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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 is unable to trigger ErbB2 activation and cancer cell migrationSchematic diagram summarizing Sema3E isoforms: the full-length precursor of 87 kDa (p87), the proteolytically cleaved fragment p61 and the mutated uncleavable molecule (Uncl-Sema3E).Migration of MDA-MB435 cancer cells assayed in Transwell inserts in the presence of 7 nM Uncl-Sema3E, or 7 nM p61-Sema3E, or mock. Migrated cells were quantified by staining with crystal violet and measuring the absorbance at 595 nm (see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).Serum-starved A549 cancer cells were treated with either purified 7 nM p61-Sema3E, or 7 nM Uncl-Sema3E, or mock, for 10 min. The induced association between endogenous PlexinD1 receptor and ErbB2 tyrosine kinase was revealed by immunoblotting. The data are representative of three independent experiments yielding consistent results.A549 carcinoma cells were treated as above, and total protein lysates were analyzed by immunoblotting with phospho-specific antibodies to detect activated forms of MAPK, as well as with the respective non-phospho-specific antibody to provide loading controls.
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fig01: Uncl-Sema3E is unable to trigger ErbB2 activation and cancer cell migrationSchematic diagram summarizing Sema3E isoforms: the full-length precursor of 87 kDa (p87), the proteolytically cleaved fragment p61 and the mutated uncleavable molecule (Uncl-Sema3E).Migration of MDA-MB435 cancer cells assayed in Transwell inserts in the presence of 7 nM Uncl-Sema3E, or 7 nM p61-Sema3E, or mock. Migrated cells were quantified by staining with crystal violet and measuring the absorbance at 595 nm (see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).Serum-starved A549 cancer cells were treated with either purified 7 nM p61-Sema3E, or 7 nM Uncl-Sema3E, or mock, for 10 min. The induced association between endogenous PlexinD1 receptor and ErbB2 tyrosine kinase was revealed by immunoblotting. The data are representative of three independent experiments yielding consistent results.A549 carcinoma cells were treated as above, and total protein lysates were analyzed by immunoblotting with phospho-specific antibodies to detect activated forms of MAPK, as well as with the respective non-phospho-specific antibody to provide loading controls.

Mentions: Sema3E signalling in tumour cells promotes invasion and metastasis, but these activities crucially depend on its proteolytic maturation into the smaller fragment p61 (Christensen et al, 2005). In fact, a mutated ‘uncleavable’ full-length Sema3E, which cannot be targeted by furins and converted into p61 (Uncl-Sema3E; Fig 1A), did not promote metastasis formation in experiments based on direct cancer cell injection in the circulation (Christensen et al, 2005). Here, we also show that, in contrast to p61, Uncl-Sema3E does not induce cancer cell migration (Fig 1B). We have recently reported that the pro-invasive and pro-metastatic activity of p61 is due to the association between PlexinD1 and the oncogenic tyrosine-kinase receptor ErbB2 as well as the induction of ErbB2 tyrosine phosphorylation and intracellular signalling (Casazza et al, 2010). We therefore asked whether the proteolytic processing of Sema3E may be required for its ability to trigger ErbB2 signalling in cancer cells. Notably, Uncl-Sema3E was unable to induce the association of ErbB2 with PlexinD1 (Fig 1C, top), nor did it promote ErbB2 phosphorylation (Fig 1C, middle) or intracellular mitogen-activated protein kinase (MAPK) signalling (Fig 1D).


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 is unable to trigger ErbB2 activation and cancer cell migrationSchematic diagram summarizing Sema3E isoforms: the full-length precursor of 87 kDa (p87), the proteolytically cleaved fragment p61 and the mutated uncleavable molecule (Uncl-Sema3E).Migration of MDA-MB435 cancer cells assayed in Transwell inserts in the presence of 7 nM Uncl-Sema3E, or 7 nM p61-Sema3E, or mock. Migrated cells were quantified by staining with crystal violet and measuring the absorbance at 595 nm (see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).Serum-starved A549 cancer cells were treated with either purified 7 nM p61-Sema3E, or 7 nM Uncl-Sema3E, or mock, for 10 min. The induced association between endogenous PlexinD1 receptor and ErbB2 tyrosine kinase was revealed by immunoblotting. The data are representative of three independent experiments yielding consistent results.A549 carcinoma cells were treated as above, and total protein lysates were analyzed by immunoblotting with phospho-specific antibodies to detect activated forms of MAPK, as well as with the respective non-phospho-specific antibody to provide loading controls.
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Related In: Results  -  Collection

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fig01: Uncl-Sema3E is unable to trigger ErbB2 activation and cancer cell migrationSchematic diagram summarizing Sema3E isoforms: the full-length precursor of 87 kDa (p87), the proteolytically cleaved fragment p61 and the mutated uncleavable molecule (Uncl-Sema3E).Migration of MDA-MB435 cancer cells assayed in Transwell inserts in the presence of 7 nM Uncl-Sema3E, or 7 nM p61-Sema3E, or mock. Migrated cells were quantified by staining with crystal violet and measuring the absorbance at 595 nm (see Materials and Methods Section). Data are given as average ± SD of two independent experiments performed in duplicate (n = 4).Serum-starved A549 cancer cells were treated with either purified 7 nM p61-Sema3E, or 7 nM Uncl-Sema3E, or mock, for 10 min. The induced association between endogenous PlexinD1 receptor and ErbB2 tyrosine kinase was revealed by immunoblotting. The data are representative of three independent experiments yielding consistent results.A549 carcinoma cells were treated as above, and total protein lysates were analyzed by immunoblotting with phospho-specific antibodies to detect activated forms of MAPK, as well as with the respective non-phospho-specific antibody to provide loading controls.
Mentions: Sema3E signalling in tumour cells promotes invasion and metastasis, but these activities crucially depend on its proteolytic maturation into the smaller fragment p61 (Christensen et al, 2005). In fact, a mutated ‘uncleavable’ full-length Sema3E, which cannot be targeted by furins and converted into p61 (Uncl-Sema3E; Fig 1A), did not promote metastasis formation in experiments based on direct cancer cell injection in the circulation (Christensen et al, 2005). Here, we also show that, in contrast to p61, Uncl-Sema3E does not induce cancer cell migration (Fig 1B). We have recently reported that the pro-invasive and pro-metastatic activity of p61 is due to the association between PlexinD1 and the oncogenic tyrosine-kinase receptor ErbB2 as well as the induction of ErbB2 tyrosine phosphorylation and intracellular signalling (Casazza et al, 2010). We therefore asked whether the proteolytic processing of Sema3E may be required for its ability to trigger ErbB2 signalling in cancer cells. Notably, Uncl-Sema3E was unable to induce the association of ErbB2 with PlexinD1 (Fig 1C, top), nor did it promote ErbB2 phosphorylation (Fig 1C, middle) or intracellular mitogen-activated protein kinase (MAPK) signalling (Fig 1D).

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