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Mechanisms of Spontaneous Melanoma Regression in Melim Pigs Florian Rambow

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Mechanisms of Spontaneous Melanoma Regression in Melim Pigs Florian Rambow Mechanisms of Spontaneous Melanoma Regression in MeLiM Pigs Florian Rambow To cite this version: Florian Rambow. Mechanisms of Spontaneous Melanoma Regression in MeLiM Pigs. Life Sciences [q-bio]. Université de Versailles-Saint Quentin en Yvelines; Ruprecht-Karls-Universität Heidelberg, 2008. English. tel-00433225 HAL Id: tel-00433225 https://tel.archives-ouvertes.fr/tel-00433225 Submitted on 18 Nov 2009 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. PhD Thesis en Co-tutelle to obtain the degree of DOCTEUR EN SCIENCES DE L’UNIVERSITÉ DE VERSAILLES SAINT-QUENTIN-EN-YVELINES and DOCTOR SCIENTIARUM HUMANARUM of the UNIVERSITY of HEIDELBERG (Medical Faculty) Discipline: Molecular Biology Presented and defended by Florian RAMBOW May 2008 Mechanisms of Spontaneous Melanoma Regression in MeLiM Pigs Thesis Director: P. CHARDON Jury: Referee: Prof Dr M. Groenen Referee: Dr A. Prévost-Blondel Examiner: Prof Dr B. Mignotte Examiner: Prof Dr F. Velten Examiner: Dr P. Chardon “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.” (Charles Darwin) Dedicated to my parents Abstract Cutaneous melanoma, the most aggressive form of skin cancer, is a tumor originating from melanocytes with rapidly increasing incidence. Patients with advanced disease have a poor prognosis since melanoma is mostly resistant to current therapies. Therefore, the development of novel strategies for preventing and treating melanoma is important. To explore novel therapies we need to find appropriate targets and for that knowledge about the biology of melanoma is important. One elegant way to do so, is to study the natural but rare phenomenon of spontaneous melanoma regression. Unfortunately, complete spontaneous regression of advanced melanoma is extremely rare, and therefore hard to investigate. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a great opportunity to study spontaneous tumor regression. MeLiM pigs, suffering from hereditary melanoma, develop tumors naturally and regress them completely without external influence. Mechanisms leading to spontaneous melanoma regression are poorly understood at present. Therefore the overall objective of this PhD was to investigate mechanisms of spontaneous melanoma regression in MeLiM pigs. In an initial study we gained a global overview of differentially expressed genes between a growing and regressing tumor by conducting Subtractive Suppression Hybridization (SSH). We also performed SSH analysis of cell cultures isolated from growing and regressing melanoma to be able to distinguish between expressional changes induced by tumor microenvironment. Only a few genes were in common between these two SSH analyses. Thus we focused on the SSH results obtained from tumor tissue. We were able to identify a gene signature for growing melanoma that demonstrated common genes with human progressive melanoma (TYR, MITF, MLANA, SDCBP, SILV, TYRP and ZFP106). Genes overexpressed at the beginning of regression were mainly involved in functional classes such as differentiation, immune system, cell cycle arrest, and tumor suppression. Two genes, CD9 and RARRES1, showed a strong upregulation during early melanoma regression on the mRNA and protein level whereas CD9 is a motility-related protein and RARRES1 a putative tumor suppressor. Since spontaneous regression is a dynamic process, we performed time dependent gene expression profiling using DNA chips in a second study. We identified significant gene signatures at different stages of regression that were able to explain the corresponding phenotype. Identified gene signatures were mainly involved in the immune response, the cell cycle, and melanocyte differentiation/pigmentation processes. Interestingly, we showed an early downregulation of cell cycle related genes that could play a role in regression. Also, we identified different immune gene signatures, suggesting a major role of the host’s immune system in eradicating melanoma cells. By using immunohistology and flow cytometry we characterized tumor infiltrating cells of the innate and adaptive immune system. During regression the infiltration occurred in two phases: an early phase consisting of antigen presenting cells (SWC3+), followed by a later infiltration of mainly cytotoxic T lymphocytes (CD8+). Furthermore the regression process is accompanied by the presence of big highly pigmented cells that we began to characterize. This work allowed us to point out similarities between the pig and human melanoma on the transcriptomic level along with the already observed parallels on the clinical and genetic level. These findings underline the utility for this model to study human melanoma. This work helped also to decipher the process of spontaneous regression on a cellular and molecular level. In this way we observed an early mitotic arrest of tumor cells, linked to regression, that has not been described so far, and an implication of the immune system. If the immune response is the real inducer of regression needs to be verified. Already, this work contributes to human anti- melanoma therapy as it provides potential targets that could be used for developing new strategies. Résumé : Le mélanome est une tumeur originaire des mélanocytes; c’est la forme la plus agressive des cancers cutanés. Son incidence s’accroît régulièrement. De plus sa résistance aux traitements actuels (chimiothérapie, radiothérapie et immunothérapie) ne laisse qu’une faible espérance de survie aux patients présentant un stade avancé de la maladie. Aujourd’hui il est nécessaire de développer de nouveaux traitements ciblés sur la cellule tumorale. Une stratégie originale consisterait à étudier le phénomène naturel de régression spontanée des mélanomes. Malheureusement chez l’homme la régression n’est que partielle et l’extrême rareté des cas de régression totale à un stade avancé de la maladie rend impossible son étude. Cependant le modèle porcin de mélanome cutané (MeLiM) présente une opportunité unique d’étudier ces mécanismes complexes de régression spontanée. En effet, les minis porcs MeLiM developpent des mélanomes héréditaires qui régressent totalement et spontanément, indépendamment de tous facteurs externes. Actuellement, les mécanismes responsables de cette régression spontanée sont peu ou mal connus. C’est pourquoi l’objectif principal de cette thèse a été l’étude des mécanismes de la régression spontanée du mélanome dans ce modèle animal. Dans un premier temps, grace à la technique d’hybridation suppressive soustractive (HSS) nous avons comparé le transcriptome d’une tumeur en croissance et d’une tumeur en tout début de régression. Dans le but de distinguer les signaux provenant du microenvironnement tumoral de ceux propres à la cellule tumorale, nous avons également réalisé une HSS à partir des cellules isolées d’une tumeur en progression et d’une tumeur au début de la régression. Nous avons trouvé très peu de gènes en commun entre les deux HSS. Nous avons donc focalisé notre étude sur les résultats issus de la HSS à partir des tumeurs. Des gènes surexprimés chez le porc pendant la phase de proliferation tumorale sont également retrouvés dans les études d’expression conduites dans le mélanome chez l’homme (TYR, MITF, MLANA, SDCBP, SILV, TYRP and ZFP106). Au tout début du phénomène de régression, on observe une surexpression des gènes impliqués dans les fonctions de la differentiation, le système immunitaire, l’arrêt du cycle cellulaire et la suppression des tumeurs. Deux gènes CD9 et RARRES1 montrent une très forte surexpression pendant la régression tant au niveau transcriptomique que protéique. Le CD9 est lié à la motilité cellulaire et RARRES1 est un gène potentiel suppresseur de tumeur. Dans un second temps, la dynamique du processus de régression a été étudiée grâce à la technologie des puces à ADN qui a permis d’établir un profil cinétique de l’expression génique. Ainsi, pour chacun des stades de la régression une corrélation entre la signature génique et son expression phénotypique a pu être établie. La signature de la régression comprend entre autres, des gènes impliqués dans la réponse immunitaire, le cycle cellulaire et la différentiation ainsi que la pigmentation des mélanocytes. Ce travail révèle que la régression des mélanomes semblerait être lié à une sous régulation précoce du cycle cellulaire. De plus le système immunitaire semble jouer un rôle majeur dans l’éradication des cellules tumorales. Par immunohistologie et cytometrie en flux, nous avons caracterisé le phenotype des cellules infiltrant les tumeurs : les cellules de l’immunité innée et acquise. On observe deux phases pendant le processus de la régression : une phase précoce consistant principalement à une infiltration par des cellules presentatrice d’antigènes (SWC3+) puis une phase d’infiltration tardive par des lymphocytes cytotoxiques (CD8+). La régression s’accompagne également de la présence de grosses cellules hyperpigmentées que nous avons commencés à caracteriser. Ce travail a permis de montrer qu’il existe
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