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From previous studies it has been demonstrated that the fusion of tumor cells with antigen-presenting cells generates hybrids that are known to induce anti-tumor immunity. With the advancement of scientific research and medicine, the need to produce cell-cell hybrids for cancer immunotherapy and for various other applications is substantial. Among the many methods used to generate these hybrid cells, electrofusion is a technique that is more widely used and recognized as a method to efficiently produce hybrids. Electrofusion requires two steps. In the first step, cells are brought into close adjacent contact either by a mechanical method like centrifugation or by dieletrophoresis using alternating current (AC). The second step includes the reversible breakdown and fusion of cell membranes induced by high voltage direct current (DC) pulses. The goal of this investigation was to study the use of electrical charge to bring cells into close contact with one another in the cell contact stage prior to delivering high voltage fusion pulses. The possibility of achieving considerable cell-cell contact was tested in two separate electrical systems. In the first system B16 murine melanoma cancer cells were subjected to a range of direct current (DC) voltages between 4 V/cm and 40 V/cm. With the use of DC from a small power source the response of the cells was tested in multiple fusion chambers consisting of two or four electrodes. The configurations of the chambers were varied by changing the distance between the electrodes, the thickness, material and type of coating on the electrodes. In the second system the movement of cells in the presence of corona charge was studied. B16 cells in a culture dish were confined by a circular grounded electrode and subjected to corona discharge for known periods of time. Application of corona charge (positive or negative) facilitated the contact of cells in the annular region between the two circular electrodes. After series of tests, final designs for fusion chambers to be used with DC and with corona were developed. Cell contact achieved with the DC fusion chamber was not substantial enough to produce a significant amount of fusion yield. The fusion chamber designed to be used with corona on the other hand produced exceptional cell contact results consequentially generating fusion yields as high as 40%.

Cancer represents the second leading cause of death in the United States. For many malignancies, currently available treatment options offer little long-lasting survival benefits to patients. However, recent studies have shown immunotherapeutic approaches to be an attractive strategy to cancer treatment. While many current immunotherapeutic strategies convey durable responses, such responses are only seen in a minority of patients. An increased understanding of the mechanisms governing tumor immunogenicity and the biology of immune responses is crucial to improving upon the efficacy of current and future cancer immunotherapies. Histone deacetylases (HDACs), enzymes classically associated with regulation of gene expression, have been therapeutic targets in various cancers for several years due to their involvement in cell growth. However, it has become increasingly clear that HDACs are intimately involved in regulating both the immunogenicity of tumor cells and immune response of leukocytes and lymphocytes. In order to expand upon this growing knowledge, the therapeutic efficacy of the pan-HDAC inhibitor LBH589 in the treatment of melanoma was studied. The results presented here demonstrate that LBH589 is a potent inhibitor of growth in a wide variety of melanomas through induction of cell cycle arrest and apoptosis. Additionally, LBH589 increases the immune visibility of melanoma cells by increasing expression of several immune associated cell surface markers (e.g. MHC I, MHC II, CD80, CD86) in addition to upregulating expression of melanoma differentiation antigens. Furthermore, LBH589 treatment of immune cells results in an enhanced pro-inflammatory phenotype of both APCs and T-cells. These combined effects result in better activation of T-cells and ultimately prolonged survival in LBH589 treated, melanoma-baring mice. To further the understanding of the role of individual HDACs in the T-cell response, the biology of the newest HDAC, HDAC11, was further assessed. To this end, it is shown that HDAC11 is differentially expressed in T-cell populations, and expression is rapidly decreased following activation. Utilizing an HDAC11 knockout (HDAC11KO) mouse strain, it is found that both CD4+ and CD8+ T-cells lacking HDAC11 have an enhanced type 1 effector function characterized by increased proliferation and secretion of IL-2, TNF and IFN-γ. Additionally, HDAC11KO CD8+ T-cells have increased expression of both granzyme B and perforin. HDAC11KO T-cells also demonstrate enhanced resistance to inhibition by Tregs and anergy formation. As a possible mechanism for the observed phenotype, it is also demonstrated that HDAC11KO T-cells produce elevated levels of the transcription factors Eomes and T-bet, both at the basal state and post-activation. In vivo, T-cells lacking HDAC11 have a more potent and robust ability to cause GvHD and mediate an enhanced anti-tumor response. Collectively, these results demonstrate that targeting of HDACs is a viable approach to cancer immunotherapy, and that targeting of specific HDACs may be an attractive strategy for optimizing immunotherapy efficacy while minimizing side effects.

Many cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic. / text

Skeletal muscle (SkM) possesses an impressive ability to regenerate in response to injury or chronic disease. This regenerative capacity is attributed to its resident mononuclear myogenic progenitors. Previous studies have identified several types of myogenic progenitors within SkM, some of which are isolated by fluorescence activated cell sorting (FACS) using cell surface markers. Studies in our laboratory have identified melanoma cell adhesion molecule (MCAM) as a cell surface marker expressed by myogenic progenitors in human fetal SkM. However, the relationship between MCAM expression and the degree of myogenic commitment of distinct MCAM+ populations has not been elucidated. In the present study, subpopulations of MCAM+ cells were purified by FACS on the basis of Hoechst 33342 dye uptake. Specifically, MCAM+ side population (SP) was isolated by Hoechst exclusion and MCAM+ main population (MP) on Hoechst incorporation. Sorted populations were first optimized for growth in vitro since SkM SP cells are difficult to maintain in culture. In particular, Invitrogen’s StemPro® MSC SFM medium was found to support propagation of human fetal SkM SP cells with minimal differentiation. Following this optimization, sorted populations were assessed for expression of myogenic markers before and after propagation and then for fusion potential in vitro and engraftment potential in vivo. The MCAM+ subpopulations were found to express myogenic markers to a significantly greater extent than MCAM- subpopulations. Furthermore, the MCAM+ subpopulations fused robustly into myotubes in vitro whereas the MCAM- subpopulations did not. Interestingly, the MCAM+ SP population exhibited the highest fusion potential in vitro and was the only MCAM+ subpopulation to engraft into dystrophic muscle in vivo following propagation. These results indicate that MCAM is associated with myogenicity and can be used to prospectively isolate a pure myogenic fraction from human fetal SkM tissue. Moreover, the MCAM+ SP retain its myogenic potential to a greater extent than MCAM+ MP after propagation. This suggests that the MCAM+ SP fraction contains a higher percentage of early myogenic progenitors compared to the MCAM+ MP fraction. Additional studies on MCAM-expressing populations in human fetal SkM may elucidate a potent population for use in cell-based therapeutic strategies for treating muscle diseases.

Το κακόηθες μελάνωμα του δέρματος είναι το αποτέλεσμα της κακοήθους εξαλλαγής των μελανοκυττάρων της επιδερμίδας και χαρακτηρίζεται απο συνεχώς αυξανόμενη επίπτωση και θνησιμότητα παγκοσμίως. Η αξιοσημείωτη δε ανθεκτικότητα που επιδεικνύει το προχωρημένο μεταστατικό μελάνωμα στα χημειοθεραπευτικά σχήματα και στην ακτινοθεραπεία κάνει επιτακτική την ανάγκη για νέους, πιο αποτελεσματικούς θεραπευτικούς παράγοντες. Ένας αυξανόμενος όγκος δεδομένων υποστηρίζει τη παρουσία και ενεργό συμμετοχή καρκινικών κυττάρων με ιδιότητες stem κυττάρων (cancer stem cells, CSCs) στην ανάπτυξη και μετάσταση του μελανώματος. Οι μεταγραφικοί παράγοντες EZH2, SOX2 και Oct4 αποτελούν μόρια – κλειδιά στον έλεγχο του ρυθμιστικού δικτύου του stemness των εμβρυϊκών stem κυττάρων (ESCs). Είναι πλέον γνωστό ότι η χρωματίνη στα ESCs περιλαμβάνει περιοχές με «αντιμαχόμενες» τροποποιήσεις ιστονών (bivalent domain), οι οποίες φυσιολογικά σχετίζονται είτε με ενεργή (Η3Κ4me3) ή με ανενεργή κατάσταση της χρωματίνης (H3K27me3), ενώ η απορρύθμιση των επιγενετικών μηχανισμών ελέγχου σε συγκεκριμένους γονιδιακούς τόπους έχει συσχετισθεί με τη καρκινογένεση στον άνθρωπο. Σημαντικό ρόλο στη ρύθμιση αυτών των bivalent domain φαίνεται να έχουν οι πρωτεΐνες της οικογένειας Polycomb, και ιδιαίτερα ο EZH2 που δρα σαν μεθυλοτρανσφεράση στην επιγενετική τροποποίηση H3K27. Πρόσφατα, μια σειρά από μελέτες έδειξαν ότι CScs στη διηθητική παρυφή του όγκου ενδέχεται να συμμετέχουν ενεργά στη καρκινογένεση. Επιπλέον, η ανακάλυψη ότι η βιολογική διαδικασία της επιθηλιο-μεσεγχυματικής μετάβασης (EMT) οδηγεί υποπληθυσμούς καρκινικών κυττάρων εντός του όγκου να αποκτήσουν ιδιότητες stem κυττάρων, φαίνεται να αποτελεί τον σύνδεσμο μεταξύ μετάστασης και κατάστασης πολυδυναμίας (stemness). Σύμφωνα λοιπόν με τη θεώρηση αυτή, είναι πιθανό τα CSCs να εντοπίζονται κυρίως στη διηθητική παρυφή ενός όγκου, ενώ επιπλέον οι ιδιότητες stem κυττάρων που έχουν αποκτήσει είναι το αποτέλεσμα κυρίως της ΕΜΤ. Σε αυτό το πλαίσιο, παρουσιάζει επομένως εξαιρετικό ενδιαφέρον η προσεκτική και στοχευμένη εκτίμηση της ανοσοϊστοχημικής έκφρασης παραγόντων που σχετίζονται με τα stem κύτταρα στην διηθητική παρυφή του μελανώματος, και αυτός ήταν ένας από τους στόχους της παρούσας διαδακτορικής διατριβής. Σκοπός. Ο σκοπός της παρούσας διδακτορικής διατριβής είναι η μελέτη της έκφρασης των μεταγραφικών παραγόντων EZH2, Oct4, SOX2 όπως επίσης και την παρουσία των επιγενετικών τροποποιήσεων H3K4me2 and H3K27me3 (bivalent domain) στο κακόηθες μελάνωμα του δέρματος. Παράλληλα, μελετήθηκε η πιθανότητα αναγνώρισης και στοχοποίησης καρκινικών κυττάρων με ιδιότητες stem κυττάρων, με ιδιαίτερη έμφαση στη διηθητική παρυφή του όγκου. Υλικό και μέθοδος. Το ποσοστό κυττάρων με ανοσοθετικότητα για τα αντισώματα έναντι των μεταγραφικών παραγόντων EZH2, SOX2 και Oct4 όπως επίσης και των επιγενετικών τροποποιήσεων H3K4me2 and H3K27me3 εκτιμήθηκε σε 89 δείγματα ιστών από 79 ασθενείς με κακόηθες μελάνωμα 250 του δέρματος, εφαρμόζοντας τη μέθοδο της ανοσοϊστοχημείας. Για την επιλογή των κατάλληλων δειγμάτων έγινε ανασκόπηση των αρχείων του εργαστηρίου Παθολογικής Ανατομικής του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών των ετών 2001 έως 2010. Από αυτό το σύνολο των 89 δειγμάτων τα 70 αφορούν πρωτοπαθές μελάνωμα δέρματος, ενώ τα υπόλοιπα 19 προέρχονται από υλικό που εξαιρέθηκε κατά τη χειρουργική εκτομή του μεταστατικού μελανώματος. Επιπλέον 14 δείγματα περιείχαν εκτός από καρκινικά κύτταρα μελανώματος και κύτταρα σπίλων. Στην παρούσα μελέτη χρησιμοποιήθηκε το σύστημα ανίχνευσης EnVision (Envision, Dako, USA) ή MACH4 Universal HRP-Polymer Detection (Biocare Medical, USA) και πρωτογενή αντισώματα έναντι των EZH2 (Novocastra Laboratories Ltd, UK), SOX2 (R&D Systems, Inc.), Oct4 (Santa Cruz Biotechnology, Inc), H3K4me2 (Cell Signaling Technology, USA) και H3K27me3 (Cell Signaling Technology, USA). Σε κάθε περιστατικό και για κάθε δείκτη εκτιμήθηκε το ποσοστό των καρκινικών κυττάρων που εμφάνιζαν θετική ανοσοχρώση (Labeling Index, LI). Η καταμέτρηση των θετικών κυττάρων πραγματοποιήθηκε σε μεγάλης μεγέθυνσης πεδίο (400X). Η στατιστική ανάλυση έγινε με τη χρήση του SPSS στατιστικού πακέτου (SPSS©, Release 19.0). Τιμές p<0.05 θεωρήθηκαν ως στατιστικά σημαντικές. Αποτελέσματα. Πυρηνική χρώση ανιχνεύθηκε για τα αντισώματα έναντι των EZH2, H3K4me2 και H3K27me3, ενώ αντίθετα βρέθηκε πυρηνική και κυτταροπλασματική έκφραση για τους παράγοντες SOX2 και Oct4. Παρατηρήθηκε ανομοιογενές προφίλ ανοσοθετικότητας στα κύτταρα μελανώματος με σημαντικά αυξημένο ποσοστό καρκινικών κυττάρων με θετική ανοσοχρώση H3K4me2 και H3K27me3 στη διηθητική παρυφή του όγκου. Αντίστοιχη τάση για αυξημένη έκφραση έδειξε και ο μεταγραφικός παράγοντας EZH2, χωρίς όμως η διαφορά να είναι στατιστικά σημαντικά, ενώ παρατηρήθηκε και σε ορισμένες μεμονωμένες περιπτώσεις με τον SOX2. Όσον αφορά τον ΕΖΗ2, βρέθηκε σημαντική αύξηση των επιπέδων του παράγοντα στα κύτταρα μελανώματος σε σχεση με τα σπιλοκύτταρα (p=0.02). H πυρηνική έκφραση SOX2 ήταν σημαντικά υψηλότερη στα κύτταρα μελανώματος σε σχέση με τα κερατινοκύτταρα της βασική στιβάδας (p=0.02), όπως επίσης και στα σπιλοκύτταρα συγκριτικά με τα κερατινοκύτταρα της βασικής (p=0.0016) και της υπερβασικής στιβάδας (p=0.027). Η συσχέτιση της πυρηνικής έκφρασης με διάφορες παραμέτρους των ασθενών έδεξε υψηλότερα επίπεδα SOX2 στα πρωτοπαθή σε σχέση με τα μεταστατικά μελανώματα (p=0.045), στα κύτταρα μελανώματος με πάχος όγκου κατά Breslow <1mm (p=0.023), χωρίς εξέλκωση (p=0.009) και με αριθμό μιτώσεων ≤6 μιτ/mm2 (p=0.016). Τα επίπεδα πυρηνικής έκφρασης Oct4 βρέθηκαν υψηλότερα στα σπιλοκύτταρα σε σχέση με τα κερατινοκύτταρα (p<0.001) αλλά και με τα κύτταρα μελανώματος (p=0.004). Η μελέτη ωστόσο της κυτταροπλασματικής ανοσοθετικότητας έδειξε σημαντική μείωση των επιπέδων Oct στα κύτταρα μελανώματος σε σχέση με τα κερατινοκύτταρα της υπερβασικής στιβάδας (p<0.001), όπως επίσης στα μεταστατικά σε σχέση με τα πρωτοπαθή μελανώματα (p=0.025). Αξιοσημείωτο είναι επίσης ότι το προφίλ έκφρασης του Oct4 βρέθηκε σε ορισμένες περιπτώσεις να αυξάνεται τοπικά στα ενδοθηλιακά κύτταρα αγγείων εντός των μελανωμάτων. Τα μελανώματα με υψηλό επίπεδο διήθησης κατά Clark (IV-V) (p=0.038) ή μεγάλο 251 πάχος όγκου κατά Breslow (>1mm) (p<0.001) εμφάνισαν χαμηλότερο ποσοστό καρκινικών κυττάρων με ανοσοθετικότητα για το αντίσωμα H3K4me2 σε σχέση με τους όγκους με μικρότερο βαθμό διήθησης. Επιπλέον, παρατηρήσαμε ότι οι μεταστατικοί όγκοι είχαν χαμηλότερα ποσοστά θετικής ανοσοχρώσης και για τις δύο επιγενετικές τροποποιήσεις, H3K4me2 και H3K27me3, συγκριτικά με τους πρωτοπαθείς όγκους (p=0.0065 και p=0.027 αντίστοιχα). Τέλος, η ανάλυση της παράλληλης ανοσοϊστοχημικής έκφρασης στα κύτταρα μελανώματος έδειξε θετική συσχέτιση των δύο επιγενετικών τροποποιήσεων (p<0.01), όπως επίσης και μεταξύ του EZH2 και της επιγενετικής τροποποίησης H3K27me3 (p=0.03). Ισχυρή συσχέτιση βρέθηκε παρομοίως μεταξύ των επιπέδων έκφρασης Oct4 και SOX2, τόσο για την πυρηνική όσο και την κυτταροπλασματική εντόπιση (p<0.001 και p<0.001 αντίστοιχα). Συμπεράσματα. Λαμβάνοντας υπόψη τη λειτουργική σημασία των τριών υπό μελέτη μεταγραφικών παραγόντων και του ρόλου των επιγενετικών μηχανισμών στην καρκινογένεση, τα ευρήματα μας εισηγούνται ότι οι ΕΖΗ2, SOX2, Oct4 όπως επίσης και οι επιγενετικές τροποποιήσεις Η3Κ4me3 και H3K27me3 αποτελούν εν δυνάμει δείκτες καρκινικών stem κυττάρων στο κακόηθες μελάνωμα, και ιδιαίτερα στη διηθητική παρυφή του όγκου. Η υπόθεση αυτή πρέπει να διεριευνηθεί περαιτέρω, καθώς θα μπορούσε να αποτελέσει, σε συνδυασμό και με άλλες μελέτες, ένα μικρό βήμα προς τη κατεύθυνση της στοχευμένης αντικαρκινικής θεραπείας του μελανώματος στα πλαίσια της Ιατρικής του μέλλοντος. / Cutaneous malignant melanoma originates from melanocytes and is characterized by constantly growing incidence and mortality rates world-wide. The substantial unresponsiveness of advanced metastatic melanomas to most forms of chemotherapy and radiation indicates an urgent need for more effective agents to overcome chemoresistance. Accumulating evidence strongly suggests the presence and involvement of cancer cells with properties of stem cells (CSCs) in the initiation, progression and metastasis of malignant melanoma. EZH2, SOX2 and Oct4 represent crucial components of the reciprocal regulatory circuit that controls stemness. Genome-wide analyses of chromatin states of embryonic stem and progenitor cells suggest a ‘bivalent’ colocalization of the activating H3K4 methylation and the repressive H3K27me3 in development-associated genes, while the misregulation of histone modifications on specific residues actively contributes to human cancer. PcG proteins and mainly EZH2 are responsible for maintaining the balance of the bivalent chromatin domain through the methylation of H3K27. Recently a number of studies have shown that cancer cells with properties of stem cells at the tumor invasion front might be involved in the development of metastasis. The discovery that the epithelial to mesenchymal transition (EMT) generates cells with properties of stem cells and a more invasive and metastatic phenotype, brings a connection between metastasis and stem-cell state. According to this model, cells with stem cell properties are located predominantly at the invasion front of the tumor and can derive through the acquisition of transient EMT phenotype. In this context, a comparative analysis of the expression profile of putative CSC markers between the invasion front and the inner tumor mass could test this hypothesis in the case of cutaneous melanoma as well. Purpose. Taking these data into account, we performed the current study in order to evaluate the immunohistochemical expression of EZH2, SOX2 and Oct4 as well as H3K4me2 and H3K27me3, which constitute stem cell-like "bivalent"domains, in cutaneous malignant melanoma, investigating besides the potential identification of cancer cells with stem cells properties at the invasion front of the tumor. Materials and methods. Expression of EZH2, SOX2, Oct4, H3K4me2 and H3K27me3 was evaluated in 89 malignant melanoma (MM) lesions, deriving from 79 patients, using immunohistochemistry, on formalin-fixed paraffin-embedded tissue sections. The analyzed cases were accessioned over the time interval 2001-2010 and retrieved from an electronic database maintained by the Department of Pathology of the University General Hospital of Patras (Rion, Greece). The sample consists of 70 primary and 19 metastatic specimens. 14 specimens contained both melanoma cells and nevus cells. Analysis and comparative studies were carried out on the expression of the proteins tested in nevus cells (where existed), melanoma cells, melanoma cells at the invasion front, basal and suprabasal 253 keratinocytes as well. Polymer based technique (Envision, Dako, USA) or MACH4 Universal HRPPolymer Detection (Biocare Medical, USA) and primary antibodies against EZH2 (Novocastra Laboratories Ltd, UK), SOX2 (R&D Systems, Inc.), Oct4 (Santa Cruz Biotechnology, Inc), H3K4me2 (Cell Signaling Technology, USA) and H3K27me3 (Cell Signaling Technology, USA) were used. In each case, the percentage of cells exhibiting positive staining was determined. Cell counts were performed at a 400X magnification. Data were analyzed using the SPSS statistical package (SPSS©, Release 19.0). The level of significance was set at p-value <0.05. Results. The three markers studied, EZH2, H3K4me2 and H3K27me3, were identified in the cell nuclei of melanoma cells, nevus cells and normal epidermal keratinocytes, while SOX2 και Oct4 showed nuclear as well as cytoplastik expression. A specific distribution pattern of H3K4me2 and H3K27me3 was found, as stronger levels were localized at the invasion front of the tumor (p=0.034 and p<0.01 respectively). A similar trend was also observed for EZH2, whithout achieving however statistical significance (p=0.08), and similarly for SOX2 in a few sporadic cases. Significantly increased EZH2 immunohistochemical expression was observed in melanoma cells with respect to nevus cells (p=0.02). Nuclear SOX2 levels were also higher in melanoma cells than basal keratinocytes (p=0.02) and in nevus cells than basal keratinocytes (p=0.0016) and suprabasal keratinocytes (p=0.027). Furthermore LIs in melanoma cells presented significantly higher values in primary with respect to metastatic malignant melanoma lesions (p=0.045) as well as in melanoma cells with low Breslow’s depth (≤1mm) (p=0.023), under the absence of ulceration (p=0.009) and with low (≤6/mm2) mitotic rate (p=0.016). As well as nuclear expression of Oct4 is concerned, it was found increased in nevus cells with compared to keratinocytes (p<0.001) and melanoma cells (p=0.004). Cytoplastic expression of Oct4 followed an opposite trend, with decreasing levels in melanoma cells with respekt to suprabasal keratinocytes (p<0.001) and in metastic compared to to primary melanoma cases (p=0.025). Remarkably occasionally increased Oct4 expression in endothelial cells of the tumor microvasculature in melanoma tissues was observed. Furthermore, H3K4me2 and H3K27me3 levels were lower in metastatic with respect to primary melanoma cases (p=0.0065 and p=0.027 respectively). Advanced melanoma demonstrated significantly lower H3K4 immunohistochemical expression than cases of lowest Clark’s level (I) (p=0.038) or low Breslow’s depth (≤1 mm) (p<0.001). Moreover, EZH2 expression in melanoma cells was higher compared to nevus cells (p=0.02). Finally statistical analysis further revealed a positive correlation in melanoma cells betwenn EZH2 and H3K27me3 (p=0.03), H3K4me2 and H3K27me3 (p<0.01), as well as between Oct4 and SOX2 for both nuclear and cytoplastik expression (p<0.001 and p<0.001 respektively). Conclusions. Our results suggest the possibility that combined immunohistochemical expression of EZH2, SOX2, Oct4, H3K4me2 and H3K27me3 might identify cancer cells with potential stem cell properties, particularly at the invasion front of this malignancy. This hypothesis should be further investigated, as many of the epigenetic changes are reversible via pharmacologic manipulations and new CSC-directed therapies, overpassing the resistance of advanced melanoma, may be developed.

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.

Melanoma is the most deadly form of skin cancers, with an incidence increasing more rapidly than any other malignant cancer in the past 40 years. Metastatic melanoma is resistant to conventional treatments, such as chemotherapy and radiation therapy. Our lab has previously demonstrated that Mcl-1 is a key contributor in protecting melanoma from therapy-induced cell death. RNAi therapeutics was employed as a novel way to silence the anti-apoptotic protein by using Mcl-1 mRNA sequence-specific siRNAs in vitro. In our hands, passive non-targeted delivery of RNAi therapy into melanoma tumours has been shown to be neither effective, nor selective in vitro and in vivo. Consequently, in this study, siRNA was linked to a delivery system which expressed a ligand specifically targeting melanoma cells. Previously shown, melanoma overexpresses the cell surface ganglioside GD3, thus it is my belief that the anti-GD3 R24 monoclonal antibody can function as a targeting molecule. The antibody was linked to coated cationic liposomes (CCLs) carrying siRNA molecules. Our first step was to confirm R24 ligation to CCLs. Untargeted CCLs showed insignificant values of antibody, whereas antibody-conjugated CCLs presented approximately 30 antibodies per liposome. I also confirmed that siRNA was internalized within CCLs using spectrometry, with an encapsulation efficiency of approximately 80%. Since liposomes need to be small to be effective in vitro and in vivo, CCLs were confirmed to be less than 100nm in diameter. In vitro studies using fluorescent microscopy demonstrated greater binding to melanoma cells with antibody-conjugated CCLs as compared to untargeted CCLs. In vivo experiments showed specific localization of targeted CCLs to induced subcutaneous mouse xenograft tumours. Western blotting demonstrated greater Mcl-1 knockdown using GD3-targeted CCLs. Taken together, these results suggest that anti-GD3 antibodies can serve as targeting molecules to deliver siRNA to melanoma cells and furthermore, GD3-targeted CCLs can promote siRNA-mediated gene silencing. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-09-02 10:29:37.944

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