Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2015. / A tese de doutorado intitulada “Avaliação dos efeitos citotóxicos e antiproliferativos da secreção cutânea e de peptídeos bioativos do anuro Physalaemus nattereri (steindachner, 1863)” desenvolvida por Andréa Cruz e Carvalho sob a orientação da profa. Dra. Mariana S. Castro teve o apoio financeiro do CNPq (processos n°. 563972/2010-6, Edital MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/PRÓ-CENTROOESTE nº 31/2010, n°. 302925/2012-0, Produtividade em Pesquisa - PQ – 2012 e n°. 407801-2013, Rede Centro-Oeste De Pós-Graduação , Pesquisa E Inovação - REDE PRÓ-CENTRO-OESTE), da FAPDF (processo n°. 193. 000.461/2011, Edital MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/PRÓ-CENTRO-OESTE nº 31/2010), do CNPq por meio da concessão de bolsa de estudos (nível doutorado), da FINEP (CT-INFRA) E DA FUB-UNB. / As secreções cutâneas de anuros são fontes naturais de peptídeos antimicrobianos. A realização de novas pesquisas nessa área tem revelado que alguns peptídeos com atividade antimicrobiana também apresentam efeitos tóxicos sobre células tumorais. O objetivo do presente estudo foi investigar o potencial terapêutico da secreção cutânea de Physalaemus nattereri sobre células tumorais e determinar os mecanismos da ação citotóxica sobre céluluas de melanoma murino da linhagem B16F10. Nossos resultados demonstraram que a secreção bruta reduz a viabilidade de células B16F10, causando alterações na morfologia celular, (p.ex., arredondamento e encolhimento), ruptura da membrana plasmática, redução do potencial de membrana mitocondrial e parada do ciclo celular na fase S; tais mudanças em conjunto sugerem que as células tumorais morreram por apoptose. A secreção cutânea também foi submetida ao fracionamento cromatográfico empregando RP-HPLC e as frações eluídas foram avaliadas quanto à suas atividades antiproliferativas e antimicrobiana. Três frações ativas exibiram componentes de massa molecular compatíveis com peptídeos. Os três componentes majoritários presentes em tais frações foram purificados por RP-HPLC e analisados por MALDI-TOF/MS. Tais peptídeos mostraram-se ativos sobre bactérias e células tumorasi e foram identificados como pertencentes ao grupo das nattererinas, peptídeos antibacterianos previamente isolados desse animal por nosso grupo. Embora, o mecanismo específico envovlido na redução da viabilidade celular e citotoxicidade após o tratamento com essa secreção bruta ainda não seja conhecido, podemos supor que a atividade de moléculas, como peptídeos, presentes nessa secreção seja efetivos contra as células de melanoma murino (B16F10). Considerando-se a necessidade crescente por novas drogas anticâncer, o presente estudo fortemente reforça a validade do uso da secreção cutânea de P. nattereri como uma rica fonte de novas moléculas anticâncer, principalmente peptídeos. / Anuran secretions are rich natural sources of antimicrobial peptides. The emergence of new research in this area has shown that some peptides with antimicrobial activity also exert toxic effects against cancer cells. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against cancer cells, and to verify the mechanisms of its cytotoxic action on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduces the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), rupture of the plasma membrane, reduction in mitochondrial membrane potential, fragmentation of DNA, and cell cycle arrest in Sphase; together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also submitted to chromatographic fractionation using RP-HPLC and the eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular masses components with a molecular range compatible to peptides. Thes major petidic components present on these fractions were purified by RP-HPLC and analysed by MALDI-TOF/MS. These peptides were active against bacteria and cancel cells, and were identified as nattererins, antibacterial peptides previously isolated from this animal by our group. Although, the specific mechanism causing the reduction in cell viability and cytotoxicity after treatment with the crude secretion is still unknown, we can consider that the activity of molecules, such as peptides, within the secretion is effective against murine melanoma (B16F10) tumor cells. Considering the growing need for new anticancer drugs, the present data strongly reinforce the validity of crude secretion of P. nattereri as a rich source of new anticancer molecules, mainly peptides.

Le mélanome est un cancer qui représente la grande majorité de la morbidité et de la mortalité causées par tout cancer de la peau en raison de son implication dans les métastases. Parmi plusieurs biomarqueurs rapportés, les récepteurs du domaine de la discoïdine (DDR) et la métalloprotéinase Matrix (MMP) sont également considérés comme des acteurs potentiels du mélanome. Des études récentes démontrent que les DDR et les MMP sont surexprimés dans les mélanomes de mauvais pronostic, ce qui nécessite le recours à des sondes théranostiques à biomarqueurs spécifiques. De nombreux progrès ont été réalisés pour réduire le risque associé au mélanome métastatique à l'aide d'anticorps monoclonaux traditionnels, mais les inconvénients tels que la complexité de la production, la variabilité d'un lot à l'autre, la courte durée de conservation, l'immunogénicité, la réactivité croisée et le coût élevé ne peuvent être négligés. Par conséquent, au cours des dernières années, les aptamères ont acquis un grand intérêt comme substitut d’anticorps pouvant être synthétisés chimiquement avec un faible coût de production et dépassant les limitations associées aux anticorps. Les aptamères sont de courts oligonucléotides à haute affinité et spécificité vis-à-vis de la molécule cible, développés par une méthode combinatoire appelée SELEX (Evolution systématique de ligands par enrichissement exponentiel). Au cours de ma thèse, nous avons réalisé une étude SELEX contre les biomarqueurs DDR1, DDR2 et HMMP14 sur la base de supports alternatifs pour le tri des candidats et le séquençage à haut débit. Un groupe d'aptamères a été sélectionné contre DDR1 et le meilleur candidat a ensuite été dopé pour effectuer davantage de tours de sélection contre DDR1. Finalement, un aptamère contre DDR1 a été sélectionné avec un Kd dans la gamme nanomolaire basse. Cet aptamère peut être utilisé comme outil théranostique dans les études sur le mélanome associé à DDR1. Comme les aptamères présentent un avantage en termes de conjugaison et de modifications aux positions désirées, nous visons en outre à conjuguer cet aptamère avec des nanoparticules pour des études in vivo. / Melanoma is a cancer that accounts for the vast majority of morbidity and mortality caused by any skin cancer due to its involvement in metastasis. Among several reported biomarkers, Discoidin Domain Receptors (DDRs) and Matrix metalloproteinase (MMPs) are also considered as potential actors in melanoma. Recent studies demonstrate that DDRs and MMPs are overexpressed in melanoma with poor prognosis which demands the need of biomarkers specific theranostics probes. Though many advances has been made in order to reduce the risk associated with metastatic melanoma with the help of traditional monoclonal antibodies, but the drawbacks like production complexity, batch to batch variability, short shelf life, immunogenicity, cross reactivity and high cost cannot be neglected. Therefore in recent years, aptamers have gained high interests as a substitute of antibodies that can be synthesized chemically with low production cost and overcomes the limitations associated to antibodies. Aptamers are short oligonucleotides with high affinity and specificity against its target molecule, raised by a combinatorial method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). During my thesis, we performed a SELEX against DDR1, DDR2 and HMMP14 biomarkers based on alternative supports for candidates sorting and high throughput sequencing. A group of aptamers were selected against DDR1 and the best candidate was further doped to perform more rounds of selection against DDR1. Finally an aptamer against DDR1 was selected with a Kd in low nano-molar range. This aptamer can be used as a theranostics tool in DDR1 associated melanoma studies. As aptamers have an advantage of conjugation and modifications at desired positions so we further aim to conjugate this aptamer with nanoparticles for In-vivo studies.

During development, neural crest (NC) cells arise from the neural plate border and are further differentiated into multiple cell types, including melanocytes. Each step of this process is controlled by a gene regulatory network (GRN), and disruption of the GRN governing melanocyte differentiation contributes to the pathogenesis of pigmentation disorders and melanoma. While many of the factors within this network have been well studied, the role of Transcription Factor Activating Enhancer-Binding Protein 2 (TFAP2) paralogs has been unclear. TFAP2A and TFAP2C are required for NC induction. Later, TFAP2A is also expressed in melanocytes, and TFAP2A mutations cause pigmentation phenotypes in humans, mice, and zebrafish. Other paralogs with high homology to TFAP2A, particularly TFAP2B in mouse and Tfap2e in zebrafish, also function redundantly with TFAP2A in the melanocyte lineage. Here, we have used ChIP-seq and expression profiling to identify direct transcriptional targets of TFAP2A in melanocytes, which include genes involved in melanin synthesis and melanosome biology. Furthermore, we show that TFAP2A directly regulates many of the same genes as Microphthalmia-associated Transcription Factor (MITF), the “master regulator” of the melanocyte lineage. MITF activity has been described as a rheostat in melanoma, with high levels promoting differentiation and lower levels promoting invasiveness. The overlap between TFAP2A and MITF transcriptional targets in melanocytes suggests that TFAP2A may influence the MITF rheostat, driving it toward the differentiated state. To study the role of other TFAP2 paralogs in NC and melanocytes, we generated zebrafish lines that are double and triple mutant for tfap2a, tfap2c, and tfap2e and confirm genetic compensation among these paralogs. We also demonstrate that melanocyte-specific inhibition of Tfap2 activity by Kctd15 affects differentiation and that Kctd15 may participate in a negative feedback loop regulating Tfap2 expression. In support of a pro-differentiation role for TFAP2A in melanocytes, we show that overexpression of tfap2a in a zebrafish melanoma model significantly delays tumor formation. Together these results indicate that, in addition to its earlier roles in the NC, TFAP2A acts within the melanocyte GRN to directly regulate differentiation genes in parallel with MITF. This, combined with the tumor-suppressor function of TFAP2A in melanoma, implicates TFAP2A and the factors that regulate it as potential targets for melanoma therapies.

The neural crest is a transient, multipotent, cell population that gives rise to several important tissues during embryonic development, including the craniofacial skeleton, peripheral nervous system, and melanocytes. The neural crest arises from the ectoderm, along with the skin and central nervous system. This process of specification is dependent on a gene regulatory network (GRN) which is made up of transcription factors that regulate each other. While we know many of the members of this GRN, the direct connections among the members are largely unsolved. Breakdown of this GRN can lead to birth defects, such as cleft lip and palate, and cancer of neural crest derivatives, such as melanoma, thus understanding the intricate details of this network is important. The transcription factor Tfap2a is an important member of the GRN, as loss of tfap2a and its paralog tfap2c leads to loss of pre-migratory neural crest and all neural crest derivatives. Despite its importance in this network little is known about how its expression is regulated. We hypothesized that, due to its importance in this network, it will have multiple enhancers that drive its expression in the neural crest. We have identified two neural crest enhancers of tfap2a. We found that one of these enhancers is responsive to WNT signals and is maintained by forming a positive feedback loop with Sox10. Our results suggest that this enhancer is important for both induction and maintenance of tfap2a expression in the neural crest. Tfap2 paralogs are important at several different stages throughout neural crest lineage specification. However, the only direct target of Tfap2a that has been identified is sox10. Thus, we wanted to determine the direct targets of Tfap2 in this network. Through the integration of several data sets, including ATAC-seq and expression profiling of tfap2a/c double mutants, we have identified several direct targets including sox9b and alx1. Melanoma is cancer of the melanocytes, a neural crest derivative. Recent studies have shown that melanoma and the neural crest share genetic similarities. TFAP2A expression is decreased in metastatic melanoma compared to primary tumors, thus we wanted to investigate the mechanism of TFAP2A in metastatic melanoma. We found that the promoter of TFAP2A is hypermethylated in some metastatic melanoma tumors. This was confirmed by samples in the TCGA database. Hypermethylation of the promoter contributes to the downregulation of TFAP2A in metastatic melanoma. In conclusion, we have further illuminated the connections among transcription factors in the GRN important for neural crest lineage specification. Further, we have identified a new mechanism regulating TFAP2A expression in metastatic melanoma. Together, these studies reveal regulatory mechanisms of TFAP2A gene expression.

Le mélanome métastatique est resté longtemps synonyme de pronostic catastrophique en raison de la faible efficacité des traitements disponibles. Depuis quelques années, de nombreuses innovations thérapeutiques ont révolutionné la prise en charge de ces patients, avec l’autorisation de mise sur le marché de traitements modulateurs de l’immunothérapie d’une part et de thérapies ciblant la voie des MAP kinases destinées aux patients porteurs de la mutation BRAF (présente dans environ 50% des mélanomes) d’autre part.Les inhibiteurs de BRAF et de MEK sont à l’origine de réponses spectaculaires, mais le contrôle de la maladie est généralement limité dans le temps, avec l’apparition de résistances au traitement entraînant une progression de la maladie. Etablir des stratégies thérapeutiques permettant de retarder ou de contourner ces résistances s’avère donc primordial. Pour ce faire, des biomarqueurs ou outils prédictifs de la réponse, in vitro et in vivo, peuvent contribuer à mieux stratifier les patients, et personnaliser leur prise en charge.Dans une première partie, nous avons comparé l’apport respectif d’un traceur de prolifération cellulaire, la 18F-FLT, et du traceur de consommation de glucose le 18F-FDG, dans l’évaluation thérapeutique d’une xénogreffe de mélanome BRAF muté traité par un inhibiteur de BRAF. Nous avons confirmé la place prépondérante du 18F-FDG, et étayé l’intérêt des index volumiques pour le suivi thérapeutique, notamment avec la 18F-FLT.Dans une seconde partie nous avons évalué in vivo la capacité d’un schéma d’administration intermittente à retarder l’apparition de résistance par rapport à un schéma d’administration continue dans des xénogreffes BRAF mutées traitées par une combinaison d’inhibiteurs BRAF/MEK. Dans nos modèles expérimentaux, il ne semblait pas y avoir de supériorité de l’administration intermittente. Toutefois nos expérimentations n’ont pas permis de trancher de façon formelle la question.Dans une troisième partie, nous avons voulu reproduire le schéma d’administration intermittent ex vivo en utilisant la technique d’histocultures. Nous avons par ailleurs évalué la possibilité pour des histocultures issues de tumeurs de patients de prédire la réponse aux thérapies ciblées chez ces mêmes patients. Les histocultures semblent un outil fiable pour guider les choix thérapeutiques. La documentation des modifications génétiques et de l’hétérogénéité moléculaire du mélanome pourrait également inciter à adapter la stratégie en fonction de l’agressivité présumée du mélanome chez un patient donné / Metastatic melanoma has long been synonymous of dismal prognosis, due to the weak efficacy of available treatments. Numerous therapeutic innovations have profoundly modified the management of these patients, with marketing authorizations of therapies targeting the MAP Kinases pathway for patient with BRAF mutated melanoma (about 50% of patients) on the one hand and immune checkpoint inhibitors on the other hand.BRAF and MEK inhibitors result in dramatic responses, but disease control is generally short-lived, with onset of drug resistance leading to disease progression. Designing therapeutic strategies allowing delaying or bypassing this resistance phenomenon is of primary importance. Thus, in vitro and vivo biomarkers or tools predictive of response could help stratifying patients, and personalize each patient’s management.In the first part, we compared the respective value of a proliferation tracer, 18F-FLT, and of the glucose consumption tracer, 18F-FDG for therapeutic evaluation of a BRAF mutated melanoma xenograft, treated by a BRAF inhibitor. We confirmed the predominant role of 18F-FDG, and backed up the potential interest of volumetric parameters for therapeutic follow-up, especially with 18F-FLT.In a second part we assessed in vivo the ability of an intermittent schedule to delay resistance onset, compared with a continuous schedule in BRAF mutated xenografts treated by a BRAF/MEK inhibitors combination. In our experimental models, there did not seem to be any superiority for the intermittent schedule. However our experiments did not allow us to draw any final conclusions on the subject.In a third part, we tried to reproduce ex vivo the intermittent schedule with histocultures device. We also assessed the possibility for histocultures with patient derived tumours to predict response to targeted therapies in the same patients. Histocultures appear as a relevant tool to guide therapeutic choice. Determination of genetic modifications and molecular heterogeneity may also prompt tailoring therapeutic strategy depending on the supposed aggressiveness of a melanoma in a given patient

Metastatic melanoma is among the most aggressive forms of cancer for which there are no effective therapies. Emerging evidence indicates that melanomas can be sensitized to chemotherapy by increasing the function of integrin transmembrane adhesion receptors. Current integrin therapies work by targeting the extracellular domain, resulting in complete gains or losses of integrin function that lead to toxicity.An attractive alternative approach is to target proteins from inside the cell, such as vinculin, that associate with the integrin cytoplasmic domains and regulate its ligand binding properties. The work presented in this thesis describes a novel reagent, denoted vinculin activating peptide or VAP, which increases integrin activity from within the cell as measured by elevated: (1) numbers of active integrins, (2) adhesion of cells to extracellular matrix ligands, (3) numbers of cell-matrix adhesions, and (4) downstream signaling. The effects of VAP are dependent on both integrins and a key regulatory residue A50 in the vinculin head domain. I further show that VAP dramatically increases the sensitivity of melanomas to chemotherapy in clonal growth assays and in vivo mouse models of melanoma. Finally, we demonstrate that the increase in chemosensitivity results from increases in DNA damage-induced apoptosis by a mechanism that requires both p53 and β1 integrin. Collectively these findings demonstrate that integrin function can be manipulated from within the cell and validate integrins as a new therapeutic target for the treatment of chemoresistant melanomas.

Le mélanome représente 8000 nouveaux cas par an et plus d'un millier de décès. Dés l'apparition de métastases, le pronostic devient très mauvais en raison de l'inefficacité de tous les traitements. Enfin, bien que des résultats encourageants aient été obtenus récemment avec des thérapies ciblées, ces réponses restent insuffisantes. L'identification de nouvelles molécules candidates est donc un élément essentiel pour le traitement du mélanome. Pendant la 1ère partie de ma thèse, je me suis intéressé à la metformine. Nous avons observé une diminution de l’invasion des cellules de mélanome dépendante de l'activation de l’axe AMPK/ p53 en réponse à la metformine. Ainsi, nous avons démontré pour la 1ère fois que la metformine peut être utilisée pour inhiber l'invasion des cellules de mélanome. Ensuite, je me suis intéressé à d'autres antidiabétiques, les TZD. Grâce à un criblage structure / activité, nous avons identifié une nouvelle molécule, le HA15, dérivé des TZD et ayant une très forte activité anti-mélanome. Nous avons montré que le HA15 induit un stress du RE (UPR), par l'intermédiaire de la protéine chaperon Bip, conduisant à l'activation de l'apoptose et de l'autophagie, entraînant ainsi la mort des cellules. Cette étude a permis d’identifier une nouvelle molécule efficace contre le mélanome et renforce l'idée que l'UPR pourrait être une cible thérapeutique pour le traitement du mélanome. / Melanoma accounts for 8000 new cases each year and more than a thousand deaths. If diagnosed early enough, wide surgical excision may be sufficient to treat. However, at the onset of metastasis, the prognosis becomes very bad due to the ineffectiveness of all treatments. Finally, although encouraging results were recently obtained with B-Raf inhibitors and immunotherapy, these responses are insufficient. So identification of new candidate molecules is an essential element for melanoma treatment. During the first part of my PhD, I was interested in metformin, the most commonly used molecule for the treatment of type 2 diabetes mellitus. It had been shown in our laboratory that metformin reduces melanoma cells viability in vitro and in vivo. Melanoma is a highly metastatic cancer, so I focused on the potential effect of metformin on melanoma cells invasion. We observed a decrease of melanoma cells invasion dependent on the activation of AMPK / p53 axis in vitro and in vivo. So we have shown for the first time that metformin can be used to inhibit melanoma cells invasion.During the second part of my PhD, I have been interested in other antidiabetic drugs, thiazolidinediones (TZD). Through a structure / activity screening we identified a molecule, HA15, derived from TZD and having a very strong anti-melanoma activity in vitro and in vivo. We showed that HA15 induced unfolded protein response (UPR), via the chaperone protein Bip, leading to autophagy and apoptosis activation, and resulting in cell death. This study identified a new molecule potentially effective against melanoma and reinforces the idea that UPR could be a therapeutic target for melanoma treatment.

Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

La galectine-3 (Gal-3), protéine de type lectine, de 29-35 kDa, étudiée comme marqueur d’aggressivité dans les gliomes, présente des caractéristiques biologiques importantes justifiant son étude dans le domaine du mélanome. En effet, la Gal-3 est une protéine qui peut se lier à la laminine, tout comme l’intégrine α6/β1 dont l’expression est réduite dans le mélanome. L’expression de cette intégrine peut d’ailleurs être modulée par la Gal-3 comme récemment montré dans des lignées cellulaires de cancer du sein (BT-549) et de glioblastome (U373). Le mélanome, véritable problème de santé publique qui est susceptible d’atteindre 1 individu sur 75 dans nos contrées, reste un tumeur mal comprise avec des évolutions parfois incertaines, et des traitements dont l’efficacité est limitée. Le diagnostic histologique du mélanome lui-même peut parfois représenter une difficulté pour le clinicien et l’expert pathologiste ou dermatopathologiste. La couleur (hyperpigmentation d’un lésion pigmentée), dont l’évaluation d’ailleurs reste subjective à défaut de standardisation, ne peut à elle seule signer la malignité d’une lésion pigmentée. Globalement l’évolution d’un patient est prédite par l’indice de Breslow qui traduit en mm l’épaisseur de la tumeur. Si cet indice dépasse 1mm, le risque métastatique augmente, justifiant la réalisation de bilans extensifs de suivi. Ceci dit, certains mélanomes épais peuvent ne pas présenter de caractéristiques d’aggressivité, alors que des mélanomes fins sont parfois mortels. L’identification de marqueurs moléculaires est donc impérative, tant pour développer des stratégies thérapeutiques ciblées, que pour affiner le diagnostic et le pronostic d’un patient. Après avoir mis en évidence par immunohistochimie une expression de Gal-3 par les mélanocytes, nous avons démontré une surexpression de cette protéine par les mélanocytes tumoraux. Nous avons démontré également sur des lésions primitives qu’à l’aggressivité mesurée selon l’indice de Breslow correspondait une diminution de cette surexpression. Cette observation a pu être confirmée par un modèle de greffe orthotopique chez la souris nude. Nous nous somme intéressés par la suite à la détection de la protéine dans le sérum, et nous avons constaté, un taux élevé de Gal-3 dans le sérum de patients en stade métastatique avancé, ce taux élevé pouvant s’expliquer tant par la charge tumorale que par la présence d’une inflammation, d’ailleurs bien connue chez le patient cancéreux en stade avancé. Le rôle antiapoptotique de la Gal-3 nous a alors amené à préciser la valeur prédictive et pronostique de cette protéine. L’hypothèse d’une potentielle action bénéfique sur la réponse immunitaire des patients atteints de mélanome qui ont été vaccinés a été rejetée. La Gal-3 sérique s’est révélée comme facteur de mauvais pronostic chez les patients métastatiques, et une analyse multivariée avec la définition d’une valeur « cut-off » de 10 ng/ml a permis de montrer une valeur pronostique indépendante, supérieure à la S100B et à la CRP.

Because of the failure so far to find effective treatment for patients with advanced stages of melanoma, increasing efforts have been made to find prognostic factors identifying patients in the risk zone for development of metastasis. This thesis investigates the prognostic powers of a few selected serological and immunohistochemical biomarkers. In the first and second study, patients operated on for localized malignant melanoma were investigated regarding the prognostic impact of angiogenic serological markers and circulating levels of S100. We concluded that the S100 assays, especially S100BB, are potential biomarkers in patients with malignant melanoma, correlated to both survival and disease free survival. However, no such conclusion could be drawn from the first study, where we found no correlation to survival and investigated angiogenic markers. In the third and fourth study four new potential immunohistochemical biomarkers where investigated in collaboration with the Swedish Human Protein Atlas Program, and those where TRP-1, galectin-1, DLG5 and syntaxin-7. We found that TRP-1 correlated inversely with tumor stage and galectin-1 correlated to Ki-67. DLG5 showed a significant inverse correlation to Ki67 and the expression of STX7 was inversely correlated to tumor stage, suggesting that decreased expression is associated with more aggressive tumors. None of the investigated markers in study III and IV correlated with disease free survival or overall survival. In the fifth and last study, we examined the expression of SOX10, a transcription factor, in different melanocytic lesions. Also, a proliferation assay was carried out in a human melanoma cell line. The results reveal the presence of SOX10 in different melanocytic lesions, with a weak inverse correlation to survival and a significant inverse correlation to T-stage. A significant decrease in proliferation rate for SOX10 silenced cells was found and our data also suggests an increased migratory response in SOX10 silenced cells.