Regulation of mTOR signaling by long non-coding RNA

Abstract

The mechanistic target of rapamycin (mTOR) is a major signaling hub that coordinates cellular and organismal responses, such as cell growth, proliferation, apoptosis, and metabolism. Dysregulation of mTOR signaling occurs in many human diseases, and there are significant ongoing efforts to pharmacologically target this pathway. Long noncoding RNAs (lncRNA), defined by a length > 200 nucleotides and absence of a long open-reading-frame, are a class of non-protein-coding RNAs. Mutations and dysregulations of lncRNAs are directly linked to the development and progression of many diseases, including cancer, diabetes, and neurologic disorders. Recent findings reveal diverse functions for lncRNA that include transcriptional regulation, organization of nuclear domains, and regulation of proteins or RNA molecules. Despite considerable development in our understanding of lncRNA over the past decade, only a fraction of annotated lncRNAs has been examined for biological function. In addition, lncRNAs have emerged as therapeutic targets due to their ability to modulate multiple pathways, including mTOR signaling. This review will provide an up-to-date summary of lncRNAs that are involved in regulating mTOR pathway.

1. Introduction

The mTOR pathway is an evolutionary conserved signaling pathway that senses and integrates extra- and intra-cellular signals to coordinate basic cellular and organismal responses such as cell growth, proliferation, apoptosis, and metabolism [1]. mTOR is one of the most frequently dysregulated signaling cascades in human diseases and malignancies [2,3]. mTOR is a serine/threonine protein kinase in the PI3K-related family that forms the catalytic subunit of two structurally and functionally distinct protein complexes known as mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) [4,5]. mTOR is activated by diverse stimuli such as growth factors [6], nutrients [7], and energy and stress signals [8] in order to control cell growth, proliferation, and survival. mTORC1 has five components: mTOR, mLST8, Raptor, PRAS40, and Deptor (Fig. 1A), and is sensitive to the immunosuppressant and anticancer drug rapamycin. mTORC1 controls gene transcription, ribosomal biogenesis, protein synthesis, autophagy, and other growth-related processes [9]. Downstream bona fide targets of mTORC1 include S6 kinase 1 (S6K1) and eIF4E-binding protein 1 (4E-BP1) [10]. mTORC2 has six components: mTOR, Rictor, mSIN1, Protor, mLST8, and Deptor (Fig. 1B). The function of mTORC2 is less well understood and is thought to promote cell survival and organization of the actin cytoskeleton [11]. mTORC2 is resistant to rapamycin and generally insensitive to nutrients and energy signals. Cell survival is mediated primarily through activation of AKT phosphorylation at Ser473 [12], as well as other members of the protein kinases A, G, and C family [13]. Activation of mTORC1 occurs by upstream regulators, such as phosphatidylinositol 3-kinase (PI3K), PDK1, TSC1-TSC2 complex, and Rheb [14]. However, the mechanism by which growth factors activate mTORC2 is not clear. The crucial role of mTOR in cell biology has generated interest in mTOR inhibitors, many of which have already undergone clinical trials for disease treatment, mainly cancer. Limitations of mTOR inhibitors, such as rapamycin and many of its analogs, include toxicity, sensitivity, and unwanted side effects such as hyperinsulinemia, glucose intolerance, and dysregulated autophagy [15]. Limitations also arise at the molecular level; for example, hyperphosphorylation of 4E-BP1, which is pathogenic in human cancer, is usually resistant to rapamycin/rapalogs [16]. Many proteins and signaling molecules are known to control mTOR activity; however, recent studies have identified long non-coding RNAs (lncRNAs) as novel regulators of the mTOR pathway.

Fig. 1.

The mTOR signaling pathway. Protein components and downstream effectors of A) mTORC1 and B) mTORC2.

Although a large number of RNA species are transcribed from the mammalian genome, only a small fraction of the total transcripts is protein-coding [17]; the rest of the transcripts are non-coding RNAs (ncRNAs). There are two major classes of regulatory ncRNAs based on their size: small ncRNAs (< 200 nt in length) represented by micro- RNAs, and long ncRNAs (lncRNAs) (> 200 nt in length) [18,19]. LncRNAs are classified into six groups, based on genomic organization relative to protein-coding genes (PCGs), which is summarized in Fig. 2. There are a large number of lncRNAs reported in different databases, such as the encode database (http://www.encode.com), noncode database (http://www.noncode.org), and LNCipedia (http://www.lncipedia.org). The number of reported lncRNA ranges from < 20,000 to over 100,000 in humans [20]. The large variation in the number of lncRNAs among databases results from the type of annotation method, whether automated and/or manual. Both types of annotations suffer from tradeoffs between quality and size, and are comprehensively reviewed by Uszczynska-Ratajczak et al. [21]. LncRNAs display several similarities to PCGs; the majority of lncRNAs are transcribed by Pol II, and harbor histone modifications associated with Pol II transcriptional elongation [22]. Furthermore, lncRNAs are 5′-capped [23] and many are polyadenylated [24]. Unlike PCGs, lncRNA do not always display evolutionary sequence conservation [25] and most have limited protein-coding potential [26]. Another distinctive feature of lncRNA is tissue- and cell type-specific expression [27]. This feature is clearly evident from studies in our laboratory focusing on lncRNAs in the mouse kidney, in which a small fraction of the annotated murine lncRNA (< 10%) were expressed in the adult mouse kidney [28]. Until recently, lncRNAs were considered to be transcriptional noise; however, it has become increasingly evident that many lncRNAs are involved in numerous biological functions and pathological processes, including development, proliferation, apoptosis, survival, differentiation, and many malignancies [29–32]. LncRNAs were found to influence cellular activities and biological pathways in different mechanisms. For example, some lncRNAs regulate gene transcription by interacting with transcriptional complexes, DNA elements, and chromatin structures [33], while others function post-transcriptionally by regulating mRNA stability, splicing and modifications, as well as protein translation [34]. lncRNA function, in part, can be deduced from the cellular localization of the transcript. For example, MALAT1, a well-known nuclear lncRNA, is involved in the assembly of nuclear speckles and transcriptional regulation of metastatic genes [35–37]. NORAD, on the other hand, is abundant in cytoplasm, and regulate genomic stability by sequestering PUMILIO proteins that repress the stability and translation of mRNAs to which they bind [38,39].

Fig. 2.

Positional classification of lncRNAs relative to nearest protein-coding-gene (PCG). Intronic lncRNAs are found within an intron of a PCG, whereas exonic lncRNAs overlap with an exon of a PCG. Both can be transcribed in a sense or anti-sense direction. Bidirectional lncRNAs share the same promoter of nearby PCG. Intergenic lncRNAs donot overlap with a PCG.

This review will summarize the latest findings that implicate lncRNAs in the regulation of mTOR signaling. We will provide a detailed description of lncRNAs that regulate mTOR signaling in an identified mechanism of action, and briefly discuss other lncRNAs that influence mTOR signaling in various conditions and diseases. We will then discuss the significance of these findings in the context of lncRNAs as potential therapeutic targets.

2. lncRNAs and mTOR signaling

In the past decade, many studies have reported dysregulation of mTOR signaling in response to altered expression of lncRNA and vice versa [40]; however, only a small number of these studies describe the mechanism of regulation. For example, some lncRNAs influence mTOR activity by directly binding to components of the mTOR complex, while others regulate proteins that are upstream or downstream of mTOR. In addition, some lncRNAs function via a lncRNA-miRNA network that affects mTOR signaling. Other studies describe correlative effects between lncRNA expression and mTOR signaling. All the lncRNAs described in this review are listed in Table 1.

Table 1.

mTOR-Associated lncRNAs.

lncRNA	Expression	Disease	Effect on mTOR	Mechanism	Reference
DLEU1	Upregulated	Endometrial cancer	Activation	Binds mTOR complex	Du et al., 2018 [46]
HAGLROS	Upregulated	Gastric cancer	Activation	1- Interacts with mTOR, Raptor, and PRAS40 2- Antagonize miR-100	Chen et al., 2018 [47]
NBR2	Downregulated	Cancer cells	Suppression	Binds to AMPK	Liu et al., 2016 [49]
H19	Downregulated	Pituitary cancer	Suppression	Binds to and prevents 4E-BP1 phosphorylation	Wu et al., 2018 [56]
FA2H-2	Downregulated	Atherosclerosis	Suppression	Represses MLKL transcription	Guo et al., 2019 [57]
LINC00152	Upregulated	Hepatic cancer	Activation	Increases EpCAM transcription	Ji et al., 2015 [61]
MALAT1	Upregulated	Hepatic cancer	Activation	Increases expression of S6K1-isoform 2	Malakar et al., 2017[68]
MetaLnc9	Upregulated	Non-small cell lung cancer	Activation	Binds to and stabilizes PGK1 expression	Yu et al., 2017 [69]
Dlk1-Gtl2 locus	Downregulated	Normal liver development	Suppression	Give rise to miRNA cluster that targets mTOR pathway	Qian et al., 2016[72]
BFAL	Upregulated	Gut microbiota-induced colorectal cancer	Activation	Regulates RHEB expression by sponging miR-155–5p and miR-200a-3p	Bao et al., 2019 [73]
DANCR	Upregulated	Lung cancer	Activation	Antagonize miR-496	Lu et al., 2018 [80]
ANRIL	Upregulated	Gastric cancer	Activation	Repression of miR-99a and miR-449a	Zhang et al., 2014 [83]
GAS5	Downregulated	Multiple cancers	Repression		Mourtada-Maarabouni et al., 2009 [87] Pickard et al., 2013 [88] Cao et al., 2014 [89] Sun et al., 2014 [90]
Hoxb3os	Downregulated	Polycystic kidney disease	Suppression		Aboudehen et al., 2018 [28]
CRNDE	Upregulated	Glioma	Activation		Wang et al., 2015 [93]
UCA1	Upregulated	Bladder cancer	Activation		Li et al., 2014[95]
NEAT1	Upregulated	1) Diabetic Nephropathy 2) Nonalcoholic fatty liver disease	Activation		Huang et al., 2019 [98] Wang et al., 2018 [77]
HOTAIR	Upregulated	1) Hepatic cancer 2) Intervertebral degeneration	Activation Suppression		Wei et al., 2017 [102] Yang et al., 2019 [103] Zhan et al., 2019 [104]
ZNNT1	Downregulated	Uveal melanoma	Suppression		Li et al., 2019 [105]
HULC	Upregulated	Human Gliomas	Activation		Zhu et al., 2016 [110]

2.1. lncRNAs that directly interacts with mTOR complex

Two lncRNAs physically interact with the mTOR complex: DLEU1 and HAGLROS. In both studies, lncRNA binding to the mTOR complex was determined by RNA immunoprecipitation. Thus, additional experiments, such as RNA/protein crosslinking-based methods, are needed to validate and determine the level of interaction.

-DLEU1 (Deleted in lymphocytic leukemia 1)

This lncRNA is located at the 13q14.3 in humans, a chromosomal region that is recurrently deleted in B-cell chronic lymphocytic leukemia (CLL) [41]. Because 13q14 is a known tumor suppressor locus that contains the Retinoblastoma (RB1) gene, DLEU1 was thought to be a tumor suppressor gene. However, the DLEU1 prompter is hyperactive in leukemia cells and causes silencing of nearby tumor suppressor genes [42]. In addition, DLEU1 functions as an oncogene in various carcinomas including colorectal, gastric, ovarian, and oral [43–45]. Evidence implicating DLEU1 in mTOR signaling is described in endometrial cancer, in which DLEU1 levels are elevated in patients with the disease compared to healthy subjects [46]. In vitro overexpression of DLEU1 results in increased cell viability, migration, invasion, and decreased apoptosis, whereas DLEU1 knockdown produces the opposite effects [46]. At the molecular level, DLEU1 overexpression results in increased mTOR phosphorylation and activation of downstream targets, including pS70K. RNA immunoprecipitation (RIP) assays reveal that DLEU1 is immunoprecipitated with an antibody against mTOR protein. The results of this study indicate that DLEU1 enhances tumorigenesis by binding mTOR protein and activating mTORC1.

-HAGLROS (HAGLR opposite strand)

Is a lncRNA that localizes to the HoxD cluster of homeotic genes and overlaps with another lncRNA called HAGLR (HoxD antisense growth-associated long-oncoding RNA) [47]. Unlike HAGLR, which is involved in the postnatal growth and development of the mouse, little is known about the role and function of HAGLROS. Recent studies reveal that overexpression of HAGLROS contributes to the malignancy of colorectal and gastric cancer cells by decreasing cell apoptosis and autophagy [48]. In gastric cancer, HAGLROS is activated as a result of increased binding of STAT3 transcription factor to the promoter region [47]. HAGLROS activation increases total and phospho-mTOR and decreases autophagy. Mechanistically, HAGLROS activates mTOR signaling and inhibits autophagy in dual manner. On one hand, HAGLROS functions as a competing endogenous RNA (CeRNA) that antagonizes miR100–5p-mediated degradation of mTOR. On the other hand, HAGLROS interacts directly with mTOR, Raptor, and PRAS40, which stabilizes mTORC1 activity [47].

2.2. lncRNAs that function upstream or downstream of mTOR signaling

A number of lncRNAs modulates mTOR pathway by regulating molecules that are upstream and downstream of mTOR complex (Fig. 3). These lncRNAs include:

Fig. 3.

lncRNAs that function upstream and downstream of mTOR signaling.

-NBR2 (Neighbor of BRCA1 gene 2)

Liu et al. identified NBR2 as a glucose starvation-induced lncRNA in various cancer cell lines, excluding Hela or A549 cells, which lack the tumor suppressor liver kinase B1 (LKB1) gene [49]. Re-expression of Lkb1 restores energy stress-induced NBR2, while AMPK inactivation lowers NBR2 induction. The data indicate that NBR2 function through the LKB1-AMPK pathway. Further analysis reveals a NBR2-AMPK feedback-forward loop, in which AMPK induces NBR2 expression and NBR2, in turn, potentiates AMPK activity. RNA-pulldown assays reveal that NBR2 interacts with AMPKα subunit and the interaction significantly increases in response to glucose starvation. AMPK is an upstream negative regulator of mTORC1 signaling that acts by phosphorylating Raptor and the TSC1-TSC2 complex. AMPK also promotes autophagy through direct phosphorylation of the autophagy regulator ULK1 [50,51]. As a result, the investigators examined whether mTOR signaling and cell autophagy are influenced by NBR2 activity. NBR2 knockdown rendered cancer cells partially resistant to energy stress-induced mTORC1 inactivation, while NBR2 overexpression repressed mTORC1 activity. In addition, energy stress-induced autophagy was defective in NBR2 deficient cells. Results of the study indicate that NBR2 represses mTORC1 activity by binding and activating AMPK.

-H19 (Homo sapiens H19 imprinted maternally expressed transcript)

H19 is an imprinted lncRNA that is exclusively expressed from the maternal allele and is in close proximity to the insulin-like growth factor 2 (IGF2), which is produced from the paternal allele [52,53]. The H19 locus also harbors a microRNA, miR-675, which controls placental growth by regulating the expression of the Igf1 gene [54]. Deletion of the H19 locus in the mouse results in an over-growth phenotype that is rescued by overexpression of H19 [55]. Wu et al. found that H19 expression is downregulated in human pituitary adenomas and expression negatively correlates with tumor progression [56]. Restoring H19 expression in tumor cells inhibits cell proliferation and tumor growth. At the molecular level, H19 suppresses 4E-BP1 phosphorylation without affecting S6K1 phosphorylation. To identify the underlying mechanism of H19 specificity towards 4E-BP1 phosphorylation, immunoprecipitation and RNA pulldown assays were performed and reveals that H19 directly binds to 4E-BP1, which prevents Raptor from binding and phosphorylating 4E-BP1.

-FA2H-2:

Guo et al. examined the role of lncRNAs in atherosclerosis by treating cells with oxidized low-density lipoprotein (OX-LDL), a model that induces inflammation and causes atherosclerosis [57]. OX-LDL treatment results in increased expression of the mixed lineage kinase domain-like protein (MLKL), which is known to attenuate autophagy [58], as well as decrased levels of the nearby FA2H-2 lncRNA. In addition, silencing of FA2H-2 or overexpression of MLKL activates inflammation and inhibits autophagy in an mTOR-dependent manner, which suggest that MLKL and FA2H-2 have antagonizing effects on mTOR signaling. Further analysis reveal that FA2H-2 knockdown increases MLKL expression, which indicates that FA2H-2 is an upstream inhibitor of MLKL. This was confirmed by performing Chromatin Isolation by RNA Purification (ChIRP) and luciferase reporter assays, which reveal binding of FA2H-2 to MLKL promoter and repression of the MLKL transcript. The results of this study indicate that FA2H-2 has a therapeutic effect during atherosclerosis by inhibiting MLKL expression and silencing mTOR signaling.

-LINC00152:

This lncRNA was initially identified as a differentially hypomethylated gene in hepatic cancer and is overexpressed in many tumors including lung and gastric cancer [59,60]. Ji et al. found mTOR signaling to be highly active in hepatic cancer cells that overexpress LINC00152 [61]. They also reveal that EpCAM, a gene located near LINC00152, is upregulated in response to LINC00152 overexpression. EpCAM is a type I transmembrane glycoprotein that is overexpressed in a large variety of human epithelial-derived neoplasms, and previous studies describe EpCAM as an upstream inducer of mTOR signaling [62–64]. Luciferase reporter gene assays reveal that knockdown of LINC00152 decreases the promoter activity of EpCAM, and tethering of LINC00152 to the EpCAM reporter stimulates the transcription of the reporter [61]. The results of the study suggest that LINC00152-dependent mTOR hyperactivation results from EpCAM overexpression, whereby LINC00152 binds to and increases EpCAM promoter activity, which causes an increase in mTOR activity.

-MALAT1 (Metastasis-associated lung adenocarcinoma transcript 1):

MALAT1 is one of the most abundantly expressed lncRNA, which was originally identified in metastatic non-small-cell lung cancer cells [65,66]. MALAT1 localizes to nuclear bodies known as nuclear speckles, a region that contains a distinct set of pre-mRNA splicing factor, and specifically interacts with SRSF1, 2, and 3 [35,67]. Malakr et al. reveal that upregulation of MALAT1 in hepatocellular carcinoma causes increased mTOR signaling via effects on the splicing factor SRSF1 [68]. Induction of SRSF1 by MALAT1 modulates SRSF1 splicing targets, one of which is S6K1. In response to activation by MALAT1, SRSF1 favors the formation of S6K1-isoform 2, a shorter spliced variant of S6K1. S6K1-isoform 2 possesses an oncogenic activity that binds and activates mTORC1, which results in increased phosphorylation of 4E-BP1.

-MetaLnc9:

MetaLnc9, also known as LINC00963, is located on chromosome 9 and has one transcript containing five exons [20]. MetaLnc9 is highly abundant in non-small cell lung cancer (NSCLC), and expression correlates with enhanced cell migration and invasion [69]. Yu et al. examined the biological function of MetaLnc9 in NSCLC, both in vitro and in vivo. They found that AKT/mTOR signaling was inhibited by MetaLnc9 knockdown and activated upon MetaLnc9 overexpression [69]. RNA-pulldown assay followed by mass spectrometry revealed that MetaLnc9 interacts with phosphoglycerate kinase 1 (PGK1) protein. This interaction prevents PGK1-mediated ubiquitination, which results in PGK1 accumulation and subsequent mTOR activation. The inhibitory effects of MetaLnc9 knockdown on AKT/mTOR signaling was rescued upon PGK1 overexpression and reversed by PGK1 knockdown. Results of this study demonstrate that MetaLnc9 directly binds to and stabilizes PGK1 protein, which leads to mTOR activation.

2.3. mTOR regulation through lncRNA-miRNA interaction

Studies identify several lncRNAs that influence mTOR signaling through a miRNA/mTOR regulatory network. These lncRNAs function in different mechanisms; for example, the Dlk1-Gtl2 locus harbors many lncRNAs that give rise to multiple miRNAs that target mTOR components. In contrast, BFAL1 and DANCR function as a competitive endogenous RNA (ceRNA) that sequester miRNAs and alleviate the mTOR-mediated repression. Other lncRNAs, such as ANRIL regulate mTOR activity through transcriptional activation or repression of miRNAs known to target mTOR.

-Dlk1-Gtl2 locus:

Is an imprinted locus, which contains three protein-coding genes (Dlk1, Rtl1, and Dio3) on the paternally inherited allele and produces multiple lncRNAs from the maternally inherited allele. These lncRNAs give rise to multiple miRNAs, including the largest miRNA mega-cluster in mammals (anti-Rtl1, which contains the miR-127/miR-136 cluster with 7 miRNAs, and Mirg, which contains the miR-379/miR-410 cluster with 39 miRNAs). miRNAs encoded within the Dlk1-Gtl2 locus have important cellular functions, such as regulation of skeletal muscle differentiation and neurogenesis [70,71]. In hematopoietic stem cells (HSCs), Dlk1-Gtl2 expression is required for the maintenance of HSC population in the mouse fetal liver, and loss of imprinting at the locus results in significant reduction of HSCs due to AKT-mTOR hyperactivation and subsequent metabolic abnormalities [72]. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. Investigators identified 21 genes that are distributed throughout the PI3K-mTOR pathway and harbors one or multiple miRNA binding sites in their respective 3′ UTRs. Regulation of these targets by the miRNA cluster was confirmed by luciferase reporter and rescue experiments [72].

-BFAL1 (Bacteroides fragilis-associated lncRNA1)

Bao et al. identified BFAL1 as a novel lncRNA in colorectal cancer (CRC) that is caused by the Enterotoxigenic Bacteroides fragilis (ETBF), a known tumor-inducing bacterium in the human gut [73]. Patients with CRC display high levels of BFAL1, which correlates with tumor size and reduce survival. Infection of CRC cells with ETBF upregulates BFAL1, and injection of BFAL1 overexpressing cells into a xenograft mouse model results in increased tumorigenesis due to mTOR hyperactivation. Further analysis reveals that BFAL1 overexpression upregulates RHEB, a Ras homolog that binds and activates the mTOR complex. Mechanistically, BFAL1 upregulates RHEB post transcriptionally by binding and sequestering two miRNAs that normally target RHEB, miR155–5p and miR200a-3p [73].

-DANCR (Anti-differentiation ncRNA)

DANCR was identified as an oncogenic lncRNA in multiple malignant tumors [74,75]. DANCER is a known regulator of multiple miRNAs, [76,77] transcription factors [78], and chromatin modifiers [79]. Lu et al. reveal that DANCR levels are upregulated in lung cancer and inversely correlate with miR-496 expression [80]. Knockdown of DANCR by siRNA increased miR-496 levels and overexpression suppressed miR-496. Investigators utilized two assays to demonstrate mir-496/DANCR interaction. First, miR-496 mimic decreased the luciferase activity of a plasmid that harbors DANCR sequence. Second, a miRNA pulldown assay utilizing biotinylated miR-496 mimic reveals that miR-496 directly interacts with DANCR. Since miR-496 is known to target mTOR [81], investigators tested the hypothesis that DANCR can modulate mTOR signaling via miR-496. In vitro assays reveal that knockdown of DANCR reduced the levels of mTOR mRNA and protein, and the effects are reversed by a miR-496 inhibitor. The results of this study indicate that DANCR modulates mTOR activity by acting as a ceRNA for miR-496.

-ANRIL (Antisense Noncoding RNA in the INK4 locus):

ANRIL is a lncRNA that is transcribed in an antisense direction to the tumor suppressor gene CDKN2B (Cyclin Dependent Kinase Inhibitor 2B). ANRIL regulates CDKN2B expression by interacting with the Polycomb proteins PRC1 and PRC2 [82]. Zhang et al. found that expression of ANRIL is upregulated in patients with gastric cancer, and ANRIL levels negatively correlate with patient survival [83]. In vitro and In vivo assays reveal that silencing of ANRIL results in decreased cell proliferation and viability. At the molecular level, ANRIL represses the expression of miR-99a and miR-449a, two miRNA that are known to target mTOR. This is due to recruitment of the PRC2 complex to the miRNA promoter region, causing a decrease in miR-99a/miR-449a expression and subsequent activation of mTOR.

2.4. lncRNAs dysregulations that correlate with mTOR signaling

Many studies describe dysregulations in lncRNA expression in response to changes in mTOR activity and vice versa; however, the mechanism(s) behind lncRNA/mTOR dysregulations have yet to be determined Some of the lncRNAs are described below.

-GAS5 (Growth arrest-specific transcript 5):

GAS5 was originally identified as a tumor suppressor lncRNA that is induced in response to serum starvation or treatment with rapamycin [84]. Despite having a short open-reading-frame, GAS5 does not possess protein-coding capacity and acts as a host-gene for snoRNAs [85]. The induction of GAS5 transcript is regulated by the interplay between the mTOR and the nonsense-mediated decay (NMD) pathway, whereby GAS5 translation in active mTOR cells is rapidly degraded by the NMD pathway [86]. Thus many cancers, such as breast [87], prostate [88], cervical [89], and gastric [90], have deceased GAS5 expression due to mTOR hyperactivation. GAS5 elicits apoptosis and reduces proliferation by regulating multiple targets, such as c-Myc, Ybx1, cIAP2, and SGK1 [86]. The molecular mechanism by which mTOR inhibition results in GAS5 upregulation is not known.

-Hoxb3os (Homeobox B3 opposite strand):

This lncRNA is located in the HoxB cluster of homeotic genes and is transcribed from the antisense strand. Hoxb3os expression is highly abundant in the kidney and is downregulated in mouse and human kidneys with autosomal dominant polycystic kidney disease (ADPKD) [28]. In situ hybridization assays demonstrate that Hoxb3os transcript is primarily cytoplasmic and localizes to different segments of the nephron. Deletion of Hoxb3os in normal kidney cells results in increased phosphorylation of mTOR protein and activation of downstream targets of mTORC1, including p70S6 kinase, RS6, and 4E-BP1 [28]. Consistent with mTROC1 activation, Hoxb3os-mutant cells display enhanced mitochondrial respiration. The Hoxb3os-mutant phenotype is rescued upon re-expressing Hoxb3os in knockout cells using a lentiviral vector. The results of this study suggest that suppression of Hoxb3os may contribute to activation of mTOR pathway and restoration of Hoxb3os may have therapeutic benefits in ADPKD.

-CRNDE (Colorectal neoplasia differentially expressed):

CRNDE shares a bi-directional promoter with iroquois homeobox 5 (IRX5) gene and gives rise to 10 different transcript variants [91]. CRNDE expression is upregulated in a number of neoplastic diseases, specifically in tissues where the gene is not normally expressed [92]. In glioma cell lines and primary glioma samples from patients, CRNDE transcript variants two and four are upregulated by as much as 50-fold [93]. Overexpression or knockdown of CRNDE affects the growth, migration, and invasion of normal and glioma cells. Interestingly, knockdown of CRNDE does not alter the levels of total or phosphorylated mTOR, but results in increased expression of P70S6K, a downstream mTOR target [93], which suggests that CRNDE can modulate the downstream activity of mTORC1 signaling.

-UCA1 (Urothelial cancer associated 1):

UCA1 was first cloned and identified from a bladder cancer cell line [94]. Studies identified oncogenic functions for UCA1 in various cancers by upregulating mTOR signaling [40]. In bladder cancer cells, UCA1 activates mTOR signaling and results in increased glycolysis, in part due to activation of Hexokinase 2 (HK2), an enzyme that catalyzes the first and irreversible step in the ATP-dependent phosphorylation of glucose to yield glucose-6-phosphate [95]. Two major signals control HK2 expression as a result of mTOR activation: a) STAT3, an mTOR downstream effector, promotes HK2 transcriptional activation and b) mTOR dependent repression of miR-143, which normally targets HK2 mRNA, restores HK2 expression at the post-transcriptional level. Results of this study indicate that UCA1 increases glucose metabolism in cancer cells through the mTOR-STAT3/miR143-HK2 signaling cascade.

- NEAT1 (Nuclear paraspeckle assembly transcript 1):

NEAT1 is adjacent to the MALAT1 locus, and is an essential architectural component of paraspeckle nuclear bodies [96]. NEAT1 is upregulated and plays an oncogenic role in many solid tumor [97]. In diabetic nephropathy (DN), NEAT1 is upregulated and correlates with increased AKT/mTOR signaling, and NEAT1 silencing improves renal function and lowers mTOR activation in vitro [98]. These data imply that NEAT1 contributes to progression of DN by activating Akt/mTOR signaling. Similar findings for NEAT1 were reported in nonalcoholic fatty liver disease (NAFLD), in which NEAT1 expression and mTOR signaling are increased in vivo models of NAFLD [99].

-HOTAIR (HOX transcript antisense RNA):

HOTAIR overlaps with the HoxC genes on chromosome 12 and is transcribed from the negative strand. HOTAIR expression is upregulated in multiple tumors such as liver [100] and breast cancer [101]. Overexpression of HOTAIR in hepatocellular carcinoma cells (HCC) causes increased glucose consumption and lactate production [102]. The HOTAIR-dependent glycolytic flux results from enhanced mTOR signaling, as well as binding to and increasing the expression of glucose transporter isoform 1 (GLUT1). Yang el al. examined the role of HOTAIR in exosomal release during HCC tumorigenesis. Their studies reveal that overexpression of HOTAIR results in increased release of exosomes, which is partly due to mTOR-dependent phosphorylation of synaptosome associated protein 23 (SNAP23) [103]. Other studies reveal that HOTAIR effects on mTOR signaling appears to be cell-type specific. For example, Zhan et al. investigated the effect of HOTAIR in Intervertebral disc degeneration (IDD) utilizing nucleus pulposus cells. Contrary to its role in HCC, HOTAIR overexpression enhances autophagy and promotes apoptosis and senescence of NP cells by increasing p-AMPK and p-ULK1 protein expression, as well as lowering p-mTOR levels [104].

-ZNNT1 (The ZNF706 gene neighboring transcript 1):

ZNNT1 is located on chromosome eight, and contains one exon. Li et al. identified ZNNT1 as a downstream target of mTOR signaling, and is induced in uveal melanoma (UV) cells treated with rapamycin, which suggest that ZNNT1 is a downstream of mTOR signaling [105]. Expression analysis followed by mass spectrometry reveals that ZNNT1 upregulates the expression of ATG12, a ubiquitin-like protein that conjugates to ATG5 to facilitate the lipidation of LC3/Atg8 on autophagosomes [106]. Further experiments demonstrate that ZNNT1-dependent upregulation of ATG12 promotes autophagy by increasing the conversion of LC3-I to LC3-II and SQSTM1 degradation; ZNNT1 inhibition, on the other hand, produced the opposite effects. The mechanism by which mTOR upregulates ZNNT1 expression is not known.

-HULC (Highly up-regulated in liver cancer):

HULC is implicated in various cellular processes and is upregulated in many human malignancies, such as esophageal cancer [107], pancreatic cancer [108] and hepatocellular carcinoma [109]. HULC is overexpressed in patients with gliomas, and silencing of HULC suppresses angiogenesis by inhibiting cells proliferation and invasion [110]. At the molecular level, HULC inhibition decreased the phosphorylation of ERK, AKT, mTOR and the downstream molecule eIF4E, which suggests that HULC is an upstream regulator of mTOR signaling in human gliomas.

3. LncRNAs as therapeutic targets

The development of RNA-targeting therapeutics provides tremendous opportunities to modulate pathogenic molecules and pathways in various diseases. One of the distinctive features of lncRNAs is their highly tissue- and cell type-specific expression which could serve as prognostic markers for diagnosing diseases, or even predicting responses to treatments. In addition, as mentioned in this review, many lncRNAs were found to regulate specific genes and signaling pathways including mTOR. Therefore, drugs that target lncRNAs, such as synthetic antisense oligonucleotides (ASOs), can be less toxic and more refined than conventional protein-targeting drugs [111]. There is an increased focus on developing ASOs for the knockdown of deleterious lncRNAs in wide range of human diseases [112]. One such example is Angelman syndrome, a neurological disorder characterized by severe intellectual and developmental disabilities, has been successfully rescued in mice by inhibiting the anti-sense transcript, Ube3a-ats, which normally represses the imprinted ubiquitin protein ligase E3a gene, Ube3a [113]. Other classes of RNA-based therapeutics are currently being developed including splice-switching oligonucleotides, such as the ASO drug Spinraza™, which is clinically approved for correcting a splice switch in the SMN2 (survival of motor neuron 2) gene in the CNS of patients suffering from spinal muscular atrophy (SMA) [114]. These recent successes with RNA-targeting therapeutics have allowed companies to focus their efforts on developing and improving oligonucleotide chemistry for less toxic, more stable and efficient targeting in vivo. For instance, locked nucleic acids (LNAs) are a new class of ASOs that mediate the degradation, as well as the blockage of lncRNA activity [27]. As of today, there are no clinical trials that target lncRNAs that modulate mTOR signaling; however, clinical trials will become more likely as more lncRNA-mTOR mechanistic studies emerge.

4. Concluding remarks

Over the last decade, there has been an exponential increase in studies that aim to investigate the role of lncRNAs in human diseases. New mechanisms of gene regulations by lncRNAs have been uncovered, and many of the signaling pathways that are dysregulated in human diseases are influenced in a lncRNA-dependent manner. A number of studies have already elucidated the molecular mechanism(s) by which lncRNAs regulate various components of mTOR signaling, and nucleic acid-based therapeutics are emerging as a promising therapeutic approach for modulating lncRNA expression. From a clinical perspective, cell-type and tissue specificity of lncRNAs expression confer significant advantage to selectively modulate mTOR signaling in affected organs without disturbing normal tissues. However, the field is still in its early stages and more research is needed before incorporating lncRNAs into the clinic.