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. 2015 Jul 7;112(27):8326-31.
doi: 10.1073/pnas.1424220112. Epub 2015 Jun 25.

The Bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation

Chiara Pastori  1 , Philipp Kapranov  2 , Clara Penas  1 , Veronica Peschansky  1 , Claude-Henry Volmar  1 , Jann N Sarkaria  3 , Amade Bregy  4 , Ricardo Komotar  4 , Georges St Laurent  5 , Nagi G Ayad  6 , Claes Wahlestedt  6
Affiliations

The Bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation

Chiara Pastori et al. Proc Natl Acad Sci U S A. .

Abstract

Bromodomain and extraterminal (BET) domain proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule inhibitors of BET bromodomain proteins reduce expression of several oncogenes required for Glioblastoma Multiforme (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression, but mechanistic insight into their expression and regulation by BET bromodomain inhibitors remains elusive. In this study, we used Helicos single molecule sequencing to comprehensively profile lncRNAs differentially expressed in GBM, and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET bromdomain inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOX transcript antisense RNA (HOTAIR) and restored the expression of several other GBM down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET151 treatment abrogates the antiproliferative activity of the BET bromodomain inhibitor. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Bromodomain Containing 4 (BRD4) to the HOTAIR promoter, suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that modulation of lncRNA networks may, in part, mediate the antiproliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.

Keywords: BRD4; I-BET151; epigenetics; glioblastoma; long noncoding RNAs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SMS reveals hundreds of lncRNAs altered in glioblastoma. (A) The heat map shows expression of the 200 lncRNAs most up- or down-regulated in GBM compared with normal brain samples. The top 100 lncRNAs up- and down-regulated in GBM are shown in the top and bottom halves, respectively. The heat map was generated with an R package using normalization across rows (tissues). (B) A list of the most up-regulated and down-regulated lncRNAs has been selected from A for validation by RT-qPCR using the same samples of the RNA sequencing experiment. (C) Validation of HOTAIR by RT-qPCR (normalized on 18S) was performed in nine control brain samples from postmortem patients, nine control samples from lobotomy in epileptic patients, and 51 GBM specimens. HOTAIR expression is undetectable in all of the control samples and in nine samples of the glioblastoma cohort.
Fig. 2.
Fig. 2.
Knockdown of HOTAIR causes apoptosis and reduces the proliferation of glioblastoma cells in vitro and in vivo. (A) LN18 cells were transfected with siRNAs targeting HOTAIR (siHOTAIR#1 and siHOTAIR#2), and after 5 d cells were incubated with EdU, fixed, permeabilyzed, and the EdU (red) was detected by Alexa 488 at the fluorescence microscope. Nuclei were marked by Hoechst (blue) and the cells in S-phase are visible in pink in the merged pictures. (B) LN18 transfected with siControl, siHOTAIR#1, and siHOTAIR#2 were harvested 5 d after transfection, washed with binding buffer, and then incubated with Annexin V and 7-AAD to be analyzed by flow cytometry. Early apoptotic cells, Annexin Vpos/7-AADneg; late apoptotis cells, Annexin Vpos/7-AADpos. (C) The U87MG GBM cell line stably expressing luciferase was transduced with lentivirus carrying shRNA of control and shRNA targeting HOTAIR. Tumor growth was assessed every week for 2 wk after surgery [14 dpo (day post operation)]. Imaging of the animals was performed with the IVIS. (D) Tumor growth of the U87MGLuc GBM cell line implanted in the striatum of nude mice was quantified by IVIS.
Fig. 3.
Fig. 3.
BET bromodomain inhibitors alter the expression of lncRNAs in glioblastoma cell lines and patient-derived cells. (A) LN18 cells have been treated with I-BET151 (1 μM) and DMSO as a control for 24 h, and RNA was extracted. The expression of some lncRNAs present in the GBM-signature was measured by RT-qPCR. (B) LN18 and U87MG cell lines were treated with I-BET151, I-BET762, and JQ1 for 6 h. RNA was extracted to measure the expression of HOTAIR by RT-qPCR. (C and D) PDX cell lines (PDX6, PDX10, PDX12, and PDX22) and GBM cell lines (U87MG, A172, LN18, and T98G) were treated with I-BET151 for 6 h (C) and for 24 h (D) to collect RNA samples and measure HOTAIR levels by RT-qPCR. Error bars represent the SD calculated over three independent experiments. *P ≤ 0.05; **P ≤ 0.01. The P value has been calculated using the Student’s t test (A, C, and D) or one-way ANOVA with Tukey HSD test (B). In B the ANOVA test gave P ≤ 0.01 for all of the samples (I-BET151, I-BET762, and JQ1; 500 nM and 1 μM) compared with DMSO, both in LN18 and U87MG experiments.
Fig. 4.
Fig. 4.
Overexpression of HOTAIR abrogates the antiproliferative effect of I-BET151 in U87MG cells. (A) U87MG cells were transduced with HOTAIR-overexpressing lentiviruses and treated with DOX (+/–DOX) for 4 d, harvested, and the RNA was extracted to measure HOTAIR expression by RT-qPCR. (B) U87MG cells were transduced with the HOTAIR-overexpressing virus, and the infected cells were treated with IBET-151 at a concentration of 0.5 μM for 96 h. HOTAIR expression was induced by DOX for 96 h. In this representative experiment, the proliferating cells were identified with the EdU labeling kit. The automated counting was done using the Termo Scientific imaging platform (Cellomics ArrayScan VTI HCS). (C) Fluorescence microscopy images (magnification, 20×) of one representative experiment show EdU-positive cells (in red; in pink in the merged picture) over the total amount of cells (nuclei, Hoechst in blue) in U87MG treated with DMSO or I-BET151 500 nM with +/–DOX for 96 h. Error bars (B) represent the SE of six independent experiments. *P ≤ 0.05; **P ≤ 0.01. The P value was calculated with a t test (A) or one-way ANOVA Tukey HSD test (B).
Fig. 5.
Fig. 5.
BRD4 directly regulates the expression of HOTAIR. (A) LN18 cells were transfected with siRNAs (20 nM) targeting BRD2, BRD3, BRD4, and control siRNA. The RNA was extracted after 96 h to measure the mRNA levels of BRD2, BRD3, and BRD4. (B) HOTAIR expression was measured by RT-qPCR after the BRD2, BRD3, and BRD4 knockdown experiments shown in A. (C) H19 and MEG3 RNA levels were measured by RT-qPCR upon BRD4 depletion. (D) ChIP was performed with anti-BRD4 antibody and anti-IgG as negative control in LN18 cells treated for 24 h with DMSO or I-BET151 (1 μM). The chromatin obtained by the pull-down was used for qPCR to amplify the promoter region of HOTAIR located ∼1 kb upstream of the transcription start site. The graph shows the fold enrichment of BRD4 on HOTAIR’s promoter normalized on the IgG signal after normalization on the respective input signal. Error bars (A–D) represent the SD calculated over three independent biological replicates. *P ≤ 0.05; **P ≤ 0.01; ns, not significant. The P value has been calculated using the Student’s t test (A and C) or one-way ANOVA Tukey HSD test (B and D). RT-qPCR data (A–C) were analyzed with Comparative Cq Method.
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References

    1. Stupp R, et al. European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987–996. - PubMed
    1. Papavassiliou KA, Papavassiliou AG. Bromodomains: Pockets with therapeutic potential. Trends Mol Med. 2014;20(9):477–478. - PubMed
    1. Filippakopoulos P, Knapp S. Targeting bromodomains: Epigenetic readers of lysine acetylation. Nat Rev Drug Discov. 2014;13(5):337–356. - PubMed
    1. Pastori C, et al. BET bromodomain proteins are required for glioblastoma cell proliferation. Epigenetics. 2014;9(4):611–620. - PMC - PubMed
    1. Sanchez R, Zhou MM. The role of human bromodomains in chromatin biology and gene transcription. Curr Opin Drug Discov Devel. 2009;12(5):659–665. - PMC - PubMed

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