\SECTION{METHODOLOGY & SUMMARY}

\subsection{Experimental approach}

\subsubsectioni{Karyotype, CGH and QM-PCR}

One starting point to identify genes involved in cancer development or progression is to

investigate genomic changes in tumors. This will help pinpoint regions on the

chromosome that represent genomic instability and are potentially responsible for tumor

progression. Some common techniques used to detect genomic changes include

karyotype studies, CGH, arrayCGH, LOH, and quantitative RT-PCR \cite{Rakha2006a}.

Karyotypic studies typically reveal gross chromosomal changes amongst tumors, but

with very little resolution. Comparative genomic hybridization, microarray, and LOH

studies have all impacted research based on their ability to identify gene targets. CGH,

with a 10-20Mb \cite{Kallioniemi1992} \cite{Squire2003} of resolution, although

significantly better than karyotypic studies, is still not sufficient resolution for identifying

individual candidate genes. As a result, further evaluation of the region using STS

markers is required. One technique that we have used, QM-PCR \cite{Richter2003}, is

able to detect one allele vs. two alleles with 97% confidence \cite{Richter2003}, and

thus, is highly advantageous for identifying gene targets that may have potential tumor

suppressor/oncogenic roles in cancer. Development and use of this technique \

cite{Richter2003}\cite{Corson2005,Marchong2004,Orlic2006} has significantly aided our

advancement in the study and identification of gene candidates involved in

retinoblastoma development.

\subsubsection{Confirming Targets}

Although QM-PCR identifies genes based on copy number with great accuracy it

is necessary to test the relevance of such genes by evaluating their expression. We

discovered two potential targets, CDH11 and CDH13 based on our QM-PCR and LOH

studies. To determine the candidate gene(s) involved we I assayed for expression of

these proteins in primary retinoblastoma, retinoblastoma cell lines and tumors of the

mouse model, TAg-RB. Cadherin-11 demonstrated promising results, as it (CDH11-i)

was found to show loss be absent in the majority of tumors. Importance of expression

studies cannot be overstated as in our case cadherin-13 was shown to have no impact

in retinoblastoma development as its protein and mRNA expression did not change from

retina to retinoblastoma both in human and TAg-RB tumors. Thus, we I sought to

investigate CDH11 and identify its role in retinoblastoma development and progression.

\subsubsection{Common Experimental Approaches}

Typical in vitro techniques used to investigate genes implicated in tumor

suppressor function include gain and loss of function assays. These usually involve

overexpressing or inhibiting the gene of interest in tumor cell lines to evaluate cell

proliferation, arrest, death, and migration. Common techniques used for in vivo study

involve the use of a tumor mouse model. One strategy is to genetically manipulate the

tumor mouse model to evaluate the function of the gene of interest. Another strategy

involves the use of an immuno-deficient mouse to test the gene of interest on tumor

growth and metastatic ability.

\subsubsection{My Experimental Approach}

At first, weI planned to use in vitro assays to test the tumor suppressor ability of

CDH11 using retinoblastoma cell lines and a viral transduction procedure. However,

availability of Cdh11 knockout mice provided an excellent opportunity to investigate the

role of Cdh11 in the healthy murine retina. This also provided an attractive strategy, to

cross Cdh11 knockout mice with TAg-RB mice, to test the tumor suppressor ability of

Cdh11 by tracking tumor development in Cdh11+/- and Cdh11-/- genotypes. As a result,

I chose to use these mouse models to investigate Cdh11 in retina and retinoblastoma

development.

\subsubsection{Tumor Suppressor Gene Criteria}

Tumor suppressor genes are typically activated upon cellular stress or DNA

damage. They function to suppress proliferation, actively halting the cell cycle, so that

DNA repair can occur. As a result the cell is appropriately arrested to ensure that it

does not divide and produce cells that contain damaged DNA. Typically, tumor

suppressor genes are transcription factors that act directly on the cell cycle; however

other known tumor suppressor genes, example CDH1 act indirectly to impact the cell

cycle \cite{Rakha2006a}.

In order to evaluate CDH11 and investigate its role as a potential tumor

suppressor, it is necessary to have criteria with which to compare. Below is a list of

criteria based on the current literature of how a tumor suppressor gene is typically

defined \cite{Rakha2006a,Toyooka2002c}.

1. Mutations are identified within the gene. These can be somatic or germline

mutations or epigenetic modifications. Most tumors show LOH at the defined

locus. Tumor suppressor genes typically follow the two hit hypothesis

developed by Knudson (1971) \cite{Knudson1971}, which implies that loss of

both alleles that code for a particular gene are rate limiting for tumor

development.

2. Tumors must show loss function of the protein, either by loss of expression by

protein and/or mRNA.

3. The protein must exhibit the ability to suppress tumor growth when

functionally present and enhance tumor growth when functionally absent.

\subsection{My Key findings}

Based on analyses presented in the previous chapters we were able to fit three of the

four stipulated criteria. In Chapter 2, we discussed chromosomal region 16q22 was a

common region of loss in retinoblastoma samples. We further narrowed down the

region of loss to a minimal region of 1.62 Mb and identified two candidates whose STS

markers were most frequently lost. By describing frequent LOH at this locus in

retinoblastoma tumors, we fulfilled Criteria #2. Based on expression studies we were

able to focus on a single gene, CDH11, where its protein isoform, CDH11-I was shown

to have reduced expression or complete loss in many retinoblastomas \

cite{Marchong2004}. More recent studies of retinoblastoma show allelic loss of CDH11

in 45\% of 20 tumors \cite{Bowles2007}, confirming our candidate, CDH11 for a tumor

suppressor role in retinoblastoma. These results fulfill Criteria #3.

Although mutational studies were a priority at the beginning of our study, preliminary

analysis of tumors that were suggestive of mutations showed no change based on sizes

of PCR products; please refer to the similar technique performed by (ref). However,

thorough sequencing and mutational detection were not performed. Our more exciting

approach to evaluate CDH11 tumor suppressor function in retinoblastoma was to

examine tumor growth in TAg-RB mice with targeted loss of Cdh11. We are convinced

that we meet the final Criteria #4, since tumors of Cdh11+/- and Cdh11-/- mice displayed

faster growth than tumors of Cdh11+/+ mice.

\SECTION{POTENTIAL LIMITATIONS}

\subsection{Limitations of QM-PCR}

QM-PCR and LOH are common techniques used to study genomic copy number

change. LOH is by far the most widely used technique \cite{Rahka2006a}, since it

detects allelic loss at specific loci based on polymorphic microsatellite markers spaced

across the region. The downside to LOH is that it is dependent on constitutional cells of

the patient being heterozygous, and thus many samples are uninformative. As a result,

in our study of 16q loss, QM-PCR was used in conjunction with LOH. This proved to be

highly effective at detecting allelic loss. The limitation of QM-PCR is that it is highly

dependent on PCR, a technique that is subject to random variation in amplification. As

a result, repetition is a necessary component of QM-PCR, in order for the technique to

be highly rigorous. As well, another limitation is the comparison to an appropriate

‘normal’ which can sometimes present to be a challenge. In this case, the ‘normal’ is

located at chromosome 10q21, a region of the genome that is found to unaltered in

most retinoblastoma cases. However, since retinoblastoma tumors all show genomic

instability and although 10q21 rarely shows genomic instability, it remains possible that

this region would express gain/loss in some rare tumors. As a result, these limitations

must be taken into consideration when identifying gene targets. Probably the most

effective way of confirming targets are through expression studies.

\subsection{Limitations of expression studies}

Observation of mRNA or protein in tumor samples is essential for identification of

phenotype and roles of particular proteins in the retina. However, expression studies

have limitations that must be addressed when a conclusion is to be made. One concern

for reverse transcriptase-PCR (RT-PCR) is contamination. Since such a technique is

highly dependent on the quality of the RNA. It is necessary to evaluate the general

trend from a large number of samples to rule out conclusions made by contamination

issues. Another limitation of this technique is PCR, which has been previously

discussed.

In terms of protein, expression does not address function of the protein. Thus, it

can be expressed, but non-functional. Proteins can appear to be co-expressed in 2-D

images, but upon examination in 3-D or confocal images, they can be in two different

parts of the cell. As a result, co-localization and co-expression studies need to be

rigorous if one is to conclude that these two proteins function together. Contamination

is also a serious concern for immunoblotting. In this case, tumor samples can

potentially be contaminated with normal tissue upon harvest, providing a wrong

conclusion. The same is true for immunohistochemistry when preparation of tumor

samples have come in contact with normal tissue.

Several immunohistochemical studies were performed in this project. Repetition

of these studies cannot be overstated, since many factors can influence expression of a

protein on a fixed tissue sample. Such factors include how the sample was fixed, the

method used for antigen retrieval, the quality and concentration of the antibody used.

As a result, it is necessary that such results are reproducible under the same conditions

before a conclusion is made. Immunohistochemical studies outlined in this thesis were

rigorously performed and repeated at least three times before any conclusion was

established.

In this study we first assayed for protein and mRNA expression of both cadherin-

11 and cadherin-13 in a panel of retinoblastoma samples, cell lines and tumors of the

TAg-RB mouse model. Since contamination is concern, these results are based solely

on the confidence that the samples were clean. This is something that cannot be

tested, but a correct conclusion can be made based on the majority. Repetition of this

assay in another panel of tumors (increasing the sample size) and getting the same

result would provide sufficient evidence to conclude a trend. In preliminary studies,

assaying for cadherin-11 and cadherin-13 expression in retinoblastoma, we assayed

thirteen retinoblastoma cell lines and primary tumors and found a general trend for

cadherin-11(i) loss (Figure .. A) and no change for cadherin-13. This trend was

confirmed when we assayed another set of tumors (nine additional samples), bringing

our sample size to 22 retinoblastoma cell lines and primary tumors (Figure ..B).

The use of a ‘normal’ sample is a difficult issue for retinoblastoma studies. Since

retinoblastoma is a developmental disease, the perfect control is a fetal retina that is of

approximately the same age as the tumor sample. Such a tissue is nearly impossible to

obtain thus for most studies, adult retina or in some cases, a fetal retina (if available)

was used as a control. Thus, conclusions made must take into consideration the

‘normal’ it is compared against.

\subsection{Limitations of mouse studies}

The use of animal studies has been critical to our understanding of retinal

development, the mechanisms involved in retinoblastoma development and

progression, as well as treatment of the disease. It is obvious that these models are not

100% representative but animal models are the best available tools models to

understand the human disease. Manipulation of mouse models through transgenic,

inducible and conditional technologies is becoming a more promising approach to

recapitulation of the human disease \cite{Cespedes2006}, \cite{Vignjevic2007}. Since

no model is perfect, their have been numerous trials to produce a model that accurately

recapitulates retinoblastoma, see Pacal et al., (2006) for review.

Cdh11 knockout mice provided by Dr. Takeichi were essential to this project. We

were able to learn about the development of the retina in Cdh11-/- mice and its

littermates Cdh11+/- and Cdh11+/+. We were also able to use these mice to track

tumorigenesis when crossed with TAg-RB mice. This investigation was

fundamentalprovided evidence to our understanding of the tumor suppressor ability of

Cdh11 in retinoblastoma. To our advantage, we were able to learn double fold about the

role of Cdh11. We observed two roles for Cdh11: 1. the model provided us with the

understanding of cadherin-11 in is important in the development of the cell of origin of

retinoblastoma, 2. as well as itsCdh11 has a tumor suppressor role in retinoblastoma

progression.

As mentioned in chapter 1, this mouse model of retinoblastoma has its limitations

because of the interaction of small and large T-Antigens with unknown proteins making

the model harder to interpret. In addition to these well known limitations, this model

does not completely recapitulate the sequence of mutational events that we hypothesize

would occur in the human disease. Typically, the study tumor development and

progression in these mice would be evaluated after RB1 loss (initiation events). In this

case, an inducible model would be more beneficial as Cdh11 loss could be controlled

with the use of the inducible system. In using such a system, the effect of Cdh11 loss

can be evaluated at both an early and/or late mutational event after RB1 loss in

retinoblastoma development and progression. REFER TO AN EXAMPLE OF

INDUCIBLE LOSS OF TSG…IE MAYBE RB OR ANOTHER MODEL ETC….

\SECTION{16Q- REGION OF LOSS AND MECHANISM OF LOSS IN CANCER }

\subsection{16q loss and target genes}

As previously discussed in Chapter 1, chromosomal arm 16q is a hallmark for loss in

many cancer types \cite{Rahka2006a}. The literature is heavily populated with reports

on 16q loss in breast cancer, and so knowledge about genomic characterization of 16q

loss is mostly with respect to breast cancer.

Chromosomal arm 16q is frequently found to be involved in structural and numerical

abnormalities due to gross rearrangements like deletions, mitotic recombination,

monosomies and unbalanced translocations. The most common translocation of 16q is

with 1q, and this is frequently reported in breast cancers. As well, 16q harbors three

common fragile sites, FRA16B, FRA16C and FRA16D, many of which have been

shown to be involved in genetic alterations in cancer. Fragile site instability is thought to

contribute to the initiation and development of cancer, when genes at that region are

deleted, translocated or amplified. The most well-known fragile site at this region is

FRA16D, located at 16q23, which lies within the large WWOX gene. WWOX has been

identified as a tumor suppressor gene, lost in breast and ovarian cancers \

cite{Iliopoulos2006}. Chromosome 16q22 is one of the most frequent alterations in

breast cancer with reported LOH frequency in 30-75\% of cases (see review Rahka et

al., (2006) \cite{Rahka2006a}. In breast cancer, this genomic change is usually

reported as the sole genetic change or in combination with few other chromosomal

changes.

CDH1 has been identified as a tumor suppressor in that region, and has been shown to

have a substantial role in the invasive properties of breast cancer, however, it has been

suggested that there are other genes at this location that are at play in the development

or initiation of less aggressive breast tumors.

Potential candidate genes suggestive of tumor advantage in various cancer types at this

location include the WWOX gene, which is an essential mediator of tumor necrosis-

factor-alpha induced apoptosis \cite{Chang2002}, CDH11, CDH13, both of which are

cell adhesion molecules, E2F4, a member of the E2F family of transcription factors

involved in cell cycle, RBL2, homologue of p130 from the mouse genome and essential

for telomere length control in human fibroblasts \cite{Kong2006}, and c-Maf, which is a

member of the MAF family of transcription factors involved in terminal differentiation \

cite{Pouponnot2006}.

As mentioned previously, all retinoblastomas show genomic aberrations defined by

gains and losses. Such genomic instability is thought to drive tumor progression via the

generation of mutations in key genes, like tumor suppressor genes and oncogenes; in

this case, loss of 16q and the potential tumor suppressors located at this region.

Evidence suggests that loss of RB1 facilitates or acts as a prelude to genomic instability

\cite{Knudsen2006}. This is due to pRB ‘s role in the regulation of S-phase and mitotic

progression \cite{Knudsen2006}. Thus it is possible to suggest that loss of 16q and

subsequent inactivation of CDH11 is a consequence of RB1 loss. In the past we have

assumed this hypothesis to be true since we consider loss of RB1 the initiating event

and thus all other mutations follow. However, the prominent role of Rb in regulating the

cell cycle and maintaining genomic stability is critical for the mutations that follow.

\subsection{cadherins in cancer}

CDH11 regulation or mechanism of action during tumorigenesis is not well

studied, thus I will discuss other cadherins that have been heavily investigated in order

to hypothesize a mechanism for CDH11 loss in retinoblastoma.

\subsubsection{CDH1}

CDH1 (epithelial-cadherin), termed for its expression in epithelial tissues, is the

most heavily studied of the cadherins. Its expression has been shown to be reduced or

lost in a significant proportion of tumors from varying organs such as colon, stomach,

pancreas, esophagus, and liver \cite{Beavon2000}. The strongest evidence supporting

its role in the pathogenesis of cancer is the discovery of germline mutations in the

CDH1 gene in diffuse-type gastric cancer \cite{Gayther1998}\cite{Guilford1998}.

Somatic mutations have also been identified in cancers of the breast, endometrium, and

ovary \cite{Hajra2002} and cadherin-1 loss of expression has also been shown to be

due to epigenetic mechanisms via promoter hypermethylation \cite{Hajra2002}. As well,

it has been shown that loss of expression of cadherin-1 correlates with enhanced tumor

aggressiveness and dedifferentiation \cite{Beavon2000,Vleminckx1991}. These

observations taken together identify CDH1 as a tumor suppressor gene that plays a

prominent role in invasion and metastasis in many types of carcinomas \

cite{Gottardi2001,Perl1998}. To further define CDH11 as a tumor suppressor gene, it is

necessary to perform mutational analysis to identify how CDH11 in mutated in

retinoblastoma. Hypermethylation of the CDH11 promoter has never been reported,

however identification of polymorphic alleles have been described in some colon cancer

cases \cite{Braungart1999}.

Current work on CDH1 regulation describe transcriptional repression via zinc

finger proteins, Slug/Snail which function to bind directly to the CDH1 promoter to

repress transcription thereby increasing cell motility and invasion in tumors \

cite{Halbleib2006}. As a result, with the loss of cell-cell adhesion and release of bound

-cateinin from the cadherin-catenin adhesion complex, there is an increase in free -

catenin within the cytoplasmic pool. In this way, -catenin can perform its function in the

Wnt signaling pathway by complexing with TCF/LEF transcription factors and activating

proto-oncogenes c-myc and cyclin D1 \cite{Christofori2003}. In terms of CDH11

transcriptional regulation, the promoter is not well defined and it is unknown by what

mechanism it is silenced in tumors \cite{Braungart1999}\cite{Zhou2000)\

cite{Kashima1999}. However, it is possible that a similar strategy of suppression can be

occurring in retinoblastoma; a mechanism that remains to be characterized.

Other cadherins located at this region and implicated as tumor suppressor genes

include CDH13 (H-cadherin) for its hypermethylation in various epithelial cancers \

cite{Toyooka2001b} \cite{Toyooka2001} \cite{Sakai2004} \cite{Kawakami1999} \

cite{Toyooka2002c}; as well as CDH3 (Pcadherin) \cite{Mueller2002} \

cite{Smythe1999} \cite{Seline1996}.

\subsubsection{CDH2}

CDH2 (Neuronal-cadherin) is preferentially expressed in tissues of neuronal

origin, but also in the heart and somites. Although it is located at Chr. 18q11.2 and not

at our region of loss at 16q, it is interesting to mention because many reports correlate

its expression with CDH11 possibly due to its amino acid similarity (53%) \

cite{Hoffmann1995} and potential functional similarity. For example in cancers, during

epithelial to mesenchymal transition (EMT), where cadherin-1 is typically

downregulated, cadherins like cadherin-11 and cadherin-2 tend to be upregulated \

cite{Hajra2002,Yanagisawa2006}; this has been found to correlate with tumor motility,

invasion and metastasis and is typically seen in breast cancers \

cite{Hazan2000,Nagi2005,Pishvaian1999}, and gastric carcinomas \cite{Shibata1996}.

Cadherin-2 has also been investigated in retinoblastoma and described to have a high

expression in retinoblastoma samples, suggestive of a role in invasiveness \

cite{Mohan2007,Van2002}. It was not described or hypothesized the mechanism by

which cadherin-2 inflicts such a response, but it is possible that the invasive nature

could be attributed to the fibroblast growth factor receptor (FGF-R) site located at the

cytoplasmic tail of cadherin-2, which is involved in the mitogenic pathway. Considering

cadherin-11 is upregulated in various cancers through EMT transition, it may be

possible that cadherin-11 has binding partners similar to cadherin-2 involved in the

mitogenic process. Investigation of cadherin-11 binding partners would be a prudent

experiment to evaluate the mechanism by which cadherin-11 overexpression in cancers

correlates with invasive and metastatic properties.

\subsection{CDH11 in cancer }

A recent study by Gratais et al., (2007) also analyzed the 16q region using LOH,

conventional and array based CGH techniques with a panel of 58 retinoblastoma

tumors \cite{Gratias2007}. They defined a 5.7 Mb minimal region of loss at 16q24 and

described a complex pattern of LOH on 16q in 18/58 samples. One tumor showed LOH

at marker 16q24. They analyzed CDH13 at this region and found that it was not

mutated, based on sequencing of its 14 exons nor was it methylated, suggesting no

involvement in retinoblastoma, confirming our previous findings \cite{Marchong2004}.

They also show that markers close to CDH11 (CDH11 was outside their boundary of

analysis) displayed lower expression in retinoblastoma vs. retina, but none showed LOH

\cite{Gratias2007}. Since they did not test CDH11 directly, it would be interesting to

investigate CDH11’s expression in their panel of tumors to further corroborate our

findings.

Gratais et al, 2007 also correlate 16q allele loss with diffuse intraocular seeding. Recent

studies of mutational events in retinoblastoma suggest CDH11 loss to be a late event

based on frequency and correlation with other genomic changes \cite{Bowles2007}. As

a result, correlation of diffuse vitreous seeding and 16q loss would concur with our

hypothesis of retinoblastoma progression.

Cadherin-11 is also reported to be lost in a fraction of colon cancers (5/23) \

cite{Braungart1999} as well as in astrocytoma cell lines \cite{Zhou2000). Studies on

osteosarcoma and cadherin-11 are quite similar to what we find in retinoblastoma.

Kashima et al., (1999) show that cadherin-11(i) is lost in tumors and cadherin-11(v) is

expressed, with a few tumors showing very high expression \cite{Kashima1999}. Some

tumors also show expression of cadherin-11(s). These authors also describe loss of

expression of cadherin-11 and cadherin-2 in osteosarcoma cell line, Dunn, and

metastatic osteosarcoma cell line, LM8. They also showed that overexpression of

cadherin-11 and cadherin-2 in these cell lines resulted in an inhibitory effect on

migration in vitro and suppression of metastasis in an in vivo model \cite{Kashima2003}.

These observations implicate a role for cadherin-11 in osteosarcoma progression and

are suggestive of a similar mechanism occurring in retinoblastoma. It is possible for

these two cancer types to share this mechanism of CDH11 loss since they both involve

the mutations of the RB1 gene. It is not understood how osteosarcoma develops, but

mutations of both pRB and p53 have been found to play major roles in its development \

cite{Kansara2007} . Analysis of ostesosarcoma genomic aberrations shows gains at

1q, 4q, 5p, 6p, 7q, 8q, 14q and 19, and losses at 2q, 3p, 6q, 8p, 10p \cite{Squire2003};

with overlapping regions of gain at 1q and 6p, to retinoblastoma. Although chromosome

arm 16q is not reported to be lost in osteosarcoma, cadherin-11 is documented to be

lost in a majority of osteosarcomas. Thus, it is possible, that mutational events are

similar in both retinoblastoma and osteosarcoma and that loss of CDH11 may be an

important step in conferring tumor advantage.

Conversely, many reports describe expression of cadherin-11 in various cancers types,

like breast, prostate, rhabydosarcoma, Wilms’ tumor, ovarian, and stomach \

cite{Bussemakers2000,Markus1999,Pishvaian1999,Ramburan2006,Shibata1996}. In

breast cancer cadherin-11(i/v) is expressed in invasive cell lines but not in non-invasive

cell lines, suggesting a role for cadherin-11 in facilitating tumor cell invasion and

metastasis \cite{Feltes2002,Pishvaian1999}. As well, in rhabdomyosarcoma and

prostate cancer, cadherin-11 is highly expressed in tumor cells, but not in normal cells \

cite{Bussemakers2000,Markus1999}, suggesting a role for cadherin-11 in progression

of these cancer types.

An interesting correlation that I have noticed of cadherin-11 expression in various

cancer types is that cadherin-11, a mesenchymal cadherin, is lost mostly in sarcomas or

tissues of the brain where cells are loosely attached, whereas it is gained mostly in

carcinomas, where cells are more tightly attached. Expand on attachement theory like

epithelial tissue mor tightly attached than mesenchymal tissue. This interesting

correlation suggests that the varying roles for cadherin-11 are dependent on tissue type.

An attractive explanation mentioned previously for cadherin-11 expression in these

invasive cancer types, is the possibility of cadherin-11 isoforms binding growth factor

molecules similar to cadherin-2 and involving a mitogenic process. However, such a

study remains to be examined.

\SECTION{CADHERINS IN RETINA}

\subsection{Cadherins in retina}

Previous studies of cell adhesion molecules in the neural retina describe expression of

certain cadherin subtypes by restricted retinal cell populations. Based on these

expression studies authors have suggested roles for these molecules in maintaining

selective neuronal associations between cells \cite{Faulkner-Jones1999a,Honjo2000}.

Since at least eleven other cadherins have been reported to be involved in retinal

development \cite{Faulkner-Jones1999,Faulkner-Jones1999a}, it is highly possible that

Cdh11 loss is not enough to observe a retinal phenotype and postulate a function in the

development of the retina. As a result, Cdh11 may play a very small role in retinal

development that is missed by our experiments.

Cdh2 has been shown to be necessary for lens differentiation, retinal lamination, and

tissue patterning of the Zebrafish retina \cite{Erdmann2003,Malicki2003,Masai2003}.

Since cadherin-11 shares 53% amino acid similarity to cadherin-2 \cite{Hoffmann1995}

it is possible that it functions in a similar but more subtle way to cadherin-2. Based on

the observation that loss of cadherin-11 delays expression of T-antigen in TAg-RB mice

we suggest that cadherin-11 plays a role in the proper sorting, positioning or

differentiation of cell types during retinogenesis. Since our assays for aberrant

proliferation, cell death and cell type numbers were all negative; it remains possible that

cadherin-11 plays a very small role in retinogenesis.

CDH7 is a Type II cadherin located on murine Chr. 1q, outside the cadherins cluster at

mouse Chr.8. It has been evaluated in the retina and has shown strong expression in

the neural retina with restricted expression in neurons of the INL and GCL. Based on

observation of retinal phenotype with cell type markers it has been suggested to have

roles in the proper differentiation of amacrine and ganglion cell types \cite{Faulkner-

Jones1999}. We performed a similar approach when investigating Cdh11 in the retina;

however no gross phenotype was observed. Thus we are consistent with suggesting

that cadherin-11 plays a subtle role.

Cadherins are also known to be key molecules in controlling dendritic morphogenesis

and synapse formation in the nervous system. Tanabe et al (2006) describe that

cadherins are required for dendrite morphogenesis of horizontal cells and the

subsequent synapse between photoreceptors and in the vertebrate retina. \

cite{Tanabe2006}.

Considering that no role has been identified for Cdh11 in the development of the

mammalian retina as of yet, it remains possible that it does not play a role. However,

since other cadherins have been identified to have very specific roles in retinal

development, it also remains possible that we have not used the proper systems or

examined all the routes by which a role for Cdh11 would be defined. Given its

expression by horizontal and Müller gila cells, this is highly suggestive of a role in the

development of these cell types. Such as the proper migration, sorting, or positioning or

perhaps synapse formation of these cell types with corresponding cell types of the

retina. Further studies are needed to make an appropriate conclusion as to the role of

Cdh11 during murine retinogenesis.

A recent report by Johnson et al., (2007) describe early retinoblastoma tumors in their

murine model to exhibit neurite extension and synapse connections characteristic of

amacrine/horizontal cell types in the retina. They describe late tumors to have lost their

differentiation and exhibit rosettes but no synapses \cite{Johnson2007}. Considering

that cadherins are implicated in synapse formation, we can suggest that cadherins play

a role in early retinoblastoma development. In light of the highly antagonistic roles of

cadherins in cancer, one hypothesis is that since CDH2 is upregulated in

retinoblastoma, it may play an important role in early retinoblastoma development and

synapse formations and when the tumor grows larger, loss of CDH11 becomes

advantageous to the tumor in development of its mobile and invasive nature. These

hypotheses can easily be explored in our TAg-RB mouse model by assaying for

expression of these proteins in early and late tumors.

\SECTION{CURRENT AND FUTURE DIRECTIONS}

\subsection{CDH11 function in Retinoblastoma}

\subsubsection{CDH11 mutation identification}

To date, the mechanism of CDH11 loss and its role in tumorigenesis is unknown.

Insights and clues into the tumor suppressor role of CDH11 was provided by Kashima et

al (1999) where they showed overexpression of cadherin-11 and cadherin-2 in

osteosarcoma cell lines to result in decreased cell motility in vitro and suppression of

metastasis in vivo. In our study, we suggest Cdh11 to be a tumor suppressor since loss

of either one allele or both alleles of Cdh11 in TAg-RB mice lead to faster growing

tumors than mice with normal Cdh11. The first step in continuing to examine CDH11 in

tumorigenesis is to identify how CDH11 expression lost. Such studies include

investigating mutations within the gene like somatic, germline or epigenetic mutations

like DNA hypermethylation.

\subsubsection{in vitro and in vivo studies to test tumor suppressor role}

The next step to studying Cdh11 in retinoblastoma tumorigenesis is to confirm our study

with a proper system since in our study Cdh11 loss affected TAg expression and

delayed tumor development from the start. Since we assume CDH11 to be a later

mutational event, the ideal mouse experiment to assay Cdh11’s role in tumorigenesis

would be the use of a conditional mouse model where Cdh11 loss could be induced

after RB1 loss. Although there are currently many mouse models of retinoblastoma, it is

important to use a mouse model, where Cdh11 does not have a developmental role (like

TAg-RB); in this way the study would only be relevant to its role in tumor development.

In using a conditional system the sequence of mutational events are somewhat kept

conserved. To further assess Cdh11 role in retinoblastoma progression, analysis of

timepoints later than 3 months of tumor development is necessary. In this way, we

could assay for Cdh11 involvement in metastasis and aggressiveness of tumor growth

compared to mice with normal Cdh11.

Another in vivo technique commonly used to study tumor suppressor gene function is a

tumorgenicity assay using a severe combined immuno-deficient (SCID) mouse. In this

assay, tumors (tumor cell lines manipulated expressing cadherin-11 and not expressing

cadherin-11) can be tested for the effect of cadherin-11 on growth rate and metastatic

ability. Several reports have outlined the successful use of transplanting retinoblastoma

tumors subcutaneously (Yan, EJC, 2000; Cowell, EJC, 1997).

Alternatively, in vitro experiments are helpful in understanding the effect of cadherin-11

in retinoblastoma cell lines. Together with the overexpression and inhibition of cadherin-

11 in retinoblastoma cell lines, we could assay for its role in cell migration and

invasiveness. All of these experiments would help us confirm CDH11 tumor suppressor

role in retinoblastoma.

The next step is to identify the mechanism by which CDH11 loss contributes to

tumorigenesis. This is a harder question to answer since the molecules and pathways

involved in maintaining proper cellular adhesion are still being revealed (as noted in

Chapter 1). Molecules like -catenin and p120 catenin, which are components of the

cadherin-catenin cell adhesion complex, both bind several proteins that are involved in

other pathways that promote cell motility and invasion. Investigation of these proteins

and their pathway proteins together with cadherin-11 loss would help identify how

cadherin-11 loss contributes to tumor progression. Since CDH1 is the most heavily

investigated, current study of CDH1 loss in tumorigenesis provides a place to start.

Investigation and definition of the CDH11 promoter would also help us understand what

molecules bind to it and regulate its transcription. As well, study of CDH11 regulation

both in tumor samples as well as normal samples would aid our understanding in the

mechanism by which CDH11 loss contributes to increased tumorigenesis.

\subsection{ CDH11 function in ‘EMT’ cancers}

Experiments described above would help identify a role for CDH11 in tumorigenesis

when it is lost. But what about its role or mechanism of action in cancers where EMT

occurs and cadherin-11 is heavily expressed? In this case, study of CDH2 has lead to

some interesting facts. Cadherin-2 has been shown to bind and directly activate

fibroblast growth factor receptor (FGFR), activating the mitogenic pathway (MAPK-ERK)

\cite{Halbleib2006}. More recently, the soluble form of cadherin-2 has been shown to

interact with FGF-R and stimulate migration \cite{Derycke2006}. Since cadherin-11 is

the most closely related to cadherin-2 and has been shown to be upregulated together

with cadherin-2 in various cancers, it would be interesting to test if the isoforms of

cadherin-11 act in a similar manner to cadherin-2, binding molecules involved in

mitogenic pathways. Reports in breast cancer and osteosarcoma have already

indicated that cadherin-11(v) is upregulated. As well, in our study of retinoblastoma, we

have also seen expression of cadherin-11(v/s) in a few samples. Evaluation of the

binding partners of cadherin-11 and its isoforms would help us understand how

expression of cadherin-11 correlates with invasive properties in these cancers.

\subsection{CDH11 developmental role in TAg-RB mice}

The role of CDH11 in the delay of TAg-expression in TAg-RB mice remains unknown.

Since other cadherin molecules have been shown to be important for tissue patterning,

differentiation of cell types and synapse formation, it is possible to suggest a role for

CDH11 in either of those functions. Evaluation of such roles would require more than

simply examining CDH11-/- mice as previously performed, since no gross role was

observed and we suggest that the role for CDH11 is a subtle one. A more careful

evaluation of the subtypes of horizontal and Müller glia cells in CDH11-/- mice may help

us understand whether or not CDH11 has a role in the maintenance/development of

these retinal cell types.

\subsection{mutational events in reitnoblastoma}

Mutational events described in retinoblastoma are pertinent to our understanding of how

tumor develops and progresses. This knowledge is important for the development of

any kind of prognostics or treatment for the disease. To ensure that the mouse model is

most similar to human retinoblastoma in terms of mutational events it is necessary to

perform a CGH study on murine retinoblastoma, similar to what was performed in

human retinoblastoma samples. Findings of this study would be important to the study

of retinoblastoma since similar events occurring in both mouse and human further

recapitulates the human disease and thus provides a more relevant and useful ‘model’

for studying the disease. If similar events are indeed present between human and

murine retinoblastoma, development of therapeutics based on these mutational events

would be the primary target for pre-clinical use of the murine model.

Another query is the order of mutational events. We assume that loss of both RB1

alleles are initiating events but how do we determine the order of the M3-Mn mutational

events? Bowles et al., (2007) have already suggested an order, based on the

frequency and correlation with other genomic changes. Obviously such order of

mutational events is based on the majority, however it should be stated that different

tumors can exhibit different mutational events, in a different order and thus it is possible

to suggest that CDH11 loss could be an early mutational event. Such tumors may

present to be the more aggressive and faster growing with extensive vitreous seeding at

an earlier age compared to the majority of cases. However, examination of early tumors

and late tumors would be an excellent opportunity to identify a general trend of early

and late mutational events. This study could be easily performed in the mouse model

and be related to human retinoblastoma.

\SECTION{CONCLUSION AND SIGNIFICANCE OF THIS WORK}The study of retinoblastoma, because it is the proto-typical model of cancer, has

profound implications to the understanding of cancer genetics and cancer biology.

Identifying and understanding the sequence of mutational events in this cancer are

essential to the development of new and better therapeutics for its treatment. This lab

has investigated the chromosomal regions that have shown instability in retinoblastoma

tumors. Further, we have identified genes at these gross chromosomal regions that are

potentially advantageous for tumor growth. This thesis focused on the gene CDH11 at

16q22 and its role in retinoblastoma progression. We suggest that CDH11 is tumor

suppressor in retinoblastoma however more work is needed to confirm our findings and

identify mutations within this gene that render it non-active. With this knowledge and

the knowledge of the other genes, we were able to hypothesize a sequence of events

that are potentially involved in tumorigenesis. As 16q loss is always accompanied by 1q

gain and 2p or 6p gain, and considering the function of the genes at these regions, it

has been proposed that 1q gain would be the first mutational event, after RB1 loss,

followed by 6p gain and then 16q loss or 2p gain. Using this knowledge clinicians can

potentially assay for retinoblastoma progression and therapeutic targets can be

developed against these genes at the varying stages for treatment of the disease. This

knowledge is already being put to use, as currently these mutational events described in

Bowles et al., (2007) are being used as a ‘retinoblastoma profile’ to examine tumor

samples where the RB1 mutation has not yet been identified (about 11% of cases) \

cite{Richter2003}. If the retinoblastoma profile of mutational events for the tumor

sample is not achieved, the tumor is suggested to be non-retinoblastoma tissue. Such

translation, although small, from our lab research to the clinic is an exciting and

important step in the goal towards curing retinoblastoma patients.

Table 1. Cadherin-11 gain/loss of expression in cancer

Expression of Cadherin-11

Cancer Type Reference

Loss Astrocytoma Zhou et al., 2000

Loss Osteosarcoma Cheng et al., 1998

Kashima et al., 1999

Kashima et al., 2003

Loss Colon Munro et al., 1995

Braungart et al., 1999

Morimoto et al., 2004

Gain Breast Pishvaian et al., 1999

Nieman et al., 1999

Feltes et al., 2002

Nagaraja et al., 2006

Gain Prostate Tomita et al., 2000

Bussemakers et al., 2000

Gain Rhabydosarcoma Markus et al., 1999

Gain Wilm’s Tumor Shulz et al., 2000

Ramburan et al., 2006

Gain Gastric Cancer Shibata et al., 1996

Braungart et al., 1999

Ovarian Sario et al., 2006

Davidson et al., 2006

Fusion protein Aneurysmal

Bone Cyst

Oliveira et al., 2004