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Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells.

Otsubo T, Hida Y, Ohga N, Sato H, Kai T, Matsuki Y, Takasu H, Akiyama K, Maishi N, Kawamoto T, Shinohara N, Nonomura K, Hida K - Cancer Sci. (2014)

Bottom Line: Targeting tumor angiogenesis is an established strategy for cancer therapy.We identified 131 genes that were differentially upregulated in mTEC.The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery II, DSP Cancer Institute, Dainippon Sumitomo Pharma Co., Ltd, Osaka, Japan.

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Functional analysis of tumor endothelial cell (TEC) markers using siRNA-mediated gene silencing. Melanoma-EC were transfected using three different siRNA for each gene. Silencing of each TEC marker 48 h after transfection was evaluated using qRT-PCR (upper panel). Cell proliferation was measured after 72 h using Alamar Blue (lower panel). Cell migration towards the chemoattractant EGM-2MV for 20 h was evaluated using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (BD Biosciences) (lower panel). siRNA-mediated effects of each TEC marker are shown in (a–e) (**P < 0.01; *P < 0.05). (a) Tmem176b; (b) Pcdhb22; (c) Nsg1; (d) Enah; and (e) Def6. NC, Negative control siRNA.
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fig04: Functional analysis of tumor endothelial cell (TEC) markers using siRNA-mediated gene silencing. Melanoma-EC were transfected using three different siRNA for each gene. Silencing of each TEC marker 48 h after transfection was evaluated using qRT-PCR (upper panel). Cell proliferation was measured after 72 h using Alamar Blue (lower panel). Cell migration towards the chemoattractant EGM-2MV for 20 h was evaluated using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (BD Biosciences) (lower panel). siRNA-mediated effects of each TEC marker are shown in (a–e) (**P < 0.01; *P < 0.05). (a) Tmem176b; (b) Pcdhb22; (c) Nsg1; (d) Enah; and (e) Def6. NC, Negative control siRNA.

Mentions: Excessive angiogenesis occurs through a series of steps including enhanced EC proliferation and migration.36 Therefore, targeting proliferation and/or migration of EC is one of the most attractive and effective strategies for treating angiogenesis-dependent disorders. We reported that mTEC grow faster and migrate better than mNEC.28 These in vitro characteristics of mTEC represent enhanced tumor angiogenesis in vivo and the genes responsible for increased proliferation or migration of mTEC may serve as ideal targets for antiangiogenic therapy. To identify such molecules, we performed loss-of-function screening of the 131 potential TEC markers in Melanoma-EC, one of the mTEC that showed high activity in the proliferation and migration assays.28 We first cotransfected Melanoma-EC with three different sequences of siRNA per gene. Cell proliferation and/or migration were inhibited by >20% compared with mock-transfected cells using siRNA targeted to 44 genes (Fig.2). Subsequently, three different siRNA specific for each of the 44 genes were used to independently transfect Melanoma-EC. We performed migration and proliferation assay using siRNA and finally selected five genes (Def6, Nsg1, Enah, Tmem176b and Pcdhb22; Table3, Fig.3) whose respective siRNA (two or more per gene) inhibited cell proliferation or migration by >30% (Figs2, 4). Cell migration was inhibited by siRNA of Tmem176b, Pcdhb22, Nsg1 and Enah, but cell proliferation was not inhibited. In contrast, cell proliferation was inhibited by Def6 siRNA. These were considered potential regulators of proliferation or migration of mTEC. There was no gene whose two or more siRNA inhibited both cell proliferation and migration by >30% (data not shown). Knockdown of each gene was confirmed using qRT-PCR 48 h after transfection (Fig.4).


Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells.

Otsubo T, Hida Y, Ohga N, Sato H, Kai T, Matsuki Y, Takasu H, Akiyama K, Maishi N, Kawamoto T, Shinohara N, Nonomura K, Hida K - Cancer Sci. (2014)

Functional analysis of tumor endothelial cell (TEC) markers using siRNA-mediated gene silencing. Melanoma-EC were transfected using three different siRNA for each gene. Silencing of each TEC marker 48 h after transfection was evaluated using qRT-PCR (upper panel). Cell proliferation was measured after 72 h using Alamar Blue (lower panel). Cell migration towards the chemoattractant EGM-2MV for 20 h was evaluated using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (BD Biosciences) (lower panel). siRNA-mediated effects of each TEC marker are shown in (a–e) (**P < 0.01; *P < 0.05). (a) Tmem176b; (b) Pcdhb22; (c) Nsg1; (d) Enah; and (e) Def6. NC, Negative control siRNA.
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fig04: Functional analysis of tumor endothelial cell (TEC) markers using siRNA-mediated gene silencing. Melanoma-EC were transfected using three different siRNA for each gene. Silencing of each TEC marker 48 h after transfection was evaluated using qRT-PCR (upper panel). Cell proliferation was measured after 72 h using Alamar Blue (lower panel). Cell migration towards the chemoattractant EGM-2MV for 20 h was evaluated using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (BD Biosciences) (lower panel). siRNA-mediated effects of each TEC marker are shown in (a–e) (**P < 0.01; *P < 0.05). (a) Tmem176b; (b) Pcdhb22; (c) Nsg1; (d) Enah; and (e) Def6. NC, Negative control siRNA.
Mentions: Excessive angiogenesis occurs through a series of steps including enhanced EC proliferation and migration.36 Therefore, targeting proliferation and/or migration of EC is one of the most attractive and effective strategies for treating angiogenesis-dependent disorders. We reported that mTEC grow faster and migrate better than mNEC.28 These in vitro characteristics of mTEC represent enhanced tumor angiogenesis in vivo and the genes responsible for increased proliferation or migration of mTEC may serve as ideal targets for antiangiogenic therapy. To identify such molecules, we performed loss-of-function screening of the 131 potential TEC markers in Melanoma-EC, one of the mTEC that showed high activity in the proliferation and migration assays.28 We first cotransfected Melanoma-EC with three different sequences of siRNA per gene. Cell proliferation and/or migration were inhibited by >20% compared with mock-transfected cells using siRNA targeted to 44 genes (Fig.2). Subsequently, three different siRNA specific for each of the 44 genes were used to independently transfect Melanoma-EC. We performed migration and proliferation assay using siRNA and finally selected five genes (Def6, Nsg1, Enah, Tmem176b and Pcdhb22; Table3, Fig.3) whose respective siRNA (two or more per gene) inhibited cell proliferation or migration by >30% (Figs2, 4). Cell migration was inhibited by siRNA of Tmem176b, Pcdhb22, Nsg1 and Enah, but cell proliferation was not inhibited. In contrast, cell proliferation was inhibited by Def6 siRNA. These were considered potential regulators of proliferation or migration of mTEC. There was no gene whose two or more siRNA inhibited both cell proliferation and migration by >30% (data not shown). Knockdown of each gene was confirmed using qRT-PCR 48 h after transfection (Fig.4).

Bottom Line: Targeting tumor angiogenesis is an established strategy for cancer therapy.We identified 131 genes that were differentially upregulated in mTEC.The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery II, DSP Cancer Institute, Dainippon Sumitomo Pharma Co., Ltd, Osaka, Japan.

Show MeSH
Related in: MedlinePlus