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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 antagonizes p61 and blocks its pro-metastatic signalling in cancer cellsCOS cells transfected to express PlexinD1 were probed with alkaline phosphatase (AP)-conjugated Uncl-Sema3E putative ligand. The binding was revealed by incubation with an AP substrate (see Supporting Information Methods). Scale bar: 30 µm.PlexinD1-expressing COS cells (as above) were incubated with AP-labelled p61-Sema3E (7 nM) in combination with increasing concentrations of Uncl-Sema3E (precisely: 3, 7, 15, 60 nM) (n = 6 for each experimental condition). Equimolar concentration of Uncl-Sema3E strongly reduced p61-Sema3E binding to PlexinD1, which was almost abrogated upon excess competition. Statistical significance was calculated by t-test with respect to p61-Sema3E binding in the absence of Uncl-Sema3E: *p = 1.3E−06; **p = 3.2E−08; §p = 1.4E−08; §§p = 1.5E−08. The binding of the independent semaphorin Sema4D to COS cells overexpressing its cognate receptor PlexinB1 provided a specificity control.Serum starved A549 cancer cells were incubated with 7 nM Uncl-Sema3E or mock for 1 h. Cells were then stimulated with mock, 7 nM p61, or 0.2 nM Heregulin-β1 (as positive control) in the presence of Uncl-Sema3E. ErbB2 tyrosine phosphorylation was revealed by immunoblotting.Migration of A549 and 4T1 cells expressing Uncl-Sema3E or control-EV, assayed in Transwell inserts (n = 4). Data shown indicate fold change versus respective controls.Fluorescently labelled A549 cells, either expressing Uncl-Sema3E or control-EV (same as above), were injected intravenously in nude mice. Forty-eight hours later the mice were sacrificed, and the lungs fixed and analyzed under a fluorescence microscope to reveal and quantify extravasated cancer cells colonizing the tissue (n = 5; see Materials and Methods Section for details). Representative images are shown on the right; scale bars: 100 µm.A549 and 4T1 cells expressing Uncl-Sema3E or control-EV were injected intravenously in nude mice (n = 5 and 6, respectively, for the two cell lines). Superficial metastatic colonies in the lungs were counted under a stereomicroscope; the graph indicates average values ± SD. Representative macroscopic images of the lungs are also shown, where red arrows indicate some of the metastatic lesions. Scale bars: 200 µm.
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fig02: Uncl-Sema3E antagonizes p61 and blocks its pro-metastatic signalling in cancer cellsCOS cells transfected to express PlexinD1 were probed with alkaline phosphatase (AP)-conjugated Uncl-Sema3E putative ligand. The binding was revealed by incubation with an AP substrate (see Supporting Information Methods). Scale bar: 30 µm.PlexinD1-expressing COS cells (as above) were incubated with AP-labelled p61-Sema3E (7 nM) in combination with increasing concentrations of Uncl-Sema3E (precisely: 3, 7, 15, 60 nM) (n = 6 for each experimental condition). Equimolar concentration of Uncl-Sema3E strongly reduced p61-Sema3E binding to PlexinD1, which was almost abrogated upon excess competition. Statistical significance was calculated by t-test with respect to p61-Sema3E binding in the absence of Uncl-Sema3E: *p = 1.3E−06; **p = 3.2E−08; §p = 1.4E−08; §§p = 1.5E−08. The binding of the independent semaphorin Sema4D to COS cells overexpressing its cognate receptor PlexinB1 provided a specificity control.Serum starved A549 cancer cells were incubated with 7 nM Uncl-Sema3E or mock for 1 h. Cells were then stimulated with mock, 7 nM p61, or 0.2 nM Heregulin-β1 (as positive control) in the presence of Uncl-Sema3E. ErbB2 tyrosine phosphorylation was revealed by immunoblotting.Migration of A549 and 4T1 cells expressing Uncl-Sema3E or control-EV, assayed in Transwell inserts (n = 4). Data shown indicate fold change versus respective controls.Fluorescently labelled A549 cells, either expressing Uncl-Sema3E or control-EV (same as above), were injected intravenously in nude mice. Forty-eight hours later the mice were sacrificed, and the lungs fixed and analyzed under a fluorescence microscope to reveal and quantify extravasated cancer cells colonizing the tissue (n = 5; see Materials and Methods Section for details). Representative images are shown on the right; scale bars: 100 µm.A549 and 4T1 cells expressing Uncl-Sema3E or control-EV were injected intravenously in nude mice (n = 5 and 6, respectively, for the two cell lines). Superficial metastatic colonies in the lungs were counted under a stereomicroscope; the graph indicates average values ± SD. Representative macroscopic images of the lungs are also shown, where red arrows indicate some of the metastatic lesions. Scale bars: 200 µm.

Mentions: Despite this apparent lack of activity, we found that Uncl-Sema3E is fully capable of binding the receptor PlexinD1 in analogy to p61-Sema3E isoform (Fig 2A). Moreover, we found that Uncl-Sema3E selectively competes with p61 for binding the receptor in the same nanomolar concentration range (Fig 2B). We then asked whether Uncl-Sema3E may potentially act as a dominant-negative molecule by interfering with p61 signalling. Indeed, ErbB2 transactivation elicited by p61 in A549 cells was strikingly reduced in the presence of Uncl-Sema3E (Fig 2C). Furthermore, data shown above in Fig 1C revealed that the basal level of ErbB2 tyrosine phosphorylation decreased in cells treated with Uncl-Sema3E, consistent with its functional competition with endogenous p61.


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 antagonizes p61 and blocks its pro-metastatic signalling in cancer cellsCOS cells transfected to express PlexinD1 were probed with alkaline phosphatase (AP)-conjugated Uncl-Sema3E putative ligand. The binding was revealed by incubation with an AP substrate (see Supporting Information Methods). Scale bar: 30 µm.PlexinD1-expressing COS cells (as above) were incubated with AP-labelled p61-Sema3E (7 nM) in combination with increasing concentrations of Uncl-Sema3E (precisely: 3, 7, 15, 60 nM) (n = 6 for each experimental condition). Equimolar concentration of Uncl-Sema3E strongly reduced p61-Sema3E binding to PlexinD1, which was almost abrogated upon excess competition. Statistical significance was calculated by t-test with respect to p61-Sema3E binding in the absence of Uncl-Sema3E: *p = 1.3E−06; **p = 3.2E−08; §p = 1.4E−08; §§p = 1.5E−08. The binding of the independent semaphorin Sema4D to COS cells overexpressing its cognate receptor PlexinB1 provided a specificity control.Serum starved A549 cancer cells were incubated with 7 nM Uncl-Sema3E or mock for 1 h. Cells were then stimulated with mock, 7 nM p61, or 0.2 nM Heregulin-β1 (as positive control) in the presence of Uncl-Sema3E. ErbB2 tyrosine phosphorylation was revealed by immunoblotting.Migration of A549 and 4T1 cells expressing Uncl-Sema3E or control-EV, assayed in Transwell inserts (n = 4). Data shown indicate fold change versus respective controls.Fluorescently labelled A549 cells, either expressing Uncl-Sema3E or control-EV (same as above), were injected intravenously in nude mice. Forty-eight hours later the mice were sacrificed, and the lungs fixed and analyzed under a fluorescence microscope to reveal and quantify extravasated cancer cells colonizing the tissue (n = 5; see Materials and Methods Section for details). Representative images are shown on the right; scale bars: 100 µm.A549 and 4T1 cells expressing Uncl-Sema3E or control-EV were injected intravenously in nude mice (n = 5 and 6, respectively, for the two cell lines). Superficial metastatic colonies in the lungs were counted under a stereomicroscope; the graph indicates average values ± SD. Representative macroscopic images of the lungs are also shown, where red arrows indicate some of the metastatic lesions. Scale bars: 200 µm.
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fig02: Uncl-Sema3E antagonizes p61 and blocks its pro-metastatic signalling in cancer cellsCOS cells transfected to express PlexinD1 were probed with alkaline phosphatase (AP)-conjugated Uncl-Sema3E putative ligand. The binding was revealed by incubation with an AP substrate (see Supporting Information Methods). Scale bar: 30 µm.PlexinD1-expressing COS cells (as above) were incubated with AP-labelled p61-Sema3E (7 nM) in combination with increasing concentrations of Uncl-Sema3E (precisely: 3, 7, 15, 60 nM) (n = 6 for each experimental condition). Equimolar concentration of Uncl-Sema3E strongly reduced p61-Sema3E binding to PlexinD1, which was almost abrogated upon excess competition. Statistical significance was calculated by t-test with respect to p61-Sema3E binding in the absence of Uncl-Sema3E: *p = 1.3E−06; **p = 3.2E−08; §p = 1.4E−08; §§p = 1.5E−08. The binding of the independent semaphorin Sema4D to COS cells overexpressing its cognate receptor PlexinB1 provided a specificity control.Serum starved A549 cancer cells were incubated with 7 nM Uncl-Sema3E or mock for 1 h. Cells were then stimulated with mock, 7 nM p61, or 0.2 nM Heregulin-β1 (as positive control) in the presence of Uncl-Sema3E. ErbB2 tyrosine phosphorylation was revealed by immunoblotting.Migration of A549 and 4T1 cells expressing Uncl-Sema3E or control-EV, assayed in Transwell inserts (n = 4). Data shown indicate fold change versus respective controls.Fluorescently labelled A549 cells, either expressing Uncl-Sema3E or control-EV (same as above), were injected intravenously in nude mice. Forty-eight hours later the mice were sacrificed, and the lungs fixed and analyzed under a fluorescence microscope to reveal and quantify extravasated cancer cells colonizing the tissue (n = 5; see Materials and Methods Section for details). Representative images are shown on the right; scale bars: 100 µm.A549 and 4T1 cells expressing Uncl-Sema3E or control-EV were injected intravenously in nude mice (n = 5 and 6, respectively, for the two cell lines). Superficial metastatic colonies in the lungs were counted under a stereomicroscope; the graph indicates average values ± SD. Representative macroscopic images of the lungs are also shown, where red arrows indicate some of the metastatic lesions. Scale bars: 200 µm.
Mentions: Despite this apparent lack of activity, we found that Uncl-Sema3E is fully capable of binding the receptor PlexinD1 in analogy to p61-Sema3E isoform (Fig 2A). Moreover, we found that Uncl-Sema3E selectively competes with p61 for binding the receptor in the same nanomolar concentration range (Fig 2B). We then asked whether Uncl-Sema3E may potentially act as a dominant-negative molecule by interfering with p61 signalling. Indeed, ErbB2 transactivation elicited by p61 in A549 cells was strikingly reduced in the presence of Uncl-Sema3E (Fig 2C). Furthermore, data shown above in Fig 1C revealed that the basal level of ErbB2 tyrosine phosphorylation decreased in cells treated with Uncl-Sema3E, consistent with its functional competition with endogenous p61.

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