ADAR1 and its implications in cancer development and treatment.

The family of adenosine deaminases acting on RNA (ADARs) regulates global gene expression output by catalyzing adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) and through interacting with RNA and other proteins. ADARs play important roles in development and disease, including an increasing connection to cancer progression. ADAR1 has demonstrated a largely pro-oncogenic role in a growing list of cancer types, and its function in cancer has been attributed to diverse mechanisms. Here, we review existing literature on ADAR1 biology and function, its roles in human disease including cancer, and summarize known cancer-associated phenotypes and mechanisms. Lastly, we discuss implications and outstanding questions in the field, including strategies for targeting ADAR1 in cancer.

Transcriptome Profiling of ADAR1 Targets in Triple-Negative Breast Cancer Cells Reveals Mechanisms for Regulating Growth and Invasion.

ADARs catalyze adenosine-to-inosine (A-to-I) editing of double-stranded RNA and regulate global gene expression output through interactions with RNA and other proteins. ADARs play important roles in development and disease, and previous work has shown that ADAR1 is oncogenic in a growing list of cancer types. Here we show that ADAR1 is a critical gene for triple-negative breast cancer cells, as ADAR1 loss results in reduced growth (viability and cell cycle progression), invasion, and mammosphere formation. Whole transcriptome sequencing analyses demonstrate that ADAR1 regulates both coding and noncoding targets by altering gene expression level, A-to-I editing, and splicing. We determine that a recoding edit in filamin B (FLNB chr3:58156064) reduces the tumor suppressive activities of the protein to promote growth and invasion. We also show that several tumor suppressor miRNAs are upregulated upon ADAR1 loss and suppress cell-cycle progression and invasion. This work describes several novel mechanisms of ADAR1-mediated oncogenesis in triple-negative breast cancer, providing support to strategies targeting ADAR1 in this aggressive cancer type that has few treatment options. IMPLICATIONS: Targeting ADAR1 and thus downstream FLNB editing and miRNA regulation represents a possible novel therapeutic strategy in triple-negative breast cancer.

An ADAR1-dependent RNA editing event in the cyclin-dependent kinase CDK13 promotes thyroid cancer hallmarks.

BACKGROUND: Adenosine deaminases acting on RNA (ADARs) modify many cellular RNAs by catalyzing the conversion of adenosine to inosine (A-to-I), and their deregulation is associated with several cancers. We recently showed that A-to-I editing is elevated in thyroid tumors and that ADAR1 is functionally important for thyroid cancer cell progression. The downstream effectors regulated or edited by ADAR1 and the significance of ADAR1 deregulation in thyroid cancer remain, however, poorly defined. METHODS: We performed whole transcriptome sequencing to determine the consequences of ADAR1 deregulation for global gene expression, RNA splicing and editing. The effects of gene silencing or RNA editing were investigated by analyzing cell viability, proliferation, invasion and subnuclear localization, and by protein and gene expression analysis. RESULTS: We report an oncogenic function for CDK13 in thyroid cancer and identify a new ADAR1-dependent RNA editing event that occurs in the coding region of its transcript. CDK13 was significantly over-edited (c.308A > G) in tumor samples and functional analysis revealed that this editing event promoted cancer cell hallmarks. Finally, we show that CDK13 editing increases the nucleolar abundance of the protein, and that this event might explain, at least partly, the global change in splicing produced by ADAR1 deregulation. CONCLUSIONS: Overall, our data support A-to-I editing as an important pathway in cancer progression and highlight novel mechanisms that might be used therapeutically in thyroid and other cancers.

ADAR1-mediated RNA editing is a novel oncogenic process in thyroid cancer and regulates miR-200 activity.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. A-to-I editing of RNA is a widespread posttranscriptional process that has recently emerged as an important mechanism in cancer biology. A-to-I editing levels are high in several human cancers, including thyroid cancer, but ADAR1 editase-dependent mechanisms governing thyroid cancer progression are unexplored. To address the importance of RNA A-to-I editing in thyroid cancer, we examined the role of ADAR1. Loss-of-function analysis showed that ADAR1 suppression profoundly repressed proliferation, invasion, and migration in thyroid tumor cell models. These observations were validated in an in vivo xenograft model, which showed that ADAR1-silenced cells had a diminished ability to form tumors. RNA editing of miRNAs has the potential to markedly alter target recognition. According to TCGA data, the tumor suppressor miR-200b is overedited in thyroid tumors, and its levels of editing correlate with a worse progression-free survival and disease stage. We confirmed miR-200b overediting in thyroid tumors and we showed that edited miR-200b has weakened activity against its target gene ZEB1 in thyroid cancer cells, likely explaining the reduced aggressiveness of ADAR1-silenced cells. We also found that RAS, but not BRAF, modulates ADAR1 levels, an effect mediated predominantly by PI3K and in part by MAPK. Lastly, pharmacological inhibition of ADAR1 activity with the editing inhibitor 8-azaadenosine reduced cancer cell aggressiveness. Overall, our data implicate ADAR1-mediated A-to-I editing as an important pathway in thyroid cancer progression, and highlight RNA editing as a potential therapeutic target in thyroid cancer.

Functional classification of long non-coding RNAs by k-mer content.

The functions of most long non-coding RNAs (lncRNAs) are unknown. In contrast to proteins, lncRNAs with similar functions often lack linear sequence homology; thus, the identification of function in one lncRNA rarely informs the identification of function in others. We developed a sequence comparison method to deconstruct linear sequence relationships in lncRNAs and evaluate similarity based on the abundance of short motifs called k-mers. We found that lncRNAs of related function often had similar k-mer profiles despite lacking linear homology, and that k-mer profiles correlated with protein binding to lncRNAs and with their subcellular localization. Using a novel assay to quantify Xist-like regulatory potential, we directly demonstrated that evolutionarily unrelated lncRNAs can encode similar function through different spatial arrangements of related sequence motifs. K-mer-based classification is a powerful approach to detect recurrent relationships between sequence and function in lncRNAs.

Genetic variation in TLR genes in Ugandan and South African populations and comparison with HapMap data.

Genetic epidemiological studies of complex diseases often rely on data from the International HapMap Consortium for identification of single nucleotide polymorphisms (SNPs), particularly those that tag haplotypes. However, little is known about the relevance of the African populations used to collect HapMap data for study populations conducted elsewhere in Africa. Toll-like receptor (TLR) genes play a key role in susceptibility to various infectious diseases, including tuberculosis. We conducted full-exon sequencing in samples obtained from Uganda (n = 48) and South Africa (n = 48), in four genes in the TLR pathway: TLR2, TLR4, TLR6, and TIRAP. We identified one novel TIRAP SNP (with minor allele frequency [MAF] 3.2%) and a novel TLR6 SNP (MAF 8%) in the Ugandan population, and a TLR6 SNP that is unique to the South African population (MAF 14%). These SNPs were also not present in the 1000 Genomes data. Genotype and haplotype frequencies and linkage disequilibrium patterns in Uganda and South Africa were similar to African populations in the HapMap datasets. Multidimensional scaling analysis of polymorphisms in all four genes suggested broad overlap of all of the examined African populations. Based on these data, we propose that there is enough similarity among African populations represented in the HapMap database to justify initial SNP selection for genetic epidemiological studies in Uganda and South Africa. We also discovered three novel polymorphisms that appear to be population-specific and would only be detected by sequencing efforts.

Genetic susceptibility to tuberculosis associated with cathepsin Z haplotype in a Ugandan household contact study.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), causes 9 million new cases worldwide and 2 million deaths annually. Genetic linkage and association analyses have suggested several chromosomal regions and candidate genes involved in TB susceptibility. This study examines the association of TB disease susceptibility with a selection of biologically relevant genes on regions on chromosomes 7 (IL6 and CARD11) and 20 (CTSZ and MC3R) and fine mapping of the chromosome 7p22-p21 region identified through our genome scan. We analyzed 565 individuals from Kampala, Uganda, who were previously included in our genome-wide linkage scan. Association analyses were conducted for 1,417 single-nucleotide polymorphisms (SNP) that passed quality control. None of the candidate gene or fine mapping SNPs was significantly associated with TB susceptibility (p > 0.10). When we restricted the analysis to HIV-negative individuals, 2 SNPs on chromosome 7 were significantly associated with TB susceptibility (p < 0.05). Haplotype analyses identified a significant risk haplotype in cathepsin X (CTSZ; p = 0.0281, odds ratio = 1.5493, 95% confidence interval [1.039, 2.320]).

Tuberculosis as a complex trait: impact of genetic epidemiological study design.

Several studies have suggested a role for human genetic risk factors in the susceptibility to developing tuberculosis (TB). However, results of these studies have been inconsistent, and one potential reason for these inconsistencies is variation in aspects of study design. Specifically, phenotype definitions and population genetic factors have varied dramatically. Since TB is a complex trait, there are many challenges in designing studies to assess appropriately human genetic risk factors for the development of TB as opposed to the acquisition of latent M. tuberculosis infection. In this review we summarize these important study design differences, with illustrations from the TB genetics literature. We cite specific examples of studies of the NRAMP1 (SLC11A1) gene and present Fisher's combined p values for different stratifications of these studies to further illustrate the impact of study design differences. Finally, we provide suggestions for the design of future genetic epidemiological studies of TB.

Multivariate association analysis of the components of metabolic syndrome from the Framingham Heart Study.

Metabolic syndrome, by definition, is the manifestation of multiple, correlated metabolic impairments. It is known to have both strong environmental and genetic contributions. However, isolating genetic variants predisposing to such a complex trait has limitations. Using pedigree data, when available, may well lead to increased ability to detect variants associated with such complex traits. The ability to incorporate multiple correlated traits into a joint analysis may also allow increased detection of associated genes. Therefore, to demonstrate the utility of both univariate and multivariate family-based association analysis and to identify possible genetic variants associated with metabolic syndrome, we performed a scan of the Affymetrix 50 k Human Gene Panel data using 1) each of the traits comprising metabolic syndrome: triglycerides, high-density lipoprotein, systolic blood pressure, diastolic blood pressure, blood glucose, and body mass index, and 2) a composite trait including all of the above, jointly. Two single-nucleotide polymorphisms within the cholesterol ester transfer protein (CETP) gene remained significant even after correcting for multiple testing in both the univariate (p < 5 x 10-7) and multivariate (p < 5 x 10-9) association analysis. Three genes met significance for multiple traits after correction for multiple testing in the univariate analysis, while five genes remained significant in the multivariate association. We conclude that while both univariate and multivariate family-based association analysis can identify genes of interest, our multivariate approach is less affected by multiple testing correction and yields more significant results.

Triglyceride levels and not adipokine concentrations are closely related to severity of nonalcoholic fatty liver disease in an obesity surgery cohort.

Although nonalcoholic fatty liver disease (NAFLD) is frequent in obesity, the metabolic determinants of advanced liver disease remain unclear. Adipokines reflect inflammation and insulin resistance associated with obesity and may identify advanced NAFLD. At the time of obesity surgery, 142 consecutive patients underwent liver biopsy and had their preoperative demographic and clinical data obtained. Liver histology was scored by the NAFLD activity score, and patients subdivided into four groups. Concentrations of retinol-binding protein 4 (RBP4), adiponectin, tumor necrosis factor-alpha (TNF-alpha), and leptin were determined approximately 1 week prior to surgery and results were related to liver histology. The prevalence of no NAFLD was 30%, simple steatosis 23%, borderline nonalcoholic steatohepatitis (NASH) 28%, and definitive NASH 18%. Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS) prevalence were 39 and 75%, respectively, and did not differ across the four histological groups (P = NS). Triglyceride (TG) and alanine transaminase (ALT) levels, strongly associated with advanced stages of NAFLD and NASH (P = 0.04). TG levels >150 mg/dl, increased the likelihood of NASH 3.4-fold, whereas high-density lipoprotein (HDL) levels predicted no NAFLD (P < 0.01). Concentrations of TNF-alpha, leptin, and RBP4 did not differ among histological groups and thus did not identify NASH; however, there was a trend for adiponectin to be lower in NASH vs. no NAFLD (P = 0.061). In summary, both TG and ALT levels assist in identification of NASH in an obesity surgery cohort. These findings underscore the importance of fatty acid delivery mechanisms to NASH development in severely obese individuals.