EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

EMMPRIN's expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2) in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2α and its translocation to the nucleus where it forms heterodimers with HIF-1β. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2α localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2α/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion

Implementation of the One Health approach to fight arbovirus infections in the Mediterranean and Black Sea Region: Assessing integrated surveillance in Serbia, Tunisia and Georgia

Background In the Mediterranean and Black Sea Region, arbovirus infections are emerging infectious diseases. Their surveillance can benefit from one health inter-sectoral collaboration; however, no standardized methodology exists to study One Health surveillance. Methods We designed a situation analysis study to document how integration of laboratory/clinical human, animal and entomological surveillance of arboviruses was being implemented in the Region. We applied a framework designed to assess three levels of integration: policy/institutional, data collection/data analysis and dissemination. We tested the use of Business Process Modelling Notation (BPMN) to graphically present evidence of inter-sectoral integration. Results Serbia, Tunisia and Georgia participated in the study. West Nile Virus surveillance was analysed in Serbia and Tunisia, Crimea-Congo Haemorrhagic Fever surveillance in Georgia. Our framework enabled a standardized analysis of One Health surveillance integration, and BPMN was easily understandable and conducive to detailed discussions among different actors/institutions. In all countries, we observed integration across sectors and levels except in data collection and data analysis. Data collection was interoperable only in Georgia without integrated analysis. In all countries, surveillance was mainly oriented towards outbreak response, triggered by an index human case. Discussion The three surveillance systems we observed prove that integrated surveillance can be operationalized with a diverse spectrum of options. However, in all countries, the integrated use of data for early warning and inter-sectoral priority setting is pioneeristic. We also noted that early warning before human case occurrence is recurrently not operationally prioritized

Rôle de la glycoprotéine EMMPRIN dans l'invasion et l'angiogenèse en général et dans le mélanome

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Extracellular matrix metalloproteinase inducer up-regulates the urokinase-type plasminogen activator system promoting tumor cell invasion.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a membrane glycoprotein overexpressed in many cancer tissues and is known for its ability to stimulate MMP expression. In this work, we show that EMMPRIN is also a regulator of the urokinase-type plasminogen activation (uPA) system of serine proteases, thus participating to the increase of the overall proteolytic function of the cancer cells. Enhanced EMMPRIN expression in a tumorigenic breast epithelial cell line NS2T2A increased the levels of uPA, uPA receptor, and the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1), as measured by quantitative reverse transcription-PCR, Western blot, and plasminogen-casein zymography. This response was down-regulated by either EMMPRIN small interfering RNA or a blocking antibody to EMMPRIN. EMMPRIN-containing purified membrane fraction from Chinese hamster ovary cells when added exogenously to NS2T2A cells induced a similar activation of the uPA/PAI-1 system. Additionally, overexpression of EMMPRIN in NS2T2A cells increased uPA levels in cocultured endothelial cells, showing a paracrine regulation loop involving a tumor-stroma interaction. EMMPRIN-expressing cells also exhibited enhanced invasive potential in vitro, and the use of amiloride (uPA inhibitor) and marimastat (MMP inhibitor) showed that the two proteolytic systems reduced alone and in combination the invasive potential mediated through EMMPRIN. These data show a novel regulatory pathway for uPA activity and suggest that EMMPRIN is involved in uPA dysregulation observed in cancer

Immunohistochemical staining of EMMPRIN, VEGFR-2 and HIF-2α in sections of human melanoma tissues.

<p><b>Left</b>, representative melanoma with lower expression of EMMPRIN. (<b>A</b>) EMMPRIN, (<b>B</b>) VEGFR-2 and (<b>C</b>) HIF-2α staining. <b>Right</b>, representative melanoma exhibiting strong expression of EMMPRIN. (<b>D</b>) EMMPRIN, (<b>E</b>) VEGFR-2, and (<b>F</b>) HIF-2α staining.</p

EMMPRIN up-regulates VEGFR-2 through HIF-2α stimulation under normoxic conditions.

<p><b>A</b>. Melanoma cells were transfected with HIF-2α siRNA or scrambled siRNA (Ctl siRNA) before treatment with 25 µg/ml of Emp for 1 h. a) HIF-2α and VEGFR-2 transcripts were quantified by qRTPCR (columns, means of relative expression to <i>PPIA</i> housekeeping gene of at least three independent experiments; <i>bars</i>, SD. * p<0.05). b) HIF-2α protein expression was evaluated by Western blotting. Cells were transfected with scrambled siRNA before Emp treatment. Actin was used as a loading control; representative blot of 3 independent experiments is shown. <b>B</b>. HIF-2α nuclear localization after EMMPRIN stimulation of M10 melanoma cells. a) Immunofluoresence analysis of HIF-2α. Cells were grown on the glass slide chamber and treated or not with Emp for 4 h before fixation. HIF-2α protein localization was visualized by confocal microscopy. b) Western blot analysis of the subcellular fractions (cytosoloic (C) and nuclear (N)) of M10 cells treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 h before being harvested. C. HIF-2α forms heterodimers with HIF-1β after EMMPRIN stimulation of M10 melanoma cells. a) HIF-2α protein expression was evaluated by Western blotting of the nuclear fraction after immunoprecipitation with HIF-1β antibody. b) In situ PLA detection of HIF-2α and HIF-1β heterodimers: HIF-1β/HIF-2α heterodimers (red) in nuclei of M10 melanoma cells after in situ PLA using antibodies against HIF-1β/HIF-2α. M10 cells were treated or not with Emp or 100 µM deferoxamine (DFO), used as positive control, for 4 hours. Nuclei were stained with DAPI (blue). The EMMPRIN and DFO panels show high magnification to clearly visualize the PLA spots representing heterodimers.</p

EMMPRIN up-regulates VEGFR-2 expression in primary melanoma cells.

<p><b>A</b>. M10 and WM278 cells were transfected for 24 hours with two different EMMPRIN siRNA (Ambion siRNA) or their corresponding scrambled control siRNA (Ctl siRNA) at 33 nmol/L concentration. EMMPRIN and VEGFR-2 protein expression was evaluated by Western blot analysis of 15 µg cell lysates respectively (actin was used as a loading control; representative blot of 3 independent experiments is shown) and conditioned medium (CM) was harvested for gelatinases and uPA zymographies. <b>B</b>. Immunofluorescence of M10 cells treated with EMMPRIN siRNA and immunostained for EMMPRIN, VEGFR-2, uPA and TIMP-1. <b>C</b>. QRT-PCR analyses of EMMPRIN, VEGFR-2 and TIMP-1 showing the mean ±SD of relative expression to <i>PPIA</i> house keeping gene of at least 3 independent experiments. <b>D</b>. M10 and WM278 cells were incubated with CHO-membranes containing or not EMMPRIN (Ctl and Emp respectively). VEGFR-2 and uPA transcript levels were quantified by qRT-PCR showing the mean ±SD of relative expression to <i>PPIA</i> house keeping gene of at least 3 independent experiments. VEGFR-2 protein expression was evaluated by Western blot analysis of 30 µg cell lysates (actin was used as a loading control; representative blot of 3 independent experiments is shown); bars, SD. *, p<0.05.</p