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The WHO classification identifies nine classes of melanocytic proliferations according to location, UV exposure, histological, and genetic features. Only a minority of lesions remain unclassified. We describe five cases that harbored either an ERBIN-RASGRF2 or an ATP2B4-RASGRF2 in-frame fusion transcript. These lesions were collected from different studies, unified only by the lack of identifiable known mutations, with a highly variable phenotype. One case was a large abdominal congenital nevus, three were slowly growing pigmented nodules, and the last was an ulcerated nodule arising on the site of a preexisting small nevus, known since childhood. The latter was diagnosed as a 4 mm thick melanoma with loss of BAP1 expression. The four other cases were compound, melanocytic proliferations with an unusual deep pattern of small dense nests of bland melanocytes encased in a fibrous background. The RASGRF2 fusion was confirmed by a break-apart FISH technique. Array CGH performed in three cases found non-recurrent secondary copy number alterations. Follow-up was uneventful. In silico analysis identified a single RASGRF2 fusion in the TCGA pan-cancer database, whereas RASGRF2 variants were stochastically distributed in all cancer subtypes.

International audience

Dans le cadre du module d’enseignement Communication Scientifique et Littérature du Master Biologie Moléculaire et Cellulaire de Lyon, les étudiants des parcours M2 Génopath et Biologie de la Peau se sont formés à l’écriture scientifique sur un sujet libre. Suite à un travail préparatoire avec l’équipe pédagogique, chaque étudiant a rédigé, conseillé par un chercheur, une Nouvelle. Le parcours M2 Génopath s’adresse aux étudiants scientifiques et médecins et les forme à la recherche fondamentale dans les domaines de la génétique, de la biologie cellulaire et de leurs applications biomédicales. Le parcours M2 Biologie de la Peau est une formation unique en France, et forme des spécialistes de la recherche en biologie cutanée qui s’inséreront dans les services de recherche et développement hospitalier ou de l’industrie dermo-cosmétique et dermo-pharmaceutique.

Due to its high proclivity to metastasize, and despite the recent development of targeted and immune therapy strategies, melanoma is still the deadliest form of skin cancer. Therefore, understanding the molecular mechanisms underlying melanoma invasion remains crucial. We previously characterized Tspan8 for its ability to prompt melanoma cell detachment from their microenvironment and trigger melanoma cell invasiveness, but the signaling events by which Tspan8 regulates the invasion process still remain unknown. Here, we demonstrated that β-catenin stabilization is a molecular signal subsequent to the onset of Tspan8 expression, and that, in turn, β-catenin triggers the direct transcriptional activation of Tspan8 expression, leading to melanoma invasion. Moreover, we showed that β-catenin activation systematically correlates with a high expression of Tspan8 protein in melanoma lesions from transgenic Nras; bcat* mice, as well as in deep penetrating naevi, a type of human pre-melanoma neoplasm characterized by a combined activation of β-catenin and MAP kinase signaling. Overall, our data suggest that β-catenin and Tspan8 are part of a positive feedback loop, which sustains a high Tspan8 expression level, conferring to melanoma cells the invasive properties required for tumor progression and dissemination.

Since the crucial role of the microenvironment has been highlighted, many studies have been focused on the role of biomechanics in cancer cell growth and the invasion of the surrounding environment. Despite the search in recent years for molecular biomarkers to try to classify and stratify cancers, much effort needs to be made to take account of morphological and nanomechanical parameters that could provide supplementary information concerning tissue complexity adaptation during cancer development. The biomechanical properties of cancer cells and their surrounding extracellular matrix have actually been proposed as promising biomarkers for cancer diagnosis and prognosis. The present review first describes the main methods used to study the mechanical properties of cancer cells. Then, we address the nanomechanical description of cultured cancer cells and the crucial role of the cytoskeleton for biomechanics linked with cell morphology. Finally, we depict how studying interaction of tumor cells with their surrounding microenvironment is crucial to integrating biomechanical properties in our understanding of tumor growth and local invasion.

Human skin is particularly vulnerable to age-related deterioration and undergoes profound structural and functional changes, reflected in the external skin appearance. Skin ageing is characterized by features such as wrinkling or loss of elasticity. Even if research advances have been done concerning the molecular mechanisms that underlie these changes, very few studies have been conducted concerning the structure stiffness of the skin organ as a whole. In this study, we showed, thanks to human skin reconstructs and the Japanese Medaka fish model, that biomechanics is a new biomarker of skin ageing. We revealed that global stiffness measurement by Atomic Force Microscopy, since modulated through ageing in these models, can be a new biomarker of skin ageing, and reflects the profound reorganization of the dermis extracellular matrix, as shown by Transmission Electron Microscopy. Moreover, our data unveiled that the Japanese Medaka fish could represent a highly relevant integrated model to study skin ageing in vivo.

// Manale El Kharbili 1, 2, 3, 4 , Clement Robert 1, 2, 3 , Tiffany Witkowski 5, 6 , Emmanuelle Danty-Berger 7 , Laetitia Barbollat-Boutrand 1, 2, 3 , Ingrid Masse 1, 2, 3 , Nicolas Gadot 8 , Arnaud de la Fouchardiere 9 , Paul C. McDonald 10 , Shoukat Dedhar 10 , Francois Le Naour 11, 12 , Francoise Degoul 5, 6 , Odile Berthier-Vergnes 1, 2, 3 1 Universite de Lyon, Lyon, France 2 Universite Lyon 1, Lyon, France 3 CNRS, UMR5534, Centre de Genetique et de Physiologie Moleculaire et Cellulaire, Villeurbanne, France 4 Current address: Department of Dermatology, University of Colorado, Aurora, Colorado, USA 5 Clermont Universite, Universite d’Auvergne, Imagerie Moleculaire et Therapie Vectorisee, Clermont-Ferrand, France 6 Inserm, U990, Clermont-Ferrand, France 7 Laboratoire CarMeN (INSERM 1060, INRA1397, INSA), Universite Lyon 1, Lyon, France 8 Universite Lyon 1, Federation de Recherche Sante Lyon-Est, ANIPATH, Faculte Laennec, Lyon, France 9 Departement de Biopathologie, Centre Leon Berard, Lyon, France 10 Department of Integrative Oncology, British Columbia Cancer Research Center, Vancouver, Canada 11 INSERM U602, Villejuif, France 12 Current address: INSERM U1193, Hopital Paul Brousse, Villejuif, France Correspondence to: Odile Berthier-Vergnes, email: odile.berthier-vergnes@univ-lyon1.fr Keywords: melanoma, matrix, integrin, tetraspanin 8, ILK Received: November 09, 2016 Accepted: January 09, 2017 Published: February 04, 2017 ABSTRACT Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with β1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced β 1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through β1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.

International audience; We report a series of 33 skin tumors harboring a gene fusion of the MAP3K8 gene, which encodes a serine/threonine kinase. The MAP3K8 fusions were identified by RNA sequencing in 28 cases and by break-apart FISH in five cases. Cases in which fusion genes were fully characterized demonstrated a fusion of the 5' part of MAP3K8 comprising exons 1-8 in frame to one of several partner genes at the 3' end. The fusion genes invariably encoded the intact kinase domain of MAP3K8, but not the inhibitory domain at the C-terminus. In 13 (46%) of the sequenced cases, the 3' fusion partner was SVIL. Other recurrent 3' partners were DIP2C and UBL3, with additional fusion partners that occurred only in a single tumor. Clinically, the lesions appeared mainly in young adults (2-59 years of age; median = 18), most commonly involving the lower limbs (55%). Five cases were diagnosed as Spitz nevus, 13 as atypical Spitz tumor, and 15 as malignant Spitz tumor. Atypical and malignant cases more commonly occurred in younger patients. Atypical Spitz tumors and malignant Spitz tumors cases tended to show epidermal ulceration (32%), a dermal component with giant multinucleated cells (32%), and clusters of pigmented cells in the dermis (32%). Moreover, in atypical and malignant cases, a frequent inactivation of CDKN2A (21/26; 77%) was identified either by p16 immunohistochemistry, FISH, or comparative genomic hybridization. Gene expression analysis revealed that MAP3K8 expression levels were significantly elevated compared to a control group of 57 Spitz lesions harboring other known kinase fusions. Clinical follow-up revealed regional nodal involvement in two of six cases, in which sentinel lymph node biopsy was performed but no distant metastatic disease after a median follow-up time of 6 months.

MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.

Tetraspanins are transmembrane proteins that interact laterally with each other and with different partners such as integrins, immunoglobulin (Ig)-domain-containing proteins, growth factors and cytokine receptors. Such tetraspanin-partner complexes help to organize dynamic membrane networks called "tetraspanin web", which trigger different signalling pathways. Despite the fact that tetraspanins seem abundantly and widely expressed, their function remained unclear. However, it is well established that they control fundamental cellular processes including cell survival, adhesion, migration, invasion or viral infection, but the underlying molecular mechanisms are not well elucidated. This review focuses on tetraspanins that are expressed in epidermis and the roles they play in normal and pathological conditions, specifically in skin cancer.

La peau humaine est particulierement sensible au vieillissement et les profonds changements structurels et fonctionnels qu’elle subit alterent rapidement son apparence. Malgre le developpement de modeles 3D in vitro pour etudier le vieillissement cutane, l’utilisation de modeles animaux reste cruciale pour caracteriser de facon integree les mecanismes moleculaires et cellulaires qui sous-tendent le vieillissement de la peau. Notre etude revele que le poisson japonais Medaka pourrait etre un modele pertinent pour etudier le vieillissement cutane in vivo. En effet, nous avons mis en evidence une augmentation du nombre de cellules senescentes (SA-βgal) avec l’âge dans la peau du Medaka ainsi qu’une modulation de l’expression de gerontogenes, comme dans la peau humaine. Nous avons egalement montre, que d’un point de vue biomecanique, la mesure de rigidite par microscopie a force atomique (AFM) constitue un nouveau biomarqueur du vieillissement cutane. Nous avons enfin determine par microscopie electronique a transmission (TEM) que cette modulation de rigidite cutanee reflete une reorganisation profonde du derme au cours du vieillissement. Ainsi, le poisson Medaka pourrait etre un modele tres pertinent pour l’etude du vieillissement cutane, en particulier via l’etude de la rigidite cutanee.

La recherche d’anomalies “driver” dans les tumeurs melanocytaires cutanees s’est considerablement amelioree suite au developpement des techniques de criblage genomique de masse sur du materiel FFPE. Nous rapportons ici une serie de 33 lesions cutanees presentant une fusion genique qui implique la serine/threonine kinase MAP3K8 et qui conduit a la conservation du domaine kinase mais a la perte de son domaine regulateur C-terminal. La grande majorite des lesions est de morphologie spitzoide, couvrant le spectre complet : naevus benin de Reed, tumeur de Spitz atypique et tumeur maligne. Dans 46 % des cas sequences, le partenaire de fusion en 3’ est SVIL, un gene codant pour la supervilline, une proteine capable de liaison au cytosquelette d’actine. Les cas MAP3K8-SVIL sont caracterises par une morphologie inhabituelle, presentant une ulceration epidermique, un composant cutane avec des cellules multinucleees geantes et des clones pigmentes dermiques focaux. Certaines de ces tumeurs melanocytaires presentant un potentiel metastatique et des fusions MAP3K8 ayant egalement ete identifiees dans d’autres types de tumeurs agressives, il apparait crucial de mieux comprendre les mecanismes moleculaires associes, afin d’envisager eventuellement des therapeutiques ciblant la voie MAPkinase.

Le melanome cutane est le plus grave des cancers de la peau en raison de sa capacite a metastaser, et ce, malgre le developpement recent de nouvelles therapies ciblees et immunes. Mieux comprendre les mecanismes moleculaires qui sous-tendent l’initiation et la progression tumorale du melanome est donc crucial pour ameliorer la survie des patients. Nous avons precedemment identifie un nouvel acteur crucial pour l’invasion precoce du melanome cutane : la Tetraspanine 8, dont l’expression est correlee au potentiel invasif des cellules de melanome (Berthier-Vergnes et al., 2011). Un criblage par interference a l’ARN nous a permis d’identifier plusieurs regulateurs transcriptionnels de Tspan8 : le membre du Complexe Mediateur LCMR1/MED19 (Agaesse et al., Oncogene 2017), le suppresseur de tumeurs p53 (Agaesse et al., Oncogenesis 2017) et la proteine b-catenine (El Kharbili et al., Oncogene 2019). Nos donnees recentes suggerent que les facteurs de transcription qui regulent la transition epithelio-mesenchymateuse seraient egalement des modulateurs de l’expression endogene de Tspan8. Nous developpons actuellement des modeles in vivo chez le poisson Medaka pour etudier l’impact de la regulation fonctionnelle de Tspan8 par ces facteurs dans la progression tumorale du melanome cutane.

Tetraspanins are transmembrane proteins that interact laterally with each other and with different partners such as integrins, immunoglobulin (Ig)-domain-containing proteins, growth factors and cytokine receptors. Such tetraspanin-partner complexes help to organize dynamic membrane networks called "tetraspanin web", which trigger different signalling pathways. Despite the fact that tetraspanins seem abundantly and widely expressed, their function remained unclear. However, it is well established that they control fundamental cellular processes including cell survival, adhesion, migration, invasion or viral infection, but the underlying molecular mechanisms are not well elucidated. This review focuses on tetraspanins that are expressed in epidermis and the roles they play in normal and pathological conditions, specifically in skin cancer.

Dietary restriction (DR) is the most universal intervention known to extend animal lifespan. DR also prevents tumor development in mammals, and this effect requires the tumor suppressor PTEN. However, the metabolic and cellular processes that underly the beneficial effects of DR are poorly understood. We identified slcf-1 in an RNAi screen for genes that extend Caenorhabditis elegans lifespan in a PTEN/daf-18-dependent manner. We showed that slcf-1 mutation, which increases average lifespan by 40%, mimics DR in worms fed ad libitum. An NMR-based metabolomic characterization of slcf-1 mutants revealed lower lipid levels compared to wild-type animals, as expected for dietary-restricted animals, but also higher pyruvate content. Epistasis experiments and metabolic measurements support a model in which the long lifespan of slcf-1 mutants relies on increased mitochondrial pyruvate metabolism coupled to an adaptive response to oxidative stress. This response requires DAF-18/PTEN and the previously identified DR effectors PHA-4/FOXA, HSF-1/HSF1, SIR-2.1/SIRT-1, and AMPK/AAK-2. Overall, our data show that pyruvate homeostasis plays a central role in lifespan control in C. elegans and that the beneficial effects of DR results from a hormetic mechanism involving the mitochondria. Analysis of the SLCF-1 protein sequence predicts that slcf-1 encodes a plasma membrane transporter belonging to the conserved monocarboxylate transporter family. These findings suggest that inhibition of this transporter homolog in mammals might also promote a DR response.

International audience; Circadian rhythms are observed in nearly all aspects of physiology and behavior. In mammals, such biological rhythms are supported by a complex network of self-sustained transcriptional loops and posttranslational modifications, which regulate timely controlled production and degradation of critical factors on a 24-h basis. Among these factors, the orphan nuclear receptor rev-erbalpha plays an essential role by linking together positive and negative regulatory loops. As an essential part of the circadian core clock mechanism, REV-ERBalpha expression shows a precisely scheduled oscillation reflecting the tight control of its production and degradation. In previous studies, we identified two alternative transcripts encoding two protein variants referred to as REV-ERBalpha1 and -alpha2. Interestingly, recent work identified structural elements present only in REV-ERBalpha1 that controls its turnover and thereby influences circadian oscillations. In the present work, we comparatively analyze the two variants and show that REV-ERBalpha2 exhibits a half-life incompatible with a circadian function, suggesting that this variant exerts different biological functions. However, our comparative study clearly indicates undistinguishable DNA-binding properties and transcriptional repression activity as well as a similar regulation mechanism. The only consistent difference appears to be the relative expression level of the two transcripts, rev-erbalpha1 being one to 100 times more expressed than alpha2 depending on tissue and circadian time. Taking this finding into consideration, we reassessed REV-ERBalpha2 turnover and were able to show that this variant exhibits a reduced half-life when coexpressed with REV-ERBalpha1. We propose that the relative expression levels of the two REV-ERBalpha variants fine-tune the circadian period length by regulating REV-ERBalpha half-life.

In Caenorhabditis elegans, the insulin/IGF-1 DAF-2 receptor controls entry into dauer and longevity. DAF-2 signaling cascade includes the PI3 kinase homolog AGE-1 and the FOXO transcription factor DAF-16. The DAF-2 pathway is downregulated by DAF-18 which is encoded by the ortholog of the human tumor suppressor gene PTEN. We have previously shown that, like PTEN, DAF-18 antagonizes the activity of PI3 kinase/AGE-1. To further explore the role of DAF-18 in the regulation of the insulin pathway, we investigated which tissue(s) DAF-18 functions in to regulate dauer formation and lifespan. Our data show that complete dauer formation requires daf-18 expression in several tissues and that the remodeling of dauer tissues depends on both cell autonomous and cell nonautonomous daf-18 function(s). Conversely, daf-18 expression increases adult lifespan in all individual tissues tested. Furthermore, we show that the role of DAF-18 in dauer and lifespan control depends on DAF-16 activation, which is regulated by both cell autonomous and cell nonautonomous DAF-18 function(s) and in a tissue-specific manner. Overall, our data strongly suggest that several tissues act as signaling centers to mediate DAF-18 function and that DAF-18 could act outside the canonical DAF-2/DAF-16 pathway to regulate dauer and lifespan.

Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3 beta, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.

GATA3 belongs to the GATA transcription factor family and is a crucial regulator of lymphocyte differentiation. More recently, GATA3 was shown to be involved in skin cell lineage determination, in morphogenesis and maintenance of hair follicle keratinocytes as well as in epidermal barrier formation in mouse. In human, the potential role of GATA3 in the regulation of interfollicular epidermal homeostasis was still poorly explored. We thus investigated whether GATA3 could play a role in the regulation of proliferation and/or differentiation processes in human primary keratinocytes. We silenced the expression of GATA3 by small interfering RNA in either proliferating or differentiated human primary keratinocytes and analyzed the effect on cell proliferation and differentiation. We showed that GATA3 inhibition increased cell number, BrdU incorporation and expression of the proliferation markers PCNA and Ki67, demonstrating that GATA3 can inhibit keratinocyte proliferation. Moreover, GATA3 seems to be able to induce keratinocyte differentiation since its silencing leads to a decrease of both early and late differentiation markers such as Keratins 1 and 10, Involucrin and Loricrin. Our results demonstrate that GATA3 transcription factor inhibits proliferation and induces differentiation of primary keratinocytes, which suggest that it may regulate human interfollicular epidermal renewal.

International audience; Although p63 and MYC are important in the control of epidermal homeostasis, the underlying molecular mechanisms governing keratinocyte proliferation or differentiation downstream of these two genes are not completely understood. By analyzing the transcriptional changes and phenotypic consequences of the loss of either p63 or MYC in human developmentally mature keratinocytes, we have characterized the networks acting downstream of these two genes to control epidermal homeostasis. We show that p63 is required to maintain growth and to commit to differentiation by two distinct mechanisms. Knockdown of p63 led to down-regulation of MYC via the Wnt/β-catenin and Notch signaling pathways and in turn reduced keratinocyte proliferation. We demonstrate that a p63-controlled keratinocyte cell fate network is essential to induce the onset of keratinocyte differentiation. This network contains several secreted proteins involved in cell migration/adhesion, including fibronectin 1 (FN1), interleukin-1β (IL1B), cysteine-rich protein 61 (CYR61), and jagged-1 (JAG1), that act downstream of p63 as key effectors to trigger differentiation. Our results characterized for the first time a connection between p63 and MYC and a cell adhesion-related network that controls differentiation. Furthermore, we show that the balance between the MYC-controlled cell cycle progression network and the p63-controlled cell adhesion-related network could dictate skin cell fate.

International audience; The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.

International audience; Circadian regulation of gene expression plays a major role in health and disease. The precise role of the circadian system remains to be clarified, but it. is known that circadian proteins generate physiological rhythms in organisms by regulating clock-controlled target genes. The estrogen receptor beta (ER beta) is, together with ER alpha, a member of the nuclear receptor superfamily and a key mediator of estrogen action. Interestingly, recent studies show that disturbed circadian rhythmicity in humans can increase the risk of reproductive malfunctions, suggesting a link between the circadian system and ER-mediated transcription pathways. Here, we identify a novel level of regulation of estrogen signaling where ER beta, but not ER alpha, is controlled by circadian clock proteins. We show that ER beta mRNA levels fluctuate in different peripheral tissues following a robust circadian pattern, with a peak at the light-dark transition, which is maintained under free-running conditions. Interestingly, this oscillation is abolished in clock-deficient BMAL1 knockout mice. Circadian control of ER beta expression is exerted through a conserved E-box element in the ER beta promoter region that recruits circadian regulatory factors. Furthermore, using small interfering RNA-mediated knockdown assays, we show that the expression levels of the circadian regulatory factors directly influence estrogen signaling by regulating the intracellular levels of endogenous ER beta.

The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.

Melanocytic tumors represent the cancers with the highest incidence in recent years. They are ranked 6th among the most frequent cancers in the world. In order to better understand and adapt treatments to different melanocytic tumors, a classification has been proposed over the years. It evolves according to the advancement of knowledge on melanocytic tumors and, thanks to the development of molecular biology analysis techniques, now takes into account the genetic abnormalities encountered. Nevertheless, this multidimensional classification remains imperfect, since some patient samples remain unclassified either from a histological point of view or from a genetic one. During my thesis work, two axes were developed. On one hand, we have chosen to characterize, using FISH, aCGH and RNAseq techniques, a subgroup of 33 Spitz tumors with common morphologies and whose main molecular abnormality is a fusion involving the MAP3K8 gene. On the other hand, we have shown that the RASGRF2 gene was involved in a fusion found in five cases of melanocytic tumors with similar morphologies. Overall, my thesis work allows to underline MAP3K8 and RASGRF2 gene fusions as important gene events during melanomagenesis and will improve the diagnosis of melanocytic pathologies as well as patients care. This work will be continued to further improve the classification of melanocytic tumors., Les tumeurs mélanocytaires représentent les cancers avec la plus forte incidence ces dernières années. Elles se classent au 6ème rang des cancers les plus fréquents dans le monde. Afin de mieux comprendre et d’adapter les traitements aux différentes tumeurs mélanocytaires, une classification a été proposée au fil des ans. Elle évolueen fonction de l’avancée des connaissances sur les tumeurs mélanocytaireset, grâceeu développement des techniques d’analyse de biologie moléculaire, prend aujourd’hui en compte les anomalies génétiques rencontrées. Malgré tout, cette classification multidimensionnelle reste imparfaite, car certains échantillons demeurent inclassés soit du point de vue histologique soit du point de vue génétique. Au cours de mon travail de thèse, deux axes ont été développés. D’une part, nous avons choisi de caractériser, grâce à des techniquesde FISH, aCGH et RNAseq, un sous-groupe de 33 tumeurs de Spitz présentant des morphologies communes et ayant pour anomalie moléculaire principale une fusion impliquant le gène MAP3K8. D’autre part, nous avons montré que le gène RASGRF2 était impliqué dans une fusion retrouvée dans cinq cas de tumeurs mélanocytaires présentant des morphologies similaires. L’ensemble de mes travaux de thèse permet à présent d’inscrire les fusions de gènes MAP3K8 et RASGRF2 comme des évènements géniques importants au cours de la mélanomagenèse et permettra d’améliorer le diagnostic des pathologies mélanocytaires des patients ainsi que leur prise en charge. Ce travail sera poursuivi pour continuer d’améliorer la classification des tumeurs mélanocytaires.

Cutaneous melanoma is the deadliest skin condition. Curing this cancer requires an early diagnosis and surgical excision of the non-invasive primary lesions. Indeed, patients with metastasis have few chances to survive this cancer since the melanoma lesions rapidly develop treatment resistances, and can disseminate metastasis in numerous organs. Crossing the skin basal membrane called the dermal epidermal junction is the first crucial step of early melanoma invasion. Our team has studied the early invasion of melanoma for many years, and demonstrated for the first time the implication of Tetraspanin (TSPAN8) in melanoma early invasion. Indeed, the expression of this gene and the protein that it codes appears with the progression of melanoma and confers the tumor cells an invasive phenotype. Several members of the tetraspanin protein family are known for their implication in various cancers, yet their transcriptional regulation remains poorly understood. In the case of TSPAN8, nothing was known regarding its transcriptional regulation in melanoma. The experiments presented in this thesis allowed us to identify the first known regulators of TSPAN8 transcriptional expression, and also to begin the functional study of the regulators impact on TSPAN8 dependent invasion of human cutaneous melanoma. Amongst these regulators are the member of the Mediator Complex MED19/LCMR1 and the tumor suppressor p53. The results presented in the present manuscript allow a better understanding of cutaneous melanoma early invasion and should help improving the treatments against this cancer in the future, Le mélanome cutané est l’affection de la peau la plus meurtrière. Afin de pouvoir être traité efficacement, ce cancer nécessite un diagnostic et une exérèse chirurgicale précoce des lésions primitives non-invasives. En effet, les patients atteints de métastases ont peu de chance de survivre car les lésions de mélanome développent rapidement des résistances aux thérapies et possèdent une forte propension à disséminer des métastases dans de nombreux organes. Le franchissement de la lame basale de la peau appelée jonction dermo épidermique est la première étape cruciale dans l’invasion précoce du mélanome cutané. Notre équipe étudie cette étape depuis plusieurs années et a démontré que l’expression de la tétraspanine 8 (TSPAN8) apparait lors de la progression de ce cancer et permet l’acquisition d’un phénotype invasif par les cellules tumorales. Plusieurs membres de la famille des protéines tétraspanines sont connus pour leurs implications dans divers cancers, mais notre connaissance de leurs régulations transcriptionnelles est encore assez réduite. Les travaux présentés dans cette thèse ont permis d’identifier les premiers régulateurs transcriptionnels connus de TSPAN8, et également de commencer l’étude fonctionnelle de ces régulations sur l’invasion dépendante de TSPAN8 par le mélanome cutané. En particulier, nous montrons que l’invasion dépendante de TSPAN8 est entre autres régulée par le membre du complexe Mediator LCMR1/MED19 et par le suppresseur de tumeur p53. Ainsi, ces travaux apportent une meilleure compréhension de l’invasion précoce du mélanome cutané, ce qui devrait permettre une meilleure prise en charge des patients à l’avenir