The long non-coding RNA HOTAIR is transcriptionally activated by HOXA9 and is an independent prognostic marker in patients with malignant glioma

Abstract

The lncRNA HOTAIR has been implicated in several human cancers. Here, we evaluated the molecular alterations and upstream regulatory mechanisms of HOTAIR in glioma, the most common primary brain tumors, and its clinical relevance. HOTAIR gene expression, methylation, copy-number and prognostic value were investigated in human gliomas integrating data from online datasets and our cohorts. High levels of HOTAIR were associated with higher grades of glioma, particularly IDH wild-type cases. Mechanistically, HOTAIR was overexpressed in a gene dosage-independent manner, while DNA methylation levels of particular CpGs in HOTAIR locus were associated with HOTAIR expression levels in GBM clinical specimens and cell lines. Concordantly, the demethylating agent 5-Aza-2'-deoxycytidine affected HOTAIR transcriptional levels in a cell line-dependent manner. Importantly, HOTAIR was frequently co-expressed with HOXA9 in high-grade gliomas from TCGA, Oncomine, and our Portuguese and French datasets. Integrated in silico analyses, chromatin immunoprecipitation, and qPCR data showed that HOXA9 binds directly to the promoter of HOTAIR. Clinically, GBM patients with high HOTAIR expression had a significantly reduced overall survival, independently of other prognostic variables. In summary, this work reveals HOXA9 as a novel direct regulator of HOTAIR, and establishes HOTAIR as an independent prognostic marker, providing new therapeutic opportunities to treat this highly aggressive cancer.

Keywords: HOTAIR; HOXA9; glioblastoma; glioma; prognosis.

Figure 1. Molecular characterization of HOTAIR in gliomas

(A) Expression levels of HOTAIR in 424 gliomas, stratified according to WHO grade, IDH and 1p/19q codeletion statuses (1 IDH-wt, 3 IDH-mut, and 3 IDH-mut and 1p/19q codeleted grade II gliomas; 1 IDH-wt, 12 IDH-mut, and 6 IDH-mut and 1p/19q codeleted grade III gliomas; 368 IDH-wt and 30 IDH-mut glioblastomas (GBM); and 10 unmatched normal brains from the TCGA microarray data). HOTAIR is highly expressed (TCGA data “level 3” values ≥ 0) in 34.2% (n = 126) of IDH-wt GBM samples and in 1 IDH-mut GBM (3%) and 1 IDH-wt grade II glioma (100%). (B) HOTAIR gene copy number status in 270 GBMs (250 IDH-wt and 20 IDH-mut) from TCGA. HOTAIR is amplified (Log₂ Copy Number Tumor/Normal ≥ 0.5) in 0.8% (n = 2; green dots), and deleted (Log₂ Copy Number Tumor/Normal ≤ –0.5) in 3.2% (n = 8; red dots) of IDH-wt GBM samples. Red dashed lines represent the normal copy number interval. (C) Heatmap representations of DNA methylation levels (TCGA β-values) of the chromosomal region encompassing HOTAIR and the 2 closest genes (HOXC12 and HOXC11) in 74 GBMs (70 IDH-wt and 4 IDH-mut) from TCGA. A total of 56 methylation probes (vertical blue bars) were assessed. CpG islands > 300bp are represented in green. ^*indicate probes whose methylation indexes are significantly inversely correlated with HOTAIR expression levels (probes cg00079219, cg18824990 and cg24895871). The color code (grades of red color corresponding to different methylation indexes) is shown below the heatmap. Each column corresponds to a probe and each row to a patient. (D) Correlation graphs between HOTAIR expression levels (TCGA “level 3” value) and DNA methylation indexes (TCGA β-values) in 70 GBM samples. Only probes whose methylation values are significantly inversely correlated with HOTAIR expression are shown (cg00079219, cg18824990 and cg24895871; marked with ^*in C). (E–F) Glioma cell lines were treated with 5 µM 5-Aza for 72 hours, upon which promoter methylation status (E) and HOTAIR expression levels (F) were evaluated. 5-Aza treatment promoted HOTAIR promoter demethylation (E) that associated with its increased expression in a cell line-dependent manner (F). qPCR levels were normalized to the expression of HPRT1 and are presented as fold-changes; methylation-specific PCR was controlled by blood DNA (NB) untreated (Control) or in vitro methylated (Met). No detectable HOTAIR expression was found for U87 (untreated or 5-Aza-treated). The results are representative of at least 2 replicates (mean ± SD). ^*p < 0.05; NB - DNA from normal blood.

Figure 2. HOXA9 transcriptionally activates HOTAIR via direct interaction with its promoter region

(A) MatInspector representation of specific binding sites for HOXA9 matrix in HOTAIR promoter region. Each matrix match is represented by a half round symbol and each color symbolizes a matrix family. Pink, blue and violet colors represent the matrixes HOXA9.02 (matrix sim = 0.899, sequence: ctgtgacaTAAAattgg and family ABDB), PBX-HOXA9.01 (matrix sim = 0.849, sequence: gaggTGGTttatgagct and family HOXC) and MEIS1B-HOXA9.01 (matrix sim = 0.837, sequence: TGCCaattttatgtc and family HOXH), respectively. Basepairs in italic appear in a position with a high conservation profile in the matrix (ci-value > 60). Basepairs in capital letters represent the core sequence used by the program. Matches represented on the top of the sequence line were found on the positive strand, while below the sequence line reside matches found on the negative strand. The red arrows represent HOTAIR putative transcription start sites (TSS). (B–C) HOTAIR and HOXA9 expression were evaluated by reverse-transcriptase PCR (B) and quantitative PCR (C) in a panel of adult and pediatric glioma-derived cell lines, and in the GBM-L18 primary GBM-derived cell culture. GBM cell line U87MG-MSCV does not present detectable levels of endogenous HOTAIR expression, which were significantly increased upon retrovirally-mediated HOXA9 overexpression (U87MG-HOXA9). White and grey bars represent HOTAIR and HOXA9 expressions, respectively. qPCR levels were normalized to the expression of HPRT1. The results are representative of triplicates (mean ± SD). ^*p = 0.029; ^**p = 0.0088. (D–E) The putative binding of HOXA9 protein to the promoter region of HOTAIR was assessed by chromatin immunoprecipitation (ChIP) analysis followed by quantitative PCR in U251 cells (D), and U87MG-HOXA9 and their HOXA9-negative counterparts (E). IgG was used as negative control for the ChIP. Chromatin immunoprecipitated with an anti-HOXA9 antibody shows direct binding of HOXA9 to the HOTAIR promoter. Relative enrichment is normalized to input DNA (not subjected to immunoprecipitation) and to the IgG background signal (D), whereas fold change occupancy is normalized to input, IgG and HOXA9-negative cells (E), from three independent experiments (mean ± SD). (D) ^***p = 0.0002; (E) ^*p = 0.0148.

Figure 3. Expression levels of HOTAIR and HOXA9 are significantly correlated in GBM clinical specimens

(A–C) Correlation graphs of HOTAIR and HOXA9 expression levels in GBMs from the Portuguese dataset (A), and IDH-wt GBMs from the French dataset (B), and IDH-wt GBMs the TCGA dataset (microarray data) (C), showing statistically significant correlations between their expressions in all datasets. (A) Pearson’s r = 0.655, p < 0.0001; (B) Pearson’s r = 0.649; p = 0.002; (C) Pearson’s r = 0.494; p < 0.0001.

Figure 4. High levels of HOTAIR expression are significantly associated with shorter survival in GBM patients

Kaplan–Meier overall survival curves of HOTAIR prognostic value in (A) 554 GBM patients from TCGA, (B) 367 IDH-wt GBM patients from TCGA and (C) 67 GBM patients from REMBRANDT dataset, showing patients whose tumors present high HOTAIR expression have a statistically significant shorter overall survival compared to those with HOTAIR-low tumors, in both independent datasets (A, p = 0.026; B, p = 0.032; C, p = 0.005; Log-rank tests).