ANRIL: A lncRNA at the CDKN2A/B Locus With Roles in Cancer and Metabolic Disease.

A primary driver of interest in ANRIL is the large body of genomic data linking the ANRIL gene with risk of human disease. Genome-wide association studies (GWAS) have identified many disease-associated SNPs in or near the ANRIL gene ( 60 ). The CDKN2A/B locus is remarkable for the large number of associated diseases, ranging from aging and frailty to cancer to metabolic disease. Perhaps surprisingly given the validated importance of the products of the CDKN2A and CDKN2B genes in cell biology, in some cases ANRIL expression shows stronger phenotype association than protein-coding CDKN2A/B locus genes ( 4 , 48 ), linking ANRIL itself to a range of important human diseases. Studies indicate that SNPs in the ANRIL gene can impact ANRIL expression and function. The CDKN2A/B locus is associated with risk of cancer, atherosclerotic disease, type 2 diabetes, stroke, aneurysm, periodontitis, Alzheimer's disease, aging, frailty, glaucoma, endometriosis, multiple sclerosis, hypertension ( 10 , 61 ). Reviewed here are only SNPs within or downstream of the ANRIL gene; broader CDKN2A/B locus disease associations have been reviewed previously ( 10 , 62 ). Integrating information from published observations and the NCBI linkage disequilibrium database ( 63 ), we find that disease-associated SNPs in the ANRIL gene that modulate locus gene expression fall into approximately six groups (Table 1 ) defined loosely by linkage block and reported effects. Exceptions outnumber the rules, however; for nearly all groups there are reports of SNPs with different or even opposite effects. Summarized here is a generalized synopsis of the majority of reports. Group A SNPs, while located in ANRIL introns, generally impact CDKN2A/B but not ANRIL biology ( 48 , 50 , 64 ). All other SNP groups have reported impacts on ANRIL itself, but reports often describe conflicting direction of change. Some SNPs are reported to fall in enhancer regions ( 43 , 49 , 64 ) or to impact ANRIL splicing ( 8 ) or secondary structure ( 69 , 74 ). The data are incomplete. A particular weakness of the field is that although tissue-specific effects are likely to determine how polymorphisms impact disease risk, in many cases the relevant primary tissue has not been tested. Disease associated SNPs in/near the ANRIL gene that modulate locus gene expression. Groups A–F are defined loosely based on linkage disequilibrium (defined as LD>0.8 in Caucasian population in LDHap) and by predicted or tested impact on ANRIL expression or structure. Intron and exon numbers are based on 21 exons . There is no consistent global pattern with respect to SNP impact on ANRIL abundance. For most SNP groups, risk-SNPs are reported that both increase and decrease ANRIL levels in different studies. Variability may be related to differences in technique used to detect ANRIL that favor one isoform over others, cell type studied, acute and chronic biology and genetic origin of the cellular material studied, and of course the individual biology of each polymorphism. Most ANRIL SNPs fall in large linkage blocks, which are variable among different human genetic groups; in many cases the SNP tested may not be the causative SNP in the linkage block, and published linkage blocks may not apply to the material tested if not carefully matched by origin. It is entirely possible that all conflicting results are correct; for example, a CAD risk-SNP could increase pro-proliferative ANRIL isoforms in endothelial, macrophage or vascular smooth muscle cells to drive atherosclerosis, whereas a diabetes risk-SNP at the same position could decrease proliferative ANRIL isoforms in beta cells to limit beta cell mass. The complexity of the human system necessitates testing the relevant ANRIL isoforms in the relevant cell type, preferably in primary cells, in tissue- and disease-specific manner. A comprehensive review of all SNP effects is beyond the scope of this review. Some ANRIL located disease-associated SNPs impact both ANRIL expression and CDKN2A/CDKN2B expression ( 14 , 48 ); others impact ANRIL but not CDKN2A or CDKN2B ( 14 , 48 , 60 ), and still others impact CDKN2A/CDKN2B but not ANRIL ( 48 , 50 , 64 ). Some SNPs are located within predicted or proven enhancer regions ( 10 , 43 , 48 , 49 , 64 , 75 ) or miRNA binding sites ( 65 , 66 ), providing possible mechanisms of cell type specific gene regulation. Beyond regulation of ANRIL transcription, polymorphisms could impact ANRIL function by influencing relative abundance of different isoforms through RNA splicing or stability, or through altering the secondary structure or interactions of any given isoform. Several studies have identified ANRIL isoform-specific effects ( 50 , 53 , 67 , 71 , 72 ); for example, four SNPs forming an atherosclerosis risk haplotype were associated with increased expression of some, but not all, ANRIL isoforms ( 44 ). SNPs may influence the relative abundance of linear compared to circular isoforms ( 8 ). Several SNPs are reported to impact ANRIL free energy of folding, resulting in a predicted change in secondary structure, with implications for function and stability ( 48 , 69 , 74 ).

The discovery of functional noncoding RNAs has opened a kaleidoscopic world of unanticipated mechanisms extending far beyond the DNA-RNA-protein paradigm; noncoding RNAs may in fact outnumber coding RNAs ( 1 ). Long noncoding RNAs (lncRNAs) have been discovered throughout the genome; scientists are working to explore their functions in health and disease. The ANRIL lncRNA was first identified in a melanoma kindred with a large (403 kb) deletion at the CDKN2A/B locus ( 2 ). ANRIL has attracted broad attention because it is located at a genomic hotspot for disease heritability, the CDKN2A/B locus. Although protein coding genes at this locus have important well-studied roles in cell cycle regulation, data suggest that some locus disease-associated single nucleotide polymorphisms (SNPs) act through effects on ANRIL itself. Intriguingly, studies suggest ANRIL not only impacts the biology of cancer, but also has cell-type-specific roles in metabolic disease. Although ANRIL has been reviewed in the past ( 3 , 4 ), knowledge has exponentially increased in recent years. Here we review advances in ANRIL SNPs, gene regulation, cell biology, and disease roles of ANRIL .

YK, C-HH, and LA wrote and revised the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

In addition to cancer, genome-wide association studies have repeatedly and confidently identified links between the genomic region containing ANRIL and risk of developing cardiometabolic disease, including type 2 diabetes and manifestations of atherosclerosis such as CAD and stroke ( 10 , 62 ). This locus influences risk not only of classic type 2 (obesity-related) diabetes, but also with related syndromes such as gestational diabetes, transplant-associated diabetes, and cystic fibrosis related diabetes, but not risk of type 1 (autoimmune) diabetes ( 10 ). Although diabetes is a clinical risk factor for atherosclerosis, the genetic influence for these conditions at the ANRIL locus is mostly non-overlapping, with atherosclerosis SNPs located throughout the ANRIL gene, and T2D SNPs located distal to the last ANRIL exon (7). One exception is a SNP located in ANRIL exon 2, rs564398, which is associated with both T2D and CAD ( 105 ). Since CDKN2A/B locus genes are known for their roles in cell cycle regulation and cancer, and not metabolism, many questions remain as to how this locus impacts metabolic disease. Since ANRIL locus SNPs influence risk of atherosclerosis, many studies have now tested whether ANRIL gene expression is related to atherosclerosis-associated diseases. In subjects with angiographically confirmed CAD in the Leipzig heart study, specific ANRIL isoforms were positively correlated with CAD risk SNP haplotype in PBMCs, whole blood, and atherosclerotic plaque tissue ( 44 ). In the Framingham heart study, ANRIL SNPs were associated with multiple CAD-related outcomes, and showed isoform-specific ANRIL correlation in leukocytes, with short isoforms predicted to contribute to CAD pathogenesis ( 50 ). CAD risk-SNPs may regulate the relative abundance of linear and circular ANRIL isoforms ( 8 ). Intriguingly, abundance of ANRIL in circulating plasma was positively correlated with in-stent restenosis ( 53 ), but in PBMCs harvested at the time of angioplasty/reperfusion, ANRIL levels were lower in subjects with myocardial infarction, but higher in subjects with older age, diabetes, hypertension. In this cohort, ANRIL levels in PBMCs improved model prediction of subsequent left ventricular dysfunction ( 106 ). ANRIL promoter methylation may mediate an epigenetic influence on future cardiac risk; higher CpG methylation at birth was associated with higher pulse wave velocity, a marker for increased arterial stiffness indicating greater cardiovascular risk, at 9 years of age ( 31 ). Mechanisms by which ANRIL impacts atherosclerotic disease remain debated. In aortic smooth muscle cells, knockdown of ANRIL using siRNA targeting exon 1 or exon 19 revealed altered gene expression networks impacting cell proliferation, apoptosis, extracellular matrix, and inflammation ( 14 ). Atherogenic gene expression networks were regulated by ANRIL via the Alu mechanism, in which Alu motifs target ANRIL to particular gene locations, recruiting PRC complexes and altering gene methylation status ( 46 ). ANRIL may impact risk of ischemic stroke by regulating the Caspase recruitment domain 8 (CARD8) gene in endothelial cells ( 52 ). A known CAD-associated miRNA, miR-92a, may mediate some ANRIL effects; ANRIL targets GATA2, MAP1B, and ARG1 were found to require miR-92a, placing this miRNA downstream of ANRIL for some atherogenic effects ( 69 ). Finally, ANRIL is related to inflammation: ANRIL is increased by pro-inflammatory factors NF-κ B and TNF-α in endothelial cells, and ANRIL was found to bind directly to the YY1 transcription factor to mediate TNF-a induction of cytokines IL-6 and IL-8 ( 26 ). Although GWAS studies do not suggest a link between CDKN2A/B locus SNPs and obesity risk in adult populations, intriguingly, ANRIL may be a genomic site of environmental epigenetic influence on obesity. The ANRIL promoter contains CpG methylation sites that are differentially regulated across samples. In human tissues taken at birth, lower CpG methylation in infancy predicted higher fat mass at 6 years of age, as well as increased bone size, mineralization and density ( 29 , 30 ). ANRIL promoter methylation was also negatively correlated with BMI in contemporaneous samples of peripheral blood from adolescents and in adipose tissue from adults ( 29 ). Methylation of these CpG sites increased tissue abundance of ANRIL RNA, in a mechanism that might include increased activity of an estrogen response element. Functional studies in a liposarcoma cell line showed that transcription factor binding to an adjacent ERE was enhanced by methylation, and estradiol increased ANRIL expression ( 29 ). Multiple SNPs in different linkage blocks at the CDKN2A/B locus are associated with T2D risk; evidence in human populations suggests these SNPs impact pancreatic islet mass or function ( 10 ). Despite the fact that the T2D risk SNPs are located in or near the ANRIL gene, the field has largely assumed the effect was mediated by the protein coding genes at the locus, due to extensive published work implicating p16INK4A in the regulation of beta cell mass ( 10 ). However, although studies have found no association between CDKN2A/B T2D SNPs and transcript level of p14ARF, p15INK4B , or p16INK4A in human islets ( 10 , 107 ), an age-dependent positive association was identified between distal T2D risk-SNPs (group F in Table 1 ) and ANRIL expression ( 68 ). On the other hand, a T2D risk-SNP in ANRIL exon 2 (group C in Table 1 ) was associated with reduced ANRIL expression, again with no change in p14ARF, p15INK4B , or p16INK4A expression ( 14 , 48 ); however, these studies were carried out in blood rather than islets. In human islets, this exon 2 SNP was shown to remove a CpG methylation site; risk allele was associated with reduced islet insulin content but no change in locus gene expression ( 108 ). Risk allele at this SNP was associated with impaired beta cell proliferation response to high glucose ( 68 ). In a study relevant to diabetic retinopathy, high glucose exposure increased ANRIL expression in human retinal epithelial cells ( 25 ). In ANRIL was found to increase expression of VEGF, a critical element of the neovascularization that is central to damage from retinopathy, via a mechanism involving PRC2 and miR200b ( 25 , 109 ).

Studies suggest the ANRIL lncRNA influences risk of a number of diseases, including many types of cancer as well as metabolic disease. Current understanding of ANRIL biology indicates the primary function of this lncRNA is to regulate gene expression, both locally at CDKN2A/B as well as across the genome, via mechanisms including chromatin modulation, transcription factor binding, and miRNA regulation. Knowledge concerning ANRIL function in cancers is more solid and advanced than for metabolic tissues. Mechanisms by which SNPs influence ANRIL abundance remain uncertain and require more study; how DNA methylation regulates ANRIL in cancers also will benefit from more study. Much remains to be learned about the structural complexity of ANRIL ; how the various identified linear and circular isoforms impact tissue biology to modulate disease risk is mostly unknown. There is an urgent need for deeper understanding of how ANRIL isoforms modulate cellular function in human organs and tissues, and to explore the differing roles of ANRIL in cancer and metabolic disease. Given the advent of RNA therapeutics, and the broad disease relevance of ANRIL , it is possible that these studies may lead to future disease prevention and treatment.

The evolutionary development of the human ANRIL gene has been studied by comparative analysis of the genomes of 27 organisms including non-mammalian vertebrates, non-placental mammals, non-primate placental mammals, and primates ( 15 ). ANRIL originated in ancestors of the Eutherian (placental) mammalian clade. Initially the gene contained only a few exons; over time, ANRIL underwent clade-specific evolution, adding exons in many mammals but losing exons in rodents. The full 21 exon gene is present only in simians. ANRIL genes contain many repeat elements, both intronic, and exonic; evidence suggests that transposon activity has mediated many of the observed evolutionary changes in exon presence or absence, location, sequence, conservation, and structure, as well as introduction of splice sites ( 15 ). Early ANRIL variants were likely not transcribed or functional ( 15 ). One hallmark of functional RNAs is splice signals at intron/exon boundaries. In simians, 191 intron/exon boundaries contained canonical splice signals, while 20 did not. In lower mammals, however, only about half of intron/exon junctions contained identifiable splice signals ( 15 ). This finding suggests that as ANRIL gained exons, and exon sequences became more conserved across species, it also increased the number of splice signals and gained functionality. Taken together, data suggest that ANRIL may be functional only in simians, and that functionality may have been introduced by transposon activity ( 15 ).

Many studies show that ANRIL functions in cells to regulate gene expression via chromatin modification. Acting in cis, ANRIL interacts with both PRC-1 and−2 to mediate epigenetic transcriptional repression of neighboring genes CDKN2A and CDKN2B , through mechanisms involving histone modification and chromatin remodeling ( 13 , 21 , 33 ). ANRIL interacts with PRC1 component CBX7 to recruit PRC1 to the p14ARF and p16INK4A loci, silencing the CDKN2A locus by H3K27-trimethylation ( 21 ). At CDKN2B, ANRIL was shown to recruit SUZ12, a subunit of the PRC2 ( 13 ). ANRIL also interacts with PRC-associated protein YY1 ( 46 ). Intriguingly, the structural conformation of the methyl-lysine binding pocket in the chromodomain of CBX7, which interacts with H3K27-trimethylation to cause chromatin compaction, is influenced by allosteric RNA-protein binding with ANRIL ( 47 ). However, despite this well-documented repression of other locus genes by ANRIL , a positive correlation between ANRIL (both short and long isoforms), CDKN2A and CDKN2B RNA abundance has been frequently reported, suggesting transcriptional co-regulation of these genes predominates in many tissues ( 8 , 10 , 14 , 16 , 44 , 48 – 51 ). ANRIL also acts in a PRC1/2 dependent mechanism to repress distant genes in trans ( 32 , 46 ). Trans regulation by ANRIL may be dependent on Alu motifs, which are found both in ANRIL transcripts and in the promoters of ANRIL target genes ( 46 ). This mechanism was shown to regulate the CARD8 gene in endothelial cells ( 52 ). Polycomb group proteins, which are highly enriched near Alu motifs across the genome, are recruited to target gene promoters upon ANRIL over-expression. In support of this concept, silencing ANRIL impacts expression of a large number of genes across the genome ( 14 ). Separate from chromatin modification, ANRIL is reported to regulate Wnt signaling by binding to SOX2, increasing transcriptional activity of the WNT/β-catenin pathway ( 39 ). ANRIL also influences gene expression via miRNA networks. ANRIL regulates miRNAs both at the epigenetic level, through regulation of miRNA transcription, and through direct binding to miRNAs, acting as a miRNA “sponge.” In gastric cancer cells, ANRIL epigenetically silences miR-99a/miR-449a through a PRC2 mechanism ( 22 ). In general, expression of ANRIL and its target miRNAs are negatively correlated in tissues and cell lines ( 22 , 53 – 57 ). ANRIL has been described as having pro-oncogenic effects by sponging miRNAs (see below for more details). On the other hand, circ ANRIL containing exons 5-6-7 was found to lack miRNA sponge activity ( 6 ). Inhibition of miRNAs can reverse the effects of ANRIL knockdown. ANRIL has broad impacts on cell biology, including influence over proliferation, senescence, apoptosis, extracellular matrix remodeling, and inflammation ( 14 ). In cancer, ANRIL -miRNA interactions regulate networks of downstream targets of miRNAs, promoting an oncogenic role for ANRIL in cell proliferation, metastasis, invasion, radio-resistance, drug-induced cytotoxicity and apoptosis, involving many different signaling pathways ( 22 , 53 – 57 ). Specifically, repression of cell cycle inhibitors p14ARF, p15INK4B, and p16INK4A increases proliferation, decreases senescence, and contributes to the DNA damage response ( 13 , 21 , 33 ). PRC-mediated epigenetic repression of Kruppel-like factor 2 (KLF2) influences proliferation and apoptosis ( 40 , 58 ). Cooperation between ANRIL and PRC-associated YY1 increases TNF-alpha dependent inflammatory mediators (IL-6, IL-8) through NF-kB ( 26 ). ANRIL influences the cellular response to oxidative stress through a miR-125a regulation of MCL-1 ( 59 ). Circular ANRIL species were found to regulate ribosome biogenesis in vascular smooth muscle cells ( 6 ).

Abundance of ANRIL species is determined by promoter transcriptional activity, splicing decisions, and RNA stability (Figure 2 ). Like other genes, ANRIL promoter activity is influenced by epigenetic control and transcription factor occupancy. Intriguingly, epidemiological findings suggest that epigenetic regulation of ANRIL , through promoter methylation, has important long-lasting consequences for tissue function ( 29 – 31 ). As such, ANRIL regulation is one mediator of the impact of early life environmental signals on adult human health. Summary of regulation and functions of the ANRIL lncRNA. (Left) Some of the known mechanisms by which ANRIL abundance is regulated, at the transcriptional and post-transcriptional levels. On the (Right) , a selection of known ANRIL cellular functions are depicted. We apologize for observations not included in this summary image. The ANRIL structural prediction in the center is of a common long-isoform of ANRIL, and was generated from Gruber et al. ( 28 ). Epidemiological and experimental findings demonstrate that methylation of the ANRIL promoter region regulates ANRIL gene expression and has functional importance. The first exons of ANRIL and p14ARF are separated by only 300 bp, in head-to-head antisense orientation; the intergenic region between them is a bidirectional promoter ( 4 , 32 , 33 ). In silico analysis of ENCODE ChromHMM data ( 34 ) revealed that this region is enriched for both promoter and enhancer activity, and DNAse I hypersensitivity, across multiple cell types, suggesting this is a regulatory region. This region is bound by CTCF, usually considered to be a transcriptional repressor, insulating promoters from enhancer activity. Oddly, CTCF binding at the ANRIL promoter was associated with active-chromatin mark histone H3K4 trimethylation ( 35 ). CTCF binding, and ANRIL and p14ARF expression, were inhibited by methylation of local CpG islands and increased by demethylation. Knockdown of CTCF prevented the demethylation-induced expression of ANRIL and p14ARF, confirming that CTCF is a methylation-sensitive positive regulator of ANRIL promoter activity ( 35 ). Additional evidence supports the functional importance of CpG sites for ANRIL promoter activity, locus gene expression and transcription factor binding. Mutagenesis of the CpG sites affects both ANRIL and p14ARF promoter activity ( 29 , 30 ). Methylation status of several CpG differentially methylated regions at ANRIL in umbilical cord tissues was positively associated with abundance of linear but not circular ANRIL , but inversely associated with p14ARF and p16INK4a expression ( 29 ). CpG methylation also affects other transcription factors binding at the ANRIL promoter to regulate downstream gene expression, such as interferon gamma, SMAD3/4 and ERα ( 29 , 30 ). Methylation status of CpG islands around the p16INK4A transcription start site was also shown to coordinate transcription of ANRIL and p16INK4A in human cells ( 36 ). Given the multiple protein complexes binding across different CpG sites, and variable quantitative impact of individual CpG region mutagenesis on ANRIL isoforms and locus gene expression, regulation appears to be complex. ANRIL expression is influenced by cellular processes such as genotoxic stress, tumorigenesis, senescence, and inflammation. Activity at the bidirectional promoter region upstream of both ANRIL and p14ARF genes is influenced by the critical cell cycle regulator E2F1 ( 32 , 33 ). In response to the genotoxic stress of DNA damage, E2F1 transcriptionally activates ANRIL in an ATM-dependent manner ( 33 , 37 ). In this case, ANRIL is thought to promote cell growth by suppressing locus INK-family inhibitors after DNA repair is complete, allowing re-entry into cell cycling ( 33 ). The ANRIL promoter was also responsive to E2F1 in cancer cells ( 32 ). In addition to E2F1, several known potent oncogenes regulate ANRIL expression in various cancers. In lung cancer, c-MYC binds to an E-box in the ANRIL promoter and induces ANRIL expression ( 38 ). In nasopharyngeal carcinoma, transcription factor SOX2 was shown to bind directly to the ANRIL promoter and activate transcription of ANRIL and its downstream effector β-catenin ( 39 ). In liver cancer, SP1 binds the ANRIL promoter and positively regulates ANRIL transcription ( 40 ). On the other hand, TET2, a tumor suppressor in human gastric cancer, binds to the promoter region of ANRIL and regulates expression of ANRIL as well as p16INK4a, p15INK4b, and p14ARF ( 41 ). Transcription regulation of ANRIL is involved not only in cell DNA damage and oncogenesis, but also in disparate processes such as cell senescence and inflammation. In senescence, oncogenic Ras was found to reduce expression of ANRIL ( 13 , 21 , 42 ). In inflammation, STAT1 activates the ANRIL locus in vascular endothelial cells has been reported; CAD-associated ANRIL SNP rs10757278, located in a known downstream enhancer region, disrupts the STAT1 binding site and modulates IFN-γ induced ANRIL expression via stimulation ( 43 ). Intriguingly, the binding of STAT1 at this enhancer exerts cell-type specific regulation of ANRIL expression: repression in lymphoblastoid cells lines, but activation in HUVEC cells ( 43 ). In sum, data support an important role for cell-type specific transcriptional regulation of the ANRIL lncRNA in a range of cellular processes and outcomes. Cell type dependent variation in abundance of different ANRIL isoforms suggests that splicing may be a point of regulation ( 8 , 44 ). Almost nothing is known about ANRIL splicing decisions. Disease-associated ANRIL gene polymorphisms have shed light on this process. In lymphocytes, the coronary artery disease (CAD) associated SNP rs10757278 (intron 12) correlates with abundance of certain circular (14-5 and 4-6) and linear (exon 1-2, but not 18-19, containing) isoforms ( 8 ). The rs10757278 A allele was found to inhibit skipping of exon 15, promoting circ ANRIL species ending in exon 14 ( 8 ). Mechanisms regulating ANRIL splicing require further study. Determinants of ANRIL transcript longevity and stability remain uncertain, but miRNAs can participate. ANRIL , downregulated following Kaposi's sarcoma associated herpesvirus (KSHV) infection, contains multiple seed matches for KSHV miRNAs. Forced miRNA expression decreased ANRIL abundance, and miRNA pull-down experiments confirmed a direct interaction. In addition, KSHV latency associated proteins vFLIP and vCyclin also decreased ANRIL abundance, suggesting post-transcriptional miRNA-dependent and independent regulation ( 45 ).