layer (ONL), made up of rod and cone photoreceptors,
the inner nuclear layer (INL), consisting of horizontal, amacrine, bipolar and Muller glia cells
and the ganglion cell layer (GCL), including ganglion cells and amacrine cells. The ONL uses
light as its stimulus and translates the information through synapses of the INL to the GCL,
which transmits the results to various targets of the brain. Just like in other structures of the
brain, glial cells provide retinal architecture and different external cues for retinal development
and apoptosis.
1.6 Retinoma – The Benign Precursor
Studies of retinoblastoma indicated early on that additional genomic aberrations after the loss of
RB1 exist in all retinoblastomas (Squire et al., 1985) suggesting further mutational events were
needed for full development of the disease. In earlier years, inactive tumors resembling
retinoblastoma seen on rare occasions were referred to as „spontaneous regression of
retinoblastoma‟. Without much evidence to support this definition, the term „retinoma‟ was
proposed to describe non-progressive retinal lesions that lacked malignant characteristics (Gallie
et al., 1982). Following, many case studies have supported the idea that retinomas are benign
precursors to malignant retinoblastoma (Balmer et al., 1991; Eagle et al., 1989; Singh et al.,
2000). This idea is supported by studies in mouse models and tumor progression models which
explore the idea that cancer is multistep process (Redston, 2003; Vogelstein and Kinzler, 1993).
8
Much evidence for a multistep process in retinoblastoma has accumulated. Alone, the loss of
RB1 (M1 and M2) is not sufficient for malignant transformation. Further genomic changes are
needed to transform the benign retinoma into malignant retinoblastoma (M3-Mn) (Dimaras et al.,
2008) (Figure 1.1). This study showed that after the loss of RB1, lesions remained
nonproliferative and also expressed senescence markers, not seen in retinoblastomas. This and
other studies have focused on defining genomic changes that drive progression of the disease to
malignant retinoblastoma.
Figure 1.1 Retinoblastoma Genetic: Multistep Model.
Retinoblastoma is initiated by the biallelic loss of the RB1 gene in a pre-mature retinal cell, mutational events M1
and M2. The loss of RB1 rendering cells RB1-/- is sufficient for retinoma, the benign form of retinoblastoma.
However, additional mutations, termed M3-Mn events are necessary to drive progression to malignancy,
retinoblastoma.
1.7 Genomic Changes Driving Progression to Retinoblastoma
Early studies to determine genomic changes in retinoblastoma focused on revealing recurrent
chromosomal abnormalities. Karyotyping studies defined consistent patterns of genomic change
in retinoblastoma samples. These studies reported most frequent gains at 6p, 1q and loss of
chromosome 16 (Benedict et al., 1983; Chaum et al., 1984; Kusnetsova et al., 1982; Pogosianz
and Kuznetsova, 1986; Squire et al., 1985; Workman and Soukup, 1984). Comparative genomic
hybridisation (CGH) technology followed 15 years later, allowing for higher resolution of
9
chromosomal aberrations. Five major CGH studies and one matrix study studied a total of 179
tumor samples to find common regions of gains and losses [reviewed in (Corson and Gallie,
2007)]. The results confirmed earlier observations and defined additional changes occurring at
lower frequency. The most common changes included gains at 1q (53%), 6p (54%), 2p (34%),
13q (16%) as well as losses at 13q (12%) and 16q (32%) (Chen et al., 2001; Herzog et al., 2001;
Lillington et al., 2003; Mairal et al., 2000; van der Wal et al., 2003; Zielinski et al., 2005).
These chromosomal regions of gain and loss suggested loci of tumor suppressor and oncogenes
and were presumed to contain target genes that contribute to the overall progression of
retinoblastoma. Several candidate genes have arisen from work in recent years, and defining
their role in retinoblastoma is essential in understanding the pathway of tumorigenesis. This is
necessary to develop therapeutics that can ultimately be used to halt retinoblastoma progression
at an early stage.
1.7.1 Gain at 1q
Genomic gain of chromosome 1q was first seen in retinoblastoma as rearrangements leading to
trisomy of 1q25-1q32 (Gardner et al., 1982). The chromosomal region of 1q31-1q32 is gained in
a variety of cancers (Baudis and Cleary, 2001; Mertens et al., 1994) and in 53% of
retinoblastoma tumors (Corson and Gallie, 2007). This strongly suggested a candidate oncogene
at this locus and QM-PCR revealed candidate genes. Only KIF14 was overexpressed in various
cancers and identified as the target gene with 20/22 retinoblastomas showing mRNA expression
levels of 100-1000 fold increase from normal retina (Corson et al., 2005). KIF14, a member of
the kinesin family, is required for efficient cytokinesis and RNAi mediated knockdown results in
multinucleated cells and acute apoptosis (Carleton et al., 2006; Gruneberg et al., 2006). KIF14
is also overexpressed in breast cancer primary tumors and cell lines, primary lung tumors and
medullablastoma cell lines (Corson et al., 2005). Further studies on KIF14 revealed that mRNA
10
expression was prognostic for patient outcome in breast and lung cancer (Corson and Gallie,
2006; Corson and Gallie, 2007). Additionally, when KIF14 was knocked down with siRNA in a
lung carcinoma cell line H1299, decreased proliferation and colony formation on soft agar was
observed (Corson et al., 2007). Recent experiments in both retinoblastoma and ovarian cancer
cell lines show similar results (Theriault, unpublished data). This gives strong evidence for
KIF14 as a prominent oncogene in multiple cancers and a potential therapeutic target.
1.7.2 Gain at 6p
Gains in regions 6p were consistently shown in retinoblastomas (Corson and Gallie, 2007;
Potluri et al., 1986; Squire et al., 1985). Recent studies looked to define the minimal region of
gain and two genes were implicated as targets, DEK and E2F3 (Grasemann et al., 2005; Orlic et
al., 2006). E2F3 is a family member of the E2F transcription factors, and has a crucial role as a
cell cycle promoter in proliferating cells (Leone et al., 1998). It was first implicated in Wilms‟
tumor (Baudry et al., 2002) and its overexpression in bladder cancer correlates to histological
grade (Hurst et al., 2008; Oeggerli et al., 2006). DEK is an abundant nuclear phosphoprotein that
shares structural similarity to E2F family members and has multiple roles in chromatin
remodeling, mRNA splicing and transcriptional regulation (Kappes et al., 2001; Waldmann et
al., 2004). It is overexpressed in numerous cancers including bladder, colon, cervical
carcinomas, melanomas and highly defined in myeloid leukemia, where it is translocated to
NUP214 (CAN) (6;9 translocation), forming a DEK-CAN fusion protein ((Carro et al., 2006;
von Lindern et al., 1992; Wu et al., 2008). In retinoblastoma, DEK and E2F3 have been
implicated to have separate roles, E2F3 in growth, and DEK in both growth and survival (Orlic
et al, submitted).
11
1.7.3 Gain at 2p
Karyotype studies consistently identified chromosomal abnormalities of gain and amplification
at chromosome 2p in retinoblastoma (Squire et al., 1985). Later CGH studies confirmed a
minimal region of gain at 2p24 in 34% of retinoblastoma (Corson and Gallie, 2007) and QM-
PCR revealed MYCN amplification in 3% of tumors and 29% of retinoblastoma cell lines
(Bowles et al., 2007). Earlier studies also reported low-level and high-level MYCN
amplification in retinoblastoma (Lee et al., 1984; Squire et al., 1986). Interestingly, a recent
transgenic mouse study revealed that murine metastatic tumors exhibit high levels of MYCN
amplification (MacPherson et al., 2007). MYCN is a nuclear protein that regulates genes
involved in proliferation and apoptosis (Cavalieri and Goldfarb, 1988; Ramsay et al., 1986).
High amplification in retinoblastoma indicates MYCN expression may be favourable for cell
growth and proliferation. MYCN is commonly amplified in tumors of neuroectodermal origin
including small lung carcinoma and most commonly in neuroblastoma (25-30%), where
amplification has prognostic significance (Brodeur, 2003; Chan et al., 1997). Recent studies
have identified a small subset of tumors in which no RB1 mutation can be found (nmf) (Richter
et al., 2003). Recent work in our lab showed that 56% of these tumors have abnormally high
frequency of MYCN amplification (Yee, unpublished data). Furthermore, these tumors do not
show typical genomic changes of retinoblastoma, including gain at 1q and 6p, or loss of 16q.
This leads to an interesting question of whether MYCN could cause retinoblastoma in patients
with no RB1 mutations.
1.7.4 Loss at 16q
Loss of chromosome 16 was seen in early karyotype studies of retinoblastoma (Potluri et al.,
1986; Squire et al., 1985). Six CGH studies later revealed that 32% (58/179) of tumors showed
loss of either the entire chromosome 16 or 16q, with a minimal region of loss at 16q22 (all but 7
12
tumors) (Corson and Gallie, 2007). Analysis by loss of heterozygosity (LOH) and QM-PCR
further minimized the region of loss to an area of 2.62Mb, including two sequence tagged sites
(STS) at 16q22.1 (lost in 54% of 71 tumors) and 16q23.3 (lost in 39% of 28) tumors, within
CDH11 and CDH13, respectively (Marchong et al., 2004). CDH13 was ruled out as a candidate
gene as it showed expression levels comparable to healthy human retina in all retinoblastoma
primary tumors and cell lines. Cadherin-11 (Cdh11) expression, however, was lost or decreased
in 91% of tumors and cell lines, compared to adult healthy retina. In addition, RT-PCR revealed
that Cdh11 mRNA expression was lost in 38% of TAg-RB mouse model primary tumors
(Marchong et al., 2004). Supporting these observations, further studies showed decreased Cdh11
expression in retinoblastoma samples, but levels of expression in adjacent retinoma were
equivalent to normal retina (Dimaras et al., 2008).
Cdh11 is a classical cadherin family member that is involved in cell-cell adhesion. It is also
known as OB-cadherin, for its original isolation from mouse osteoblasts (Okazaki et al., 1994).
It is known to be involved in differentiation and migration of different cell types (Kii et al.,
2004; McCusker et al., 2009; Monahan et al., 2007; Zhou and Snead, 2008).
16q21-22 loss has been implicated in a variety of cancers, strongly suggestive of tumor
suppressor genes located at this region. This might be due to the cluster of cadherin molecules
located at this locus (Figure 1.2). Cadherins are important for a wide variety of roles including
adhesion, signaling pathways, tissue morphology and maintenance of cell integrity, and so it is
not unexpected that the loss of these molecules would lead to progressive cancer phenotypes.
Cadherin genes located at this locus include CDH8, CDH11, CDH1, CDH3, CDH5, and CDH16.
16q loss has been seen in chromosomal aberration studies of cancers of the breast (Loo et al.,
2004), prostate (Saramaki and Visakorpi, 2007), lung (Sato et al., 1994), ovary (Kawakami et al.,
13
1999), fallopian tubes (Snijders et al., 2003), pediatric medullablastomas (Lo et al., 2007),
neuroendocrine tumors (Kim do et al., 2008), prognosis of Wilms‟ tumors (Davidoff, 2009),
rhabdomyosarcoma (Visser et al., 1997), acute myeloid leukemia (Mrozek, 2008), astrocytomas
(Zhou and Skalli, 2000), and hepatocellular carcinomas (Herath et al., 2006).
Figure 1.2 Chromosome 16: Positioning of CDH11 and Cadherin Cluster.
Ideogram of chromosome 16 is from the website: http://www.genecards.org. Diagram is not drawn to scale and
represents the cadherin cluster at 16q21-22.1.
1.8 Cadherin Biology
1.8.1 Cadherin Structure
Cadherin molecules make up a superfamily of transmembrane glycoproteins that have been
implicated in many roles such as embryonic development, tissue morphogenesis, cellular
maintenance, differentiation, migration, and cancer progression (Behrens, 1999; Jeanes et al.,
2008; Tepass et al., 2000). This is due to their abilities to adhere to neighboring cells through
interactions via their extracellular domains and their cell signaling capabilities through their
interactions with catenin molecules via their cytoplasmic domains (Takeichi, 1995) (Figure
1.3A). Cell to cell interactions are usually homophilic, although heterophilic contacts have been
14
observed (Volk et al., 1987). The cadherin superfamily is very large of >100 members, and
contains very diverse protein structures. However, they all share the characteristic extracellular
cadherin repeats (ECs) (Nollet et al., 2000).
The cadherin superfamily is split into many different subfamilies, which include the classical
cadherins. This subfamily is the most extensively studied, and includes approximately 20
members, that are split into two types, Type I and Type II. These types differ solely on the
presence of an HAV (Histidine-Alanine-Valine) motif in the Type I, and a QAV (Glutamine-
Alanine-Valine) motif in Type II (Blaschuk et al., 1990). Their protein structures are very
similar, with five extracellular domain (EC) repeats, a transmembrane domain and a cytoplasmic
domain that is highly conserved among cadherin subtypes (Halbleib and Nelson, 2006). In
classical cadherins, the cytoplasmic domain associates with α-catenin, β-catenin, γ-catenin, and
p120 (Kemler, 1993; Reynolds et al., 1996).
Homophilic interactions occur in the presence of calcium, which binds to the EC domains, and
regulates stability of the extracellular domain (Harrison et al., 2005; Hirano et al., 1987). Upon
cell-cell contact, cadherin molecules bind directly to β-catenin, γ-catenin, or p120. β-catenin
recruits α-catenin to the complex (Aberle et al., 1994), an actin filament binding protein and
links the complex to the cytoskeleton (Kobielak and Fuchs, 2004). This complex, known as the
cadherin cell adhesion complex (CAM), or the adherens junction (AJ), is necessary to maintain
cell-cell adhesion and cellular architecture (Aberle et al., 1996). These junctions are dynamic
and the structure and signaling provided by the complex ultimately determines the cellular
phenotype and behaviour (Wheelock and Johnson, 2003).
15
1.8.2 Cadherin Regulation and Expression
Regulation of cadherin molecules is involved in normal tissue development and it is now clear
that classical cadherin dysfunction is a major contributor to cancer progression (Birchmeier and
Behrens, 1994; Thiery, 2002). Regulation happens at many levels, including gene expression,
transport to, and protein turnover at the cell surface (Figure 1.3B). Cadherin transcription is
directly regulated by DNA methylation and repression of promoter activity. Methylation of the
E-cadherin promoter is associated with reduced E-cadherin expression, disease progression and
metastasis in diffuse-type gastric cancer and ductal breast carcinoma (Strathdee, 2002).
Transcriptional repression of E-cadherin has been observed by zinc finger proteins of the
Slug/Snail family, resulting in decreased cell-cell adhesion and an increase in cell migration
(Halbleib and Nelson, 2006).
At the cell surface, cadherins are regulated by phosphorylation, ubiquitination and proteolysis.
Phosphorylation and dephosphorylation of various proteins in the AJ can positively or negatively
influence the structural integrity of the complex. Phosphorylation of the cadherin itself or β-
catenin, can affect and alter the binding affinity of the complex, or even target the cadherin for
endocytosis, resulting in degradation by the proteosome (Fujita et al., 2002; Roura et al., 1999;
Stappert and Kemler, 1994). In developmental processes, cells have to rapidly adapt to changes
in morphogenesis and tissue structure. This is often reflected in the dynamics of cadherin cell
adhesion, which can be regulated by protein turnover. One study showed that N-terminal
cleavage of Cdh11 by ADAM9 and 13 was essential for the migration of cells during the
development of the cranial neural crest, in vivo (McCusker et al., 2009). Furthermore, C-
terminal cleavage of N-cadherin by ADAM10, allows the cytoplasmic fragment to activate
CREB-mediated transcription, thought to have significance in neuronal growth and survival
(Marambaud et al., 2003).
16
Receptor tyrosine kinases (RTKs) play important roles in development and cancer, and interact
with cadherins to influence cell-cell adhesion and signaling pathways (Pece and Gutkind, 2000).
For example, fibroblast growth factor receptors (FGFRs) are implicated in promoting epithelial
to mesenchymal transition (EMT) events in cancer progression (Thiery, 2002). In a mouse
pancreatic tumor model, N-cadherin forms a multi-protein complex with FGFR, leading to loss
of adhesion, dissemination of tumor cells and increased invasive properties (Cavallaro et al.,
2001). Mechanistically, binding inhibits internalisation of the cadherin and activates MAPK
signaling, leading to proliferation and migration (Suyama et al., 2002).
Cadherins are also regulated through the cytoplasmic proteins they bind to (Figure 1.3B). p120
has dual roles in the cell as it acts as a member of the AJ, and interacts with the Rho family of
GTPases in the cytoplasm. It has been seen to regulate and stabilize cadherin strength and
turnover, its significance shown by experiments where ablation of p120 in mice led to down-
regulation of E-cadherin levels (Davis et al., 2003). Upon loss of cadherins, p120 accumulates
in the cytoplasm, repressing RhoA and activating Cdc42 and Rac1, which modulate the
cytoskeleton and increase migration and invasiveness (Christofori, 2006; Stemmler, 2008). β-
catenin also has a dual role in cell adhesion and as the intracellular transducer in the canonical
Wnt signaling pathway, acting as a transcriptional coactivator in the nucleus (Gumbiner, 1995).
In the presence of cadherins, newly synthesized β-catenin saturates the pool of the AJs, and is
never available for signaling. Changes in cell-cell adhesion lead to excess β-catenin, where the
cytoplasmic pool is regulated by Wnt signaling. If the pathway is inactive, the
APC/AXIN/GSK-3β complex phosphorylates β-catenin. It is then ubiquitinated, and targeted for
destruction by the proteosome (Aberle et al., 1997). Upon activation of Wnt ligands, the
complex is inhibited and β-catenin accumulates and transports to the nucleus where it binds to
transcription factors T cell factor/ lymphocyte enhancer binding factor (LEF/TCF) affecting
17
transcription of target genes (Behrens et al., 1996; Ikeda et al., 2000). Wnt signaling ultimately
activates transcriptional programs and there is no limit to the type of biological event that may be
controlled (Clevers, 2006). Wnt signaling has been implicated in many cancers. For instance,
some direct target genes include proto-oncogenes c-myc and cyclinD1, and have been the focus
of many studies. Furthermore, in some cancers, one or more of the Wnt signaling proteins are
mutated, leading to aberrant signaling, and malignant phenotypes (Kinzler and Vogelstein, 1996;
Morin et al., 1997). Wnt signaling has even been implicated as having tumor suppressor
functions in retinoblastoma (Tell et al., 2006).
Cadherin switching is a prominent feature in morphogenetic function and cell sorting during
development (Wheelock and Johnson, 2003). The same phenomenon highly occurs in tumor
progression (Christofori, 2003). In a number of cancer types, E-cadherin expression is lost, and
occasionally, de novo expression of another cadherin, N-cadherin or Cdh11, has been observed
(Li and Herlyn, 2000; Tomita et al., 2000). This recapitulates what is seen during EMT in
development; for example when cells switch from E-cadherin to N-cadherin in primordial germ
cells when migrating to populate the genital ridge (Bendel-Stenzel et al., 2000). Based on
numerous studies, cadherin switching seems to be a hallmark of the transition from a benign to
malignant form of cancer, and correlates with invasiveness and poor prognosis (Cavallaro and
Christofori, 2004; Christofori, 2003; Halbleib and Nelson, 2006).
18
Figure 1.3 Classical Cadherin Structure and Regulation.
A. This depicts a classical cadherin involved in the Adherins Junction, mediating cell-cell adhesion. In the presence
of Calcium, the extracellular domains bind to cadherin molecules on neighbouring cells in homophilic interactions.
The cytoplasmic domains interact with various catenin molecules, ϐ-catenin, α-catenin and p120, which link the
complex to the actin cytoskeleton, mediating cell-cell adhesion and intracellular signalling pathways. B. Signalling
Pathways may be affected with the down-regulation of cadherin molecules. p120 has a dual role in cell adhesion
and interacting with Rho-GTPases. Upon loss of cell-cell adhesion, p120 interacts with Cdc42 and Rac1, involved
in promoting cell motility and invasiveness. ϐ-catenin also has dual roles in cell adhesion and as a main player in
Wnt signalling. Upon loss of cell-cell adhesion, ϐ-catenin accumulates in the cytoplasm, and when Wnt signalling is
active, translocates to the nucleus interacting with transcription factors to regulate expression of many genes,
including proto-oncogenes, cyclin-D1 and c-myc. When Wnt is not activated, the free ϐ-catenin in the cytoplasm is
phosphorylated, targeting it for degradation.
1.8.3 Cadherins in the Mammalian Retina
Cadherin molecules have major roles in development, suggested by their tightly regulated
expression patterns and numerous functional studies (Gumbiner, 1996; Huber et al., 1996). They
have a role in the CNS, in establishing and maintaining synaptic connections and in
19
differentiation of the cranial neural crest (Colman, 1997; Zhou and Snead, 2008). The retina, in
common with the CNS, develops from the neuroepithelium, and the processes that govern this
process are similar to that of the brain (Honjo et al., 2000). Earlier studies examining the
cadherin expression patterns in the developing mouse retina suggest that cadherin expression
coupled with temporal regulation suggested roles for these molecules in cell migration, synaptic
formation, and selective cell associations (Faulkner-Jones et al., 1999b). Cumulatively, studies
showed eleven different cadherin molecules to be expressed in the postnatal mouse and chick
retinas. These include N-cadherin (CDH1), E-cadherin (CDH2), P-cadherin (CDH3), R-cadherin
(CDH4), VE-cadherin (CDH5), CDH6, CDH7, CDH8, CDH10, OB-cadherin (CDH11), and
CDH12 (Faulkner-Jones et al., 1999a; Faulkner-Jones et al., 1999b; Honjo et al., 2000; Wohrn et
al., 1998). Out of these, N-cadherin has been the most extensively studied in the mammalian
retina. Studies show that early in development all undifferentiated cells express N-cadherin,
leading to axonal outgrowth (Kljavin et al., 1994), and downregulated later in development,
where it is localized to the INL (Honjo et al., 2000). Deregulation leads to deficits in visual
behaviour of Drosophila correlated to disruptions in photoreceptor connectivity (Lee et al.,
2001). Further studies in zebrafish reported crucial roles for N-cadherin in retinal lamination and
retinal organization (Erdmann et al., 2003; Masai et al., 2003). Cadherin-6 has also been
implicated to have a role in formation of the retina as knockdown zebrafish embryos showed
severely disrupted differentiation of retinal cell layers (Liu et al., 2008). In Xenopus embryos,
loss-of-function studies reported a decrease in cell proliferation, retinal lamination defects and
disruptions in structural organization (Ruan et al., 2006). Other cadherins have not been as
extensively studied, but are suggested to have various roles during retinogenesis, based on
restrictions in expression patterns throughout development. For instance, both R-cadherin and
cadherin-6 are concentrated in the GCL and amacrine layers, suggestive of a role for these
20
cadherins in interneural connections between restricted pairs of neurons in retinogenesis (Honjo
et al., 2000). The role of Cdh11 in retinal development is still unclear, as studies in knockout
mice show no obvious phenotype (Marchong, Yurkowski, et al, PLOs Genetics (in press)). It is
postulated that this could be due to compensation by expression of other cadherins.
1.8.4 Cadherin-11 and its Isoforms
Cadherin-11 is a member of the Type II classical cadherins. Type I cadherins usually have broad
distributions segregated by embryonic germ layer or tissue type (Nishimura et al., 1999), while
Type II cadherins are often found in overlapping and tightly regulated patterns of expression, in
loosely associated tissues and in weaker intercellular adhesion (Takeichi, 1995). Cdh11 is
primarily expressed in tissues derived from the mesoderm (MacCalman et al., 1996; Shibata et
al., 1996). Cdh11 is a 796 amino acid protein that exists in three different isoforms: intact,
variant and secreted. It was originally cloned from mouse osteoblasts, but was first found in
nervous tissue (Okazaki et al., 1994; Tanihara et al., 1994). It resides in the human genome on
Chr. 16, and is a 120-kD protein, with a 97% homology to its mouse counterpart, on Chr. 8 in the
mouse genome (Hoffman and Balling, 1995; Okazaki et al., 1994). The variant form shows a
179bp insertion in the transmembrane domain, leading to a frameshift and alternative splicing,
resulting in an altered cytoplasmic region and an 85-kd protein (Kawaguchi et al., 1999). It also
lacks cell-cell adhesion properties, but assists the intact form in adhesion. The secreted form is
an 80-kd protein and thought to be a result of proteolysis of the intact form as seen in
osteosarcoma samples, resulting in downregulation of Cdh11 (Kashima et al., 1999) (Figure 1.4).
21
Figure 1.4 Cadherin-11 and its Isoforms.
A. Human cadherin-11 exists in three isoforms: an intact form, an alternatively spliced variant form and a secreted
form, thought to be derived from the intact form via proteolysis. B. Mouse cadherin-11 is located on chromosome 8
and is 97% homologous to human cadherin-11. Also shown is the inactive form of cadherin-11 found in cadherin-
11 knockout mice generated by using a targeting vector. A PGKNeopA gene cassette replaced the last 56 AA of the
extracellular domain and some of the transmembrane domain (Horikawa et al, 1999).
1.8.5 Cadherin-11: Implicated Roles
Cadherin-11 knockout mice were previously used to analyze its role in different tissues. In
Cdh11 null mice, a significant decrease in bone density was observed at specific parts of the
skeleton, suggesting a role in osteoblast differentiation and mineralization of the osteoid matrix
(Kawaguchi et al., 2001). Another study observed that mice null for Cdh11 showed reduced fear
22
and anxiety responses in the brain, as well as increased long-term potentiation, suggesting Cdh11
junctions are necessary for normal development of synaptic organization in the brain (Manabe et
al., 2000). Additionally, developing somites of mice with disrupted Cdh11 genes were observed,
but showed no structural anomalies. However, mice null for both Cdh11 and N-cadherin
resulted in greatly fragmented somites, a more dramatic phenotype than in N-cadherin null mice
alone, suggesting the two cadherins worked together in somite morphogenesis (Horikawa et al.,
1999).
These and further roles for Cdh11 have since been studied. Cdh11 is expressed in motor and
sensory neurons in the developing mouse embryo, implicating a role in growing motor axons,
controlling growth and interactions of the growth cone (Marthiens et al., 2005). Additionally,
roles have been observed for Cdh11 in differentiation of osteoblasts and chondroblasts from
mesenchymal cells (Kii et al., 2004), as well as in human trophoblast cells (Getsios and
MacCalman, 2003). Recent studies have focused on identifying the role for Cdh11 in formation
and organization of the synovium (Kiener et al., 2006; Valencia et al., 2004). Mice null for
Cdh11 showed a resistance to inflammatory arthritis (Lee et al., 2007), and L cells transfected
with Cdh11 showed an increase in invasive capabilities in chronic synovitis and rheumatoid
arthritis (Kiener et al., 2009). Lastly, Cdh11 has been observed to have roles in cranial neural
crest differentiation and migration. One study showed human embryonic stem (ES) cells
positive for Cdh11 expression, were multi-progenitor cells capable of differentiating into fates
associated with the cranial neural crest (Zhou and Snead, 2008). Cdh11 extracellular cleavage
by a disintegrin and metalloprotease (ADAM) promoted migration of CNC cells through
modifying cell-cell adhesion (McCusker et al., 2009). These studies have proven the wide
variety of roles cadherin molecules can have. Cdh11 itself has multiple roles ranging from
development to differentiation and migration in different tissue types.
23
1.8.6 Cadherins in Cancer Progression
1.8.6.1 Cadherin-11
Allelic loss of 16q21-22 has been seen in a variety of cancers. In retinoblastoma, we observed
loss of Cdh11 expression in 91% of cancer cell lines and retinoblastoma primary tumors
(Marchong et al., 2004). Cdh11 has also been implicated in the progression of a variety of
tumors over the past decade. Like retinoblastoma, similar patterns of loss have been observed in
osteosarcoma. Original studies showed altered expression of Cdh11 in human osteosarcomas
(Kashima et al., 1999). High expression of the alternatively spliced variant form of Cdh11
suppressed the intact form (Kawaguchi et al., 1999). Additionally, the secreted form was most
prevalent and was suggested to disrupt cell-cell adhesion. It was hypothesized that down-
regulation of Cdh11 was related to morphology and metastatic potential (Kashima et al., 1999).
The same group then showed that while there was strong expression of Cdh11 in normal
osteoblasts, there was faint expression in osteosarcoma. Their study showed overexpression of
Cdh11 in osteosarcoma cell line LM8 led to a marked reduction pulmonary metastasis in vivo
(Kashima et al., 2003). More recently, two cell lines developed from the primary tumor and
metastasis of an osteosarcoma patient, showed decreased expression of Cdh11 in the metastatic
cell line, suggesting a role in the metastatic process (Zou et al, 2008). These results are
paralleled by a recent study where expression of Cdh11 in normal osteoblast cell lines was
followed by marked decreases in expression in primary tumors cell lines and again in metastatic
cell lines (Nakajima et al., 2008). This same study revealed Cdh11 expression was significantly
correlated to the patient‟s survival and was suggested as a prognostic marker for osteosarcoma.
Besides osteosarcoma, decreased Cdh11 expression has been observed in a subset of colon
cancers (5 of 23) (Braungart et al., 1999), and as a frequent molecular event with the transition of
normal astrocytes to astrocytomas (Zhou and Skalli, 2000). However, Cdh11 has also been
24
observed to be increased in a variety of cancers. This is evident in numerous studies that
implicated Cdh11 in the progression of breast cancer, suggestive of Cdh11 as a marker of
invasive and aggressive forms (Bellahcene et al., 2007; Pishvaian et al., 1999; Sarrio et al.,
2008). It also has been seen to be up-regulated in rhabdomyosarcomas (Markus et al., 1999),
signet ring carcinomas (Shibata et al., 1996), nephroblastomas (Ramburan et al., 2006), and in
prostate cancer cell lines (Bussemakers et al., 2000), tumor samples (Tomita et al., 2000), and
metastasis (Chu et al., 2008). The observation that Cdh11 is lost in some cancers and gained in
others suggests a role based on tissue type and microenvironmental signals, although the
mechanisms are still unclear. Differences in adhesion strength, extracellular binding partners or
alterations in cell signaling via the cytoplasmic domains are all possibilities.
1.8.6.2 E-Cadherin
E-cadherin and its role in cancer progression is the most intensely studied of any of the
cadherins. It is down-regulated in a variety of cancers such as gastric carcinomas, breast, colon,
esophagus, liver, and pancreatic cancers (Beavon, 2000; Munro et al., 1995; Oka et al., 1993;
Tamura et al., 1996a; Tamura et al., 1996b). Its function in the progression of cancer was best
shown by abrogation of invasive phenotype when different cells of different cancer types were
transfected with E-cadherin cDNA (Chen and Obrink, 1991; Luo et al., 1999; Vleminckx et al.,
1991). Its tumor suppressive function was highlighted by revealing its prominent role in driving
numerous cancers to invasive and metastatic forms (Beavon, 2000; Gottardi et al., 2001;
Nawrocki-Raby et al., 2003; Perl et al., 1998). The mechanism behind its suppression is not yet
fully known, but many studies have supported its tumor suppressor abilities through the finding
of different mutations, and biallelic inactivation in breast and diffuse gastric tumors (Becker et
al., 1996; Becker et al., 1994). Germline mutations have been identified in gastric cancer
samples (Gayther et al., 1998; Guilford et al., 1998), as well as somatic mutations in gastric,
25
breast, endometrium and ovary cancers (Becker et al., 1996; Hajra and Fearon, 2002). Missense
mutations lead to increased cell motility and decreased cell adhesion (Handschuh et al., 1999;
Handschuh et al., 2001). Promoter hypermethylation has been seen in acute leukemia and
numerous carcinomas including breast, gastric, hepatocellular, esophageal and renal cell (Corn et
al., 2001; Corn et al., 2000; Hu et al., 2002; Kanai et al., 1997; Nojima et al., 2001; Tamura et
al., 2001; Zhao et al., 2007). Transcriptional repression via proteins such as Slug and Snail, have
been suggested by binding to the CDH1 promoter (Halbleib and Nelson, 2006). Other
mechanisms recently reported include post-transcriptional mechanisms such as phosphorylation
by tyrosine kinases, which induce endocytosis and subsequently target E-cadherin for
ubiquitination and degradation (Christofori, 2006; Fujita et al., 2002). Phosphorylation of
catenins such as β-catenin has can lead to invasion through disassembly of the complex and
induction of migration (Hu et al., 2001). Posttranslational studies looking at truncation of the E-
cadherin protein present another argument in down-regulation in cancers. Studies show both
extracellular cleavage through metalloproteinases and cytoplasmic cleavage yields truncated
proteins that are inactivated, inhibit cell aggregation, and enhance tumorigenesis and metastasis
(Noe et al., 2001; Rashid et al., 2001; Rios-Doria et al., 2003).
1.8.6.3 N-Cadherin
N-cadherin (CDH2) is usually expressed in tissues of neuronal origin. N-cadherin is a hallmark
of EMT and is usually up-regulated in cancers, especially when E-cadherin is down-regulated,
leading to an increased affinity for mesenchymal cells (Yilmaz and Christofori, 2009). The gain
of N-cadherin provokes increased cell migration, invasion and metastasis (Hulit et al., 2007;
Nieman et al., 1999). An up-regulation of N-cadherin is typically seen in cancers such as breast,
prostate and gastric carcinomas (Gravdal et al., 2007; Hazan et al., 2000; Hazan et al., 2004;
Nagi et al., 2005). An increase in N-cadherin expression and presence of an N-cadherin/catenin
26
complex is observed in invasive retinoblastoma samples (Mohan et al., 2007; Van Aken et al.,
2002). Mechanisms leading to up-regulation of N-cadherin are undefined, but several proteins
have been linked to its increased expression. Twist, a transcriptional repressor, was seen to be
up-regulated in certain cancers, and led to the induction of N-cadherin expression (Alexander et
al., 2006; Yang et al., 2007). N-cadherin interacts with different signal transduction pathways.
For example, it interacts with the fibroblast growth factor receptor (FGFR), activating the MAPK
pathway. As a result, increased proliferation and matrix metalloproteinase (MMP) secretion
promote invasiveness in N-cadherin expressing cells (Suyama et al., 2002).
1.9 Cadherin-11 as a Tumor Suppressor in Retinoblastoma Progression
As a candidate gene in retinoblastoma progression, preliminary assessment of the tumor
suppressor role of Cdh11 was done in TAg-RB mice (Figure 1.5). Immunohistochemical
staining revealed Cdh11 was lost in 38% of primary tumors of TAg-RB mice, over a time period
of five months, paralleling human retinoblastoma genomic changes. When TAg-RB+/-
mice were
crossed with Cdh11+/+
and Cdh11-/-
mice to examine tumorigenesis, tumor volumes were
calculated per single TAg positive cell at post-natal day 8 (PND8), to a large tumor at PND84.
Volumes were larger overall in Cdh11-/-
mice compared to mice with normal Cdh11 alleles and
tumor growth was faster when quantified between PND28 and PND84 (Marchong, Yurkowski et
al, PLOs Gentics (in press)). This suggested Cdh11 was acting as a tumor suppressor. Tumor
growth can be defined as a fine balance between cell proliferation and cell death and therefore,
both possibilities were considered. Preliminary experiments showed no statistical differences in
the expression of proliferation marker PCNA, but showed a statistical difference in activated
caspase-3 staining as a marker of apoptosis when extrapolated to the entire tumor. This
preliminary data suggested that Cdh11 has a role in apoptosis and led to two hypotheses. These
hypotheses are addressed in the specific aims for this study and are discussed in section 1.11.
27
Figure 1.5 Cadherin-11 as a Tumor Suppressor in Retinoblastoma Progression.
A. Cadherin-11 expression in normal murine retina resides in the INL, where human and murine retinoblastomas
initiate from. B. Cadherin-11 expression was lost in 38% of TAg-RB tumors analyzed by RT-PCR (Marchong et al,
2004). C. When quantifying tumor growth in TAg-RB tumors from post natal day (PND) 28 to PND84, growth was
faster in tumors of mice null for Cdh11. D. No statistical difference was seen between TAg-RB tumors null and
wildtype for Cdh11 in staining for proliferation marker, PCNA. Statistical difference was seen between genotypes
in staining for caspase-3, a pro-apoptotic marker, with mice null for Cdh11 showing decreased caspase-3 staining.
1.10 Apoptosis
Apoptosis is an evolutionary conserved programmed cell death mechanism that is crucial in
development and homeostasis (Hengartner, 2000). It is characterized by typical morphological
28
and biochemical hallmarks such as cell shrinkage, DNA fragmentation and membrane blebbing
(Hengartner, 2000; Wyllie et al., 1980). The genetic basis of apoptosis implies that cell numbers
can be regulated by cell death, in addition to proliferation and differentiation, and like any other
metabolic or developmental program, can be affected by mutation (Thompson, 1995). Studies
first showed large percentages of cell loss seen in tumors was due to apoptosis (Kerr et al.,
1972). Subsequent studies revealed a higher frequency of apoptosis in spontaneously regressed
tumors and in tumors treated with cytotoxic anticancer agents (Kerr et al., 1994). In recent
years, numerous studies have reported deregulation of apoptosis in the progression of multiple
cancers (Kerbauy and Deeg, 2007). Research has also focused on what factors trigger this
deregulation, such as imbalances of growth and survival factors, loss of cell-cell adhesion,
hypoxia and radiation. Imbalances in internal factors such as DNA damage, telomere
malfunction, or inappropriate proliferation signals can also lead to apoptosis (Hartland et al.,
2009; King and Cidlowski, 1995; Lowe and Lin, 2000).
1.10.1 Extrinsic Pathway
It is now well accepted that there are two distinct pathways of cell death, the extrinsic and
intrinsic pathways (Figure 1.6). The extrinsic pathway is initiated by „death receptors‟, members
of the tumor necrosis factors (TNF) receptor gene superfamily. This consists of more than 20
proteins which have a wide range of biological functions such as cell death, survival,
differentiation and immune regulation (Walczak and Krammer, 2000). The best characterized
death receptors include CD95 (Apo-1/Fas), TNF receptor 1 (TNFR1), and TNF-related
apoptosis-inducing ligand-receptor 1 (TRAIL-1) and TRAIL-2 (Walczak and Krammer, 2000).
Upon binding of their ligands the receptors are trimerized and signal transducing molecules are
recruited (Walczak and Krammer, 2000). For example, upon TRAIL binding to its receptor, the
Fas-associated death domain (FADD) is recruited along with the initiator caspase-8 (Sprick et
29
al., 2000). Caspase-8 cleaves itself to its activated form, which initiates a protease cascade,
cleaving cellular targets and resulting in cell death (Cohen, 1997). This signaling can be
negatively regulated by proteins recruited to receptor cytoplasmic domains, like cellular caspase-
8 (FLICE)-like inhibitory protein (cFLIP). Recruitment of FLIP instead of procaspase-8 can
block caspase activity (Krueger et al., 2001).
1.10.2 Intrinsic Pathway
The intrinsic pathway of apoptosis is initiated by activating permeabilization of the outer
mitochondrial membrane. This is achieved by numerous cytotoxic and pro-apoptotic signals
converging on the outer membrane, including proteins of the Bcl-2 family (Green and Kroemer,
2004). BH3-containing molecules activate mitochondrial permeability transition by inactivation
of anti-apoptotic Bcl-2 family members, like Bcl-2 or Bcl-Xl, or through activation of pro-
apoptotic members, such as BAX (Ghiotto et al., 2009). This results in the release of proteins
into the cytosol that activate the central apoptotic pathway. Cytosolic cytochrome c interacts
with apoptotic-protease-activating factor 1 (APAF1) and caspase-9, which forms a heptameric
complex, the apoptosome (Acehan et al., 2002). The formation of this complex initiates a
protease cascade, similar to described above, leading to widespread cleavage of apoptotic
substrates (Li et al., 1997).
Apoptosis cannot be simply described by two parallel pathways. Firstly, studies in mice showed
that activation by death receptors was highly variable depending on cell type and specific to
microenvironmental stimuli (Hakem et al., 1998; Yoshida et al., 1998). Secondly, links between
the mitochondrial and death receptor pathway exist leading to „cross-talk‟ on different levels.
For example, caspase-8 activation through death receptors can lead to cleavage and activation of
30
BID (tBID), a member of the Bcl-2 family thought to facilitate release of cytochrome c from the
mitochondria (Billen et al., 2008).
Figure 1.6 Apoptotic Pathways.
A. The extrinsic pathway is initiated by death receptors of the tumor necrosis (TNF) death receptor gene
superfamily activating caspase-8 and initiating the protease cascade leading to cell death. B. The intrinsic pathway
is initiated by permeability of the mitochondrial membrane, leading to release of proteins and cytochrome c,
interacting with caspase-9 and APAF1 to form the apoptosome, initiating a protease cascade. Cross-talk also occurs
through caspase-8 activation of BID, a Bcl-2 family member that releases cytochrome c from the mitochondria.
1.11 Project Aims and Hypothesis
1.11.1 To examine if Cadherin-11 is promoting apoptosis in TAg-RB tumors and define the
acting apoptotic pathway
1.11.2 To determine if the loss of Cadherin-11 expression leads to invasive and aggressive
tumors through expression of invasion markers
31
Chapter 2
Methods and Materials
2.1 Mouse Models
Cdh11 knockout mice (Cdh11-/-
), background strain 129, were originally provided by Dr. M
Takeichi (Horikawa et al., 1999; Kawaguchi et al., 2001). One generation crosses were made
between Cdh11-/-
129 and Cdh11+/+
C57Bl-6 to get a mixed background of 129/C57Bl-6.
Cdh11-/-
littermates were used to make one generational crosses with SV40 TAg-RB (TAg+/-
),
background strain C57Bl-6, which were available in the lab and first provided by Joan O‟Brien
(Windle et al., 1990). These littermate, double heterozygotes Cdh11+/-
TAg+/-
, on a 129/C57Bl-6
background, were further crossed with Cdh11-/-
, Cdh11+/-
or Cdh11+/+
of a 129/C57Bl-6 mixed
background. These crosses yielded the three genotypes used for this study: Cdh11-/-
;TAg+/-
,
Cdh11+/-
;TAg+/-
and Cdh11+/+
;TAg+/-
. These mice were sacrificed at post natal day 84 (PND84),
a timepoint of three months.
2.2 Genotyping
Genotyping of Cdh11-/-
mice and their littermates were carried out using genomic DNA isolated
from mouse tail. PCR conditions were previously described by Dr. M Takeichi (Horikawa et al.,
1999). PCR reactions were performed in a RoboCycler Gradient 96 thermal cycler (Stratagene).
This included 940C, 2 min, 1 cycle [94
0C, 30s, 50
0C, 30s, 72
0C, 30s] 35 cycles, 72
0C 10 min, and
400C, cool block. Primers used were: forward, 5‟ to 3‟ (21bp): TTC AGT CGG CAG AAG CAG
32
GAC and backward, 5‟ to 3‟ (19bp): GTG TAT TGG TTG CAC CAT G, and neo 5‟ to 3‟ (23bp):
TCT ATC GCC TTC TTG ACG AGT TC. The sizes of expected PCR products were: Cdh11+/+
:
240bp, Cdh11+/-
: 480bp and 240bp, and Cdh11-/-
: 480bp. The reaction mixture included 2.5 µl
of 2 mM dNTPs, 1.5 µl 25 mM MgSO4, 2.5 µl KOD 10X PCR buffer (Novagen), 0.5 µl forward
5‟ primer, 1.0 µl reverse 3‟ primer and 0.5 µl neo primer, 0.25 µl KOD hot start DNA
polymerase (Novagen), 1.0 µl template, up to 25 µl with ddH2O. Genotyping of TAg mice and
littermates were carried out using PCR conditions: 940C, 2 min, 1 cycle, [94
0C, 1 min, 58
0C, 1
min, 720C, 1min] 30 cycles, 72
0C, 10 min, 1 cycle and 4
0C, cool block. Primers used were:
forward, 5‟ to 3‟ (30bp): GAC TTT GGA GGC TTC TGG GAT GCA ACT GAG and backward: 5‟
to3‟ (30bp): GGC ATT CCA CCA CTG CTC CCA TTC ATC AGT. The size of the expected PCR
product was 420bp. The reaction mixture included 2.5 µl 10XPCR buffer (100 mMTris-HCl pH
8.3; 500 mM KCl), 0.5 µl 2 mM dNTPs, 2 µl 25 mM MgCl2, 0.5 µl each of forward and reverse
primers, 0.5 µl Taq polymerase isolated from E.coli that has been transformed with pTaq
plasmid, 1.0 µl template up to 25 µl total volume. All animals were treated in accordance with
protocols approved by the Animal Care Committee of the Ontario Cancer Institute.
2.3 Histology and Slide Selection
Mouse eyes were dissected and fixed in freshly prepared 4% PFA for 24hrs and then stored in
70% EtOH at 40C. Eyes were then paraffin embedded and sectioned at 0.5 µm (Pathology,
Hospital for Sick Children, ON, Canada). For littermates Cdh11+/+
;TAg+/-
, Cdh11+/-
;TAg+/-
and
Cdh11-/-
;TAg+/-
, horizontal serial sections were specifically made through the entire eye and
optic nerve averaging approximately 270-420 sections per eye with 5-7 sections per slide and
approximately 60 slides per eye. To estimate apoptosis and proliferation throughout the entire
tumor, every 10th
slide was chosen for analysis, averaging to 6 slides per eye. Every section on
each slide was used in the analysis. One eye was analyzed per mouse and 4-6 mice were
33
analyzed in each experiment. To examine the histology, slides were stained with hematoxylin
and eosin and then scanned using the Aperio ScanScope XT.
2.4 Immunohistochemistry
Slides that were chosen for analysis were processed through 100% xylene (2 times 10 min
incubations), 100% ethanol washes (2 X 5 min incubations), and 2 min each in 95%, 70% and
50% ethanol. This was followed by a rinse in 1X TBS for 1 x 5 min incubation. Slides were
placed into PBS citrate solution and heat treated for antigen retrieval. This was performed in a
pressure cooker in the microwave at power level 10 for 15 min and then power level 7 for an
additional 5 min. Slides were removed after reaching room temperature, rinsed in 1X TBS for 2
min and treated with Triton-X-100 for 10 min. Afterwards, the slides were rinsed in 1X TBS for
5 min, and then protein blocked using 10% DakoCytomation Protein Block (Ref#X0909,
Lot#1001228) for 30 min. Slides were incubated in primary antibodies made in 10%
DakoCytomation Antibody Diluent (Ref# S3022, Lot# 036205) and 1% BSA/TBST overnight at
40C. These were used at various dilutions, depending on the specific antibody. The next day the
slides were washed in 0.1% BSA/TBST for 3 X 10 min incubations. To visualize pro and anti-
apoptotic stained cells, a Biotin/Streptavidin protocol was used. Slides were incubated with
biotinylated secondary antibodies, either anti-mouse, anti-rabbit, or anti-goat, used at a dilution
of 1:200 in 1% BSA/TBST and 10% DakoCytomation Antibody Diluent, for 1 h at room
temperature. Slides were then again washed with 0.1% BSA/TBST for 3 X 10 min incubations.
Slides were then incubated with either Streptavidin-Alexa488 or Streptavidin-Alexa594, used at
1:200 dilution made in 1X TBS, for 15 min at room temperature. Slides were briefly washed in
1X TBS and incubated for 15 min in 4‟,6-diamidino-2-phenylindole (DAPI), used at 1:50
dilution in 1X TBS, at room temperature. After another brief wash in 1X TBS, slides were
mounted using DakoCytomation Fluorescent Mounting Medium (Ref#S3023, Lot#10027230).
34
Slides were analyzed under a fluorescent microscope and quantified for apoptotic expression.
Slides stained for BrdU were first treated in 2M HCl for 20 min at room temperature, followed
by incubation with 2M sodium borate for 2 min. This was followed by a 5 min wash in PBS and
then treated as previously described until detection of the antibody. To analyze T-antigen and
BrdU stained cells, an Immunopure metal-enhanced diaminobezidine (DAB) Substrate Kit
(Thermoscientific) was used. Slides were incubated for 1h in ABC solution (Vectastain ABC
Elite, Vector Laboraties). The solution was prepared one half hour before incubation by adding
1 drop of each reagent A and reagent B, in 2.5 ml 1X TBS. After incubation slides were washed
for 5 min in 1X TBS, and stained cells visualized after treatment for 3-10 min in DAB Substrate
solution (Thermoscientific) prepared fresh with 10% DAB/Metal Concentrate, 10x (Lot#
KA129317, Prod#1856090) made in 1X Stable Peroxide Buffer (Lot#JA119477, Prod#
1855910). Slides were scanned and analyzed on computer (Section 2.6). Every 15th
slide
sectioned was also stained for Hematoxylin and Eosin (H&E) and analyzed using light
microscopy. Table 2.1 provides a complete list of all antibodies used in experimental analysis.
35
Table 1 Antibodies used in Experimental Analysis.
Antibody Name Company Dilution Used
OB-Cadherin (anti-Cadherin-11)
rabbit polyclonal
ZYMED Laboratories Cat#71-7600,
Lot#389746A
1:100
BrdU (purified anti-bromodeoxy-
uridine) mouse monoclonal
BD Biosciences Pharmigen
Cat#555627, Lot#52817
1:200
Caspase-3 rabbit polyclonal Promega
Cat#G7481, Lot#242477
1:100
Caspase-8 rabbit polyclonal Abnova Antibody Innovation
Cat#PAB0246, Lot#30476849
1:1000
Caspase-9 rabbit polyclonal Cell Signaling Technology
Cat#9509, Lot#83
1:100
BAX (B-9) mouse monoclonal Santa Cruz Biotechnology, Inc
Cat#sc-7480
1:100
TRAIL (K-18) goat polyclonal Santa Cruz Biotechnology, Inc
Cat#sc-6079, Lot#E3008
1:100
Anti-Bid mouse monoclonal Novus Biologicals Cat#NB100-
94174, Lot#B1249056
1:50
FLIPs/l (G-11) mouse monoclonal Santa Cruz Biotechnology, Inc
Cat#sc-5276, Lot#F0208
1:100
FAS (X-20)-G goat polyclonal Santa Cruz Biotechnology, Inc
Cat#sc-1024-G, Lot#K037
1:100
TNF alpha rat monoclonal [MP6-
XT22]
Abcam Cat#ab39542 1:100
Bcl-xL (H-5) mouse monoclonal Santa Cruz Biotechnology, Inc
Cat#sc-8392, Lot#A079
1:100
Bcl-2 rat monoclonal Santa Cruz Biotechnology, Inc
Cat#sc-578, Lot#C187
1:100
36
2.5 BrdU Incorporation
To analyze proliferating cells at PND84, mice were given an intraperitoneal (IP) injection with
bromodeoxyuridine (BrdU) reagent (5-bromo-2‟-deoxyuridine and 5-fluoro-2‟-deoxyuridine,
10:1, used at 1 ml reagent per 100g body weight, Cat#555627, Lot#33076 , BD Pharmigen).
After 4 h, the mice were sacrificed and eyes fixed as previously mentioned.
2.6 Image Analysis
To analyze proliferation within the tumors at PND84, the amount of BrdU staining was
quantified by scanning representative slides at the Advance Optical Microscopy Facility
(AOMF) at the Ontario Cancer Institute using an Aperio ScanScope CS. Images were retrieved
using ImageScope software and all analyzed as TIFF images using ImageJ: Image Processing
and Analysis in Java software, downloadable from http://rsb.info.nih.gov.ij/. The amount of
proliferating cells determined by BrdU positive staining was determined by previous methods
used in the lab (Dimaras et al., 2009). The BrdU positive cells were traced and the area was
measured in pixels. The traced areas were converted into an 8-bit scale. By a manually selected
threshold tool, the area of proliferating cells (DAB stained) was measured by the program in
pixels. The area of BrdU positive, proliferating cells in pixels, was compared to the total tumor
area, also measured in pixels (area of BrdU cells in pixels/tumor area in pixels)*100 (Figure 3.6).
24 sections were analyzed per eye to account for differences throughout the tumor. To quantify
the amount of apoptotic proteins, images were analyzed using a 40x inverted microscope (Leica
DMLB). Selected slides were analyzed under 40x to 100x to precisely identify positively stained
apoptotic cells. Double and triple staining was performed with additional antibodies and with
DAPI to confirm cellular staining. To quantify the amount of binding of each antibody, slides
were manually scanned through with the microscope and individual positively stained cells were
37
counted. This was done for each section on the selected slides to total 36 sections per eye.
Representative slides were selected throughout the entire eye to account for any differences that
may be occurring throughout the tumor. From a total number of apoptotic cells counted from
each section, an average was taken of positively stained cells. This led to a total number of
apoptotic cells that could be found on any section within the tumors (Figure 3.3).
2.7 Statistical Analysis
A total of 5 Cdh11-/-
;Tag+/-
and 5 Cdh11+/+
;Tag+/-
mice were sacrificed at PND84 for the analysis
of apoptotic protein expression. A total of 6 mice per genotype were sacrificed at PND84 for
BrdU injection and analysis of proliferation within the tumors. The student‟s t-test was used to
assess results as significant, using a 95% confidence level. This was accessed from
http://www.graphpad.com/quickcalcs/ttest1.cfm.
2.8 Tissue Culture and Cell Lines
All cell lines used were grown in a humidified 370C incubator with 10% CO2 concentration.
They were grown in their respective tissue culture media (Tissue Culture Media Facility,
OCI/PMH, ON, CA). Cell lines used for analysis included T+532 (22wks), T+539b (20wks),
T+572a (22wks), T+794a (22wks), T+818a (19wks), and T+827b (20wks). These lines were all
derived from primary TAg-RB tumors, at their respective time points, as shown in brackets.
Each of these lines were grown in cell culture media DMEM-H21 (Gibco) with the addition of
100 U/ml penicillin and 0.1 mg/ml streptomycin (Wisent Bioproducts, QC, Canada), 15% Horse
Serum (Gibco, Cat#200-6050), 5% FBS (Wisent Bioproducts, 080-350) and 10 mM HEPES.
To study the role of Cdh11 in the progress of these tumors, cell lines were harvested for protein
and RNA and analyzed for Cdh11 expression. Additionally, Cdh11 in these cell lines was
38
knocked down using Invitrogen stealth siRNA and analyzed for differences in apoptotic protein
expression and expression of different invasion markers.
2.9 RNA Isolation
Total RNA was isolated from cell lines after knockdown of Cdh11 with Invitrogen stealth
siRNA. RNA was extracted from cell lines through the use of TRIzol Reagent (Invitrogen,
Cat#15596-026), through the manufacturer‟s instructions. RNA was redissolved in 20 µl ddH20
and the concentration found using a Nanodrop-1000 spectrophotometer (Thermo Scientific). For
cDNA synthesis, 1 µg total of RNA was reverse transcribed using random primers (Fermentas),
and Superscript II Reverse Transcriptase (Invitrogen). Gene expression analysis was done by
PCR using a RoboCycler Gradient 96 thermal cycler (Stratagene). Conditions for PCR was the
same for all the primers used, and is as follows: 940C, 2 min, 1 cycle, [94
0C, 30s, 60
0C, 30s,
680C, 30s] 35 cycles, 68
0C, 10 min, 1 cycle, and 4
0C, cool block. The reaction mixture included
2.5 µl KOD PCR 10X buffer (Novagen), 2.5 µl 2 mM dNTPs, 1 ul 25 mM MgSO4, 0.5 µl each
of forward and reverse primers, 1.0 µl template, 0.5 µl KOD hot start DNA polymerase
(Novagen), up to 25 µl total volume. Primers used were all designed according to the mouse
genome sequence. They are listed below with expected product sizes in Table 2.2.
39
Table 2 Primers used with Expected Product Sizes.
Gene Primer Sequence Expected Size
(bp)
Cdh11 5‟-AGGAGTATATGCCCGACGTG-3‟
5‟TCGTCCACATCCACACTGTT-3‟ 504
Twist 5‟-ACGACAGCCTGAGCAACAG-3‟ 5‟-
CATCTTGGAGTCCAGCTCGT-3‟ 488
SNai2 (Slug) 5‟-AACATTTCAACGCCTCCAAG-3‟ 5‟-
CAGTGAGGGCAAGAGAAAGG-3‟ 631
Ctnnb1 (B-catenin) 5‟-CAAGATGATGGTGTGCCAAG-3‟ 5‟-
CTGCACAAACAATGGAATGG-3‟ 502
Lef1 5‟-CTCATCACCTACAGCGACGA-3‟ 5‟-
CGTGCACTCAGCTACGACAT-3‟ 498
RhoA 5‟-AAGGACCAGTTCCCAGAGGT-3‟ 5‟-
ACAAGATGAGGCACCCAGAC-3‟ 582
MMP2 5‟-ATCTACTTGCTGGACATCAGGG-3‟
5‟- TGGCTCGAAATTCACAAGGTCC-3‟ 493
MMP9 5‟-GAAGGCAAACCCTGTGTGGT-3‟ 5‟-
GGCTTAGAGCCACGACCATA-3‟ 497
2.10 Protein Isolation
TAg-RB cell lines were collected and resuspended in cold TNE buffer (2% NP-40, 20 mM Tris
pH8.0, 150 mM NaCl, 5 mM EDTA, 2 mM NaN3, 0.1 mM phenylmethylsulfonyl fluoride, 2
µg/ml leupeptin, and 20 µg/ml aprotinin). Samples were incubated for 1 h at 40C on a rotor
mixer. After centrifugation at 12,600 g for 20 min, supernatants were collected and protein
concentration was determined using the BioRad Protein Assay (BioRad, Cat#500-0006), in a
Beckman Coulter DU640B spectrophotometer. Proteins (50 µg) were separated using SDS-
40
PAGE, via a 4-20% Tris-Glycine gradient gel (Lonza, Cat#58511) at 125V. Following
separation, proteins were transferred to a PVDF membrane (BioRad, Cat#162-0177) at 100V for
1 h. Membranes were blocked using 5% Blotto (BioRad) in TBS overnight at 40C. The
following day, membranes were incubated with the proper primary antibody in TBS, 0.05%
Tween-20 for 0.5 h. The respective dilutions for all primary antibodies used are as follows:
Cdh11 (1:750, ZYMED), caspase-3 (1:200, Promega), caspase-8 (1:1000, Abnova), caspase-9
(1:1000, Cell Signalling Technology), β-catenin (1:1000, BD Biosciences) and β-tubulin
(1:1000, Sigma-Aldrich, TO198). Primary antibodies were followed by 3X 10 min washes in
TBS, 0.05% Tween-20, and incubated for 1 h in the respective secondary antibody. The
dilutions for the antibodies are as follows: anti-mouse-HRP (1:10000, Invitrogen, Cat#G21040)
and anti-rabbit -HRP (1:10000, Santa Cruz, sc-2004). This incubation was followed by 3X 10
min washes in TBS, 0.05% Tween-20. HyGLO chemiluminescence Detection Reagent
(Denville) and HyBlot autoradiography film (Denville) was used to detect the proteins.
2.11 Stealth RNAi
To analyze apoptotic protein expression and changes in invasion markers, Cdh11 was knocked
down in TAg-RB cell line of choice, T+539b (20wks). Stealth RNAi (siRNA) was provided by
Invitrogen (Cat# 1320003). Control siRNA was the GL-2 vector (Qiagen). T+539b cells were
transfected with the siRNA targeting mouse Cdh11, at time of plating in triplicate, in media
without the addition of penicillin and streptomycin. The procedure included transfection of 125
pmol of each siRNA oligo in Lipofectamine 2000 (Invitrogen), in a total of 2 ml plating medium.
Cells were left for time periods of 24 h, 48 h, 72 h, 5 d, 7 d or 10 d. Knockdown was confirmed
by Western blot analysis of Cdh11 expression.
41
Chapter 3
Results
3.1 Cadherin-11 acts a tumor suppressor in TAg-RB tumors through
promotion of apoptosis
3.1.1 Active Apoptotic Pathway in TAg-RB Mice
Immunohistochemical analysis was performed on 5 Cdh11-/-
;TAg+/-
and 5 Cdh11+/+
;TAg+/-
mice
sacrificed at PND84, to analyze the apoptotic activity within TAg-RB tumors. Mice were
sacrificed at this timepoint as tumors at this time are well defined and have a distinct phenotype
and differences in tumor volume are observed (Marchong et al, submitted). Slides were stained
with antibodies to both pro-apoptotic and anti-apoptotic proteins and within both the extrinsic
and intrinsic pathways of apoptosis. The methods and materials section contains a full list of all
antibodies used in this experiment (Table 2.1). Positive staining occurred with pro-apoptotic
proteins, BAX, TRAIL, caspase-8, caspase-9, caspase-3, and anti-apoptotic proteins, Bcl-xL,
FLIP (Figure 3.1). This list includes proteins involved in both the intrinsic and extrinsic
pathways. Activation of the pro-apoptotic protein BID (tBID) was observed, and was the
common meeting point for the two pathways in our model.
42
Figure 3.1 Expression of Apoptotic Proteins within TAg-RB Tumors.
Immunohistochemistry was performed on TAg-RB tumors wildtype for Cdh11 at PND84. Analysis of a variety of
pro and anti-apoptotic proteins was done to discover the acting apoptotic pathway (x100 and x400 magnification).
Positive staining was shown with Caspase-3, 8, 9, TRAIL, BAX, Bcl-xl, FLIP and BID. Positive staining was
confirmed with double staining with both DAPI and additional cell death markers.
43
3.1.2 Quantification of Apoptotic Protein Expression in TAg-RB Tumors
The expression of the apoptotic proteins previously mentioned was quantified to assess the
difference in apoptotic activity between Cdh11+/+
;TAg+/-
and Cdh11-/-
;TAg+/-
tumors at PND84.
Initial data led to the idea that apoptosis may be affected by loss of Cdh11 in TAg-RB mice
(Section 1.9) (Marchong, Yurkowski, et al, PLOs Genetics (in press)). Therefore, detailed
analysis and quantification of apoptotic activity within these tumors was pursued in order to find
a mechanism of suppressive function for Cdh11. For this experiment, mice were sacrificed at
PND84 and the eyes paraffin fixed and horizontally sectioned onto slides. One eye from each of
the 5 Cdh11-/-
;TAg+/-
and 5 Cdh11+/+
;TAg+/-
mice was used in analysis. Upon sectioning, an
average of ~60 slides is obtained per mouse eye, and for this experiment, every 10th
slide was
chosen to get a good representative sample of the entire tumor. This equaled to using 6 slides
per eye (mouse) in analysis. Five proteins that had previously been described to be involved in
the TAg-RB apoptotic pathway (Figure 3.2) were used in the quantification and included BAX,
TRAIL, caspase-3, caspase-8, and caspase-9. Slides are produced with 6-7 0.5µm sections on
each slide and a total of 36 sections were used in the analysis. For each section, positively
stained cells were manually counted under the microscope. A total number of positively stained
cells were obtained for the entire tumor and then an average per section was derived from
dividing by the 36 sections used in analysis. This analysis yielded a true number of apoptotic
cells from anywhere within the tumor. Upon quantification, a very significant difference in
apoptotic activity was observed. In comparison, Cdh11-/-
;TAg+/-
mice were revealed to have
anywhere from 5 to 10 times less apoptotic activity happening within the tumors than
Cdh11+/+
;TAg+/-
mice. This was true for every cell death marker assessed (caspase-3, p=0.014;
caspase-8, p=0.029; caspase-9, p=0.014; TRAIL, p=0.008; and BAX, p=0.029) (Figure 3.3).
This gave very strong evidence that Cdh11 is promoting apoptosis within TAg-RB tumors.
44
Figure 3.2 Acting Apoptotic Pathway in Retinal Tumors of TAg-RB Mice.
Immunohistochemical staining implicated extrinsic proteins (A) and intrinsic proteins (B) in the apoptotic pathway
in TAg-RB mice. Cross talk occurs through activation of BID and both pathways lead to activation of caspase-3.
45
Figure 3.3 Differences in Apoptotic Protein Expression in TAg-RB Tumors at PND84.
Cells were manually counted and an average number of apoptotic cells per section were calculated in order to
quantify expression of five apoptotic proteins (caspase 3, 8, 9, TRAIL and Bax) active in TAg-RB tumors at
PND84. When averaged, results revealed between five to ten times less apoptotic activity in TAg-RB tumors null
for Cdh11 (Student‟s T-test: p values = 0.014, 0.029, 0.014, 0.008, and 0.029, respectively).
3.1.3 Analysis of Cadherin-11 in TAg-RB Derived Cell Lines
To support the in vivo data from the TAg-RB tumors, in vitro work was performed with cell lines
previously described (Section 2.8), derived from primary TAg-RB tumors. Various cell lines
were harvested to isolate protein and Western Blot analysis was performed to determine Cdh11
expression. Six candidate cell lines were chosen to analyze for Cdh11 expression, including
46
T+532, T+539b, T+572a, T+794a, T+818a, and T+827b. Protein analysis revealed varied Cdh11
expression, supporting evidence that TAg-RB tumors parallel human retinoblastoma genomic
and expression changes (Marchong, Yurkowski, et al, PLOs Genetics (in press), Dimaras et al.,
2008), as 4 of 6 or 67% of cell lines showed a decrease, little or no Cdh11 expression (Figure
3.4).
Figure 3.4 Cdh11 Protein Expression in TAg-RB Derived Cell Lines.
Cdh11 protein expression was analyzed via Western blot using six different cell lines all derived from primary TAg-
RB tumors. Expression was varied in all six cell lines ranging from abundant Cdh11 expression, to decreased or
little Cdh11 expression, paralleling human retinoblastoma cell lines.
3.1.4 Apoptotic Protein Expression in a TAg-RB Derived Cell Line
In order to support the idea that the suppressive mechanism of Cdh11 in TAg-RB tumors
involved the promotion of apoptosis, cell line T+539b, derived from primary TAg-RB tumors
(Section 2.10) was chosen as a candidate line to use in knockdown experiments, on the basis that
it showed abundant Cdh11 expression via Western blot (Figure 3.4). This experiment was done
in order to establish and confirm a direct effect of Cdh11 on apoptosis, by directly inhibiting
47
Cdh11 by siRNA (Sigma, MSS202865-87). After 10 days treatment, Western blot analysis was
done to look the change in pro-apoptotic protein, caspase-3 expression. Effective knockdown of
Cdh11 was assessed by Western Blot and showed significant knockdown by siRNA, by 67.8%
(siRNA MSS202867) (Figure 3.5). Accordingly, when comparing the expression of these
proteins to the siRNA control lane, caspase-3 expression was also decreased by 53.1%. This
data indicated Cdh11 had an effect on apoptosis in vitro, and showed support for the
observations seen in vivo (Figure 3.3), that Cdh11 is promoting apoptosis within TAg-RB
tumors.
Figure 3.5 Cdh11 Knockdown Leads to Decreased Caspase-3 Expression.
Cdh11 was knocked down using stealth siRNA (MSS202867, Invitrogen) in a cell line derived from TAg-RB
tumors, T +539. Upon knockdown of Cdh11, caspase-3 expression was significantly decreased compared to
control.
48
3.1.5 Cdh11 and Proliferation in TAg-RB Tumors
Evidence supports the notion that Cdh11 is promoting apoptosis within TAg-RB tumors.
However, development of tumors is often distinguished as a fine balance between apoptosis and
proliferation, and so experiments were pursued to determine the effect of loss of Cdh11 on
proliferation within TAg-RB tumors. Therefore, these experiments were necessary to specify the
role of Cdh11 in the progression of these tumors. Preliminary evidence suggested Cdh11 was
not affecting proliferation in TAg-RB tumors through immunohistochemistry using PCNA as a
marker for proliferation (Marchong, Yurkowski, et al, PLOs Genetics (in press)), and so to
expand and confirm these initial results, quantification with BrdU was performed. Mice were
sacrificed at PND84 (3 months), the same timepoint used for apoptotic protein expression
experiments. For these experiments 6 Cdh11-/-
;TAg+/-
and 6 Cdh11+/+
;TAg+/-
mice were used in
the analysis. Four hours before sacrificing these mice, an IP injection of BrdU was given. Eyes
were sectioned as previously described and every 10th
slide was chosen in analysis. This gave a
total of 24 sections measured and analyzed per mouse. Immunohistochemistry was used to look
at proliferating cells by staining for BrdU. The area of the positively stained cells (examples
outlined in blue), measured in pixels, was taken and used as a percentage of tumor area (outlined
in yellow), as described by previous methods used in the lab (Figure 3.6) (Dimaras et al., 2009).
Upon quantification, no significant difference was seen in the amount of BrdU positive cells
between the two genotypes (Figure 3.7), suggesting that Cdh11 was not affecting proliferation
within the tumors. By these results, Cdh11 appears to be affecting tumor growth and exerting a
suppressive function specifically through affecting apoptotic activity.
49
Figure 3.6 Quantifying the Percentage of BrdU Proliferating Cells as a Function of Tumor Area.
A) Eyes were horizontally sectioned onto slides and every 10th
slide was chosen to use in analysis. A total of 24
sections were analyzed. B) Selected sections were stained for BrdU using DAB for visualization in bright field.
Area of proliferating cells was found by using Image J software. Images were analyzed in an 8-bit grey scale, and
the area of BrdU positive cells (red) was found by measuring in pixels. Area of proliferating cells was taken as a
percentage of total tumor area (in yellow).
50
Figure 3.7 Average Area of Proliferating Cells Per Tumor Area at PND84.
6 Cdh11+/+
;TAg+/-
and 6 Cdh11-/-
;TAg+/-
PND84 eyes were stained for BrdU. Proliferation was analyzed by
measuring BrdU positive cells (in pixels) as a percentage of tumor area. An average was found for each genotype.
The percentage of BrdU positive cells as a function of tumor area is shown with no significant difference in the
amount of staining for the proliferation marker seen between the two genotypes (p value = 0.121).
51
3.2 Loss of Cdh11 in TAg-RB Tumors Increases Invasion Potential
3.2.1 Knockdown of Cdh11 in a TAg-RB Cell Line Leads to an Increase of EMT
Markers and Metalloproteases
It was confirmed by the above experiments, that Cdh11 is acting as a tumor suppressor in TAg-
RB tumors through promotion of apoptosis, and that when Cdh11 is lost, tumors grow at a faster
rate due primarily to decreased cell death. We then questioned if the loss of Cdh11 would affect
the invasion potential of these tumors, as when Cdh11 was lost, if these tumors would take on a
more aggressive role. It is well established that altered expression of cadherin molecules affect
tumorigenesis and influence invasion. Often the loss of cell-cell adhesion and the alteration of
signaling transduction pathways lead to malignant transformation as well as enhanced migration,
invasion and metastasis (Cavallaro and Christofori, 2004; Yilmaz and Christofori, 2009). To
examine if Cdh11 would have a similar role in retinoblastoma, RT-PCR was performed to
analyze mRNA expression levels in vitro using candidate TAg-RB cell lines to observe the
mRNA expression of various EMT markers, and MMPs upon knockdown of Cdh11 using
siRNA. The cell line T+539b, positive for Cdh11 as described above (Figure 3.4, Section 2.8),
was chosen for these experiments. Cdh11 was knocked down in line T+539b and at a timepoint
of 10 days, RNA was isolated and RT-PCR was done in order to analyze the levels of the
different invasion markers. Cdh11 knockdown was confirmed via Western blot as described
above (Figure 3.5). Two different EMT markers were analyzed; TWIST and the SNAi2, the
mouse homolog of Slug. End-point RT-PCR revealed both of these markers had low expression
in line T+539b and when treated with the control siRNA, but were upregulated after knockdown
with siRNA (Figure 3.8A). Both these molecules have been widely published in the literature to
increase during EMT and essential to metastasis formation and invasion (Kurrey et al., 2005;
Yang et al., 2004). End-point RT-PCR for MMPs also revealed an up-regulation in MMP-2 and
MMP-9 upon knockdown of Cdh11 in T+539b (Figure 3.8B). A recent study saw up-regulation
52
of both MMP-2 and MMP-9 in human samples of retinoblastoma, in a higher percentage of
invasive tumors compared to non-invasive tumors (Adithi et al., 2007). Our results demonstrate
up-regulation of two MMPs, and two EMT markers, indicating an increase in invasion potential
of TAg-RB tumors, upon the loss of Cdh11.
Figure 3.8 Knockdown of Cdh11 Leads to Increased mRNA Expression of EMT and Invasion
Markers.
Cdh11 was knocked down with siRNA (Invitrogen) and RT-PCR analysis was done to look at mRNA expression
levels of invasion markers after 10 days treatment. Both EMT markers analyzed, TWIST and SNAi2 showed
upregulation after knockdown. Additionally, two MMPs analyzed, MMP-2 and MMP-9 showed upregulation after
Cdh11 knockdown.
53
3.2.2 β-catenin mRNA and Protein Levels Increase Upon Knockdown of Cdh11
The loss of Cdh11 was seen to lead to an up-regulation of certain invasion markers in vitro. A
question remained to how Cdh11 loss could be linked to both this increase of invasion markers
and promotion of apoptosis. β-catenin is the primary protein that interacts with Cdh11. It is well
established to have dual roles within the cell, in adhesion and in a variety of downstream
signaling events, most notably in Wnt signaling. The loss of Cdh11 cell-cell adhesion could
quite possibly affect the levels of free ϐ-catenin within the cell, and lead to altered downstream
signaling. Therefore, to us to look at ϐ-catenin levels upon the loss of Cdh11, Cdh11 was
knocked down in cell line T+539b, and the mRNA and protein levels of ϐ-catenin were analyzed,
by RT-PCR and Western blot, respectively. At 10 days with siRNA, mRNA and protein samples
showed increased expression of ϐ-catenin and the knockdown of Cdh11 (Figure 3.9A, B). Many
downstream effects could result from this increase and would warrant further study.
54
Figure 3.9 Cdh11 Knockdown Leads to Upregulation of ϐ-catenin mRNA and Protein Expression
Levels.
A) Cdh11 was knocked down using three different stealth siRNAs (Invitrogen) targeted to Cdh11, in TAg-RB
derived cell line T+539. Upon knockdown of Cdh11 (MSS202865 – siRNA #1, MSS202866 – siRNA #2,
MSS202867 – siRNA #3) expression levels of ϐ-catenin analyzed via Western blot were increased in 2 out of 3
Cdh11 siRNA treated samples. B) Cdh11 was knocked down with siRNA (Invitrogen). Following knockdown, RT-
PCR performed showed an increase in ϐ-catenin mRNA levels in TAg-RB cell line T+539.
55
Chapter 4
Discussion
4.1 Cdh11 as a Tumor Suppressor in Retinoblastoma Progression
Earlier studies have implicated involvement of Cdh11 in Retinoblastoma progression. Early
cytogenetic studies showed recurrent chromosomal abnormalities in human retinoblastoma, and
chromosome 16 was the most common genomic loss [reviewed in(Potluri et al., 1986)]. More
recent CGH studies corroborate these findings, and revealed that 32% (58/179) of tumors
showed loss of either the entire chromosome 16 or 16q, with all but 7 tumors showing a minimal
region of loss at 16q22 [reviewed in(Corson and Gallie, 2007)]. Marchong et al., showed copy
number loss for Cdh11 in retinoblastoma based on LOH and QM-PCR techniques. In addition,
some tumors analyzed showed exonic deletion of Cdh11 and three tumors showed potential
intragenic mutations, although no further analysis was done past this point. Copy number loss
was confirmed by another recent study in which allelic loss of Cdh11 was observed in 45% of 20
tumors (Bowles et al., 2007). Expression analysis of Cdh11 revealed loss or decrease in 91% of
tumors and cell lines. The same study also revealed decreased expression in advanced TAg-RB
tumors (Marchong et al., 2004). While early tumors of TAg-RB mice showed high expression of
Cdh11, tumors by 21 weeks showed decreased Cdh11 expression, and 38% of TAg-RB
advanced tumors showed complete loss/decrease of Cdh11 mRNA expression. Supporting the
link of loss of Cdh11 associating with malignancy in retinoblastoma, levels of expression in
adjacent retinoma were equivalent to normal retina (Dimaras et al., 2008). The observation of
loss of Cdh11 expression in late stage tumors supports the hypothesis that Cdh11 loss is a late
event based on frequency and correlation with other genomic changes (Bowles et al., 2007).
56
CDH13 was also listed as a potential tumor suppressor as the second most frequently deleted
marker was in intron 2 of the CDH13 gene. However, it was ruled out as a candidate gene as it
showed normal levels of expression in all retinoblastoma primary tumors and cell lines. Another
study analyzed the 16q region through LOH and CGH techniques, studying 58 retinoblastoma
tumors (Gratias et al., 2007), and defined a minimal region of loss at 16q24. They analyzed
Cdh13 and found no mutations, suggesting no involvement in retinoblastoma, confirming the
previous findings (Marchong et al., 2004).
As a candidate gene in retinoblastoma progression, preliminary assessment of the tumor
suppressor role of Cdh11 was done in TAg-RB mice. When TAg-RB+/-
mice were crossed with
Cdh11+/+
and Cdh11-/-
mice to examine tumorigenesis, tumor volumes were calculated from a
single cell at post-natal day 8 (PND8), to a large tumor at PND84. Volumes were larger overall
in Cdh11-/-
mice compared to those with normal Cdh11 alleles and tumor growth was faster
when quantified between PND28 and PND84 (Marchong, Yurkowski, et al, PLOs Genetics (in
press)). This suggested Cdh11 was acting as a tumor suppressor. Tumor growth can be defined
as a fine balance between cell proliferation and cell death and therefore, both possibilities were
considered. Preliminary experiments showed no statistical differences in PCNA, and did show a
statistical difference in caspase-3 staining when extrapolated to the entire tumor. These studies
required further work to 1) confirm the role of Cdh11 in Retinoblastoma as a tumor suppressor
and 2) to define the mechanism by which this was occurring. The work presented in the present
study achieved both of these goals.
4.2 Cdh11 Acts as a Tumor Suppressor by Promoting Apoptosis
The current study focused on defining the mechanism of suppressive action of Cdh11 in
retinoblastoma and to further identify its suppressive role in late progression of the disease.
57
Further information was necessary to confirm that this suppressive role was tied to apoptotic
activity. Every experiment in the present study used the TAg-RB murine model. These mice
develop tumors resembling human retinoblastomas and past studies in the lab have shown that
expression changes in this model parallel human retinoblastoma (Dimaras et al., 2008; Marchong
et al., 2004). Even cell lines derived from primary TAg-RB tumors parallel expression changes
in vitro, with 67% of lines tested showing decreased or no expression of Cdh11 (this study).
This model is also the most widely used model for pre-clinical studies, making it an ideal model
for our purposes, in which crossing it with Cdh11 knockout mice allowed us to study the effect
of Cdh11 on late stage tumor development and progression.
Our analysis began with mapping out the acting apoptotic pathway in TAg-RB mice. This was
done by analyzing the presence of various pro and anti-apoptotic factors, verified by
immunohistochemical positive staining. Our first inclination was to assume a purely extrinsic
mechanism, due to the inactivation of p53 in this model. In contrast, we saw proteins activated
involved in both the intrinsic mitochondrial apoptotic pathway and the extrinsic death receptor
activated pathway. Apoptosis is initiated via the extrinsic pathway, through the initiation of
TRAIL, a TNF death receptor superfamily member. TRAIL mediated induction of apoptosis has
been shown to be preferential in a wide variety of tumor cells, but not in normal cells, in vitro
(Falschlehner et al., 2009). Limited studies looking at apoptosis have been done in human
retinoblastoma cells, which show a varied pattern of apoptosis. Both p53 dependent and
independent mechanisms have been reported in different tumors, with the majority of cases
leading to activation of caspase-3 (Sitorus et al., 2009). We speculate the same idea is paralleled
in TAg-RB tumors, where we also see activation of both pathways. In TAg-RB mice, we
observed merging of the two pathways, through caspase-8 cleavage and activation of tBID.
58
Since tumor “growth” results from an imbalance between cell death and proliferation, we
examined in detail the contributions of cell proliferation (Figure 3.7) and cell death (Figure 3.3)
in TAg-RB mice with normal Cdh11 alleles vs. mice with mutated Cdh11 alleles. Our data
indicate that when Cdh11 is lost, cell death is deficient by five to ten times the amount, while
proliferation remains unchanged; suggesting that the tumor suppressor function of Cdh11 is
mediated through apoptosis rather than cell proliferation. This idea is further supported by our in
vitro data, where we showed a direct link between apoptosis and Cdh11, as there was a
significant decrease in caspase-3 expression of 67.8% in Cdh11 knockdown experiments (Figure
3.5), in lines derived from primary TAg-RB tumors. To examine proliferation, BrdU was used
as a marker in vivo, assayed by immunohistochemistry to look at differences between genotypes.
Quantification revealed no significant differences between TAg-RB mice with normal Cdh11
alleles, and mice null for Cdh11. These results further confirmed our hypothesis that Cdh11 was
acting as a tumor suppressor through promoting apoptosis, and not through inhibition of cell
division. Furthermore, these results support and coincide with a previous report that yielded
similar results in TAg-RB mice when proliferation and apoptosis were examined using PCNA
and caspase-3 as markers, respectively (Marchong, Yurkowski, et al, PLOs Genetics (in press)).
However, we must remember that while apoptosis is a linear process, proliferation is
exponential. Therefore, experiments to quantitate proliferation over time might reveal
significant effect of loss of Cdh11, as suggested by a trend in our studies of markers of
proliferating cells at isolated time points.
Cadherin molecules are well known to manipulate various cellular functions, including
apoptosis. The majority of studies have focused on N-cadherin, which has been seen to be
upregulated during tumorigenesis in many different tumor types (Yilmaz and Christofori, 2009).
Studies have shown that N-cadherin mediated cell adhesion actually prevents apoptosis in
59
different cells types, and loss of this cell-cell adhesion via N-cadherin results in an increase of
apoptotic signaling and cell death (Erez et al., 2004; Hermiston and Gordon, 1995; Peluso, 1997;
Peluso et al., 1996). However, E-cadherin, which is often lost during tumorigenesis in a variety
of tumor types, has been seen to have a similar effect on apoptosis as presented in this study. A
recent study showed that E-cadherin restoration in human melanoma cell lines, led to increased
levels of activated caspase-3 and caspase-8, and sensitization of apoptosis (Kippenberger et al.,
2006). Current literature does not reveal studies directly linking Cdh11 to apoptotic activity.
My thesis then presents a novel function for Cdh11, in promoting apoptosis. We speculate that
both cadherin type and tissue type may lead to differential roles for cadherins in apoptotic
activity. Interestingly, most studies have implicated a role for N-cadherin in preventing
apoptosis, a cadherin molecule usually upregulated in cancer. Kippenberger et al, implicated E-
cadherin in promoting apoptosis, a molecule typically lost in tumorigenesis. Cdh11 has been
seen to be both lost and gained in the progression of different cancers, and in retinoblastoma, this
thesis shows that it acts to promote apoptosis, leading to the Cdh11 tumor suppressor role in this
cancer. Further studies done in cell lines and tissues of different origin then the retina could
clear up if the role of Cdh11 is context dependent.
4.3 Loss of Cdh11 and Invasion
After defining the mechanism of suppressive action of Cdh11 in the progression of
retinoblastoma, we were curious to further define its role in tumorigenesis. Experiments were
performed to analyze Cdh11 in invasion of retinoblastoma, and determine if this loss would lead
to more aggressive tumors. Our results indicate an upregulation of various invasion markers,
Twist and SNai2, as well as MMPs, MMP-2 and MMP-9. Often the loss of cell-cell adhesion
and the alteration of signaling transduction pathways lead to malignant transformation as well as
enhanced migration, invasion and metastasis (Cavallaro and Christofori, 2004; Yilmaz and
60
Christofori, 2009). For example, downregulation of E-cadherin has been observed in invasive
and metastatic forms of many cancers, such as breast and gastric carcinomas (Margineanu et al.,
2008). Furthermore, recent data has correlated this change in phenotype to an upregulation in
MMPs (Llorens et al., 1998; Nawrocki-Raby et al., 2003). An increase in MMPs has been seen
in retinoblastoma, where expression of MMP-2 and MMP-9 were significantly correlated to
invasive tumors (Adithi et al., 2007). In addition, more invasive phenotypes correlate with an
upregulation of EMT markers, a hallmark in malignant transformation and metastasis formation
(Karreth and Tuveson, 2004), which include a wide variety of molecules from transcription
factors like Twist and Slug to growth factors and candidates in downstream effector pathways
(Yang et al., 2004; Yilmaz and Christofori, 2009).
A recent study supports our hypothesis that Cdh11 acts as a tumor suppressor in retinoblastoma
progression and that loss of Cdh11 leads to increased invasion potential (Laurie et al., 2009).
Cell lines were generated from a Chx10-Cre;RbLox/+;p107-/-;p53Lox/Lox murine model of
retinoblastoma. In culture, two of these cell lines, SJmRBL-3 and SJmRBL-8, underwent
genetic changes affecting cell adhesion. Gene expression microarrays showed changes in Cdh11
and N-cadherin mRNA and protein expression. Xenografts of both newborn rats and SCID mice
revealed highly invasive tumors, invading the optic nerve, anterior chamber, choroid and
subretinal space upon transplantation of SJmRBL-3 and SJmRBL-8. Further experiments
injected both Y79 and SJmRBL-8 cells, into adult SCID mice. These cells had been transfected
with Cdh11/N-cadherin or a control vector. Only eyes in xenografts with the control vector
revealed extensive optic nerve invasion. Either cadherin was sufficient to cause an increased
cell-adhesion phenotype resulting in decreased invasion properties of transfected tumor cells.
This established a functional significance in murine retinoblastoma invasion, mediated by
cadherin cell-adhesion. Furthermore, FISH performed on human retinoblastoma whole eye
61
sections showed selective loss of Cdh11 in some samples with invasion of the optic nerve and
choroid.
4.4 Cdh11 in Cancer Progression
Cdh11 has an interesting role in cancer progression, as it has been seen to be both down
regulated and up regulated in different cancers. In retinoblastoma, Cdh11 acts as a tumor
suppressor, as implicated by earlier studies (Marchong et al., 2004; Marchong, Yurkowski, et al,
PLOs Genetics (in press)), and further shown by evidence presented in the current study. Here,
we have provided data showing that this effect is mediated through the promotion of apoptosis.
Additionally, the loss of Cdh11 correlates to up-regulation of EMT markers and MMPs,
increasing the invasion potential of these tumors. Studies done in osteosarcoma have shown a
similar tumor suppressor role for Cdh11. Multiple studies witnessed decreased expression of
Cdh11 in cell lines of primary tumors and even lower expression in metastatic cell lines, where
the loss of Cdh11 was even suggested as a prognostic marker (Nakajima et al., 2008; Zou et al.,
2008). Decreased Cdh11 expression has also been seen in astrocytomas and a subset of colon
cancers (Braungart et al., 1999; Zhou and Skalli, 2000). These types of changes in Cdh11
expression are reminiscent of changes seen in E-cadherin expression in a variety of cancers,
where the loss of E-cadherin is often correlated to aggressive and invasive cancer (Prasad et al.,
2009; von Burstin et al., 2009).
Upregulation of Cdh11 is also seen in certain cancers, such as breast and prostate cancers,
including invasive and metastatic forms (Chu et al., 2008; Nagaraja et al., 2006; Sarrio et al.,
2008). In this context, the role of Cdh11 is similar to the role of N-cadherin in cancer, which is
upregulated in a variety of cancers, especially those undergoing EMT (Gravdal et al., 2007; Nagi
et al., 2005). This could suggest binding partners for Cdh11 similar to N-cadherin in these
62
cancers. Additionally it is often correlated with a downregulation of E-cadherin (Yilmaz and
Christofori, 2009). Interestingly, in retinoblastoma, we observe decreased expression of Cdh11
(Marchong et al., 2004) and in invasive retinoblastoma, an increased expression of N-cadherin
(Mohan et al., 2007).
These observations lead us to speculate why Cdh11 has such opposing roles in cancer
progression, having either a suppressive or oncogenic function. One possible explanation is that
the role of Cdh11 depends on tissue type and microenvironment, two ideas now heavily studied
in tumorigenesis. It appears that Cdh11 has more of a suppressive role in tumors that develop in
looser attached tissues, while an oncogenic role emerges in tighter attached tissues. This idea is
supported by a study in which cell density was observed to affect the expression of Cdh11 in
MDA-MB-231 cells, a mesenchymal-like breast cancer cell line. In this study, the expression of
Cdh11 depended on the density of cells plated. At increased cell densities, Cdh11 expression
was increased in comparison to low density (Farina et al., 2009).
Tumor microenvironment has emerged to become a hugely discussed topic in the field of
tumorigenesis. The tumor microenvironment is a completely unique environment that emerges
in the course of tumor progression as a result of interactions with the host. It is created and
shaped by the tumor which orchestrates molecular and cellular events taking place in
surrounding tissues. This leads to the implication that unique differences in tumor
microenvironment could be responsible for regulation of expression of various molecules, like
cadherins, leading to altered signaling pathways, cell-cell adhesion and the extracellular matrix.
For instance, a recent study showed that stable ectopic expression of interleukin 6 (IL-6) in
MCF-7 breast adenocarcinoma cells actually led to an EMT phenotype, with the downregulation
of E-cadherin, upregulation of N-cadherin, and an upregulation of EMT markers Snail and Twist
63
(Sullivan et al., 2009). Other studies demonstrated that collagen type I, produced by pancreatic
tumors, influences the invasiveness of pancreatic carcinoma cells (Koenig et al., 2006; Shintani
et al., 2006). One study focused on the influence collagen type I had on disrupting the E-
cadherin adhesion complex within these cells which led to increased cell proliferation (Koenig et
al., 2006), and the other observed a response to collagen type I through signaling of JNK1
leading to an upregulation of N-cadherin and increased motility (Shintani et al., 2006). Other
factors have been implicated to have similar roles as well, including TGFϐ, reducing E-cadherin
expression in colon cancer cells (Bates and Mercurio, 2005). The implication of these and
additional microenvironmental factors being involved in regulation of cadherin molecules points
to multiple mechanisms impeding cell-cell adhesion in tumorigenesis.
4.5 Future Directions
4.5.1 The Mechanism of Influence of Cdh11 on Apoptosis and Invasion Potential
Cadherin levels have been heavily discussed in the literature as having numerous cellular effects
in both healthy tissue, developing tissue and in tumorigenesis. However, the direct link of
Cdh11 to apoptotic activity is rather novel. Generally, studies have looked at N-cadherin levels
and linked the loss of cadherin mediated cell-cell adhesion to an increase in apoptotic activity
(Erez et al., 2004). However, loss of E-cadherin in cancer has been shown to have the opposite
effect, and restoration of E-cadherin levels actually increased the levels of activated caspase-3
and caspase-8, sensitizing cells to apoptosis (Kippenberger et al., 2006). This study showed a
significant decrease of apoptosis upon the loss of Cdh11.
Cadherin molecules have numerous roles and huge impact in tissues and disease largely due to
their catenin binding partners, linking them to a wide variety of intracellular signaling pathways,
as well as their attachment to the cytoskeleton and their ability to bind to neighbouring cells and
64
interact with other cell surface molecules. These characteristics enable multiple pathways, which
may include cross-talk, to be severely altered upon loss or gain of these molecules. Additionally,
micro-environmental factors add in another level of endless possibilities to the effects of
cadherins on cellular processes. These are reasons why it would be interesting to take the next
step in evaluating how the loss of Cdh11 cell-cell adhesion actually influences apoptosis and
invasion potential in retinoblastoma. Because Cdh11 binds to various catenin molecules in order
to assemble the AJs, various pathways could be altered that cumulatively lead to our
observations. One idea is that the loss of Cdh11 would increase ϐ-catenin levels, leading to
increased Wnt signaling, transcription of target genes, and decreased apoptosis or even increased
invasion potential. Wnt proteins have been linked to apoptosis, through both ϐ-catenin
dependent and independent mechanisms, inhibiting it through downstream signaling and by an
increase in Bcl-2 expression (Almeida et al., 2005). My preliminary results looking at
expression of ϐ-catenin after Cdh11 knockdown indeed show an increase in both protein and
mRNA levels. Whether this would affect Wnt signaling or not needs to be determined. Studies
have shown increased level of ϐ-catenin does not always indicate activation of Wnt, as shown
recently in a study in osteosarcoma (Cai et al., 2009). Furthermore, even if Wnt signaling was
activated due to increased ϐ-catenin, we have to keep in mind a recent study showing that Wnt
signaling, while enhancing tumorigenesis in multiple cancers, has tumor suppressive properties
in retinoblastoma (Tell et al., 2006). In addition, it is possible other cellular pathways are
affected by the loss of Cdh11, and in turn, may be linked to the increase we see in expression of
β-catenin. Cellular pathways such as the PI3K/Akt have been linked to β-catenin stabilization
(Fang et al., 2007), and are suggested to have roles in various cancers (Agarwal et al., 2005;
Shukla et al., 2007). Furthermore, deregulations of this pathway have been linked to cell
survival [reviewed in (Song et al., 2005)]. It is also plausible that Cdh11 could be affecting the
65
levels of other molecules it interacts with like p120, known to interact with RhoGTPases. Upon
loss of cadherins, p120ctn accumulates in the cytoplasm, repressing RhoA and activating Cdc42
and Rac1, which modulate the cytoskeleton and increase migration and invasiveness
(Christofori, 2006; Stemmler, 2008). To further understand how Cdh11 is actually affecting
apoptotic activity and invasion potential, future studies have to be done. Experiments involved
in directly look at downstream targets of pathways such as Wnt signaling, as well as microarray
analysis after shRNA inhibition of Cdh11 would provide clues into this mechanism.
Understanding these mechanisms will give important insight into cadherin molecules and their
links to various cellular pathways, as well as insight into tumorigenesis of retinoblastoma and
other cancers, where similar pathways may be affected.
4.5.2 Mechanism of Cdh11 Downregulation in Retinoblastoma
It is thought that the loss of RB1 facilitates and acts as a prelude to genomic instability, linked to
the role of pRB in regulation of S-phase and mitotic progression (Knudsen et al., 2006). Thus, it
is plausible that the loss of 16q and Cdh11 is a consequence of genomic instability secondary to
RB1 loss. In order to further understand Cdh11 in retinoblastoma tumorigenesis, the next step is
to identify how it is lost. To date, the mechanism of Cdh11 loss is unknown, apart from a
reduction in copy number from 2 to 1. Previous work in our lab has suggested that the loss of
Cdh11 is a late event in retinoblastoma progression (Bowles et al., 2007; Dimaras et al., 2008).
Some insight can be taken from what has been seen as mechanisms for downregulation of E-
cadherin. Germline, somatic, and missense mutations identified in the E-cadherin gene have
been observed in a variety of cancers, including gastric and breast (Gayther et al., 1998; Hajra
and Fearon, 2002). Epigenetic modifications such as DNA hypermethylation of the promoter is
observed in numerous carcinomas (Hu et al., 2002; Zhao et al., 2007), and transcriptional
repression via Snail and Slug have been suggested (Halbleib and Nelson, 2006). Post-
66
transcriptional modifications such as phosphorylation of ϐ-catenin leading to disassembly of the
AJ (Hu et al., 2001), and cleavage through MMPs have also been implicated in enhancing
tumorigenesis and metastasis (Noe et al., 2001). To date, studies have shown some
hypermethylation in colon cancer cases (Braungart et al., 1999), but the promoter is not well
defined, so the mechanism of silencing is still unknown (Kashima et al., 1999; Zhou and Skalli,
2000). Some studies looking into the mechanism of loss of Cdh11 could include mutational
analysis within the gene, looking for somatic, germline or epigenetic modifications. Cytogenetic
studies could reveal translocations.
4.6 Summary and Significance
Our lab has contributed significantly to understanding tumorigenesis toward retinoblastoma. As
a proto-typical model of cancer, understanding the progression of retinoblastoma could have
profound implications in the study of cancer genetics and biology. In the recent years, our lab
has been successful in taking information from earlier genomic studies and defining candidate
genes that have oncogenic and tumor suppressive properties in retinoblastoma. My thesis
focused on defining the tumor suppressor role of Cdh11 in retinoblastoma progression. Earlier
evidence had supported the idea that Cdh11 acts as a tumor suppressor, and my work focused
around defining the specific mechanism in which it exerted this suppressive function. I found
that Cdh11 was promoting apoptosis within these tumors since when it was lost, a significant
decrease in apoptotic activity resulted, supported by both in vitro and in vivo analysis.
Furthermore, I saw no significant effect on proliferation, specifying the mechanism to promoting
apoptosis. I also explored the idea that since the loss of Cdh11 leads to faster growing tumors, if
they may be more aggressive and possibly lead to invasion. I showed the expression of various
invasion markers increased after specific knockdown of Cdh11. I also showed increased levels
of ϐ-catenin expression after Cdh11 knockdown, which opens the door for new studies to link
67
Cdh11 to apoptosis and invasion. Identifying and specifying the tumor suppressive role of
Cdh11 in retinoblastoma, will lead not only to further understanding of retinoblastoma
tumorigenesis, but also of the role of Cdh11, perhaps in other cancers. Additionally, this study
could lead to developing more ideas of how cadherin molecules in general affect different
cellular mechanisms, signaling pathways and tissue specific diseases. As a cell surface
molecule, Cdh11 has great potential for therapy consideration once its role and interactions in
tumorigenesis are fully understood.
68
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