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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Research Article Oncology

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 3

ITGA1 3′-UTR promotes metastasis through its miR-181b–binding site.

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ITGA1 3′-UTR promotes metastasis through its miR-181b–binding site. (A) ...
(A) RNA-Seq analysis of predicted miR-181b targets in 393P_ZEB1 cells and 393P_vector cells expressed as a ratio (ZEB1/Vec). (B) miR-181b and ITGA1 mRNA copy numbers. (C) EGFP reporters fused to ITGA1 coding sequence (CDS) or 3′-UTR containing wild-type (WT) or mutated (MT) miR-181b–binding sites. Western analysis (bottom) of EGFP in 344SQ cells cotransfected with EGFP reporters and miR-181b, miR-181c, or vector (Vec). (D) Constructs containing MS2 binding sites (12X) fused downstream of a WT (UTR-WT-MS2) or mutated (UTR-MT-MS2) ITGA1 3′-UTR. MS2-based RIP (bottom). MS2-UTR–associated miR-181b and miR-200b (negative control) quantified as fold enrichment relative to MS2. (E) Fluorescence microscopic images of lung metastases in mice injected with the 344SQ_RFP transfectants (see Supplemental Figure 5, A and B). Scatter plot: numbers of lung metastases per mouse (dots). (F) Boyden chamber migration assays. 393P cells transfected with ITGA1 3′-UTR (WT or MT) or EGFP. Migratory cells were photographed (images) and counted (bar graph). Scale bar: 200 μm. (G) Numbers of viable 393P transfectants on low-adhesion plates (bar graph). Western blotting (gels) to detect cleaved PARP in transfectants on adhesive or low-adhesion (suspension) plates. (H) Numbers of lung metastases in mice injected by tail vein with mCherry-labeled 393P cells transfected with ITGA1 3′-UTR (WT or MT) or control (EGFP). (I) Boyden chamber assays to quantify migratory H1299 cells cotransfected with a shRNA (shCTL or shITGA1) and a vector that expresses EGFP or ITGA1 3′-UTR (WT or MT). *P < 0.05 and **P = 0.005. (J) Boyden chamber assays. 393P cells cotransfected with ITGA1 3′-UTR or EGFP and miR-181b or vector (LVX). (K) Boyden chamber assays. 344SQ cells cotransfected with ITGA1 siRNA or control (siCTL) and antagomiR-181b or anti-NC. *P < 0.05 and **P < 0.01. Values are mean ± SD. n = 3, unless otherwise indicated. P values, Dunnett’s test. Results were replicated (n ≥ 2 experiments).