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. 2009 Apr 15;69(8):3272-7.
doi: 10.1158/0008-5472.CAN-08-4886. Epub 2009 Apr 7.

The L6 Protein TM4SF1 Is Critical for Endothelial Cell Function and Tumor Angiogenesis

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Free PMC article

The L6 Protein TM4SF1 Is Critical for Endothelial Cell Function and Tumor Angiogenesis

Shou-Ching Shih et al. Cancer Res. .
Free PMC article

Abstract

Transmembrane-4-L-six-family-1 (TM4SF1) was originally described as a cancer cell protein. Here, we show that it is highly expressed in the vascular endothelium of human cancers and in a banded pattern in the filopodia of cultured endothelial cells (EC). TM4SF1 knockdown prevented filopodia formation, inhibited cell mobility, blocked cytokinesis, and rendered EC senescent. Integrin-alpha5 and integrin-beta1 subunits gave a similar staining pattern and interacted constitutively with TM4SF1, whereas integrin subunits often associated with angiogenesis (alphaV, beta3, beta5) interacted with TM4SF1 only after vascular endothelial growth factor (VEGF)-A or thrombin stimulation. TM4SF1 knockdown substantially inhibited maturation of VEGF-A(164)-induced angiogenesis. Thus, TM4SF1 is a key regulator of EC function in vitro and of pathologic angiogenesis in vivo and is potentially an attractive target for antiangiogenesis therapy.

Figures

Figure 1
Figure 1
TM4SF1 expression in the vascular endothelium of human cancers and in cultured EC. A, in situ hybridization shows strong TM4SF1 expression in the blood vessels of colon, kidney, ovary, and breast carcinomas. Images shown are representative of those taken from four different patients with each type of cancer. Bright field reveals histology and dark field shows mRNA expression. Arrows, TM4SF1-positive blood vessels. B, immunofluorescence staining of a typical colon cancer with antibodies against TM4SF1 (green) and either (i) VE-cadherin or (ii) anti-human α-smooth muscle actin (red). C, TM4SF1 expression in several types of cultured EC (left) and in HUVEC stimulated with indicated growth factors (right). *, P < 0.05. D, TM4SF1 (green) and F-actin (red) double-staining of HUVEC reveals that TM4SF1 is localized to the plasma membrane, perinuclear vesicles (arrow), and particularly to filopodia (a). Filopodia exhibited a banded staining pattern (b and c). Arrows, filopodia branching (c). Scale bars, 100 μm (A and B) and 10 μm (D).
Figure 2
Figure 2
TM4SF1 KD rendered HUVEC senescent. A, TM4SF1 KD efficiency at various times after transfection with 50-moi Ad-hTM4-shRNA or Ad-Con-shRNA. B, day 3 TM4SF1-KD HUVEC lose TM4SF1 staining and develop stress fibers (i), lose filopodia (i and ii), increase cells in G2-M by 4.3× (iii), and express β-galactosidase activity (iv). C, loss of TM4SF1 staining in a spontaneously senescent HUVEC. D, 3 d TM4SF1-KD HUVEC migrate poorly in a wound healing assay, fail to form tubes on Matrigel.
Figure 3
Figure 3
TM4SF1-integrin expression and interactions. A, integrin expression profile in HUVEC ± TM4SF1 KD. B, immunofluorescence staining of integrin α5, αV, and β1 subunits in HUVEC 3 d after 50-moi transfection with either Ad-hTM4-shRNA or Ad-Con-shRNA. Scale bars, 10 μm. C, control HUVEC or HUVEC stimulated for 6 h with VEGF-A (20 ng/mL) or thrombin (THR; 1.5 u/mL). Cell lysates, prepared before and after immunoprecipitation (IP) with anti-TM4SF1 antibody, were immunoblotted with indicated antibodies. IgG and β-actin were loading controls. The anti-TM4SF1 antibody effectively immunoprecipitated native TM4SF1 but reacted poorly with denatured TM4SF1. Expression of β3 and β5 were below detection levels in whole cell lysates.
Figure 4
Figure 4
Expression of TM4SF1 and integrin subunits in Ad-VEGF-A164–induced angiogenesis; effect of TM4SF1 on angiogenesis. A, mRNA expression levels of TM4SF1 and integrin subunits in mouse ears at indicated times after injection of 2.5 × 107 plaque-forming unit Ad-VEGF-A164. B, in situ hybridization localization of TM4SF1 mRNA to VM in day 11 Ad-VEGF-A164–injected mouse ears (top). Hematoxylin-stained VM illustrating smooth muscle cell coat (green arrows, bottom). C, angiogenic response in mouse ears at indicated times after Ad-VEGF-A164 injection. Moi (109) of Ad-mTM4-shRNA or Ad-Con-shRNA were injected into the right and left ears, respectively, 1 d before Ad-VEGF-A164 injection. The experiment was repeated four times. D, quantification of angiogenesis with the Evan's Blue dye 19 d after Ad-VEGF-A164 injection.

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