Functional interplay between long non-coding RNAs and Breast CSCs

There are several ways in which the DNA sequence is not altered but epigenetic regulation affects gene expression; they include histone modifications, methylation of DNA, and genomic imprinting [81, 82]. LncRNAs are essential modulators of the epigenetic state of the human genome (Fig. 1). Chromatin remodeling and modification complexes are attracted to particular places by certain lncRNAs [83]. Examples of lncRNAs that can influence carcinogenesis include EPB41L4A-AS2 [84], BLAT1 [85], BCLIN25 [86], ANRASSF1 [87], H19 [88], and piR-823 [89] which can control DNA methylation. Han and his group found that BLAT1 expression is controlled by lowering the degree of promoter DNA methylation of CpG islands [90]. They showed that cancer patients with tumors that are BLAT1-hypomethylated have a decreased chance of surviving long-term. The high BLAT1 expression with hypomethylation at CpG sites in BC may be associated with the aggressiveness of the disease. BCLIN25 reduces miR-125b expression and elevates BC risk by increasing CpG methylation at the miR-125b promoter region, increasing ERBB2 expression [86]. Calanca et al. found that the tumor suppressor RASSF1A's epigenetic suppression has been linked to the lncRNA ANRASSF1. They proved that ANRASSF1 also is a therapeutic target with the potential to restore the restrictive chromatin changes generated by PRC2 at the promoter of RASSF1A, potentially leading to upregulation of RASSF1A in BC [91]. In addition, a lncRNA known as H19 suppresses the maternal allele at the H19/IGF2 gene via the methylation process in BC, resulting in a more aggressive phenotype [88]. A recent study demonstrated that lncRNA piR-823 overexpression in luminal BC cells activated Wnt signaling and induced cancer cell stemness by increasing DNMTs (DNMT1, DNMT3A, DNMT3B) expression and promoting adenomatous polyposis coli (APC) methylation [89].

In addition, lncRNA decreases gene transcription by recruiting proteins that modify histones or remodel chromatin. Histone regulators interact with the lncRNA HOTAIR, which results in transcriptional gene suppression in chromatin dynamics [92]. For example, HOTAIR contributes to the silencing of miR-205 by altering the balance of histone modifications on the miR-205 promoter between H3K4me3 and H3K27me3, which controls the production of cyclin J (CCNJ) [93]. Furthermore, HOTAIR inhibits miR-7 expression, leading to increased SETDB1 expression in BC stem cells, inducing EMT [94]. Moreover, HOTAIR promotes EMT by acetylating histone H3K27 to methylate the E-cadherin promoter, inhibiting E-cadherin production [95].

One of the most well-studied lncRNAs is Xist, active in the early phases of X chromosomal inactivation in female embryos. First instances of lncRNA directly engaged in the production of repressive chromatin are found in Xist [96]. Furthermore, Xist through particular RNA sequences, organizes the anchoring of chromatin modifiers to one of the two X chromosomes, enabling transcriptional silencing [97].

Several noncoding RNA molecules function as ligands and form complexes with transcription factors to regulate gene transcription [98, 99]. cis and trans regulation are two mechanisms used by lncRNAs to control gene transcription. Classifying lncRNAs into four categories by Wang and Chang namely; (decoy molecules, guiding molecules, scaffolding molecules, and signal molecules) contributed significantly to the progress of the lncRNA research area in the past few years [100]. The involvement of lncRNAs as a decoy, guiding, scaffolding, and signal molecules at the transcriptional level promotes or suppresses gene expression in BC metastasis (Fig. 1). For example, lncRNAs Lethe, NORAD, and PANDA work as "decoys," mimicking and competing with miRNAs or proteins in the nucleus [28, 98, 101] (Fig. 1a). Furthermore, the “guiding lncRNAs,” such as HOTAIR [102] and lincRNA-p21 [103], may directly act on transcriptional factors or chromatin modifiers and protein complexes and attract them to find particular target gene(s) to alter the transcription process (Fig. 1b) [104]. Moreover, LncRNAs MALAT1 [105], HOTAIR [106], and LINP1 [107] can form ribonucleoprotein complexes and regulate gene expression as scaffold molecules (Fig. 1c). In addition, to limit the activities of regulatory miRNAs, they also function as decoy microRNA-binding sites, like ceRNAs [108]. Additionally, various studies have revealed that lncRNAs can be used as molecular signals since they are transcribed at very particular times and locations, allowing cells to integrate developmental clues, read the cellular environment, or respond to a variety of stimuli as chemical signals [100, 109] (Fig. 1d). According to Wang and his colleagues, the lncTCF7 promotes TCF7 expression by recruiting SWI/SNF to TCF7's promoter. These steps may cause Wnt signaling to be activated, which might lead to cancer stem cells self-renewing and tumor cell proliferation [110]. Besides, the specific targets of guide lncRNAs are promoted by RNA–RNA, RNA–protein, and RNA–DNA interactions (Table 2).

LncRNAs influence the translation of mRNAs and regulate their integrity at the posttranscriptional stage by creating double-stranded RNA with mRNAs or via binding to proteins. Tang and his group found that proliferation, colony formation, and development of orthotopic xenograft tumors were all reduced by the depletion of PVT1. Through KLF5/beta-catenin signaling, they also showed that lncRNA PVT1 controls TNBC [111]. Antisense lncRNAs are the most common lncRNAs engaged in mRNA post-transcriptional regulation. There are various ways in which lncRNAs might alter the splicing process of pre-mRNA, either alone or with other splicing factors. For example, the lncRNA RP1-506.5 interacts with eIF4E and inhibits eIF4E from binding to eIF4G, inhibiting p27kip1 translation and adversely regulating Snail levels in BC cells [75]. Likewise, Beta-catenin signaling is increased due to lncRNA PVT1 binding to KLF5 and increasing its stability via BAP1, which increases BC tumorigenesis [111]. The biological processes of malignancies are influenced by lncRNA dysfunction, which has been linked to tumor prognosis. The lncRNA encoded by metallothionein 1 J (MT1JP) has been linked to carcinogenesis, and its expression is downregulated in different types of cancer [112‐114]. Meanwhile, in vivo and in vitro studies recently showed that MT1JP inhibition increases miRNA-214 gene expressions in BC cells by modulating miRNA-214/RUNX3 Axis [115]. Moreover, it was also shown that lncRNA treRNA interacts with ribonucleoproteins (RNPs) (PUF60, SF3B3, FXR1, FXR2, PUF60, and hnRNP K) to produce a treRNA-RNP complex that inhibits E-cadherin translation by targeting eIF4G1 [116] (Fig. 2).

It has been found that RNAs can interact with each other in a novel way (ceRNA networks), and that these interactions are critical to the formation of cancers. They have the potential to act as therapeutic targets in addition to being diagnostic and prognostic markers. According to literature studies, numerous lncRNAs act as ceRNAs in BrCSCs, targeting miRNAs and altering the EMT process. For instance, linc-ROR affects the expression of the stemness factors Nanog, Oct4, and SOX2 and regulates the maintenance of human embryonic stem cells (hESCs) via sponging miR-145 [72]. Further, upregulation of linc-ROR in BC resulted in a higher stem cell phenotype and greater mammosphere development, indicating that the Linc-ROR-miR-145 pathway is critical in cancer stemness [117, 118]. In addition, one of the most well-known and investigated ceRNA mechanisms is the lncRNA H19/Let-7 miRNA [119]. BrCSCs and breast tumors had a significant level of lncRNA H19 expression. Increasing the amount of H19 in tumor cells permits them to survive treatments [120], also H19 is involved in controlling BrCSCs' division by sponging microRNA let-7 which provide potential strategies for stem cell invasion and mammosphere formation [121]. Likewise, lncRNA HOTAIR (homeobox antisense transcript antisense RNA) is associated with the growth and spread of BC cells [122]. BrCSC derived from MCF7 or MB231 have high levels of HOTAIR up-regulation, which affects BrCSC growth, migration, and self-renewal. HOTAIR is also known to influence miR-34a, which in turn promotes Sox2 expression, according to a recent study [61]. HOTAIR can reduce the expression of miR-7 by regulating HoxD10 in MCF-7 and MDA-MB-231 BrCSCs [94]. Additionally, LUCAT1 promotes BrCSC stemness by sponging miR-5582-3p and promoting TCF7L2 and the Wnt/β-catenin pathway. On the other hand, BrCSCs self-renewal was hindered by LUCAT1 downregulation, which enhanced miR-5582-3p expression [123]. Moreover, lncCCAT1 is highly expressed in BrCSCs and is linked to poor patient consequences. LncCCAT1 overexpression promotes BrCSC proliferation, stemness, migration, and invasion capabilities. Depends on the above results, lncRNAs can be used as a marker of the patient's prognosis and the risk of their tumor recurrence which may apply in clinical translation. Meanwhile, miR-204/211, miR-148a/152, and ANXA2 can all interact with LncCCAT1, which in turn can activate TCF4 or promote the translocation of β-catenin to the nucleus and activate Wnt signaling [124]. Additionally, in a recent study, Song and his team found that lncRNA SPRY4-IT1 enhances BC cell proliferation and stemness, as well as BrCSC renewal and stemness maintenance, via promoting the expression of TCF7L2 through targeting miR-6882-3p [125].

Besides, LincK also controls ZEB1 through the sponging of miR-200, which contributes to breast tumorigenesis [126]. In a recent study, lncRNA LSINCT5 has increased cell motility by sponging miR-30a from the Wnt/β-catenin pathway. TCF4 and c-Myc expression was likewise downregulated in cells in which LSINCT5 was knocked out, resulting in decreased proliferation, motility, and EMT [127]. Furthermore, by promoting the miR-185-3p/E2F1/Nanog axis, lncRNA LINC00511 leads to BC tumorigenesis and stemness [128]. Similarly, In TNBC models, LINC01133 acts as a direct mediator of the MSC-triggered miR-199a-FOXP2 axis, enhancing phenotypic and growth characteristics of CSC-like cells [129]. Lastly, evidence supports that the lncRNA, HOTTIP (HOXA transcript at the distal tip), is upregulated in a variety of malignancies, including BC. HOTTIP is also involved in several biological activities, such as maintaining stem cell viability. As a molecular sponge for miR-148a-3p, HOTTIP controls the CSC-like features of BrCSCs, enhancing WNT1 transcription and providing a novel therapeutic target for BC patients [130] (Fig. 3).

Table 2 provides additional examples of lncRNA that regulate BrCSCs through ceRNAs mechanism.

Overall, in terms of lncRNA aberrant expression and the causative factors, lncRNAs may be useful prognostic biomarkers in BrCSCs. Additionally, lncRNAs have the potential to be targeted to reverse the process of carcinogenesis, making them valuable therapeutic targets for treatment of cancer.

Since most of lncRNAs/miRNAs axes (ceRNA axes) have been only assessed in one paper, there is no way to compare the results of studies. However, it is possible that a single lncRNA acts as a molecular sponge for more than one miRNA. In fact, it is possible to identify several lncRNAs/miRNAs axes in which some of the contributing ncRNAs have common roles.

As it turns out, hypoxia influences a specific subset of long noncoding RNAs. Hypoxia-responsive lncRNAs (HRLs) such as NORAD, LncHIFCAR, AC020978, KB-1980E6.3, RAB11B-AS1 may have a role in cancer cell survival and disease progression [101, 136, 137]. HRLs may be classified into two categories: those dependent on hypoxia-inducible factors (HIF) and those not dependent on HIF. Specifically, the hypoxia response element (HRE), located in the promoter regions of HRLs, may directly bind to HIF in hypoxic tumor microenvironments and control their expression by modulating their transcription. Conversely, the HRLs that are excessively expressed may trigger a tumor-specific molecular profile and contribute to the development of tumor characteristics [138]. The molecular properties of HRLs in BC, however, remain unsolved.

Recently, in an in vitro and in vivo studies undertaken in a hypoxic condition, Zhu and colleagues found that the KB-1980E6.3 enhanced BrCSC self-renewal and tumorigenesis [139].

They observed that HIF-1α might interact directly with the HRE in the KB-1980E6.3 promoter to control its transcription activities in hypoxia conditions. Furthermore, Because of its capacity to bind to IGF2BP1, lncRNA KB-1980E6.3 was critical for BrCSC stemness because it increases the stability of c-Myc mRNA and enhances it BC cells' responsiveness to hypoxia [139].

The estrogen receptor (ER) regulates the Hh, Wnt, and Hippo pathways. The dysregulation of these pathways is linked to the fundamental hallmarks of BrCSCs, such as self-renewal, cancer resistance, metastasis, and recurrence [140, 141]. In addition, components in the tumor microenvironment, including hypoxia, angiogenesis, and inflammatory responses, have a main role in modulating BrCSCs [142, 143]. The Hh pathway components (PTCH1, Gli1, and Gli2) are abundantly expressed in normal mammary stem/progenitor cells and stimulated in BrCSCs. For the self-renewal of human BrCSCs, the Hh pathway and Bmi-1 are found to play critical roles highlighting the relevance of the Hh pathway and Bmi-1 in the controlling CSCs, hence applying techniques focused on blocking both pathways provide a helpful therapeutic strategy [144]. Chemotherapy has been demonstrated to be ineffective against BrCSCs, and the stemness of CSCs can be preserved through the Hh pathway. Using MCF-7 cells from BC patients as a model, Miao and his team discovered that the Hh signaling pathway is involved in drug responsiveness [145]. The Hh ligand secreted by CSCs can control the response of cancer-associated fibroblasts (CAFs). Indeed, the CAFs provide elements that encourage CSC growth and self-renewal [146]. Thus, inhibiting this signaling system might be a unique treatment method for BC therapy. Furthermore, SMO protein, transmembrane receptors (PTCH 1), (GLI 1–3), and extracellular Hh ligands are components of this complicated signaling cascade. Also, the Hh ligands activate signal transduction events in the cell, which interact with PTCH. To activate the transcription factor and allow it entry into the cell nucleus, the Hh ligand binds to PTCH, causing changes in its physical conformation and removing the inhibition of SMO. This results in increased cell function, growth, and differentiation [147, 148]. According to recent data, there is a clear relationship between lncRNAs and the cellular proliferation of BrCSCs through different signaling pathways (Table 2). For instance, LINC00617 increases the fraction of a phenotypic stem cell CD44( +)/CD24(-) subpopulation, enhancing BC metastasis. LINC00617 promotes EMT via rising the SOX2 expression in BC cells [50, 149]. Furthermore, lncRNA XIST expression in BC patients is linked to a higher risk of brain metastases. Reduced XIST expression promotes tumor cell stemness via stimulating EMT transition and activating c-Met through moesin MSN-mediated protein stabilization. In mouse mammary glands, knocking down XIST promotes the formation of primary tumors and brain metastases [150]. In addition, there is a clear relationship between EMT and the cellular proliferation of tumor cells. lncRNA-Hh, a lncRNA related to the Hh pathway, has been shown to directly bind GAS1 (a hedgehog signaling enhancer) and promote the activities of SOX2 and OCT4 [59]. Furthermore, lncROPM is a marker for BC patients' malignant grade, stage, and poor prognosis. Studies on the function gain and loss of lncROPM have shown that it is necessary to regulate BrCSC characteristics in vitro and in vivo studies. Through directly binding to the 3'-UTR of PLA2G16 (Group XVI phospholipase A2), lncROPM controls the expression of PLA2G16 by increasing the mRNA stability. Higher PLA2G16 enhances phospholipid metabolism and the generation of free fatty acids [151], particularly arachidonic acid, in BrCSCs, promoting PI3K/AKT, Wnt/-catenin, and Hippo/YAP pathways and ultimately contributing to the regulation of BrCSC stemness [152]. An animal investigation in BrCSCs found that H19 increased stemness via sponging Let-7c and activating its ESR1 and Wnt pathway targets [153]. Additionally, inhibition of symmetric division of BrCSCs by H19 or Let-7c miRNA increases non-CSCs. lncRNA-Hh is linked with the Shh-GLI1 pathway and is transcriptionally controlled by Twist, directly targets GAS1 to enhance Hh signaling activation. Increased GLI1 expression and increased SOX2 and OCT4 transcription are all associated with the activation of Hh, which is thought to play a regulatory function in CSC development [154]. Additionally, lncRNA-Hh stimulates the Hh pathway protein Hh to increase OCT4 and SOX2 expression for BrCSC homeostasis [59]. Similarly, Han et al. showed that lncRNA HOTTIP modulates the miR-148a-3p/WNT1 axis, which allows BrCSCs retain their CSC-like features and promote BC development [130]. In contrast, overexpression of LINC00968 in BC cells reduces the development of resistance by decreasing the stimulation of the Wnt2-β-catenin pathway by suppressing Wnt2 [155] (Fig. 4).

Altogether, these data imply that the lncRNAs (lncRNA-Hh, H19, LINC00617, lncROPM, XIST, PLA2G16) play a critical role in the regulation of CSCs and are associated with the invasive and migration characteristics of BC cells. They showed that these molecules might be useful in the prognostic and diagnostic method in metastatic BC patients.

Despite advances in cancer treatment, drug resistance continues to be a significant challenge for people with BC. A comprehensive evaluation of off-target effects, possible toxicity, and drug delivery/precision targeting is necessary for the lncRNA silencing approaches to be considered effective therapeutic strategies. Many lncRNA transcript variations also present a considerable challenge for developing techniques to target molecules. It is possible that not all transcript variations will be targeted by the lncRNA silencing technique, which may reduce the treatment's effectiveness if that variant is functional. Moreover, CRISPR/Cas9 may have challenges or limited utility for the knockout of non-coding genes [179, 180]. Despite their equivalent molecular weight, there are no open reading frames (ORPs) in lncRNAs, unlike protein-coding genes. Furthermore, the roles of most lncRNAs remain unknown, making the creation of efficient medicines and delivery methods much more difficult. A comparison study in clinical trials would be required to determine whether or not lncRNAs are better targets than protein-coding genes [181].

As previously stated, CSCs have responsibility for drug resistance and recurrence, all of which have an impact on anticancer therapeutic potential. The use of lncRNAs or related pathways to target CSCs as a possible therapy for cancer is a novel approach [182]. In BC, down-regulation of the oncogenic lncRNA CCAT1 is linked to an increase in radiosensitivity through altering the miR-148b, which is responsible for cancer progression [183]. Interestingly, Dendrosomal curcumin (DNC), a natural chemical, can be used to treat cancer by reducing the expression of tumor suppressor genes Tusc7 and GAS5 lncRNA in BC cells MCF7, MDA-MB231, and SKBR3. Furthermore, Esmatabadi and his team found that down-regulating GAS5 in BC cells can reduce many characteristics of DNC's anti-cancer activities. This suggests that combining DNC with GAS5 over-expression could be a clinically effective tool for drug-resistance in BC cells [184].

Recent study reveals that MEG3 interacts directly with miRNA-421 to regulate a variety of CSC characteristics, including self-renewal and invading abilities [185]. However, as a result of the methylation that occurs in its promoter region, the expression of the tumor suppressor lncRNA MEG3 is downregulated in cancer cells. A new nanotechnology-based preparation of natural curcumin known as dendrosomal curcumin increased the expression of MEG3 by down-regulating DNA methyltransferase (1A, 3A, and 3B) expression through promoting of miR-29a and miR-185 [186].

Chemoprevention is the focus of new strategies to enhance clinical outcomes and reduce the toxicity of anticancer medications. In the case of advanced ovarian cancer and BC, for example, Oxaliplatin (Oxa) is a platinum medication of the third generation that is used either alone or in combination with other treatments [187, 188]. However, several mechanisms contribute to resistance to Oxa, including DNA damage repair, inhibition of apoptosis, deregulation of signaling pathways, and increased detoxifying efficiency. Combination therapy has been proposed as an emerging approach to solving this problem [189]. It was found that DNC alone or in combination with Oxa has synergistically downregulated several oncogenic lncRNAs such as GAS5, MALAT1, FAL1, ANRIL, ABO73614, CCAT2, LSINCT5 in different types of cancers such as BC, NSCLC, and ovarian cancer through suppressing cell proliferation, prompting cell death, and reducing therapeutic resistance [184, 188, 190, 191]. They also found that DNC or curcumin combined with anti-cancer drugs had a more significant inhibitory effect than monotherapy. Curcumin reduces the lncRNA H19-induced EMT [192].

In addition, CRISPR interference (CRISPRi) or knockout (KO), a technology that may be applied to any genomic site, can be used to inhibit the activity of lncRNAs, which are crucial for the survival or self-renewal of cancer cells [193]. Complete excision of the whole gene, excision of the promoter and transcriptional start point, removing exon/exon junctions, or excision of the transcriptional terminal site are all strategies for lncRNAs knocking out [193]. For instance, CRISPR/Cas9 technology was used by Peng and colleagues to modify LncROR expression in BC cell lines. Researchers found that lncROR increased estrogen production through this new approach and triggered the MAPK/ERK axis in BC. CRISPR/cas9 technique was used in this study to investigate lncRNA loss of function and gives evidence for lncROR might be a possible target in ER⁺ BC patients [194]. Thus, it is a promising technique to reduce drug resistance, such as tamoxifen resistance of ER⁺ BC, and can be used as a therapeutic strategy in BC patients. In addition, lncRNA BC200 functions as an oncogene and has an important role in cancer proliferation and drug resistance [195]. Singh and colleagues published a notable study elucidating that oncogenic LncBC200 is elevated in BC [196]. The amount of LncBC200 in breast tumor tissues is greater in ER⁺ tumors than in ER⁻ tumors. They used CRISPR/Cas9 technology to knock off LncBC200 expression to understand more about the function of ER-regulated LncBC200 expression. According to the findings LncBC200 was shown to have a significant role in the development of cancer. Ultimately, oncogenic lncAK023948 which is essential for cancer cell survival has been used as a target for CRISPR/Cas9 to reduce tumor growth in different types of BC cells [197]. These findings offer a scientific basis for the concept that lncRNAs play an important part in the progression of cancer and could be used as therapeutic targets.