Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

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Abstract

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction.

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Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells

LA Liotta, S Aznavoorian, H Krutzsch … (1990)

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell- recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)

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Anti-LRP/LR-specific antibody IgG1-iS18 significantly reduces adhesion and invasion of metastatic lung, cervix, colon and prostate cancer cells.

Aadilah Omar, Uwe Reusch, Stefan Knackmuss … (2012)

The 37-kDa/67-kDa laminin receptor [laminin receptor precursor/high-affinity laminin receptor (LRP/LR)] is thought to play a major role in invasion and adhesion, key components of metastatic cancer. Lung cancer, cervical cancer, colon cancer and prostate cancer are among the top 10 cancer types worldwide. Here, we report that LRP/LR levels on the surface of lung cancer cells, cervical cancer cells, colon cancer cells and prostate cancer cells are significantly increased compared to non-tumorigenic fibroblasts. Adhesion of lung cancer cells, cervical cancer cells, colon cancer cells and prostate cancer cells to laminin-1 is significantly reduced, employing the anti-LRP/LR-specific antibody IgG1-iS18. Invasion of these cell lines into the Matrigel™ matrix was significantly impeded with IgG1-iS18. The Pearson's correlation coefficient proves a correlation between LRP/LR cell-surface levels and invasion potential, as well as adhesion and invasion, respectively. Our findings suggest that IgG1-iS18 antibody might act as alternative therapeutic tool for treatment of various metastatic cancer types. Copyright © 2012 Elsevier Ltd. All rights reserved.

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Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor.

Eckhard Wolf, Melvyn Little, Harald Lahm … (2008)

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.

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Author and article information

Contributors

Corinne Ida Lasmezas: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2014

Publication date (Electronic): 5 May 2014

Volume: 9

Issue: 5

Electronic Location Identifier: e96268

Affiliations

[1 ]School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, Gauteng, The Republic of South Africa (RSA)

[2 ]Affimed Therapeutics AG, Technologiepark, Im Neuenheimer Feld, Heidelberg, Baden-Wuerttemberg, Germany

The Scripps Research Institute Scripps Florida, United States of America

Author notes

* E-mail: stefan.weiss@ 123456wits.ac.za

Competing Interests: S.F.T.W. is currently a PLOS ONE Editorial Board Member. U.R., S.K. and M.L. are affiliated with or employed by Affimed Therapeutics A.G., a commercial company which produces therapeutic antibodies for the treatment of cancer and inflammatory diseases. Furthermore, the anti-LRP/LR antibodies used in this study for the blockade of invasion and adhesion have been described in two international patents as potential therapeutic anticancer tools. Namely patent, EP0984987, entitled “A soluble laminin receptor precursor and methods to inhibit its interactions” has claims directed to a pharmaceutical composition comprising a soluble laminin receptor precursor or functional derivative or fragment thereof and is owned by the University of the Witwatersrand. This patent has been validated in the United Kingdom and Germany. The second patent, EP1670826, is co-owned by the University of the Witwatersrand and Affimed Therapeutics AG and is entitled “Single chain antibody acting against 37 kDa/67 kDa laminin receptor as tools for the diagnosis and therapy of prion diseases and cancer, production and use thereof”. This granted European patent was validated in the United Kingdom, France, Germany, Switzerland and Austria. The claims are directed to a single chain antibody molecule specifically targeting LRP/LR for the treatment of prion diseases or cancer. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Conceived and designed the experiments: SFTW. Performed the experiments: CC. Analyzed the data: CC BDCD. Contributed reagents/materials/analysis tools: UR SK ML. Wrote the paper: CC. Edited the manuscript: TK ML BDCD.

Article

Publisher ID: PONE-D-13-49627

DOI: 10.1371/journal.pone.0096268

PMC ID: 4010454

PubMed ID: 24798101

SO-VID: 9463fcb7-a7dd-44a3-952e-918465c0d428

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 25 November 2013

Date accepted : 4 April 2014

Page count

Pages: 10

Funding

This work was supported by the National Research Foundation, the Republic of South Africa and the Medical Research Council, the Republic of South Africa. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s), and therefore, the National Research Foundation does not accept any liability in this regard thereto. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

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Most cited references 17

Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells

Anti-LRP/LR-specific antibody IgG1-iS18 significantly reduces adhesion and invasion of metastatic lung, cervix, colon and prostate cancer cells.

Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor.

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