Angiopoietin-like protein 1 suppresses SLUG to inhibit cancer cell motility
Tsang-Chih Kuo, … , Jen-Liang Su, Min-Liang Kuo
Tsang-Chih Kuo, … , Jen-Liang Su, Min-Liang Kuo
Published February 22, 2013
Citation Information: J Clin Invest. 2013;123(3):1082-1095. https://doi.org/10.1172/JCI64044.
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Research Article

Angiopoietin-like protein 1 suppresses SLUG to inhibit cancer cell motility

Abstract

Angiopoietin-like protein 1 (ANGPTL1) is a potent regulator of angiogenesis. Growing evidence suggests that ANGPTL family proteins not only target endothelial cells but also affect tumor cell behavior. In a screen of 102 patients with lung cancer, we found that ANGPTL1 expression was inversely correlated with invasion, lymph node metastasis, and poor clinical outcomes. ANGPTL1 suppressed the migratory, invasive, and metastatic capabilities of lung and breast cancer cell lines in vitro and reduced metastasis in mice injected with cancer cell lines overexpressing ANGPTL1. Ectopic expression of ANGPTL1 suppressed the epithelial-to-mesenchymal transition (EMT) by reducing the expression of the zinc-finger protein SLUG. A microRNA screen revealed that ANGPTL1 suppressed SLUG by inducing expression of miR-630 in an integrin α1β1/FAK/ERK/SP1 pathway–dependent manner. These results demonstrate that ANGPTL1 represses lung cancer cell motility by abrogating the expression of the EMT mediator SLUG.

Authors

Tsang-Chih Kuo, Ching-Ting Tan, Yi-Wen Chang, Chih-Chen Hong, Wei-Jiunn Lee, Min-Wei Chen, Yung-Ming Jeng, Jean Chiou, Pei Yu, Pai-Sheng Chen, Ming-Yang Wang, Michael Hsiao, Jen-Liang Su, Min-Liang Kuo

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Figure 4

ANGPTL1 represses SLUG protein expression through inducing miRNA-630.

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ANGPTL1 represses SLUG protein expression through inducing miRNA-630. (A...
(A) Western blot analysis of SLUG in ANGPTL1-overexpressing CL1-5 cells after treatment with 10 μM MG132. SLUG levels were arbitrarily assigned a value of 1 in CL1-5/pcDNA3.1 cells. Numbers above the blots represent SLUG levels. (B) Western blot analysis of SLUG in ANGPTL1-overexpressing CL1-5 cells after treatment with 10 μg/ml cycloheximide (CHX). SLUG levels of CL1-5/pcDNA3.1 and CL1-5/ANGPTL1 were arbitrarily assigned a value of 100% at 0 hours. (C) Schematic selection of candidate miRNAs. Using overexpression of ANGPTL1 in CL1-5 cells as a model in miRNA microarrays, 10 candidate miRNAs (merged microarray data with software predictive results from 3 online computational algorithms, TargetScan, PicTar, and miRanda) were selected. (D) qRT-PCR analysis of differential expression of 10 miRNAs in ANGPTL1-overexpressing CL1-5 cells and ANGPTL1 knockdown CL1-0 cells. The dashed line indicates that the qPCR value of CL1-5/ANGPTL1 was divided by CL1-5/pcDNA3.1. (E) Schematic diagram presents the predicted miR-545– and miR-630–binding sequences or mutated versions (top). Luciferase activity (bottom) of wild-type SNAI2 3′-UTR (WT-3′-UTR) or mutated-type SNAI2 3′-UTR (MT-3′-UTR) reporter genes in 293T cells transfected with miR-545 and miR-630 at different ratios. (F) qRT-PCR analysis of miR-545 and miR-630 expression in CL1-5 cells treated with 50 ng/ml rANGPTL1. (G and H) Western blot analysis of SLUG and measurement of the migration and invasion in ANGPTL1-overexpressing CL1-5 cells transiently transfected with (G) anti–miR-545 and (H) anti–miR-630. Numbers above the blots represent SLUG levels. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 (2-tailed Student’s t test).