The long non‐coding RNA landscape in triple‐negative breast cancer

Abstract Triple‐negative breast cancer (TNBC) is a type of breast cancer that has a higher risk of distant recurrence and metastasis, leading to a relatively aggressive biological behaviour and poor outcome. So far, the clinical management of TNBC is challenging because of its heterogeneity and paucity of specific targeted therapy. Recently, various studies have identified a lot of differently expressed long non‐coding RNAs (lncRNAs) in TNBC. Those lncRNAs have been reported to play important roles in the multistep process of TNBC tumorigenesis. Here, we review the biological characteristics of lncRNAs, and present the current state of knowledge concerning the expression, function and regulation of lncRNAs in TNBC. Accumulating studies explored the potential lncRNAs‐based therapeutics in TNBC, including the techniques of genetic modification using antisense oligonucleotides, locked nucleic acid and RNA nanotechnology. In current review, we also discuss the future prospects of studies about lncRNAs in TNBC and development of lncRNA‐based strategies for clinical TNBC patients.


| INTRODUC TI ON
Breast cancer (BC) is the most frequently diagnosed malignancy and the leading cause of cancer death in females worldwide. 1 Triple-negative breast cancer (TNBC) is a subgroup of breast cancers that lack the expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2). 2 The risk of distant recurrence and metastasis in TNBC patients is substantially higher than in non-TNBC patients. 3 The clinical management of TNBC is challenging because of the relatively aggressive biologic behaviour and paucity of specific targeted therapy. 4 Thus, a better understanding of the regulations and mechanisms of tumorigenesis in TNBC cells and the identification of effective biomarkers for diagnosis and prognosis of TNBC patients are consequently keenly awaited.
LncRNAs, with a length exceeding 200 nucleotides, are non-protein-coding transcripts. 5 Accumulating studies report that lncRNAs expression is dysregulated in various types of cancer including breast cancer, ovarian cancer, hepatocellular carcinoma and many others. [6][7][8][9][10] Moreover, several lncRNAs have been reported to play crucial roles in various biological processes, including cell proliferation, apoptosis, invasion, differentiation and development. [11][12][13][14] In TNBC, various studies have identified a lot of dysregulated lncRNAs that play important roles in the process of tumorigenesis through diverse mechanisms. For instance, lncRNAs can act as miRNA 'sponges' and compete miRNA-targeted mRNAs, thereby affecting the miRNA-mediated gene regulation. 15,16 This competing endogenous RNAs (ceRNA) mechanisms and network construction, by sequestering miRNAs and sparing their protein-coding counterparts from post-translational regulation, have been mainly studied to act as the main molecular mechanism of lncRNA biological function. 15 Some lncRNAs were reported to assemble with mRNAs to protect them from miRNA action and increase their stability. Some lncRNAs are named scaffold lncRNAs, which could serve as a central platform to assemble with different molecular components such as proteins and RNAs and promote their intermolecular interactions. Moreover, signal lncRNAs have also been reported to interact with transcription factors (TFs) or histone-modifying enzymes to cis-regulate or trans-regulate the expression of their target genes. 8 Thus, lncRNAs promise potential diagnostic and prognostic biomarkers, therapeutic targets and improve the clinical benefits for TNBC patients.
Accumulating studies have explored the potential ln-cRNAs-based therapeutics in TNBC, including the techniques of genetic modification using antisense oligonucleotides (ASOs), locked nucleic acid (LNA) and RNA nanotechnology. Such as, Jin et al designed eight ASOs targeting LncRNA TROJAN and transfected TNBC cells with ASOs without using any transfection reagents to simulate in vivo conditions. They observed that lung metastasis nodules were significantly reduced in anti-TROJAN ASO-treated group than the control group, and the ASO toxicity was limited after detecting the murine blood biochemical indexes. 17 Hu et al reported that treatment with LINK-A LNAs could repress cell proliferation in TNBC cells and increase the sensitivity of mammary gland tumours to immunotherapy. 18 In current review, we accumulated literature to the understanding of lncRNAs biogenesis and function, as well as the latest findings of novel lncRNAs-based therapeutics in TNBC. We also present the current state of knowledge concerning the expression and regulation of lncRNAs in TNBC, and discuss the future development of lncRNA-based strategies for clinical TNBC patients.

| B IOLOG IC AL CHAR AC TERIS TIC S OF LN CRNA S
LncRNAs are functionally defined as transcripts >200 nt in length with no protein-coding potential, many of which are uniquely expressed in differentiated tissues or specific cancer types. 19 Distinguishing lncRNAs from other protein-coding mRNAs is not a trivial process. H19, the first lncRNA reported by Brannan et al in 1990, was just defined as not a classical mRNA, and the product of H19 gene was described to be an RNA molecule. 20 In fact, lncR-NAs were first described during the large-scale sequencing of fulllength cDNA libraries in the mouse. 21 The number of lncRNAs was reported to outnumber protein-coding genes, and their sequences cover a larger fraction of the human genome. 22 LncRNAs may be located within nuclear or cytosolic fractions, and are overlapping with, or interspersed between, multiple coding and non-coding transcripts. 23, 24 Based on their genomic proximity to neighbouring transcripts, they are classified five categories ( Figure 1): (a) sense, overlapping one or more exons of a protein-coding gene on the same strand; (b) antisense, overlapping one or more exons of a protein-coding gene on the opposite strand; (c) bidirectional, initiating its expression in close genomic proximity at <1000 base pairs away to a neighbouring coding transcript on the opposite strand; (d) intronic, deriving from an intron of a second transcript; or (e) intergenic, acting as an independent unit within the genomic interval between two genes. 25,26 LncRNAs were initially thought to be the products of an inconsequential transcription resulting from low RNA polymerase fidelity. 27 It is now widely recognized that lncRNAs could identify cellular pathologies such as cancer, provide prognostic value, or even inform therapeutic options for cancer patients, by serving as signals of specific cellular states or readouts of active cellular programmes. 28 Recent studies have shown that lncRNAs can regulate gene expression at different levels, including chromatin modification, transcription and post-transcriptional regulation. 29 LncRNAs were reported to regulate several biological processes such as cell proliferation, apoptosis, cell cycle, cell invasion and metastasis, cellular differentiation, chromatin modification and nuclear-cytoplasmic trafficking. 30 It has been suggested that the involvement of lncRNAs in human diseases could be far more prevalent than previously known. 31 Recently, lncRNAs-related studies in cancer increased dramatically and have become one of the hottest topics in RNA biology.

| PROFILE S OF LN CRNA S E XPRE SS I ON IN TNBC
Recently, abnormal expression of many lncRNAs has been found in almost all tumours in humans, including TNBC. However, our understanding of lncRNAs remains significantly less mature than mRNAs, or even miRNAs. Next generation sequencing (NGS) is a DNA sequencing technology, which could perform sequencing of millions of small fragments of DNA in parallel. These fragments are then pieced together by mapping the individual reads to the human reference genome. 32 NGS is now used to sequence entire genomes or constrained to specific areas of interest to get the population genomic and gene expression differences in a large array of organisms. 32 Thus, NGS technologies may help researchers to accelerate the identification and characterization of important, yet-to-be-annotated functional lncRNAs in TNBC.
In recent years, researchers got a lot of abnormally expressed lncRNAs in TNBC patients or cells using public databases based on the NGS technologies. Tian et al found a total of 1034 dysregulated lncRNAs in the two TNBC microarrays from the Gene Expression Omnibus (GEO) database. Among them, 537 lncRNAs were significantly correlated with 451 protein-coding genes, which were mainly enriched in terms including cell division, cell cycle, and involved in PI3K-Akt, MAPK, ErbB family and p53 signalling pathways. 33 In addition, further literatures related to the lncRNA expressions profiles also detected a series of dysregulated lncRNAs in TNBC. [34][35][36][37][38][39][40] Previous studies have shown that lncRNAs can act as miRNA 'sponges' and compete miRNA-targeted mRNAs, thereby affecting the miRNA-mediated gene regulation. 15,16 This crosstalk forms a complex post-transcriptional regulatory network including mRNAs, lncRNAs, The Cancer Genome Atlas (TCGA) database and two GEO databases. 41 As a result, they identified differentially expressed 4565 mRNAs, 427 miRNAs and 4852 lncRNAs, and constructed ceRNA network using 37 lncRNAs, 28 miRNAs and 16 mRNAs. On the basis of establishing the ceRNA network, they found that two mRNAs expression are correlated with prognosis of TNBC patients. 41 Similarly, Liu et al also constructed a ceRNA network based on analysis of differentially expressed RNAs between 150 TNBC tissues and 823 non-TNBC tissues downloaded from TCGA database. 42 They identified 190 differentially expressed lncRNAs, 48 differentially expressed mRNAs and 13 differentially expressed miRNAs in this ceRNA network. They concluded that eight lncRNAs and one mRNA could act as prognostic factors in TNBC, using survival analysis and receiver operating characteristic (ROC) curve creation in the network. 42 Additionally, they found that lncRNA OSTN-AS1 was primarily related to immunologic function, including immune cell infiltration and immune-related markers co-expression. 42 45 They also found that the prognostic and predictive accuracy of this ceRNA signature was better than clinicopathological parameters to predict tumour recurrence and the benefit of taxane chemotherapy in TNBC. 45 Taken together, the ceRNA co-regulatory network could help us understand the potential charac-

| ROLE S OF LN CRNA S IN TNBC
To date, numerous lncRNAs have been identified to dysregulated express and play an important role in the biological function of TNBC, including cellular proliferation, apoptosis, cell cycle, migration, invasion, angiogenesis and drug resistance (Table 1). In this chapter, we will provide an overview of lncRNA biological function in TNBC ( Figure 2).

| lncRNAs involved in the regulation of cell proliferation and apoptosis
Cancer has been considered to be the result of accumulated gene mutations, which led to uncontrolled cell proliferations. And deregulated cell proliferation and inhibition of cell apoptosis lie at the heart of tumour development. 46 The role of lncRNAs in the regulation of TNBC cell proliferation and apoptosis has also been widely investigated. ceRNA mechanisms and network construction, by sequestering miRNAs and sparing their protein-coding counterparts from post-translational regulation, have been mainly studied to act as the main molecular mechanism of lncRNA biological function. 15 For example, lncRNA GAS5 was reported to promote apoptosis and inhibit proliferation of TNBC cells by targeting miR-196a-5p and miR-378a-5p/SUFU signalling. [47][48][49] LINC00096 promoted cell proliferation by sponging miR-383-5p and regulating RBM3 expression in TNBC. 50 LncRNA HEIH was shown to regulate cell proliferation and apoptosis through miR-4458/SOCS1 axis in TNBC. 51 LncRNA HCP5 could also promote cell proliferation and inhibit cell apoptosis as a ceRNA to regulate BIRC3 by sponging miR-219a-5p. 52 LINC00339 promoted cell proliferation and inhibited cell apoptosis through miR-377-3p/ HOXC6 signalling pathway. 53 Knockdown of lncRNA HOST2 could inhibit the proliferation of TNBC cells via regulation of the let-7b/ CDK6 axis. 54 There are also other molecular mechanisms of LncRNAs. Some lncRNAs assemble with mRNAs to protect them from miRNA action and increase their stability. Some lncRNAs are named scaffold lncRNAs, which could serve as a central platform to assemble with different molecular components such as proteins and RNAs and promote their intermolecular interactions. Signal lncRNAs have also been reported to interact with transcription factors (TFs) or histone-modifying enzymes to cis-regulate or trans-regulate the expression of their target genes. 8 For instance, in TNBC, Tariq et al revealed that LINC02095 promotes breast cancer proliferation by facilitating the expression of oncogenic transcription factor, SOX9. 55 LncRNA DANCR was reported to bind with RXRA and increase its serine 49/78 phosphorylation, leading to activating PI3K/ Akt signalling and TNBC cell proliferation. 56 Shen et al demonstrated that LINC00152 obviously enhanced NEDD4-1-mediated ubiquitination and degradation of PTEN protein in TNBC. 57 Meanwhile, Wu et al also revealed that LINC00152 could enhance TNBC tumorigenesis by inactivation of the BRCA1/PTEN through DNA methyltransferases. 58 Besides, LncRNA PDCD4-AS1 was reported to stabilize PDCD4 RNA by forming RNA duplex and regulate the interaction between PDCD4 RNA and RNA decay promoting factors such as HuR. 59 Several studies have indicated that LncRNAs could also play an important role in the TNBC cell proliferation and apoptosis process by regulating other LncRNAs. LncRNA DRHC was shown to inhibit TNBC cells proliferation by down-regulating the expression of ln-cRNA HOTAIR, while HOTAIR did not affect the expression level of DRHC. 60 Similarly, LncRNA PTCSC3 overexpression led to down-regulated lncRNA H19 in TNBC cells, while H19 overexpression did not affect PTCSC3 expression. 61  was also shown to contribute to accelerate cell cycle progression through modulating miR-5702. 64 Besides, Wang et al reported that LINC00339 inhibited cell cycle arrest at G 0 /G 1 phase by sponging to miR-377-3p and activating miR-377-3p/HOXC6 signalling pathway in TNBC. 53 Shin et al revealed that LncRNA NEAT1 conferred oncogenic role by regulating cell cycle progression in TNBC cells. 75 LncRNA RMST was also shown to induce the block of G 0 /G 1 phase in TNBC. 76

| lncRNAs involved in the regulation of cell invasion and metastasis
Metastasis is the major cause of cancer-related deaths. It has been increasingly recognized that lncRNAs play important roles in tumour invasiveness and metastasis. Overexpression of GAS5 was shown to undermine the tumour promotion effect induced by ectopic expression of miR-196a-5p, including cell invasion and FOXO1/PI3K/Akt signal pathway activation. 49  NAMPT-AS could recruit POU2F2 to activate the transcription of NAMPT, or serve as a ceRNA to rescue NAMPT degradation from miR-548b-3p in TNBC. 79 Epithelial-mesenchymal transition (EMT) has been involved in carcinogenesis and confers metastatic properties upon cancer cells by enhancing cell mobility, invasion and resistance to apoptotic stimuli. 80 Zhang et al revealed that AFAP1-AS1 could activate Wnt/β-catenin pathway to promote tumorigenesis and cell invasion by inducing the expression of c-myc and EMT-related molecules in TNBC. 81 MiR-34a was reported to implicate in certain EMT-associated signal pathways to repress tumorigenesis, cancer progression and metastasis. 82 LncRNA PAPAS was shown to promote migration and invasion of TNBC cells by down-regulating miR-34a. 83 LncRNA sONE was also reported to repress endothelial nitric oxide synthase (eNOS)-induced nitric oxide (NO) production, regulating TP53 and c-Myc proteins levels and finally altering the levels of a panel of tumour-suppressor miRNAs, including miR-34a, miR-15, miR-16 and let-7a. 84  The results of our studies also showed that lncRNA androgen

| lncRNAs involved in the regulation of drug resistance
Emerging evidences suggest that lncRNAs could implicate in regu-  111  Importantly, blocking LINP1 could increase the sensitivity of the tumour-cell response to radiotherapy in TNBC. 117 Additionally, knockdown of lncRNA PCAT6 promoted the radiosensitivity of TNBC cells through regulating miR-185-5p/TPD52 axis. 118 Taken together, these studies evoke the potential of altering lncRNAs expression in future to represent a novel therapeutic approach to reverse drug resistance or radiotherapy resistance in TNBC patients. However, further studies and mechanistic investigations of the regulation mechanism of lncRNAs-mediated drug resistance in TNBC are needed in the future.

| Others
Several recent studies also demonstrated that lncRNAs could im-

| LN CRNA AC TS A S B I OMARK ER FOR D IAG NOS IS AND PROG NOS IS IN TNBC
Since various lncRNAs have been found to be differentially expressed epigenome-wide association study (EWAS) and identified that LINC00299 is high methylated in TNBC patients' peripheral blood, making hypermethylation of LINC00299 a useful circulating biomarker for TNBC patients. 136 LncRNAs were also be reported to predict responses to therapy, including chemotherapy, radiotherapy and immunotherapy. One study has showed that circulating lncRNA H19 was high expressed and could predict the response to neoadjuvant chemotherapy (NAC) in TNBC patients. 137 They found patients with a pathological complete response (pCR) had lower pre-therapeutic levels of lncRNA H19 compared with the non-complete responders. Meanwhile, patients with higher degree of downstaging of initial tumours had lower baseline levels of lncRNA H19 among non-complete responders. 137 Those data suggested that circulating lncRNA H19 may be a useful marker for predicting the response to neoadjuvant chemotherapy. Another study determined that lncRNA LINK-A could predict immunosuppression and immunotherapy resistance. 18 138 They also found higher lncAFAP1-AS1 expression was correlated with poor disease-free survival and overall survival of TNBC patients. 138 These results demonstrated that high lncAFAP1-AS1 expression is associated with radio-resistance of TNBC patients, and the expression level of lncAFAP1-AS1 in tumour tissues could be used to predict the outcome of TNBC radiotherapy.
In conclude, it was noticeable that lncRNAs might be more reliable diagnostic and prognostic biomarkers for TNBC patients as a result of its aberrant expression in tumorigenesis ( Figure 6). However, in the future, lncRNA diagnosis and prognosis biomarker studies will need to specify more focus on the serum circulating lncRNA and predicting the response to therapy, including chemotherapy, radiotherapy, targeted therapy and immunotherapy. Additionally, further investigation of a larger patient population is necessary to confirm the diagnostic and prognostic evaluation of lncRNAs in TNBC patients.

| LN CRNA-BA S ED THER APEUTI C S IN TNBC
Since lncRNAs play crucial roles in the biological processes and tumorigenesis and abnormal expression of lncRNAs occur in multiple human cancers, this presents with lncRNA-based therapeutics possibility to correct this dysregulation. Recently, accumulating studies indicating the significance of lncRNAs in the regulation of TNBC development and drug resistance accelerated the investigation to explore the potential lncRNAs-based therapeutics in TNBC ( Figure 6).
Antisense oligonucleotides (ASOs) were able to modulate RNA processing and protein expression through different mechanisms, making them able to serve as a variety of molecular targets. 139 Recently, advancements of ASO structure and chemical F I G U R E 6 lncRNA act as biomarker for diagnosis and prognosis in TNBC modifications greatly improved the advantage and effectiveness of ASOs to act as precious tools to understand disease mechanisms and as valuable therapeutics for disease intervention. 140 Moreover, many ASOs are undergoing clinical trials, taking advantage of the various mechanisms and synthetic structures now available for the design of ASOs-based therapies. 140 ASOs also were used to inhibit the expression of lncRNAs for lncRNA-based therapeutics in TNBC.
LncRNA TROJAN was shown to promote TNBC proliferation and metastasis, and correlated with poor patient survival. 17 148 They demonstrated that the combination of low-frequency ultrasound (LFUS) irradiation and nanobubble complexes was regarded as an efficient and safe method for siRNA transfection. 148 Recently, another study engineered a reduction-responsive nanoparticle (NP) platform for effective lncAFAP1-AS1 siRNA (siAFAP1-AS1) delivery, and reported that systemic delivery of siAFAP1-AS1 with the reduction-responsive NPs can synergistically reverse radio-resistance by scavenging intracellular glutathione, leading to a dramatically enhanced radiotherapy effect in both xenograft and metastatic TNBC tumour models. 138 Overall, these results demonstrate that this RNA nanoparticle-based targeted therapy by nanoparticle-mediated modulation of onco-lncRNAs is a promising approach that utilizes chemically modified RNPs for tumour-specific targeting and lncRNA inhibition that will be beneficial in TNBC and other cancers setting where lncRNA knockdown is desired for a better clinical output. 138,147,148

| CON CLUS I ON S AND FUTURE PROS PEC TS
Overall, recent evidences suggest that many lncRNAs were abnor- In the future, (a) lncRNA diagnosis and prognosis biomarker studies will need to specify more focus on the serum circulating lncRNA and predicting the response to therapy, including chemotherapy, radiotherapy, targeted therapy and immunotherapy.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
WZ, XG and JT conceived the study and wrote the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.