Stressing out Over Long Noncoding RNA☆

Biochimica et Biophysica Acta 1859 (2016) 184–191

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Biochimica et Biophysica Acta

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Review Stressing out over long noncoding RNA☆

Timothy E. Audas, Stephen Lee ⁎

Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA article info abstract

Article history: Genomic studies have revealed that humans possess far fewer protein-encoding genes than originally predicted. Received 2 April 2015 These over-estimates were drawn from the inherent developmental and stimuli-responsive complexity found in Received in revised form 17 June 2015 humans and other mammals, when compared to lower eukaryotic organisms. This left a conceptual void in many Accepted 19 June 2015 cellular networks, as a new class of functional molecules was necessary for “ﬁne-tuning” the basic proteomic Available online 2 July 2015 machinery. Transcriptomics analyses have determined that the vast majority of the genetic material is tran-

Keywords: scribed as noncoding RNA, suggesting that these molecules could provide the functional diversity initially sought Long noncoding RNA from proteins. Indeed, as discussed in this review, long noncoding RNAs (lncRNAs), the largest family of noncod- Stress Response ing transcripts, have emerged as common regulators of many cellular stressors; including heat shock, metabolic Cancer deprivation and DNA damage. These stimuli, while divergent in nature, share some common stress-responsive pathways, notably inhibition of cell proliferation. This role intrinsically makes stress-responsive lncRNA regulators potential tumor suppressor or proto-oncogenic genes. As the list of functional RNA molecules continues to rapidly expand it is becoming increasingly clear that the signiﬁcance and functionality of this family may someday rival that of proteins. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxon- omy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. © 2015 Elsevier B.V. All rights reserved.

1. Introduction have revealed that only 2% of the genetic material contains protein- encoding sequences [5,6]. Additionally, humans, chickens, pufferfish and It is a mystery why it has taken so long for the scientific community to worms share a similar number of protein-encoding genes [7–12], leaving embrace the concept of functional RNA molecules. The existence of these a conceptual void in the increased developmental complexity and envi- transcripts was proposed by Francis Crick in the 1950s, when he hypoth- ronmental adaptability found in the higher organisms [13,14]. This sug- esized about “metabolic RNA”,despite“meager” evidence at the time to gests that the capacity to regulate organismal complexity may fall to support his notion [1]. The proof he sought arrived only a few years another class of molecules. The RNA transcriptome appears to be a later with the identification of the first noncoding RNAs: transfer RNA prime candidate to accomplish this task, as studies have shown that the (tRNA), ribosomal RNA (rRNA) and small nuclear RNA (snRNA) [2,3]. vast majority of the genome is actively transcribed [15].Longnoncoding These transcripts were shown to be prominent and indispensable factors RNAs (lncRNAs) represent the single largest family of non-protein- in their ribonucleoprotein complexes. Unfortunately, their discoveries did coding transcripts in mammals (70–90% of the human genome) [16]. not usher in an era of acceptance of RNA as functional ribozymes; instead This enormous group of molecules has been arbitrarily lumped together the central dogma of gene expression [4] remained for many decades, (lengths greater than 200 nucleotides), with some sub-categories emerg- with every basic biology class teaching that DNA codes for RNA that ing based on the location of the gene with respect to protein-encoding codes for proteins. This ensured that for several generations, scientists loci. However, this system appears to be insufficient as more transcripts would view RNA as a mere messenger for the genetic material. are identified and characterized, revealing a diversity for this group that Today noncoding RNAs are emerging as important biological mole- may eventually rival or surpass the proteome. Given their novelty, func- cules, outside of the context of protein synthesis. The classic proteo- tionality and sheer abundance, it is clear that lncRNAs are destined to centric view of polypeptides as the predominant functional and regulatory have a tremendous impact on many areas of cell biology. factors within the cell may in fact be faulty. Genome sequencing results 2. The cellular response to stress ☆ This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. One emerging field in lncRNA biology is deciphering the role these ⁎ Corresponding author at: Department of Biochemistry & Molecular Biology, Sylvester transcripts play in the cellular response to stress. Higher eukaryotes Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL 33101-6129, USA. possess extremely sophisticated molecular networks to monitor intra- E-mail address: [email protected] (S. Lee). and extra-cellular conditions and allow cells to maintain homeostasis

http://dx.doi.org/10.1016/j.bbagrm.2015.06.010 1874-9399/© 2015 Elsevier B.V. All rights reserved. T.E. Audas, S. Lee / Biochimica et Biophysica Acta 1859 (2016) 184–191 185 in suboptimal growth environments. Perturbation activates these path- pathwaysareactivatedbythepresenceofdamaged,unfoldedoraggre- ways, as the cell attempts to mitigate the strain and repair any gated proteins, which is a major consequence of elevated temperatures. associated damages. However, if these conditions are too harsh for To alleviate this form of proteotoxic stress cells: 1) induce the expression recovery to occur apoptotic programs are induced, eliminating dam- of heat shock proteins (hsp), a family of proteases and chaperones, 2) aged cells and ensuring organismal viability. The pro-survival or pro- inhibit the transcription/translation of proteins, thus reducing the burden apoptotic fate of a cell is entirely dependent upon its ability to elicit on the folding machinery and 3) impair major metabolic activities includ- the appropriate response to the specific stimuli. Recent evidence ing; RNA splicing, nucleo-cytoplasmic transport and DNA synthesis to suggests that lncRNAs are key factors in these pathways, allowing for focus the cellular energy/resources on stress recovery [19–21]. All of greater diversity and “fine tuning” of the cellular stress response. these pathways utilize lncRNA to elicit the appropriate response. Within an organism, individual cells can be exposed to numerous adverse conditions including: high temperatures (proteotoxic stress), metabolite deprivation (nutrient deficiency and hypoxia) and DNA 3.1. Induction of heat shock proteins damaging agents. Regardless of the detrimental stimulus, the cellular response follows a basic three step process: sense the insult, transduce The lncRNA molecule heat shock RNA-1 (HSR-1) has been suggested a signal to the site of action and effect the appropriate adjustments to to be an important factor in the ribonucleoprotein complex that acti- cellular metabolism [17]. Here, we will review these environmental vates the hsp family in response to heat shock [22]. In cooperation stimuli and highlight the essential roles lncRNAs play in regulating the with three molecules of heat shock transcription factor-1 (HSF-1) and cellular stress response (Table 1). For the sake of space we will limit the translation elongation factor eEF1A, the HSR-1 complex binds DNA our review primarily to mammalian lncRNAs and analyzing the effects responsive elements to activate gene expression [22]. The authors proof these transcripts in human cancers. posed that this lncRNA may act as a mammalian RNA thermosensor, regulating the activity of the complex. While the work on HSR-1 has 3. The lncRNA-mediated heat shock response been called into question (some suggest this lncRNA is the product of bacterial contamination [23]), the concept of RNA thermosensors is in- Many consider high temperature exposure, termed heat shock, to be triguing. Bacterial and drosophila mRNAs have been shown to possess the founding and preeminent cellular stress [18]. Not surprisingly, the thermo-sensitive base-pairing in the untranscribed regions of some cell does not appear to possess a simple thermostat, detecting unaccept- mRNA, which regulate the rate of translation of these molecules able temperature and eliciting a cellular response. Instead, heat shock [24–31]. As the secondary structure of noncoding RNA molecules is

Table 1 Examples of long noncoding RNAs associated with stress and cancer.

Stimulus lncRNA lncRNA response to stress Function Linked to References cancer

HSR-1 No change in RNA levels Complex with HSF-1 and eEF1A to activate hsp genes No [22] (acts as a thermosensor?) – Heat shock Alu Up-regulation Inhibit global RNA Pol II transcription No [34 40] Sat III Up-regulation Inhibition of global RNA processing by sequestration No [50–58] of RNA splicing components into nuclear stress bodies

IGSRNA Up-regulation (stress-speciﬁc Protein immobilization in the nucleolar detention center No [62–67] IGSRNAs are induced) to regulate HIF levels and ribosomal RNA biogenesis

pRNA Up-regulation Epigenetic regulation of ribosomal RNA biogenesis, No [80,83,84] through NoRC and DNMT3b recruitment

PAPAS Up-regulation Regulation of ribosomal RNA biogenesis by chromatin compaction No [81,82] – Metabolite Gas5 Up-regulation (RNA stabilization) Suppression of glucocorticoid receptor responsive genes Yes [71 78,120] deprivation (including inhibitors of apoptosis) NEAT1 Up-regulation Regulates hypoxic paraspeckle formation and reduces apoptosis Yes [90]

lncRNAEFNA3 Up-regulation Mediates hypoxic accumulation of Ephrin-A3 Yes [91] lncRNA-LET Down-regulation Negative regulator of HIF-1a protein levels Yes [88]

lincRNA-ROR Up-regulation Positive regulator of HIF-1a protein levels Yes [89,100] RNA levels not examined Translational suppressor of p53

lincRNA-p21 Up-regulation Mediates binding of the repressive hnRNP-K to the promoters of Yes [92,101] apoptotic genes

PANDA Up-regulation Inhibits the activity of the transcription factor NF-YA, Yes [102,103] repressing the apoptotic pathway

7SK No change in RNA levels (complex Global inhibitor of RNA Pol II transcription elongation No [105–108] DNA damage dissociated by DNA damage)

ncRNACCND1 Up-regulation Through its association with TLS, this lncRNA inhibits epigenetic No [109] activators of the cyclin D1 gene

Pint Up-regulation Recruits the epigenetically repressive PRC2 complex to cell cycle Yes [101,117] regulator genes

TUG1 Up-regulation Recruits the epigenetically repressive PRC2 complex to cell cycle Yes [110,115,119] regulator genes

APTR Down-regulation DNA damage represses this constitutive PRC2-recruiting Yes [116] epigenetic-silencer of p21 186 T.E. Audas, S. Lee / Biochimica et Biophysica Acta 1859 (2016) 184–191 tightly linked to function it is extremely appealing to speculate that this stimuli (extracellular acidosis and ribosomal stress) [62,65,66]. What form of regulation may exist. distinguishes this form of targeting from other subcellular trafficking events is the change in protein dynamics associated with nucleolar DC 3.2. Inhibition of global gene expression localization [62,63,67]. Unlike classic sequestration, where the steady state residency time is simply increased (proteins remain free to diffuse Approximately thirty years ago, the quintessential and ubiquitous in and out of the region) [68–70], IGSRNA-mediated detention repre- “junk DNA” sequences of the human (Alu elements) and mouse (B2 sents a new form of post-translational regulation, altering the localiza- elements) genomes [12,32,33] were shown to be highly upregulated tion and mobility of proteins in the nucleolus [64]. To date, a number (10–40 fold) in response to heat shock conditions [34–37]. The signifi- of molecules have been shown to be targets of the nucleolar detention cance of this induction remained a mystery until recently, when it was pathway, affecting multiple cellular networks including proteasomal ac- shown that Alu/B2 lncRNA molecules associated with RNA polymerase tivity and ribosomal RNA biogenesis [63,64]. The mechanism by which II machinery and regulated its activity [38–40]. When present, Alu/B2 IGSRNAs facilitate both protein trafficking and immobilization is an RNA directly and tightly associates with RNA polymerase II [38,39]. important and outstanding question. Protein aggregates associate with This prevents the formation of a proper pre-initiation complex at the the site of IGSRNA expression on the ribosomal DNA (rDNA) cassette, DNA promoter [40], impairing transcription [38–40].Thesefindings suggesting the transcripts act as a bridging molecule between the DNA provide an lncRNA-mediated mechanism for global gene silencing in and targeted cellular factors [62]. However, the means by which these response to elevated temperatures. lncRNAs convert mobile proteins to immobile aggregates is unclear.

3.3. The formation of subnuclear bodies to disrupt cellular function 4. Adaptation to metabolite deprivation by lncRNA Unlike organelles within the cell, subnuclear regions are not delin- To survive and maintain homeostasis cells must acquire or produce a eated by the presence of lipid bilayers. The biogenesis of these bodies, myriad of vital metabolites. Deprivation of these essential small mole- while poorly understood, is mediated by the translocation and stable as- cules (serum components, sugars, amino acids and oxygen) can signifi- sociation of molecules at specific nuclear coordinates [41–43].LncRNAs cantly alter metabolic pathways, potentially leading to growth arrest play a prominent and essential structural role in the formation of and death. While some of these molecules can be synthesized by the these cellular regions. One of the first temperature-inducible lncRNA cell, at an energetic cost, others cannot (oxygen) and the cell must molecules to be identified was the hsr-omega (hsrω) transcript adapt to their absence in order to survive. LncRNAs are crucial molecular [44,45], which is responsible for omega speckle formation [46,47] and regulators influencing cell survival and metabolic activity under these thermo-tolerance [48] in drosophila. The human functional analogs conditions. of omega speckles are the nuclear stress bodies [49], which contain the heat shock-inducible satellite III (SatIII) noncoding transcripts. This heterogeneous family of lncRNA [50] is expressed from the repeti- 4.1. Starvation induced growth arrest tive pericentromeric heterochromatin of chromosomes 9, 12 and 15 [50–52]. In response to elevated temperatures, HSF-1 triggers a Growth arrest-specific5(Gas5) is an alternately spliced 200–600 10,000–100,000 fold up-regulation of SatIII RNAs [50,51,53–55].These nucleotide lncRNA [71] induced in response to serum starvation transcripts remain associated with their site of expression [50–52] and [72–74]. Unlike many transcripts, levels of this lncRNA are regulated mediate the targeting of proteins into this subnuclear region. While post-transcriptionally [74,75], with nuclear run-on assays demonstrat- the function of nuclear stress bodies has not been fully established, ing that Gas5 is transcribed at similar levels in growing and starved based on the content of targeted protein most believe they are cells [74]. The primary mode of induction is enhanced RNA stability connected to RNA processing [56–58]. This would be in line with the [76]. Gas5 is short-lived under standard growth conditions and stabi- observation that heat shock represses global splicing events [59,60]. lized during serum starvation [75,77]. Its main function appears to be What is unclear is whether the targeted RNA processing factors are as a regulator of glucocorticoid receptor (GR) activity [78]. This lncRNA functionally active within nuclear stress bodies, or simply sequestered inhibits the activity of GR, through binding to the DNA-binding domain away from their downstream effectors. Since the lncRNA transcripts of the hormone receptor and impairing its association with DNA ele- are known to bind splicing components [56–58] and the repetitive ments [78]. This “decoy” effect represses the expression of GR down- nature of the SatIII loci and sequence variability of SatIII RNA [50] stream targets, including apoptotic inhibitors and metabolic genes [78]. make the bioinformatics identification of exon/intron junctions The ribosomal DNA cassettes appear to be a hotbed of lncRNA tran- impossible, it is difficult to establish whether active RNA processing scription activity in response to cellular stress. This makes intuitive is occurring in these subnuclear domains. Regardless, a reduction in sense, given that these repeats form the scaffolding of the nucleolus, a the nuclear pool of available spliceosome components would elicit region thought to be a stress-sensing hub within the cell [79].Inaddi- the desired effect on global RNA processing rates during periods of tion to the heat shock/extracellular acidosis-induced IGSRNA (described thermo-stress. above) [62], several other lncRNAs have been discovered in the rDNA In 1984 Welch and Feramisco demonstrated that heat shock protein cassette [80–82]. Promoter-associated RNA (pRNA) mediates the re- 70 kDa (Hsp70) translocates to the nucleolus in response to elevated cruitment of two distinct epigenetic complexes, the nucleolar remodel- temperatures [61]. This was one of the earliest pieces of data linking ing complex and DNA methyltransferase 3B, to rDNA in response to the most visible subnuclear structure to the heat shock response. glucose levels [80,83,84]. Targeting of DNA methyltransferase 3B is par- Recently, a mechanism and function for this translocation were ticularly intriguing as recruitment of this gene silencer is mediated by shown, with thermo-stress triggering a rapid lncRNA-mediated re- the formation of a DNA:RNA triplex, a potentially new and ubiquitous organization of the nucleolus, culminating in the formation of a new pathway for targeting epigenetic factors [83].Severalpromoter and electron-dense subnucleolar region, termed the nucleolar detention pre-rRNA antisense (PAPAS) lncRNAs were also shown to be expressed center (DC) [62–64]. This process is regulated by a family of lncRNA from the rDNA cassette in response to serum starvation or growth arrest molecules derived from the ribosomal RNA intergenic spacer (IGSRNA) [81,82]. PAPAS transcript induction triggers chromatin compaction by

[62]. Two of these transcripts IGS16RNA and IGS22RNA (the numbers tri-methylation of lysine 20 on histone H4 [81]. While these stimuli represent the distance in kilobases from the ribosomal RNA transcrip- and lncRNA are dissimilar they accomplish a common goal, down- tional start site) collaborate to recruit proteins during heat shock condi- regulation of the energetically intense process of synthesizing rRNA in tions [62,63], while additional IGSRNAs are induced in response to other a metabolite-starved environment. T.E. Audas, S. Lee / Biochimica et Biophysica Acta 1859 (2016) 184–191 187

Fig. 1. The cellular IncRNA-mediated response to low oxygen availability (hypoxia). Several IncRNAs have been shown to act as upstream (IGS28RNA and IncRNA-LET) or downstream

(NEAT1 and IncRNAEFNA3) effectors of the hypoxia inducible factor-alpha (H1Fα) family of proteins to regulate the hypoxic stress response.

4.2. LncRNAs regulate the hypoxic response of the extracellular milieu (a consequence of glycolysis), is known to prevent degradation of the HIF proteins [65,67]. Nuclear enriched abun- The response to hypoxia differs from other metabolite deprivation dant transcript 1 (NEAT1), long intergenic noncoding RNA upstream of pathways (glucose, amino acids, nucleotides, etc.), as the cell cannot p21 (lincRNA-p21) and lncRNAEFNA3 are all established HIF downstream synthesize oxygen and must simply compensate for its absence. The targets [90–92]. Activation of NEAT1, the paraspeckle scaffolding major consequence of this stressor is a reduction in energy generation molecules [93], is believed to reduce apoptosis during periods of low associated with the transition from aerobic to anaerobic respiration. oxygen [90], while lincRNA-p21 promotes glycolysis to augment ATP The hypoxia-inducible factors (HIF-1α and HIF-2α) are prominent generation [92]. transcription/translation factors that facilitate this transition by inducing the expression of genes associated with angiogenesis, glucose 5. DNA damage associated lncRNA transport and glycolysis [85–87]. Several lncRNAs have been identified as upstream or downstream effectors of the HIF proteins (Fig. 1). Unlike the heat shock response, where elevated temperatures are in- Hypoxic repression of lncRNA low expression in tumor (lncRNA-LET) directly assessed through the presence of denatured proteins, the DNA [88] and activation of the large intergenic noncoding RNA-regulator of damage response (DDR) pathway directly detects disruptions in DNA reprograming (lincRNA-ROR) [89] lead to enhanced HIF-1α protein structure and integrity. Various extracellular (genotoxic chemicals, ul- synthesis. Post-translationally, HIF-2α stability can be regulated by traviolet and ionizing radiation) and intracellular (DNA synthesis the IGSRNA-mediated nucleolar detention pathway described above errors) events lead to genomic instability [94], though screens using [62,65]. Sequestration of E3 ubiquitin ligases, in response to acidification multiple DNA damage-causing agents show a similar pattern of lncRNA 188 T.E. Audas, S. Lee / Biochimica et Biophysica Acta 1859 (2016) 184–191 up- and down-regulation, regardless of the DDR-inducer or cell line Numerous additional lncRNAs act as epigenetic regulators examined [95,96]. These results highlight some of the commonalities [110–114]. These transcripts mediate the targeting of chromatin modi- within the DDR network, though the authors did not delve into the fying structures, such as the Polycomb repressive complex 2 (PRC2), to mechanistic effects of DDR-specific alterations in lncRNA expression specificgenomicloci[110–114]. Here, our focus is on the stress respon- levels. sive lncRNAs, specifically p53 induced noncoding transcript (Pint) [101], taurine upregulated gene1 (TUG1) [115] and Alu-mediated p21 5.1. Regulating the master regulator p53 transcriptional regulator (APTR) [116]. DNA damage triggers induction of Pint and TUG1 [101,115], which recruits PRC2 to specific genomic P53 is expressed at low levels in untreated cells and induced loci and facilitates the silencing of cell proliferative genes by tri- upon stress treatment, suggesting this “master regulator” is under the methylation of lysine 27 on histone H3 [110,117]. Conversely, APTR control of other cellular factors. Post-translational stabilization and up- levels are down-regulated by DNA damage and heat shock treatment regulated protein synthesis appear to be key pathways enhancing its [116]. In normal cells APTR constitutively recruits the PRC2 epigenetic DNA damage-mediated expression [97,98].LincRNA-ROR– a transcript machinery to the p21 locus, repressing this cell cycle inhibitor. Stress linked to cellular pluripotency [99] – negatively regulates p53 translation stimuli repress this transcript, alleviating epigenetic silencing to activate by associating with heterogeneous nuclear ribonucleoprotein I (hnRNP p21 [116]. I) [100]. This lncRNA-mediated effect is regulated by a small region of lincRNA-RoR (28 nucleotides of a 2590 base transcript) and is limited 6. LncRNA and cancer to conditions of DNA damage [100]. As we've seen above, down-regulation of apoptotic genes and cell cycle suppression are common themes in the cellular response to 5.2. LncRNA as DNA damage-mediated transcription factors numerous stress stimuli. Many of the lncRNA described here facilitate these goals, with their aberrant expression leading to common cancer Recently, lncRNA tiling arrays were used to identify molecules asso- hallmarks, such as replicative immortality, resistance to cell death or ciated with the DDR [101,102]. The PANDA (p21 associated noncoding evasion of growth suppression [118]. Of the molecules discussed RNA DNA damage activated) and lincRNA-p21 transcripts, respectively above PANDA [102], lincRNA-p21 [92], Pint [117],TUG1[119],APTR located approximately 5 and 15 kilobases upstream of the cell cycle in- [116], Gas5 [120], lncRNA-LET [88], lincRNA-ROR [89],NEAT1[90] and hibitor p21, were shown to be induced in a p53-dependent manner in lncRNAEFNA3 [91] have all been linked to tumorigenesis, with varying response to the DNA damaging agent doxorubicin [101,102]. Both of levels of in vitro and in vivo data supporting these associations. these lncRNA have minimal effect on cellular proliferation rates, but sig- fi ni cantly impaired DDR induced apoptosis [101,102]. While their roles 6.1. Looking beyond the established players in cancer are similar, their mode of action appears to be divergent. The 5′ end of lincRNA-p21 binds to the heterogeneous nuclear ribonucleoprotein K Shortly after the identification of XIST [121] and H19 [122], cancer (hnRNP-K) and facilitates docking on DNA elements [101]. This docking became an emerging theme in lncRNA research. Groundbreaking represses expression of apoptotic genes, though the mechanism is works by pioneers within the field identified a number of transcripts, unclear. PANDA binds to the alpha subunit of nuclear transcription such as HOTAIR [123], ANRIL [124], MALAT1/NEAT2 [125],PCAT-1 factor Y (NF-YA), a protein known to induce the expression of apoptotic [126,127] and PCGEM1 [128,129], which regulate various tumorigenic genes [103]. The formation of a PANDA/NF-YA complex prevents bind- pathways. The roles these established transcripts play in cancer ing of the transcription factor to cell death genes, impairing the apopto- progression, and their mechanism of action, have been reviewed thor- tic pathway [102]. oughly in the past [130–133] and will not be discussed here. Instead, Transcription elongation is regulated by the cyclin dependent kinase we will focus on a couple of new fields of lncRNA in tumorigenesis, P-TEFb, which phosphorylates RNA polymerase II to ensure productive as the study of lncRNAs in cancer appears to be surging within the elongation of full length RNA transcripts [104]. Under normal growth scientific community, with publication rates increasing at a rate greater conditions, a major population (~50%) of this general transcription than exciting topics from the 1980s (oncogenes), 1990s (integrins) and factor is held in an inactive state by its association with the lncRNA 2000s (epigenetics) (Fig. 2). 7SK (the core molecule of a large ribonucleoprotein complex) [105,106]. Removal of 7SK negates its repressive effect, ensuring 6.2. LncRNA as biomarkers with altered expression in cancer transcriptional elongation [107] and making this lncRNA an important gatekeeper of the transcriptional pathway. Treatment of cells with The recent advent of high-throughput RNA sequencing (RNA-seq) ultraviolet light triggers a release of P-TEFb from the inhibitory 7SK technologies has revolutionized the field of lncRNA research. As a conse- complex, causing transcriptional up-regulation in response to DNA quence of this technical breakthrough, numerous groups have used damage-induction [105,108]. This suggests that 7SK may be a stress- RNA-seq to map global changes to the lncRNA landscapes of normal response lncRNA capable of regulating global gene expression. and cancerous cells. These studies have generated a wealth of data on the up- and down-regulation of lncRNA in specific cancer cell types 5.3. DNA damage-mediated epigenetic effects [127,134–140]. The natural progression of this research has been to cor- relate these lncRNA expression profiles with patient prognosis. This has The first DNA damage responsive lncRNA transcript was found up- occurred for several established (HOTAIR [123] and MALAT1 [141])and stream of the cyclin D1 promoter (ncRNACCND1) [109]. In response to novel transcripts (NBAT-1 [142], SChLAP1 [143], GAPLINC [144],FAL1 ionizing radiation, this extremely low copy number (4 per cell) mole- [145], lncRNA-ATB [146] and NKILA [147]), linking these lncRNAs to cule binds to the protein translocated in liposarcoma (TLS) and alters metastasis and mortality in highly divergent tumor types. Remarkably, its conformation. In this open state TLS is capable of associating with a these findings are quickly transitioning from the realm of basic research histone acetyltransferase complex, inhibiting its function and conse- to translational medicine, as some of these RNA are being utilized as quently repressing the expression of the nearby cell cycle controlling biomarkers. Several groups have begun developing non-invasive gene cyclin D1 [109]. This lncRNA is a prime example of the virtues blood/urine tests to detect tumor-specific lncRNA in cancer patients inherent to utilizing RNA transcripts over polypeptides. By forgoing [148–150]. What remains to be seen, when assessing these large scale the protein synthesis step, cells can more rapidly respond to a stimulus transcriptomic analyses, is whether the changes in lncRNA are causative and tightly regulate the copy number of a critical molecule. or merely correlative to cancer progression. T.E. Audas, S. Lee / Biochimica et Biophysica Acta 1859 (2016) 184–191 189

Oncogenes Integrins Epigenetics LncRNA + Cancer 3000 3000 3000 1000

2500 2500 2500 800

2000 2000 2000 600 1500 1500 1500 400 1000 1000 1000

200 500 500 500 Number of Publications Number of Publications Number of Publications Number of Publications

0 0 0 0 1995 2000 2005 2010 2015 2020 1990 1995 2000 2005 2010 2015 1980 1985 1990 1995 2000 2005 1985 1990 1995 2000 2005 2010 Publication Year Publication Year Publication Year Publication Year

Fig. 2. Publications, by year, for innovative research topics over the last four decades. The number of PubMed entries per year for the search terms; oncogenes, integrins, histone and chromatin or IncRNA and cancer. Highlighted regions (green) represent periods of strong growth in the ﬁeld.

6.3. LncRNA and chemotherapeutic drug resistance Conﬂict of interest statement

Resistance of tumor cells to therapeutic treatment is a crucial prob- The authors declare that they have no conflict-of-interest and no lem in the field of cancer biology. Adaptation to drug treatment likely competing financial interests. relies upon: 1) increased expulsion of the hazardous agent or 2) de- activation of cell death pathways induced by the drug. LncRNAs are Acknowledgments known to be differentially expressed to drug-sensitive and drug- resistant cells [151,152], suggesting these molecules play a prominent This work was supported by funds provided by the Sylvester role in chemotherapeutic-resistance. P-glycoprotein (P-gp), a trans- Comprehensive Cancer Center. We apologize to all authors whose membrane efflux pump with broad substrate specificity, is thought to work could not be cited due to space constraints. facilitate drug elimination in resistant cancer cells [153]. 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