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Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor |
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| Authors: | Zuber Chantal Knackmuss Stefan Zemora Georgeta Reusch Uwe Vlasova Ekaterina Diehl Daniela Mick Vera Hoffmann Karin Nikles Daphne Fröhlich Thomas Arnold Georg J Brenig Bertram Wolf Eckhard Lahm Harald Little Melvyn Weiss Stefan |
| Institution: | 1 Laboratorium für Molekulare Biologie—Genzentrum—Institut für Biochemie der LMU München, Feodor-Lynen-Str. 25, D-81377 München, Germany 2 Affimed Therapeutics AG, Technologiepark, Im Neuenheimer Feld 582, 69120 Heidelberg, Germany 3 Institut für Molekulare Tierzucht und Biotechnologie, Genzentrum, LMU München, Feodor-Lynen-Str. 25, D-81377 München, Germany 4 Laboratory for Functional Genome Analysis (LAFUGA, Genzentrum, LMU München, Feodor-Lynen-Str. 25, D-81377 München, Germany 5 Tierärztliches Institut der Universität Göttingen, Burckhardtweg 2, D-37077 Göttingen, Germany |
| Abstract: | The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.6-fold higher cell-surface levels of LRP/LR. We show that anti-LRP/LR tools interfere with the invasive potential of HT1080 cells. Anti-LRP/LR single-chain variable fragment antibody (scFv) iS18 generated by chain shuffling from parental scFv S18 and its full-length version immunoglobulin G1-iS18 reduced the invasive potential of HT1080 cells significantly by 37% and 38%, respectively. HT1080 cells transfected with lentiviral plasmids expressing small interfering RNAs directed against LRP mRNA showed reduced LRP levels by approximately 44%, concomitant with a significant decrease in the invasive potential by approximately 37%. The polysulfated glycans HM2602 and pentosan polysulfate (SP-54), both capable of blocking LRP/LR, reduced the invasive potential by 20% and 35%, respectively. Adhesion of HT1080 cells to laminin-1 was significantly impeded by scFv iS18 and immunoglobulin G1-iS18 by 60% and 68%, respectively, and by SP-54 and HM2602 by 80%, suggesting that the reduced invasive capacity achieved by these tools is due to the perturbation of the LRP/LR-laminin interaction on the cell surface. Our in vitro data suggest that reagents directed against LRP/LR or LRP mRNA such as antibodies, polysulfated glycans, or small interfering RNAs, previously shown to encompass an anti-prion activity by blocking or downregulating the prion receptor LRP/LR, might also be potential cancer therapeutics blocking metastasis by interfering with the LRP/LR-laminin interaction in neoplastic tissues. |
| Keywords: | CMD carboxymethylated dextran LRP/LR laminin receptor precursor/laminin receptor GAG glycosaminoglycan IgG1 immunoglobulin G1 scFv single-chain variable fragment antibody HEK human embryonic kidney PBS phosphate-buffered saline HSPG heparan sulfate proteoglycan FACS fluorescence-activated cell sorting GST glutathione S-transferase MMP matrix metalloproteinase DMEM Dulbecco's modified Eagle's medium HRP horseradish peroxidase EDTA ethylenediaminetetraacetic acid |
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