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Abstract
Blood borne metastatic tumor cell adhesion to endothelial cells constitutes a critical rate-limiting step in hematogenous cancer metastasis. Interactions between cancer associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag) and endothelium-expressed galectin-3 (Gal-3) have been identified as the leading molecular mechanism initiating tumor/endothelial cell adhesion in several types of cancer. However, it is unknown how these rather weak and transient carbohydrate/lectin mediated interactions are stabilized. Here, using Western blot and LC tandem mass spectrometry analyses of pull-downs utilizing TF-Ag loaded gold nanoparticles, we identified Gal-3, endothelial integrin α3β1, Src kinase, as well as 5 additional molecules mapping onto focal adhesion pathway as parts of the macromolecular complexes formed at the endothelial cell membranes downstream of TF-Ag/Gal-3 interactions. In a modified parallel flow chamber assay, inhibiting α3β1 integrin greatly reduced the strength of tumor/endothelial cell interactions without affecting the initial cancer cell adhesion. Further, the macromolecular complex induced by TF-Ag/Gal-3/α3β1 interactions activates Src kinase, p38, and ERK1/2, pathways in endothelial cells in a time- and α3β1-dependent manner. We conclude that, following the initial metastatic cell attachment to endothelial cells mediated by TF-Ag/Gal-3 interactions, endothelial integrin α3β1 stabilizes tumor/endothelial cell adhesion and induces the formation of macromolecular signaling complex activating several major signaling pathways in endothelial cells.
Highlights
Metastasis is a major cause of cancer-related morbidity and mortality
To identify integrin molecules and other proteins interacting with Thomsen-Friedenreich antigen (TF-Ag)/Gal-3 complexes at the endothelial cell membrane we have performed a pulldown assay using gold nanoparticles bearing multiple TFAg moieties (TF-Au) covalently attached to the particles through the polyethylene glycol (PEG) linker and control nanoparticles, on which PEG linkers are terminated with the OH groups (PEG-Au) as described in Materials and Methods
Probing the membranes with the panel of antibodies directed against α3, α4, α5, αV, β1, β3, β4, and β5 integrins revealed the presence of α3 and β1 integrin subunits (i.e. α3β1 integrin) in TF-Au, but not in untreated endothelial cell or PEG-Au samples (Fig. 1, A) indicative of α3β1 interaction with TF-Ag/Gal-3 complexes
Summary
Metastasis is a major cause of cancer-related morbidity and mortality. The initial arrest of blood borne metastatic tumor cells in distant organ microvasculature constitutes an important rate-limiting step in hematogenous cancer metastasis [1,2,3]. TF-Ag expressing cancer cells interact with cell surface clustered endothelial Gal-3 to initiate metastatic cell arrest in distant organ vasculature [9, 17,18,19,20]. These carbohydrate/ lectin mediated adhesive interactions are rather weak and transient in nature. Gal-3 does not contain a transmembrane domain and, lacks means of being anchored at the endothelial cell surface without the involvement of additional molecules capable of providing such anchorage It is currently unknown which molecules stabilize tumor/endothelial cell adhesion downstream of TF-Ag/Gal-3 interactions
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