Identification and modulation of molecular pathways implicated in cutaneous melanoma aggressiveness

Résumé : Le mélanome est un cancer cutané très agressif caractérisé par une forte capacité métastatique. La forte plasticité des cellules de mélanome limite le potentiel thérapeutique des thérapies ciblées. En effet, malgré des taux de réponse élevés et une diminution du volume tumoral chez la majorité des patients, la quasi-totalité de ces derniers rechutent 6 à 12 mois après le début du traitement. Ces rechutes peuvent être causées par l’apparition de mutations oncogéniques mais également par la reprogrammation transcriptionnelle (plasticité) des cellules de mélanome au cours du traitement. En utilisant la technologie CRISPR-SAM (cribles gain de fonction à l’échelle du génome), nous avons identifié des gènes, dont SMAD3, impliqués dans la survie initiale des cellules de mélanome au cours du traitement ainsi que dans la reprise de la croissance tumorale, qui conduit les patients vers la rechute. Ces gènes constituent des cibles thérapeutiques dont l’inhibition pourrait permettre d’exploiter pleinement le potentiel des thérapies ciblées actuelles. Le deuxième objectif principal de cette thèse a été d’étudier plus spécifiquement les mécanismes participant à la plasticité des cellules de mélanome. Le facteur de transcription MITF est le régulateur central de ce mécanisme. Alors que sa régulation transcriptionnelle est très étudiée, sa régulation post-transcriptionnelle l’est beaucoup moins. Nous démontrons que la protéine de liaison aux ARN HuR régule positivement l’expression de MITF, participant ainsi à la plasticité des cellules de mélanome. L’ensemble de ces résultats ouvrent donc de nouvelles perspectives sur la compréhension de la plasticité des cellules de mélanome, mécanisme à l’origine de l’agressivité caractéristique de ce cancer ainsi que de la résistance aux thérapies.Melanoma is a very aggressive skin cancer, characterized by a high metastatic capability. Cell plasticity limits therapeutic potential of targeted therapies. Indeed, despite a high response rate and tumor volume decreased in the majority of patients, almost all patients relapse within 6 to 12 months after treatment start. Relapses can be due to the acquisition of new oncogenic mutations but can also be caused by transcriptional reprogramming (plasticity) of melanoma cells during treatment. Using CRISPR-SAM (gain-of-function genome wide screens), we identified genes implicated in melanoma cells survival during treatment but also tumor growth restart (leading to patient relapses). These genes are new potential therapeutic targets. Inhibition of them could allow patient to fully benefit from current targeted therapies. The second main goal of this thesis was to study more specifically the regulation of the transcription factor MITF in melanoma cell plasticity. MITF is known to be the main regulator of melanoma cell plasticity. Whereas its transcriptional regulator is quite well described, its post-transcriptional regulation has been far less studied. We showed here that the RNA binding protein HuR regulates positively MITF expression, being thus implicated in melanoma cells plasticity. Altogether, these results open new perspectives in the comprehension of melanoma cells plasticity, a mechanism implicated in melanoma aggressiveness and resistance to therapies

Human TYRP1: two functions for a single gene?

International audienceIn the animal kingdom, skin pigmentation is highly variable between species, and it contributes to phenotypes. In humans, skin pigmentation plays a part in sun protection. Skin pigmentation depends on the ratio of the two pigments pheomelanin and eumelanin, both synthesized by a specialized cell population, the melanocytes. In this review, we explore one important factor in pigmentation: the tyrosinase-related protein 1 (TYRP1) gene which is involved in eumelanin synthesis via the TYRP1 protein. Counterintuitively, high TYRP1 mRNA expression is associated with a poor clinical outcome for patients with metastatic melanomas. Recently, we were able to explain this unexpected TYRP1 function by demonstrating that TYRP1 mRNA sequesters microRNA-16, a tumor suppressor miRNA. Here, we focus on actors influencing TYRP1 mRNA abundance, particularly transcription factors, single nucleotide polymorphisms (SNPs), and miRNAs, as they all dictate the indirect oncogenic activity of TYRP1

Inflammasomes involvement in Staphylococcus aureus infection of human osteoblast-like cells MG-63

Inflammation is a coordinated immune response to infections or tissue damage. The inflammasome is a multi-protein signaling platform that assembles after recognition of danger signals and/or pathogens by a family of cytosolic receptors called NLRs (nucleotide-binding domain and leucine rich repeats containing receptors) or PYHIN protein family members. Once assembled, inflammasomes initiate signaling by activation of downstream proteases, most notably Caspase-1 and Caspase-11, which then proteolytically mature pro-IL-1β, pro-IL-18, and pro-IL-33, and promote their secretion from the cell. Furthermore, inflammasome activation triggers pyroptosis, an inflammatory form of cell death. Staphylococcus aureus is a highly adaptive and versatile gram-positive bacterium that has major importance to human and animal health. S. aureus can cause life-threatening infections such as bacteremia, pneumonia, meningitis, endocarditis and sepsis. S. aureus are also the predominant cause of bone infections worldwide. Comprehending the mechanisms by which staphylococci interact with and damage bone is critical to the development of new approaches to meet this challenge. While the role of inflammasomes formed in the different types of phagocytes during S. aureus infection was widely investigated, the involvement of inflammasomes in osteoblast cells have not been studied. Objective: To understand the mechanisms of Bone Joint Infection we investigated the involvement of inflammasomes in the model of persistent infection of human osteoblast-like cells.Materials and Methods: CRISPR/Cas9 technology was used to prepare Caspase-1 deficient line of MG-63 cells. Western blot analysis and ELISA were employed for the detection of activated cytokines.Results: An employment of wild type vs Caspase-1 deficient MG-63 osteoblast-like cells allow demonstrating the involvement of inflammasomes during S. aureus infection. Using deletion mutants and complemented S. aureus strains, we determined the role of its most important virulence factors for their capacity to activate Caspase-1 and produce IL-1β.Conclusions: Non-phagocytic osteoblast-like MG-63 cells form inflammasomes in the response to S. aureus infection, however the time of inflammasomes formation was different compared to the professional phagocytes. Many virulence factors induce the inflammasomes assemblage

Key role of caspase-1 in bacterial clearance during S. aureus infection of osteoblasts-like cells.

International audienceStaphylococcus aureus (S. aureus), a versatile Gram-positive bacterium, is the main cause of bone and joint infections, which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected non-professional phagocytes, human osteoblast-like MG-63 cells, has not yet been fully investigated. To investigate the role of inflammasomes in S. aureus-infected MG-63 cells, the establishement of CASP1–/–MG-63 cell line was carried out using the CRISPR-Cas9 gene editing system. We and others showed that S. aureus can be internalized and survive within professional phagocytes, such as macrophages as well as within non phagocytic epithelial cells or osteoblasts. To study the capacity of intracellular S. aureus to induce inflammasomes formation in MG-63 cells, we confirmed the presence of intracellular bacteria using transmission electron microscopy We show here that S. aureus-infected MG-63 cells but not caspase-1 knock-out CASP1–/–MG-63 cells activate the inflammasome as monitored by the release of mature IL-1β. The effect was strain-dependent. The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr) regulates the expression of many virulence factors including the expression of most S. aureus toxin genes. Of note, the expression of PSMs encoding genes (PSMα 1 to 4, PSMβ 1 and 2, and δ-toxin sometimes called PSMƔ) is tightly controlled by the Agr system. The use of S. aureus LAC (USA300) wild-type strain, its deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated that PSM toxins are involved in inflammasomes-related IL-1β production by infected MG-63 cells. Furthermore, we found that the lack of caspase-1 in CASP1–/–MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1–/– MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance

Involvement of caspase-1 in inflammasomes activation and bacterial clearance in S. aureus-infected osteoblast-like MG-63 cells

International audienceStaphylococcus aureus, a versatile Gram-positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1 beta and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1 beta and in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. We show here that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knock-out CASP1( -/-)MG-63 cells, which were generated using CRISPR-Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1 beta. The effect was strain-dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1 beta production. Furthermore, we found that the lack of caspase-1 in CASP1( -/-)MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1( -/-) MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance

Inflammasome activation and an involvement of Caspase-1 in a bacterial clearance during S. aureus infection.

International audienceStaphylococcus aureus is a highly versatile Gram-positive bacterium that is carried asymptomatically by up to fifty percent of healthy people, while being a major cause of hospital-acquired infections including bone and joint infections, such as osteomyelitis, which are prone to recurrence. S. aureus-caused bovine mastitis is also significant problem in veterinary medicine. The innate immune response plays a pivotal role in the defense against S. aureus. This response is initiated through pattern recognition receptors, and involves an activation of multi-protein signaling complexes known as inflammasomes. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. We investigated the role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected osteoblast-like MG-63 cells. Using CRISPR-Cas9 technology, we demonstrated that MG-63 but not caspase-1 knock-out CASP1−/−MG-63 cells, activate the inflammasome as monitored by the release of mature IL-1β. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1β production. Furthermore, we investigated the role of caspase-1in S. aureus clearance. We found that the lack of caspase-1 in CASP1−/−MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1−/− MG-63 compared to wild-type cells.Using a flow cytometric approach we isolated only S. aureus-bearing cells from mixed populations that allows to identify signals specific to intracellular infection. After RNA-seq and KEGG and Reactome pathway enrichment analysis, the remodeled transcriptomic profile of infected cells revealed exacerbated immune and inflammatory responses, as well as metabolic and epigenetic dysregulations that likely influence the intracellular life of bacteria. Collectively, our results demonstrate that human osteoblast-like cells induce an immune response against S. aureus through inflammasomes activation and processing of IL-1β. The outcome of the infection depends on the balance between the host immune response and the action of main S. aureus virulence factors, such as PSMs, whose production differ among the S. aureus strains. Our results showed that the active caspase-1 prevents exacerbated intracellular replication of S. aureus in non-professional phagocytes in addition to professional phagocytes, suggesting the crucial role of caspase-1 in S. aureus clearance independently from the type of cells. Furthermore, our results provide an atlas of deregulated host genes and biological pathways and identify novel markers and potential candidates for prophylactic and therapeutic approaches

Inflammasome activation and an involvement of Caspase-1 in a bacterial clearance during S. aureus infection.

International audienceStaphylococcus aureus is a highly versatile Gram-positive bacterium that is carried asymptomatically by up to fifty percent of healthy people, while being a major cause of hospital-acquired infections including bone and joint infections, such as osteomyelitis, which are prone to recurrence. S. aureus-caused bovine mastitis is also significant problem in veterinary medicine. The innate immune response plays a pivotal role in the defense against S. aureus. This response is initiated through pattern recognition receptors, and involves an activation of multi-protein signaling complexes known as inflammasomes. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1β and IL-18. We investigated the role of inflammasomes and caspase-1 in the secretion of mature IL-1β and in the defence of S. aureus-infected osteoblast-like MG-63 cells. Using CRISPR-Cas9 technology, we demonstrated that MG-63 but not caspase-1 knock-out CASP1−/−MG-63 cells, activate the inflammasome as monitored by the release of mature IL-1β. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1β production. Furthermore, we investigated the role of caspase-1in S. aureus clearance. We found that the lack of caspase-1 in CASP1−/−MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1−/− MG-63 compared to wild-type cells.Using a flow cytometric approach we isolated only S. aureus-bearing cells from mixed populations that allows to identify signals specific to intracellular infection. After RNA-seq and KEGG and Reactome pathway enrichment analysis, the remodeled transcriptomic profile of infected cells revealed exacerbated immune and inflammatory responses, as well as metabolic and epigenetic dysregulations that likely influence the intracellular life of bacteria. Collectively, our results demonstrate that human osteoblast-like cells induce an immune response against S. aureus through inflammasomes activation and processing of IL-1β. The outcome of the infection depends on the balance between the host immune response and the action of main S. aureus virulence factors, such as PSMs, whose production differ among the S. aureus strains. Our results showed that the active caspase-1 prevents exacerbated intracellular replication of S. aureus in non-professional phagocytes in addition to professional phagocytes, suggesting the crucial role of caspase-1 in S. aureus clearance independently from the type of cells. Furthermore, our results provide an atlas of deregulated host genes and biological pathways and identify novel markers and potential candidates for prophylactic and therapeutic approaches

CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance

International audienceMost genetic alterations that drive melanoma development and resistance to targeted therapy have been uncovered. In contrast, and despite their increasingly recognized contribution, little is known about the non-genetic mechanisms that drive these processes. Here, we performed in vivo gain-of-function CRISPR screens and identified SMAD3, BIRC3, and SLC9A5 as key actors of BRAFi resistance. We show that their expression levels increase during acquisition of BRAFi resistance and remain high in persister cells and during relapse. The upregulation of the SMAD3 transcriptional activity (SMAD3-signature) promotes a mesenchymal-like phenotype and BRAFi resistance by acting as an upstream transcriptional regulator of potent BRAFi-resistance genes such as EGFR and AXL. This SMAD3-signature predicts resistance to both current melanoma therapies in different cohorts. Critically, chemical inhibition of SMAD3 may constitute amenable target for melanoma since it efficiently abrogates persister cells survival. Interestingly, decrease of SMAD3 activity can also be reached by inhibiting the Aryl hydrocarbon Receptor (AhR), another druggable transcription factor governing SMAD3 expression level. Our work highlights novel drug vulnerabilities that can be exploited to develop long-lasting antimelanoma therapies

Unleashing the power of inflammasomes and trained immunity: promising strategies in the fight against Staphylococcus aureus infection

International audienceStaphylococcus aureus causes life-threatening diseases such as pneumonia and osteomyelitis. Pathogens activate inflammasomes that trigger protease activation, particularly caspase-1, which promotes the secretion of mature IL-1β and IL-18. Trained immunity (TI) enhances the immune response to subsequent unrelated challenges through epigenetic reprogramming of transcriptional pathways and alteration of cell metabolism. Our objectives include a comparison of IL-1β production by monocyte–like ThP1 cells vs osteoblast-like MG-63 cells and comprehending the role of inflammasomes effector, caspase-1, investigating the development of TI in non-immune MG-63 and epithelial lung A549 cells, in the context of S. aureus infection.The role of inflammasomes and Caspase-1 was examined by a comparison of MG-63 cells and caspase-1 knock-out CASP1−/−MG-63 cells that were produced using CRISPR-Cas9 technology. We developed an in vitro TI model using MG-63 and A549 cells and investigated the involvement of ROS using the scavenger N-acetylcysteine (NAC). ThP1 cells produced a higher amount of IL-1β compared to MG-63 cells. Infected MG-63 cells release mature IL-1β, while CASP1−/−MG-63 cells didn’t. PSMs were identified as key contributors to IL-1β production using deletion and complemented phenol-soluble modulins (PSMs) S. aureus mutants. Caspase-1 deficiency impaired cell defense, resulting in decreased bacterial clearance in CASP1−/−MG-63 cells. β-glucan training of MG-63 and A549 cells increased IL-6/IL-8 production upon a stimulation with S. aureus. Interleukin production positively correlated with Histone 3 acetylation (H3K27), indicating epigenetic reprogramming. NAC addition, prior to β-glucan training before S. aureus infection, inhibited IL-6/IL-8 production, thereby supporting the involvement of ROS in the induction of TI. Cell exposure to bacterium with probiotic properties, Lactococcus lactis, before S. aureus infection elevated IL-6/IL-8 production, accompanied by H3K27 acetylation, suggesting its ability to induce TI. Our results demonstrate two host strategies against S. aureus infection with therapeutic potential