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. 2017 Mar;108(3):419-426.
doi: 10.1111/cas.13157.

Vasohibin-2 Is Required for Epithelial-Mesenchymal Transition of Ovarian Cancer Cells by Modulating Transforming Growth Factor-β Signaling

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Free PMC article

Vasohibin-2 Is Required for Epithelial-Mesenchymal Transition of Ovarian Cancer Cells by Modulating Transforming Growth Factor-β Signaling

Rie Norita et al. Cancer Sci. .
Free PMC article

Abstract

Vasohibin-2 (VASH2) is a homolog of VASH1, an endothelium-derived angiogenesis inhibitor. Vasohibin-2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial-mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor-β (TGF-β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF-β type I receptor, namely activin receptor-like kinase 5. Transforming growth factor-β1-induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF-β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF-β1-induced reduced expression of epithelial markers including E-cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF-β1-induced EMT. In accordance with these results, the effects of TGF-β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF-β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.

Keywords: EMT; ALK5; TGF-β; ovarian cancer; vasohibin-2.

Figures

Figure 1
Figure 1
Vasohibin‐2 (VASH2) knockdown reduces the expression of activin receptor‐like kinase 5 (ALK5). (a) Quantitative real‐time RTPCR analysis of VASH2, ALK5, and transforming growth factor‐β type II receptor (TβRII) expression in DISS cells transfected with either control or VASH2 siRNA was carried out 24 h after transfection. Values were normalized to the β‐actin mRNA level. (b) Western blotting of ALK5 in DISS cells transfected with either control (si‐Cont) or VASH2 siRNA was carried out 24 h after transfection. β‐Actin in cell lysate was used as a loading control. (c) Quantitative real‐time RTPCR analysis of VASH2, ALK5, and TβRII expression in SKOV3 cells transfected with either control or VASH2 siRNA was undertaken 4 days after transfection. Values were normalized to the β‐actin mRNA level. (d) Western blotting of ALK5 in SKOV3 cells transfected with either control (si‐Cont) or VASH2 siRNA was undertaken 4 days after transfection. α‐Tubulin in cell lysates was used as a loading control. (e) Quantitative real‐time RTPCR analysis of VASH2, ALK5, and TβRII expression in DISS cells transfected with mock or shVASH2 was carried out. (f) Western blotting of ALK5 in DISS cells transfected with control mock or shVASH2 was carried out. β‐Actin in cell lysates was used as a loading control. The intensity of each band was determined by densitometry. Values indicate the fold change of ALK5 level normalized to β‐actin. (a,c,e) Mean and SDs are shown (*< 0.05).
Figure 2
Figure 2
Vasohibin‐2 (VASH2) is required for transforming growth factor‐β (TGF‐β) signaling. (a) SKOV3 cells were cotransfected with the (CAGA)9‐Luc construct and either control siRNA or VASH2 siRNA. Three days after this procedure, cells were treated or not with TGF‐β1 for 24 h, and the (CAGA)9‐Luc reporter activity was then quantified. (b) SKOV3 cells transfected with control siRNA or VASH2 siRNA. Three days after this procedure, cells were treated or not with TGF‐β1 for 24 h. Thereafter, quantitative real‐time RTPCR analysis of plasminogen activator inhibitor type 1 (PAI‐1) expression was carried out. Values were normalized to the β‐actin mRNA level. (c) Control mock or shVASH2 transfected cells established from DISS were transfected with the (CAGA)9‐Luc construct. Twenty four hours after this procedure, cells were treated or not with TGF‐β1 for 24 h, and the (CAGA)9‐Luc reporter activity was then quantified. (d) DISS cells transfected with mock or shVASH2 were treated or not with TGF‐β1 for 24 h. Quantitative real‐time RTPCR analysis of PAI‐1 expression was then carried out. Values were normalized to the β‐actin mRNA level. (e) DISS cells transfected with mock or shVASH2 were treated with TGF‐β1 for the indicated times then Western blotting for pSmad2, total Smad2, pSmad3, and total Smad3 was undertaken. α‐Tubulin in the cell lysates was used as a loading control. The intensity of each band was determined by densitometry. Values indicate the fold change of pSmad2 and pSmad3 levels normalized to total Smad2 and Smad3, respectively. Mean and SDs are shown (*< 0.05). N.S., not significant.
Figure 3
Figure 3
Downregulation of vasohibin‐2 (VASH2) blocks transforming growth factor‐β1 (TGF‐β1)‐induced gene regulation in relation to epithelial–mesenchymal transition. (a) DISS cells transfected with mock or shVASH2 were treated or not with TGF‐β1 for 24 h. Thereafter, quantitative real‐time RTPCR analysis of E‐cadherin, fibronectin, vimentin, SM22α, ZEB2, and Snail2 expression was carried out. Values were normalized to the β‐actin mRNA level. (b) DISS cells transfected with mock or shVASH2 were treated or not with TGF‐β1 for 24 h. Thereafter, Western blotting of E‐cadherin, fibronectin, and vimentin was carried out. β‐Actin in the cell lysate was used as a loading control. The densitometric analysis of Western signals is shown in the column graph. The bars indicate the fold change of E‐cadherin, fibronectin, and vimentin expression, normalized to β‐actin and relative to untreated mock. (c) DISS cells transfected with control siRNA or VASH2 siRNA were treated or not with TGF‐β1 for 24 h. Thereafter, quantitative real‐time RTPCR analysis of E‐cadherin, fibronectin, and SM22α expression levels was carried out. Values were normalized to the β‐actin mRNA level. (d) DISS cells were treated or untreated (control) with 5 μM SB431542 (SB) for 48 h. Thereafter, quantitative real‐time RTPCR analysis of E‐cadherin, fibronectin, SM22α, ZEB2, and Snail2 expression levels was carried out. Values were normalized to the β‐actin mRNA level. (e) Cells were infected with AdLacZ or AdALK5‐TD and incubated for 48 h, then Western blotting of hemagglutinin (HA)‐tag and activin receptor‐like kinase 5 (ALK5) was carried out. (f) Cells were infected with AdLacZ or AdALK5‐TD and incubated for 48 h, then quantitative real‐time RTPCR analysis of E‐cadherin, fibronectin, and SM22α expression was carried out. Values were normalized to the β‐actin mRNA level. (a,c,d,f) Mean and SDs are shown (*< 0.05).
Figure 4
Figure 4
Downregulation of vasohibin‐2 (VASH2) blocks transforming growth factor‐β1 (TGF‐β1)‐induced protein modulation in relation to epithelial–mesenchymal transition. Immunofluorescence staining of E‐cadherin (green) and SM22α (red) was compared between mock and shVASH2 transfectant cells established from DISS, or between siControl (Ctrl) and siVASH2. Nuclei were stained with DAPI (blue). Scale bar = 20 μm.
Figure 5
Figure 5
Downregulation of vasohibin‐2 (VASH2) blocks transforming growth factor‐β1 (TGF‐β1)‐induced morphological changes, cell migration, and invasion. (a,b) DISS cells transfected with mock or shVASH2 were treated or not with TGF‐β1 for 24 h. (a) Cells were observed under an optical microscope. Scale bar = 100 μm. (b) Transwell migration assay was carried out. Total number of cells that had migrated were stained with DAPI and counted in duplicate. (c) DISS cells transfected with mock or shVASH2 were treated or not with TGF‐β1 for 48 h then the Matrigel invasion assay was undertaken. Total number of cells that invaded the gel were stained with DAPI and counted in duplicate. (d) DISS cells transfected with mock or shVASH2 were treated or not with TGF‐β1 for 24 h, then quantitative real‐time RTPCR analysis of MMP2 expression was carried out. Values were normalized to the β‐actin mRNA level. Mean and SDs are shown (*< 0.05). (b,c) Data shown are representative of three independent experiments.

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