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 5

ITGA1 and ADCY9 are miR-181b–regulated ceRNAs.

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ITGA1 and ADCY9 are miR-181b–regulated ceRNAs. (A) qPCR of mRNAs in 393P...
(A) qPCR of mRNAs in 393P transfectants (antagomiR-181b [anti-181b] or control [anti-NC]). Levels of 32 predicted miR-181b targets that were approximately equimolar with ITGA1 in 393P_ZEB1 cells expressed as a ratio (anti-181b/anti-NC). Red, significantly upregulated more than 2-fold. *P < 0.05 and ***P < 0.001. (B) Schema, 3′-UTRs of ADCY9, ONECUT2, TGFBR3, and BHLHE41 with locations of predicted miR-181b–binding sites. Bar graph: luciferase activity in 344SQ cells cotransfected with miR-181b or control (miR-NC) and a 3′-UTR reporter with WT or mutant (MT) miR-181b–binding sites. ONECUT2 reporters with 3′-UTR portions (ONECUT2-1, -2, and -3). (C) AGO2 RIP performed on lysates from 344SQ transfectants (ITGA1 siRNA [siITGA1] or control [siCTL]). AGO2-associated ITGA1, TGFBR3, ONECUT2, and ADCY9 mRNAs normalized on the basis of IgG control and expressed as fold enrichment relative to siCTL. (D) AGO2 RIP on 393P cells transfected with WT or mutant (MT) ITGA1 3′-UTR or EGFP. AGO2-associated ITGA1 3′-UTR and ADCY9 mRNA normalized on the basis of IgG control and expressed as fold enrichment relative to EGFP transfectants. (E) qPCR of ADCY9 mRNA in 393P cells transfected with ITGA1 3′-UTR (WT or MT) or EGFP. (F) Luciferase activities in 393P cells cotransfected with ADCY9 3′-UTR reporters and ITGA1 3′-UTR (WT or MT) or EGFP. ZEB1 3′-UTR reporter included as negative control. (G and H) ITGA1 and ADCY9 levels in human lung adenocarcinomas (n = 541) in The Cancer Genome Atlas (TCGA) as groups (top and bottom quartiles). (I) Pan-cancer analysis examining correlations between ZEB1, ITGA1, and ADCY9 mRNA levels for each of 32 different cancer types in TCGA. See Methods (Statistics section) for list of tumor types and sample sizes. Values are mean ± SD. n = 3, unless otherwise indicated. P values, 2-tailed Student’s t test and Dunnett’s test for 2-group and more-than-2-group comparisons, respectively. Results were replicated (n ≥ 2 experiments).