The epithelial-to-mesenchymal transition activator ZEB1 initiates a prometastatic competing endogenous RNA network
Xiaochao Tan, … , Ignacio I. Wistuba, Jonathan M. Kurie
Xiaochao Tan, … , Ignacio I. Wistuba, Jonathan M. Kurie
Published January 11, 2018
Citation Information: J Clin Invest. 2018;128(4):1267-1282. https://doi.org/10.1172/JCI97225.
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The epithelial-to-mesenchymal transition activator ZEB1 initiates a prometastatic competing endogenous RNA network

Abstract

Epithelial tumor cells undergo epithelial-to-mesenchymal transition (EMT) to gain metastatic activity. Competing endogenous RNAs (ceRNAs) have binding sites for a common set of microRNAs (miRs) and regulate each other’s expression by sponging miRs. Here, we address whether ceRNAs govern metastasis driven by the EMT-activating transcription factor ZEB1. High miR-181b levels were correlated with an improved prognosis in human lung adenocarcinomas, and metastatic tumor cell lines derived from a murine lung adenocarcinoma model in which metastasis is ZEB1-driven were enriched in miR-181b targets. ZEB1 relieved a strong basal repression of α1 integrin (ITGA1) mRNA, which in turn upregulated adenylyl cyclase 9 mRNA (ADCY9) by sponging miR181b. Ectopic expression of the ITGA1 3′-untranslated region reversed miR-181b–mediated metastasis suppression and increased the levels of adenylyl cyclase 9 protein (AC9), which promoted tumor cell migration and metastasis. In human lung adenocarcinomas, ITGA1 and ADCY9 levels were positively correlated, and an AC9-activated transcriptomic signature had poor-prognostic value. Thus, ZEB1 initiates a miR-181b–regulated ceRNA network to drive metastasis.

Authors

Xiaochao Tan, Priyam Banerjee, Xin Liu, Jiang Yu, Don L. Gibbons, Ping Wu, Kenneth L. Scott, Lixia Diao, Xiaofeng Zheng, Jing Wang, Ali Jalali, Milind Suraokar, Junya Fujimoto, Carmen Behrens, Xiuping Liu, Chang-gong Liu, Chad J. Creighton, Ignacio I. Wistuba, Jonathan M. Kurie

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Figure 2

ZEB1 suppresses the prometastatic activity of antagomiR-181b.

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ZEB1 suppresses the prometastatic activity of antagomiR-181b. (A) Boyden...
(A) Boyden chamber assays on KP cells with low endogenous ZEB1 levels (393P and 307P) or high endogenous ZEB1 levels (344SQ and 344P) after transfection with antagomiR-181b or control antagomirs (anti-NC). Migratory cells were photographed (images) and counted (bar graph). Results expressed relative to controls (anti-NC). Scale bars: 200 μm. (B) Numbers of viable antagomiR-181b– and anti-NC–transfected cells seeded on low-adhesion plates and counted 24–48 hours later. n = 4. (C) Western analysis to detect cleaved PARP in transfectants seeded on adhesive or low-adhesion (suspension) plates. β-Actin included as loading control. (D) Soft agar colony formation by antagomiR-181b– and anti-NC–transfected cells. Colonies were photographed (images) and counted (bar graph). Results expressed relative to controls. Scale bar: 100 μm. (E) 3′-UTR activity assays of a known miR-181b target, BCL2. 393P and 344SQ cells were transfected with a BCL2 3′-UTR luciferase reporter and antagomiR-181b or anti-NC. Luciferase activity was normalized and expressed as a ratio (antagomiR-181b/anti-NC). ZEB1 3′-UTR included as a negative control. n = 4. (F) Western analysis of BCL2 levels in 393P and 344SQ cells transfected with antagomiR-181b or anti-NC. The numbers indicate the normalized expression levels of BCL2 protein. (G) Boyden chamber assays on antagomir-181b– and anti-NC–transfected cells. Migratory and invasive cells were photographed (images) and counted (bar graph). Results expressed relative to anti-NC. Scale bars: 200 μm. **P < 0.01 and ***P < 0.001. Values are mean ± SD. n = 3, unless otherwise indicated. P values, 2-tailed Student’s t test. Results were replicated (n ≥ 2 experiments).