The guanine exchange factor SWAP70 mediates vGPCR-induced endothelial plasticity.

BACKGROUND: The viral G protein-coupled receptor (vGPCR) is proposed to act as one of the predominant mediators of Kaposi's sarcoma (KS), a human herpes virus 8 (HHV8)-elicited disease. The actions of vGPCR manifest pathogenesis, in part, through increased permeability of endothelial cells. Endothelial cell-cell junctions have indeed emerged as an instrumental target involved in the vasculature defects observed within the tumor microenvironment. The pathway leading to adherens junction destabilization has been shown to involve the activation of the small GTPase Rac, in the context of either latent infection or the sole expression of vGPCR. However, the precise molecular mechanisms governed by vGPCR in vascular leakage require further elucidation. FINDINGS: Guanine exchange factors (GEFs) function as critical molecular switches that control the activation of small GTPases. We therefore screened the effects of 80 siRNAs targeting GEFs on vGPCR-driven endothelial permeability and identified switch-associated protein 70 (SWAP70) as necessary for its elevating effects. Pull-down experiments further showed that Rac activation by vGPCR was dependent on SWAP70. Examination of tissues and cells from HHV8-positive patients revealed that SWAP70 was ubiquitously expressed. Furthermore, SWAP70 was found to be crucial for vGPCR-driven endothelial tube formation and endothelial sprouting in vitro. CONCLUSIONS: SWAP70 appears to act as a molecular intermediate between vGPCR and endothelial activation. Because of the important role of vGPCR-mediated endothelial plasticity in KS pathogenesis, inhibition of SWAP70 function could be of interest for blocking vGPCR-driven activities in HHV8-defined diseases.

Super-enhancers: drivers of cells' identities and cells' debacles.

Precise spatiotemporal regulations of gene expression are essential for determining cells' fates and functions. Enhancers are cis-acting DNA elements that act as periodic transcriptional thrusters and their activities are cell type specific. Clusters of enhancers, called super-enhancers, are more densely occupied by transcriptional activators than enhancers, driving stronger expression of their target genes, which have prominent roles in establishing and maintaining cellular identities. Here we review the current knowledge on the composition and structure of super-enhancers to understand how they robustly stimulate the expression of cellular identity genes. We also review their involvement in the development of various cell types and both noncancerous and cancerous disorders, implying the therapeutic interest of targeting them to fight against various diseases.

Tracking Targets of Dynamic Super-Enhancers in Vitro to Better Characterize Osteoclastogenesis and to Evaluate the Effect of Diuron on the Maturation of Human Bone Cells.

BACKGROUND: Osteoclasts are major actors in the maintenance of bone homeostasis. The full functional maturation of osteoclasts from monocyte lineage cells is essential for the degradation of old/damaged bone matrix. Diuron is one of the most frequently encountered herbicides, particularly in water sources. However, despite a reported delayed ossification in vivo, its impact on bone cells remains largely unknown. OBJECTIVES: The objectives of this study were to first better characterize osteoclastogenesis by identifying genes that drive the differentiation of CD14+ monocyte progenitors into osteoclasts and to evaluate the toxicity of diuron on osteoblastic and osteoclastic differentiation in vitro. METHODS: We performed chromatin immunoprecipitation (ChIP) against H3K27ac followed by ChIP-sequencing (ChIP-Seq) and RNA-sequencing (RNA-Seq) at different stages of differentiation of CD14+ monocytes into active osteoclasts. Differentially activated super-enhancers and their potential target genes were identified. Then to evaluate the toxicity of diuron on osteoblasts and osteoclasts, we performed RNA-Seq and functional tests during in vitro osteoblastic and osteoclastic differentiation by exposing cells to different concentrations of diuron. RESULTS: The combinatorial study of the epigenetic and transcriptional remodeling taking place during differentiation has revealed a very dynamic epigenetic profile that supports the expression of genes vital for osteoclast differentiation and function. In total, we identified 122 genes induced by dynamic super-enhancers at late days. Our data suggest that high concentration of diuron (50µM) affects viability of mesenchymal stem cells (MSCs) in vitro associated with a decrease of bone mineralization. At a lower concentration (1µM), an inhibitory effect was observed in vitro on the number of osteoclasts derived from CD14+ monocytes without affecting cell viability. Among the diuron-affected genes, our analysis suggests a significant enrichment of genes targeted by pro-differentiation super-enhancers, with an odds ratio of 5.12 (ρ=2.59×10-5). DISCUSSION: Exposure to high concentrations of diuron decreased the viability of MSCs and could therefore affect osteoblastic differentiation and bone mineralization. This pesticide also disrupted osteoclasts maturation by impairing the expression of cell-identity determining genes. Indeed, at sublethal concentrations, differences in the expression of these key genes were mild during the course of in vitro osteoclast differentiation. Taken together our results suggest that high exposure levels of diuron could have an effect on bone homeostasis. https://doi.org/10.1289/EHP11690.

Ewing sarcoma from molecular biology to the clinic.

In Europe, with an incidence of 7.5 cases per million, Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children, adolescents and young adults, after osteosarcoma. Since the 1980s, conventional treatment has been based on the use of neoadjuvant and adjuvant chemotherapeutic agents combined with surgical resection of the tumor when possible. These treatments have increased the patient survival rate to 70% for localized forms, which drops drastically to less than 30% when patients are resistant to chemotherapy or when pulmonary metastases are present at diagnosis. However, the lack of improvement in these survival rates over the last decades points to the urgent need for new therapies. Genetically, ES is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family. In 85% of cases, the chromosomal translocation found is (11; 22) (q24; q12), between the EWS RNA-binding protein and the FLI1 transcription factor, leading to the EWS-FLI1 fusion protein. This chimeric protein acts as an oncogenic factor playing a crucial role in the development of ES. This review provides a non-exhaustive overview of ES from a clinical and biological point of view, describing its main clinical, cellular and molecular aspects.

The p53 Family Members p63 and p73 Roles in the Metastatic Dissemination: Interactions with microRNAs and TGFß Pathway.

TP53 (TP53), p73 (TP73), and p63 (TP63) are members of the p53 transcription factor family, which has many activities spanning from embryonic development through to tumor suppression. The utilization of two promoters and alternative mRNA splicing has been shown to yield numerous isoforms in p53, p63, and p73. TAp73 is thought to mediate apoptosis as a result of nuclear accumulation following chemotherapy-induced DNA damage, according to a number of studies. Overexpression of the nuclear ΔNp63 and ΔNp73 isoforms, on the other hand, suppresses TAp73's pro-apoptotic activity in human malignancies, potentially leading to metastatic spread or inhibition. Another well-known pathway that has been associated to metastatic spread is the TGF pathway. TGFs are a family of structurally related polypeptide growth factors that regulate a variety of cellular functions including cell proliferation, lineage determination, differentiation, motility, adhesion, and cell death, making them significant players in development, homeostasis, and wound repair. Various studies have already identified several interactions between the p53 protein family and the TGFb pathway in the context of tumor growth and metastatic spread, beginning to shed light on this enigmatic intricacy.

Implication of the p53-Related miR-34c, -125b, and -203 in the Osteoblastic Differentiation and the Malignant Transformation of Bone Sarcomas.

The formation of the skeleton occurs throughout the lives of vertebrates and is achieved through the balanced activities of two kinds of specialized bone cells: the bone-forming osteoblasts and the bone-resorbing osteoclasts. Impairment in the remodeling processes dramatically hampers the proper healing of fractures and can also result in malignant bone diseases such as osteosarcoma. MicroRNAs (miRNAs) are a class of small non-coding single-strand RNAs implicated in the control of various cellular activities such as proliferation, differentiation, and apoptosis. Their post-transcriptional regulatory role confers on them inhibitory functions toward specific target mRNAs. As miRNAs are involved in the differentiation program of precursor cells, it is now well established that this class of molecules also influences bone formation by affecting osteoblastic differentiation and the fate of osteoblasts. In response to various cell signals, the tumor-suppressor protein p53 activates a huge range of genes, whose miRNAs promote genomic-integrity maintenance, cell-cycle arrest, cell senescence, and apoptosis. Here, we review the role of three p53-related miRNAs, miR-34c, -125b, and -203, in the bone-remodeling context and, in particular, in osteoblastic differentiation. The second aim of this study is to deal with the potential implication of these miRNAs in osteosarcoma development and progression.

BET bromodomains' functions in bone-related pathologies.

Throughout life, bones are subjected to the so-called 'bone-remodeling' process, which is a balanced mechanism between the apposition and the resorption of bone. This remodeling process depends on the activities of bone-specialized cells, namely the osteoblasts and the osteoclasts. Any deregulation in this process results in bone-related pathologies, classified as either metabolic nonmalignant diseases (such as osteoporosis) or malignant primary bone sarcomas. As these pathologies are not characterized by common targetable genetic alterations, epigenetic strategies could be relevant and promising options. Recently, targeting epigenetic regulators such as the bromodomains and extraterminal domains (BET) readers have achieved success in numerous other pathologies, including cancers. In this review, we highlight the current state of the art in terms of the diverse implications of BET bromodomain proteins in the bone's biology and its defects. Consequently, their role in bone-related pathologies will also be developed, especially in the context of the primary bone sarcomas.

MicroRNA-17-5p Reduces Inflammation and Bone Erosions in Mice With Collagen-Induced Arthritis and Directly Targets the JAK/STAT Pathway in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

OBJECTIVE: We undertook this study to examine microRNA (miRNA) expression across rheumatoid arthritis (RA) phenotypes, along with the effects and mechanisms of action of miRNA-17-5p (miR-17). METHODS: A miRNA array was performed in synovial tissue biopsied from patients with naive erosive RA (n = 3) and patients with nonerosive RA (n = 3). MicroRNA-17 lipoplex was delivered intraarticularly in the murine collagen-induced arthritis model. Clinical, histologic, and structural effects were studied over the course of arthritis. In-depth studies of the mechanisms of action of miR-17 were performed in primary RA fibroblast-like synoviocytes (FLS) isolated from synovial tissue. RESULTS: Fifty-five miRNAs including miR-17 were reduced in erosive RA. The miR-17 transfection into arthritic paws reduced the clinical inflammation score between day 2 and day 7 (2.8 versus 1.9; P = 0.03). Synovial B cell, T cell, macrophage, and polynuclear neutrophil infiltration was significantly reduced. Structural damage was also decreased, as shown by a reduction in the number of osteoclasts detected using tartrate-resistant acid phosphatase staining (osteoclast surface/bone surface 32% versus 18%; P = 0.005) and erosion score by computed tomography analysis (2.9 versus 1.7; P = 0.023). Proinflammatory cytokines from the interleukin-6 (IL-6) family and IL-1ß expression were also significantly reduced, but tumor necrosis factor was not. MicroRNA-17 directly targeted the 3'-untranslated regions of STAT3 and JAK1. STAT3 and JAK1 messenger RNA (mRNA) and protein expression were reduced in RA FLS following miR-17 transfection. STAT3 and JAK1 mRNA and activation of STAT3, as assessed by immunohistochemistry, were also reduced in injected paws (% stained area 93% versus 62%; P = 0.035). CONCLUSION: We demonstrate an antiinflammatory and antierosive role of miR-17 in vivo. This effect involves the suppression of the IL-6 family autocrine-amplifying loop through the direct targeting of JAK1 and STAT3.

Loss of miR-198 and -206 during primary tumor progression enables metastatic dissemination in human osteosarcoma.

The metastatic dissemination is a complex multistep process by which tumor cells from a primary site enter into the systemic circulation to finally spread at distant sites. Even if this mechanism is rare at the tumor level, it remains the major cause of Osteosarcoma-patients' relapse and mortality. MicroRNAs (miRNAs) have recently been described as novel epigenetics' genes' expression regulators actively implicated in cancer progression and dissemination. The understanding of their implication in the metastatic spreading could help clinicians to improve the outcome of osteosarcoma. We established the miRNA's expression-profile between primary bone-tumors (PTs), circulating tumor cells (CTCs) and lung metastatic (META) samples from in vivo mice xenograft models. Our results show that the expression level of the miR-198 and -206 was decreased in META samples, in which the expression of the metastasis-related receptor C-Met was up-regulated. Those expression variations were validated in osteosarcoma patient biopsies from matching primary tumors and lung metastasis. We validated in vitro the endogenous miRNAs inhibitory effects on both migration and invasion, as well as we confirmed by luciferase assays that the C-Met receptor is one of their bona-fide targets. The anti-metastatic effect of these miRNAs was also validated in vivo, as their direct injections into the tumors reduce the number of lung-metastases and prolongs the overall survival of the treated animals. All together, our results suggest the absence of the miR-198 and -206 as powerful predictive biomarkers of the tumor cell dissemination and the rationale of their potential therapeutic use in the treatment of Osteosarcoma.

Implication of molecular vascular smooth muscle cell heterogeneity among arterial beds in arterial calcification.

Vascular calcification is a strong and independent predictive factor for cardiovascular complications and mortality. Our previous work identified important discrepancies in plaque composition and calcification types between carotid and femoral arteries. The objective of this study is to further characterize and understand the heterogeneity in vascular calcification among vascular beds, and to identify molecular mechanisms underlying this process. We established ECLAGEN biocollection that encompasses human atherosclerotic lesions and healthy arteries from different locations (abdominal, thoracic aorta, carotid, femoral, and infrapopliteal arteries) for histological, cell isolation, and transcriptomic analysis. Our results show that lesion composition differs between these locations. Femoral arteries are the most calcified arteries overall. They develop denser calcifications (sheet-like, nodule), and are highly susceptible to osteoid metaplasia. These discrepancies may derive from intrinsic differences between SMCs originating from these locations, as microarray analysis showed specific transcriptomic profiles between primary SMCs isolated from each arterial bed. These molecular differences translated into functional disparities. SMC from femoral arteries showed the highest propensity to mineralize due to an increase in basal TGFß signaling. Our results suggest that biological heterogeneity of resident vascular cells between arterial beds, reflected by our transcriptomic analysis, is critical in understanding plaque biology and calcification, and may have strong implications in vascular therapeutic approaches.

A Disintegrin And Metalloproteinase 12 produced by tumour cells accelerates osteosarcoma tumour progression and associated osteolysis.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour in children and adolescents for whom the prognosis remains unfavourable despite treatment protocols that combine chemotherapy and surgery. Metalloproteinases decisively contribute to cancer development and promotion by regulating cell growth, angiogenesis or inflammation. However, their role in osteosarcoma remains still unknown. METHODS: A screening of a large panel of metalloproteinases and their inhibitors, carried out in osteolytic (K7M2 and POS-1) or osteoblastic (MOS-J) mouse osteosarcoma models, shows that a member of a family of cell surface metallopeptidases, A Disintegrin And Metalloproteinase 12 (ADAM12), is highly expressed in the K7M2 and POS-1 cell lines and weakly expressed in the MOS-J cell line. To investigate whether ADAM12, involved in several pathologic conditions characterised by abnormal cell growth, plays a role in osteosarcoma tumour growth, ADAM12 was overexpressed in MOS-J and downregulated in K7M2 cells. RESULTS: In vivo experiments demonstrated that ADAM12 favours tumour growth, leading to a significant modification in animal survival. In vitro assays showed that ADAM12 knockdown in K7M2 cells slows cell proliferation. In addition, the study of microarchitectural parameters, assessed by micro-computed tomography (CT) analysis, showed that ADAM12 favours bone osteolysis, as demonstrated both in an ADAM12 overexpressing (MOS-J) and a knockdown (K7M2) model. Histological analysis showed that ADAM12 inhibited osteoblast activity and therefore enhanced bone resorption. CONCLUSIONS: Our study demonstrates that ADAM12 expression not only favours tumour growth but also associates enhanced osteolysis with a significant reduction in animal survival, suggesting that ADAM12 could be a new therapeutic target in osteosarcoma.

Modulatory effects of proteoglycans on proteinase activities.

Proteoglycans (PGs), composed of a core protein and one or more covalently attached sulfated glycosaminoglycan (GAG) chains, interact with a wide range of bioactive molecules, such as growth factors and chemokines, to regulate cell behaviors in normal and pathological processes. Additionally, PGs, through their compositional diversity, play a broad variety of roles as modulators of proteinase activities. Interactions of proteinases with other molecules on the plasma membrane anchor and activate them at a specific location on the cell surface. These interactions with macromolecules other than their own protein substrates or inhibitors result in changes in their activity and/or may have important biological effects. Thus, GAG chains induce conformational changes upon their binding to peptides or proteins. This behavior may be related to the ability of GAGs to act as modulators for some proteins (1) by acting as crucial structural elements by the control of proteinase activities, (2) by increasing the protein stability, (3) by permitting some binding to occur, exposing binding regions on the target protein, or (4) by acting as coreceptors for some inhibitors, playing important roles for the acceleration of proteinase inhibition. Understanding the modulatory effects exerted by PGs on proteinase activities is expected to lead to new insights in the understanding of some molecular systems present in pathological states, providing new targets for drug therapy.