Long Noncoding RNAs Involved in the Endocrine Therapy Resistance of Breast Cancer

Long noncoding RNAs (lncRNAs) are defined as RNAs longer than 200 nucleotides that do not encode proteins. Recent studies have demonstrated that numerous lncRNAs are expressed in humans and play key roles in the development of various types of cancers. Intriguingly, some lncRNAs have been demonstrated to be involved in endocrine therapy resistance for breast cancer through their own mechanisms, suggesting that lncRNAs could be promising new biomarkers and therapeutic targets of breast cancer. Here, we summarize the functions and mechanisms of lncRNAs related to the endocrine therapy resistance of breast cancer.


Introduction
For women worldwide, breast cancer is the most common cancer, and one in eight to ten women will develop breast cancer during their lifetime [1,2]. Although the endocrine therapies that target sex hormone receptor signaling pathways are effective treatment for breast cancer, therapy resistance and cancer recurrence remain important issues, and novel therapeutic strategies are required. Recent transcriptome analyses have revealed that a large number of long noncoding RNAs (lncRNAs), which are RNAs that are longer than 200 nucleotides in length and do not encode proteins, are expressed in humans [3][4][5]. LncRNAs play key roles in various biological process and diseases, including cancers [6][7][8][9][10]. In breast cancer, some lncRNAs exert oncogenic or tumor-suppressive functions to control breast cancer pathophysiology, such as invasion and metastasis, and drug resistance; these findings are summarized in a recent review article [11]. In terms of endocrine therapy, selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders or downregulators (SERDs), and aromatase inhibitors, are mainly used as drugs to suppress estrogen signaling. For an experimental model of aromatase inhibitor-resistant breast cancer, cells that can obtain the ability to grow under long-term estrogen deprivation (LTED) conditions are preferentially used. Here, we extensively focus on endocrine therapy resistance-associated lncRNAs in breast cancer by including these drugs and experimental models, and describe the recent findings on their functions and mechanisms.

Breast Cancer
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide [1,2]. According to the GLOBOCAN 2018 database of the International Agency for Research on Cancer, which estimates the incidence and mortality of several cancers,

LncRNAs
Together with the advancement of technologies of cDNA cloning and RNA sequencing,~70-90% of mammalian genomes are shown to be transcribed to produce huge numbers of noncoding RNAs (ncRNAs), while less than 3% of these genomes are translated to proteins, suggesting the importance of ncRNAs in biological processes [25][26][27]. ncRNAs are classified by their length, i.e., ncRNAs shorter than 200 nucleotides are classified as small ncRNAs, while longer ncRNAs are defined as lncRNAs. MicroRNAs (miRNAs) belong to the small ncRNA category and are involved in translational repression and mRNA destabilization in cooperation with various proteins, including argonaute (AGO) proteins [28]. As it has been shown that miRNAs play key roles in numerous biological processes and diseases, including various types of cancers, their clinical application has been studied [10,29]. Moreover, lncRNAs have been suggested to be essential for cell physiology. Previous studies have identified a large number of lncRNA genes in mammals. For example, the GENCODE project, which is part of the ENCODE project and aims to annotate all gene features in the mouse and human genomes, has identified 13,197 and 17,952 lncRNA genes in mice and humans, respectively [30]. Moreover, a previous transcriptome study reported 58,648 lncRNA genes in humans [5]. Although most lncRNAs remain to be studied, it has been gradually elucidated that some lncRNAs play important roles in multiple biological phenomena, such as cell differentiation and organogenesis and diseases [6][7][8].
The expression of lncRNAs tends to be highly cell type-and tissue-specific [3], implying that lncRNAs are good candidate biomarkers and therapeutic targets for diseases. Intriguingly, the expression of some lncRNAs is deregulated in cancers, and these lncRNAs exert oncogenic or tumor-suppressive functions via various mechanisms, such as regulating the transcription or translation of target genes and modulating signal transduction [9,10]. Furthermore, some lncRNAs are involved in breast cancer progression via controlling some processes of breast cancer pathophysiologies, such as invasion and metastasis, and drug resistance (reviewed in [11]). Thus, lncRNAs may be promising biomarkers and therapeutic targets of cancers, including breast cancer. As mentioned above, endocrine therapy resistance is one of the major therapeutic problems for breast cancer. Intriguingly, some lncRNAs regulate the endocrine therapy resistance of breast cancer, and may be key factors for the treatment of breast cancer with endocrine therapy resistance. In the following sections, we introduce the studies of lncRNAs using models of breast cancer cells that are sensitive or resistant to drugs used for endocrine therapy (e.g., tamoxifen as the SERM, ICI182,780 as the SERD, and anastrozole as the aromatase inhibitor), models of breast cancer cells under LTED conditions, and clinical specimens of breast tumors, and intensely describe the functions and mechanisms of lncRNAs in the endocrine therapy resistance of breast cancer, as revealed by these studies.

LncRNAs Involved in the Endocrine Therapy Resistance of Breast Cancer
LncRNAs can be classified into one or more of five categories: (A) sense lncRNAs that overlap the neighboring protein-coding gene on the same strand; (B) antisense lncRNAs that overlap the neighboring protein-coding gene on the opposite strand; (C) bidirectional lncRNAs, which are transcribed from the opposite strand within 1 kb from the nearest protein-coding gene; (D) intronic lncRNAs that are derived wholly from intronic regions of protein-coding genes; or (E) intergenic lncRNAs, or long intergenic noncoding RNAs (lincRNAs) that are transcribed from the genomic interval between two genes ( Figure 1) [31]. For example, among the lncRNAs related to the endocrine therapy of breast cancer, HOX transcript antisense RNA (HOTAIR) is classified as an antisense lncRNA, while urothelial cancer associated 1 (UCA1) is a lincRNA. In addition, several endocrine therapy resistance-related lncRNAs belong to multiple categories. In the following section, we introduce lncRNAs involved in the endocrine therapy resistance of breast cancer based on these categories.

Antisense LncRNAs
The HOTAIR lncRNA enhances resistance to tamoxifen [32]. HOTAIR is a~2.2 kb lncRNA, and its gene overlaps the homeobox C11 (HOXC11) gene on the opposite strand [33]. A previous study using an ER-positive breast cancer cell line, MCF7, showed that HOTAIR binds to the estrogen receptor α (ERα), and the overexpression of HOTAIR enhances ER signaling by upregulating ERα expression levels and promoting the chromatin binding of the ERα, even under hormone-starved conditions. These results suggest that HOTAIR activates ligand-independent ER signaling, which may contribute to tamoxifen resistance ( Figure 2) [32]. Moreover, HOTAIR has been demonstrated to promote breast cancer progression via transcriptional regulation. HOTAIR binds to the polycomb repressive complex 2 (PRC2) with its 5 side, and regulates the PRC2-mediated trimethylation of H3K27 in trans at the HOXD locus on chromosome 2, which leads to transcriptional repression of the HOXD locus [33]. Furthermore, HOTAIR induces selective re-targeting of PRC2 and trimethylated H3K27 genome-wide, thus promoting the invasion of breast cancer cells [34]. Consistently, high expression of HOTAIR is associated with a short duration of metastasis-free and overall survival in patients with breast cancer [34,35]. Moreover, the 3 side of HOTAIR binds to corepressor for element-1-silencing transcription factor (CoREST)/repressor element-1 silencing transcription factor (REST) complexes, including lysine-specific demethylase 1 (LSD1), which mediates the demethylation of dimethylated H3K4 (H3K4me2). HOTAIR can bind simultaneously to PRC2 and the LSD1/CoREST/REST complexes, to coordinate the targeting of these complexes to hundreds of genes for coupled H3K27 methylation and H3K4 demethylation [36]. Furthermore, the HOTAIR-LSD1 complex is involved in transcriptional activation mediated by c-Myc. In addition, the hepatitis B X-interacting protein (HBXIP) binds directly to c-Myc on target genes and recruits LSD1 via interaction with HOTAIR, which enhances the transcription of c-Myc target genes, possibly through the LSD1-mediated demethylation of H3K4me2 [37]. HOTAIR, HBXIP, and LSD1 promote breast cancer proliferation, highlighting the function of HOTAIR as a critical effector of c-Myc in cooperation with HBXIP and LSD1 [37]. Thus, HOTAIR plays important roles in the epigenetic regulation of gene expression. However, a recent study has proposed that PRC2 is dispensable for the HOTAIR-mediated trimethylation of H3K27 and gene silencing [38], suggesting that further studies are necessary for elucidating the precise mechanisms by which HOTAIR epigenetically controls gene expression. In addition, HOTAIR acts as a competing endogenous RNA (ceRNA) that specifically sponges the target miRNAs and inhibits their activities. For example, HOTAIR is a ceRNA for miR-206 and increases the expression of a miR-206 target gene, the Bcl-w/Bcl-2 like protein 2 (BCL2L2) gene, thus promoting breast cancer proliferation [39]. Moreover, HOTAIR functions as a ceRNA for miR-20a-5p and upregulates a miR-20a-5p target, the high mobility group AT-hook 2 (HMGA2) gene, which enhances the proliferation, survival, migration, and invasion of breast cancer cells, and the growth of breast tumors [40]. Thus, HOTAIR controls breast cancer progression via multiple pathways of regulation of gene expression. Thymopoietin antisense transcript 1 (TMPO-AS1) is an lncRNA that was demonstrated recently to enhance tamoxifen resistance in breast cancer [41]. TMPO-AS1 is a~3.2 kb lncRNA, and its gene overlaps the thymopoietin (TMPO) gene on the opposite strand. High expression of TMPO-AS1 is associated with short distant-metastasis-free and overall survival in patients with breast cancer [41]. In addition, the upregulation of TMPO-AS1 is observed in MCF7-derived, tamoxifen-resistant cells (OHTR) and MCF7-derived LTED cells, which is associated with poor relapse-free survival in patients with breast cancer treated with tamoxifen [41]. TMPO-AS1 is induced by estrogen in MCF7 cells and another ER-positive breast cancer cell line, T47D. The purification experiments of TMPO-AS1 from these cell lines by using its antisense oligonucleotide probes suggest that TMPO-AS1 binds to the 3 untranslated region (UTR) of the estrogen receptor 1 (ESR1) mRNA, which encodes the ERα protein, through an RNA-RNA interaction. Moreover, this RNA-RNA interaction results in the stabilization of the ESR1 mRNA. Thus, TMPO-AS1 upregulates ESR1 expression and ER signaling pathways, contributing to cell proliferation and tamoxifen resistance ( Figure 2) [41]. Therefore, TMPO-AS1 is a promising biomarker and therapeutic target for ER-positive breast cancer. In other cancers, it has been suggested that TMPO-AS1 interacts with RNAs other than the ESR1 mRNA and promotes disease progression [42][43][44]. For example, in cervical cancer, TMPO-AS1 functions as a ceRNA via the sponging of miR-577 and upregulates a miR-577 target, RAB14, to promote cell proliferation, survival, and migration [42]. In osteosarcoma, TMPO-AS1 increases the expression of WNT7B by sponging miR-199a-5p, which promotes cell proliferation and survival [43]. In addition, TMPO-AS1 stabilizes the TMPO mRNA to promote the proliferation, survival, migration, and invasion of non-small cell lung cancer (NSCLC) cells. Mechanistically, TMPO-AS1 may stabilize the TMPO mRNA through their interaction [44]. Thus, TMPO-AS1 exerts oncogenic effects in various cancers by forming RNA duplexes with some target RNAs.
Conversely, the ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) antisense RNA 2 (ADAMTS9-AS2) lncRNA decreases tamoxifen resistance [45]. ADAMTS9-AS2 is an antisense transcript of the tumor-suppressor ADAMTS9 gene. It has been suggested that low expression of ADAMTS9-AS2 is associated with poor prognosis in patients with several types of cancers, such as lung, colorectal, and gastric cancers, while high ADAMTS9-AS2 expression is associated with poor prognosis in patients with some cancers, such as bladder cancer and salivary adenoid cystic carcinoma [46][47][48][49][50]. ADAMTS9-AS2 is downregulated in tamoxifen-resistant cells derived from MCF7, and downregulation of ADAMTS9-AS2 is also observed in breast cancer tissues, especially in breast tumors with grade III-IV or a tumor size larger than 2 cm [45]. From the knockdown or overexpression experiments of ADAMTS9-AS2 in an MCF7-derived tamoxifen-resistant cell line, it is shown that ADAMTS9-AS2 acts as a ceRNA by sponging miR-130a-5p to promote the expression of a miR-130a-5p target gene, phosphatase and tensin homolog (PTEN), which is a well-known tumor-suppressor and enhances tamoxifen sensitivity ( Figure 2) [45].
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a lincRNA that has been suggested to be involved in the tamoxifen resistance of breast cancer [63]. MALAT1 was initially reported as an lncRNA that is highly expressed in stage I NSCLC tumors that subsequently metastasize, and high expression of MALAT1 is associated with short overall survival in patients with NSCLC [64]. Moreover, dysregulation of MALAT1 expression has been indicated in various cancers, including breast cancer [65]. MALAT1 is an~8.7 kb lincRNA, and its gene is located on human chromosome 11q13.1. The MALAT1 primary transcript contains a tRNA-like structure at the 3 end [66]. RNase P and RNase Z, which are endonucleases that cleave the 5 or 3 side of a tRNA precursor [67,68], cleave both sides of this tRNA-like structure, resulting in the 3 -end maturation of MALAT1 [66]. The excised tRNA-like RNA (MALAT1-associated small cytoplasmic RNA (mascRNA) is added with CCA trinucleotides at the 3 end and exported to the cytoplasm [66].
Although the function of mascRNA is not well understood, it is suggested that mascRNA is an immune regulator in monocytes that is involved in innate immunity in cardiomyocytes [69]. Moreover, the 3 end of mature MALAT1 contains two U-rich sequences and the associated A-rich sequences, and these sequences form a triple-helical structure that enhances the stability of MALAT1 [70,71]. MALAT1 is localized in the nucleus, especially in nuclear bodies, which are termed nuclear speckles or SC35 domain and are enriched for splicing factors [72]. Regarding tamoxifen resistance, high expression of MALAT1 is associated with a short recurrence-free survival in patients with ER-positive breast cancer treated with tamoxifen [63]. In addition, high MALAT1 expression is associated with poor recurrence-free survival in patients with ER-negative breast cancer, indicating the importance of the ER-independent functions of MALAT1 [63]. The roles of MALAT1 in breast cancer are complicated, because both oncogenic and tumor-suppressive roles of MALAT1 in breast cancer have been reported. For example, MALAT1 acts as a ceRNA for some miRNAs, such as miR-124, miR-1, miR-129-5p, miR-204, and miR-339-5p, thus promoting breast cancer progression [73][74][75][76][77]. In contrast, MALAT1 functions as a ceRNA for miR-20a to inhibit the growth and metastasis of breast cancer [78]. Moreover, MALAT1 regulates transcriptional and posttranscriptional events in ways other than sponging miRNAs. For instance, MALAT1 interacts with the promoter of the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene and upregulates EEF1A1 expression by enhancing the trimethylation of H3K4, which promotes the proliferation and invasion of breast cancer [79]. In addition, MALAT1 forms a complex with the serine/arginine-rich splicing factor 1 (SRSF1), the inhibitor of the DNA binding 4, HLH protein (ID4), and mutant p53, and regulates the alternative splicing of the vascular endothelial growth factor A (VEGFA) mRNA precursor (pre-mRNA), which increases the angiogenic potential of breast cancer cells [80]. Conversely, MALAT1 binds to an RNA-binding protein, Hu antigen R (HuR), and interacts with the CD133 gene to downregulate CD133, thus suppressing the epithelial-to-mesenchymal transition (EMT) and migration activity of breast cancer cells [81]. Although the mechanisms via which MALAT1 exerts both oncogenic and tumor-suppressive functions are not well understood, its functions may depend on context, such as cell type and environment. Furthermore, the dual roles of MALAT1 in cancer progression have been suggested by studies using Malat1 knockout (KO) mice [82,83]. Arun et al. reported that Malat1 KO suppresses the lung metastasis of mammary tumors generated in mouse mammary tumor virus (MMTV)-polyomavirus middle T antigen (PyMT) mice [82]. Inversely, Kim et al. later demonstrated that Malat1 KO enhances the dissemination and lung metastasis of mammary tumors in MMTV-PyMT mice, and that this phenotype can be rescued by the transgenic expression of Malat1 from the ROSA26 locus [83]. Although it is not clear why there is a discrepancy between those results, it may be partly attributed to differences in the methodology for Malat1 KO. In the former study, Malat1 KO was accomplished by deletion of a~3 kb genomic region containing the 5 end of the Malat1 gene and its promoter using Cre-mediated recombination technology, while in the latter study, Malat1 was depleted by inserting the lacZ gene and polyadenylation sequences 69 bp downstream of the transcriptional start site of Malat1. These genomic rearrangements in Malat1 KO mice might affect the chromosomal conformation and some nuclear events of gene expression differently, resulting in differential phenotypes. Based on their findings, the manner in which the expression of lncRNAs is suppressed may be important for elucidating lncRNA functions.
The large intergenic noncoding RNA-regulator of reprogramming (lincRNA-ROR) also upregulates tamoxifen resistance [84][85][86]. LincRNA-ROR was originally identified as an lncRNA that is upregulated in induced pluripotent stem cells (iPSCs) compared with embryonic stem cells (ESCs), and has been shown to modulate reprogramming [87]. LincRNA-ROR promotes the proliferation and invasion of MCF7 and a TNBC cell line, MDA-MB-231, by regulating the TGF-β signaling pathway, and high expression of lincRNA-ROR is associated with a poor prognosis in patients with breast cancer [88]. Regarding the mechanisms by which lincRNA-ROR regulates tamoxifen resistance, lincRNA-ROR knockdown experiments in BT474 suggest that lincRNA-ROR enhances tamoxifen resistance by inhibiting autophagy ( Figure 3B) [85]. In addition, a previous study using lincRNA-ROR-KO MCF7 cells suggests that lincRNA-ROR promotes the degradation of an extracellular signal-regulated kinase (ERK)-specific phosphatase-the dual specificity phosphatase 7 (DUSP7)-under estrogen-deprived conditions, which results in the ligand-independent activation of ERα mediated by the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. As a result, lincRNA-ROR promotes estrogen-independent growth and tamoxifen resistance in breast cancer ( Figure 3B) [86]. Moreover, from a previous study using an MCF7-derived tamoxifen-resistant cell line, lincRNA-ROR is suggested to act as a ceRNA by sponging miR-205-5p to upregulate the miR-205-5p target zinc finger E-box binding homeobox 1/2 (ZEB1/2), thus enhancing EMT and tamoxifen resistance ( Figure 3B) [84]. In MDA-MB-231, lincRNA-ROR acts as a ceRNA for another miRNA, miR-145, to upregulate its targets, i.e., ADP ribosylation factor 6 (ARF6) and mucin 1, which control the subcellular localization of E-cadherin and the metastasis of TNBC [89,90]. In addition to these findings, the genotypes of the rs4801078 SNP in lincRNA-ROR are associated with the risk of breast cancer [91], suggesting that lincRNA-ROR is both a promising biomarker and a therapeutic target of breast cancer.
Furthermore, the lincRNA termed lncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1) enhances tamoxifen resistance [92]. LINP1 was initially identified as an lncRNA that is highly expressed in TNBC. In TNBC, LINP1 forms a complex with Ku autoantigen, 80kDa (Ku80) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and activates the NHEJ pathway, which repairs double-stranded breaks in DNA [93]. Consistent with these findings, LINP1 enhances the resistance to radiation and chemotherapeutic drugs that cause DNA damage, such as 5-fluorouracil and doxorubicin [93,94]. LINP1 also promotes the proliferation of ER-positive MCF7 breast cancer cells [94]. In addition, LINP1 expression is negatively regulated by estrogen, and is upregulated in ER-positive breast cancer cell lines, MCF7 and T47D, under estrogen-deprived or tamoxifen-treated conditions, as well as in tamoxifen-resistant breast cancer cells derived from these cell lines [92]. From the knockdown and overexpression experiments of LINP1 in MCF7 and T47D, it is suggested that LINP1 inhibits the ER signaling pathway by downregulating ERα, which may be involved in tamoxifen resistance [92].
Recently, it has been reported that the cytoskeleton regulator (CYTOR)/LINC00152 lincRNA is involved in tamoxifen resistance [95]. CYTOR promotes the proliferation and migration of breast cancer cells, and high expression of CYTOR is associated with relapse-free survival in patients with breast cancer. CYTOR regulates the epidermal growth factor receptor and mTOR signaling pathways and control the organization of the filamentous actin cytoskeleton [96]. Moreover, CYTOR is upregulated in tamoxifen-resistant breast cancer cell lines derived from MCF7, and CYTOR functions as a ceRNA by sponging miR-125a-5p, and upregulates a target of miR-125a-5p, the serum response factor (SRF), which promotes the tamoxifen resistance and cell proliferation [95]. Consistent with these data, CYTOR expression is higher in breast tumors from tamoxifen-resistant patients [95]. In addition, CYTOR is associated with poor prognosis in patients with TNBC, and induces the ubiquitination-mediated degradation of PTEN in TNBC [97].
Although there few studies have addressed lncRNAs that regulate the resistance to aromatase inhibitors, there is some evidence of this phenomenon. The lncRNA MIR2052HG lincRNA enhances the resistance to aromatase inhibitors [98,99]. LncRNA MIR2052HG is a~2 kb lncRNA, and its gene is located on human chromosome 8q21.11-q21. 13. This lncRNA upregulates the expression of ERα. From the knockdown experiments of MIR2052HG in ER-positive CAMA-1 cells, MCF7 cells stably transfected with the cytochrome P450 family 19 subfamily A member 1 (CYP19A1) gene that is an aromatase inhibitor target, and MCF7-derived cells resistant to an aromatase inhibitor anastrozole, it has been shown that MIR2052HG increases the expression level of the lemur tyrosine kinase 3 (LMTK3), which in turn regulates the expression of both ESR1 mRNA and ERα protein, and contributes to the resistance to anastrozole ( Figure 3C) [99,100]. For regulating the ESR1 mRNA, LMTK3 decreases the activity of protein kinase C (PKC), which suppresses Ser 473 phosphorylation and the activation of AKT mediated by PKC. As AKT phosphorylates and induces the proteasome-mediated degradation of forkhead box O3 (FOXO3), a transcription factor that controls ESR1 expression, the MIR2052HG/LMTK3/PKC/AKT axis stabilizes FOXO3, thus upregulating ESR1 transcription [99,100]. Conversely, LMKT3 suppresses the activity of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK/ribosomal S6 kinase 1 (RSK1) signaling pathway through the downregulation of PKC activity, which results in a decrease in the Ser 167 phosphorylation level of ERα as well as its stabilization ( Figure 3C) [99,100]. In addition, single-nucleotide polymorphisms (SNPs) located near or within the MIR2052HG gene locus are associated with the recurrence of breast cancer in patients treated with aromatase inhibitors as adjuvant therapy, suggesting that these SNPs in MIR2052HG are promising biomarkers that can be used to identify patients in whom aromatase inhibitors would be an appropriate therapy [98].
Moreover, a recent study indicated that the lncRNA LINC00309 is associated with poor disease-free survival in patients with breast cancer treated with endocrine therapy using aromatase inhibitors, which suggests that LINC00309 plays important roles in the acquisition of resistance to these therapeutic agents [101].

LncRNAs Belonging to Multiple Categories
The growth-arrest specific 5 (GAS5) lncRNA is downregulated in tamoxifen-resistant breast cancer cells, and low GAS5 expression enhances resistance to tamoxifen [102]. GAS5 was originally isolated as a gene that is preferentially expressed in growth-arrested NIH3T3 cells [103]. The GAS5 gene has two alternative promoters, as well as multiple exons and introns. As alternative choices of these exons and alternative promoter usage produce multiple GAS5 variants, GAS5 can be defined as an antisense lncRNA overlapping a protein-coding gene, the zinc finger and BTB domain containing 37 (ZBTB37) gene, on the opposite strand or as a bidirectional lncRNA [104]. GAS5 is a host gene of a class of small noncoding RNAs termed box C/D small nucleolar RNAs (SNORDs); 10 SNORDs are encoded within the GAS5 intronic regions [104]. These snoRNAs are transcribed as part of the GAS5 primary transcript, and are then excised and matured. Regarding endocrine therapy resistance, a previous study using an MCF7-derived tamoxifen-resistant cell line suggests that GAS5 acts as a ceRNA by sponging miR-222 and upregulates PTEN, which is a target of miR-222 and weakens the tamoxifen resistance of breast cancer cells (Figure 4) [102].
In addition to downregulating tamoxifen resistance, GAS5 exerts tumor-suppressive effects in breast cancer via several pathways. For example, GAS5 acts as a ceRNA by sponging miR-21 and upregulates the expression of miR-21 targets programmed cell death 4 (PCDC4) and PTEN, both of which are tumor-suppressor genes [105]. Moreover, GAS5 acts as a ceRNA for miR-196a-5p and downregulates the forkhead box O1 (FOXO1)/phosphoinositide 3-kinase (PI3K)/AKT pathway, thus suppressing the invasion of TNBC cells [106]. In addition to its functions as a ceRNA, GAS5 is involved in transcriptional regulation. GAS5 suppresses glucocorticoid-induced transcription and sensitizes breast cancer cells to apoptosis [107,108]. Exon 12 of GAS5 contains a hairpin structure with two sequences similar to the GR target sequence, termed glucocorticoid response element (GRE). This hairpin structure is called the GAS5 GRE-mimic, and it interacts directly with the DNA-binding domain of GR and suppresses the transcriptional activation of GR target genes, including antiapoptotic genes like cellular inhibitor of apoptosis 2 (cIAP2) and serum-and glucocorticoid-regulated kinase 1 (SGK1), which facilitate stress-inducible apoptosis [107]. In addition to the GR, GAS5 and the GAS5 GRE-mimic bind to other 3-keto steroid receptors, such as the mineralocorticoid, progesterone, and androgen receptors, and inhibit their transcriptional activities [107,108]. Interestingly, the GAS5 GRE-mimic alone can increase apoptosis in breast cancer cells, suggesting that the oligonucleotides of the GRE-mimic may be applicable to breast cancer therapy [108]. Consistent with these findings, GAS5 is downregulated in breast tumors compared with normal tissues, and low expression of GAS5 is associated with poor overall survival in patients with breast cancer and TNBC [102,104,106]. Moreover, a recent study showed that an insertion (ins)/deletion (del) polymorphism located within the GAS5 promoter (rs145204276 AGGCA/-) affects the risk of breast cancer [109]. In that study, the rs145204276 ins/del and del/del genotypes, as well as the del allele, were associated with a reduced risk of breast cancer [109]. As GAS5 expression is significantly higher in patients with breast cancer carrying the rs145204276 ins/del and del/del genotypes versus the rs145204276 ins/ins genotype carriers, and since the rs145204276 del allele increases the transcription of GAS5, this polymorphism may affect the risk of breast cancer by modulating GAS5 expression levels [109]. The Down syndrome cell adhesion molecule antisense RNA 1 (DSCAM-AS1) is an intronic antisense lncRNA that is transcribed from the opposite strand of the Down syndrome cell adhesion molecule (DSCAM) gene, and is wholly derived from the intronic region of DSCAM. DSCAM-AS1 promotes tamoxifen resistance [110,111] and is upregulated in breast cancer tissues compared with normal tissues [110,112]. Moreover, DSCAM-AS1 expression is higher in luminal and HER2-positive breast cancers, and particularly in the luminal B subtype [110,112]. Importantly, previous studies have demonstrated that DSCAM-AS1 is an ERα target gene [110,112], and is important for cell proliferation and the invasion of MCF7 and T47D cells [110,112,113], as well as for the growth and liver metastasis of T47D cells xenografted into immunodeficient mice [110]. Moreover, DSCAM-AS1 expression is elevated in tamoxifen-resistant breast cancer tissues, and the knockdown and overexpression experiments of DSCAM-AS1 in breast cancer cell lines, such as MCF7 and T47D, suggest that DSCAM-AS1 promotes tamoxifen resistance [110,111]. Consistent with these results, a high expression of DSCAM-AS1 has been associated with a short disease-free survival for patients with luminal breast cancer and those with luminal breast cancer treated with endocrine therapy [113]. Although the manner in which DSCAM-AS1 functions in breast cancers remains unclear, the RNA-binding protein heterogeneous nuclear ribonucleoprotein L (hnRNPL) is required for DSCAM-AS1 activity in MCF7 and T47D cells [110]. DSCAM-AS1 interacts with hnRNPL via its 3 region, which contains CACA-rich RNA sequences [110]. Furthermore, a previous study using MCF7-derived, tamoxifen-resistant cells suggests that DSCAM-AS1 acts as a ceRNA by sponging miR-137, which increases the expression of epidermal growth factor receptor pathway substrate 8 (EPS8), thus contributing to tamoxifen resistance (Figure 4) [111]. In addition, it was reported recently that DSCAM-AS1 functions as a ceRNA for miR-204-5p in the breast cancer susceptibility gene 1 (BRCA1)-mutated TNBC cell line HCC1937, to promote tumor growth via the upregulation of ribonucleotide reductase M2 (RRM2) [114].
ESR1 locus enhancing and activating noncoding RNAs (Eleanors) were identified as a group of lncRNAs that are transcribed from inside and around the ESR1 locus, and could consist of lncRNAs of all categories [115]. Previous studies have shown that Eleanors play important roles in ER-positive breast cancer progression under estrogen-deprived conditions. Eleanors are specifically expressed in ER-positive breast cancer tissues and MCF7 cells, and are increased in MCF7 cells cultured under LTED conditions [115]. u-Eleanor is an Eleanor that is transcribed from~40 kb upstream of the canonical promoter of ESR1 and upregulates the transcription of the ESR1 mRNA and other Eleanors to promote the proliferation of LTED cells (Figure 4) [115]. A chromatin immunoprecipitation-sequencing (ChIP-seq) analysis showed that the u-Eleanor locus in LTED cells is enriched for monomethylated H3K4, rather than trimethylated H3K4, suggesting that the u-Eleanor locus functions as an enhancer. Clinically, the upregulation of u-Eleanor has been reported to be negatively associated with increasing breastfeeding duration [116]. u-Eleanor tends to be upregulated in healthy women without a history of breastfeeding and women with a breastfeeding duration of 1-6 months. Epidemiological studies have demonstrated that breastfeeding experiences play a protective role against breast cancer in women, while a lack or a short duration of breastfeeding increases breast cancer risk [117][118][119]. Therefore, u-Eleanor may be used as a biomarker of breast cancer at early stages [116]. Furthermore, a recent study has revealed the function of another Eleanor called promoter-associated Eleanor (pa-Eleanor), which is transcribed from the region proximal to the transcriptional start site of ESR1 [120]. In the nucleus, chromosomes fold into domains called topologically associating domains (TADs), which exhibit intra-chromatin interactions [121]. The chromosome conformation capture combined with high-throughput sequencing (4C-seq) analysis, reported in a recent study, reveals that a TAD including Eleanor-expressing regions (Eleanor TAD) resides on human chromosome 6q25.1, and that Eleanor TAD contains the ESR1 gene and three other genes: coiled-coil domain containing 170 (CCDC170), chromosome 6 open reading frame 211 (C6orf211), and required for meiotic nuclear division 1 homolog (RMND1) [120]. pa-Eleanor upregulates genes within Eleanor TAD and promotes the proliferation of LTED cells (Figure 4). In addition, pa-Eleanor upregulates u-Eleanor, whereas u-Eleanor does not affect pa-Eleanor expression, suggesting that pa-Eleanor upregulates the transcription of the ESR1 mRNA through u-Eleanor [120]. Moreover, pa-Eleanor enhances an intra-chromosomal interaction between the ESR1 promoter region and the region near the FOXO3 locus on human chromosome 6q21 [120]. This chromosomal interaction may affect the expression of genes within Eleanor TAD. FOXO3 is a transcription factor that induces apoptosis through the transcriptional regulation of apoptosis-associated genes, and its expression is elevated in LTED cells. The knockdown of pa-Eleanor decreases ESR1 expression levels (but does not affect the expression of FOXO3) and induces the apoptosis of LTED cells. Therefore, pa-Eleanor may promote the survival of LTED cells by regulating the balanced expression of ESR1 and FOXO3 [120]. Thus, the functions of u-Eleanor and pa-Eleanor suggest that the regulation of Eleanor expression may represent a new treatment strategy for breast cancer adapted to estrogen-deprived conditions. Consistent with this idea, resveratrol and glyceollin I, phytoalexins that are synthesized in plants under stress conditions, decrease the expression of Eleanors to induce apoptotic death in LTED cells [122].

Antisense Oligonucleotides (ASOs) in Clinical Use
Considering that lncRNAs play essential roles in endocrine therapy resistance, intervention against lncRNAs may be promising for breast cancer treatment. Antisense oligonucleotides (ASOs) are used for regulating the stability and activity of RNAs. Some chemically modified ASOs targeting transcripts of protein-coding genes have been approved for clinical use by the U.S. Food and Drug Administration (FDA) [124]. For example, a 2 -O-(2-methoxyethyl) (MOE) phosphorothioate (PS) ASO called nusinersen is used for the treatment of spinal muscular atrophy (SMA). [125,126]. SMA is an autosomal-recessive neuromuscular disorder with degeneration of the motor neurons in the anterior horn of the spinal cord, leading to atrophy of the voluntary muscles of the limbs and trunk [125]. SMA is caused by deletions or loss-of-function mutations of survival of motor neuron 1, telomeric (SMN1) gene and the consequent reduced expression of survival of motor neuron (SMN) proteins from SMN1 transcripts. Although there is a homologue of SMN1 gene called survival of motor neuron 2, centromeric (SMN2), SMN proteins are not efficiently produced from SMN2 transcripts. The SMN2 gene has an identical coding sequence but differs from SMN1 gene by 11 nucleotides. The different sequences between these genes contain a C-to-T mutation on exon 7, which is a synonymous mutation but affects SMN protein expression by promoting the skipping of exon 7. Due to this mutation, 80%-90% of SMN2 mRNAs lack exon 7 and are translated into truncated SMN proteins, which are rapidly degraded ( Figure 5A). Therefore, the SMN2 gene does not fully compensate for the loss-of-function of the SMN1 gene [125], and modulating the splicing pattern of SMN2 pre-mRNA to produce the full-length SMN proteins is one of therapeutic strategies of SMA. Nusinersen is an ASO complementary to a site within intron 7 of the SMN2 pre-mRNA called intronic splicing silencer-N1 (ISS-N1), which is involved in the skipping of exon 7 and blocks the activity of ISS-N1 to facilitate the inclusion of the exon 7, resulting in the synthesis of the functional, full-length SMN proteins and the rescue of motor neurons ( Figure 5A) [124,125]. Another oligonucleotide drug, mipomersen, is used to treat homozygous familial hypercholesterolemia, an autosomal disorder of the lipid metabolism characterized by elevated levels of low-density lipoprotein (LDL) cholesterol [124,127]. Mipomersen targets the transcripts of the apolipoprotein B (APOB) gene. The middle region of mipomersen shows DNA-like properties and induces the cleavage of these transcripts mediated by ribonuclease H (RNase H), which cleaves RNAs that form heteroduplexes with DNA. As the apoB-100 protein, encoded by APOB gene, is a component of LDL cholesterol, mipomersen-mediated downregulation of APOB decreases the circulating levels of LDL cholesterol ( Figure 5B) [123,126,128]. In addition to ASOs, a small interfering RNA (siRNA)-based drug, patisiran, was recently approved by the FDA [124]. Therefore, the targeting lncRNAs with ASOs and siRNAs may be translated into new therapies for breast cancer.

Conclusions
In this review, we describe the functions and mechanisms of lncRNAs related to the endocrine therapy resistance of breast cancer (Table 1), and their potential as therapeutic targets. Additionally, LncRNAs may hold promise as biomarkers of breast cancer. Importantly, the quantification of the prostate cancer antigen 3 (PCA3) lncRNA in urine samples has been developed as a diagnostic test for prostate cancer [129], suggesting that lncRNAs may be applicable to the analysis of non-invasive liquid biopsies for the diagnosis of cancers, including breast cancer. Thus, lncRNAs are potential key factors in the development of new strategies of breast cancer treatment, and further studies of lncRNAs in the context of breast cancer are required. ER-positive breast tumors from patients who were primarily treated with tamoxifen as the unique endocrine therapy [123] RP11-259N19.1 Up-regulation KLF3-AS1 Down-regulation PDCD4-AS1 Down-regulation PP14571 Up-regulation RP11-69E11. 4 Down-regulation LincRNAs

Conflicts of Interest:
The authors declare no conflict of interest.