RNA regulation and cancer development

Cancer Letters 246 (2007) 12–23 www.elsevier.com/locate/canlet

Mini-review

RNA regulation and cancer development Eva Scholzova´ a,*, Radek Malı´k b, Jan Sˇevcˇ´ık a, Zdeneˇk Kleibl a a

First Medical Faculty, Institute of Biochemistry and Experimental Oncology, Charles University, U Nemocnice 5, 128 53 Prague 2, Czech Republic b Laboratory of Protein Dynamics and Signaling, Eukaryotic Transcriptional Regulation Section, National Cancer Institute, Frederick, MD, USA Received 20 January 2006; received in revised form 20 March 2006; accepted 24 March 2006

Abstract Cancer is viewed as a genetic disease. According to the currently accepted model of carcinogenesis, several consequential mutations in oncogenes or tumor suppressor genes are necessary for cancer development. In this model, mutated DNA sequence is transcribed to mRNA that is finally translated into functionally aberrant protein. mRNA is viewed solely as an intermediate between DNA (with ‘coding’ potential) and protein (with ‘executive’ function). However, recent findings suggest that (m)RNA is actively regulated by a variety of processes including nonsense-mediated decay, alternative splicing, RNA editing or RNA interference. Moreover, RNA molecules can regulate a variety of cellular functions through interactions with RNA, DNA as well as protein molecules. Although, the precise contribution of RNA molecules by themselves and RNA-regulated processes on cancer development is currently unknown, recent data suggest their important role in carcinogenesis. Here, we summarize recent knowledge on RNA-related processes and discuss their potential role in cancer development. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Nonsense-mediated decay; RNA editing; Alternative splicing; RNA interference; microRNA; Cancer

1. Introduction Cancer is viewed as a genetic disease. According to the established model of oncogenesis several consequential mutations in coding regions of oncogenes and tumor suppressor genes, regulating fundamental cellular processes like proliferation, differentiation, and apoptosis, are necessary for cancer development

Abbreviations: ADAR, adenosine deaminases acting on RNAs; BRCA1, breast cancer 1; dsRNA, double stranded RNA; EJC, exon junction complex; miRNA, microRNA; ncRNA, non-coding RNA; NMD, non-sense mediated decay; PTC, premature termination codon; RNAi, RNA interference; siRNA, small interfering RNA; SWI/SNF, mating-type switch/sucrose nonfermenting. * Corresponding author. Tel.: C420 22496 5745; fax: C420 22496 5742. E-mail address: [email protected] (E. Scholzova´).

[1]. To become a cancer cell, several essential alterations in cellular physiology have to occur: autonomous proliferation, evasion of apoptosis, resistance to growth-inhibitory signals, unlimited replicative potential, angiogenesis and invasion and metastasis [2]. It has emerged recently that cancer may arise from organ-specific or system-specific stem cells [3], but alternative origins of cancer are also possible [4]. Stem cells have extended replicative potential that is necessary to their function—refilling the pool of tissue-specific progenitor cells, which are responsible for the replacement of terminally differentiated cells. The extended lifespan and strong mitotic potential of stem cells greatly increase their chance of acquiring mutations. Such mutations are transmitted to their progenitors, thus multiplying the number of cells harboring a particular genetic defect. The subsequent

0304-3835/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.03.021

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accumulation of somatic mutations irreversibly condemns the cell to malignant transformation [5]. However, it was postulated that functional characteristics of tumor cells including potential to metastasize might be already defined early in tumorigenesis [6]. Changes leading to cancer development were supposed to be caused by DNA sequence alterations (mutations, deletions/insertions or gross chromosomal re-arrangements) and conclusive production of functionally aberrant protein. Besides sequence alterations, several epigenetic events may affect gene expression and thus quantitatively influence the level of ‘physiological’ protein(s). Alteration of the methylation status of promoters and/or covalent modification of histones were shown to affect targeted gene expression [7]. Different regulation and/or usage of promoters (in otherwise ‘intact’ genes without any DNA sequence alterations) can influence gene expression as well, as was shown, e.g. for BRCA1 [8] and Dicer [9] genes. Up to now, in all these scenarios (m)RNA is viewed as a ‘passive’ intermediate product between DNA (with coding potential) and proteins (with executive potential). However, recent evidence suggests that different types of RNA molecules play important roles in gene regulation not only at the level of mRNA processing and regulation of mRNA stability but also at the level of gene transcription: (1) In the nucleus, newly synthesized pre-mRNAs are subjected to ‘quality control’ processes including ‘capping’, polyadenylation, alternative splicing, nonsense-mediated decay (NMD), RNA editing or RNA export from the nucleus. All these RNA processing mechanisms influence each other and are linked in a ‘multi-component’ machine encompassing DNA polymerase II [10]. Thus, the ‘quality’ of (m)RNA is actively checked and tightly regulated. (2) In the cytoplasm, (m)RNAs metabolism is further regulated by controlling (m)RNAs’ turnover [11,12] and mRNA translation efficiency [13,14]. Deregulation of these processes may significantly contribute toward carcinogenesis [14]. (3) MicroRNAs (miRNAs), a large family of 21–22 bp small non-coding RNAs (ncRNAs), are able to interfere with mRNA translation and downregulate or even forbid specific gene expression [15]. The same molecules are involved in DNA methylation [16–18], consequently regulating gene expression at multiple levels. (4) There are increasing numbers of non-coding RNAs (ncRNAs), which are produced either as individual

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transcripts or during transcription and processing of specific protein-coding sequences (introns, flanking sequences transcribed by a read-through mechanism). They play an important role in the regulation of genes expression, mainly by interfering with transcription through an antisense mechanism [19]. Moreover, ncRNAs participate in diverse processes including DNA methylation, transcription, (m)RNA processing and translation [16,20,21]. Enclosing ncRNAs into regulatory circuits supplies better and faster control of mRNA/protein expression than solely proteinbased regulation [22]. Predicted and proven functions of ncRNAs are discussed in details in recently published papers describing the construction of non-coding RNA’s databases [23,24]. In this review, we focus on alternative splicing, nonsense-mediated decay, RNA editing and RNA interference as mechanisms that potentionally influence tumorigenesis and may be promise targets in cancer treatment. 2. Alternative splicing As revealed by sequencing the human genome, human cells contain approximately 30,000 genes, a number that is much lower than was previously expected [25]. Alternative splicing, potentially generating a large number of mRNAs from a single gene, is a mechanism that increases the number of proteins and their isoforms coded by a constrained number of genes [26]. Newly synthesized pre-mRNAs are immediately processed and intron sequences are cleaved out to form mature mRNAs. This process is mediated by a large multi-protein complex called the spliceosome [27]. Besides indispensable cis regulatory motifs such as 5 0 and 3 0 splice sites and branch points, there are other cis regulatory sequences called exonic or intronic splicing enhancers and silencers [28,29]. These sequences are recognized by a number of regulatory proteins, represented, for example, by serine/arginine-rich (SR) proteins, which bind RNA with limited sequence specificity [30]. Different combinations of positively and negatively-acting cis and trans regulators influence the final decision of whether a particular exon is included in the mature mRNA or not. As a result of premRNA splicing, different combinations of exons may arise and may be a ‘natural’ cause of errors in gene expression by introducing premature termination codons (PTC; similarly as nonsense mutations) or

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altering protein structure (protein domains composition) leading to changes in spectrum of interacting proteins, intracellular localization, protein stability or posttranslational modification [26]. Cancer is often associated with a predominance of a splice form that is not seen in the normal surrounding tissue. The existence of cancer-specific protein isoforms has been documented for human estrogen [31] and progesterone [32] receptors, BRCA1 tumor suppressor gene participating in the pathogenesis of hereditary breast and/or ovarian cancers [29], CD44 antigen [33], vascular endothelial growth factor [34] and many others (for review see [35]). What is the cause of the production of cancer-related predominant splice forms? A common reason may be mutations in cis splicing regulating sequences, which might shift production to mRNA with cancer-prone potential. As an example, TOG transversion in the codon 64 of BRCA1 protein was originally predicted to change a conserved cysteine residue to glycine in a RING finger domain. Yang et al. [36] showed, however, that this mutation disrupts an exonic splicing enhancer and leads to production of null protein due to aberrant splicing. But, can cancer-prone isoforms be produced without mutations in genomic DNA, influenced only by deregulation of alternative splicing in trans? The answer is probably yes. Alternative splicing is regulated, for example, by the STAR (signal transduction and RNA binding) protein family [37]. RNA binding protein Sam68 (Src-associated in mitosis), a member of the STAR family of proteins, is phosphorylated by ERK (extracellular signal-regulated kinase) on several residues. ERK-dependent phosphorylation of Sam68 leads to the specific inclusion of exon v5 in CD44 mRNA [38]. Sam68 influences alternative splicing in cooperation with Brahma (Brm), a component of SWI/SNF chromatin remodeling complex [39]. The recruitment of alternative exons is promoted by Brmmediated slowdown of DNA Polymerase II elongation rate and assembly of complex splicing machinery at the suboptimal splicing sites [39]. Signal transductiondependent changes in alternative splicing due to the modification of trans acting splicing factors were proposed also for other regulatory proteins [37,40–42]. The exact spatial and temporal regulation of spliceosome and the process of alternative splicing is still not well understood [43]. An increasing number of examples describing the control of alternative splicing by signal transduction pathways favor the model, where the loss of the accurate control of alternative splicing may lead to preferential expression of cancer-prone

protein isoforms even without any disruption in genetic information [44], (Fig. 1). 3. Nonsense-mediated decay Nonsense-mediated decay (NMD) is an evolutionally conserved mechanism that is responsible for degradation of mRNAs bearing a premature termination codon (PTC) and is functionally tightly connected with alternative splicing [45] and other RNA-related processes such as stimulation of translation [46] or telomerase regulation [47]. As a result of pre-mRNA splicing, a protein complex called exon junction complex (EJC) is deposited w20–24 nucleotides upstream of splicing-generated exon–exon junctions [48]. During the so-called ‘pioneer round of translation’ each newly synthesized mRNA is scanned for the presence of stop codons and EJCs. It is still unclear whether the initial round of translation takes place in the cytoplasm (where the bulk of translation takes place) or in the nucleus. According to the current model of NMD, mRNA is subjected to the pioneer round of translation in the nucleus or soon after its transportation to the cytoplasm, probably during transport through the nuclear pore [45,49]. Recognition of the stop codon that is followed by the EJC complex attached to the mRNA more than 50 nucleotides downstream of this stop codon activates NMD machinery and particular mRNA is degraded [50,51]. It was proposed that up to 35% of all human tran scripts contain PTCs and are potential targets of NMD [52]. PTCs may result from nonsense mutations, aberrant pre-RNA splicing involving cryptic or mutated splice sites or gene rearrangements generating frameshifts [53]. The essential role of NMD in suppression and degradation of mRNAs coding for potentially deleterious proteins was recognized in many autosomal dominant diseases [53]. Muscular dystrophy is a prototype genetic disease in which NMD influences the genotype–phenotype pattern of disease. Mutations in the dystrophin gene introducing PTCs cause the severe form of disease (Duchenne type) as the whole transcript is eliminated by NMD. Mutations and large deletions without PTC occurrence result in the milder Becker’s form [54]. It can be speculated that NMD plays a role also in cancer development albeit the precise role is not known yet. Deregulation of NMD may lead to translation of PTC containing mRNA(s) producing functionally aberrant proteins that may influence many cellular processes, including tumorigenesis. A new strategy named ‘gene identification by NMD inhibition’ (GINI)

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Fig. 1. Schematic representation of RNA-based mechanisms and their potential regulatory role in cancer development. According to the current model of carcinogenesis, DNA mutations in the coding regions of oncogenes and tumor suppressor genes are necessary for carcinogenesis. Protein produced by the translation of mutated mRNA is functionally aberrant (looses its physiological functions or gains dominant-negative functions) and may govern the cell to transformation. RNA-based mechanisms like RNA interference (RNAi), RNA editing, alternative splicing and nonsensemediated decay (NMD) may, at least theoretically, regulate the expression of these mutated transcripts, but probably are not able to prevent or stop the cell transformation (left part). However, functional deregulation of these RNA-based mechanisms may influence the expression of ‘normal’ genes bearing no mutation (red arrows). Such aberrant function of RNA-based mechanisms may be the first step in the process of carcinogenesis, before the occurrence of mutations in DNA will ultimately ‘fix’ the cell transformation process (see the main text for detailed discussion).

was used for the identification of genes containing nonsense mutations [55]. GINI was tested on a colon cancer cell line, which contained previously described nonsense mutations in the MLH1 and hexoaminidase B (HEXB) genes. Treatment of the cells with the translation-blocking drug emetine resulted in elevated mRNA levels of different transcripts including MLH1 and HEXB. Ionov et al. [56] used a similar approach for the identification of gene mutations in cancer cells using cDNA arrays. In this study, a combination of emetine and actinomycin D (inhibitor used to prevent emetine-induced up-regulation of stress-induced transcripts) was used to ‘stabilize’ mutant transcripts in a colorectal cell line showing microsatellite instability. Stabilized mRNAs were subsequently detected on a cDNA microarray. Several potential PTC-bearing transcripts were discovered and new mutations in UV resistance-associated gene (UVRAG) and p300 genes

were confirmed. In another study, NMD was shown to down-regulate the majority of BRCA1 mRNAs bearing non-sense mutations [57]. Taken together, NMD modulates the expression of cancer-related mRNA transcripts in vivo. Such down-regulation seems to be beneficial because of eliminating potentially harmful transcripts. However, not all PTC-bearing transcripts impair cellular functions. In these cases NMD may eliminate mRNAs that would otherwise result in the production of partly or fully functional truncated protein. Can NDM influence tumorigenesis without mutations in DNA? Is deregulation of the factors responsible for NMD capable of wrongly degrading ‘good’ transcripts? Three up-frameshift mutation (UPF) proteins are key components of NMD machinery. Recently, a Staufen1-mediated mRNA decay mechanism involving UPF1 protein and stop codon

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recognition, but independent of splicing (deposition of EJC complex), was described [58]. Both these decay mechanisms are regulated by phosphorylation of their key components UPF1 [59] and Staufen1 [58], respectively. Despite the limited experimental evidence, NMD is probably regulated by signal transduction pathway(s) through phosphorylation of its components. Thus, signal transduction-dependent deregulation of NMD may be a potential mechanism promoting the expression of cancer-prone mRNA transcripts. But, what are the targets of NMD if there are no mutations (and thus no PTCs) present? Besides its well-established role in the regulation of PTCbearing mRNAs, NMD is proposed to play a more general role by regulating the ‘transcriptome’ and reducing ‘genomic noise’ [60]. In the study of Mendell et al. [60], they knocked down the NMD pathway in HeLa cells and compared differences in the transcription profile of those cells with wild type HeLa cells by microarray analysis. Their results revealed that nearly 5% of genes were up-regulated, including those with upstream open reading frames in the 5 0 untranslated region, transcripts in which non-sense codons or frameshifts were introduced by alternative splicing, those that contain introns in the 3 0 untranslated region, transcripts derived from ancient transposons and endogenous retroviruses and transcripts harboring selenocysteine codons and, interestingly, mRNAs participating in amino acid metabolism. Proteins involved in PTC-mediating mRNA decay may exert more general function on mRNA metabolism. NMD factors promote translation and direct efficient translation termination of normal mRNAs in mammalian cells [46]. Taken together, NMD is an important post-transcriptional regulatory event physiologically regulating many mRNA transcripts and not merely functioning in RNA surveillance pathway. Deregulation of NMD may have critical consequences on (m)RNA metabolism (Fig. 1). 4. RNA interference and microRNAs RNA interference (RNAi) is a phylogeneticallyconserved mechanism of double-stranded RNAmediated mRNA silencing (for review see [61,62]). The physiological role of RNAi in mammals is not known at present but it is possible that RNAi plays a role in such diverse processes like over-all mRNA regulation [63], defense against viruses [64] as well as DNA methylation [17,16]. MicroRNAs (miRNAs) are abundant w21–22 bp ncRNAs. In humans, miRNAs are encoded in the

genome as stem-loop containing precursors (pri-premiRNA), which are transcribed mainly by DNA Polymerase II [65]. Nuclear RNase III-like endonuclease Drosha, as a part of the multi-protein microprocessor complex [66], cuts off a stem-loop structure from pri-premiRNA precursors liberating a w70 nt pre-miRNA. PremiRNA is then transported from the nucleus to the cytoplasm by an Exportin-5-dependent mechanism [67]. In the cytoplasm, another RNase III-like endonuclease Dicer, in cooperation with dsRNA-binding proteins [68], cleaves pre-miRNA and produces mature miRNA [69]. Mature miRNA associate with Dicer, TRBP (human immunodeficiency virus transactivating response RNAbinding protein), Gemin 3 and Gemin 4, and Argonaute family proteins eIF2C1-4 in a large 15S miRNP complex [68,70]. This complex concentrates target mRNAs to cytoplasmic P bodies, which contain untranslated mRNAs and can serve as a sites of mRNA degradation [71]. Detailed mechanisms of miRNA/siRNA biogenesis, dsRNA-induced silencing complex (RISC) assembly and mechanisms of miRNA/siRNA function were currently reviewed [61,62]. It was proposed that there are about 200–250 miRNAs coded in the human genome, an amount corresponding to w1% of protein-coding genes, which potentially regulate the expression of w10% human protein-coding genes. However, recent results indicate that both these predictions may be underestimated [72,73]. The role of miRNAs in a variety of physiological processes becomes rapidly elucidated concurrently with the prediction and/or experimental verification of potential miRNA-regulated targets [73–75]. MiRNAs were shown to regulate proliferation [76], differentiation [63] and apoptosis [76]; processes well known to be involved in cancer pathogenesis and development. Deregulation of several miRNAs was reported in colorectal cancer (for review see [77,78]). Many oncogenes and tumor suppressor genes were predicted to be regulated by miRNAs. Among others, Ras oncogene, implicated in the pathogenesis of many solid tumors is the target for the regulation by the let-7 family of miRNAs. Human Ras genes contain multiple potential let-7 binding sites allowing let-7 to regulate Ras expression [79]. Moreover, the expression of let-7 and Ras was inversely correlated in samples from lung tumors, which is consistent with the study of Takamizawa et al. [80], where reduced expression of let-7 in human lung cancers was associated with shortened postoperative survival. MiR-16, which expression is frequently downregulated in chronic lymphocytic leukemia [81], was

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implicated in the regulation of mRNA stability through AU-rich (ARE) sequences present in 3 0 -UTR regions of a variety of short-lived mRNAs, including many oncogenes and tumor suppressor genes [82]. The destabilizing function of ARE is regulated by AREbinding proteins and is mediated by the exosome. It was proposed that the exosome is also required for the decay of mRNAs targeted by RISC [83]. It is tempting to speculate that miRNA may regulate mRNA outcome not ‘solely’ by inhibiting a translation through the RISC complex but also by cooperating with other mRNAregulating machineries, like NMD or ARE-mediated mRNA decay. In experiments directly addressing the role of miRNAs in cancer, He et al. [84] analyzed the expression of miR-17-92 polycistron, which is located in a region of DNA that is amplified in human B-cell lymphomas. Overexpression of the miR-17-92 locus, in cooperation with c-Myc, accelerates tumor development in a mouse B-cell lymphoma model. Absence of apoptosis distinguished these tumors from solely c-Myc-induced lymphomas. The mechanism by which the miR-17-92 locus inhibits apoptosis and promotes lymphoma development was suggested by O’Donell et al. [85] c-Myc was shown to directly bind to the promoter of miR-17-92 locus on chromosome 13 and stimulate its expression. Interestingly, two miRNAs within this cluster, miR-17-5p and miR-20a, negatively regulate E2F1 transcription factor, additional direct target of c-Myc that promotes cell cycle progression. A more general role of miRNAs in tumorigenesis can be anticipated based on the results of Lu et al. [86]. Using a bead-based flow cytometric miRNA expression profiling method they systematically analyzed the expression of 217 mammalian miRNAs from samples of multiple human cancers. Global down-regulation of miRNA expression was observed. Moreover, the miRNA profiles reflected the developmental lineage and differentiation state of the tumors much better than mRNA profiles. Expression of proteins involved in miRNA processing and function are de-regulated in cancers as well. Reduced expression of Dicer in lung cancer was associated with poor prognosis after surgical treatment [87]. The human EIF2C1 gene, a component of the RISC complex, is located on the short arm of chromosome 1 in the region 1p34-p35. This genomic region is frequently lost in human cancers such as Wilms tumors, neuroblastoma, and carcinomas of the breast, liver, and colon. Little is known about the regulation of RNAi by signaling pathways. Two recent reports addressed this

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question. Hanson et al. [88] used a neuroblastoma cell line NBP2-PN25 stably transfected with short-lived green fluorescent protein (d2EGFP) under the control of CMV promoter. They transfected these cells with a retroviral plasmid expressing U6 promoter-driven shRNA targeted to d2EGFP or with synthetic siRNA. In both cases, elevation of cAMP (after treatment either with adenylate cyclase activator or cyclic nucleotide phosphodiesterase inhibitor) increased siRNA activity. The siRNA activity was increased also by inhibition of phosphatidylinositol-3-kinase (PI3K), a natural target of cAMP regulation, but was not affected by protein kinase C activation or inhibition. These results suggest that cAMP affects siRNA efficacy in neuroblastoma cells in PI3K-dependent manner. Dresios et al. [89] studied the RNA binding motif protein 3 (Rbm3), which has been postulated to facilitate protein synthesis under cold-stress conditions. Surprisingly they observed that over-expression of c-Myc-tagged Rbm3 protein leads to changes in the amount of miRNAcontaining complexes. Thus, Rbm3 increases protein synthesis via down-regulation of miRNA under stress conditions when global protein synthesis might be less efficient. Results of both of these reports as well as overall complexity of RNAi and its interconnectivity with other RNA-related processes indicate that RNAi is actively controlled process connected to signal transduction pathways whose de-regulation may alter (m)RNA function (Fig. 1). 5. RNA editing RNA editing is another mechanism capable of changing genetic information outside genomic DNA [90]. Deamination reactions are the major types of base modification in mammals. Conversion of cytosine (C) or adenosine (A) to uracyl (U) or inosin (I), respectively, might change coding potential of mRNA(s) and result in the synthesis of protein isoform(s) not predictable from genomic DNA sequence. Deamination is catalyzed by a superfamily of RNA-dependent deaminases. APOBEC-1, the first described and up-to-now best characterized member of CDAR (cytidine deaminase acting on RNA) subfamily, acts in a multi-subunit complex, whereas ADARs (adenosine deaminases acting on RNAs) might function as homodimers or ‘single’ polypeptides. APOBEC-1-mediated modification of apolipoprotein B mRNA in the small intestine was the first RNA editing reaction discovered in mammals. Deamination of C to U changes the glutamine codon (CAA) to a stop codon (UAA) and results in the translation of a shorter

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protein termed apoB48 [91]. APOBEC-1 is homologous to AID (activation-induced cytidine deaminase), a protein potentiating diversification of immunoglobulin gene through cytidine deamination of DNA [92]. Harris et al. [93] investigated whether APOBEC-1 by itself and other APOBEC-1 homologues have similar potential to change DNA sequence besides their role in modification of (m)RNAs. They found that at least three members of APOBEC family; APOBEC-1, -3C, and -3G are able to mutate DNA in Escherichia coli assays. Thus, DNA-directed activity of APOBEC family proteins may play a significant role also in cancer development. ADARs deaminate A to I either site-specifically or promiscuously. The best characterized example of sitespecific editing is an A to I change in mRNA encoding a subunit of an ionotropic glutamate receptor (GluR) [94]. Physiologically important editing of the Q/R site in the GluR2 subunit of the AMPA receptor changes a glutamine codon (CAG) to an arginine one (CIG); the edited arginine residue is located in a channel-forming domain and decreases the channel permeability for Ca2C ions. Editing by ADARs requires a dsRNA structure around the editing site, which can be formed by neighboring exonic and/or intronic sequences, which indicates that editing takes place in the nucleus and precedes splicing. Deregulation of ADARs in cancer might result in alterations in their enzymatic activity and substrate preferences as was shown in malignant neuroblastomas [95]. Promiscuous hyper-editing by ADARs occurs in extended, perfectly double stranded dsRNAs in which up to 50% of adenosines are converted to inosine. These hyper-edited RNAs are bound by a protein complex containing inosine-specific binding protein p54nrb, the protein associated splicing factor (PSF) and inner nuclear matrix structural protein matrin 3, which prevents export of hyper-edited RNAs from nucleus to cytoplasm [96]. Since ADAR editing takes place in dsRNA regions, which are also important for siRNA biogenesis and RNAi, possible antagonism between these processes was anticipated. Scadden and Smith [97] showed that RNAi is inhibited in Drosophila cytoplasmic extracts if the triggering dsRNA is first deaminated by ADAR2. Yang et al. [98] examined the connection between RNAi and RNA editing in mammalian system. They showed that ADAR1 and ADAR2, but not ADAR3, bind to siRNAs with high affinity and compromise efficiency of RNAi. A dsRNAbinding domain of ADAR1, which has the highest affinity for siRNAs, is required for RNAi inhibition [98]. Recently, they showed that editing of specific

adenosine residues in the miR-142 precursor by ADAR1 and ADAR2 might alter its recognition by Drosha and subsequent miR-142 maturation and function [99]. But the interplay between RNA editing and RNAi seems not to be simple antagonistic. Tudor-SN, a RISC subunit, was shown to interact specifically with hyperedited RNAs and promote their cleavage in Xenopus laevis [100]. Interestingly, a mammalian homolog of Tudor-SN, a p100 protein, was shown to be a coactivator for STAT6 transcription factor; p100 was proposed to bridge STAT6 to the basal transcription machinery. Another unsuspected observation comes from Wang et al. [101]. They identified vigilin as a component of the protein complex bound to hyper-edited RNAs. Moreover, they showed that vigilin binds not only ADAR1 but also RNA helicase A and the Ku86/70 catalytic subunit of DNA-dependent protein kinase; both proteins being involved in numerous nuclear processes including DNA repair. Finally, binding of vigilin to primary heterochromatin regions (the a- and b- satellite sequences) was confirmed by chromatin immunoprecipitation. Thus, the RNA editing machinery may act independently or in cooperation with RNAi and other RNA-related processes in the regulation of heterochromatin assembly and DNA metabolism, e.g. DNA damage response as discussed in Fernandez et al. [102]. Mechanisms of functional regulation of RNA editing factors were reported recently. RNA editing activity of ADAR1 is inhibited by sumoylation [103] and the activity of IAD is regulated by protein kinase A-mediated phosphorylation [104]. ADAR2 is regulated by inositol hexakisphosphate (IP6), an abundant signaling molecule, which is a part of ADAR2 catalytic domain and is necessary for its proper folding and function [105]. Control of RNA editing by diverse cellular signals, however, currently just being discovered, is an important pre requisite for potential deregulation during cancer development (Fig. 1). 6. Can RNA cause cancer? Not only mutations in genomic DNA are responsible for deregulation of gene expression. Epigenetic alterations in chromatin structure may influence gene expression independently of structural alterations/mutations in DNA and may directly lead to cancer cell transformation or at least importantly modify this process [7].

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mRNA surveillance mechanisms not only check mRNA ‘quality’ but are inter-connected with other cellular processes including DNA metabolism. NMD shares common regulation with DNA repair through the action of an ATM-related kinase, hSMG-1 [106]. hSMG-1 phosphorylates hUPF-1, a key protein in NMD machinery [59] as well as many proteins involved in DNA damage response due to its kinase activity similar to ATM. DNA damage response is activated early in tumorigenesis, before detectable genomic instability, to delay or prevent cancer development [107]. This brings RNA molecules more closely to the ‘heart’ of cancer generation. Thus, DNA damage response and mRNA surveillance processes may be intimately connected and mutually influence each other. Another functional connection between DNA and RNA comes from ncRNAs. Only approximately 5% of the genome output (‘transcriptome’) consists of protein-coding mRNAs [108]. Besides tRNA, rRNA and several structural RNAs with well-defined roles in processes like translation or splicing, there are variety of ncRNAs with so far poorly understood functions [24,23]. A possible connection of ncRNAs with tumorigenesis and other RNA-related processes can be documented by XIST. XIST is critically involved in the process of X chromosome inactivation and heterochromatin constitution [109]. Surprisingly, the DNA damage checkpoint protein BRCA1 was shown to be necessary for XIST action [110]. Breast and ovarian carcinoma cells lacking BRCA1 had a defect in Xi heterochromatin structure. Reconstitution of wtBRCA1 expression led to the re-appearance of focal XIST RNA staining, suggesting possible Xi perturbations and silent state destabilization in female BRCA1-deficient cells. The main BRCA1 function is to regulate doublestranded DNA break repair. Among many other proteins, BRCA1 interacts with RNA helicase A, a protein connecting DNA damage response to RNAi, RNA editing and heterochromatin regulation [101], thus illustrating the comprehensive interconnection among DNA- and RNA-based mechanisms. It is tempting to speculate that together with uncovering more ncRNAs and disclosure of their function, ncRNAs may become fundamental players in many cellular processes, including those involved in cancer. Connection between RNA and DNA-based machineries is favored by a co-localization of their particular components. The nucleus is the only site where DNA is located and where regulation of DNA metabolism takes place. Are components of RNA-regulating machineries present in the nucleus as well? The answer is yes; (1)

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RNAi, a predominantly cytoplasmic process regulating (m)RNA translation, was shown to be functional in the nucleus of mammalian cells in the process called transcriptional gene silencing and it also regulates DNA methylation [17,20]; (2) NMD, though driving PTCcontaining mRNAs for degradation mainly in the cytoplasm, begins its action in the nucleus in a process called the ‘pioneer round of translation’ [45,49,50]; (3) translation, whose bulk is performed by ribosomes in the cytoplasm, is partially localized to the nucleus as well [49,111]. RNA- and DNA-based mechanisms are spatially and functionally co-localized. How can these regulatory circuits be altered early in tumorigenesis? The answer might come from an unexpected source. Genetic elimination of T and B lymphocytes in a transgenic mouse model of inflammation-associated epithelial carcinogenesis (e.g. K14-HPV16 mice) blocked the recruitment of innate cells, subsequent tissue remodeling, angiogenesis and arrested tumorigenesis at the stage of epithelial hyperplasias [112]. This effect was abrogated by adoptive transfer of B lymphocytes or serum from wild-type HPV19 mice. These findings suggest that B lymphocytes establish chronic inflammatory states and promote de novo tumorigenesis. The underlying cellular and molecular mechanisms are not clear at present, but B lymphocyte-induced deregulation of several signaling pathways may be anticipated. It is now evident that RNA is an active player in the mechanisms of tumorigenesis. But, several fundamental questions regarding the role of RNA remain unresolved. Is mRNA, a product of DNA transcription, only an intermediate for protein synthesis or does it posses also a regulatory function? What is the exact role of (nc)RNA(s) in the regulation of DNA- and RNAbased processes? Are RNA surveillance mechanisms and their deregulation able to substantially change transcriptome output? How are these processes altered in the early stages of tumorigenesis? Are the aberrant processing of pre-(m)RNAs, the functional genome output, responsible per se for a cancer development? These questions are hard to answer at this time, but it is certain that RNA is involved in many processes, which are able to influence cancer rise and output. 7. Conclusions and perspectives Is cancer solely a genetic disease? Deregulation of fundamental cellular pathways controlling cell proliferation, differentiation, and apoptosis is central in tumorigenesis. Mutations or epigenetic alterations leading to altered expression of oncogenes and tumor

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suppressor genes are the main causes of this deregulation. However, recent evidence suggests that ncRNAs are important regulators of gene expression [72–74,76]. Also cellular mechanisms regulating coding RNA (mRNA) expression including NMD, RNAi, RNA editing and (alternative) splicing may significantly influence final protein expression pattern and genes’ penetrance. Taken together, RNA molecules are able to influence the regulation of overall gene expression and thus modify cancer cell developmental fate. All these consequences may influence future clinical therapy [113]. Since mutational analysis of coding regions of oncogenes and tumor suppressor genes is still central to cancer research, it is now apparent that non-coding regions of the human genome play an important role as well. Functional analysis of disruptions in these sequences will be necessary to fully understand their potential involvement in tumorigenesis and cancer progression. NcRNAs may be targets as well as tools in antitumor therapy. The rationale of RNA-based therapeutics is a modification (mainly down-regulation) of protein expression using RNA-targeted drugs. The most successful in vitro RNA-based approaches in cancer treatment were using either antisense oligonucleotides [114] or engineered ribozymes [115]. But results of clinical trails, especially phase III ones, were largely disappointing. New encouragement arose shortly after the discovery of RNAi and its therapeutic potentials [116,117]. As to other RNA-based therapeutics, there are still unresolved problems with specificity and delivery of these agents to the specific sites of action, i.e. cancer cells [116,118,119]. Nevertheless, despite of these difficulties, RNAi became the major player in this field, giving the most promising perspectives. To keep in mind its intrinsic role in cancer, it is not surprising that alternative splicing is also a possible target for cancer therapy [120]. Antisense-mediated modification of alternative splicing is one of the interesting possibilities how to change the pattern of alternative splicing toward the production of nonpathogenic mRNAs [121]. Therapeutic influence on NMD process may be useful in a treatment of many diseases including cancer. However, exact balance between the physiological benefits of NMD and detrimental effects in cases of specific genetic alterations have to be considered [53]. Cytostatic therapy currently available is nonspecific, has severe side effects and has low efficiency, especially in advanced tumors. Target-specific therapy as represented by, for example, proteasome inhibitor

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