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 4

ZEB1 upregulates ITGA1 expression through intermediates.

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ZEB1 upregulates ITGA1 expression through intermediates. (A) Top: locati...
(A) Top: locations of GC repeats (red hash marks) and CpG island (blue shading). Middle: ITGA1 promoter. First nucleotide in 5′-UTR (+1) and CpG island (CpG). Bottom: bisulfite sequencing. Methylated (black circles) and unmethylated (white circles) CpG repeats (rows) in replicate reactions (columns). (B) qPCR of mRNAs after vehicle (CTL) or 5-azacytidine (5-Aza) treatment. (C) qPCR of mRNAs in 393P_ZEB1 cells (ZEB1) and 393P_vector cells (Vec). *P < 0.05 and ***P < 0.001. (D) qPCR of mRNAs in 393P transfectants. Vec, empty vector. (E) qPCR of mRNAs in 344SQ transfectants (scrambled [shCTL] or TET2 [shTET2] shRNAs). *P < 0.05 and **P < 0.01. (F) Schema, ITGA1 promoter constructs; SP1-, ARNT-, and RUNX1-binding sites (squares, ovals, and diamonds, respectively). Solid, WT; open, mutated. Bar graph: luciferase activity in 393P cells cotransfected with ZEB1 or empty (Vec) expression vectors and empty (pGL3) or ITGA1 promoter reporter. ***P < 0.001. (G) ITGA1 chromatin immunoprecipitation assays before (input) or after immunoprecipitation with anti-ARNT (ARNT) or IgG. Gel: PCR of ARNT-binding site (+200) or control ITGA1 promoter locus (–1k) or water. Arrows, PCR primer locations. (H) qPCR of mRNAs in 344SQ transfectants (siCTL or ARNT siRNAs). *P = 0.02 and ***P < 0.001. (I) Luciferase activity in 393P cells cotransfected with ZEB1 or empty (Vec) expression vectors, siARNT or siCTL, and an ITGA1 promoter reporter (WT or mutant [MT] ARNT-binding sites). ***P < 0.001. (J) Top: ITGA1 3′-UTR reporter. Bottom left: luciferase activity in 344SQ cotransfectants (reporter and miRs or negative control miR-NC). Bottom right: luciferase activity in 344SQ cotransfectants (miR-148a or miR-NC and ITGA1 3′-UTR reporter containing miR-148a–binding sites [WT or MT]). (K) qPCR of mRNAs in 344SQ cells transfected with miRs or empty vector (Vec). (L) Working model. Values are mean ± SD. n = 3. 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).