A Novel Monoclonal Antibody to Human Laminin α5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521

Abstract

Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

Identification of integrin α3 as a molecular marker of cells undergoing epithelial–mesenchymal transition and of cancer cells with aggressive phenotypes

“…Expression of laminin‐332 is induced at the wounded edge of epidermis and at the leading edge of invading carcinomas. Additionally, laminin‐332 and ‐511 enhance migration and scattering of the cells through binding to integrin α3β1, and blockade of these interactions by mAb strongly inhibited α3β1 integrin‐mediated adhesion of cancer cells . In the present study, ITGA3 was shown to be associated with invasion of phospho‐ERK‐positive cells (Figs a,f).…”

“…Cancer cell invasion was recently classified into two categories, so‐called “collective cancer cell invasion” and “fibroblast‐led collective invasion” . In the former, cancer cells invade without the assistance of other cells, whereas the latter highlights the importance of certain fibroblasts that initiate a collective invasion chain of cancer cells .…”

Platelets store laminins 411/421 and 511/521 in compartments distinct from α‐ or dense granules and secrete these proteins via microvesicles

“…Mouse monoclonal antibodies (mAbs) to human laminin α4 (clones 3H2 and 6C3) and α5 (clones 4B5 and 4B12) chains are described in our previous studies . Laminin β1 and β2 chains were detected with mAbs DG10 and C4, respectively .…”

Chain-specific antibodies for laminin-511

“…In a recent report, five antibodies were developed which recognized the LMa5 chain (Wondimu et al, 2013). Those antibodies were useful for immunohistochemistry; however, one of the antibodies (8G9) inhibited integrin-dependent adhesion and migration of several cancer lines on LM-511.…”

“…Used in series, antibodies that recognize individual chains of LM-511 could be employed to specifically purify LM-511 from complex tissues such as human placenta. A recent report described a LMa5 specific monoclonal antibody (mAb) 8G9, with a specificity different to mAb 4C7, which is capable of inhibiting LM-511-dependent cell adhesion and migration of several cancer cell types (Wondimu et al, 2013).…”