MITF is the master melanocyte transcription factor and has a complex role in melanoma. Both gain- and loss-of function mutations in MITF have been identified in melanoma, although its’ role in melanoma development and the effects of targeting MITF are unknown. Using a temperature-sensitive mitf zebrafish mutant I show that low levels of MITF are oncogenic with BRAFV600E in melanoma progression. By pathology and MITF target gene expression, BRAFV600Emitfavc7 tumours are distinct from BRAFV600Ep53M214K tumours, and represent two melanoma subtypes. Melanomagenesis can also be driven independently of BRAFV600E, in a transgenic zebrafish with mutations in mitf and p53, representing a new melanoma model. Abrogating MITF activity in BRAFV600Emitfavc7 melanoma leads to regression of the tumour, characterised by macrophage infiltration and increased apoptosis. This result confirms the dependence on MITF activity in BRAFV600Emitfavc7 melanomas and highlights the role of MITF as a therapeutic target for melanoma. Exome and transcriptome sequencing has been carried out to gain insight into the expression and genomic mutational landscape that is driven by these melanoma transgenic models and results in these genotype-phenotype specific subtypes observed.

La protéine SHP2 est une tyrosine phosphatase cytosolique impliquée dans la régulation des phénomènes de prolifération, migration et survie cellulaire. Elle contrôle l'état d'activation des voies de signalisation des récepteurs à tyrosine kinase faisant intervenir en particulier les protéines kinases MAPK/ERK, PI3K/AKT. Des mutations germinales et somatiques de SHP2 sont à l'origine de syndromes et de néoplasies. Au cours de ce travail, nous avons mis en évidence une nouveau partenaire pour SHP2: la protéine CDCP1. CDCP1 est une protéine transmembranaire dont la surexpression et la tyrosine phosphorylation par les kinases de la famille de SRC dans de nombreux carcinomes permet une augmentation des propriétés métastatiques des cellules. Nous avons de plus mis en évidence la régulation de la phosphorylation de CDCP1 et sa présence à la membrane cellulaire par SHP2. Dans une seconde partie de ce travail de thèse, notre attention s'est portée sur la protéine adaptatrice GAB2, partenaire et substrat principal de SHP2, dont la surexpression dans les mélanomes primaires et métastatiques commence à être décrite. Nous mettons en évidence la présence d'une boucle de régulation entre GAB2, dont l'expression est régulée par le facteur de transcription MITF et le récepteur KIT, dont l'activation, dépendante de l'expression de GAB2 régule l'activation de MITF et la motilité des cellules de mélanome.L'ensemble de ce travail met ainsi en évidence la présence d'un nouvel interacteur de SHP2, et d'une nouvelle boucle de régulation dans les cellules de mélanome. / SHP2 is a protein tyrosine phosphtase which regulates cell proliferation, mirgration and survival. It controls MAPK/AKT and PI3K/AKT signaling pathways activated by tyrosine kinase receptors. Germinal and somatic mutations in SHP2 lead to syndromes and neoplasia. We show a novel interaction between SHP2 and the protéine CDCP1. CDCP1 is a transmembrane protein which overexpression and tyrosine phosphorylation by SRC family kinases in carcinomas lead to an increase of the tumor cells metastatic properties. We also demonstrated that SHP2 regulates CDCP1 tyrosine phosphorylation level and its presence at the cell membrane.In a second part of this work, we focused on GAB2 anchor protein, which is one of the most important partner and substrate for SHP2. GAB2 is overexpressed in primary and metastatic melanoma. We show the presence of a regulation loop, involving GAB2 regulation by the transcription factor MITF, KIT receptor and its activation depending on GAB2, which controls MITF activation and melanoma cell motility.This thesis work demonstrate the presence of a novel partner for the protein SHP2 and of a new regulation pathway in melanoma cells.

Les protéines et les voies impliquées dans la réponse aux dommages de l'ADN (DDR), permettant de maintenir la stabilité génétique et de préserver la fidélité de la réplication, agissent non seulement comme l'initiation de la cancérogenèse mais peuvent également jouer un rôle majeur dans la progression tumorale et dans la résistance aux thérapies. La voie FANC joue un rôle central dans la stabilité génétique lors d'un stress réplicatif. La perte de fonction de cette voie est la cause d'un syndrome de fragilité chromosomique et de prédisposition au cancer appelé Anémie de Fanconi (FA).Nous avons démontré que les protéines FANC était surexprimées et suractivées dans les mélanome métastatiques exprimant le facteur de transcription MITF, un oncogène exprimé dans 80% des cas de mélanome. Nous avons identifié MITF comme un régulateur majeur de l'expression des transcrits codant les protéines de la voie FANC dans les cellules de mélanomes et montré que les cellules déplétées pour MITF présentaient les caractéristiques des cellules FA, i.e., une hypersensibilité aux agents pontants l'ADN. De plus, la voie FANC module également la migration des cellules de mélanome. Nos observations montrent le rôle central de cette voie de réparation dans la résistance des cellules de mélanomes aux dommages de l'ADN. Cette voie serait donc une nouvelle cible thérapeutique dans le traitement du mélanome.Nous avons également observé que la perte de fonction de la voie FANC augmente l'expression de MiTF dans des cellules FA et des cellules déplétées pour les protéines FANC. Nous avons montré que la voie FANC régule négativement l'expression de MITF au niveau transcriptionnel. Des résultats préliminaires montre que FANCD2 pourrait être associés au promoteur de MiTF au niveau d'un site de fixation de NF-kB, où il pourrait empêcher son action. La déplétion de MiTF conduit à une sensibilité aux agents pontants l'ADN dans des cellules contrôles mais pas dans des cellules FA, suggérant que MITF jouerait un rôle dans la DDR en régulant l'expression des protéines FANC. Enfin, nous avons montré que l'expression de MiTF est induite en réponse aux stimuli pro-inflammatoire. Enfin, l'expression altérée de MITF pourrait expliquer des défauts de pigmentation et la microphthalmie rapportés chez les patients FA. L'ensemble de ces données, à la fois validée et préliminaire, supporte l'existence d'une relation épistatique entre MiTF et la voie FANC. Cet voie aurait un rôle important à la fois dans la résistance au mélanome métastatique et dans certaines caractéristiques pathologiques de l'anémie de Fanconi. / Proteins and pathways involved in DNA damage response (DDR), maintaining genetic stability and safeguarding DNA replication, act not only as caretakers against cancer initiation but also play a major role in sustaining cancer progression and resistance to pharmacological-based therapies. The FANC pathway is central in maintaining genetic stability under conditions of replication stress and its loss-of-function is causative of the cancer predisposition and chromosome fragility syndrome Fanconi Anemia (FA).We demonstrate here that FANC proteins are over-expressed and over-activated in metastatic melanoma cells expressing the oncogenic microphthalmia-associated transcription factor (MiTF), which high expression is maintained in 80% of melanoma cases. We identified MiTF as a critical regulator of the expression of the mRNAs coding key proteins of the FANC pathway in melanoma cells and demonstrated that MiTF-silenced cells display the primary characteristics of FA cells, i.e., the cellular and chromosomal hypersensitivity to DNA interstrand crosslink- inducing agents. Moreover, FANC pathway also modulates melanoma cell migration. Our observations point to a central role of the FANC pathway in cellular and chromosomal resistance to DNA damage in melanoma cells. Thus, the FANC pathway appears as a promising new therapeutic target for melanoma treatment.Inversely, we observed that FANC pathway loss-of-function is associated to increased expression of MiTF in both FA patient-derived and siRNA-downregulated cells. We demonstrated that the FANC pathway negatively regulates MiTF expression at the mRNA level and have obtained preliminary data suggesting that FANCD2 associates to the MiTF promoter, impeding the action of the NF-kB transcription factor. MiTF depletion increases MMC sensitivity in FANC pathway proficient cells, but does not modify the sensitivity of FA cells, supporting the hypothesis that MiTF acts on the DDR by regulating the expression of FANC proteins. Finally, we demonstrated that MiTF expression is induced in response to inflammatory stimuli, like TNF-a. Thus, altered MiTF expression in FA could be involved in the pigmentation defects and microphthalmia reported in patients.In conclusion, we will present a corpus of both validated and yet preliminary data that strongly supports the existence of an epistatic relationship between MiTF and the FANC pathway. This circuitry appears to have an important role in melanoma resistance to chemotherapies and in some FA pathological traits.

Neural crest cells are a transient population of cells which differentiate into multiple derivatives. How these derivatives become specified is not well understood but Sox10 is known to be important in many of them. We are interested in defining the precise role of Sox10 in zebrafish melanophores. Current evidence suggests that the only vital function that Sox10 performs in melanophores is to induce expression of the melanocyte master regulator mitfa (Elworthy et al. 2003). We explored a model for Sox10 function in melanophores, based upon a model for Sox10’s role in mouse sympathetic neurons (Kim et al. 2003), and tested the following predictions: as well as inducing expression of mitfa, Sox10 will repress expression of genes downstream of Mitfa thus, Sox10 must be downregulated, via Mitfa, to allow melanophore differentiation. We observed derepression of melanophore marker genes in sox10t3 mutants, supporting the hypothesis that Sox10 represses these genes in wild type melanophores. We documented Sox10/sox10 downregulation in developing melanophores and generated transgenic lines to test whether this is necessary for differentiation. Unfortunately our experimental lines did not express our transgene so we were unable to test this hypothesis. However, transgenic lines, generated as controls, which express CFP in melanophores or xanthophores will be useful tools in their own right. Finally we conducted RNA injection experiments to explore regulation of melanophore genes by Sox10 and Mitfa. We found that injection of mitfa induces expression of all our melanophore markers whereas co-injection of mitfa and sox10 does not. We also found that the 7.2 kb sox10 promoter contains six Mitf binding sites and is Mitfa responsive. Our data broadly support our original model but also suggest that it does not describe the complete network. We propose a modified model for the role of Sox10 in the genetic regulatory network controlling melanophore development.

No description available.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic syndrome characterized by the growth of benign tumors in multiple organ systems including brain, lung, kidney, skin, and heart. Kidney angiomyolipoma (AML) are benign, slow growing renal tumors that are seen in about 80% of TSC patients, but also occur sporadically. Although heterogeneous in nature, AMLs have a relatively low somatic mutation rate compared to most other cancers, with biallelic loss of either TSC1 or TSC2 gene considered as the primary and sufficient driver for tumor development. We hypothesized that epigenetic alterations of the AML chromatin landscape change the transcriptional dynamics of the underlying genetic system that supports and gives rise to the tumor-cell phenotype. Our data have identified microphthalmia-associated transcription factor (MITF) to be an orchestrating gene in AML development, as 6 out of the top 10 differentially expressed genes in AML are putative MITF-target genes. Integrative analysis of RNA Seq (n=28), H3K27ac ChIP Seq (n=25) and MITF ChIP Seq data (n=3), obtained from fresh-frozen kidney AML specimens, has enabled us to characterize components of a tumor-specific regulatory network under the transcriptional control of MITF. This novel approach has the potential to identify a variety of therapeutic targets, as well as provide unprecedented insight into the mechanisms behind angiomyolipoma development. / 2021-06-07T00:00:00Z

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.

Melanoma is the most lethal type of skin cancer with over 132,000 cases occurring globally each year and continually rising incidence. BRAFV600E inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance. Evidence from human melanoma indicates that the melanocyte lineage is critical for melanoma survival and contributes to therapeutic resistance. MITF is a highly conserved “master melanocyte transcription factor” with a complex role in melanoma. Our lab has previously developed a temperature sensitive BRAFV600E mitfavc7 zebrafish melanoma model carrying a human oncogene and mitfavc7 splice site mutation that enables the conditional control of its endogenous activity by changes to water temperature. As part of my PhD project, I characterized and compared two new models developed since then: a very aggressive BRAFV600E mitfavc7p53M214K melanoma model with three driving mutations and a slower developing BRAF-independent mitfavc7p53M214K. I showed that the MITF activity is crucial for melanocyte survival in both models and that both mutated BRAF and p53 deficiency are oncogenic with low levels of MITF, and result in fish nevi and melanoma resembling the pathology of human disease. Both models are also relevant to a low-MITF subclass of human melanomas that emerged from a recent classification by The Cancer Genome Atlas Network. In addition, I established that, similarly to the BRAFV600Emitfavc7, complete inhibition of MITF activity leads to rapid tumour regression, but once its activity is restored the melanomas recur at the same site as the original tumour. I used histopathology studies and melanocyte lineage transgenes to identify and visualize subpopulations of cells remaining at the site of regression in these new zebrafish melanoma models. I hypothesised that these are the cells of origin for tumour recurrence (melanoma stem or progenitor cells), showed that some of them express a cancer stem cell marker aldehyde dehydrogenase, and attempted to target these subpopulations using 5-nitrofurans (a prodrug NFN1, shown previously by our lab to target ALDHhigh subpopulations in context of melanoma) in fish after melanoma regression. Finally, I also developed and described a new primary zebrafish melanoma cell line that I derived from one of these zebrafish tumours. This study is still in progress, but the cell line will be a useful tool for further investigation of these proposed melanoma progenitor cells in vitro, with potential applications for lineage tracing and transplantations.

MITF (MIcrophthalmia-associated Transcription Factor) contrôle de multiples aspects de la physiopathologie du lignage mélanocytaire. Par des techniques de génomique haut débit (ChIP-seq, RNA-seq), nous avons montré que MITF active un ensemble de gènes impliqués dans la réplication et la réparation de l’ADN ainsi que la mitose pour stimuler la prolifération des cellules de mélanome, et réprime des gènes contrôlant leur caractère invasif. Pour étudier le mécanisme d’action de MITF, son interactome a été déterminé par spectrométrie de masse mettant en évidence de nombreux partenaires à activité co-activateur ou co-répresseur (bcaténine, complexes de remodelage de la chromatine BRG1 et NURF) ainsi que des facteurs intervenant dans le cycle de l’ubiquitination et de déubuquitination (HERC2 et USP11). Une caractérisation fonctionnelle de HERC2 et USP11 suggère qu’ils agissent comme des cofacteurs transcriptionnels de MITF essentiels pour la prolifération des cellules de mélanome. / MITF (MIcrophthalmia-associated Transcription Factor) controls multiple aspects of the physiopathology of the melanocyte lineage. Using high throughput genomics techniques (ChIP-seq, RNA-seq), we show that MITF activates a set of genes involved in DNA replication and repair as well as mitosis to promote melanoma cell proliferation, while repressing genes involved in promoting their invasion. To better understand how MITF acts both as a transcriptional activator and repressor, we characterized the MITF interactome by tandem immuno-affinity purification and mass-spectrometry. A complex set of partners with coactivatoror co-repressor properties were identified (b-catenin, the BRG1 and NURF chromatin remodeling complexes) as well as novel factors with ubiquitin E3 ligase (HERC2) and ubiquitin-specific protease (USP11) activities. Functional characterization of HERC2 and USP11 suggests that they act as transcriptional cofactors for MITF essential for melanoma cell proliferation.

Zebrafish (Danio rerio) show a unique pigmentation pattern comprised of three pigment cell types: melanophores, iridophores and xanthophores. Other pigmented cells include the retinal pigmented epithelium (rpe) which absorbs excess light in the eye and maintain the extracellular environment around the photoreceptors. While previous mutations in mitfa showed a role in regulating trunk melanophores, the rpe was not affected. TALENs and CRISPR-Cas9 systems were used to generate mutant zebrafish for tfec, a transcription factor expressed in both neural crest and rpe. Embryos with tfec mutations showed a loss of iridophore pigmentation, and delays in the pigmentation of xanthophores and rpe, showing positive regulation of multiple pigment cells. Double mutants for tfec and mitfa displayed greater losses of iridophore, xanthophore and rpe pigmentation with noncircular globes, suggesting cooperative roles for these transcription factors.