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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 endothelial cell migration and tube formationThe haptotactic migration of HUVEC was assayed in Transwell inserts. In the presence of Uncl-Sema3E or p61-Sema3E (7 nM) endothelial cells migration was inhibited, in PlexinD1-dependent manner (PLXND1 knock-down was achieved by shRNA expression, see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).HUVEC were grown in Matrigel-coated wells for 24 h (see Materials and Methods Section) in the presence or absence of purified p61-Sema3E or Uncl-Sema3E. Representative phase-contrast microscopy images are shown on the left (scale bar: 200 µm). The assay was repeated several times (n = 4) and quantified by counting bifurcations of vessel-like tubular structures in multiple microscopic fields (the graph on the right shows mean values ± SD from at least five separate wells for each condition).HUVEC spheroids (containing approximately 400 cells each) were implanted in collagen gels, in the presence or absence of bFGF (see Materials and Methods Section for details). Moreover, either Uncl-Sema3E or p61 (1 µg/ml each) were included with bFGF in some of the wells (n = indicated in figure below respective bars). Spheroids were photographed after 24 h (representative images are shown; scale bar: 100 µm) and endothelial sprouts were quantified as described (Shraga-Heled et al, 2007).
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fig04: Uncl-Sema3E inhibits endothelial cell migration and tube formationThe haptotactic migration of HUVEC was assayed in Transwell inserts. In the presence of Uncl-Sema3E or p61-Sema3E (7 nM) endothelial cells migration was inhibited, in PlexinD1-dependent manner (PLXND1 knock-down was achieved by shRNA expression, see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).HUVEC were grown in Matrigel-coated wells for 24 h (see Materials and Methods Section) in the presence or absence of purified p61-Sema3E or Uncl-Sema3E. Representative phase-contrast microscopy images are shown on the left (scale bar: 200 µm). The assay was repeated several times (n = 4) and quantified by counting bifurcations of vessel-like tubular structures in multiple microscopic fields (the graph on the right shows mean values ± SD from at least five separate wells for each condition).HUVEC spheroids (containing approximately 400 cells each) were implanted in collagen gels, in the presence or absence of bFGF (see Materials and Methods Section for details). Moreover, either Uncl-Sema3E or p61 (1 µg/ml each) were included with bFGF in some of the wells (n = indicated in figure below respective bars). Spheroids were photographed after 24 h (representative images are shown; scale bar: 100 µm) and endothelial sprouts were quantified as described (Shraga-Heled et al, 2007).

Mentions: Consistent with its potent regulatory activity on integrin function and cytoskeletal dynamics, we found that Uncl-Sema3E strongly inhibited endothelial cell migration in a PlexinD1-dependent manner (Fig 4A); notably, this activity paralleled that of the proteolytic fragment p61 (Casazza et al, 2011). Moreover, we found that this inhibitory effect of Sema3E in endothelial cells is dependent on the expression of the intracellular adaptor molecule Rnd2 (Supporting Information Fig 5B). We further assessed the activity of Uncl-Sema3E in HUVEC engaged in vascular tube formation. In fact, HUVEC grown in vitro on a basement membrane matrix (Matrigel) undergo spontaneous alignment into hollow tubes, forming capillary-like networks within 24 h (Grant et al, 1989). However, we found that endothelial tubule formation and stability was significantly impaired in the presence of Uncl-Sema3E compared to untreated cultures (Fig 4B). Moreover, when HUVEC were grown as spheroids in a three-dimensional collagen matrix in the presence of basic fibroblast growth factor (bFGF), they formed elongated sprouts, which were significantly reduced in the presence of either Uncl-Sema3E or the proteolytic fragment p61 (Fig 4C). These data further indicated that full-length uncleaved Sema3E is not an inactive precursor molecule, since it can elicit a potent PlexinD1-mediated inhibitory response in endothelial cells. Moreover, they demonstrated that the endothelial-repelling function of Sema3E pre-exists to, and it is not significantly blocked by, the proteolytic processing of the molecule. Taken together, these findings characterize Uncl-Sema3E as a partial agonist of PlexinD1 as compared to the cleaved mature p61 fragment. In fact, Uncl-Sema3E is able to bind to Plexin-D1 and elicits endothelial cell repulsion and anti-angiogenic activity in vitro, but unlike p61, it is unable to trigger the ErbB2-dependent pathway in cancer cells, and instead, it antagonizes this major signal promoting invasion and metastasis.


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 endothelial cell migration and tube formationThe haptotactic migration of HUVEC was assayed in Transwell inserts. In the presence of Uncl-Sema3E or p61-Sema3E (7 nM) endothelial cells migration was inhibited, in PlexinD1-dependent manner (PLXND1 knock-down was achieved by shRNA expression, see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).HUVEC were grown in Matrigel-coated wells for 24 h (see Materials and Methods Section) in the presence or absence of purified p61-Sema3E or Uncl-Sema3E. Representative phase-contrast microscopy images are shown on the left (scale bar: 200 µm). The assay was repeated several times (n = 4) and quantified by counting bifurcations of vessel-like tubular structures in multiple microscopic fields (the graph on the right shows mean values ± SD from at least five separate wells for each condition).HUVEC spheroids (containing approximately 400 cells each) were implanted in collagen gels, in the presence or absence of bFGF (see Materials and Methods Section for details). Moreover, either Uncl-Sema3E or p61 (1 µg/ml each) were included with bFGF in some of the wells (n = indicated in figure below respective bars). Spheroids were photographed after 24 h (representative images are shown; scale bar: 100 µm) and endothelial sprouts were quantified as described (Shraga-Heled et al, 2007).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376853&req=5

fig04: Uncl-Sema3E inhibits endothelial cell migration and tube formationThe haptotactic migration of HUVEC was assayed in Transwell inserts. In the presence of Uncl-Sema3E or p61-Sema3E (7 nM) endothelial cells migration was inhibited, in PlexinD1-dependent manner (PLXND1 knock-down was achieved by shRNA expression, see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).HUVEC were grown in Matrigel-coated wells for 24 h (see Materials and Methods Section) in the presence or absence of purified p61-Sema3E or Uncl-Sema3E. Representative phase-contrast microscopy images are shown on the left (scale bar: 200 µm). The assay was repeated several times (n = 4) and quantified by counting bifurcations of vessel-like tubular structures in multiple microscopic fields (the graph on the right shows mean values ± SD from at least five separate wells for each condition).HUVEC spheroids (containing approximately 400 cells each) were implanted in collagen gels, in the presence or absence of bFGF (see Materials and Methods Section for details). Moreover, either Uncl-Sema3E or p61 (1 µg/ml each) were included with bFGF in some of the wells (n = indicated in figure below respective bars). Spheroids were photographed after 24 h (representative images are shown; scale bar: 100 µm) and endothelial sprouts were quantified as described (Shraga-Heled et al, 2007).
Mentions: Consistent with its potent regulatory activity on integrin function and cytoskeletal dynamics, we found that Uncl-Sema3E strongly inhibited endothelial cell migration in a PlexinD1-dependent manner (Fig 4A); notably, this activity paralleled that of the proteolytic fragment p61 (Casazza et al, 2011). Moreover, we found that this inhibitory effect of Sema3E in endothelial cells is dependent on the expression of the intracellular adaptor molecule Rnd2 (Supporting Information Fig 5B). We further assessed the activity of Uncl-Sema3E in HUVEC engaged in vascular tube formation. In fact, HUVEC grown in vitro on a basement membrane matrix (Matrigel) undergo spontaneous alignment into hollow tubes, forming capillary-like networks within 24 h (Grant et al, 1989). However, we found that endothelial tubule formation and stability was significantly impaired in the presence of Uncl-Sema3E compared to untreated cultures (Fig 4B). Moreover, when HUVEC were grown as spheroids in a three-dimensional collagen matrix in the presence of basic fibroblast growth factor (bFGF), they formed elongated sprouts, which were significantly reduced in the presence of either Uncl-Sema3E or the proteolytic fragment p61 (Fig 4C). These data further indicated that full-length uncleaved Sema3E is not an inactive precursor molecule, since it can elicit a potent PlexinD1-mediated inhibitory response in endothelial cells. Moreover, they demonstrated that the endothelial-repelling function of Sema3E pre-exists to, and it is not significantly blocked by, the proteolytic processing of the molecule. Taken together, these findings characterize Uncl-Sema3E as a partial agonist of PlexinD1 as compared to the cleaved mature p61 fragment. In fact, Uncl-Sema3E is able to bind to Plexin-D1 and elicits endothelial cell repulsion and anti-angiogenic activity in vitro, but unlike p61, it is unable to trigger the ErbB2-dependent pathway in cancer cells, and instead, it antagonizes this major signal promoting invasion and metastasis.

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