Tumour growth inhibition and anti-metastatic activity of a mutated furin-resistant Semaphorin 3E isoform.
Bottom Line: Uncl-Sema3E also acts independently as a potent anti-angiogenic factor.It activates a PlexinD1-mediated signalling cascade in endothelial cells that leads to the inhibition of adhesion to extracellular matrix, directional migration and cell survival.In summary, we conclude that Uncl-Sema3E is a novel inhibitor of tumour angiogenesis and growth that concomitantly hampers metastatic spreading.
Affiliation: Institute for Cancer Research and Treatment (IRCC), University of Torino Medical School, Candiolo, Italy.Show MeSH
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Mentions: Further investigating the functional relevance of uncleaved Sema3E, we observed that it could trigger the typical semaphorin-driven ‘collapsing’ response in COS cells expressing PlexinD1 (unpublished observation). Moreover, Uncl-Sema3E strongly and rapidly induced the retraction of cellular processes and the rounding of human umbilical vein-derived endothelial cells (HUVEC) (Figure 3A and Supporting Information Movies 1A–C). These effects have been reported for several semaphorins and are currently explained by the inhibitory activity mediated by plexins on integrin-based focal adhesive complexes (Barberis et al, 2004). Notably, a recent study showed that Sema3E triggers integrin-beta1 internalization leading to reduced cell-substrate adhesion (Sakurai et al, 2010). We therefore investigated the ability of Uncl-Sema3E to inhibit integrin function and intracellular signalling in endothelial cells. We found that as little as 5 min of treatment with Uncl-Sema3E were sufficient to trigger a dramatic disassembly of focal adhesion complexes (Fig 3B); these structures connect integrins with the cytoskeleton and are pivotally involved in the protrusion of cellular processes as well as in cell migration dynamics. Moreover, the presence of integrin-beta1 molecules in active conformation on the cell surface was significantly reduced upon treatment with Uncl-Sema3E (Fig 3C and Supporting Information Fig 4), further indicating reduced adhesion to the extracellular matrix. We also found that pre-treating endothelial cells with antibodies directed against the active conformation of integrin-beta1, which forcibly stabilizes cell-substrate adhesion, was sufficient to prevent cell contraction induced by Uncl-Sema3E (Supporting Information Fig 5A). This indicated that the loss of cell-substrate adhesion is crucial for the inhibitory activity of Uncl-Sema3E in endothelial cells. Notably, several studies underlined that substrate adhesion is a major requirement for the viability of endothelial cells, and its default leads to programmed cell death by anoikis, both in culture and in vivo (reviewed by Cheresh & Stupack, 2008). We found that focal adhesion disassembly triggered by Uncl-Sema3E further inhibited downstream intracellular signalling cascades, especially the activation of focal adhesion kinase (FAK) and the associated upregulation of phosphorylated MAPK/ERK (Fig 3D). It was shown previously that molecules interfering with FAK and subsequent MAPK activation in endothelial cells lead to an apoptotic response (Lu & Rounds, 2011). In fact, we observed an increased number of active-caspase 3-positive apoptotic endothelial cells upon treatment with Uncl-Sema3E as assessed by two independent methods (Fig 3E and F).
Affiliation: Institute for Cancer Research and Treatment (IRCC), University of Torino Medical School, Candiolo, Italy.