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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 anti-angiogenic activity leads to tumour hypoxia and apoptosisTissue sections from tumours formed by MDA-MB435 control-EV cells, or cells overexpressing Uncl-Sema3E or p61-Sema3E, were stained with pimonidazole (PIMO, green) to reveal hypoxic areas (n = 5; see Materials and Methods Section) and for the endothelial marker CD31 (red); nuclei were stained with DAPI. Data are given as percent fraction of PIMO+ area/total area.Sections of the same tumours as above were immunostained for cleaved caspase 3 (green), CD31 (red) and DAPI. Uncl-Sema3E and p61 expression enhanced the apoptotic index (number of cleaved Casp3 positive cells/total cell number). The graph indicates mean values ± SD (n = 5); ***p = 5.9E−11; **p = 0.0046; §§§p = 1.9E−09; §§p = 0.0025.Sections of the same tumours as above were analyzed by immunohistochemistry to reveal the mitotic nuclear marker Ki67, followed by hematoxilin counterstaining. No significant difference was detected between samples. Scale bars (throughout this figure): 100 µm.Superficial metastatic colonies in the lungs of mice bearing the same tumours as above (n = 5 per each experimental group) were counted under a stereomicroscope after airways infusion with ink. Unlike what seen with p61, Uncl-Sema3E did not promote tumour metastatic spreading.
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fig06: Uncl-Sema3E anti-angiogenic activity leads to tumour hypoxia and apoptosisTissue sections from tumours formed by MDA-MB435 control-EV cells, or cells overexpressing Uncl-Sema3E or p61-Sema3E, were stained with pimonidazole (PIMO, green) to reveal hypoxic areas (n = 5; see Materials and Methods Section) and for the endothelial marker CD31 (red); nuclei were stained with DAPI. Data are given as percent fraction of PIMO+ area/total area.Sections of the same tumours as above were immunostained for cleaved caspase 3 (green), CD31 (red) and DAPI. Uncl-Sema3E and p61 expression enhanced the apoptotic index (number of cleaved Casp3 positive cells/total cell number). The graph indicates mean values ± SD (n = 5); ***p = 5.9E−11; **p = 0.0046; §§§p = 1.9E−09; §§p = 0.0025.Sections of the same tumours as above were analyzed by immunohistochemistry to reveal the mitotic nuclear marker Ki67, followed by hematoxilin counterstaining. No significant difference was detected between samples. Scale bars (throughout this figure): 100 µm.Superficial metastatic colonies in the lungs of mice bearing the same tumours as above (n = 5 per each experimental group) were counted under a stereomicroscope after airways infusion with ink. Unlike what seen with p61, Uncl-Sema3E did not promote tumour metastatic spreading.

Mentions: The disruption of vessel networks usually correlates with deficient tissue oxygenation. In fact, we found that Uncl-Sema3E-expressing tumours were diffusely hypoxic as revealed by pimonidazole (PIMO) staining (Fig 6A) and heavily apoptotic compared to controls (Fig 6B); this likely accounted for the observed shrinkage of the tumours, as the mitotic index was not significantly affected by the treatment (Fig 6C). Notably, tumours expressing Uncl-Sema3E did not give rise to increased secondary metastatic foci with respect to controls (Fig 6D), whereas we have previously shown that either wild-type cleavable Sema3E or the mature fragment p61 are actively pro-metastatic in vivo (Casazza et al, 2010).


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 anti-angiogenic activity leads to tumour hypoxia and apoptosisTissue sections from tumours formed by MDA-MB435 control-EV cells, or cells overexpressing Uncl-Sema3E or p61-Sema3E, were stained with pimonidazole (PIMO, green) to reveal hypoxic areas (n = 5; see Materials and Methods Section) and for the endothelial marker CD31 (red); nuclei were stained with DAPI. Data are given as percent fraction of PIMO+ area/total area.Sections of the same tumours as above were immunostained for cleaved caspase 3 (green), CD31 (red) and DAPI. Uncl-Sema3E and p61 expression enhanced the apoptotic index (number of cleaved Casp3 positive cells/total cell number). The graph indicates mean values ± SD (n = 5); ***p = 5.9E−11; **p = 0.0046; §§§p = 1.9E−09; §§p = 0.0025.Sections of the same tumours as above were analyzed by immunohistochemistry to reveal the mitotic nuclear marker Ki67, followed by hematoxilin counterstaining. No significant difference was detected between samples. Scale bars (throughout this figure): 100 µm.Superficial metastatic colonies in the lungs of mice bearing the same tumours as above (n = 5 per each experimental group) were counted under a stereomicroscope after airways infusion with ink. Unlike what seen with p61, Uncl-Sema3E did not promote tumour metastatic spreading.
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Related In: Results  -  Collection

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fig06: Uncl-Sema3E anti-angiogenic activity leads to tumour hypoxia and apoptosisTissue sections from tumours formed by MDA-MB435 control-EV cells, or cells overexpressing Uncl-Sema3E or p61-Sema3E, were stained with pimonidazole (PIMO, green) to reveal hypoxic areas (n = 5; see Materials and Methods Section) and for the endothelial marker CD31 (red); nuclei were stained with DAPI. Data are given as percent fraction of PIMO+ area/total area.Sections of the same tumours as above were immunostained for cleaved caspase 3 (green), CD31 (red) and DAPI. Uncl-Sema3E and p61 expression enhanced the apoptotic index (number of cleaved Casp3 positive cells/total cell number). The graph indicates mean values ± SD (n = 5); ***p = 5.9E−11; **p = 0.0046; §§§p = 1.9E−09; §§p = 0.0025.Sections of the same tumours as above were analyzed by immunohistochemistry to reveal the mitotic nuclear marker Ki67, followed by hematoxilin counterstaining. No significant difference was detected between samples. Scale bars (throughout this figure): 100 µm.Superficial metastatic colonies in the lungs of mice bearing the same tumours as above (n = 5 per each experimental group) were counted under a stereomicroscope after airways infusion with ink. Unlike what seen with p61, Uncl-Sema3E did not promote tumour metastatic spreading.
Mentions: The disruption of vessel networks usually correlates with deficient tissue oxygenation. In fact, we found that Uncl-Sema3E-expressing tumours were diffusely hypoxic as revealed by pimonidazole (PIMO) staining (Fig 6A) and heavily apoptotic compared to controls (Fig 6B); this likely accounted for the observed shrinkage of the tumours, as the mitotic index was not significantly affected by the treatment (Fig 6C). Notably, tumours expressing Uncl-Sema3E did not give rise to increased secondary metastatic foci with respect to controls (Fig 6D), whereas we have previously shown that either wild-type cleavable Sema3E or the mature fragment p61 are actively pro-metastatic in vivo (Casazza et al, 2010).

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