Lncrna pvt1 links estrogen receptor alpha and the polycomb repressive complex 2 in suppression of pro-apoptotic genes in hormone-responsive breast cancer

ABSTRACT RNA-based therapeutics highlighted novel approaches to target either coding or noncoding molecules for multiple diseases treatment. In breast cancer (BC), a multitude of deregulated

long noncoding RNAs (lncRNAs) have been identified as potential therapeutic targets also in the context of antiestrogen resistance, and the RNA binding activity of the estrogen receptor α

(ERα) points additional potential candidates to interfere with estrogenic signaling. A set of lncRNAs was selected among ERα-associated RNAs in BC cell nuclei due to their roles in processes

such as transcriptional regulation and epigenetic chromatin modifications. Native immunoprecipitation of nuclear ERα-interacting RNAs coupled to NGS (RIP-Seq) was performed in MCF-7 cells,

leading to the identification of essential lncRNAs interacting with the receptor in multi-molecular regulatory complexes. Among these, PVT1, FGD5-AS1 and EPB41L4A-AS1 were selected for

further investigation. Functional assays and transcriptome analysis following lncRNA knock-down indicated PVT1 as the master modulator of some of the most relevant BC hallmarks, such as cell

proliferation, apoptosis, migration and response to hypoxia. In addition, targeted experiments identified PVT1 as a key factor in the composition of PRC2-ERα network involved in

downregulation of tumor suppressor genes, including BTG2. SIMILAR CONTENT BEING VIEWED BY OTHERS LNC-DC PROMOTES ESTROGEN INDEPENDENT GROWTH AND TAMOXIFEN RESISTANCE IN BREAST CANCER Article

Open access 25 October 2021 LINC00355 REGULATES P27KIP EXPRESSION BY BINDING TO MENIN TO INDUCE PROLIFERATION IN LATE-STAGE RELAPSE BREAST CANCER Article Open access 13 April 2022 LNCRNA

LY6E-DT AND ITS ENCODED METASTATIC-RELATED PROTEIN PLAY ONCOGENIC ROLES VIA DIFFERENT PATHWAYS AND PROMOTE BREAST CANCER PROGRESSION Article 19 December 2023 INTRODUCTION The increasing

knowledge of the broad spectrum of functional roles exerted by different classes of RNAs allowed, in recent years, the advancement of RNA therapeutics from a hypothetical concept to clinical

reality, thus providing a novel way to target coding as well as noncoding RNAs (ncRNAs) for the customized treatment of multiple diseases. Indeed, deregulated gene expression may be

controlled, either permanently or transiently, by directly targeting specific RNAs or proteins with the aid of small molecules endowed with therapeutic action. Therefore, RNA-binding

proteins (RBPs) and their connected molecules play crucial roles in this context [1]. RBPs form ribonucleoprotein complexes that specifically modulate gene expression through alternative

splicing, RNA decay, translocation or translation [2]. Moreover, their aberrant activity was described to contribute to cancerous transformation and/or resistance to therapies [3]. In this

view, the disruption of RNA–protein networks represents a promising avenue for cancer therapeutics, especially in the absence of “druggable” molecules, or to counteract the occurrence of

drug resistance [1]. In breast cancer (BC), the discovery that the estrogen receptor α (ERα), the main hallmark of luminal-like hormone-responsive BC subtype, acts as a non-canonical RBP,

brought out a new oncogenic signature under the control of this factor [4]. It has been well established that, upon estrogen stimulation, ERα is recruited, together with a host of

transcriptional coregulators, onto specific target sites within the chromatin for modulation of target genes expression. Indeed, ERα can interact with epigenetic readers, writers and erasers

within multimolecular complexes able to regulate gene expression in a combinatorial manner [5, 6]. More recent evidences demonstrated also the ability of ERα to bind several RNA species,

among which a crucial functional role is likely to be exerted by ncRNAs [4]. Furthermore, a multitude of deregulated ncRNAs has been described as novel potential “druggable” targets for BC

treatment [7, 8]. Among these, the role of long ncRNAs (lncRNAs) have been investigated for their involvement in both physiological and pathological processes [9,10,11]. These are RNA

molecules more than 200 nucleotides long that are transcribed by RNA polymerase II, 5’ capped, polyadenylated and, in most cases, lack an open reading frame (ORF) and therefore

protein-coding ability [11, 12]. Recently, lncRNAs received growing interest for their involvement in multiple cellular processes and for their functional roles in epigenetics,

transcriptional and post-transcriptional events, making them attractive target candidates for improvement of cancer treatment efficiency [13, 14]. The blockade of the estrogenic signaling

through ERα inhibition using selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs) or aromatase inhibitors (AIs) represented, for many years, the

first line approach for hormone-based therapy in patients suffering with ERα-positive BC [15]. The main problem, occurring in ~30% of the cases, is the acquisition of resistance to hormone

therapies, nowadays representing the main cause of death for these patients [16]. Despite recent advancements in treatments based, for example, on new targeted and chemotherapies, BC remains

a significant threat for women’s health, and this points to the urgent need to find novel targets to overcome this hindrance [2, 7]. The role of ERα as RBP is still not well defined and

understood, although representing a new central piece of the puzzle. In this study, we investigated whether the receptor would be enrolled in modulating the expression of target genes,

important for tumor sustainment and neoplastic transformation, through the association, within the nuclear compartment, with specific lncRNAs. Firstly, ERα-interacting lncRNAs were

identified by using RNA-immunoprecipitation coupled to sequencing (RIP-Seq), then, once identified the most informative interacting RNAs, we evaluated the functional effects of their

silencing on cell proliferation, induction of apoptotic cell death and modulation of the estrogenic signaling after RNA knock-down with antisense-oligonucleotides (ASOs). Out of three

lncRNAs selected among those previously demonstrated to be essential for BC cell growth [17], PVT1 emerged as the most significantly associated with BC prognosis, since its overexpression

correlated with worse overall survival in TCGA and TARGET Pan Cancer patients. Moreover, it emerged as a key component of the estrogenic signaling acting as a bridge factor between Polycomb

Repressive Complex 2 (PRC2) activity and ERα transcriptional modulation. In particular, the experimental results identified PVT1 as a core molecule in the composition of a transcriptional

repressive complex allowing the functional cooperation of PRC2 and ERα through PVT1-mediated association of EZH2 and the receptor with the transcription unit of tumor suppressive genes such

as BTG2 (BTG family member 2/NGF-inducible anti-proliferative protein PC3). Targeting PVT1 by means of ASO-based silencing may thus represent an alternative way to clinically interfere with

estrogenic signaling and activate the apoptotic cascade in BC. MATERIALS AND METHODS For detailed methods, see Supplementary Material. RNA IMMUNOPRECIPITATION For ERα-associated RNA

immunoprecipitation (RIP), 7 μg of anti-ERα or anti-IgG Isotype Control were conjugated overnight at 4 °C with 100 μl of Dynabeads M-280 Sheep AntiRabbit IgG (Thermo Fisher). Cells were

washed twice with cold PBS supplemented with 0.1% of EDTA (500 mM), harvested by scraping and collected in a tube. After a centrifugation at 3000 rpm for 5 min at 4 °C, the nuclear fraction

was extracted by resuspending the pellet in nuclear isolation Buffer (NIB) (1.28 M Sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2 and 4% Triton X-100) supplemented with 100 U/ml RNAse inhibitor

(RiboLock RNase Inhibitor, Invitrogen) and proteinase inhibitors (1 mM PMSF and 1× PIC). Samples were incubated on ice for 20 min and then centrifuged at 2500 × _g_ at 4 °C for 15 min. Once

removed the supernatant, nuclear pellets were resuspended in 400 μl of RIP Buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT e 0.5% NP40) supplemented with 100 U/ml RNAse and

proteinase (1 mM PMSF and 1x PIC) inhibitors. Cell nuclei were sonicated for 10 cycles (15” ON and 15” OFF) using Bioruptor (Diagenode, Denville, New Jersey, USA) and subsequently

centrifuged at 13,000 rpm for 10 min at 4 °C. The resulting supernatant represented the nuclear protein extract whose concentration was measured with Bradford assay. Then, 2.5 mg of nuclear

extract was incubated at 4 °C for 2 h with the conjugated beads/antibodies. After the binding, beads were washed 3 times with RIP Buffer in rotation for 5 min at 4 °C, and twice quickly.

Once discarded all the supernatant, 1 ml of TRIzolTM (Life Technologies, Thermo Fisher) was added directly to the beads and RNA extraction was performed according to the manufacturer’s

guidelines. RNA SEQUENCING Libraries preparation for transcriptome profiling was performed by using the TruSeq Stranded Total RNA Library Prep Gold (Cat. 20020599, Illumina, San Diego,

California, USA) for RIP-Seq and silencing experiments, while the lllumina Stranded Total RNA prep Ligation with Ribo-Zero Plus kit (Cat. 20040529, Illumina) was employed for Nascent-Seq,

according to manufacturers’ guidelines. RIP-Seq libraries were sequenced on NextSeq 500 (Illumina) using 2 × 75 bp paired end mode. Total RNA-Seq and Nascent-RNA Seq libraries were sequenced

on Novaseq 6000 platform (Illumina) using 2 × 100 bp paired end mode. CHORIOALLANTOIC MEMBRANE (CAM) ASSAY CAM assay was performed as previously described by Bianco et al. [18] with minor

modifications. Briefly, fertilized chicken eggs were equilibrated at 37 °C in a humidified incubator from the 1st until the 8th day of gestation. Then an artificial air sac was designed by

piercing the eggshell with a needle into the air sac and near the allantoic vein. Subsequently, a vacuum was applied to the air sac hole to detach CAM from the eggshell and a perforation of

1 cm2, near the allantoic vein, was performed to expose the CAM. MCF-7 cells, 6 h post ASO transfection, were inoculated at 5 × 106 cells/each CAM. In details, transfected cells were

detached from the culture dish, counted and suspended in 40 μl of a 1:1 mixture composed of growth medium and BME (Cat. #3533-005-02, R&D Systems, Minneapolis, USA). Cells were

inoculated in an 8 mm sterile Teflon ring placed on the membrane to prevent cells spreading. Embryos were incubated for 4 days after which tumors were excised from the site of inoculation.

Surface measurements were performed by averaging the areas (width*width) of each tumor. DATA ANALYSIS Nascent, Total RNA-Seq and RIP-Seq data analysis was performed as follows: Fastq were

generated from bcl files using bcl2fastq (Illumina v2.20.0.422), while the quality check was assessed using FastQC (v0.11.9) [19]. The adapters were removed using cutadapt (v 3.3) [20] and

the resulted fastq were aligned on human genome (hg38) using STAR [21] (v 2.7.8a) with assembly of GENCODE v37 as GTF file. The raw counts were generated using featurecounts (v2.0.1) [22].

Differential expression analysis and the normalized counts were produced using DESeq2 (v 1.38.3) on R (4.2.2). Transcripts were considered differentially expressed if they showed |FC| ≥ 1.5

and adjusted _p_-value ≤ 0.05 or enriched if they showed |FC| > 1. Volcano plots were generated using EnhancedVolcano (v 1.16.0) while Gene Ontology circos with GOplot (v 1.0.2). All

sequencing data are available in ArrayExpress database with the following accession numbers: E-MTAB-14147, E-MTAB-14148, E-MTAB-14149. Furthermore, boxplots with TGCA data were produced on

GEPIA2 [23] while TCGA and TARGET Pan Cancer BRCA data available on UCSC Xena tool were used for the survival plot [24]. All the barplots, the heatmaps and the related statistics were made

using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. All other plots and statistics were produced on R with an alpha value set for

_p_ < 0.05. Predictions of lncRNAs interaction with DNA were calculated with the online version of LongTarget (www.gaemons.net) [25] using the default parameters and the entire hg38 for

genome scale prediction. Sequence Searcher [26] was used to identify the presence of ERE or imperfect ERE (allowing a maximum of 2 mismatches) as explained by Driscoll et al. [27]. Chip and

CUT&Tag data, for EZH2 and H3K27me3 respectively, were downloaded from GEODataset (GSE201262) produced by Tian et al. [28], while ERα Clip-Seq was obtained from supplementary material of

Xu et al. [4] and converted to hg38 using liftOver from UCSC [29]. CHIA-PET long-range chromatin interactions data was downloaded from GEODataset (GSE176821). ERα Chip-Seq was re-analyzed

from data previously published by Nassa et al. [30] and converted to hg38 using liftOver from UCSC. RESULTS IDENTIFICATION OF ERΑ-ASSOCIATED RNAS IN BC CELL NUCLEI Previous work from our

group, by applying Tandem Affinity Purification (TAP) coupled to mass spectrometry (MS) followed by in vitro RNAse digestion, demonstrated quantitative changes in ERα association with a

large subset of its nuclear interactors in MCF-7 BC cell nuclei [31]. These ERα molecular partners include enzymes and transcription regulators mainly demonstrated to be part of

chromatin-associated multiprotein complexes required for ERα activity [31]. Among others, we noticed a reduction of ERα co-immunoprecipitation with epigenetic modulator proteins such as the

Bromodomain Adjacent To Zinc Finger Domain 1B (BAZ1B), the scaffold protein menin 1 (MEN1) and the Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), previously identified to

be involved with ERα in controlling key BC hallmarks (Fig. 1A) [32, 33]. This strongly suggested the potential involvement of RNA(s) in ERα-associated nuclear multimolecular complexes

assembly, which may act as functional mediators of the receptor transcriptional activity in the modulation of specific target genes expression. To investigate this hypothesis, a native ERα

immunoprecipitation coupled to RNA sequencing (RIP-Seq) was performed in nuclear extracts from exponentially growing MCF-7 cells, leading to the identification of 2212 ERα-interacting RNAs

(Fig. 1B, Supplementary Table S1). Among these, 2071 (93.6%) are protein coding while 141 (6.4%) are noncoding RNAs (Fig. 1B) and in them, lncRNAs (107) were selected for our investigation

(Fig. 1C). Considering the growing interest in lncRNAs in cancer biology, we directed our focus on those found to be essential for BC growth and/or survival (defined as fitness lncRNAs) by

Liu et al. [17], that identified, through CRISPRi screening, fitness lncRNAs in multiple tumor cell lines including some of the more representative BC ones. The comparison between this

dataset and the generated list of ERα-interacting ncRNAs identified five of them as essential in ERα-positive BC cells (Fig. 1D, Supplementary Table S2). Among these, FGD5-AS1, EPB41L4A-AS1

and PVT1 (Fig. 1D) were selected for further investigation, based on published literature, fitness score and the predicted biological meaning of their functional cooperation with ERα [17,

34,35,36,37,38]. The interaction between ERα and these three lncRNAs was confirmed in MCF-7 cells by RIP coupled to RT-qPCR using as negative control the lncRNA PRNCR1, which was not found

here to bind ERα (Fig. 1E, Supplementary Table S1). This result was validated also with a different primers set and in both MCF-7 and T-47D cells (Supplementary Fig. S1A and B). FUNCTIONAL

CHARACTERIZATION OF ERΑ-INTERACTING LNCRNAS The expression levels of the selected lncRNAs were analyzed in BC RNA-Seq data from TCGA (Fig. 2A) and measured by RT-qPCR in ERα + BC cell lines

(Fig. 2B). Results showed that FGD5-AS1 expression level is not significantly different in normal vs cancer tissues and in luminal-like (MCF-7, T-47D and ZR-75-1), triple negative

(MDA-MB-231 and Hs-578T) BC and immortalized epithelial (MCF-10A) breast cell lines. On the contrary, EPB41L4A-AS1, previously described as a tumor suppressor [34], resulted downregulated in

tumor tissues and in BC cells, compared to normal ones, while PVT1, previously described as a pan-oncogene [35,36,37], resulted overexpressed in both. Furthermore, in native conditions, the

three RNAs displayed a different localization within the cell compartments. Specifically, EPB41L4A-AS1 and FGD5-AS1 appear more abundant in the cytoplasm whereas PVT1 prevails in the

nuclear compartment (Fig. 2C). In order to investigate their relationship with the estrogenic signaling, lncRNAs expression levels were evaluated in hormone-deprived MCF-7 cells stimulated

with a mitogenic dose of estradiol (E2 10 nM) for 24 h (Fig. 2D). Interestingly, the results obtained showed a significant decrease of EPB41L4A-AS1 and an increase in PVT1 expression level

upon hormonal treatment, in line with the presence of ER-binding motifs (EREs) within its transcription unit (Fig. 2D, Supplementary Fig. S2A). To further confirm that the observed

modulation might be linked to ERα, the expression levels of the three lncRNAs were evaluated in MCF-7 cells following siRNA-mediated ERα gene (ESR1) knock-down. Results showed that ERα kd

resulted in a decrease of both PVT1 and FGD5-AS1 levels. On the other hand, EPB41L4A-AS1 expression was unaffected by receptor downregulation, indicating that this lncRNA, although

responding to estrogen stimulation, does not appear to be a direct ER target (Fig. 2E). Chromatin associated ERα-RNA immunoprecipitation (CARIP) coupled to RT-qPCR, confirmed an enrichment

of the three molecules investigated (Fig. 2F). This indicated that, although in the presence of a different nuclear concentration among them, since PVT1 results highly expressed in this

compartment compared to the others, all three lncRNAs result associated with chromatin, suggesting that all exert a functional role in the genome of these BC cells. Furthermore, given the

knowledge that different lncRNAs are likely to act together by assembling in multi-modular complexes for gene expression modulation [14], we first pursued to identify putative lncRNA-DNA

binding motifs within MCF-7 cells genome and then compared common sites with ERα binding sites previously identified in the same experimental conditions [31]. This allowed the identification

of a set of commonly occupied genes, whose expression was predicted to be regulated by a complex involving both ERα and these lncRNAs (Supplementary Fig. S2B, Supplementary Table S3), thus

suggesting an involvement of these molecules in estrogen-responsive regulation of gene expression in luminal-like cells. A functional characterization of FGD5-AS1, EPB41L4A-AS1 and PVT1 was

then performed through silencing experiments, to evaluate whether this would affect gene transcription and cell proliferation rate. Specifically, despite the three molecules were located in

both cytoplasmic and nuclear compartments, isoform-specific antisense oligonucleotides (ASOs) were employed for all the experiments of this study, mainly because of their better targeting in

the nucleus where the estrogen-mediated transcriptional regulation of gene expression takes place (Supplementary Fig. S3A) [38]. Transcriptome profiling following FGD5-AS1, EPB41L4A-AS1 and

PVT1 knock-down identified, respectively, 1445, 818 and 1128 differentially expressed genes, compared to the effects of a scramble oligonucleotide used as negative control (Fig. 3A left,

Supplementary Table S4). Among these, 304 were in common between all conditions and 86 hold at least one ERα binding site as defined by Nassa et al. [30] (Supplementary Table S5), suggesting

a regulatory mechanism in which these molecules might be involved (Fig. 3A right). Furthermore, the involvement of the latter subset of deregulated genes in pathway crucial for BC

progression and invasion (Fig. 3B) corroborates the possibility to consider them as putative therapeutic targets against this tumor [39, 40]. Moreover, lncRNA silencing resulted in reduction

of cell proliferation rate, observed only in estrogen-responsive, ERα-positive BC cell lines MCF-7, T-47D and ZR-75-1 but not in ERα-negative MDA-MB-231 (TNBC) and MCF-10A (mammary

epithelial) cells (Fig. 3C, Supplementary Fig. S3A–E). The observed functional effect was also accompanied by ERα protein reduction strongly detected particularly after silencing of PVT1

(Fig. 3D). Contrarily to what expected, based on screen score shown in Fig. 1D and on what already observed by others [41], we experienced a neglectable effect on the proliferation rate of

triple negative MDA-MB-231 cells following PVT1 silencing, probably due to the isoform retrieved among ERα-enriched RNAs that was specifically targeted. Finally, the influence of targeted

lncRNA silencing on the transcriptional activity of ERα was evaluated with a trans-activation assay in hormone-deprived MCF-7 cells before and after 24 h E2 stimulation (10 nM). This

resulted in a marked decrease of ERα trans-activating efficiency, once again more pronounced following PVT1 silencing (Fig. 3E), suggesting this as a possible master co-regulator of

ERα-mediated transcriptional activity. On the contrary, the silencing of EPB41L4A-AS1 determined an increase of ERα activity (Fig. 3E), correlating with the demonstrated oncosuppressive role

of this lncRNA [36, 41]. THE LNCRNA PVT1 AFFECTS MULTIPLE BC HALLMARKS The comparison between nuclear ERα RIP-Seq and cytoplasmatic ERα CLIP-Seq datasets [4] allowed the identification of

17 shared lncRNAs (Supplementary Table S6), including PVT1 and FGD5-AS1, a result demonstrating their direct association with the receptor also in the cytoplasm of MCF-7 BC cell line. In

order to evaluate whether this interaction would be mediated by the ERα RNA binding domain (RBD) identified by Xu et al. [4], the same constructs generated and kindly provided by these

authors were used here by applying RIP coupled to RT-qPCR in a system composed of Hs-578T (ERα-negative BC cell line) stably expressing full-length-3xFlag-ESR1 (Flag-ERα) with or without

mutation in its RBD. This model was chosen to avoid misleading interpretation due to hetero-dimerization between endogenous and exogenous receptor in ERα-positive cell lines. The RIP results

showed a reduction of lncRNAs enrichment after nuclear flagged ERα immunoprecipitation in cells expressing mutant ERα RBD and this was significant in particular for PVT1 (Fig. 4A). We thus

decided to further investigate the role of this lncRNA in regulation of estrogen-dependent gene expression, supported by the evidence that its high expression correlated with worse overall

survival in BC patients (Fig. 4B). To this purpose, we went back to evaluate those genes whose expression was specifically affected by PVT1 silencing. The comparison between whole

transcriptome data at 72 h of PVT1 silencing and nascent RNA profiles at 48 h after silencing identified 391 (103 up and 288 down) deregulated genes in common between the two conditions that

were thus likely to be affected already in the early phases of RNA synthesis (Fig. 4C, Supplementary Table S7). These were mainly involved in pathways significant for BC progression and

survival such those related to migration, response to hypoxia and cell death (Fig. 4D). Of note, the observation of HIF-1α protein reduction corroborated the hypothesis of a deregulation of

hypoxia signaling by PVT1, which has been already demonstrated to have a key role in HIF-1α stabilization in nasopharyngeal carcinoma [42]. Likewise, the reduction of the antiapoptotic

protein Bcl-xL and the increase of the pro-apoptotic BAX mRNA asserted the activation of cell death response (Fig. 4E, F). In addition, a decrease in BC cell motility was also observed in

the wound healing assay following PVT1 knock-down (Fig. 4G). To further confirm the in vitro results, we employed the chicken chorioallantoic membrane (CAM) as extraembryonic in vivo model

[18, 43]. On day 8th of embryo development, to achieve PVT1 silencing MCF-7 cells were in-plate transfected for 6 h, to allow the maximal effect of liposomes, as detailed in methods section,

then they were detached and 5 ×106 cells were inoculated into the CAMs; the tumor formation was then evaluated after 4 days. The treatment reduced both the weight and the area of the tumors

(Fig. 5A). Furthermore, PVT1 silencing modulated apoptosis, as demonstrated by the increase of BAX mRNA and decrease of Bcl-xL protein levels, and hypoxia through the reduction of HIF-1α

protein (Fig. 5B, C). The results were globally strengthened by a parallel reduction of ERα protein level (Fig. 5C) and confirmed after immunohistochemistry staining of FFPE samples obtained

in parallel from the same tumors (Fig. 5D). THE COOPERATION BETWEEN PRC2 COMPLEX, PVT1 AND ERΑ IS RESPONSIBLE FOR MODULATION OF TARGET GENES EXPRESSION Transcriptome analysis following PVT1

silencing displayed, among deregulated ones, the pathway related to PRC2 activity (Fig. 4D). Since it was previously described the association between PVT1 and the PRC2-subunit EZH2 [44,

45] and given that the interaction between EZH2 and ERα was demonstrated to be mediated by RNAs (Fig. 1A), we hypothesized a nuclear cooperating machinery involving ERα, EZH2 and the lncRNA

PVT1. Based on the knowledge that EZH2-mediated trimethylation of H3K27 is a marker of gene silencing, we speculated a repression machinery involving these three factors. For this reason, we

searched among genes downregulated following estrogen stimulation and those upregulated following PVT1 silencing, identifying 124 common targets involved in BC-related hallmarks such as

hypoxia and oxidative stress response, p53 and K-Ras pathway (Supplementary Fig. S4A-B, Supplementary Table S8). Among them, 64 displayed at least one ERα binding site in their transcription

unit, 41 of which holding a perfect/imperfect ERE and 15 an EZH2 binding site (Fig. 6A). We then investigated whether PVT1 could affect the level of the main protein components of the PRC2

complex (JARID2, EZH2, SUZ12 and PHF1), but no differences were observed before and after its silencing (Supplementary Fig. S4C). Interestingly, a strong reduction in H3K27Me3, the histone

modification specifically induced by EZH2 was observed following PVT1 knock-down (Fig. 6B), indicating that indeed, as predicted by functional analysis, this lncRNA is likely to modulate

PRC2 activity by affecting this histone mark. Among the genes commonly occupied by ERα and EZH2, the tumor suppressor BTG2 was selected for validation, based on its crucial role as cancer

antagonist [46, 47]. Analysis of publicly available data from CUT&Tag experiments [28], performed in cell models and experimental conditions comparable to ours, showed an abundance of

H3K27Me3 markers both upstream and downstream BTG2 gene, which is more pronounced in correspondence of the ERα-bound imperfect ERE motif (Fig. 6C). Furthermore, CHIA-PET experiments

(GSE176821) focusing on the long-range chromatin interactions show a good interaction score (79) between BTG2 and the ERE motif in the investigated genomic region, despite the presence of

another ERα binding site in BTG2 upstream region. The hypothesized hormone depending trans-repression complex induces a downregulation of BTG2 mRNA following estrogen stimulation

(Supplementary Fig. S4D), while the blockade of either EZH2 or ERα, using GSK126 and Tamoxifen respectively, causes an overexpression of the transcript (Supplementary Fig. S4E), confirming

that both these factors are needed for BTG2 trans-repression. Moreover, also PVT1 silencing induces an upregulation of BTG2 transcript in cell lines and in the CAM model (Fig. 6D,

Supplementary Fig. S4F). In this view, we aimed to investigate whether the observed modulation was mediated by the lncRNA PVT1. To avoid confounding results depending upon PVT1-induced

endogenous ERα modulation, we employed a MCF-7 cell clone expressing an exogenous Flag-ERα whose expression was mostly unaffected by PVT1 silencing, as also demonstrated by the neglectable

effect observed on cell proliferation (Fig. 6E, F, Supplementary Fig. S4G). Using this approach, the immunoprecipitation of Flag-ERα following PVT1 silencing indicated a reduced recruitment

of the receptor on BTG2 gene compared to TFF1 promoter, used as control (Fig. 6G). In addition, the trimethylation of H3K4, that is a marker of transcriptionally active chromatin, was

significantly more pronounced on BTG2 following PVT1 silencing (Fig. 6H). Based on the results described here, we propose a mechanistic model for E2 depending BTG2 transcriptional repression

in which ERα needs the recruitment of PRC2 for EZH2-mediated H3K27Me3 and chromatin closure and PVT1 acts as a bridging factor between the two functional components (Fig. 6I). DISCUSSION

ERα is a mitogenic transcription factor in BC onset and progression by modulating the expression of responsive target genes in luminal-like BC cells [48]. Wide dissection of

chromatin-associated ERα multiprotein complexes has been performed in several experimental conditions leading to partial definition of the nuclear dynamics associated with standard therapies

response or resistance in hormone-dependent BCs [49, 50]. Nevertheless, the discovery of the receptor’s ability to act as an RNA-binding protein [4] has opened new perspectives in the

possibility to identify new molecules bridging protein-protein interactions in transcriptional modulation of gene expression that would guide alternative multi-molecule complexes formation

in transcriptional activation and silencing. Starting from these evidences and from our previous studies, demonstrating the existence of a RNA-mediated ERα interactome [31], we performed

native nuclear ERα-associated RNA immunoprecipitation and sequencing to identify novel molecules involved in transduction of the estrogenic signaling to chromatin. Among ERα interacting RNAs

identified, we focused on lncRNAs because of their known roles in transcriptional modulation of gene expression. Indeed, although the known functional relationship between ERα and several

lncRNAs in BC [7], there was no clear evidence demonstrating physical association of the receptor with these regulatory molecules so far. Comparing the dataset generated here with previously

published data identifying functional lncRNAs in multiple cancer models [17], FGD5-AS1, EPB41L4A-AS1 and PVT1 were selected for further experimental evaluations based on their fitness score

demonstrated in BC cell models, average expression and ERα-enrichment in our experimental models (Fig. 1D, E). After a first functional evaluation, indicating specific expression and

intracellular compartmentalization for each molecule, we observed that all of them were able to associate with BC cell chromatin and participate in transcriptional transduction of the

estrogenic signaling, since their silencing determined deregulation of estrogen-responsive genes and inhibition of ERα-positive BC cell proliferation (Fig. 3A–C). The stronger effect

observed for PVT1, its positive correlation with ERα protein expression and trans-activating function and the significant inverse correlation with BC patients’ overall survival, suggested

this as a nodal factor from the mechanistic point of view (Figs. 3D, E and 4B). Indeed, although PVT1 has been functionally studied and demonstrated to act as a pan-oncogene in several

cancer models, including BC, its direct and nuclear functional association with ERα and the estrogenic signaling has not been investigated so far. By comparing the functional effects

elicited by PVT1 silencing in our models with what already known, we hypothesized that this lncRNA could act as a core molecule in the functional cooperation between ERα-mediated

transcriptional regulation and the activity of the PRC2 complex in suppression of specific genes. Indeed, it has been recently demonstrated that PVT1 acts as a molecular partner of PRC2 in

multiple myeloma through its physical interaction with the enzymatic subunit EZH2, that catalyzes H3K27 trimethylation [38], and it was previously shown that PVT1 is involved in non-small

cell lung cancer metastasis through EZH2 [51]. On the other hand, EZH2 expression has been demonstrated to be modulated by ERα in BC [52], where both are involved within a transcriptional

axis with GREB1 in induction of tamoxifen resistance [53]. Moreover, EZH2 was among ERα partners whose association with the receptor was found to be RNA-dependent [31] (Fig. 1A). To

strengthen previous observations, “PRC2 methylase histone and DNA” was among the functional pathways significantly affected when considering ERα target gene deregulation induced by PVT1

silencing (Fig. 4D). To investigate the possible existence of a multi-modular transcriptional repression complex involving ERα, PRC2 and PVT1, we focused on ERα down-regulated genes whose

expression was affected by PVT1 kd and that were characterized from the presence of both ERα and EZH2 binding sites within their transcription units (Fig. 6A). Among them, BTG2 caught our

attention, as it has been demonstrated to be an estrogen/ERα down-regulated tumor suppressor significantly associated with low survival rate in luminal-like BC, and proposed as possible

molecular target for the treatment of these tumors [46, 54]. We speculated that, as previously observed in different cancer models, PVT1 binds to and carries PRC2 complex to target genomic

regions to suppress transcription of specific genes. In hormone-responsive BC cells, this would be achieved through the coordinated recruitment of EZH2 involving ERα-associated

multi-molecules complexes to down-regulate pro-apoptotic and/or tumor suppressor genes such as BTG2, thus allowing cell proliferation and invasion reduction. Given that, in the proposed

model, EZH2 and ERα proteins are both required for target genes modulation, disrupting their association through PVT1 inhibition might be a useful tool for blocking anti-estrogen resistant

cell proliferation, since both PVT1 and EZH2 have been already involved in resistance to therapy in several models [55,56,57]. The recognized therapeutic value of ASO administration will now

make it possible to consider PVT1 silencing as a novel pharmacological tool to be used alone or in combination with standard therapies in the treatment of ERα-positive BCs. DATA

AVAILABILITY The datasets generated and/or analyzed in the current study are available in the ArrayExpress repository with the following accession numbers: E-MTAB-14147, E-MTAB-14148, and

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RNA PVT1 reverses multidrug resistance in colorectal cancer cells. Mol Med Report. 2018;17:8309–15. CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The results shown here are in

part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. We would like to thank the Advance Histopathology Facility of TIGEM for tissue processing and

embedding and developing customized IHC protocols for protein detection. FUNDING This work was supported by Italian Association for Cancer Research [grant number: IG-23068], the University

of Salerno (fondi FARB 2024, CUP: ORSA244332), Ministry of University and Research [PNRR-MUR NextGenerationEU PRIN 2022 grant numbers: 2022A7HJEM, 2022Y79PT4 and 202282CMEA; PNRR-MUR

NextGenerationEU PRIN-PNRR 2022 grant number: P2022N28FJ] and Italian Ministry of Health [Young Researcher Grant: GR-2021-12373937]. LP is a PhD Student of the Research Doctorate in

“Translational Medicine for Development and Active Ageing” (DOT1328517) of the University of Salerno. AS and NB are residents of the Postgraduate School in Clinical Pathology and Clinical

Biochemistry of the University of Salerno. AUTHOR INFORMATION Author notes * These authors contributed equally: Viola Melone, Domenico Palumbo. AUTHORS AND AFFILIATIONS * Laboratory of

Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, 84081, Baronissi, SA, Italy Viola Melone, Domenico Palumbo,

 Luigi Palo, Noemi Brusco, Annamaria Salvati, Giorgio Giurato, Francesca Rizzo, Giovanni Nassa, Alessandro Weisz & Roberta Tarallo * Genome Research Center for Health, 84081, Baronissi,

SA, Italy Luigi Palo & Alessandro Weisz * Department of Translational Medical Sciences, Federico II University, 80131, Naples, Italy Antonietta Tarallo * Telethon Institute of Genetics

and Medicine, 80078, Pozzuoli, Italy Antonietta Tarallo * Medical Genomics Program and Division of Oncology, AOU ‘S. Giovanni di Dio e Ruggi d’Aragona’ University of Salerno, and Rete

Oncologica Campana, 84131, Salerno, Italy Alessandro Weisz Authors * Viola Melone View author publications You can also search for this author inPubMed Google Scholar * Domenico Palumbo View

author publications You can also search for this author inPubMed Google Scholar * Luigi Palo View author publications You can also search for this author inPubMed Google Scholar * Noemi

Brusco View author publications You can also search for this author inPubMed Google Scholar * Annamaria Salvati View author publications You can also search for this author inPubMed Google

Scholar * Antonietta Tarallo View author publications You can also search for this author inPubMed Google Scholar * Giorgio Giurato View author publications You can also search for this

author inPubMed Google Scholar * Francesca Rizzo View author publications You can also search for this author inPubMed Google Scholar * Giovanni Nassa View author publications You can also

search for this author inPubMed Google Scholar * Alessandro Weisz View author publications You can also search for this author inPubMed Google Scholar * Roberta Tarallo View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization: RT and AW; methodology: VM and LP; investigation: VM, LP, NB, AS and AT; formal

analysis: DP and GG; data curation: DP; writing and original draft preparation: VM, DP, LP and RT; writing, review, and editing: FR, GN RT and AW; visualization: DP, VM, GN; supervision: RT;

funding acquisition: RT, GN and AW. CORRESPONDING AUTHORS Correspondence to Alessandro Weisz or Roberta Tarallo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE All methods were performed in accordance with the relevant guidelines and regulations concerning good scientific practice. The study did

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Palumbo, D., Palo, L. _et al._ LncRNA PVT1 links estrogen receptor alpha and the polycomb repressive complex 2 in suppression of pro-apoptotic genes in hormone-responsive breast cancer.

_Cell Death Dis_ 16, 80 (2025). https://doi.org/10.1038/s41419-025-07423-4 Download citation * Received: 27 June 2024 * Revised: 13 January 2025 * Accepted: 03 February 2025 * Published: 08

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