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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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Systemic delivery of Uncl-Sema3E achieves a concomitant inhibition of tumour growth and metastatic spreadingA-D. cDNA constructs expressing either wild-type processable Sema3E, or Uncl-Sema3E (or a mock control) were hydroporated in nude mice (see Materials and Methods Section), achieving systemic delivery of the proteins in the circulation at comparable concentrations (see text). 4T1 carcinoma cells were then implanted subcutaneously in the engineered mice (n = 5, per each experimental group). Both Sema3E isoforms inhibited tumour growth (panels A and B) and tumour angiogenesis (revealed by CD31 staining, panel C; scale bar: 100 µm). However, while the occurrence of spontaneous metastasis in the lungs was significantly reduced in the presence of Uncl-Sema3E, wild-type Sema3E expression promoted metastatic spreading (panel D).
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fig08: Systemic delivery of Uncl-Sema3E achieves a concomitant inhibition of tumour growth and metastatic spreadingA-D. cDNA constructs expressing either wild-type processable Sema3E, or Uncl-Sema3E (or a mock control) were hydroporated in nude mice (see Materials and Methods Section), achieving systemic delivery of the proteins in the circulation at comparable concentrations (see text). 4T1 carcinoma cells were then implanted subcutaneously in the engineered mice (n = 5, per each experimental group). Both Sema3E isoforms inhibited tumour growth (panels A and B) and tumour angiogenesis (revealed by CD31 staining, panel C; scale bar: 100 µm). However, while the occurrence of spontaneous metastasis in the lungs was significantly reduced in the presence of Uncl-Sema3E, wild-type Sema3E expression promoted metastatic spreading (panel D).

Mentions: We finally asked whether Uncl-Sema3E could be delivered systemically in mice as a therapeutic drug to interfere with tumour progression. To this end, we analyzed multiple overtly metastatic models and applied diverse strategies for the systemic delivery of Uncl-Sema3E in tumour-bearing mice: either based on gene transfer in vivo or on the administration of a purified recombinant protein. Hydrodynamic delivery of naked DNA expression plasmids in the circulation is commonly used to achieve transgene expression in mice, especially in the liver, and sustained plasmatic levels of secreted factors (Liu et al, 1999). We therefore compared the potential tumour suppressing activity of wild-type Sema3E that can be freely processed into p61 and its mutated counterpart Uncl-Sema3E by intravenously injecting immunodeficient mice with the expression constructs encoding either protein. The next day, we transplanted the mice with 4T1 mammary carcinoma cells. Moreover, we verified by enzyme-linked immunosorbent assay (ELISA) that the average blood concentration of Sema3E isoforms in the two groups of treated mice was comparable: i.e. 185 ± 88 µg/ml for Sema3E wt, and 179 ± 75 µg/ml for Uncl-Sema3E (vs. an endogenous level of approximately 6 ng/ml Sema3E in control mice). Primary tumours grew significantly less in the presence of either of the two Sema3E isoforms (Fig 8A and B) and vessel area was comparably reduced (Fig 8C), consistent with the fact that the proteolytic processing of this semaphorin does not impact on its anti-angiogenic activity. However, while metastases were increased with respect to controls following treatment with wild-type cleavable Sema3E, they were remarkably reduced in the presence of Uncl-Sema3E (Fig 8D) consistent with its ability to compete with endogenous p61. We further validated the tumour suppressing and anti-metastatic activity of systemic Uncl-Sema3E in another metastatic tumour model expressing endogenous Sema3E, i.e. lung carcinoma cells A549, achieving similar results (Supporting Information Fig 11). Notably, a histopathological analysis of liver, kidneys, heart and adrenal glands of mice treated with Uncl-Sema3E (delivered by different approaches) did not reveal significant signs of toxicity or any differences to tissues of mock-treated animals (Supporting Information Fig 12).


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)

Systemic delivery of Uncl-Sema3E achieves a concomitant inhibition of tumour growth and metastatic spreadingA-D. cDNA constructs expressing either wild-type processable Sema3E, or Uncl-Sema3E (or a mock control) were hydroporated in nude mice (see Materials and Methods Section), achieving systemic delivery of the proteins in the circulation at comparable concentrations (see text). 4T1 carcinoma cells were then implanted subcutaneously in the engineered mice (n = 5, per each experimental group). Both Sema3E isoforms inhibited tumour growth (panels A and B) and tumour angiogenesis (revealed by CD31 staining, panel C; scale bar: 100 µm). However, while the occurrence of spontaneous metastasis in the lungs was significantly reduced in the presence of Uncl-Sema3E, wild-type Sema3E expression promoted metastatic spreading (panel D).
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Related In: Results  -  Collection

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fig08: Systemic delivery of Uncl-Sema3E achieves a concomitant inhibition of tumour growth and metastatic spreadingA-D. cDNA constructs expressing either wild-type processable Sema3E, or Uncl-Sema3E (or a mock control) were hydroporated in nude mice (see Materials and Methods Section), achieving systemic delivery of the proteins in the circulation at comparable concentrations (see text). 4T1 carcinoma cells were then implanted subcutaneously in the engineered mice (n = 5, per each experimental group). Both Sema3E isoforms inhibited tumour growth (panels A and B) and tumour angiogenesis (revealed by CD31 staining, panel C; scale bar: 100 µm). However, while the occurrence of spontaneous metastasis in the lungs was significantly reduced in the presence of Uncl-Sema3E, wild-type Sema3E expression promoted metastatic spreading (panel D).
Mentions: We finally asked whether Uncl-Sema3E could be delivered systemically in mice as a therapeutic drug to interfere with tumour progression. To this end, we analyzed multiple overtly metastatic models and applied diverse strategies for the systemic delivery of Uncl-Sema3E in tumour-bearing mice: either based on gene transfer in vivo or on the administration of a purified recombinant protein. Hydrodynamic delivery of naked DNA expression plasmids in the circulation is commonly used to achieve transgene expression in mice, especially in the liver, and sustained plasmatic levels of secreted factors (Liu et al, 1999). We therefore compared the potential tumour suppressing activity of wild-type Sema3E that can be freely processed into p61 and its mutated counterpart Uncl-Sema3E by intravenously injecting immunodeficient mice with the expression constructs encoding either protein. The next day, we transplanted the mice with 4T1 mammary carcinoma cells. Moreover, we verified by enzyme-linked immunosorbent assay (ELISA) that the average blood concentration of Sema3E isoforms in the two groups of treated mice was comparable: i.e. 185 ± 88 µg/ml for Sema3E wt, and 179 ± 75 µg/ml for Uncl-Sema3E (vs. an endogenous level of approximately 6 ng/ml Sema3E in control mice). Primary tumours grew significantly less in the presence of either of the two Sema3E isoforms (Fig 8A and B) and vessel area was comparably reduced (Fig 8C), consistent with the fact that the proteolytic processing of this semaphorin does not impact on its anti-angiogenic activity. However, while metastases were increased with respect to controls following treatment with wild-type cleavable Sema3E, they were remarkably reduced in the presence of Uncl-Sema3E (Fig 8D) consistent with its ability to compete with endogenous p61. We further validated the tumour suppressing and anti-metastatic activity of systemic Uncl-Sema3E in another metastatic tumour model expressing endogenous Sema3E, i.e. lung carcinoma cells A549, achieving similar results (Supporting Information Fig 11). Notably, a histopathological analysis of liver, kidneys, heart and adrenal glands of mice treated with Uncl-Sema3E (delivered by different approaches) did not reveal significant signs of toxicity or any differences to tissues of mock-treated animals (Supporting Information Fig 12).

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