A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

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.

Simulation of melanoblast displacements reveals new features of developmental migration.

To distribute and establish the melanocyte lineage throughout the skin and other developing organs, melanoblasts undergo several rounds of proliferation, accompanied by migration through complex environments and differentiation. Melanoblast migration requires interaction with extracellular matrix of the epidermal basement membrane and with surrounding keratinocytes in the developing skin. Migration has been characterized by measuring speed, trajectory and directionality of movement, but there are many unanswered questions about what motivates and defines melanoblast migration. Here, we have established a general mathematical model to simulate the movement of melanoblasts in the epidermis based on biological data, assumptions and hypotheses. Comparisons between experimental data and computer simulations reinforce some biological assumptions, and suggest new ideas for how melanoblasts and keratinocytes might influence each other during development. For example, it appears that melanoblasts instruct each other to allow a homogeneous distribution in the tissue and that keratinocytes may attract melanoblasts until one is stably attached to them. Our model reveals new features of how melanoblasts move and, in particular, suggest that melanoblasts leave a repulsive trail behind them as they move through the skin.

Modeling and analysis of melanoblast motion.

Melanoblast migration is important for embryogenesis and is a key feature of melanoma metastasis. Many studies have characterized melanoblast movement, focusing on statistical properties and have highlighted basic mechanisms of melanoblast motility. We took a slightly different and complementary approach: we previously developed a mathematical model of melanoblast motion that enables the testing of biological assumptions about the displacement of melanoblasts and we created tests to analyze the geometric features of cell trajectories and the specific issue of trajectory interactions. Within this model, we performed simulations and compared the results with experimental data using geometric tests. In this paper, we developed the associated mathematical model and the main focus is to study the crossings between trajectories with new theoretical results about the variation of number of intersection points with respect to the crossing times. Using these results it is possible to study the random nature of displacements and the interactions between trajectories. This analysis has raised new questions, leading to the generation of strong arguments in favor of a trail left behind each moving melanoblast.

UVB represses melanocyte cell migration and acts through ß-catenin.

The exposure of skin to ultraviolet (UV) radiation can have both beneficial and deleterious effects: it can lead, for instance, to increased pigmentation and vitamin D synthesis but also to inflammation and skin cancer. UVB may induce genetic and epigenetic alterations and have reversible effects associated with post-translational and gene regulation modifications. ß-catenin is a main driver in melanocyte development; although infrequently mutated in melanoma, its cellular localization and activity are frequently altered. Here, we evaluate the consequence of UVB on ß-catenin in the melanocyte lineage. We report that in vivo, UVB induces cytoplasmic/nuclear relocalization of ß-catenin in melanocytes of newborn mice and adult human skin. In mouse melanocyte and human melanoma cell lines in vitro, UVB increases ß-catenin stability, accumulation in the nucleus and cotranscriptional activity, leading to the repression of cell motility and velocity. The activation of the ß-catenin signalling pathway and its effect on migration by UVB are increased by an inhibitor of GSK3ß, and decreased by an inhibitor of ß-catenin. In conclusion, UVB represses melanocyte migration and does so by acting through the GSK3-ß-catenin axis.

Any route for melanoblasts to colonize the skin!

Melanocytes arise from the fourth embryonic layer, the neural crest. They emerge from the roof plate of the neural tube and migrate throughout the body. In mammals, these cells have the capacity to migrate in any type of environment and use various pathways and mechanisms to colonize the skin and hair, and for their maintenance throughout the life of the animal.

Somatic loss of p53 leads to stem/progenitor cell amplification in both mammary epithelial compartments, basal and luminal.

Mammary epithelium comprises a layer of luminal cells and a basal myoepithelial cell layer. Both mammary epithelial compartments, basal and luminal, contain stem and progenitor cells, but only basal cells are capable of gland regeneration upon transplantation. Aberrant expansion of stem/progenitor cell populations is considered to contribute to breast tumorigenesis. Germline deletions of p53 in humans and mice confer a predisposition to tumors, and stem cell frequency is abnormally high in the mammary epithelium of p53-deficient mice. However, it is unknown whether stem/progenitor cell amplification occurs in both, basal and luminal cell populations in p53-deficient mammary tissue. We used a conditional gene deletion approach to study the role of p53 in stem/progenitor cells residing in the mammary luminal and basal layers. Using two- and three-dimensional cell culture assays, we showed that p53 loss led to the expansion of clonogenic stem/progenitor cells in both mammary epithelial cell layers. Moreover, following p53 deletion, luminal and basal stem/progenitor cells acquired a capacity for unlimited propagation in mammosphere culture. Furthermore, limiting dilution and serial transplantation assays revealed amplification and enhanced self-renewal in the basal regenerating cell population of p53-deficient mammary epithelium. Our data suggest that the increase in stem/progenitor cell activity may be, at least, partially mediated by the Notch pathway. Taken together, these results strongly indicate that p53 restricts the propagation and self-renewal of stem/progenitor cells in both layers of the mammary epithelium providing further insight into the impact of p53 loss in breast cancerogenesis.

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.

Myc is required for ß-catenin-mediated mammary stem cell amplification and tumorigenesis.

BACKGROUND: Basal-like breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Recent studies have linked activation of the Wnt/ß-catenin pathway, and its downstream target, Myc, to basal-like breast cancer. Transgenic mice K5ΔNßcat previously generated by our team present a constitutive activation of Wnt/ß-catenin signaling in the basal myoepithelial cell layer, resulting in focal mammary hyperplasias that progress to invasive carcinomas. Mammary lesions developed by K5ΔNßcat mice consist essentially of basal epithelial cells that, in contrast to mammary myoepithelium, do not express smooth muscle markers. METHODS: Microarray analysis was used to compare K5ΔNßcat mouse tumors to human breast tumors, mammary cancer cell lines and the tumors developed in other mouse models. Cre-Lox approach was employed to delete Myc from the mammary basal cell layer of K5ΔNßcat mice. Stem cell amplification in K5ΔNßcat mouse mammary epithelium was assessed with 3D-culture and transplantation assays. RESULTS: Histological and microarray analyses of the mammary lesions of K5ΔNßcat females revealed their high similarity to a subset of basal-like human breast tumors with squamous differentiation. As in human basal-like carcinomas, the Myc pathway appeared to be activated in the mammary lesions of K5ΔNßcat mice. We found that a basal cell population with stem/progenitor characteristics was amplified in K5ΔNßcat mouse preneoplastic glands. Finally, the deletion of Myc from the mammary basal layer of K5ΔNßcat mice not only abolished the regenerative capacity of basal epithelial cells, but, in addition, completely prevented the tumorigenesis. CONCLUSIONS: These results strongly indicate that ß-catenin-induced stem cell amplification and tumorigenesis rely ultimately on the Myc pathway activation and reinforce the hypothesis that basal stem/progenitor cells may be at the origin of a subset of basal-like breast tumors.

The proto-oncogene Myc is essential for mammary stem cell function.

The mammary epithelium comprises two major cell lineages: basal and luminal. Basal cells (BCs) isolated from the mammary epithelium and transplanted into the mouse mammary fat pad cleared from the endogenous epithelium regenerate the mammary gland, strongly suggesting that the basal epithelial compartment harbors a long-lived cell population with multipotent stem cell potential. The luminal cell layer is devoid of the regenerative potential, but it contains cells with clonogenic capacity, the luminal progenitors. Mammary BCs and luminal progenitors express high levels of the transcription factor Myc. Here, we show that deletion of Myc from mammary basal epithelial cells led to impaired stem cell self-renewal as evaluated by limiting dilution and serial transplantation assays. Luminal progenitor population was significantly diminished in mutant epithelium suggesting control by the BC layer. Colony formation assay performed with isolated BCs showed that clonogenic capacity was abolished by Myc deletion. Moreover, transplanted BCs depleted of Myc failed to produce epithelial outgrowths. Stimulation with ovarian hormones estrogen (E) and progesterone (P) partially rescued the repopulation capacity of Myc-depleted BCs; however, the Myc-deficient mammary epithelium developed in response to E/P treatment lacked stem and progenitor cells. This study provides the first evidence that in the mammary gland, Myc has an essential nonredundant function in the maintenance of the self-renewing multipotent stem cell population responsible for the regenerative capacity of the mammary epithelium and is required downstream from ovarian hormones, for the control of mammary stem and progenitor cell functions.

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.

A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

Luminal lipid regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis.

The membrane glycoprotein CD36 binds nanomolar concentrations of long chain fatty acids (LCFA) and is highly expressed on the luminal surface of enterocytes. CD36 deficiency reduces chylomicron production through unknown mechanisms. In this report, we provide novel insights into some of the underlying mechanisms. Our in vivo data demonstrate that CD36 gene deletion in mice does not affect LCFA uptake and subsequent esterification into triglycerides by the intestinal mucosa exposed to the micellar LCFA concentrations prevailing in the intestine. In rodents, the CD36 protein disappears early from the luminal side of intestinal villi during the postprandial period, but only when the diet contains lipids. This drop is significant 1 h after a lipid supply and associates with ubiquitination of CD36. Using CHO cells expressing CD36, it is shown that the digestion products LCFA and diglycerides trigger CD36 ubiquitination. In vivo treatment with the proteasome inhibitor MG132 prevents the lipid-mediated degradation of CD36. In vivo and ex vivo, CD36 is shown to be required for lipid activation of ERK1/2, which associates with an increase of the key chylomicron synthesis proteins, apolipoprotein B48 and microsomal triglyceride transfer protein. Therefore, intestinal CD36, possibly through ERK1/2-mediated signaling, is involved in the adaptation of enterocyte metabolism to the postprandial lipid challenge by promoting the production of large triglyceride-rich lipoproteins that are rapidly cleared in the blood. This suggests that CD36 may be a therapeutic target for reducing the postprandial hypertriglyceridemia and associated cardiovascular risks.

CLEC12B Decreases Melanoma Proliferation by Repressing Signal Transducer and Activator of Transcription 3.

The potential role of CLEC12B, a gene predominantly expressed by skin melanocytes discovered through transcriptomic analysis, in melanoma is unknown. In this study, we show that CLEC12B expression is lower in melanoma and melanoma metastases than in melanocytes and benign melanocytic lesions and that its decrease correlates with poor prognosis. We further show that CLEC12B recruits SHP2 phosphatase through its immunoreceptor tyrosine-based inhibition motif domain, inactivates signal transducer and activator of transcription 1/3/5, increases p53/p21/p27 expression/activity, and modulates melanoma cell proliferation. The growth of human melanoma cells overexpressing CLEC12B in nude mice after subcutaneous injection is significantly decreased compared with that in the vehicle control group and is associated with decreased signal transducer and activator of transcription 3 phosphorylation and increased p53 levels in the tumors. Reducing the level of CLEC12B had the opposite effect. We show that CLEC12B represses the activation of the signal transducer and activator of transcription pathway and negatively regulates the cell cycle, providing a proliferative asset to melanoma cells.

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.

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.

[Analytical characteristics of Vitros 3600 analyzer]. / Evaluation des performances analytiques de l'analyseur Vitros 3600.

The newly developed, high throughput, Vitros 3600 immunoassay system associates different technologies on board: MicroWell (analytes immunocapture), MicroSensor (visible interference analysis), and Intellicheck (real time process control for analytical error detection). During a three months period, Vitros 3600 system, ease of use, practicability and MicroSensor technology efficiency have been tested using the following assays: betaHCG, troponin, ferritin, aHBcT, HBsAg, aHCV, AFP and CEA. Within-day and within-batch, between-calibration precisions are acceptable. Overall assay correlations versus the methods in use in our laboratory are good, despite small numerical differences that never affect clinical interpretation of the results. We have appreciated the system reliability, reinforced by the ease of use (simple software, on-board VDocs, continuous access to reagent and sample loading, reagent stability, very low noise (60 dB)), the quality of the results, MicroSensor and Intellicheck technologies efficiency, the short maintenance time and its traceability, and the very low liquid waste volume (5 L of liquid waste for 1600 tests). In conclusion and considering its practicability, the Vitros 3600 system is fully validated and well suited for our laboratory.

TET2-Dependent Hydroxymethylome Plasticity Reduces Melanoma Initiation and Progression.

Although numerous epigenetic aberrancies accumulate in melanoma, their contribution to initiation and progression remain unclear. The epigenetic mark 5-hydroxymethylcytosine (5hmC), generated through TET-mediated DNA modification, is now referred to as the sixth base of DNA and has recently been reported as a potential biomarker for multiple types of cancer. Loss of 5hmC is an epigenetic hallmark of melanoma, but whether a decrease in 5hmc levels contributes directly to pathogenesis or whether it merely results from disease progression-associated epigenetic remodeling remains to be established. Here, we show that NRAS-driven melanomagenesis in mice is accompanied by an overall decrease in 5hmC and specific 5hmC gains in selected gene bodies. Strikingly, genetic ablation of Tet2 in mice cooperated with oncogenic NRASQ61K to promote melanoma initiation while suppressing specific gains in 5hmC. We conclude that TET2 acts as a barrier to melanoma initiation and progression, partly by promoting 5hmC gains in specific gene bodies. SIGNIFICANCE: This work emphasizes the importance of epigenome plasticity in cancer development and highlights the involvement of druggable epigenetic factors in cancer.

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.

Downstream-of-FGFR is a fibroblast growth factor-specific scaffolding protein and recruits Corkscrew upon receptor activation.

Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. We have performed a functional characterization of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, we demonstrate that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, we identify two regions important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, our results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response.