Class 3 semaphorins (Sema3) is a family of secreted proteins that regulate axon guidance and other important processes such as tumor progression and angiogenesis. Recently it has been shown that Sema3A and Sema3F inhibited tumor cell motility and growth, impaired experimental and in vivo angiogenesis and, interestingly, controlled vascular remodeling by integrin inhibition through autocrine loops. Stemming from these observations we sought to investigate the role of Sema3 in angiogenesis during tumor progression employing transgenic mouse models of spontaneous tumorigenesis of pancreas (RipTag2) and uterine cervix (HPV16/E2). Real-time RT-PCR analysis revealed that Sema3a and, to a lesser extent, Sema3e and Sema3f genes were up-regulated in pre-malignant stages and strongly down-regulated in invasive tumors compared to normal pancreatic islets and cervices. By confocal microscopy we observed a strong Sema3A localization in vessels and, to a lesser extent, in epithelial cells of pre-malignant lesions. Based on previous studies showing that class 3 semaphorins inhibited integrins function, we assessed the role of integrin activation during tumor progression in both mouse models. Interestingly we observed that, increased α5β1 integrin activation in tumor versus dysplastic vessels, was inversely correlated with endogenous Sema3A levels. To evaluate the effect of Sema3A on tumor growth we set up a new delivery system employing adeno-associated virus (AAV) to deliver Sema3A directly into pancreas through the abdominal aorta of RipTag2 mice. We injected AAV8-Sema3A in tumor-bearing RipTag2 aimed to assess the effect of Sema3A in reducing tumor volume and impairing angiogenesis. After a month of treatment we noticed a reduction of tumor volume by 65% in treated mice compared to controls. By confocal analysis we observed that Sema3A significantly reduced tumor blood vessel density (by 41%) and branching (by 53%) in treated mice. Surprisingly we detected an increased pericytes vessel coverage (by 45%) and a normalized vascular morphology in AAV8-Sema3A-treated tumors compared to controls. Finally, we observed that Sema3A treatment strongly inhibited β1 integrin activation in tumor endothelial cells, but not in pericytes. Remarkably, Sema3A over-expression did not affect the normal vasculature. To better understand the effect of sema3A in inhibiting tumor growth we performed a short Regression Trial (RT) and we sacrificed treated mice two weeks after AAV-Sema3A delivery. In this case, we observed increased vascular endothelial cells apoptosis. In contrast, in a four weeks long treatment we observed apoptosis only in tumor cells. Finally, to better demonstrate the role of sema3A as endogenous angiogenic inhibitor we treated the mice during the angiogenic switch using a specific Sema3A inhibitor (SM-216289) and osmotic mini-pumps to assess whether inhibiting sema3A at this stage would lead to an increased angiogenesis. Notably, RipTag2 mice treated for two weeks with SM-216289 showed an increase in the number of angiogenic islets and enhanced tumor growth, compared to untreated mice. Our data unveiled for the first time anti-angiogenic and anti-tumor properties of Sema3A during spontaneous tumorigenesis. Since Sema3A inhibited tumor angiogenesis normalizing the vasculature, this molecule holds promise as a new target to design more efficient anti-angiogenic therapies.

Semaphorin 3A Regulates Angiogenesis and Tumor Progression in Mouse Models of Tumorigenesis

MAIONE, FEDERICA;MEDA, CLAUDIA MARIA;SERINI, Guido;BUSSOLINO, Federico;GIRAUDO, Enrico
2009-01-01

Abstract

Class 3 semaphorins (Sema3) is a family of secreted proteins that regulate axon guidance and other important processes such as tumor progression and angiogenesis. Recently it has been shown that Sema3A and Sema3F inhibited tumor cell motility and growth, impaired experimental and in vivo angiogenesis and, interestingly, controlled vascular remodeling by integrin inhibition through autocrine loops. Stemming from these observations we sought to investigate the role of Sema3 in angiogenesis during tumor progression employing transgenic mouse models of spontaneous tumorigenesis of pancreas (RipTag2) and uterine cervix (HPV16/E2). Real-time RT-PCR analysis revealed that Sema3a and, to a lesser extent, Sema3e and Sema3f genes were up-regulated in pre-malignant stages and strongly down-regulated in invasive tumors compared to normal pancreatic islets and cervices. By confocal microscopy we observed a strong Sema3A localization in vessels and, to a lesser extent, in epithelial cells of pre-malignant lesions. Based on previous studies showing that class 3 semaphorins inhibited integrins function, we assessed the role of integrin activation during tumor progression in both mouse models. Interestingly we observed that, increased α5β1 integrin activation in tumor versus dysplastic vessels, was inversely correlated with endogenous Sema3A levels. To evaluate the effect of Sema3A on tumor growth we set up a new delivery system employing adeno-associated virus (AAV) to deliver Sema3A directly into pancreas through the abdominal aorta of RipTag2 mice. We injected AAV8-Sema3A in tumor-bearing RipTag2 aimed to assess the effect of Sema3A in reducing tumor volume and impairing angiogenesis. After a month of treatment we noticed a reduction of tumor volume by 65% in treated mice compared to controls. By confocal analysis we observed that Sema3A significantly reduced tumor blood vessel density (by 41%) and branching (by 53%) in treated mice. Surprisingly we detected an increased pericytes vessel coverage (by 45%) and a normalized vascular morphology in AAV8-Sema3A-treated tumors compared to controls. Finally, we observed that Sema3A treatment strongly inhibited β1 integrin activation in tumor endothelial cells, but not in pericytes. Remarkably, Sema3A over-expression did not affect the normal vasculature. To better understand the effect of sema3A in inhibiting tumor growth we performed a short Regression Trial (RT) and we sacrificed treated mice two weeks after AAV-Sema3A delivery. In this case, we observed increased vascular endothelial cells apoptosis. In contrast, in a four weeks long treatment we observed apoptosis only in tumor cells. Finally, to better demonstrate the role of sema3A as endogenous angiogenic inhibitor we treated the mice during the angiogenic switch using a specific Sema3A inhibitor (SM-216289) and osmotic mini-pumps to assess whether inhibiting sema3A at this stage would lead to an increased angiogenesis. Notably, RipTag2 mice treated for two weeks with SM-216289 showed an increase in the number of angiogenic islets and enhanced tumor growth, compared to untreated mice. Our data unveiled for the first time anti-angiogenic and anti-tumor properties of Sema3A during spontaneous tumorigenesis. Since Sema3A inhibited tumor angiogenesis normalizing the vasculature, this molecule holds promise as a new target to design more efficient anti-angiogenic therapies.
2009
Mouse Models of Cancer”, AACR special conference in Cancer Research
San Francisco, CA
12-15 gennaio 2009
Mouse Models of Cancer
AACR
1
1
18
18
Maione F; Molla F; Meda C; Latini R; Zentilin L; Giacca M; Serini G; Bussolino F; Giraudo E.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/138902
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