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The FOXN3-NEAT1-SIN3A repressor complex promotes progression of hormonally responsive breast cancer
Wanjin Li, … , Jing Liang, Yongfeng Shang
Wanjin Li, … , Jing Liang, Yongfeng Shang
Published August 14, 2017
Citation Information: J Clin Invest. 2017;127(9):3421-3440. https://doi.org/10.1172/JCI94233.
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Research Article Cell biology Endocrinology

The FOXN3-NEAT1-SIN3A repressor complex promotes progression of hormonally responsive breast cancer

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Abstract

The pathophysiological function of the forkhead transcription factor FOXN3 remains to be explored. Here we report that FOXN3 is a transcriptional repressor that is physically associated with the SIN3A repressor complex in estrogen receptor–positive (ER+) cells. RNA immunoprecipitation–coupled high-throughput sequencing identified that NEAT1, an estrogen-inducible long noncoding RNA, is required for FOXN3 interactions with the SIN3A complex. ChIP-Seq and deep sequencing of RNA genomic targets revealed that the FOXN3-NEAT1-SIN3A complex represses genes including GATA3 that are critically involved in epithelial-to-mesenchymal transition (EMT). We demonstrated that the FOXN3-NEAT1-SIN3A complex promotes EMT and invasion of breast cancer cells in vitro as well as dissemination and metastasis of breast cancer in vivo. Interestingly, the FOXN3-NEAT1-SIN3A complex transrepresses ER itself, forming a negative-feedback loop in transcription regulation. Elevation of both FOXN3 and NEAT1 expression during breast cancer progression corresponded to diminished GATA3 expression, and high levels of FOXN3 and NEAT1 strongly correlated with higher histological grades and poor prognosis. Our experiments uncovered that NEAT1 is a facultative component of the SIN3A complex, shedding light on the mechanistic actions of NEAT1 and the SIN3A complex. Further, our study identified the ERα-NEAT1-FOXN3/NEAT1/SIN3A-GATA3 axis that is implicated in breast cancer metastasis, providing a mechanistic insight into the pathophysiological function of FOXN3.

Authors

Wanjin Li, Zihan Zhang, Xinhua Liu, Xiao Cheng, Yi Zhang, Xiao Han, Yu Zhang, Shumeng Liu, Jianguo Yang, Bosen Xu, Lin He, Luyang Sun, Jing Liang, Yongfeng Shang

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Figure 4

The assembly of the FOXN3-NEAT1-SIN3A complex on transcriptional targets.

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The assembly of the FOXN3-NEAT1-SIN3A complex on transcriptional targets...
(A) MCF-7 cells were infected with lentiviruses carrying the indicated shRNAs for qChIP analysis on the selected promoters using antibodies against the indicated proteins/histone modification. Error bars represent mean ± SD for triplicate experiments. Knockdown efficiency was verified by qPCR or Western blotting. (B) qPCR and Western blotting analyses of the expression of GATA3 and TJP1 (ZO1) in MCF-7 cells transfected with the indicated siRNAs. In A and B, error bars represent mean ± SD for triplicate experiments (*P < 0.05, **P < 0.01; 2-way ANOVA). (C) RIP-qPCR analysis of FOXN3 RIP, SIN3A RIP, or SAP18 RIP for the enrichment of NEAT1 in MCF-7 cells infected with lentiviruses carrying the indicated shRNAs. (D) Prediction of RNA-binding residues (RBRs) in SAP18 by RBRDetector. The predicted RBRs (red) in the 3D structure of SAP18 are visualized by Jmol (www.jmol.org/). (E) Coimmunoprecipitation assays in MCF-7 cells with antibodies against SAP18 followed by IB with antibodies against the indicated proteins. (F) Schematic diagrams of GST/His-SAP18, GST/His-SAP18Δ17–29, and GST/His-SAP18Δ139–149. The results of RNA pull-down assays with NEAT1 fragment (704–903 nt) and GST/His-SAP18, GST/His-SAP18Δ17–29, or GST/His-SAP18Δ139–149 are shown. (G) RIP-qPCR analysis of FLAG RIP for the enrichment of NEAT1 in MCF-7 cells transfected with FLAG-SAP18, FLAG-SAP18Δ17–29, or FLAG-SAP18Δ139–149 (left). RIP-qPCR analysis of SIN3A RIP for the enrichment of NEAT1 in a FOXN3-knockout MCF-7 cell line established by the CRISPR/Cas9 system (right). In C and G, error bars represent mean ± SD for triplicate experiments (*P < 0.05, **P < 0.01; 1-way ANOVA).
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