Search

International audience; CD146 involvement was recently described in skin fibrosis of systemic sclerosis through its regulation of the Wnt pathway. Because the interaction between Wnt and ROS signaling plays a major role in fibrosis, we hypothesized that in systemic sclerosis, CD146 may regulate Wnt/ROS crosstalk. Using a transcriptomic and western blot analysis performed on CD146 wild-type or knockout mouse embryonic fibroblasts, we showed a procanonical Wnt hallmark in the absence of CD146 that is reversed when CD146 expression is restored. We found an elevated ROS content in knockout cells and an increase in DNA oxidative damage in the skin sections of knockout mice compared with those of wild-type mice. We also showed that ROS increased CD146 and its noncanonical Wnt ligand, WNT5A, only in wild-type cells. In humans, fibroblasts from patients with systemic sclerosis presented higher ROS content and expressed CD146, whereas control fibroblasts did not. Moreover, CD146 and its ligand were upregulated by ROS in both human fibroblasts. The increase in bleomycin-induced WNT5A expression was abrogated when CD146 was silenced. We showed an interplay between Wnt and ROS signaling in systemic sclerosis, regulated by CD146, which promotes the noncanonical Wnt pathway and prevents ROS signaling, opening the way for innovative therapeutic strategies.

Background: Triple Negative Breast Cancers (TNBC) are the most aggressive breast cancers and lead to poor prognoses. This is due to a high resistance to therapies, mainly because of the presence of Cancer Stem Cells (CSCs). Plasticity, a feature of CSCs, is acquired through the Epithelial to Mesenchymal Transition (EMT), a process that has been recently shown to be regulated by a key molecule, CD146. Of interest, CD146 is over-expressed in TNBC. Methods: The MDA-MB-231 TNBC cell line was used as a model to study the role of CD146 and its secreted soluble form (sCD146) in the development and dissemination of TNBC using in vitro and in vivo studies. Results: High expression of CD146 in a majority of MDA-MB-231 cells leads to an increased secretion of sCD146 that up-regulates the expression of EMT and CSC markers on the cells. These effects can be blocked with a specific anti-sCD146 antibody, M2J-1 mAb. M2J-1 mAb was able to reduce tumour development and dissemination in a model of cells xenografted in nude mice and an experimental model of metastasis, respectively, in part through its effects on CSC. Conclusion: We propose that M2J-1 mAb could be used as an additional therapeutic approach to fight TNBC.

International audience; Objective Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic- and inflammation-related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc. Methods We performed in vitro experiments, including RNA-Seq and antibody arrays, and in vivo experiments using animal models of bleomycin-induced SSc and hind limb ischemia. Results Multiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM-10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5-13-sCD146 and I10-sCD146, were identified. Of interest, I5-13-sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10-sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10-sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase C epsilon signaling pathways, respectively, whereas I5-13-sCD146 displayed profibrotic effects through the Wnt-1/beta-catenin/WISP-1 pathway. Conclusion Variants of sCD146, and in particular the novel I5-13-sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis- or fibrosis-related disorders.

Background: Triple Negative Breast Cancers (TNBC) are the most aggressive breast cancers and lead to poor prognoses. This is due to a high resistance to therapies, mainly because of the presence of Cancer Stem Cells (CSCs). Plasticity, a feature of CSCs, is acquired through the Epithelial to Mesenchymal Transition (EMT), a process that has been recently shown to be regulated by a key molecule, CD146. Of interest, CD146 is over-expressed in TNBC. Methods: The MDA-MB-231 TNBC cell line was used as a model to study the role of CD146 and its secreted soluble form (sCD146) in the development and dissemination of TNBC using in vitro and in vivo studies. Results: High expression of CD146 in a majority of MDA-MB-231 cells leads to an increased secretion of sCD146 that up-regulates the expression of EMT and CSC markers on the cells. These effects can be blocked with a specific anti-sCD146 antibody, M2J-1 mAb. M2J-1 mAb was able to reduce tumour development and dissemination in a model of cells xenografted in nude mice and an experimental model of metastasis, respectively, in part through its effects on CSC. Conclusion: We propose that M2J-1 mAb could be used as an additional therapeutic approach to fight TNBC.

Additional file 1. Supplementary methods:Peptides and antibodies. Plasmids, siRNA and cell transfection. Western-blot and Immunoprecipitation assays. ELISA experiments. Immunofluorescence experiments. Flow cytometry experiments. Reverse Transcription-quantitative PCR (RT-qPCR). Cell migration assays. Cell Proliferation assays. Transwell invasion assays. Crispr/Cas9 deletion of genes. Experiments on animals and imaging. Immunohistochemistry. Patient cohort. Statistical analysis. Supplementary figures and tables: Supplementary Table 1: Absence of significant impact of age, KPS and steroid dose on progression-free survival or overall survival. Supplementary Table 2: references and application condition of the different antibodies used in the study. Supplementary Figure 1: Expression of CD146 on various types of cancers and effect on overall survival in patients with GBM. Supplementary Figure 2: Glioblastoma cell lines characterization. Supplementary Figure 3: Concentration of VEGF and sCD146 in the culture media of U87, U373, and U118 glioblastoma cell lines. Supplementary Figure 4: Avastin induces proliferation, sCD146 secretion, and EMT/CSC markers in U373 cells. Supplementary Figure 5: Avastin has no effect on CD146-negative U118 glioblastoma cells. Supplementary Figure 6: sCD146 induces U373 cell proliferation, migration and invasion in-vitro and promotes CSC and EMT markers. Supplementary Figure 7: Effect of sCD146 and VEGF on CD146-negative glioblastoma cells. Supplementary Figure 8: sCD146 binds integrin αvβ3 on U373 cells. Supplementary Figure 9: Soluble CD146 induces EMT in U118 cells transfected with integrin αvβ3. Supplementary Figure 10: Knocking-down integrin αvβ3 inhibits sCD146-induced EMT in U87 cells. Supplementary Figure 11: sCD146 mediated its effects on U87 cells through a signalosome containing CD146, αvβ3, and VEGFR2. Supplementary Figure 12: sCD146 mediates its effects on U373 cells through a signalosome containing CD146, αvβ3, and VEGFR2. Supplementary Figure 13: Validating gene knock out in U87 and U373 cells. Supplementary Figure 14: Integrin αvβ3 associates with membrane CD146 and VEGFR2 on HUVECs and binds sCD146. Supplementary Figure 15: CD146/VEGFR2/integrin αvβ3 signalosome mediates sCD146 and VEGF effects in U87 cells and activates common signaling pathways. Supplementary Figure 16: CD146/VEGFR2/integrin αvβ3 signalosome mediates sCD146 and VEGF effects in U373 cells and activates common signaling pathways. Supplementary Figure 17: CD146, integrin αvβ3, and VEGFR2 are essential for mediating sCD146 and VEGF effects on U87 cells. Supplementary Figure 18: CD146, integrin αvβ3, and VEGFR2 are essential for mediating sCD146 and VEGF effects on U373 cells. Supplementary Figure 19: Humanized anti-sCD146 antibody mucizumab significantly decreases U373 cell proliferation, migration and invasion and hampers CSC and EMT in-vitro. Supplementary Figure 20: Humanized anti-sCD146 mucizumab significantly decreases human sCD146 and human VEGF in two different mouse models of glioblastoma. Supplementary Figure 21: Illustrative summary describing the mechanism of resistance to bevacizumab in CD146-positive glioblastoma cells.

Cardiac fibrosis constitutes irreversible necrosis of the heart muscle as a consequence of different acute (myocardial infarction) or chronic (diabetes, hypertension, …) diseases but also due to genetic alterations or aging. Currently, there is no curative treatment that is able to prevent or attenuate this phenomenon that leads to progressive cardiac dysfunction and life-threatening outcomes. This review summarizes the different targets identified and the new strategies proposed to fight cardiac fibrosis. Future directions, including the use of exosomes or nanoparticles, will also be discussed.

International audience; CD146 is an endothelial junctional adhesion molecule, which expression is increased in human glomerular diseases. However, the pathological significance of this overexpression remains unknown. Induction of glomerulonephritis in mice, by using nephrotoxic serum, showed that CD146 expression was highly induced within damaged glomeruli and was associated with renal inflammation and fibrosis. Interestingly, 2 weeks after glomerulonephritis induction, CD146 knockout mice showed preserved renal function as proteinuria and blood urea nitrogen levels were significantly lower compared with wild-type littermates. Furthermore, renal structure was considerably conserved, since crescents formation, tubular dilation, monocyte and lymphocyte infiltration, and interstitial renal fibrosis were highly reduced. Colocalization with markers for different types of glomerular cells showed that CD146 expression was mainly increased within the injured endothelium of the glomerular tuft. Consequently, we generated a new transgenic strain in which CD146 was specifically deleted in the vascular endothelium. Similarly to CD146 knockout, these mice showed preservation of renal structure and function after the induction of glomerulonephritis compared with wild-type animals. These data show that endothelial CD146 plays a major role in glomerulonephritis and may represent a novel therapeutic target to reduce glomerular damage and the progression of renal disease.

International audience; CD146 is a cell adhesion molecule expressed on endothelial cells, as well as on other cells such as mesenchymal stem cells and Th17 lymphocytes. This protein also exists in a soluble form, whereby it can be detected in biological fluids, including the serum or the cerebrospinal fluid (CSF). Some studies have highlighted the significance of CD146 and its soluble form in angiogenesis and inflammation, having been shown to contribute to the pathogenesis of many inflammatory autoimmune diseases, such as systemic sclerosis, mellitus diabetes, rheumatoid arthritis, inflammatory bowel diseases, and multiple sclerosis. In this review, we will focus on how CD146 and sCD146 contribute to the pathogenesis of the aforementioned autoimmune diseases and discuss the relevance of considering it as a biomarker in these pathologies.

International audience; Initially discovered in human melanoma, CD146/MCAM is expressed on many tumors and is correlated with cancer progression and metastasis. However, targeting CD146 remains challenging since it is also expressed on other cell types, as vessel cells, where it displays important physiological functions. We previously demonstrated that CD146 is shed as a soluble form (sCD146) that vectorizes the effects of membrane CD146 on tumor angiogenesis, growth and survival. We thus generated a novel monoclonal antibody, the M2J-1 mAb, which specifically targets sCD146, but not membrane CD146, and counteracts these effects. In our study, we analyzed the effects of sCD146 on the dissemination and the associated procoagulant phenotype in two highly invasive human CD146-positive cancer cell lines (ovarian and melanoma). Results show that sCD146 induced epithelial to mesenchymal transition, favored the generation of cancer stem cells and increased the membrane expression of tissue factor. Treatment of cancer cells with sCD146 in two experimental models (subcutaneous xenografting and intracardiac injection of cancer cells in nude mice) led to increased tumor dissemination and procoagulant activity. The M2J-1 mAb drastically reduced metastasis but also procoagulant activity, in particular by decreasing the number of circulating tumor microparticles, and blocked the relevant signaling pathways as demonstrated by RNA expression profiling experiments. Thus, our findings demonstrate that sCD146 mediates important pro-metastatic and procoagulant effects in two CD146-positive tumors. Targeting sCD146 with the newly generated M2J-1 mAb could constitute an innovative strategy for preventing dissemination and thromboembolism in many CD146-positive tumors.