ABSTRACT
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-2alpha and its translocation to the nucleus where it forms heterodimers with HIF-1beta. 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-2alpha localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2alpha/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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Basigin/metabolism , Melanoma/pathology , Up-Regulation , Vascular Endothelial Growth Factor Receptor-2/genetics , Active Transport, Cell Nucleus , Animals , Apoptosis/genetics , Autocrine Communication/genetics , Basigin/genetics , CHO Cells , Cell Line, Tumor , Cell Movement/genetics , Cell Nucleus/metabolism , Cell Proliferation , Cricetinae , Cricetulus , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Melanoma/genetics , Neoplasm Invasiveness/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is thought to promote tumor angiogenesis mostly through its protease-inducing function and more recently by its ability to increase tumor cell expression of vascular endothelial growth factor (VEGF). In this study, we present evidence that EMMPRIN can promote angiogenesis by a direct effect on endothelial cells through a paracrine regulation of the VEGF/VEGF-receptor (VEGFR) system. Using human microvascular endothelial cell line-1 endothelial cells, we show that EMMPRIN selectively increased the soluble VEGF isoforms (121 and 165), but not the matrix-bound VEGF 189 form. In addition, EMMPRIN up-regulated the expression of VEGFR-2 without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of endothelial cells. EMMPRIN's effects, which were matrix metalloproteinase and urokinase-type plasminogen activator independent, were mediated primarily through hypoxia-inducible factor-2alpha expression, also up-regulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the up-regulation of hypoxia-inducible factor-2alpha, VEGFR-2, and the soluble forms of VEGF in endothelial cells, thus directly regulating the angiogenic process.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Basigin/pharmacology , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basigin/genetics , Basigin/metabolism , CHO Cells , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cells, Cultured , Cricetinae , Cricetulus , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Immunoblotting , Mice , Mice, Nude , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Solubility , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
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.
