Loss of Dicer in Newborn Melanocytes Leads to Premature Hair Graying and Changes in Integrin Expression.

Premature hair graying occurs owing to the depletion of melanocyte stem cells in the hair follicle, which can be accelerated by stress caused by genetic or environmental factors. However, the connection between stress and melanocyte stem cell loss is not fully understood. MicroRNAs are molecules that control gene expression by regulating mRNA stability and translation and are produced by the enzyme Dicer, which is repressed under stress. In this study, using 2 mouse genetic models and human and mouse cell lines, we found that the inactivation of Dicer in melanocytes leads to misplacement of these cells within the hair follicle, resulting in a lack of melanin transfer to keratinocytes in the growing hair and the exhaustion of the melanocyte stem cell pool. We also show that miR-92b, which regulates ItgaV mRNA and protein levels, plays a role in altering melanocyte migration. Overall, our findings suggest that the Dicer-miR92b-ItgaV pathway serves as a major signaling pathway linking stress to premature hair greying.

Guidelines on human factors in critical situations 2023.

OBJECTIVE: To provide guidelines to define the place of human factors in the management of critical situations in anaesthesia and critical care. DESIGN: A committee of nineteen experts from the SFAR and GFHS learned societies was set up. A policy of declaration of links of interest was applied and respected throughout the guideline-producing process. Likewise, the committee did not benefit from any funding from a company marketing a health product (drug or medical device). The committee followed the GRADE® method (Grading of Recommendations Assessment, Development and Evaluation) to assess the quality of the evidence on which the recommendations were based. METHODS: We aimed to formulate recommendations according to the GRADE® methodology for four different fields: 1/ communication, 2/ organisation, 3/ working environment and 4/ training. Each question was formulated according to the PICO format (Patients, Intervention, Comparison, Outcome). The literature review and recommendations were formulated according to the GRADE® methodology. RESULTS: The experts' synthesis work and application of the GRADE® method resulted in 21 recommendations. Since the GRADE® method could not be applied in its entirety to all the questions, the guidelines used the SFAR "Recommendations for Professional Practice" A means of secured communication (RPP) format and the recommendations were formulated as expert opinions. CONCLUSION: Based on strong agreement between experts, we were able to produce 21 recommendations to guide human factors in critical situations.

Derivation and Use of Cell Lines from Mouse Models of Melanoma.

The establishment of consistent genetically modified mouse melanoma models and cell lines is of paramount importance for prevention and treatment. In this study, we review the different mouse melanoma cell lines that have been established. After careful molecular characterization of the established mouse melanoma cell lines, modification of the genome, microenvironment, or even the environment using appropriate in cellulo and in vivo assays may reveal novel genetic and nongenetic changes. These murine melanoma cell lines with defined genetic mutations allow the testing of innovative therapies based on chemistry, physics, and biology using alternative methods. In addition to the fundamental aspects, these results are important for humans because of the relevance of these murine melanoma cell lines to human disease.

Targeting GPCRs and Their Signaling as a Therapeutic Option in Melanoma.

G-protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G-proteins, which induce cellular signaling through various pathways. Such signaling modulates numerous essential cellular processes that occur during melanomagenesis, including proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden melanoma treatment options in the future.

Adipocyte Extracellular Vesicles Decrease p16INK4A in Melanoma: An Additional Link between Obesity and Cancer.

Obesity is a recognized factor for increased risk and poor prognosis of many cancers, including melanoma. In this study, using genetically engineered mouse models of melanoma (NrasQ61K transgenic expression, associated or not with Cdkn2a heterozygous deletion), we show that obesity increases melanoma initiation and progression by supporting tumor growth and metastasis, thereby reducing survival. This effect is associated with a decrease in p16INK4A expression in tumors. Mechanistically, adipocytes downregulate p16INK4A in melanoma cells through ß-catenin-dependent regulation, which increases cell motility. Furthermore, ß-catenin is directly transferred from adipocytes to melanoma cells in extracellular vesicles, thus increasing its level and activity, which represses CDKN2A transcription. Adipocytes from individuals with obesity have a stronger effect than those from lean individuals, mainly owing to an increase in the number of vesicles secreted, thus increasing the amount of ß-catenin delivered to melanoma cells and, consequently, amplifying their effect. In conclusion, in this study, we reveal that adipocyte extracellular vesicles control p16INK4A expression in melanoma, which promotes tumor progression. This work expands our understanding of the cooperation between adipocytes and tumors, particularly in obesity.

Stabilization of ß-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

The canonical Wnt/ß-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, ß-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of ß-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of ß-catenin in cells expressing tyrosinase. Constitutive activation of ß-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating ß-catenin at various stages of development (E8.5-E11.5), we showed that the activation of ß-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/ß-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, ß-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

BRN2 is a non-canonical melanoma tumor-suppressor.

While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600E PtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.

Efficacy of Targeted Radionuclide Therapy Using [131I]ICF01012 in 3D Pigmented BRAF- and NRAS-Mutant Melanoma Models and In Vivo NRAS-Mutant Melanoma.

PURPOSE: To assess the efficiency of targeted radionuclide therapy (TRT), alone or in combination with MEK inhibitors (MEKi), in melanomas harboring constitutive MAPK/ERK activation responsible for tumor radioresistance. METHODS: For TRT, we used a melanin radiotracer ([131I]ICF01012) currently in phase 1 clinical trial (NCT03784625). TRT alone or combined with MEKi was evaluated in three-dimensional melanoma spheroid models of human BRAFV600E SK-MEL-3, murine NRASQ61K 1007, and WT B16F10 melanomas. TRT in vivo biodistribution, dosimetry, efficiency, and molecular mechanisms were studied using the C57BL/6J-NRASQ61K 1007 syngeneic model. RESULTS: TRT cooperated with MEKi to increase apoptosis in both BRAF- and NRAS-mutant spheroids. NRASQ61K spheroids were highly radiosensitive towards [131I]ICF01012-TRT. In mice bearing NRASQ61K 1007 melanoma, [131I]ICF01012 induced a significant extended survival (92 vs. 44 days, p < 0.0001), associated with a 93-Gy tumor deposit, and reduced lymph-node metastases. Comparative transcriptomic analyses confirmed a decrease in mitosis, proliferation, and metastasis signatures in TRT-treated vs. control tumors and suggest that TRT acts through an increase in oxidation and inflammation and P53 activation. CONCLUSION: Our data suggest that [131I]ICF01012-TRT and MEKi combination could be of benefit for advanced pigmented BRAF-mutant melanoma care and that [131I]ICF01012 alone could constitute a new potential NRAS-mutant melanoma treatment.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

C57BL/6 congenic mouse NRASQ61K melanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo.

RAS is frequently mutated in various tumors and known to be difficult to target. NRASQ61K/R are the second most frequent mutations found in human skin melanoma after BRAFV600E . Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRASQ61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRASQ61K mouse melanoma that can be used in vitro and in vivo in an immune-competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRASQ61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses.

Molecular and cellular basis of depigmentation in vitiligo patients.

Vitiligo is a chronic skin disease characterized by the appearance of zones of depigmentation. It is mostly described as an autoimmune disease in which the immune system destroys the melanocytes. Consistent with this origin, genetic studies have implicated genes encoding proteins mediating the immune response targeting melanocytes in the aetiology of this disease, together with proteins specific to these cells. However, the destruction of melanocytes by the immune system is neither global nor complete, because the patients do not display total depigmentation. The etiopathology of vitiligo is clearly complex and cannot be simply reduced to an autoimmune reaction directed against pigmented cells. Intrinsic changes have been observed in the melanocytes, keratinocytes and dermal cells of vitiligo patients. Identification of the molecular and cellular changes occurring in normally pigmented skin in vitiligo patients, and an understanding of these changes, is essential to improve the definition of trigger events for this disease, with a view to developing treatments with long-term efficacy. This review focuses on the early events identified to date in the non-lesional regions of the skin in vitiligo patients and discusses the process of repigmentation from melanocyte stem cells.

A histopathological classification system of Tyr::NRASQ61K murine melanocytic lesions: A reproducible simplified classification.

Genetically engineered mouse models offer essential opportunities to investigate the mechanisms of initiation and progression in melanoma. Here, we report a new simplified histopathology classification of mouse melanocytic lesions in Tyr::NRASQ61K derived models, using an interactive decision tree that produces homogeneous categories. Reproducibility for this classification system was evaluated on a panel of representative cases of murine melanocytic lesions by pathologists and basic scientists. Reproducibility, measured as inter-rater agreement between evaluators using a modified Fleiss' kappa statistic, revealed a very good agreement between observers. Should this new simplified classification be adopted, it would create a robust system of communication between researchers in the field of mouse melanoma models.

New Functional Signatures for Understanding Melanoma Biology from Tumor Cell Lineage-Specific Analysis.

Molecular signatures specific to particular tumor types are required to design treatments for resistant tumors. However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures. We developed similarity core analysis (SCA), a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines. We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases. The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion. About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7) and miRNAs (211-5p, 221-3p, and 10a-5p). The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.

A caveolin-dependent and PI3K/AKT-independent role of PTEN in ß-catenin transcriptional activity.

Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour. How PTEN loss mediates these effects is unknown. Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of ß-catenin independent of the PI3K-AKT-GSK3ß axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent ß-catenin transcriptional modulation in vitro, cooperates with NRAS(Q61K) to initiate melanomagenesis in vivo and induces efficient metastasis formation associated with E-cadherin internalization. The CAV1-ß-catenin axis is mediated by a feedback loop in which ß-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells. These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of ß-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.

Altered E-Cadherin Levels and Distribution in Melanocytes Precede Clinical Manifestations of Vitiligo.

Vitiligo is the most common depigmenting disorder resulting from the loss of melanocytes from the basal epidermal layer. The pathogenesis of the disease is likely multifactorial and involves autoimmune causes, as well as oxidative and mechanical stress. It is important to identify early events in vitiligo to clarify pathogenesis, improve diagnosis, and inform therapy. Here, we show that E-cadherin (Ecad), which mediates the adhesion between melanocytes and keratinocytes in the epidermis, is absent from or discontinuously distributed across melanocyte membranes of vitiligo patients long before clinical lesions appear. This abnormality is associated with the detachment of the melanocytes from the basal to the suprabasal layers in the epidermis. Using human epidermal reconstructed skin and mouse models with normal or defective Ecad expression in melanocytes, we demonstrated that Ecad is required for melanocyte adhesiveness to the basal layer under oxidative and mechanical stress, establishing a link between silent/preclinical, cell-autonomous defects in vitiligo melanocytes and known environmental stressors accelerating disease expression. Our results implicate a primary predisposing skin defect affecting melanocyte adhesiveness that, under stress conditions, leads to disappearance of melanocytes and clinical vitiligo. Melanocyte adhesiveness is thus a potential target for therapy aiming at disease stabilization.

A subpopulation of smooth muscle cells, derived from melanocyte-competent precursors, prevents patent ductus arteriosus.

BACKGROUND: Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/ß-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.

Modeling melanoblast development.

Melanoblasts are a particular type of cell that displays extensive cellular proliferation during development to contribute to the skin. There are only a few melanoblast founders, initially located just dorsal to the neural tube, and they sequentially colonize the dermis, epidermis, and hair follicles. In each compartment, melanoblasts are exposed to a wide variety of developmental cues that regulate their expansion. The colonization of the dermis and epidermis by melanoblasts involves substantial proliferation to generate thousands of cells or more from a few founders within a week of development. This review addresses the cellular and molecular events occurring during melanoblast development. We focus on intrinsic and extrinsic factors that control melanoblast proliferation. We also present a robust mathematical model for estimating the doubling-time of dermal and epidermal melanoblasts for all coat color phenotypes from black to white.

Efficient gene expression profiling of laser-microdissected melanoma metastases.

Comparing the transcriptomes of primary and metastatic tumour tissues is a useful strategy for studying tumour progression. One factor limiting the interpretation of tissue-based transcriptomic data is the lack of cell-type purity. Laser capture microdissection (LCM) has been shown to be useful for overcoming this limitation. We established an efficient protocol for gene expression profiling of LCM and matched metastatic melanomas using a transgenic mouse model. This optimized workflow combines microsurgical recovery of mouse lungs, appropriate tissue freezing, laser microdissection of homogeneous tumour cell populations from cryosections, isolation of high-quality RNA and gene expression analysis. The RNA isolated from laser-microdissected material was not contaminated by stroma cells, was of excellent quality, and the synthesis of cDNAs was homogeneous and highly reproducible. Subsequent custom-based Taqman-low-density-array (TLDA)-based gene expression profiling identified stronger expression of five genes (M-MITF, TYR, STAT3, CCND1 and PAX3) in primary than metastatic melanoma. We detected only minor transcriptomic differences between primary and metastatic melanoma tissue. This optimized workflow could be very valuable for various studies requiring cell type-specific transcriptomic analysis.

A phosphatidylinositol 3-kinase-Pax3 axis regulates Brn-2 expression in melanoma.

Deregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/ß-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways.