Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion.

The intrinsic biomechanical properties of cancer cells remain poorly understood. To decipher whether cell stiffness modulation could increase melanoma cells' invasive capacity, we performed both in vitro and in vivo experiments exploring cell stiffness by atomic force microscopy (AFM). We correlated stiffness properties with cell morphology adaptation and the molecular mechanisms underlying epithelial-to-mesenchymal (EMT)-like phenotype switching. We found that melanoma cell stiffness reduction was systematically associated with the acquisition of invasive properties in cutaneous melanoma cell lines, human skin reconstructs, and Medaka fish developing spontaneous MAP-kinase-induced melanomas. We observed a systematic correlation of stiffness modulation with cell morphological changes towards mesenchymal characteristic gains. We accordingly found that inducing melanoma EMT switching by overexpressing the ZEB1 transcription factor, a major regulator of melanoma cell plasticity, was sufficient to decrease cell stiffness and transcriptionally induce tetraspanin-8-mediated dermal invasion. Moreover, ZEB1 expression correlated with Tspan8 expression in patient melanoma lesions. Our data suggest that intrinsic cell stiffness could be a highly relevant marker for human cutaneous melanoma development.

RASGRF2 gene fusions identified in a variety of melanocytic lesions with distinct morphological features.

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.

Biomechanical Properties of Cancer Cells.

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.

Stiffness measurement is a biomarker of skin ageing in vivo.

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.

Tspan8 Drives Melanoma Dermal Invasion by Promoting ProMMP-9 Activation and Basement Membrane Proteolysis in a Keratinocyte-Dependent Manner.

Melanoma is the most aggressive skin cancer with an extremely challenging therapy. The dermal-epidermal junction (DEJ) degradation and subsequent dermal invasion are the earliest steps of melanoma dissemination, but the mechanisms remain elusive. We previously identified Tspan8 as a key actor in melanoma invasiveness. Here, we investigated Tspan8 mechanisms of action during dermal invasion, using a validated skin-reconstruct-model that recapitulates melanoma dermal penetration through an authentic DEJ. We demonstrate that Tspan8 is sufficient to induce melanoma cells' translocation to the dermis. Mechanistically, Tspan8+ melanoma cells cooperate with surrounding keratinocytes within the epidermis to promote keratinocyte-originated proMMP-9 activation process, collagen IV degradation and dermal colonization. This concurs with elevated active MMP-3 and low TIMP-1 levels, known to promote MMP-9 activity. Finally, a specific Tspan8-antibody reduces proMMP-9 activation and dermal invasion. Overall, our results provide new insights into the role of keratinocytes in melanoma dermal colonization through a cooperative mechanism never reported before, and establish for the first time the pro-invasive role of a tetraspanin family member in a cell non-autonomous manner. This work also displays solid arguments for the use of Tspan8-blocking antibodies to impede early melanoma spreading and therefore metastasis.

Melanocytic tumors with MAP3K8 fusions: report of 33 cases with morphological-genetic correlations.

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.

[Probiotics in the treatment of atopic dermatitis: the induction of tolerogenic dendritic cells]. / Traiter la dermatite atopique par les probiotiques - Induction de cellules dendritiques tolérogènes.

TITLE: Traiter la dermatite atopique par les probiotiques - Induction de cellules dendritiques tolérogènes. ABSTRACT: 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.

Tspan8-ß-catenin positive feedback loop promotes melanoma invasion.

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 ß-catenin stabilization is a molecular signal subsequent to the onset of Tspan8 expression, and that, in turn, ß-catenin triggers the direct transcriptional activation of Tspan8 expression, leading to melanoma invasion. Moreover, we showed that ß-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 ß-catenin and MAP kinase signaling. Overall, our data suggest that ß-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.

Tetraspanin 8 is a novel regulator of ILK-driven ß1 integrin adhesion and signaling in invasive melanoma cells.

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 ß1 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 ß1 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 ß1 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.

MicroRNA-23b-3p regulates human keratinocyte differentiation through repression of TGIF1 and activation of the TGF-ß-SMAD2 signalling pathway.

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 in cutaneous physiopathology]. / Les tétraspanines dans la physiopathologie de la peau.

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.

Transforming growth factor-ß increases interleukin-13 synthesis via GATA-3 transcription factor in T-lymphocytes from patients with systemic sclerosis.

INTRODUCTION: Transforming growth factor (TGF)-ß and interleukin (IL)-13 play a crucial role in the pathogenesis of systemic sclerosis (SSc), partly through activation of collagen production that leads to fibrosis. The aim of the present study was to determine whether TFG-ß alters IL-13 production in T lymphocytes from patients with SSc from that seen in those of healthy donors. METHODS: IL-13 mRNA and protein synthesis under TFG-ß exposure was measured in circulating T lymphocytes from healthy donors and patients with SSc and also in the Jurkat Th2 T-cell line, using quantitative real-time PCR and fluorescence-activated cell sorting analysis, respectively. The involvement of Smad and GATA-3 transcription factors was assessed by using specific inhibitors and small interfering RNA, and the binding capacity of GATA-3 to the IL-13 gene promoter was evaluated by chromatin immunoprecipitation assay. RESULTS: TGF-ß induced a significant decrease in IL-13 mRNA and protein levels in lymphocytes from healthy donors (mean [±SD] inhibition of 30% ± 10% and 20% ± 7%, respectively; p < 0.05). In contrast, TGF-ß promoted a significant increase in IL-13 mRNA levels and IL-13 synthesis by CD4(+) and CD8(+) T-cell subtypes from patients with SSc, with respective increases of 2.4 ± 0.3-fold, 1.6 ± 0.05-fold and 2.7 ± 0.02-fold. The involvement of the Smad signaling pathway and upregulation of GATA-3 binding capacity on the IL-13 promoter in lymphocytes from patients with SSc contributed to the effect of TGF-ß on IL-13 production. CONCLUSIONS: These results demonstrate that TGF-ß upregulates IL-13 synthesis through GATA-3 expression in the T lymphocytes of patients with SSc, confirming that the GATA-3 transcription factor can be regarded as a novel therapeutic target in patients with SSc.

GATA3 inhibits proliferation and induces expression of both early and late differentiation markers in keratinocytes of the human epidermis.

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.

p63 regulates human keratinocyte proliferation via MYC-regulated gene network and differentiation commitment through cell adhesion-related gene network.

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/ß-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-1ß (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.

Pyruvate imbalance mediates metabolic reprogramming and mimics lifespan extension by dietary restriction in Caenorhabditis elegans.

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.

Rev-erbalpha2 mRNA encodes a stable protein with a potential role in circadian clock regulation.

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.

A novel role for the SMG-1 kinase in lifespan and oxidative stress resistance in Caenorhabditis elegans.

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.

Expression levels of estrogen receptor beta are modulated by components of the molecular clock.

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 (ERbeta) is, together with ERalpha, 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 ERbeta, but not ERalpha, is controlled by circadian clock proteins. We show that ERbeta 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 ERbeta expression is exerted through a conserved E-box element in the ERbeta 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 ERbeta.

Lifespan and dauer regulation by tissue-specific activities of Caenorhabditis elegans DAF-18.

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.