MALAT1 and HOTAIR Long Non-Coding RNAs Play Opposite Role in Estrogen-Mediated Transcriptional Regulation in Prostate Cancer Cells

Abstract

In the complex network of nuclear hormone receptors, the long non-coding RNAs (lncRNAs) are emerging as critical determinants of hormone action. Here we investigated the involvement of selected cancer-associated lncRNAs in Estrogen Receptor (ER) signaling. Prior studies by Chromatin Immunoprecipitation (ChIP) Sequencing showed that in prostate cancer cells ERs form a complex with the endothelial nitric oxide synthase (eNOS) and that in turn these complexes associate with chromatin in an estrogen-dependent fashion. Among these associations (peaks) we focused our attention on those proximal to the regulatory region of HOTAIR and MALAT1. These transcripts appeared regulated by estrogens and able to control ERs function by interacting with ERα/ERβ as indicated by RNA-ChIP. Further studies performed by ChIRP revealed that in unstimulated condition, HOTAIR and MALAT1 were present on pS2, hTERT and HOTAIR promoters at the ERE/eNOS peaks. Interestingly, upon treatment with17β-estradiol HOTAIR recruitment to chromatin increased significantly while that of MALAT1 was reduced, suggesting an opposite regulation and function for these lncRNAs. Similar results were obtained in cells and in an ex vivo prostate organotypic slice cultures. Overall, our data provide evidence of a crosstalk between lncRNAs, estrogens and estrogen receptors in prostate cancer with important consequences on gene expression regulation.

Figure 1. Schematic representation of the peaks of eNOS by ChIP–Seq, estrogen-dependent LncRNAs expression and eNOS/ERs recruitment in prostate and breast cancer cells.

(a,b) Integrated Genome Viewer (IGV 2.3) screenshots showing peaks of eNOS identified by ChIP–Seq at the genomic regions encoding HOTAIR and MALAT1 in two prostate cancer cell lines, LNCaP and C27IM, and in the human endothelial cell line HUVEC, in the absence (NT) or presence of 17β-estradiol (10 nM E2). Region amplified in panel e and f are indicated as red circles. (c,d) Quantification of HOTAIR and MALAT1 expression by qRT-PCR in normal HUVEC, prostate hyperplastic C17IM, BCa (MCF7, MDA-MB 361) and PCa (C27IM, LNCaP, PC3) cells in basal condition (E2 0 h) and after 3 h, 6 h and 24 h of treatment with E2. The results are plotted as fold induction (+/−E2) and represent the average of 5 independent experiments. *p < 0,05 vs E2 0 h. (e,f) Recruitment of Estrogen Receptors (ER) and eNOS, on the promoter region of HOTAIR and MALAT1 by ChIPs in the presence or absence of E2 in HUVEC, breast (MCF7) and prostate (C27IM and LNCaP) cells. The immunoprecipitations were performed using anti ERα (in HUVEC and MCF7), ERβ (in C27IM and LNCaP) and eNOS or no antibody (NoAb) as a negative control. Values are represented as Fold of Induction (+/−E2) and as mean +/−SEM of 4 independent experiments. *p < 0,05 +E2 vs −E2.

Figure 2. In vivo analysis of HOTAIR, MALAT1 or ANRIL interaction with ERs and eNOS and recruitment on estrogen–target genes promoter before and after estrogen treatment.

(a–c) In vivo HOTAIR (a), MALAT1 (b) or ANRIL (c) interaction with ERs or eNOS before and after 17β-estradiol (10 nM E2, 45 min) detected by RNA-ChIP assays. RNA-ChIPs were performed using antibodies specific to ERbeta (ERb), in PCa, primary tumor- or metastatic-derived cells, C27IM and LNCaP respectively, or to ERalpha (ERa), in MCF7, or to eNOS or in the absence of Ab (NoAb) as negative control. Immunoprecipitated RNA was recovered and analysed by qRT-PCR. Results are mean +/−SEM of 5 independent experiment. *p < 0.05 E2 vs NT. (d) In vivo interaction between ERb or eNOS with HOTAIR was detected in PCa (LNCaP) cells before and after 17β-estradiol (10 nM E2, 45 min) by ChIRP assays. ChIRP was performed using DNA antisense oligos specific for HOTAIR or LacZ as negative control. Protein-bound were recovered and analysed by Dot Blot with antibodies specific for ERb or total eNOS or phosphorylated form of eNOS (p-eNOS). White lines indicate samples spotted in noncontiguous lanes (*lower exposure). (e) Recruitment of HOTAIR and MALAT1 on the promoter region of estrogen target genes (pS2, hTERT and HOTAIR) or region without eNOS-peak (chromosome 5) by ChIRPs in the presence or absence of estrogen (10 nM E2). LacZ served as a negative control. Values are calculated % Input and data are represented as mean +/−SEM of 5 independent experiments. *p < 0.05 E2 vs NT.

Figure 3. ChIPs analysis of histone tails and methylases/demethylases recruitment modification on estrogen-target genes promoter before and after estrogen treatment and effect of interference for HOTAIR on estrogen responsiveness.

(a) Recruitment of H3K9me3, H3K9me2 and H3K27me3 on pS2, hTERT and HOTAIR promoters by ChIPs in PCa cells in the presence or absence of estrogen (E2). No Antibody (NoAb) served as negative control. Data are represented as relative enrichment in Arbitrary Unit (A.U.) of 5 independent experiments. *p < 0,05 E2 vs NT. (b) Recruitment of transcriptional suppressor CBX4, methylases SETDB1, demethylases KDM1A (LSD1) and KDM4D (JMJD2D) on promoters as in a. No Antibody (NoAb) or IgG served as negative control. Data are represented as relative enrichment in Arbitrary Unit (A.U.) of 5 independent experiments. (c) pS2 and hTERT mRNA levels quantified by qRT-PCR in LNCaP cells transfected with HOTAIR or scramble gapmers, in basal condition (NT) and after 10 nM 17β-Estradiol treatment of 1, 3 or 24 hours. The results are plotted as fold induction vs “scramble_NT” +/−SEM of 5 independent experiments. *p < 0.05 E2 vs NT.

Figure 4. Effect of interference for MALAT1 on estrogen responsiveness in PCa cells and organotypic slice cultures of Prostate Tumors and model for HOTAIR and MALAT1 function in estrogen signaling.

(a) hTERT and pS2 mRNA levels in PCa cells (C27IM, left, and LNCaP, right) quantified by qRT-PCR in PCa cells transfected with MALAT1 or scramble gapmers. The results are plotted as fold induction vs scramble +/−SEM of 5 independent experiments. *p < 0.05 vs scramble. (b) pS2 and PSA mRNA levels quantified by qRT-PCR PCa-organotypic slices cultures (OSC) transfected with MALAT1 Gapmer for 4 days. Data represent results of 7 different PCa-patients and 2 Hyperplasia (BPH) and are plotted as Fold induction vs mock (mean +/−SEM, *p < 0,05 vs mock). (c) Role of HOTAIR and MALAT1 in the ER/eNOS dependent signaling on estrogen-responsive genes in aggressive Prostate Cancer.