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Nck is as an adaptor protein previously implicated in actin cytoskeleton reorganization and by our laboratory, in translation and cell response to stress. Our primary objective was to determine the expression levels of Nck isotypes (Nck-1 and Nck-2) during cancer progression. We have performed western blot analysis of the Nck isotypes expression levels profile in various human cancer cell lines, at different stages of progression. Our data show significantly higher expression levels of Nck-2 protein in metastatic melanomas compared to non-metastatic melanomas and normal melanocytes. Using semi-quantitative RT-PCR, we demonstrated that this increase in Nck-2 expression can be also seen at the transcriptional level. The Ras/RAF/MEK/ERK pathway is often spontaneously activated in melanomas causing hyperactivation of ERK. By downregulating the expression of Nck-2 using siRNA, we have established a strong correlation between increased expression levels of Nck-2 and activated ERK. Furthermore, we have demonstrated the involvement of Nck-2 in cell proliferation and adhesion in metastatic melanomas, revealing that Nck-2 acts as a new player in this disease.

Uveal Melanoma (UM) is the most common malignant intra-ocular tumor in adults. Despite the high accuracy of clinical diagnosis and advances in local treatment, more than 50% of UM patients develop metastasis within ten years of initial diagnosis. NM23 is a human metastasis suppressor gene. Reduced Nm23-H1 expression is correlated with high metastatic potential in a variety of different cancers including melanoma. C-Met is a receptor tyrosine kinase (RTK) that has been known to stimulate the invasive growth and increase the metastatic potential of cancer cells. Expression of c-Met is correlated with high mortality rate in UM patients. Treatment with CQX-2 inhibitors showed promise as an adjuvant therapy in adenocarcinoma of the colon. A previous report from our laboratory showed that topical treatment with Nepafenac (a CQX-2 inhibitor) delayed the progression of the primary tumor and the formation of metastasis in the experimental rabbit model of UM. / The purpose of this thesis is to investigate the expression levels ofNm23-H1 in UM cell lines with different metastatic potentials, in paraffin embedded tissues from primary tumors of UM patients, and in an experimental rabbit model. In addition, the aim of this thesis is to determine whether treating human uveal melanoma cell lines with Nepafenac would increase the expression levels of Nm23-H1 and decrease the expression levels of c-Met in vitro (UM cell lines) and in vivo (experimental rabbit model). / To achieve our goal, we used several types of assays in our UM cell lines and paraffin embedded tissues from patient samples and experimental rabbit model, including quantitative immunostaining, quantitative Real-time PCR, and small interference RNA (siRNA). / The Real-time PCR results of five human uveal melanoma cell lines showed that expression of Nm23-HI is higher in cell lines with low metastatic potential compared to those with high metastatic potential. The invasive ability of the uveal melanoma cell lines increased after silencing Nm23-H1 expression with siRNA. The increased immunostaining intensity of Nm23-H1 in patient samples is associated with better survival rate. Moreover, treatment with Nepafenac resulted in increase of Nm23-H1 levels and decrease of c-Met levels in both the UM cell lines and the experimental rabbit model. / In conclusion, Nm23-H1 is a potent prognostic marker to predict the survival rate of UM patients and it has the potential to identify high-risk patients. To the best of our knowledge, this is the first study to show that treatment with COX-2 inhibitor causes an upregulation of Nm23-H1 and downregulation of c-Met in UM. Therefore, treatment with COX-2 inhibitors may be a useful strategy as an adjuvant therapy for UM patients.

Uveal Melanoma (UM) is the most common malignant intra-ocular tumor in adults. Forty-five percent of UM patients develop metastasis within fifteen years of the initial diagnosis. Cutaneous Melanoma (CM) is a highly metastatic cancer that accounts for the majority of skin cancer deaths. Current treatments are not especially effective for the metastatic phase of the disease. Therefore, the identification of new molecular targets that can be exploited in the clinic are needed. / KISS1 is a putative human metastasis suppressor gene. The purpose of this study was to investigate the expression of KISS1 in melanoma and its potential value as a prognostic marker. / From results in vitro and in vivo we were able to characterize KISS1 in UM for the first time as well as its expression at the protein level, in CM. The correlation between KISS1 expression and UM survival rate suggests an important role for KISS1 as a prognostic marker in this tumor.

Uveal melanoma is the most common primary intraocular malignant tumour in adults. Despite improvements in the diagnosis and treatment of the primary tumour, patients continue to have the same mortality rate as several decades ago, reflecting our poor understanding of the mechanisms behind the formation of metastases in this disease. The purpose of this study was therefore to characterize an animal model of uveal melanoma and use this model to study the transcriptional changes that cells undergo from culture to intraocular tumour, to circulation and finally to the formation of a metastatic nodule. / Using microarrays we identified 314 changes in transcript abundance between the intraocular tumour and metastatic lesions. Principal Components Analysis was used to cluster these transcripts into four distinct groups. A further 61 gene transcripts showed statistically significant changes between re-cultured cells isolated from the model, with the circulating malignant cells representing an intermediate step between cells isolated from intraocular tumours and metastatic lesions. We have produced a detailed analysis of the molecular changes that take place as human uveal melanoma cells evolve from a primary tumour to metastasis in an animal model, including the decrease in expression of specific melanoma markers. These changes were verified using quantitative real time polymerase chain reaction and three different functional assays. / In addition we sought to describe the genetic changes that are present in these cells. Using comparative genomic hybridization arrays we were able to successfully describe the deletions and amplifications that are present in genomic DNA extracted from paraffin embedded sections of the primary tumour. This represents the first time that archival tissue has successfully been used for this sort of analysis in uveal melanoma. We identified several genomic amplifications and deletions including an area of amplification of Wnt2, which is involved in beta-catenin regulation and C-Met, which plays a role in tumour cell homing to the liver in patients. / To the best of our knowledge, this is the first time that a detailed genetic analysis has been carried out on the progression of uveal melanoma from intraocular tumour, to circulation, to the formation of metastases.

Uveal Melanoma is the most common primary intraocular tumor in adults. Despite the advances in numerous ophthalmic techniques leading to the increased accuracy of diagnosing this malignancy, the ten-year mortality rate for patients has remained unchanged at approximately fifty percent. Knowing this, further understanding of the specific steps that occur within the metastatic cascade of uveal melanoma is required. / Our laboratory utilizes five human uveal melanoma cell lines (92.1, SP6.5, MKT-BR, OCM-1, UW-1) of known proliferative, invasive, and metastatic potential. We used four methods to characterize the presence and roles of the chemotactic factors CXCL12, CXCL8, CXCL1 and HGF in these five cell lines. We also used a novel peptide inhibitor (TN14003) to block the biological action of CXCL12 on its receptor CXCR4. / With the results obtained from this thesis, we were able to establish the novel presence and importance of the four chosen factors for this malignancy. We were also able to display the positive effects TN14003 had on inhibiting uveal melanoma cell migration in vitro. This may lead to a future therapeutic target, which ultimately may delay or inhibit the metastatic process in uveal melanoma patients, improving the present unaffected ten-year mortality rate.

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