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Regulation of mTOR Signaling by Semaphorin 3F-Neuropilin 2 Interactions In Vitro and In Vivo.

Nakayama H, Bruneau S, Kochupurakkal N, Coma S, Briscoe DM, Klagsbrun M - Sci Rep (2015)

Bottom Line: Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood.Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase.In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [2] Transplant Research Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [3] Department of Surgery, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [4] Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan.

ABSTRACT
Semaphorin 3F (SEMA3F) provides neuronal guidance cues via its ability to bind neuropilin 2 (NRP2) and Plexin A family molecules. Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood. Here, we analyze SEMA3F-NRP2 signaling responses in human endothelial, T cell and tumor cells using phosphokinase arrays, immunoprecipitation and Western blot analyses. Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase. We also find that the expression of vascular endothelial growth factor, as well as mTOR-inducible cellular activation responses and cytoskeleton stability are inhibited by SEMA3F-NRP2 interactions in vitro. In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts. Taken together, SEMA3F regulates mTOR signaling in diverse human cell types, suggesting that it has broad therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

SEMA3F inhibits human tumor growth in xenografts in vivo.A, Parental U87MG cells (Mock)and human SEMA3F stable clones (S3F) were implanted into nude mice subcutaneously (1 × 106 cells/injection). The insert shows Western blot analysis of SEMA3F expression in each cell line. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. B, Representative immunohistochemical anti-CD31 staining of tumors harvested after 24 days. C, U87MG cells (1 × 106 cells/injection) were administrated subcutaneously into nude mice. After 2 days, control (Ad-Cont) or human SEMA3F-His (Ad-3F)-recombinant adenovirus (1 × 109 pfu) were injected intravenously via the tail vein. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. Mice were sacrificed on day 14. The insert shows SEMA3F expression within the liver (on day 14) by Western blot analysis using an anti-His antibody. D, Representative immunohistochemical staining of tumors with anti-CD31. E, Western blot analysis of Akt/mTOR signaling pathway within tumor samples. Panels B,D,E are representative results of 3 independent experiments.
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f5: SEMA3F inhibits human tumor growth in xenografts in vivo.A, Parental U87MG cells (Mock)and human SEMA3F stable clones (S3F) were implanted into nude mice subcutaneously (1 × 106 cells/injection). The insert shows Western blot analysis of SEMA3F expression in each cell line. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. B, Representative immunohistochemical anti-CD31 staining of tumors harvested after 24 days. C, U87MG cells (1 × 106 cells/injection) were administrated subcutaneously into nude mice. After 2 days, control (Ad-Cont) or human SEMA3F-His (Ad-3F)-recombinant adenovirus (1 × 109 pfu) were injected intravenously via the tail vein. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. Mice were sacrificed on day 14. The insert shows SEMA3F expression within the liver (on day 14) by Western blot analysis using an anti-His antibody. D, Representative immunohistochemical staining of tumors with anti-CD31. E, Western blot analysis of Akt/mTOR signaling pathway within tumor samples. Panels B,D,E are representative results of 3 independent experiments.

Mentions: To next evaluate the in vivo relevance of our signaling studies, we examined the effect of SEMA3F on tumor growth in a well-established xenograft model54445. In one approach, parental U87MG cells or U87MG cells that were engineered to constitutively overexpress SEMA3F were implanted subcutaneously into nude mice (1 × 106/mouse); tumor size (mm3) was measured at the indicated time points over a period of 3 weeks. We found that tumor growth was essentially absent when SEMA3F-producing cells were implanted vs. parental cells (p < 0.0005, Fig. 5A). Furthermore, immunohistochemical analysis of CD31-expressing EC demonstrated numerous blood vessels in parental U87MG tumors (Fig. 5B); in contrast, capillaries within the U87MG/SEMA3F-derived tumors were constricted and were without discernable lumens (Fig. 5B).


Regulation of mTOR Signaling by Semaphorin 3F-Neuropilin 2 Interactions In Vitro and In Vivo.

Nakayama H, Bruneau S, Kochupurakkal N, Coma S, Briscoe DM, Klagsbrun M - Sci Rep (2015)

SEMA3F inhibits human tumor growth in xenografts in vivo.A, Parental U87MG cells (Mock)and human SEMA3F stable clones (S3F) were implanted into nude mice subcutaneously (1 × 106 cells/injection). The insert shows Western blot analysis of SEMA3F expression in each cell line. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. B, Representative immunohistochemical anti-CD31 staining of tumors harvested after 24 days. C, U87MG cells (1 × 106 cells/injection) were administrated subcutaneously into nude mice. After 2 days, control (Ad-Cont) or human SEMA3F-His (Ad-3F)-recombinant adenovirus (1 × 109 pfu) were injected intravenously via the tail vein. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. Mice were sacrificed on day 14. The insert shows SEMA3F expression within the liver (on day 14) by Western blot analysis using an anti-His antibody. D, Representative immunohistochemical staining of tumors with anti-CD31. E, Western blot analysis of Akt/mTOR signaling pathway within tumor samples. Panels B,D,E are representative results of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496725&req=5

f5: SEMA3F inhibits human tumor growth in xenografts in vivo.A, Parental U87MG cells (Mock)and human SEMA3F stable clones (S3F) were implanted into nude mice subcutaneously (1 × 106 cells/injection). The insert shows Western blot analysis of SEMA3F expression in each cell line. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. B, Representative immunohistochemical anti-CD31 staining of tumors harvested after 24 days. C, U87MG cells (1 × 106 cells/injection) were administrated subcutaneously into nude mice. After 2 days, control (Ad-Cont) or human SEMA3F-His (Ad-3F)-recombinant adenovirus (1 × 109 pfu) were injected intravenously via the tail vein. Tumor size was measured using a standard calipers at the indicated time points. Numbers in parentheses represent the number of animals in each group. Mice were sacrificed on day 14. The insert shows SEMA3F expression within the liver (on day 14) by Western blot analysis using an anti-His antibody. D, Representative immunohistochemical staining of tumors with anti-CD31. E, Western blot analysis of Akt/mTOR signaling pathway within tumor samples. Panels B,D,E are representative results of 3 independent experiments.
Mentions: To next evaluate the in vivo relevance of our signaling studies, we examined the effect of SEMA3F on tumor growth in a well-established xenograft model54445. In one approach, parental U87MG cells or U87MG cells that were engineered to constitutively overexpress SEMA3F were implanted subcutaneously into nude mice (1 × 106/mouse); tumor size (mm3) was measured at the indicated time points over a period of 3 weeks. We found that tumor growth was essentially absent when SEMA3F-producing cells were implanted vs. parental cells (p < 0.0005, Fig. 5A). Furthermore, immunohistochemical analysis of CD31-expressing EC demonstrated numerous blood vessels in parental U87MG tumors (Fig. 5B); in contrast, capillaries within the U87MG/SEMA3F-derived tumors were constricted and were without discernable lumens (Fig. 5B).

Bottom Line: Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood.Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase.In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [2] Transplant Research Program, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [3] Department of Surgery, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 [4] Division of Cell Growth and Tumor Regulation, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan.

ABSTRACT
Semaphorin 3F (SEMA3F) provides neuronal guidance cues via its ability to bind neuropilin 2 (NRP2) and Plexin A family molecules. Recent studies indicate that SEMA3F has biological effects in other cell types, however its mechanism(s) of function is poorly understood. Here, we analyze SEMA3F-NRP2 signaling responses in human endothelial, T cell and tumor cells using phosphokinase arrays, immunoprecipitation and Western blot analyses. Consistently, SEMA3F inhibits PI-3K and Akt activity, and responses are associated with the disruption of mTOR/rictor assembly and mTOR-dependent activation of the RhoA GTPase. We also find that the expression of vascular endothelial growth factor, as well as mTOR-inducible cellular activation responses and cytoskeleton stability are inhibited by SEMA3F-NRP2 interactions in vitro. In vivo, local and systemic overproduction of SEMA3F reduces tumor growth in NRP2-expressing xenografts. Taken together, SEMA3F regulates mTOR signaling in diverse human cell types, suggesting that it has broad therapeutic implications.

No MeSH data available.


Related in: MedlinePlus