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Tumour growth inhibition and anti-metastatic activity of a mutated furin-resistant Semaphorin 3E isoform.

Casazza A, Kigel B, Maione F, Capparuccia L, Kessler O, Giraudo E, Mazzone M, Neufeld G, Tamagnone L - EMBO Mol Med (2012)

Bottom Line: Here, we show that a mutated, uncleavable variant of Sema3E (Uncl-Sema3E) binds to PlexinD1 like p61-Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Research and Treatment (IRCC), University of Torino Medical School, Candiolo, Italy.

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Uncl-Sema3E inhibits integrin-mediated adhesion and intracellular signalling in endothelial cellsHUVEC were stained with FITC-phalloidin to reveal F-actin (in green) and 4,6-diamidino-2-phenylindole (DAPI) to highlight nuclei. Upon incubation with 7 nM Uncl-Sema3E for 20 min, endothelial cells totally disassembled F-actin stress fibers, and cellular processes were retracted, leading to the typical collapsed phenotype (see Barberis et al, 2004). Scale bar: 50 µm.HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to immunofluorescence analysis by staining with anti-Paxillin antibodies (revealed in red) and with FITC-falloidin to detect F-actin (in green). On the right, images of cellular processes are magnified to reveal focal adhesions (FA) and the percent fraction of FA-containing cells is indicated aside. Scale bars: 20 and 10 µm in the magnified fields shown on the right.The graph summarizes the results of immunofluorescence analysis shown in Supporting Information Fig 4, and indicates the amount of integrin-β1 in active conformation on the surface of HUVEC, either untreated or treated with 7 nM Uncl-Sema3E for 5 min; n = 4 (each condition).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and their protein lysates were subjected to immuno-blotting analysis with antibodies directed against Y397 phosphorylated focal adhesion kinase (P-FAK), serine/threonine phosphorylated MAPK and AKT and vinculin (as protein loading control).HUVEC grown on glass coverslips were treated with 7 nM Uncl-Sema3E (n = 4) or with 200 nM staurosporin (STS; n = 4) for 1 h, or left untreated (n = 4). The early apoptotic marker activated-caspase 3 was revealed by immunofluorescence (shown in red); nuclei were counterstained with DAPI; scale bar: 50 µm. The graph indicates the average percent fraction of apoptotic cells in each condition ± SD; *p = 0.008; **p = 0.0002 (vs. control).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to cytofluorimetric analysis with antibodies directed against activated-caspase 3. The experiment was repeated three times with consistent results.
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fig03: Uncl-Sema3E inhibits integrin-mediated adhesion and intracellular signalling in endothelial cellsHUVEC were stained with FITC-phalloidin to reveal F-actin (in green) and 4,6-diamidino-2-phenylindole (DAPI) to highlight nuclei. Upon incubation with 7 nM Uncl-Sema3E for 20 min, endothelial cells totally disassembled F-actin stress fibers, and cellular processes were retracted, leading to the typical collapsed phenotype (see Barberis et al, 2004). Scale bar: 50 µm.HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to immunofluorescence analysis by staining with anti-Paxillin antibodies (revealed in red) and with FITC-falloidin to detect F-actin (in green). On the right, images of cellular processes are magnified to reveal focal adhesions (FA) and the percent fraction of FA-containing cells is indicated aside. Scale bars: 20 and 10 µm in the magnified fields shown on the right.The graph summarizes the results of immunofluorescence analysis shown in Supporting Information Fig 4, and indicates the amount of integrin-β1 in active conformation on the surface of HUVEC, either untreated or treated with 7 nM Uncl-Sema3E for 5 min; n = 4 (each condition).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and their protein lysates were subjected to immuno-blotting analysis with antibodies directed against Y397 phosphorylated focal adhesion kinase (P-FAK), serine/threonine phosphorylated MAPK and AKT and vinculin (as protein loading control).HUVEC grown on glass coverslips were treated with 7 nM Uncl-Sema3E (n = 4) or with 200 nM staurosporin (STS; n = 4) for 1 h, or left untreated (n = 4). The early apoptotic marker activated-caspase 3 was revealed by immunofluorescence (shown in red); nuclei were counterstained with DAPI; scale bar: 50 µm. The graph indicates the average percent fraction of apoptotic cells in each condition ± SD; *p = 0.008; **p = 0.0002 (vs. control).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to cytofluorimetric analysis with antibodies directed against activated-caspase 3. The experiment was repeated three times with consistent results.

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).


Tumour growth inhibition and anti-metastatic activity of a mutated furin-resistant Semaphorin 3E isoform.

Casazza A, Kigel B, Maione F, Capparuccia L, Kessler O, Giraudo E, Mazzone M, Neufeld G, Tamagnone L - EMBO Mol Med (2012)

Uncl-Sema3E inhibits integrin-mediated adhesion and intracellular signalling in endothelial cellsHUVEC were stained with FITC-phalloidin to reveal F-actin (in green) and 4,6-diamidino-2-phenylindole (DAPI) to highlight nuclei. Upon incubation with 7 nM Uncl-Sema3E for 20 min, endothelial cells totally disassembled F-actin stress fibers, and cellular processes were retracted, leading to the typical collapsed phenotype (see Barberis et al, 2004). Scale bar: 50 µm.HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to immunofluorescence analysis by staining with anti-Paxillin antibodies (revealed in red) and with FITC-falloidin to detect F-actin (in green). On the right, images of cellular processes are magnified to reveal focal adhesions (FA) and the percent fraction of FA-containing cells is indicated aside. Scale bars: 20 and 10 µm in the magnified fields shown on the right.The graph summarizes the results of immunofluorescence analysis shown in Supporting Information Fig 4, and indicates the amount of integrin-β1 in active conformation on the surface of HUVEC, either untreated or treated with 7 nM Uncl-Sema3E for 5 min; n = 4 (each condition).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and their protein lysates were subjected to immuno-blotting analysis with antibodies directed against Y397 phosphorylated focal adhesion kinase (P-FAK), serine/threonine phosphorylated MAPK and AKT and vinculin (as protein loading control).HUVEC grown on glass coverslips were treated with 7 nM Uncl-Sema3E (n = 4) or with 200 nM staurosporin (STS; n = 4) for 1 h, or left untreated (n = 4). The early apoptotic marker activated-caspase 3 was revealed by immunofluorescence (shown in red); nuclei were counterstained with DAPI; scale bar: 50 µm. The graph indicates the average percent fraction of apoptotic cells in each condition ± SD; *p = 0.008; **p = 0.0002 (vs. control).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to cytofluorimetric analysis with antibodies directed against activated-caspase 3. The experiment was repeated three times with consistent results.
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fig03: Uncl-Sema3E inhibits integrin-mediated adhesion and intracellular signalling in endothelial cellsHUVEC were stained with FITC-phalloidin to reveal F-actin (in green) and 4,6-diamidino-2-phenylindole (DAPI) to highlight nuclei. Upon incubation with 7 nM Uncl-Sema3E for 20 min, endothelial cells totally disassembled F-actin stress fibers, and cellular processes were retracted, leading to the typical collapsed phenotype (see Barberis et al, 2004). Scale bar: 50 µm.HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to immunofluorescence analysis by staining with anti-Paxillin antibodies (revealed in red) and with FITC-falloidin to detect F-actin (in green). On the right, images of cellular processes are magnified to reveal focal adhesions (FA) and the percent fraction of FA-containing cells is indicated aside. Scale bars: 20 and 10 µm in the magnified fields shown on the right.The graph summarizes the results of immunofluorescence analysis shown in Supporting Information Fig 4, and indicates the amount of integrin-β1 in active conformation on the surface of HUVEC, either untreated or treated with 7 nM Uncl-Sema3E for 5 min; n = 4 (each condition).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and their protein lysates were subjected to immuno-blotting analysis with antibodies directed against Y397 phosphorylated focal adhesion kinase (P-FAK), serine/threonine phosphorylated MAPK and AKT and vinculin (as protein loading control).HUVEC grown on glass coverslips were treated with 7 nM Uncl-Sema3E (n = 4) or with 200 nM staurosporin (STS; n = 4) for 1 h, or left untreated (n = 4). The early apoptotic marker activated-caspase 3 was revealed by immunofluorescence (shown in red); nuclei were counterstained with DAPI; scale bar: 50 µm. The graph indicates the average percent fraction of apoptotic cells in each condition ± SD; *p = 0.008; **p = 0.0002 (vs. control).HUVEC were treated with 7 nM Uncl-Sema3E for the indicated times (or left untreated) and then subjected to cytofluorimetric analysis with antibodies directed against activated-caspase 3. The experiment was repeated three times with consistent results.
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).

Bottom Line: Here, we show that a mutated, uncleavable variant of Sema3E (Uncl-Sema3E) binds to PlexinD1 like p61-Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Research and Treatment (IRCC), University of Torino Medical School, Candiolo, Italy.

Show MeSH
Related in: MedlinePlus